XXIV ciclo – NEUROGLOBIN: MOLECULAR, CELLULAR AND...

UNIVERSITÁ DEGLI STUDI ROMA TRE

Facoltà di Scienze Matematiche, Fisiche e Naturali

Dipartimento di Biologia Scuola Dottorale in Biologia

Sezione di Biologia Applicata alla Salute dell’Uomo

– XXIV ciclo –

NEUROGLOBIN: MOLECULAR, CELLULAR AND BIOMEDICAL ASPECTS

NEUROGLOBINA: ASPETTI MOLECOLARI,

CELLULARI E BIOMEDICI

PhD student: Dr. Elisabetta De Marinis

Tutors: University of Roma Tre CSIC Instituto Cajal Prof. Paolo Ascenzi Prof. Luis Miguel Garcia-Segura

Prof. Maria Marino Dr. Maria Angeles Arevalo

Coordinator of Biology applied to Human health section: Prof. Paolo Visca

A.A. 2010/2011

INDEX

SUMMARY……………………………………………………………........i RIASSUNTO................................................................................................iv 1. BACKGROUND …...…………………………………………………...1 1.1 Globins: an overview ……………………………….……….….….….1 1.2 Neuroglobin, an hexa-coordinated globin ..………………….………3

1.2.1 Neuroglobin localization ………………………………..……….5 1.2.2 Neuroprotective functions of neuroglobin ……….………..…….6 1.2.3 Cellular mechanisms underlying neuroglobin neuroprotective effects…………………………………………………………….…….8

2. AIM……………………………………………………………………..14 3. SEX STEROID HORMONES AS ENDOGENOUS MODULATORS OF NEUROGLOBIN LEVELS IN NEURONAL CELLS ……………16 3.1 Introduction ………………………………………………………….16 3.2 Results ……………..………………………...………………..............19

3.2.1 17β-estradiol effect on neuroglobin protein levels…………….19 3.2.2 Androgen effect on neuroglobin levels ………………………..22 3.2.3 Estrogen receptor involvement in 17β-estradiol-induced neuroglobin levels …….……………………………………………..23 3.2.4 Mechanisms involved in the 17β-estradiol-induced increase of neuroglobin levels ….………………………………………………..26

3.3 Discussion ……………………………………………………...……..32

4. INVOLVEMENT OF NEUROGLOBIN IN 17β-ESTRADIOL-INDUCED PROTECTION AGAINST NEUROTOXICITY ................35 4.1 Introduction ………………………………………………………….35 4.2 Results ..……………………………………………………………….36

4.2.1 Neuroglobin is involved in 17β-estradiol-induced protection against H2O2-mediated apoptosis ...………………………………….36 4.2.2 17β-estradiol changes neuroglobin intracellular localization …39 4.2.3 17β-estradiol promotes neuroglobin-cytochrome c association.42

4.3 Discussion .……………………………………………………………47 5. INVOLVEMENT OF NEUROGLOBIN IN 17β-ESTRADIOL ANTI-INFLAMMATORY EFFECTS ....………………………………………50 5.1 Introduction .…………………………………………………………50

5.2 Results …………………………………………………….…………..51 5.2.1 17β-estradiol effect on neuroglobin protein levels in mouse primary cortical astrocytes ..…………………………………………51 5.2.2 Effect of lipopolysaccharide on neuroglobin protein levels ......54 5.2.3 Neuroglobin involvement in 17β-estradiol-mediated anti-inflammatory effects against lipopolysaccharide ……………...……58

5.3 Discussion .……………………………………………………………60 6. CONCLUSION ……………..…………………………………………62 REFERENCES ………….....…………………………………………….66 ACKNOWLEDGMENTS .........................................................................84 APPENDIX A. Materials and Methods .............................................(available on CD-ROM) APPENDIX B. Peer reviewed publications .......................................(available on CD-ROM)

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SUMMARY Although globins are among the best-investigated vertebrate proteins,

no other distinct types of globins have been identified so far in this taxon. In 2000, it was identified a third globin type in humans and rodents.

This protein was predominantly expressed in the brain, and therefore they have called it neuroglobin (Ngb).

The discovery of Ngb aroused a great interest among scientific community inducing to consider heme-globins not only as mere O2 storage/delivery proteins.

Ngb is a highly conserved protein, with an evolutionary rate that is about threefold slower than that of myoglobin and hemoglobin. Thus, Ngb has remained largely unchanged during evolution, pointing to an important role of this protein.

In particular, an important role in neuroprotection has been addressed to Ngb, especially against ischemia and oxidative stress-related neurodegenerative diseases, but many divergences between in vivo and in vitro experimental approaches still render unclear the biological role of this novel globin.

Several mechanisms underlying Ngb neuroprotective effects have been proposed. Indeed, Ngb has been hypothesized: (i) to act as an O2 buffer, (ii) to facilitate O2 diffusion to the mitochondria, (iii) to catalyze the formation and the decomposition of reactive nitrogen and/or oxygen species, and (iv) to be part of intracellular signaling pathways by inhibiting the dissociation of GDP from Gα proteins and triggering the release of the Gβγ complex, and by reducing cytochrome c. Although it is unlikely that Ngb has so many distinct roles, there is no doubt that Ngb displays a protective function(s) in the brain.

The emerging neuroprotective role of Ngb arises the challenge to investigate the mechanisms able to modulate its expression. Indeed, a significant contribution to highlight the role played by Ngb in neuroprotection could derive from the identification of Ngb endogenous modulator(s) (e.g., neuroactive hormones and neurotransmitters), but, as far as we know, no Ngb involvement in the hormone signal transduction pathways has been identified yet. Thus, aim of this project is to approach to the knowledge of Ngb physiological role (i) identifying Ngb endogenous modulator(s), (ii) identifying the molecular mechanisms responsible of Ngb expression and induction, and (iii) the role played by Ngb in neuroprotective signaling pathways.

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In the first part of the project was evaluated if sex steroid hormones may act as endogenous modulators of Ngb levels in SK-N-BE human neuroblastoma cell line and in mouse primary hippocampal neurons. The reported results indicate that physiological concentration of the estrogen 17β-estradiol (E2), but not androgens, acts as endogenous modulator of Ngb in both cell models. This effect is mediated by estrogen receptor β (ERβ) via genomic and extranuclear signals involving p38/MAPK pathway.

In the second part, the involvement of Ngb in the neuroprotective effects of E2 against H2O2-induced toxicity has been investigated in SK-N-BE cells.

Indeed, E2 exerts a protective effect against the H2O2-induced injury, and requires ERβ. E2 pretreatment impairs H2O2-induced caspase-3 and PARP activation, enhancing cell viability. However, in Ngb-silenced SK-N-BE cells E2 was unable to counteract the H2O2-induced decrease in cell number and the activation of the pro-apoptotic cascade suggesting that Ngb can be regarded as part of signals activated by E2 to exert neuroprotective effects, definitely validating the role played by Ngb as an anti-apoptotic neuroprotective globin.

Thus, it has been clarified the Ngb sub-cellular localization to understand how this novel globin can intercept the apoptotic pathway.

Therefore, in SK-N-BE cells has been demonstrated that Ngb is expressed in the nucleus, mitochondria and is scattered in the cytoplasm.

E2 reallocates Ngb mainly at mitochondria strengthened the hypothesis that Ngb directly interferes with the intrinsic pathway of apoptosis, being mitochondria just the starting site of this process. Indeed, it has been assessed that Ngb co-immunoprecipitates with cytochrome c in mitochondrial fraction and this association is enhanced pretreating SK-N-BE cells with E2, suggesting that E2-induced reallocation of Ngb facilitates Ngb-cytochrome c interaction. Remarkably, E2 pretreatment before the addition of H2O2 strongly enhances Ngb co-immunoprecipitation with cytochrome c. This E2 effect is stronger during oxidative stress condition rather than in basal condition and requires ERβ activity. Thus, the mechanism underlying Ngb protection against H2O2 stress is the interception of the intrinsic pathway of apoptosis interfering directly with cytochrome c release.

In the light of Ngb neuroprotective potential, linked with E2-mediated signals, the third part of the project was aimed to characterize the E2-mediated regulation of Ngb levels in astrocytes, where E2 exerts a well known anti-inflammatory effect. In mouse primary cortical astrocytes E2 affects Ngb expression at physiological concentration. The effect of E2 on Ngb levels specifically requires ERβ, confirming also in astrocytes the

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direct involvement of ERβ in Ngb modulation, as already reported for human neuroblastoma SK-N-BE and mouse hippocampal neurons. Although it has been established that Ngb is an E2-inducible protein and that, from a functional point of view, the E2-mediated Ngb upregulation allows to promote the E2-induced outcomes, also a putative role of Ngb as a compensatory protein responding to challenging stimuli, must be considered. The finding that lipopolysaccharide (LPS) is able to increase Ngb protein levels, although with a lesser degree compared with E2, further provides an additional contribution to understand the role of Ngb also as offsetting protein. Interestingly, although both E2 and LPS are able to increase Ngb protein levels, a negative cross-talk between ERs and LPS-induced signal (i.e., NFκB) seems to be present. In fact, ERα-activated signals (which are not involved in E2-mediated Ngb upregulation) block LPS-mediated Ngb increase, whereas on the other hand, LPS impairs the ERβ-induced upregulation of Ngb protein levels. Therefore, the co-activation of ERα and ERβ is pivotal to regulate Ngb expression in presence of LPS-activated signals (i.e., NFκB).

Despite LPS, via NFκB, is able to increase Ngb levels, the role of this globin is not addressed to promote LPS effects, as observed for E2. Indeed, Ngb seems to be pivotal to mediate the E2 anti-inflammatory effects (i.e., inhibition of IL-6 and IP-10 synthesis), since Ngb knocking down prevents the protective effect of E2.

As a whole, the well known neuroprotective effects elicited by E2 may, at least in part, be explained by an enhanced Ngb expression in neurons and astrocytes. The principal role played by Ngb in the brain could be the reduction of neuronal death by resetting the trigger level of apoptosis and inhibition of pro-inflammatory molecules expression, leading to the onset of physiological response to stress. E2 acts to accelerate Ngb neuroprotective effect rapidly enhancing its protein levels in both neurons and astrocytes.

In addition, the possibility that other hormones and neurotransmitters may upregulate Ngb levels in brain a potential new opportunity for the development of neuroprotective strategies and drugs against stroke damage, inflammation, neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s disease), excitotoxicity, and injuries related to oxygen or glucose deprivation.

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RIASSUNTO Sebbene le globine siano le proteine più studiate tra i vertebrati e siano

note molte varianti funzionali della emoglobina e della mioglobina, per molto tempo non sono state identificate altre tipologie di globine.

Nel 2000 è stata identificata una terza globina nell’uomo e nelle specie murine. Tale proteina è stata chiamata neuroglobina (Ngb) in quanto identificata inizialmente nel sistema nervoso.

La scoperta della Ngb ha fatto sorgere un forte interesse nella comunità scientifica, portando a considerare le eme-globine non soltanto come proteine atte al trasporto o al mantenimento dei livelli intracellulari di O2.

La Ngb è una proteina altamente conservata, presenta infatti un tasso di evoluzione circa tre volte più lento di quelle delle più note mioglobina ed emoglobina. Quindi la Ngb è rimasta largamente immutata durante l’evoluzione, indicando un ruolo importante di tale proteina.

In particolare, è stato evidenziato un importante ruolo della Ngb nella neuroprotezione, specialmente nei confronti dell’ischemia e delle patologie legate allo stress ossidativo. Tuttavia i meccanismi alla base di tale ruolo neuroprotettivo sono ancora poco chiari, a causa delle molte divergenze tra gli approcci sperimentali in vivo e in vitro ad oggi adottati.

Tra i meccanismi alla base della funzione neuroprotettiva è stato suggerito che la Ngb (i) possa agire come sensore dell’ O2; (ii) possa facilitare la diffusione di O2 ai mitocondri; (iii) possa catalizzare la sintesi e la detossificazione delle specie reattive dell’ossigeno e dell’azoto; e (iv) possa far parte delle vie di segnalazione intracellulari inibendo la dissociazione del GDP dalle proteine Gα, e quindi determinare rilascio del complesso Gβγ. Inoltre, è stato ipotizzato che la Ngb possa agire riducendo il citocromo c ossidato che è alla base dell’attivazione della via intrinseca dell’apoptosi. Sebbene sia improbabile che la Ngb possa avere così tanti e distinti meccanismi d’azione, non v’è dubbio sulle sue funzioni protettive nel cervello.

La funzione neuroprotettiva emergente della Ngb spinge a comprendere quali siano i meccanismi in grado di modularne l’espressione. Infatti, un significativo contributo a comprendere il ruolo della Ngb nella neuroprotezione potrebbe derivare dall’identificazione di possibili modulatori endogeni (ad esempio neurotrasmettitori o ormoni neuroattivi) che al momento non sono noti.

Pertanto, lo scopo di questo progetto è quello di approfondire il ruolo fisiologico della Ngb (i) ricercando possibili modulatori endogeni; (ii) identificando i meccanismi molecolari alla base dell’espressione e

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induzione della Ngb, e (iii) identificando il ruolo della Ngb nelle vie di segnalazione alla base della neuroprotezione.

Nella prima parte del progetto è stato valutato se gli ormoni sessuali steroidei potessero agire come modulatori endogeni della Ngb nella linea cellulare SK-N-BE (neuroblastoma umano) e in neuroni primari murini di ippocampo. I risultati indicano che concentrazioni fisiologiche di 17β-estradiolo (E2), il più attivo fra gli estrogeni, inducono un aumento di Ngb in entrambi i modelli cellulari. Tale effetto però non è mediato dagli androgeni.

E2 aumenta i livelli di Ngb attraverso l’isoforma β del recettore degli estrogeni (ERβ) che agisce attraverso meccanismi genomici e non genomici, tra i quali risulta coinvolta la via di segnale p38/MAPK.

Nella seconda parte, è stato studiato il coinvolgimento della Ngb negli effetti neuroprotettivi di E2 verso la tossicità indotta da H2O2 nelle cellule SK-N-BE.

Infatti E2, attraverso ERβ, esercita un effetto protettivo rispetto a un danno da H2O2. Il pretrattamento con E2 inibisce l’attivazione delle proteine pro-apoptotiche caspasi-3 e della proteina PARP indotte da H2O2, aumentando così la sopravvivenza cellulare. Tuttavia, nelle cellule in cui la Ngb è silenziata l’effetto di E2 viene meno, indicando che la Ngb esercita un ruolo chiave nei meccanismi di protezione di E2, inibendo, in particolare, l’attivazione delle vie apoptotiche.

Per comprendere come la Ngb possa interferire nella via apoptotica è stata innanzitutto esaminata la sua localizzazione intracellulare.

Nelle cellule SK-N-BE la Ngb è espressa nel nucleo, nei mitocondri e nel citosol. Il trattamento con E2 induce una rilocalizzazione della Ngb soprattutto a livello mitocondriale, rafforzando l’idea che la Ngb possa interferire direttamente con la via intrinseca dell’apoptosi, essendo il mitocondrio proprio il sito di innesco di tale processo.

Infatti la Ngb co-immunoprecipita con il citocromo c nella frazione mitocondriale; tale associazione aumenta in presenza di E2, e in maniera ancor più significativa quando E2 è somministrato prima di un trattamento pro-apoptotico, come H2O2. Anche questo effetto dell’E2 è mediato da ERβ.

Quindi, il meccanismo alla base della protezione della Ngb dallo stress indotto da H2O2 è l’inibizione della via intrinseca dell’apoptosi interferendo direttamente con il rilascio al citosol del citocromo c che attiverebbe la via intrinseca dell’apoptosi.

Alla luce del potenziale neuroprotettivo della Ngb, associato con le vie di segnale attivate da E2, la terza parte del progetto è stata indirizzata a caratterizzare la regolazione della Ngb, mediata da E2, negli astrociti, dove E2 ha un ruolo ben noto nella protezione dall’infiammazione.

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Negli astrociti corticali primari murini E2, a concentrazioni fisiologiche, aumenta i livelli proteici di Ngb. Anche in questo tipo cellulare l’effetto di E2 è mediato specificatamente da ERβ, come visto anche nelle cellule SK-N-BE e nei neuroni primari di ippocampo.

Sebbene sia stato stabilito che la Ngb sia una proteina inducibile e che, da un punto di vista funzionale, l’aumento di Ngb promuova gli effetti protettivi di E2, anche un possibile ruolo della Ngb come proteina regolatoria, che risponda a segnali di stress, deve essere considerato. Per contribuire alla comprensione di tale ruolo della Ngb, gli astrociti primari sono stati trattati con la molecola pro-infiammatoria lipopolisaccaride (LPS). I risultati indicano che LPS è in grado di aumentare i livelli proteici di Ngb.

Sebbene sia E2 che LPS siano in grado di aumentare i livelli di Ngb, è interessante notare la presenza di una interazione contrastante tra gli ERs e i segnali attivati da LPS (NFκB). Infatti l’attivazione di ERα blocca l’aumento di Ngb indotto da LPS; d’altra parte, LPS inibisce l’induzione di Ngb mediata da ERβ. Quindi, la co-attivazione di ERα e ERβ risulta essere fondamentale per regolare l’espressione di Ngb in presenza delle vie di segnale attivate da LPS.

Nonostate LPS, attraverso NFκB, sia in grado di aumentare i livelli di Ngb, la funzione di tale globina non risulta essere quella di promuovere gli effetti di LPS, come osservato nel caso di E2. Infatti il silenziamento della Ngb negli astrociti previene gli effetti anti-infiammatori di E2 (inibizione della sintesi delle molecole pro-infiammatorie IL-6 e IP-10).

In conclusione, i ben noti effetti neuroprotettivi esercitati da E2 possono dipendere, almeno in parte, dall’aumentata espressione di Ngb nei neuroni e negli astrociti. Il principale meccanismo protettivo della Ngb nel cervello può essere ascritto alla riduzione della morte neuronale, attraverso l’inibizione della via intrinseca dell’apoptosi, e all’inibizione dell’espressione delle principali molecole pro-infiammatorie, garantendo l’attivazione dei meccanismi fisiologici di risposta allo stress. E2 agisce accelerando la comparsa degli effetti neuroprotettivi della Ngb aumentandone i livelli proteici sia nei neuroni che negli astrociti.

Inoltre, la possibilità che anche altri ormoni e neurotrasmettitori possano aumentare i livelli proteici di Ngb nel cervello costituisce una nuova opportunità per lo sviluppo di interventi terapeutici e farmacologici in grado di contrastare l’ischemia, la neuroinfiammazione, e le malattie neurodegenerative correlate allo stress ossidativo o alla deprivazione di ossigeno o glucosio.

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1. BACKGROUND

1.1 Globins: an overview

Globins are small globular metalloproteins typically consisting of about 150 amino acids. They are phylogenetically ancient molecules whose intricate adaptive evolution is demonstrated by their widespread occurrence in bacteria, fungi, plants, invertebrate and vertebrate animals. Most known globins fulfill respiratory functions, supplying the cell with adequate amounts of O2 for aerobic energy production via the respiratory chain. Together with O2 transport and storage, these proteins display also well documented (pseudo-)enzymatic properties such as cytoprotection against reactive oxygen species and NO scavenging (Bolognesi et al., 1997; Ascenzi et al., 2007; Hoy and Hargrove, 2008; Vinogradov and Moens 2008).

Globins contain a heme prosthetic group (Fe-protoporphyrin IX) which is a chemically highly active group that has been involved in biological processes as soon as life appeared. To remain soluble and active heme needs to be surrounded by a hydrophobic environment that is obtained by a few structural 3D protein arrangements.

Heme participates in many biochemical functions among which are the binding and transport of gaseous ligands (O2, NO, CO), scavenging of free oxidant species, oxido-reduction, or oxygen-sensing. The specificity of these functions is directed by the structure of the protein to which it is associated, and also by the hexa- or penta-coordination of iron atom. The most recognized is the penta-coordinated ligation (e.g., in hemoglobin; Hb, and in myoglobin; Mb), but also iron hexa-coordination is widespread, occurring in some plant and bacterial Hbs, and also in invertebrate and vertebrate nerve globins (Trent and Hargrove, 2002, Dewilde et al., 2001).

Despite the enormous diversity in their primary and quaternary structures (amino acid sequences and aggregation states) globin proteins exhibit a characteristic tertiary structure (the “globin fold”) suggesting a common ancestry (Weber and Fago, 2004). The ancestral globin gene appears to have evolved 18,000 million years ago, when O2 started to accumulate in the atmosphere suggesting that the protein’s original function may have been to scavenge toxic O2, CO and NO gases (Hardison, 1999). The evolutionary, proteome-related implication is that globins provide opportunity to trace structure-function relations in a single protein family throughout the five Kingdoms of living organisms.

Indeed, the classical globin molecule is characterized by the canonical three-on-three α-helical Mb fold. This molecule, ca 150 residues long, is

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characterized by a heme group surrounded by 8 helices designated A through H from the N to the C terminal. Helices A, B, C and E are on distal side of the heme and helices F, G and H on the proximal side. Moreover, an important feature of this structure is represented also by a pattern of 37 hydrophobic residues at conserved and solvent-inaccessible positions (Wajcman et al., 2009, and literature therein).

The tetrameric (e.g., Hb) and monomeric (e.g., Mb) vertebrate globins stand in contrast with the enormous variation in structure and function encountered in non-vertebrate globins. Microorganism globins form three families: (a) chimeric flavoproteins, where heme-carrying globin domains are linked to oxido-reductive FAD-dependent domains, (b) truncated Hbs with short polypeptide chains, and (c) bacterial Hbs (Weber and Vinogradov, 2001; Wajcman and Kiger, 2002). Plant Hbs comprise symbiotic Hbs (“legHbs”) from root nodules of leguminous plants that harbor symbiotic nitrogen-fixing bacteria, as well as non-symbiotic Hbs, that may be involved in several metabolic pathways (Arredondo-Peter et al., 1998).

Despite the large variation in structure and sequence, the tertiary structures of truncated Hbs and mini-Hb (from nemertean) are subeditings of the three-on-three a-helical sandwich, highlighting the striking structural plasticity of the globin fold.

Invertebrate Hbs illustrate phenomenal structural and functional diversity varying from single-chain monomers with molecular masses of 11.2 kDa, to multisubunit and multidomain crustacean Hbs, extracellular (3600 kDa) annelid Hbs where each molecule consists of 144 O2 binding globin chains and a number of heme-free “linker” chains, and include even larger (12,000 kDa) complexes found in some bivalve mollusks.

These proteins serve a wide range of functions apart from transporting and storing O2, such as controlling in vivo O2 levels, protection against sulphide, and enzymatic (oxidase and peroxidase-like and superoxide dismutase) activities (Weber and Vinogradov, 2001).

Notably, several intracellular globins have been found in the invertebrate and vertebrate nervous system possibly sustaining the consume of large amounts of metabolic energy, which requires a continuous supply of O2 (Geuens et al., 2004; Burmester and Hankeln, 2008). The role of globins in the brain seems to be pivotal to support nervous system functions during temporary periods of hypoxia, which may follow environmental or pathologic insults, therefore avoiding serious damages to the nervous system (Burmester et al., 2007; Williams et al., 2008; Cheng et al., 2009; Mitz et al., 2009; Avivi et al., 2010).

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Invertebrate nerve globins have been observed in several Phyla such as Annelida, Arthropoda, Echiura, Mollusca, Nemertea, and Nematoda (Weber and Vinogradov, 2001; Burmester et al., 2002). The first to observe a globin in nerves was Lankester in 1872, when he recorded the brilliant red color of the ganglia of the polychaetous annelid Aphrodite aculeata (Wittemberg et al., 2002). Since then, globins have been found in or associated with nervous tissues of several other invertebrates and are now referred to as nerve globins. They are, however, not common and can be present or absent in closely related species (Yonetani et al., 2002; Wittemberg and Wittemberg, 2003).

Although the coordination of the heme iron atom differs being penta- (e.g., nerve globin of Aplysia spp.) or hexa-coordinated (e.g., nerve globin of Tellina alternata), the oxygen affinities of the invertebrate nerve globins are all quite moderate and similar to those of vertebrate Mb (Wittemberg et al., 1965; Wittemberg, 1992; Geuens et al., 2004).

In some species, nerve globins reach a millimolar local concentration, which is likely sufficient to facilitate O2 diffusion and storage (Vandergon and Riggs, 2008; Geuens et al., 2004; Hundahl et al., 2006a; Burmester and Hankeln, 2008).

Only in 2000, the first vertebrate nerve globin, named neuroglobin (Ngb), has been identified in neuronal tissues of mice and humans (Burmester et al., 2000). More recently, cytoglobin (Cygb), globin E, globin X, and α- and β-chains of Hb have been reported to be expressed in the vertebrate nervous system (Burmester et al., 2002; Kugelstadt et al., 2004; Roesner et al., 2005; Fuchs et al., 2006; Biagioli et al., 2009). 1.2 Neuroglobin, a hexa-coordinated globin

Although globins are among the best-investigated vertebrate proteins and several functional variants of the Hb subunits are known, no other distinct types of globins have been identified so far in this taxon.

In 2000, it was identified a third globin type in humans and rodents. This protein was predominantly expressed in the brain, and therefore they have called it neuroglobin (Ngb) (Burmester et al., 2000). The human Ngb gene (NGB), located on chromosome 14q24, has a unique exon-intron structure.

In the databases of anonymous mouse and human complementary DNAs, Burmester and coworkers found partial globin-like sequences that do not correspond to any known Hb or Mb. They cloned and sequenced the coding regions of the human and mouse cDNAs and the genomic region of

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the human gene. The mouse and human gene each code for proteins of 151 amino acids (17kDa) that are 94% identical; this homology is higher than the conservation between the orthologous Hbs or Mbs of these species (77±85% identity) and within the uppermost range of more than 1100 proteins compared between man and mouse (Makalowski et al., 1996). Although the proteins clearly belong to the globin superfamily, they share little amino-acid sequence similarity with vertebrate Mbs (ca 21% identity) and Hbs (ca 25% identity), suggesting a distinct evolution and function. Moreover, Hbs and Mbs of vertebrates are thought to have diverged about 550 million years ago (Goodman et al., 1975), but the lineage leading to the vertebrate Ngb must be older. A distinct position of Ngb is also suggested by its unique exon-intron structure. There is weak but consistent support for an association of Ngb with the intracellular globins of the annelids Aphrodite aculeata (Dewilde et al., 1996) and Glycera dibranchiata (Zafar et al., 1990). The Aphrodite globin displays the highest similarity with Ngb (30% amino-acid identity). Taking into account the nerve-based expression of both the Aphrodite globin and the vertebrate Ngb, both may be functionally and Ngb represents a distinct protein family that diverged early in metazoan evolution, probably before the Protostomia/Deuterostomia split.

Reliable sequences of Ngb from 11 mammals, 1 bird and 4 teleost fish species have been determined (Burmester et al., 2004); in all species apart from the trout, Ngb seems to be present as a single-copy gene. Structural analysis showed that Ngb sequences are consistent with the globin fold template, given the conservation of aminoacids involved in heme binding and ligand interactions, i.e., PheB10, PheCD1, TyrCD3, ValE11, LeuF4, ValFg1, ValFG3 and PheG5 (Pesce et al., 2003; Vallone et al., 2004).

Ngb promoter region contains several putative Sp1-binding sites and at least three transcription starting points, but lacks a TATA box motif (Burmester et al., 2000).

The NGB gene has three introns in correspondence of nucleotides encoding the helix B (position B12-2), of helix E (E11-0) and of helix G (G7-0). The introns at helix B and G are conserved in Hbs and Mbs of vertebrates and many other taxa (Dixon and Pohajdak, 1992; Hardison, 1996), but the central intron at helix E is unprecedented. On the basis of protein structural considerations, however, the presence of a central intron in ancient globin genes was previously proposed to be exactly at this position (Go, 1981). Therefore, the vertebrate globin gene ancestor might have displayed a 3 intron / 4 exon structure. Alternatively, the central E11-0 intron of neuroglobins may represent a case of independent intron acquisition (Hankeln et al., 1997; Logsdon et al., 1998).

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Figure 1.1 Structure of human Ngb. His E7 and His F8 coordinating the Fe atom are highlighted (Pesce et al., 2003; PDB code 1OJ6). Molecular graphic image was produced using the UCSF chimera package (Pettersen et al., 2004)

1.2.1 Neuroglobin localization As described above, Ngb was so-named due to its first identification in

neuronal cells (Burmester et al, 2000). Indeed Ngb expression is widespread among brain areas and in peripheral nervous system.

The expression pattern of Ngb mRNA and protein in mammal brain and the intracellular location have been debated, and the lack of consistency in the observations has been attributed to technical differences.

Ngb is found at relatively high concentrations in highly metabolically active cells and certain specialized cells, such as neurons of the hypothalamus, and particularly in retinal cells where its concentration has been estimated to be up to 100 μM (Schmidt et al., 2003; Bentmann et al., 2005; Fago et al., 2008; Hundahl et al., 2010).

In the central nervous system (CNS) Ngb is ubiquitously distributed in the olfactory bulb, the cerebral cortex (layers II-VI), subcortical regions (e.g., hippocampus, thalamus, hypothalamus, amygdala), the brain stem and the cerebellum, revealing that the expression of Ngb is a general feature of nerve cells (Reuss et al., 2002; Hankeln et al., 2004; Hundal et al., 2010).

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The spinal cord also expresses Ngb in its gray substance and Ngb mRNA is also found in the peripheral nervous system, in sensory and autonomic ganglia (Reuss et al., 2002).

However, Ngb mRNA and/or protein expression was recognized also in non-neuronal tissues such as the gastrointestinal tract and some normal and tumoral tissues (e.g., breast, lung, kidney, lymphocytes) and endocrine tissues (Burmester et al., 2000; Moens and Dewilde, 2000; Reuss et al., 2002; Wystub et al., 2003; Burmester and Hankeln, 2004; Hankeln et al., 2004; Hankeln et al., 2005; Ostojić et al., 2006; Fordel et al., 2007a; Emara et al., 2010; Oleksiewicz et al., 2011).

In all CNS areas Ngb is expressed in neurons, but in contrast with previously reported data (Sun et al., 2001; Laufs et al., 2004; Hankeln et al., 2004) some groups demonstrated the presence of Ngb also in astrocytes and glioblastoma cells (Chen et al., 2005; Emara et al., 2009; Mitz et al., 2009; Dong et al., 2010; DellaValle et al., 2010; Emara et al., 2010). Intriguingly, Ngb expression increases in reactive astrocytes, within regions associated with the most severe pathology and the astroglial scar, in murine models of traumatic brain injury, cerebral malaria, and autoimmune encephalitis (DellaValle et al., 2010).

Among authors, a debate about the subcellular localization of Ngb has arisen. Until few years ago Ngb was recognized only in cytoplasmatic regions: in neurons Ngb mRNA and protein were consistently detected in perikarya and axonal processes (Zhang et al., 2002; Reuss et al., 2002; Wystub et al., 2003; Geuens et al., 2003) as well as perimitochondrially in axonal varicosities and terminal synapses (Schmidt et al., 2003; Hankeln et al., 2004). However, using immunolocalizion and electron microscopy, recent studies demonstrate that in neurons Ngb is also expressed in the inner wall of mitochondria and in the cell nucleus (Hundahl et al., 2010).

1.2.2 Neuroprotective functions of neuroglobin Emerging experimental works suggest that Ngb expression is

protective against hypoxic/ischemic injury in brain. Alteration of gene expression approaches were applied to address whether Ngb is neuroprotective. The first report showed that antisense-mediated knock-down of Ngb rendered cortical neurons more vulnerable to hypoxia, whereas overexpression of Ngb conferred protection of cultured neurons against hypoxia (Sun et al., 2001). Similar effects were observed in human neuroblastoma cell lines SH-SY5Y: Ngb overexpression enhanced cell

7

survival under condition of anoxia or glucose deprivation (Fordel et al., 2007b; Shao et al., 2009).

In animal stroke models, intracerebral administration of a Ngb-overexpressing adeno-associated virus vector, significantly reduced infarct size in rats after medial cerebral artery occlusion (MCAO), and the outcome was reversed when Ngb antisense oligonucleotide was applied (Sun et al., 2003).

Using Ngb-overexpressing transgenic (Ngb-Tg) mice, it was shown that the cerebral infarct size after MCAO was reduced by 30% compared with wild type, and the same protective effect was observed also in transient focal cerebral ischemia where hypoxia-induced mitochondrial aggregation and neuronal death were abolished (Khan et al., 2006; Khan et al., 2008). Reduction of brain infarction in Ngb-Tg mice can be sustained up to 14 days after ischemia compared with wild type controls (Wang et al., 2008), suggesting that Ngb overexpression is neuroprotective against transient focal cerebral ischemia, although the possible mechanisms need to be further characterized (Yu et al., 2009).

Moreover, the neuroprotective role of endogenous Ngb has been supported by its knockdown in cell cultures, which renders cortical neuronal cultures more susceptible to hypoxia (Jin et al., 2008) and decreases viability of neuroblastoma cells under oxidative stress (Ye et al., 2009).

However, some questions have been raised concerning the capacity of Ngb to provide general protection to neurons in vivo (Hundahl et al., 2006b). Contradictions in data derived from in vivo experiments almost certainly arise, at least in major part, from differences in the nature, severity, and duration of challenge used in the various studies. Any proposed mechanism of action of Ngb, in neurons, must thus consider not only its capacity to provide a level of protection to many cell types in the brain but also account for its very nonuniform distribution in the brain.

In addition, using progeny of crosses between Ngb-Tg mice and transgenic mice expressing a mutant form of human amyloid precursor protein (APPSw,Ind) associated with familial Alzheimer’s Disease, it has been shown that Ngb overexpression inhibits Alzheimer’s disease-related raft aggregation and membrane polarization, and associated neuronal death. Moreover, these double-transgenic mice produce reduced amounts of amyloid-β peptides Aβ(1-40) and Aβ(1-42), and amyloid plaques in the brain (Khan et al., 2007). These findings indicate also an important protective role of Ngb in neurodegenerative processes.

It has been well documented that Ngb expression in the brain is confined to metabolically active, oxygen-consuming cell types and mitochondria comprise a central locus for energetic perturbations and

8

oxidative stress (Burmester et al., 2004). Experimental works have shown that overexpression of Ngb promotes cell survival against Aβ toxicity, Aβ- and hypoxia-induced mitochondrial dysfunction and aggregation, and neuron death (Khan et al., 2008; Liu et al., 2009).

Moreover, the influence of Ngb on cell death after oxidative stress was also evaluated, indicating that Ngb overexpression protects against hydrogen peroxide (H2O2)-induced cell death, attenuating H2O2-induced reactive oxygen species (ROS) / reactive nitrogen species (RNS) accumulation and lipid peroxidation, decreasing H2O2-induced mitochondrial dysfunction and apoptosis, and promoting overall cell survival (Fordel et al., 2006; Li et al., 2008).

All these processes are at least in part associated with the prevention of apoptotic cell death and are closely related to recent hypothesis and evidences that indicate a possible interaction between Ngb and cytochrome c and thus a direct involvement of this globin in the apoptotic pathway (Fago et al., 2008; Brittain et al., 2010a; Brittain et al., 2010b; Raychaudhuri et al., 2010).

The emerging neuroprotective role of Ngb issues the challenge to investigate the physiological factors and mechanisms able to modulate its expression. Indeed, a significant contribution to highlight the role played by Ngb in neuroprotection could derive from the identification of Ngb endogenous modulator(s) (e.g., neuroactive hormones and neurotransmitters), but, as far as we know, no Ngb involvement in the hormone signal transduction pathways has been identified yet.

1.2.3 Cellular mechanisms underlying neuroglobin neuroprotective effects

Although Ngb is the best-investigated ‘novel’ globin type, its exact

physiological role is still uncertain. As reported above, Ngb has remained largely unchanged during evolution, pointing to an important role of this protein. Several functions of Ngb have been proposed: (i) Ngb may exert a Mb-like role, enhancing O2 supply to the mitochondria of the metabolically active neurons; (ii) Ngb may scavenge damaging ROS or RNS, which are generated for example by the respiratory chain; (iii) Ngb may detoxify harmful excess of NO to nitrate at normoxia or produce NO for signaling functions from nitrite at hypoxia for the control of blood pressure; (iv) Ngb may be involved in a signal transduction pathway, e.g. by inhibiting the dissociation of GDP from G protein α; (v) Ngb may be part of a redox

9

process that is instrumental in preventing apoptosis via reduction of cytochrome c (Burmester and Hankeln, 2009).

All these functions have been supported by some experimental data or based on analogy with other globins. However, it is unlikely that Ngb could fulfill so many distinct roles.

Initially, Ngb was suggested to be an O2 storage and transport protein, performing a function in neural tissue similar to that of Mb in muscle (Burmester et al., 2000). Indeed, Ngb expression is upregulated under hypoxic conditions (Sun et al., 2001; Sun et al., 2003; Schmidt-Kastner et al., 2006; Li et al., 2006), as is also observed for Mb and Hb.

Many in vivo studies performed in Ngb-overexpressing transgenic mice, in primary neurons, and in cultured cell lines sustain the neuroprotective role played by Ngb, although the cellular mechanisms remain poorly defined and still controversial.

Until recently, it has generally been accepted that any intracellular globin has a similar function to that of Mb, either storing O2 for hypoxic phases (e.g. in diving mammals) or facilitating O2 diffusion from the capillaries to the respiratory chain in the mitochondria. However, within the past years it has become evident that Ngb may carry out various other functions (Burmester and Hankeln, 2009).

The heme iron in both the ferrous and the ferric forms is directly bound not only to the proximal HisF8 (as canonical in all globins), but also to the distal HisE7 (figure 1.1). This internal coordination on the distal side implies that only upon rupture of the bond with HisE7 an externally added ligand (e.g., O2) can be bound; and therefore competition with the internal ligand is an additional component involved in determining the overall affinity for the gaseous ligands.

Therefore, ligand binding to the heme-Fe atom of Ngb needs the formation of the transient penta-coordinated HisF8-Fe species; the reversible intramolecular hexa-to-penta-coordination transition of the heme-Fe atom modulates exogenous ligand-binding properties of Ngb (Kiger et al., 2004; Pesce et al., 2004; Brunori and Vallone, 2006).

The O2 affinity of Ngb depends on several factors, including the relative binding rates of O2 with the heme sixth position and the competing distal histidine residue (Trent et al., 2001; Dewilde et al., 2001), and the redox state of the cell which controls the formation or cleavage of an internal disulfide bond between Cys46 CD7 (seventh residue on the inter-helix region between helices C and D) and Cys55 D5 (Hamdane et al., 2003; Hamdane et al., 2004). The presence of the S–S bond could perturb the three-dimensional structure of the CD-D (inter-helix region between helices C and D, extending to the end of helix D) region and affect the

10

location of the neighboring E-helix, thus modulating the binding of the endogenous HisE7 ligand to the heme group (Hamdane et al., 2003; Pesce et al., 2003). As a consequence, in ferrous Ngb, the distal histidine residue dissociation rate increases by a factor of 10 with respect to the protein form without the disulfide bond (Hamdane et al., 2003; Smagghe et al., 2006), leading to an effective increase in O2 affinity by the same factor (Fago et al., 2004). Moreover, determination of the O2 affinity of Ngb is particularly difficult due to its tendency to autooxidize with a half time, in air, that varies from 20 to 3 min depending on pH (Trent et al., 2001; Dewilde et al., 2001; Fago et al 2004).

However, the O2 half saturation pressure (P50) was estimated from kinetics and also by direct measurements to range between 2 and 20 torr depending on pH, temperature, and the redox state of the cell (Nienhaus and Nienhaus, 2007; Burmester and Hankeln, 2009, and literature therein).

The low average concentration of Ngb in the brain (ca 1 μM), its tendency to autooxidize and its relatively low O2 affinity under physiological conditions seem to suggest a primary role other than O2 storage and/or facilitated diffusion (Brunori and Vallone, 2007).

Moreover, O2 storage or diffusion to or from the heme group of Ngb may be assisted by the presence of a large protein matrix cavity/tunnel (approx. 120Å3), located between the heme distal site and the EF interhelical hinge and connected to the protein surface (Pesce et al., 2003; Vallone et al., 2004). The cavity may be reshaped after ligand binding, which may allow trapping of harmful ROS or RNS (Nicolis et al., 2007).

In summary, three observations are in conflict with the Ngb role in O2 storage and transport: (i) The high autooxidation rate of ferrous Ngb argues against a function as an O2 storage protein; (ii) The reported P50 values in the range of 2-20 torr also shed doubt on this conjecture; under normal conditions, the oxygen pressure in the brain varies between 8 and 40 torr, and normal intracellular oxygen pressure of the neurons is maintained as long as the tissue pressure is kept above 10 torr (Nienhaus and Nienhaus, 2007), thus to supply O2 to the tissues, the protein should have a P50 below normal tissue pressures; (iii) Finally, the average Ngb concentration in the brain is in the micromolar range which is way too low to contribute to the oxygen supply. For comparison, Mb concentrations are much higher in heart and red muscle tissue, about 200 μM and 100-400 μM, respectively. However, locally high Ngb concentrations of up to 100 mM have been found in the retina, where they could contribute significantly to the O2 supply (Schmidt et al., 2003).

Ngb binds also other gaseous ligands (e.g., NO, and CO), and displays (pseudo-)enzymatic properties (e.g., O2-mediated NO detoxification). In

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particular, the increase in the concentration of NO up to the low micromolar range as observed under ischemic conditions (Lipton, 1999; Nicolis et al., 2007), may be contrasted with its reaction with ferrous oxyNgb (oxygenated Ngb, NgbFeIIO2), yielding metNgb (ferric-Ngb, NgbFeIII) andNO3− through a hame-bound ONOO− (peroxynitrite) intermediate (Brunori et al., 2005):

NgbFeIIO2 + NO → NgbFeIIIOONO− →NgbFeIII + NO3− (1) Also metNgb reacts with NO (Herold et al., 2004) to generate the

stable NgbFeIINO species by reductive nitrosylation. This form of Ngb is a good scavenger of the ONOO−, which is produced in significant amounts under ischemic conditions by the reaction of NO with O2 (Lipton, 1999), as follows (Herold et al., 2004):

NgbFeIII + NO → NgbFeIINO (2) NgbFeIINO + ONOO− → NgbFeIIINO (3) NgbFeIIINO → NgbFeIII + NO (4) The association rate of HbNO and ONOO− is almost two orders of

magnitude smaller, which suggests that NgbNO may function in vivo as an efficient scavenger of this harmful compound (Herold et al., 2004).

Most importantly, it has been reported that metNgb, unlike metMb and metHb, does not react with hydrogen peroxide (H2O2) or ONOO− to the ferryl form (Herold et al., 2004; Nicolis et al., 2007), which is extremely oxidizing. This property is obviously beneficial under conditions of oxidative stress.

However, at low O2 conditions ferrous Ngb may react with NO2– which is normally present at fairly high concentrations within mammalian tissues (0.1-10 M) (Bryan et al., 2005) where it is a major end-product of the cellular messenger NO. This reaction results in the formation of NO and metNgb (Petersen et al., 2008):

NgbFeII + NO2− + H+ → NgbFeIIINO + OH− → NgbFeIII + NO + OH−

(5) Therefore, the protective role of Ngb against NO in vivo is

controversial (Giuffrè et al., 2008; Jin et al., 2008; Kakar et al., 2010). Although in vitro Ngb does not react with H2O2 (Herold et al., 2004; Nicolis et al., 2007) and the Ngb-NO2– adduct reacts with H2O2 facilitating the nitration of aromatic substrates (Nicolis et al., 2007), the correlation

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between ROS and RNS formation/decomposition and Ngb expression in vivo is debated (Fordel et al., 2007a; Burmester and Hankeln, 2009).

The intermediate O2 affinity, which is in the range of the partial pressures measured in the brain, suggests yet another function for Ngb, namely that of an oxygen sensor that reports hypoxic conditions to signal transduction chains, which trigger processes that protect the cell against the detrimental effects of low oxygen pressure. The Morishima group reported that human Ngb acts as a redox-coupled regulator of signal transduction (Wakasugi et al., 2003). Ferric human Ngb (but not NgbO2) was found to associate with the GDP-bound Gα subunit of heterotrimeric G proteins. Ferric Ngb was proposed to act as a guanine-nucleotide dissociation inhibitor (GDI) for Gα (Wakasugi et al., 2003), preventing exchange of GDP by GTP, which is required for Gβγ subunit reassociation with the Gα subunit. The free Gβγ subunit then activates signal transduction pathways that protect against oxidative stress. In particular, it has been demonstrated that Ngb binds two members of the Rho GTPase family, Rac1 and Rho A, as well as the Pak1 kinase, a key regulatory assembly and Rho-GDI-GTPase signaling complex activity under hypoxia. Thus, it has been hypothesized that Ngb may play a protecting role in neuronal cells by inhibiting the dissociation of the GTPase Rac-1 from its endogenous GDI, thus preventing the hypoxia-induced actin polymerization and microdomain aggregation (Khan et al., 2008).

Although intriguing, the Ngb-Gα interaction was questioned by Burmester and coworkers (Burmester and Hankeln, 2004) who failed to find sequence similarities between Ngb and other regulators of G proteins. On the other hand, Wakasugi and Morishima (Wakasugi and Morishima, 2005) found that zebra fish ferric Ngb is inactive toward G proteins, which argues against the general relevance of a GDI function. Ngb was also reported to interact with flotillin-1, a lipid raft microdomain-associated protein, and with the cysteine protease inhibitor cystatin C, suggesting that Ngb modulates the intracellular transport of cystatin C to protect against cell death caused by oxidative stress (Wakasugi et al., 2005). For these interactions, the in vivo relevance was disputed because of limited overlap of expression between these proteins (Hankeln et al., 2004; Nienhaus and Nienhaus, 2007).

Another possible involvement in cellular signaling has been proposed by Fago and coworkers (Fago et al., 2006). During ischemic episodes, cells can enter an apoptotic pathway via the partial release of ferric mitochondrial cytochrome c into the cytoplasm. Ferric cytochrome c is a required component of the caspase-cascade activating apoptosome (Nienhaus and Nienhaus 2007, and literature therein). Ngb may be able to reduce small

13

amounts of cytochrome c leaking from damaged mitochondria, thereby suppressing apoptosis (Fago et al., 2008; Brittain et al., 2010a; Brittain et al., 2010b; Raychaudhuri et al., 2010).

At present the physical association between Ngb and cytochrome c has been only suggested by computer modeling approach (Brittain et al., 2010a; Brittain et al., 2010b).

Although it is unlikely that Ngb has so many distinct roles, there is no doubt that Ngb displays a protective function(s) in the brain (Burmester and Hankeln, 2009).

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2. AIM

Hb and Mb are without doubt the best-known proteins, and most intensively studied in biology. They have the ability to bind O2 for the purpose of transport or storage, thereby enhancing the O2 carrying capacity of the blood (Hb) or the availability of O2 inside muscle cells (Mb). For a long time, Hb and Mb were considered as the only globins found in vertebrates.

The discovery of Ngb in 2000 by Burmester and colleagues (Burmester et al., 2000) aroused a great interest among scientific community inducing to consider heme-globins not only as mere O2 storage/delivery proteins.

Until recently, it has generally been accepted that any intracellular globin has a similar function to that of Mb, either storing for hypoxic phases (e.g. in diving mammals) or facilitating O2 diffusion from the capillaries to the respiratory chain in the mitochondria. However, within the past ten years it has become evident that certain globins may carry out various other functions. For example, maize globin regenerates NAD+, which is used to promote glycolysis in low oxygen environments (Sowa et al., 1998). Some prokaryotic globin and globin domains are involved in O2 sensing (Hou et al., 2001). Escherichia coli flavoHb decomposes NO, conferring resistance to NO poisoning and aconitase inactivation (Gardner et al., 1998). A similar function has been demonstrated for mammalian Mb, which scavenges NO and thus protects cellular respiration (Flögel et al., 2001). Mb may also be involved in decomposition of harmful ROS (Flögel et al., 2004).

Ngb is a highly conserved protein, with an evolutionary rate that is about threefold slower than that of Mb and Hb (Burmester et al., 2004). Thus Ngb has remained largely unchanged during evolution, pointing to an important role of this protein.

In particular, an important role in neuroprotection has been addressed to Ngb, especially against ischemia and oxidative stress-related neurodegenerative diseases, but many divergences between in vivo and in vitro experimental approaches still render unclear the biological role of this novel globin (Hundahl et al., 2006b).

The emerging neuroprotective role of Ngb arises the challenge to investigate the mechanisms able to modulate its expression. Indeed, a significant contribution to highlight the role played by Ngb in neuroprotection could derive from the identification of Ngb endogenous modulator(s) (e.g., neuroactive hormones and neurotransmitters), but, as far as we know, no Ngb involvement in the hormone signal transduction pathways has been identified yet.

15

Thus, aim of this project is to approach to the knowledge of Ngb physiological role by i) identifying Ngb endogenous modulator(s); ii) identifying the molecular mechanisms responsible of Ngb expression and induction and iii) assessing the role played by Ngb in neuroprotective signaling pathways.

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3. SEX STEROID HORMONES AS ENDOGENOUS MODULATORS OF NEUEROGLOBIN LEVELS IN NEURONAL CELLS

3.1 Introduction

The last decade has seen an exponential increase in evidence for structural, cellular, and molecular sex differences in the brain that can be described as true dimorphisms, defined as the occurrence of two forms in the same species. These include regions of human and animal brains that are important for cognition, memory, and affect, such as the hippocampus, amygdala, and cortex, and for regions controlling sensorimotor and reward systems (Gillies and McArthur, 2010, and literature therein). Although these biological sex differences are clearly important from a physiological point of view to maintain homeostasis, if the system is challenged by external factors, such as stress and disease, different organizations in circuitries in male and female brains will respond differently to environmental challenges (endogenous or exogenous) and emerge as different vulnerabilities to behavioral and neurological disorders. Several neurodegenerative and mental diseases differ markedly in their prevalence, symptomatology, progression, and/or severity between the sexes. Indeed, beneficial effects of estrogens are reported in several mental, namely schizophrenia and depression (Cyr et al., 2000; Cyr et al., 2002; Kulkarni et al., 2002; Osterlund et al., 2005; Morisette et al., 2008) and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, multiple sclerosis and ischemic stroke (Henderson, 1997; Cyr et al., 2000; Kompoliti, 2003; Wooten et al., 2004; Czlonkowska et al., 2006; Shulman and Bhat, 2006). In contrast, in the onset and progress of these diseases postmenopausal women fare worse than men (Swaab, 2004; Morisette et al., 2008; Gillies and McArthur, 2010).

Without doubt, 17β-estradiol (E2), the most active and relevant estrogen, has been pinpointed as a critical protective factor in females that gives them the advantage in diseases prevalent in men, whereas its rapid decline after menopause may forfeit this advantage.

E2 exerts its effect through two cognate estrogen receptors (ERs), referred to as ERα and ERβ. Both ER subtypes are found in the brain with a differential distribution, in neurons and glia (Laflamme et al., 1998; Behl, 2002). In addition 20 ERα and 10 ERβ splice variants have been reported (Zhao et al., 2005; Morisette et al., 2008). ERs are members of a large family of nuclear receptors acting as ligand-activated transcription factors (Ascenzi et al., 2006; Acconcia and Marino, 2011, and literature therein).

17

Although there are contradictory reports on the relative contributions of ERα and ERβ to the neuroprotective effects of estrogens in most disease models (Brann et al., 2007; Suzuki et al., 2009), evidence is emerging that both ERs have protective capacity, but they operate via different mechanisms and possibly in different time frames. For example, in ischemic brain injury and experimental autoimmune encephalomyelitis, ERα expression is induced early, whereas ERβ is induced later (Suzuki et al., 2002; Tiwari-Woodruff and Voskuhl, 2009).

In summary, E2 might directly control the transcription of genes that code for proteins that modulate neuronal survival. These proteins might enhance neurotrophic support, suppress apoptosis and affect neuronal structure (McEwen, 2002; Marin et al., 2008; Morisette et al., 2008; Vasudevan and Pfaff, 2008; Marin et al., 2009; Gillies and McArthur, 2010).

An important role in neuroprotection is also played by the membrane-bound fraction of ERs. E2 can induce cellular events that are not mediated by a direct or indirect interaction with DNA. These non-genomic actions induced by E2 are often very rapid (within minutes or even seconds) and could be associated with activation of second messenger pathways via a direct interaction of E2 with palmitoylated and plasma-associated ERs existing in a variety of neural and extraneural targets to mediate steroid action (Levin, 2002; Acconcia et al., 2004; Acconcia et al., 2005a; Marino et al., 2005; Ascenzi et al., 2006; Marino and Ascenzi, 2006; Morisette et al., 2008, Acconcia and Marino, 2011). Association between E2 and membrane-bound ER involves the rapid stimulation of Src-protein tyrosine kinase, mitogen-activated protein kinase (MAPK) pathways, and phosphatidylinositol-3 kinase (PI3K)/AKT pathways (Singer et al., 1999; Dhandapani and Brann, 2002). Moreover, genomic and non-genomic actions of ER signaling may not act independently but are shown to converge on target genes (Bjornstrom and Sjoberg, 2005).

By modulating intracellular signaling processes, E2 could indirectly and rapidly affect the transcription of genes that are controlled by various downstream effectors. Some of the signal transduction pathways that are targets of E2 and ERs are directly involved in the control of neuronal survival.

The predominant circulating sex hormone in male after puberty is testosterone, which is common precursor, in both male and female, of E2 and the most active androgen dihydrotestosterone (DHT) that derive from aromatization (catalyzed from CYP19A1 aromatase) and reduction (catalyzed from 5α-reductase) of testosterone, respectively (Nguyen et al., 2010, and literature therein).

18

Both testosterone and DHT bind to androgen receptor (AR), which exists in several splicing isoforms, and belongs to the nuclear receptor superfamily. Beyond genomic effects, AR, as well as ERs, can have actions that are independent of their interactions with DNA. ARs interacts with signal transduction proteins (e.g., MAPKs) in the cytoplasm causing rapid changes in cell function independent of changes in gene transcription, such as changes in ion transport. Regulation of signal transduction pathways by AR can indirectly lead to changes in gene transcription, for example, by leading to phosphorylation of other transcription factors (Nguyen et al., 2010, and literature therein).

Epidemiological studies indicate that men experience a significant decrease in levels of testosterone in blood and brain due to normal aging (Nguyen et al., 2010, and literature therein). Age-related androgen loss in men adversely affects muscle and bone mass, sexual arousal, sperm production, and brain functions such as mood, memory, and cognition (Nguyen et al., 2010, and literature therein). Recent data also suggest that low levels of testosterone in aging men may be one of several risk factors for the development of Alzheimer’s disease (AD) (Rosario et al., 2004; Pike et al., 2009). Androgens have many beneficial actions in the CNS and the decrease in their levels may contribute to age-related neurological deficits and Alzheimer’s disease. For example, testosterone decreases levels of Aβ (Gouras et al., 2000; Ramsden et al., 2003a). In addition, androgens are positive regulators of neuronal plasticity in the spinal nucleus of the bulbocavernosus, excitability in the CA1 region of hippocampus, and spine density in hippocampus (Nguyen et al., 2010, and literature therein).

Androgens also prevent retraction or increase the length and size of neurites from motor neurons. Other neurotrophic effects of testosterone include cell differentiation, neurogenesis, and development of neurons in the hippocampus, and motor and autonomic systems (Nguyen et al., 2010, and literature therein). In addition, androgens increase the speed of regeneration of injured axons of motor neurons in young and adult rats (Nguyen et al., 2010, and literature therein). Androgens are also endogenous regulators of viability in neurons challenged with toxic insults in adult animals. Adult male rats and mice depleted of endogenous androgens by orchidectomy exhibit increased vulnerability to hippocampal neuron loss induced by excitotoxins, an effect that can be reversed by treatment with DHT (Ramsden et al., 2003b). In primary neuron culture paradigms, testosterone and related androgens attenuate cell death induced by serum deprivation, Aβ, and H2O2 (Nguyen et al., 2010, and literature therein). However, androgens can fail to protect neurons and even exacerbate injury in response to some forms of injury

19

such as ischemia, mitochondrial toxin 3-nitropropionic acid, and muscimol-induced excitotoxicity (Nguyen et al., 2010, and literature therein). Why androgens are neuroprotective against some insults, but not others is unclear. Indeed, androgen neuroprotection is not well characterized in terms of either specificity or mechanism (Nguyen et al., 2010, and literature therein).

Moreover, often the protective effect of androgens were ascribed to testosterone, without the evaluation of which testosterone metabolite (i.e., E2 or DHT) were the effective protective molecule (Nguyen et al., 2010, and literature therein). Thus, it must be taken into account that many of the effects of androgen actually could be exerted by E2.

As a whole, these results sustain a neuroprotective role of sex steroid hormones.

Aim of this part of the project is to evaluate if sex steroid hormones may act as endogenous modulators of Ngb levels. Two different cell models have been utilized: human neuroblastoma cell line SK-N-BE and mouse primary hippocampal neurons. Indeed, these cells are sex-hormone sensitive since both models express ERs and AR. Moreover, hippocampus is one of the most dimorphic brain areas, and primary hippocampal neurons could represent a model more physiologically and metabolically related to the in vivo condition.

3.2 Results 3.2.1 17β-estradiol effect on neuroglobin protein levels

To assess the responsiveness of the selected cell models, the expression of ERs has been evaluated. In both SK-N-BE cells and in primary hippocampal neurons ERα and ERβ were expressed. (figure 3.1). In SK-N-BE, ERβ subtype was more expressed compared with ERα, whereas in primary neurons, ERs were equally expressed.

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Figure 3.1 Characterization of ERs in SK-N-BE human neuroblastoma cell line and mouse primary hippocampal neurons. ER subtype (ERα and ERβ) levels in non-stimulated cells compared to recombinant proteins (5 ng) in SK-N-BE (a) and in primary hippocampal neurons (b). The amount of protein was normalized to tubulin levels. The data are typical Western blots of five independent experiments.

E2 stimulation induced a time- and dose-dependent increase in Ngb

levels in human neuroblastoma SK-N-BE cells (figure 3.2). E2 (10 nM) effect on Ngb levels started 30 minutes after stimulation, being significant after 1 hour, and remained constant 24 hours after hormone stimulation. The E2 dose-response curve was bell-shaped with a maximum effect at physiological E2 concentrations (i.e., 1-10 nM; 24 hours after stimulation). These data were confirmed in mouse primary hippocampal neurons (figure 3.3). Indeed, in these primary neurons, the E2 effect was rapid (1 hour) and persistent (24 hours) (figure 3.3a). In addition, also in mouse hippocampal neurons, 10 nM E2 increased Ngb levels, which remained significantly elevated in comparison to control cells even at higher E2 concentrations (figure 3.3b).

In line with the slight differences found in the E2 concentration to obtain the maximum effect in both cell types, 1 and 10 nM were used in the consecutive experiments to stimulate SK-N-BE cell line and hippocampal neurons, respectively.

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Figure 3.2 Effect of E2 on Ngb protein levels in SK-N-BE cell line. a, Time-course analysis of E2 treatment (10 nM) on Ngb levels. b, E2 dose-dependent (0.1-1000 nM) effect on Ngb levels (24 hours of stimulation). The amount of protein was normalized to tubulin levels. Top panels are typical Western blots of five independent experiments. Bottom panels represent the results of the densitometric analysis. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. a, (*) significant difference vs. 0 hour and (°) vs. 0.5 hour; b, (*) significant difference vs. 0, (°) vs. 0.1, and (§) vs. 1 nM E2.

Figure 3.3 Effect of E2 on Ngb protein levels in mouse primary hippocampal neurons. a,Time-course analysis of E2 treatment (10 nM) on Ngb levels. b, E2 dose-dependent (0.1-1000 nM) effect on Ngb levels (24 hours of stimulation). The amount of protein was normalized to with tubulin levels. The data are typical Western blots of five independent experiments.

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3.2.2 Androgen effect on neuroglobin levels

Also AR was expressed in non-stimulated SK-N-BE cells (figure 3.4). DU145 human prostate cancer cell line was used as a positive control of AR expression (Alimirah et al., 2006). AR expression was previously reported in primary hippocampal neurons (Nguyen et al., 2009, and literature therein). However, the E2 effect on Ngb expression was specific since neither dihydrotestosterone (DHT) nor the common precursor of E2 and DHT, testosterone, were able to increase Ngb levels at any tested concentration in both SK-N-BE neuroblastoma cells and primary hippocampal neurons (figure 3.5). Thus, since only E2 was able to modify Ngb expression, no further studies on DHT and testosterone were performed.

Figure 3.4 Characterization of AR in SK-N-BE human neuroblastoma cell line. Analysis of AR expression levels in non-stimulated cells compared to AR-positive DU145 human prostate cancer cell line. The amount of protein was normalized to tubulin levels. The data are typical Western blots of five independent experiments.

Figure 3.5 Effect of DHT and testosterone on Ngb protein levels in SK-N-BE cell line (a, b) and in primary hippocampal neurons (c, d). a, c, Effect of different doses of DHT (0.1-1000 nM) on Ngb levels (24 hours of stimulation). b, d, Effect of different doses of testosterone (0.1-1000 nM) on Ngb levels (24 hours of stimulation). The amount of protein was normalized to tubulin levels. The data are typical Western blots of five independent experiments.

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3.2.3 Estrogen receptor involvement in 17β-estradiol-induced neuroglobin levels

The pretreatment of SK-N-BE cells with the pure E2 antagonist, ICI 182,870 (ICI), completely prevented the E2 effect on Ngb levels (figure3.6), suggesting an ER-mediated mechanism.

Figure 3.6 Impact of ERs on Ngb protein levels in SK-N-BE human neuroblastoma cell line. Western blot analysis of Ngb levels in cells stimulated for 24 hours with either vehicle or E2 (1 nM) and/or the ER inhibitor ICI (1 μM). ICI was administrated 30 min before E2. The amount of protein was normalized to tubulin levels. The figure represents a typical Western blot of five independent experiments.

As SK-N-BE cells contain both ERβ and ERα (figure 3.1a), cells were

stimulated with either the specific ERα agonist 4,4’,4’’-(4-propyl [1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or the specific ERβ agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) to discriminate the role of each ER isoform in the E2-induced Ngb level increase. Only 1 and 10 nM DPN mimicked the E2 effect on Ngb levels (figure 3.7b), whereas PPT was unable to increase Ngb levels, at any concentration investigated (figure 3.7a). This result was confirmed by cell pretreatment with the specific ERβ inhibitor (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC), which completely prevented the E2 effect (figure 3.7b).

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Figure 3.7 Impact of ERα and ERβ on Ngb protein levels in SK-N-BE human neuroblastoma cell line. a, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (1 nM), or the ERα agonist PPT (1-100 nM). b, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (1 nM), the ERβ agonist DPN (1-100 nM), or ERβ selective antagonist (THC 1μM). The amount of proteins was normalized to tubulin levels. Top panels are typical Western blots of five independent experiments. Bottom panels represent the results of the densitometric analysis. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. a, (*) significant difference vs. vehicle; b, (*) significant difference vs. vehicle, (°) vs. E2 , (§) vs. 1 nM, and (#) vs. 10 nM DPN.

Furthermore, the decrease of ERβ protein level by ERβ short

interfering RNA (siERβ) transfection caused an impairment of the E2 ability to increase Ngb levels (figure 3.8a).

A further confirmation of the ERβ involvement in the effect of E2 derives from the results obtained using the flavonoid naringenin (Nar) (figure 3.8b). Indeed, it has been previously reported that this flavonoid is a partial antagonist of E2 in the presence of ERα (Galluzzo et al., 2008) and an E2 mimetic in the presence of ERβ (Totta et al., 2004). Like E2, 0.1 μM Nar was sufficient to increase Ngb levels (figure 3.8b). This effect persisted at Nar high concentrations (i.e., 1 and 10 μM).

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Figure 3.8 Impact of ERβ silencing and of ERβ agonist naringenin on Ngb levels in SK-N-BE human neuroblastoma cell line. a, Analysis of Ngb and ERβ levels in cells transfected with either MOCK (control) or ERβ small interference RNA (siERβ) in the absence or presence of E2 (1 nM). b, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (1 nM) or naringenin (Nar; 0.01–10 μM). The amount of proteins was normalized to tubulin levels. Left panel is a typical Western blot of five independent experiments. Right panel represents the results of the densitometric analysis. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. (*) significant difference vs. vehicle, (°) vs. E2, (§) vs. 0.01, and (#) vs. 0.1 μM Nar.

ERβ was also necessary for the E2-induced Ngb increase in mouse

primary neurons. In these cells, containing a similar amount of ERα and ERβ (figure 3.1b), ICI prevented the E2 effect (figure 3.9), DPN mimicked the E2 effect (figure 3.10b), whereas PPT was unable to increase Ngb levels (figure 3.10a). Cell pretreatment with the specific ERβ inhibitor, THC, further confirmed this specificity (figure 3.10c).

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Figure 3.9 Impact of ERs on Ngb protein levels in primary hippocampal neurons. Western blot analysis of Ngb levels in cells stimulated for 24 hours with either vehicle or E2 (1 nM) and/or the ER inhibitor ICI (1 μM). ICI was administrated 30 min before E2. The amount of protein was normalized to tubulin levels. The figure represents a typical Western blot of five independent experiments.

Figure 3.10 Impact of ERα and ERβ on Ngb protein levels in mouse primary hippocampal neurons. a, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (10 nM) or the ERα agonist PPT (0.1-100 nM). b, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (10 nM) or the ERβ agonist DPN (0.1-100 nM). c, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (10 nM) and/or the ERβ-selective antagonist THC (1 μM). THC was administrated 30 min before E2. The amount of protein was normalized to tubulin levels. Data are representative of typical Western blots from five independent experiments. 3.2.4 Mechanisms involved in the 17β-estradiol-induced increase of neuroglobin levels

In the absence of ligand, the classical intranuclear ER exists in the cytosol as a monomer (inactive state) and forms a multiprotein complex comprising immunophilins and heat-shock protein. Activation of ER by E2

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causes phosphorylation of several distinct serine/threonine residues of the ER inducing dissociation from heat-shock protein. Activated ERs then homo- or heterodimerize, translocate to the nucleus and interact with specific palindromic DNA sequences (estrogen responsive element, ERE) within the promoter regions of target genes to alter gene transcription (Ascenzi et al., 2006; Acconcia and Marino, 2011, and literature therein). ER can also modulate genes devoid of ERE by ER tethering and coactivation of other DNA-bound transcription factor complexes such as nuclear factor (NF)κB, CREB, specificity protein 1 (Spl) site, or by interaction with the fos/jun transcription factors thereby regulating gene transcription via the activators protein-1 (AP-1) (Ascenzi et al., 2006; Morisette et al., 2008, Acconcia and Marino, 2011 and literature therein).

NGB promoter sequence analysis (accession No. 12,581 from Transcriptional Regulatory Element Database http://rulai.cshl.edu/cgibin/ TRED/tred.cgi?process=home) indicates that no canonical ERE consensus is present. However, there are some ERE-like sequences indicating that direct transcriptional mechanisms can be activated to promote the transcription of Ngb. In addition, several half-ERE sequences are recognized along the promoter together with AP-1 and Sp1 binding sites suggesting a mechanism of indirect transcription mediated by ER. Moreover, the rapidity of E2 effect on Ngb expression (i.e., 30 min) indicates that also rapid membrane-starting signals of ER can be involved in Ngb upregulation.

Thus, we evaluated the impact on the E2-induced Ngb levels of the transcription inhibitor actinomycin (Act), the translation inhibitor cycloheximide (Cxm), and the ER membrane localization inhibitor 2-bromohexadecanoid acid (2Br) (Acconcia et al., 2005a).

SK-N-BE cell pretreatment for 24 hours with Act completely prevented the E2 effect on Ngb levels (figure 3.11a). Similarly, Cxm (figure 3.11b) and 2Br (figure 3.11c) impaired the increase of Ngb levels induced by E2. These findings suggest that both direct and indirect transcriptional as well as extranuclear mechanisms contribute to E2 effects.

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Figure 3.11 Mechanisms underlying E2 effects on Ngb protein levels in SK-N-BE human neuroblastoma cells. a, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (1 nM) and/or the transcription inhibitor actinomycin D (Act, 1 μg/ml). b, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (1 nM) and/or the translation inhibitor cycloheximide (Cxm, 10 μg/ml). c, Analysis of Ngb levels in cells stimulated for 24 hours with either vehicle, E2 (1 nM) and/or the palmitoylacyl transferase inhibitor 2-bromohexadecanoic acid (2Br; 10 μM). Act and 2Br were administrated 30 min before E2; Cxm was administrated 1 hour before E2. The amount of proteins was normalized to tubulin levels. Representative Western blots from five independent experiments are shown.

These data prompted us to evaluate which signal transduction cascade was activated by E2 in neurons. After 15 minutes of stimulation with 1 nM E2, an increase of AKT and p38/MAPK (p38) phosphorylation in SK-N-BE cells was observed (figure 3.12). The E2-induced activation of these kinases was still present 1 hour after E2 stimulation (figure3.12a), but only the E2-induced p38 phosphorylation persisted 24 hours after hormone stimulation (figure 3.12b). By 30 minutes after 1 nM E2 stimulation, the ERK1/2/MAPK (ERK1/2) phosphorylation status decreased (figure 3.12a), in agreement with previous data obtained in cortical neurons (Numakawa et al., 2007).

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Figure 3.12 Rapid signal transduction pathways activated by E2 in SK-N-BE human neuroblastoma cell line. a, Time-course analysis of phosphorylated (P) AKT, p38, and ERK1/2 in cells stimulated for 0, 15, 30, and 60 min with E2 (1 nM). The amount of proteins was normalized to tubulin levels. Left panel is a typical Western blot of five independent experiments. Right panel represents the results of the densitometric analysis. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant difference vs. 0, (°) vs. 15, and (§) vs. 30 min. b, Analysis of phosphorylated (P) p38 levels in cells stimulated for 1, 4 and 24 hours with E2 (1 nM).The amount of protein was normalized to total p38 (p38 tot) levels. Representative Western blots from five independent experiments are shown.

Cell pretreatment with either AKT or p38 inhibitors suggested that

AKT was not involved in the E2-induced increase of Ngb levels (figure 3.13a), whereas p38 activation was required for both rapid (i.e., 1 hour) (figure 3.13b) and long term (i.e., 24 hours) E2 effects on Ngb levels (figure 3.13c). Similarly, only p38 inhibitor prevented an E2-induced Ngb level increase in hippocampal neurons (figure 3.14).

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Figure 3.13 Impact of E2-dependent rapid signal inhibitors on Ngb protein levels in SK-N-BE human neuroblastoma cell line. a, Analysis of Ngb levels in cells stimulated for 1 hour with either vehicle, or E2 (1 nM), and/or AKT inhibitor (AKT inh). b,c, Analysis of Ngb levels in cells stimulated for 1 or 24 hours with either vehicle, E2 (1 nM), and/or the p38 inhibitor SB-203580 (SB; 5 μM). AKT inh and SB were administrated 30 min before E2. Representative Western blots from five independent experiments are shown.

Figure 3.14 Impact of E2-dependent rapid signal inhibitors on Ngb protein levels in mouse primary hippocampal neurons. a, Analysis of Ngb levels in cells stimulated for 1 hour with either vehicle, E2 (1 nM), and/or the p38 inhibitor SB-203580 (SB; 5 μM), and/or the AKT inhibitor (AKT inh), and/or the palmitoylacyl transferase inhibitor 2-bromohexadecanoic acid (2Br; 10 μM). AKT inh, SB, and 2Br were administrated 30 min before E2. Representative Western blots from five independent experiments are shown.

Notably, SK-N-BE cell transfection with siERβ reduced both ERβ

levels and the E2 ability to induce p38 phosphorylation (figure 3.15). On the other hand, siERβ did not impair the E2-induced AKT activation (figure

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3.15), suggesting that ERα could be the molecular mediator of AKT activation in these cells.

Figure 3.15 Effect of ERβ silencing on the E2-activated rapid signals in SK-N-BE human neuroblastoma cell line. a, Analysis of phosphorylated (P) AKT and p38, and ERβ levels in cells transfected with either MOCK (control) or ERβ small interference RNA (siERβ) in the absence or presence of E2 (1 nM). The amount of proteins was normalized to tubulin levels. Representative Western blots from five independent experiments are shown.

Since several actions of E2 in the nervous system involve cross-talk

between ERα and the IGF-1 receptor (Marin et al., 2009), we evaluated the possibility that the ERβ-dependent E2-induced p38 phosphorylation and the Ngb-increased levels were dependent on the ERα-IGF-1 receptor cross-talk. IGF-1 was more efficient than E2 to activate AKT phosphorylation, and cell pretreatment with picropodophyllin (PPP), the IGF-1 receptor inhibitor, strongly prevented both IGF-1 and E2 effects on AKT activation (figure 3.16a), confirming that the cross-talk between IGF-1 receptor and ERs was important for AKT activation. However, IGF-1 was unable to increase p38 phosphorylation and PPP did not prevent E2-induced p38 activation (figure 3.16a). In addition, IGF-1 did not modify Ngb levels in SK-N-BE cells (figure 3.16b), further sustaining the high specificity of the E2 effect.

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Figure 3.16 Effect of IGF-1 on phosphorylated AKT and p38, and Ngb levels in SK-N-BE human neuroblastoma cell line. a, Western blot analysis of phosphorylated (P) AKT and p38 levels in cells treated for 24 hours with either vehicle, E2 (1 nM), IGF-1 (100 ng/ml) or the IGF-1 receptor inhibitor PPP (100 μM). PPP was administrated 30 min before IGF-1 or E2. b, Analysis of Ngb protein levels in cells treated for 24 hours with either vehicle, E2 (1 nM) or IGF-1 (100 ng/ml). The amount of proteins was normalized to tubulin levels. Representative Western blots from five independent experiments are shown. 3.3 Discussion

Aim of this part of the project was to identify an endogenous modulator of Ngb. The reported results indicate that E2, but not androgens, act as an endogenous modulator of Ngb.

E2 increases Ngb levels of about 300% in both human neuroblastoma cell line and in mouse primary hippocampal neurons. Although this data have been obtained with a qualitative technique (i.e., Western blot), the effect is conspicuous in that the well-known E2 effect on cyclin D1 expression, playing a relevant role in E2-induced cell proliferation, is only of about 50-70% (Marino et al., 2002). In addition, the E2-induced Ngb increase is rapid (1 hour), persistent (24 hours), and specific, being not mimicked by either the male sex steroid hormone DHT, or by the common precursor testosterone, or by IGF-1, another well-known neuroprotective hormone. These data represent the first evidence for steroid hormone modulation of globin levels in cells. Recently, it has been reported that Hb is specifically expressed in neurons, its expression being upregulated by erythropoietin and accompanied by enhanced brain oxygenation under physiologic and hypoxic conditions (Schelshorn et al., 2009). At present, the relationship between Hb and Ngb in neurons is still unclear.

Although Hb β-chains and Ngb are expressed in the same nerve cells, Ngb levels are not increased by erythropoietin (Schelshorn et al., 2009). It is therefore unlikely that these two globins have a tightly linked function, e.g.

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in facilitated oxygen transport; however, Hb and Ngb could fulfill independent tasks in neurons (De Marinis et al., 2010; De Marinis et al., 2011).

It is now well known that sex steroid hormones have numerous effects on the brain throughout the lifespan, beginning during early gestation and continuing on into senescence (Swaab, 2007). However, the inability of Ngb to react with androgens renders Ngb a new E2 target that should be added to the variety of E2-specific actions in the brain which include mood, locomotor activity, pain sensitivity, vulnerability to epilepsy, attentional mechanisms, and cognition (McEwen, 2002; De Marinis et al., 2010; De Marinis et al., 2011).

The E2 effect on Ngb levels is rapid and dose-dependent with the maximum effect at E2 physiological concentration (i.e., 1-10 nM). Notably, the E2 dose-response curve results to be bell-shaped. This is typical for E2 and other hormones that typically interact with plasma membrane receptors (Galluzzo et al., 2009). The lack of effect at higher concentrations could be considered the consequence of a receptor downregulation phenomenon, by which the cells protect themselves against high hormone levels; moreover in the case of E2 the two ERs can regulate each other their expression, affecting also in this way the cell response to the hormone.

Accordingly, the plant-derived flavonoid Nar, which partially blocks the rapid activities of ERα (Galluzzo et al., 2008), increases Ngb levels with a plateau at 1μM concentration. This result suggests a functional antagonism between the activities of ERα and ERβ in neurons, as it has been reported in other cell types (Matthews and Gustafsson, 2003).

Although human neuroblastoma cell line and mouse primary hippocampal neurons express different levels of both ER isoforms, the effect of E2 on Ngb levels specifically requires ERβ activities in both cell types. In fact, ERβ extranuclear and genomic signals cross-talk each other to guarantee both the rapid (1 hour) and the persistent (24 hours) E2 effects. In particular, the rapid (15 minutes) and persistent (24 hours) ERβ-mediated p38 activation is required for E2-induced Ngb increase. The E2-dependent activation of p38 seems to represent a conserved pathway in ERβ-based E2 rapid signals. Indeed, the E2-induced ERβ-mediated activation of the p38 occurs in ERβ-transfected HeLa cells and in ERβ-containing rat myoblasts and colon adenocarcinoma cells (Acconcia et al., 2005b; Galluzzo et al., 2007; Galluzzo et al., 2009). This signaling pathway transduces different E2 effects depending on cell context. In fact, p38 activation is required for E2-induced apoptosis of cancer cells (Acconcia et al., 2005b; Galluzzo et al., 2007), for E2-induced gene transcription (Galluzzo et al., 2007) and for E2-

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induced protection against oxidative stress in rat myoblasts (Marino, unpublished results).

Thus, although NGB promoter does not contain any canonical ERE, it is not surprising that the transcription inhibitor Act completely prevents the increase of the E2-induced Ngb levels. The sequence analysis (accession No. 12,581 from Transcriptional Regulatory Element Database http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home) indicates that several half- or non-canonical ERE sites are present in the NGB promoter along with a responsive element for other transcription factors such as Sp1 (GC boxes) or AP-1. These results suggest that the E2-induced Ngb transcription could be mediated by tethered interactions of ER with other transcription factors to activate gene expression (i.e., indirect genomic mechanism) (Ascenzi et al., 2006). The hormone rapidly induces p38 and AKT activation, and ERK1/2 dephosphorylation in neuroblastoma cells. These effects are still detected in ERα-containing but ERβ-silenced cells. Although these kinases are not directly involved in E2-induced Ngb increase, which is mediated by p38 signals, they could contribute to the E2 effects in neurons, since AKT activation has been associated with the increase of the antiapoptotic protein Bcl-2 and to the E2-induced cell survival (Acconcia et al., 2005b), while E2 protected cortical neurons against oxidative stress by reducing H2O2-induced activation of ERK1/2 (Numakawa et al., 2007).

Therefore, the integration between extranuclear and genomic ERβ-mediated events is required to provide plasticity for this neuronal response to E2 (De Marinis et al., 2010; De Marinis et al., 2011).

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4. INVOLVEMENT OF NEUROGLOBIN IN 17β-ESTRADIOL-INDUCED PROTECTION AGAINST NEUROTOXICITY

4.1 Introduction

E2 protective mechanisms in the brain range from antiapoptotic, neurotrophic, and neurogenic actions. These effects suppress neuroinflammation and oxidative processes that accompanies progression or initiation of many pathological brain conditions, including Parkinson’s disease, Alzheimer’s disease, stroke, and multiple sclerosis (McEwen, 2002; Marin et al., 2008; Morisette et al., 2008; Vasudevan and Pfaff, 2008; Marin et al., 2009; Gillies and McArthur, 2010).

Studies in several in vitro systems have indicated that E2 might have many roles in neuroprotection. It protects cultured neurons and glia against a wide range of insults (Bishop and Simpkins, 1994; Behl et al., 1995; Green and Simpkins, 2000; Wise et al., 2001), including the neurotoxic effects of the excitatory neurotransmitter L-glutamate, the viral protein gp120 and the Aβ. It also decreases astrocytosis and neuroinflammatory processes in astrocytes (Arevalo et al., 2010), and reduces cell death in response to the deprivation of nutritional and trophic factors and to various oxidative insults, such as H2O2.

Interestingly, the mechanisms by which many of these neurotoxic agents affect neurons converge on their ability to trigger downstream oxidative processes (Behl, 2002).

Oxidative stress is a well-known hallmark of neurodegenerative disorders that include both acute (cerebral stroke, head trauma) and chronic degeneration (Alzheimer’s disease, Parkinson’s disease) and is also a by-product of inflammatory processes in the brain. It is caused by the generation and accumulation of two major ROS produced by living tissue, the superoxide anion (O2

−) and H2O2. Although H2O2 is not a free radical and has a limited reactivity, it can cross biological membranes, whereas O2

− needs an anion channel for its transport (Fordel et al., 2007b). H2O2 can react with intracellular metal ions such as iron or copper to form highly toxic hydroxyl radicals, which cause DNA modification. In addition, H2O2 can cause membrane damage by increasing the release of arachidonic acid from the cell membrane (Fleming et al., 1992). Thus, H2O2 is a more important contributor to pathological events than O2

− (Fleming et al., 1992). Several studies have shown that H2O2 induces apoptotic neuronal cell death via both extrinsic (death receptor mediated) and intrinsic (mitochondrial dependent) pathways (Ruffels et al., 2004; Fordel et al., 2007b).

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As the brain is particularly vulnerable to changes in the oxidative environment, the beneficial action of E2 might be related to its capacity to interfere with oxidative neurotoxic insults.

Over recent years Ngb has been shown to provide a protective function with respect to Aβ toxicity (Khan et al., 2007) and Alzheimer’s disease (Szymanski et al., 2008), whilst also protecting from cell death associated with hypoxic ischemia both in vitro and in vivo (Sun et al., 2001; Sun et al., 2003; Khan et al., 2006; Wang et al., 2008). All these processes are at least in part associated with oxidative stress and apoptotic cell death.

In some cell lines it has been demonstrated the protective effect of Ngb overexpression against an oxidative insult. Indeed, H2O2 concentration was found to be negatively correlated with Ngb expression level in a hypoxia/reoxygenation cell model (Fordel et al., 2007b), and overexpression of Ngb was associated with improved cell survival following H2O2 treatment (Fordel et al., 2006). Moreover, overexpression of wild type Ngb, but not of mutant Ngb, significantly attenuated H2O2-induced ROS/RNS accumulation and lipid peroxidation, decreased H2O2-induced mitochondrial dysfunction and apoptosis, and promoted overall cell survival in rat pheochromocytoma PC12 cell line (Li et al., 2008). These observations further lend support to the hypothesis that Ngb may play a protective role in ROS/RNS scavenging during ischemia/reperfusion injury.

In addition, preliminary computer modeling analysis show an intriguing role of Ngb in resetting the trigger level for apoptosis (Brittain et al., 2010a; Raychaudhuri et al., 2010). Aim of this part of the project is to evaluate this hypothesis. In particular, the involvement of Ngb in E2-induced protection against H2O2-mediated neurotoxicity and the mechanisms underlying are assessed in human neuroblastoma cell line (i.e., SK-N-BE cells).

4.2 Results 4.2.1 Neuroglobin is involved in 17β-estradiol-induced protection against H2O2-mediated apoptosis 1 nM E2 stimulation did not modify SK-N-BE cell number (figure 4.1a) but, as expected (Numakawa et al., 2007), 24 hours E2 pretreatment reduced the number of apoptotic condensed nuclei caused by 50μM H2O2 injury (24 hours of stimulation), respect to H2O2 treatment alone (figure 4.1b). In agreement with these data, E2 pretreatment reduced the activation of pro-apoptotic cascade reducing by 50% the H2O2-induced decrease in cell number (figure 4.2a) as well as the increase of the 17 kDa active

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caspase-3 subunit and the cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) (figure 4.2b). Staurosporin (2 μM for 24 hours) was used as positive control of caspase activation (figure 4.2b). Notably, the E2 protective effect against H2O2-induced neuronal toxicity required ERβ, since the cell pretreatment with the specific ERβ inhibitor, THC (1 μM, 30 min before E2 administration), completely prevented E2 effects (figure 4.2).

Figure 4.1 E2 effect on SK-N-BE human neuroblastoma cell line viability. a, Cells were grown in the presence of either vehicle or E2 (1 nM) and counted at the indicated time. Data are mean ±SD of five independent experiments carried out in duplicate. b, Nuclear morphology of SK-N-BE cells 24 hours after H2O2 injury (50 μM). Cells were pretreated 24 hours before the addition of H2O2 with either vehicle or E2 (1 nM). Fixed and permeabilized cells were stained with DAPI. Apoptotic nuclei were detected as condensed nuclei (arrows) (fluorescence microscopy, original magnification 40×). Data are means ± SD of five independent experiments carried out in duplicate. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant difference vs. vehicle -H2O2, (°) vs. vehicle +H2O2.

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Figure 4.2 E2 and THC effects on SK-N-BE human neuroblastoma cell line viability. a, Cells were grown for 24 hours in the presence of either vehicle, E2 (1 nM) or E2 in the presence of the ERβ-selective antagonist THC (1 μM). After 24 hours, cells were stimulated with 50 μM H2O2 (24 hours of stimulation), and counted. Data are means ± SD of five independent experiments carried out in duplicate. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant difference vs. vehicle -H2O2, (°) vs. vehicle +H2O2, (§) vs. E2 -H2O2, (#) vs. E2 +H2O2, and (a) vs. THC + E2 -H2O2. b, Western blot analysis of caspase-3 activation and PARP cleavage were performed on cells stimulated with either the vehicle or pretreated with E2 (1 nM) for 24 hours in the presence or absence of THC pretreatment, and then treated with H2O2 50 μM (24 hours of stimulation). Staurosporin (2 μM for 24 hours) was used as positive control of caspase activation. The amount of proteins was normalized to tubulin levels. Representative Western blots from five independent experiments are shown.

Intriguingly, E2 was unable to counteract the H2O2-induced decrease in

cell number (figure 4.3a) and the activation of the pro-apoptotic cascade (i.e., caspase-3 activation and PARP cleavage) (figure 4.3b) in SK-N-BE cells transfected with Ngb short interfering RNA (siNgb).

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Figure 4.3 Effect of siNgb on E2-induced cell viability in SK-N-BE cells. a, Western blot analysis of Ngb protein levels in cells transfected with either MOCK (control) or with Ngb small interference RNA (siNgb) in the absence or presence of E2 (1 nM). The amount of proteins was normalized to tubulin levels. Representative Western blots from three independent experiments are shown in the upper panel. Bottom panel represents the results of the densitometric analysis. Cells transfected with either MOCK (empty bars) or siNgb (filled bars) were grown for 24 hours in the presence of either the vehicle or E2 (1 nM), stimulated with 50 μM H2O2 (24 hours of stimulation), and counted. Data are means ± SD of three independent experiments carried out in duplicate. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant difference vs. vehicle -H2O2, (°) vs. vehicle +H2O2, (§) vs. E2 +H2O2, (#) vs. vehicle -H2O2 siNgb, and (a) vs. E2-H2O2 siNgb. b, Western blot analysis of caspase-3 activation and PARP cleavage were performed on cells transfected with either MOCK or siNgb and incubated with either vehicle or 50 μM H2O2 in the presence or absence of E2 (1 nM) (24 hours pretreatment). The amount of proteins was normalized to tubulin levels. Representative Western blots from three independent experiments are shown. 4.2.2 17β-estradiol changes neuroglobin intracellular localization

Ngb can take part in redox reactions with other redox active proteins, such as cytochrome c, which is one of the most important effectors in the intrinsic apoptotic pathway. It has been proposed that the Ngb-cytochrome c complex can undergo a redox reaction, where ferric cytochrome c is

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reduced to a non-apoptotic ferrous form, and the redox reaction is very rapid (Fago et al., 2006; Bønding et al., 2008; Fago et al., 2008), lending argument for the hypothesis that the protective role provided by Ngb could arise from its intervention in the mitochondrial pathway of apoptosis. Actually, the possibility that Ngb can directly interferes with cytochrome c oxidation state was only recently evaluated using an in silico approach, through computer modeling analysis (Brittain et al., 2010a; Raychaudhuri et al., 2010).

The finding that Ngb is an E2-inducible protein opens the possibility that Ngb could be part of E2-induced signals and protective effects in neuronal cells.

To better understand how Ngb plays such an important role in the E2-induced anti-apoptotic pathway, first of all the intracellular localization of Ngb has been evaluated. In non-treated SK-N-BE cells, Ngb distribution appeared ubiquitous, in both nucleus and cytosol (figure 4.4a). This localization was further confirmed in Ngb-free HeLa cells transiently transfected with pcDNA flag-Ngb plasmid, showing a widespread distribution of fluorescent signal (figure 4.4b).

Figure 4.4 Localization of Ngb in SK-N-BE cells and in flag-Ngb transfected HeLa cells. a, Fluorescence analysis of SK-N-BE cells. Cells were fixed and permeabilized, and stained with anti-Ngb antibody (green, right panel) and co-stained with DAPI (left panel) (original magnification 40×). b, fluorescence analysis of Hela cells non transfected (NT, left panel) or transfected with pcDNA-flag-Ngb plasmid (flag-Ngb, right panel). Cells were fixed, permeabilized and then stained with anti-flag M2®antibody (red) (original magnification 40×). Representative images from five different experiments are shown.

To confirm this Ngb wide distribution and to evaluate in particular the

distribution rate of Ngb among the three main compartments of the cell (i.e., nucleus, mitochondria and cytosol), SK-N-BE cells were fractionated. Cell fractionation showed that Ngb was more expressed in cell nucleus and in mitochondria, than in cytosolic fraction. PARP, cytochrome c oxidase-4

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(COX-4) and protein phosphatase-2A (PP2A) were used as cell fraction purity markers of nucleus, mitochondria and cytosol, respectively (figure 4.5).

Figure 4.5 Localization of Ngb in SK-N-BE cell fractions. Western blot analysis of Ngb expression in nuclear, cytosolic and mitochondrial fractions of SK-N-BE cells. The purity of the nuclear, mitochondrial and nuclear fractions was assessed with PARP, cytochrome c oxidase-4 (Cox-4) and protein phosphatase 2A (PP2A), respectively. Left panel shows representative Western blots from five independent experiments. Densitometric analysis (right panel) of Ngb distribution rate in the three fractions was calculated respect to whole protein amount. Data are means ± SD of five different experiments.

E2 was able to modify Ngb intracellular localization just 1 hour after

stimulation, and the effect was persistent and more evident after 24 hours of stimulation. Indeed, we observed a reduced presence of Ngb in cell nucleus 1 and 24 hours after 1 nM E2 stimulation (figure 4.6a). In agreement with these data, SK-N-BE subcellular fractionation indicated that E2-mediated (i.e., 1 nM and 24 hours of stimulation) nuclear Ngb reduction was paralleled by a strong increase of Ngb localization in mitochondria, and by a moderate but significant rise in the cytosolic fraction (figure 4.6b).

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Figure 4.6 Effect of E2 on Ngb intracellular localization in SK-N-BE cells. a, Confocal microscopy analysis shows Ngb distribution in cells treated either with vehicle or E2 (1 nM) for 1 or 24 hours (original magnification 63×). Representative images from three different experiments are shown. b, Western blot analysis of Ngb expression in nuclear, cytosolic and mitochondrial fractions of SK-N-BE cells treated either with vehicle or E2 (1 nM for 24 hours). The purity of the nuclear, mitochondrial and nuclear fractions was assessed with PARP, cytochrome c oxidase-4 (Cox-4) and protein phosphatase 2A (PP2A), respectively. The amount of protein was normalized to actin levels. Upper panel shows representative Western blots from five independent experiments. Densitometric analysis (bottom panel) of Ngb distribution in the three fractions was calculated respect to whole protein amount. Data are means ± SD of five independent experiments carried out in duplicate. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant difference vs. vehicle (mitochondria), (°) vs. vehicle (nucleus), (§) vs. vehicle (cytosol). 4.2.3 17β-estradiol promotes neuroglobin-cytochrome c association

The E2-induced increase of Ngb levels in mitochondria prompted us to

evaluate if an increase in Ngb-cytochrome c association was present after H2O2 injury.

In non-stimulated SK-N-BE whole cell lysate the co-immunoprecipitation of Ngb with cytochrome c was only slightly detectable (figure 4.7). However, narrowing down to mitochondrial fraction, the

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association between Ngb and cytochrome c was clearly recognizable (figure 4.8), and E2 treatment (i.e., 1 nM for 24 hours) increased the amount of Ngb co-immunoprecipitated with cytochrome c. Intriguingly, in presence of H2O2 insult (i.e., 50 μM for 24 hours) the Ngb-cytochrome c association was enhanced, and the pretreatment with E2 (i.e., 1 nM, 24 hours before H2O2 stimulus) significantly raised this association (figure 4.8).

Figure 4.7 Ngb association with cytochrome c (Cyt-c). Total lysates of SK-N-BE cells were subjected to Cyt-c immunoprecipitation (IP), followed by Western blot (WB) with anti-Ngb or anti-Cyt-c antibodies. Representative Western blot from five independent experiments.

Figure 4.8 Effect of E2 and H2O2 on Ngb association with cytochrome c (Cyt-c) in mitochondrial fraction. SK-N-BE cells were grown for 24 hours in the presence of either vehicle or E2 (1 nM). After 24 hours, cells were stimulated with 50 μM H2O2 (24 hours of stimulation), and then were fractionated and only the mitochondrial fraction was subjected to Cyt-c immunoprecipitation (IP), followed by Western blot (WB) with anti-Ngb or anti-Cyt-c antibodies. Representative Western blot (top panel) from five independent experiments. Densitometric analysis (bottom panel) of Ngb association with Cyt-c. Data are means ± SD of five independent experiments carried out in duplicate. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant vs. vehicle -H2

O2, (°) vs. vehicle +H2 O2.

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In its reduced FeII form, cytochrome c is seized into mitochondria. In mitochondria, cytochrome c is located between the inner and the outer membrane and functions to transfer electrons from Complex III (ubiquinone-cytochrome c reductase) to Complex IV (COX-4) of the electron transport chain. It mediates electron transfer through the heme group, which switches between the reduced FeII form and the oxidized FeIII state. Moreover, in healthy cells, cytochrome c inhibits reactive oxygen species formation, thus preventing cell oxidative stress (Ascenzi et al., 2011 and literature therein).

Cytochrome c displays also a central apoptotic role. Cytochrome c release into the cytosol is particularly associated with activation of the intrinsic apoptotic pathway, which responds to intracellular stimuli such as DNA damage and oncogene activation. When stress/damage signals are activated, the mitochondrial membrane is permeabilized, leading to deterioration of the bioenergetic functions of mitochondria, overproduction of ROS, as well as to the release of cytochrome c (i.e., in FeIII form) into the cytosol. Once in the cytosol, in the presence of ATP (and more efficiently in the presence of deoxyATP), oxidized cytochrome c mediates the allosteric activation and hepta-oligomerization of the adaptor molecule apoptosis-protease activating factor-1 (Apaf-1), generating the complex known as apoptosome. Each apoptosome can recruit seven dimers of caspase 9 favoring proteinase activation. These events, tightly regulated by several heat shock proteins, allow the catalytic maturation of caspase 3 and other caspases, which eventually mediate the biochemical and morphological features of apoptosis (Ascenzi et al., 2011, and literature therein). Remarkably, at least 15% of mitochondrial cytochrome c is bound to cardiolipin, an unusual lipid largely confined to the inner mitochondrial membrane. The interaction with cardiolipin, which induces allosteric modification in cytochrome c tertiary structure, is pivotal for switching cytochrome c function(s) from mitochondrial respiration to apoptosis (Ascenzi et al., 2011, and literature therein).

It can be possible that Ngb associates to cytochrome c also in the cytosolic fraction to promote its reduction into the cytosol and, in turn, the switch off of apoptotic signaling. Indeed, upon H2O2 injury the amount of cytochrome c increased into the cytosol and decreased in mitochondria (figure 4.9). Notably, in parallel with the induction of Ngb increase in mitochondrial and cytosolic fraction, E2 treatment reduced the amount of basal level of cytosolic cytochrome c in non-injured cells, and, most importantly, E2, when is administrated before H2O2, decreased the level of cytosolic cytochrome c, which is linked with increased cytochrome c levels in mitochondria (figure 4.9).

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Figure 4.9 Effect of E2 and H2O2 on Ngb and cytochrome c (Cyt-c) levels comparing mitochondrial and cytosolic fraction in SK-N-BE human neuroblastoma cells. Cells were grown for 24 hours in the presence of either vehicle or E2 (1 nM). After 24 hours, cells were stimulated with 50 μM H2O2 (24 hours of stimulation), and then were fractionated. a, Analysis of Ngb and Cyt-c levels in mitochondria (left panel) and cytosol (right panel). Cox-4 and PP2A were used to normalize the total amount of protein in mitochondrial and cytosolic fractions, respectively. Typical Western blot of five independent experiments are shown. b, Densitometric analysis of Cyt-c levels in mitochondrial and cytosolic fraction. Data are means ± SD of five independent experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test: (*) significant difference vs. vehicle -H2O2, (°) vs. vehicle +H2O2.

ERs were involved in the E2-mediated increased association between Ngb and cytochrome c. Indeed, as reported in other cell models (Simpkins et al., 2008; Arnold and Beyer, 2009, and literature therein), both ER

a

b

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subtypes were expressed in nucleus, mitochondria and cytosol in SK-N-BE cells (figure 4.10). The pretreatment with the pure ER antagonist, ICI (i.e. 1 μM 30 min before E2 administration), prevented the E2 effect on mitochondrial Ngb-cytochrome c association in presence of H2O2 challenge (figure 4.11). In particular, THC pretreatment (i.e. 1 μM 30 min before E2 administration) impaired the E2 promotion of Ngb-cytochrome c association, indicating that ERβ was specifically involved in this mechanism (figure 4.11).

Figure 4.10 Localization of ERs in SK-N-BE cell fractions. Western blot analysis of ERα and ERβ expression in nuclear, cytosolic and mitochondrial fractions of SK-N-BE cells. The purity of the nuclear, mitochondrial and nuclear fractions was assessed with PARP, cytochrome c oxidase-4 (Cox-4) and protein phosphatase 2A (PP2A), respectively. Representative Western blots from five independent experiments are shown.

Figure 4.11 Effect of E2, THC and ICI on Ngb association with cytochrome c (Cyt-c) in the mitochondrial fraction under H2O2 stimulus. SK-N-BE cells were grown for 24 hours in the presence of either vehicle, E2, and/or THC, and/or ICI (1μM). After 24 hours, cells were stimulated with 50 μM H2O2 (24 hours of stimulation), and then were fractionated. Mitochondrial fraction was subjected to Cyt-c immunoprecipitation (IP), followed by Western blot (WB) with anti-Ngb or anti-Cyt-c antibodies. Representative Western blot from five independent experiments.

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4.3 Discussion

It has recently shown that the elevation of human Ngb expression in neurons prior to insult with H2O2 enhances cell viability and results in a significant decrease in oxidative stress and an increased intracellular ATP concentration (Antao et al., 2010). In addition, a linkage of Ngb to oxidative metabolism has been proposed (Mitz et al., 2009).

Exposure to H2O2 induces a robust increase of ROS in cells (followed by oxidation of lipids, proteins, and DNA), intracellular calcium increase, glutathione depletion, mitochondrial dysfunction, and caspase-3 activation followed by apoptotic cell death (Wang et al., 2006). It has been demonstrated that E2 exerts protective effects on several of these cellular events, including potent attenuation of lipid peroxidation, attenuated ATP depletion, alleviated intracellular calcium elevation, ablated mitochondrial calcium loading (with the subsequent mitochondrial membrane potential maintenance), reduced caspase-3 activation, and enhanced cell survival (Wang et al., 2006).

The results reported here strongly indicated that Ngb is part of the E2 response to H2O2-induced toxicity (De Marinis et al., 2010; De Marinis et al., 2011). In fact, exposure to 50 μM H2O2 induces neuroblastoma cell death (about 50%) which is accompanied by a dramatic increase in caspase-3 activation. The cell pretreatment with E2 (1 nM) decreases cell death and reduces caspase-3 activation triggered by exposure to H2O2 in good accordance with literature (Behl et al., 1997; Sawada et al., 1998; Green and Simpkins, 2000; Wang et al., 2006; Numakawa et al., 2007). This E2 effect against H2O2 toxicity is completely prevented by treatment with THC, the ERβ inhibitor, and by knocking out Ngb.

Clarifying Ngb sub-cellular localization is pivotal to understand how this novel globin can intercept the apoptotic pathway. In fact, using different techniques, only two groups recently demonstrated that Ngb is widespread in the cells, being localized into the cytosol, mitochondria, and nucleus (Bentmann et al., 2005; Hundahl et al., 2010), although until now Ngb was commonly considered only as a cytoplasmatic protein, mainly expressed in perimitochondrial regions (Zhang et al., 2002; Reuss et al., 2002; Wystub et al., 2003; Geuens et al., 2003; Schmidt et al., 2003; Hankeln et al., 2004). Also in SK-N-BE cells Ngb is expressed in the nucleus, mitochondria and is scattered in the cytoplasm, confirming the widespread distribution that has been reported in other cell models (Bentmann et al., 2005; Hundahl et al., 2010). Moreover, it has been postulated that this localization further addresses to Ngb a role as scavenging molecules in neurons (Hundahl et al., 2010).

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The finding that E2 reallocates Ngb mainly at mitochondria strengthened the hypothesis that Ngb directly interferes with the intrinsic pathway of apoptosis, being mitochondria involved in this process. Furthermore, in silico simulations suggested that a major physiological role for Ngb is the interception of the mitochondrial pathway of apoptosis interfering directly with oxidized cytochrome c release to cytosol (Fago et al., 2006; Bønding et al., 2008; Fago et al., 2008; Brittain et al., 2010a; Brittain et al., 2010b).

It has been clearly demonstrated that only the oxidized form of cytochrome c takes part in apoptosome assembly (Borutaite and Brown, 2007). As hypothesized previously, the mechanisms underlying Ngb inhibition of intrinsic apoptotic cascade can be related to the observation that reduced Ngb (i.e., FeII) rapidly complexes with oxidized cytochrome c released from mitochondria and rapidly reduces it to the FeII form which is no longer proapoptotic (Fago et al., 2008). Indeed, at basal condition Ngb-cytochrome c is slightly detectable in mitochondrial fraction, since cytochrome c is in its reduced form and thus it is associated with cardiolipin into mitochondria. On the other hand, upon H2O2 challenge (50 μM for 24 hours) the association between Ngb and cytochrome c increases significantly, suggesting a role of Ngb in the prevention, or at least in the rapid reduction, of cytochrome c oxidation and cytosolic release directly in mitochondria.

The pivotal role of Ngb in the protective effect of E2 against H2O2 toxicity seems to be strictly related to Ngb-cytochrome c association. Indeed, the association of Ngb with cytochrome c is enhanced pretreating SK-N-BE cells with E2 (i.e., 1 nM for 24 hours), suggesting that E2-induced reallocation of Ngb facilitates Ngb-cytochrome c interaction. From a functional point of view this effect can be important in the regulation of E2 function; in fact, E2, as an anabolic hormone, could induce an intracellular production of ROS as side effect of increased metabolic rate of the cell. However, in normal conditions, the ROS increase does not trigger a toxic effect due to the parallel mitochondrial increase of Ngb which prevents oxidized cytochrome c release and the activation of apoptotic cascade. Thus, at physiological condition the E2-induced Ngb-cytochrome c interaction can be considered as an E2 self-regulatory mechanism. Remarkably, the E2 effect in the increase of Ngb-cytochrome c association is stronger during oxidative stress condition (i.e., E2 1 nM 24 hours before H2O2 administration) than under basal conditions.

In agreement with these results, since E2 promotes Ngb-cytochrome c association in mitochondria, especially after an oxidative insult, the total

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amount of cytosolic cytochrome c appears to be reduced in SK-N-BE pretreated with E2 before H2O2 administration.

As a whole, Ngb co-immunoprecipitates with cytochrome c in both mitochondria and cytosol, and thus it can be envisioned that after an oxidative stress, such as H2O2, Ngb binds to mitochondrial cytochrome c impairing, or rapidly reconverting, the oxidation of cytochrome c. At the same time, in cytosol, Ngb can participate in the reduction of oxidized cytochrome c, preventing caspase activation and thus resetting the trigger level for intrinsic pathway of apoptosis. Indeed, as indicated by computational studies, the impact of Ngb may arise from both its binding to cytochrome c and its subsequent redox reaction, although the binding alone is sufficient to block caspase activation (Fago et al., 2006; Bønding et al., 2008; Fago et al., 2008; Brittain et al., 2010a; Brittain et al., 2010b).

E2 effect on the promotion of Ngb-cytochrome c interaction during oxidative condition also requires ERβ activity, being impaired by the pretreatment with ER pure antagonist, ICI, and in particular by ERβ specific antagonist, THC. This specificity further highlights the role of mitochondrial ERβ signaling in prevention of apoptosis, which remains still debated, although most of the recent studies on E2 neuroprotective mechanisms are emphasizing the pivotal role of ERs localized in mitochondria (Simpkins et al., 2008; Flynn et al., 2008; Yang et al., 2009, and literature therein).

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5. INVOLVEMENT OF NEUROGLOBIN IN 17β-ESTRADIOL ANTI-INFLAMMATORY EFFECTS

5.1 Introduction

Astrocytes are the most abundant glial cells in the brain. In humans, they exceed neurons by a ratio of 10-50 astrocytes to each neuron. In recent years, astrocytes have been shown to play important roles in the central nervous system, including signal transduction (e.g., by calcium waves) and cellular communication (e.g., via gap junctions), and in controlling synapse numbers (Kast, 2001; Hansson and Ronnback, 2003; Parri and Crunelli, 2003).

Despite the level of interest generated by the discovery of Ngb, the research has been primarily focused on its roles in neurons due to the scarce presence of Ngb in astrocytes (Reuss et al., 2002). More recently, the presence of functional Ngb in cultured astrocytes has been demonstrated (Chen et al., 2005; Dong et al., 2010). In these cells Ngb antisense treatment, reducing Ngb expression, enhanced apoptosis under ischemia (Chen et al., 2005; Dong et al., 2010).

Ngb in astrocytes may be involved in the regulation of oxygen homeostasis and in the regulation of inflammation processes. Moreover, in neuropathological models of traumatic injury, infectious, autoimmune, and excitotoxic pathogeneses, Ngb positive astrocytes were found within regions associated with most severe pathology and in the astroglial scar (DellaValle et al., 2010). The distribution of Ngb positive astrocytes in these models suggests that Ngb is upregulated in astrocytes that are exposed to significant stress from the necrotic core. Thus, Ngb upregulation in this cell population may be functional to promote directly cell survival or to scavenge ROS/RNS when heavily exposed to surrounding cells necrosis.

For more than two decades it has been known that astrocytes are also one of the cellular targets of E2 in the brain (Arevalo et al., 2010). The hormone regulates the morphology of astrocytes and the expression of different molecules which are relevant for the physiological and pathological responses of these cells (Arevalo et al., 2010, and literature therein). These include apolipoprotein E (ApoE), heat shock proteins, aquaporin-4, neurotransmitter transporters and a variety of growth factors and cytokines (Arevalo et al., 2010, and literature therein). Furthermore, E2 may act on astrocytes regulating respiratory chain activity in the mitochondria (Araújo et al., 2008) and the synthesis of other steroids, such as progesterone (Sinchak et al., 2003).

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Under pathological conditions, astrocytes release a number of pro-inflammatory cytokines and chemokines that attract macrophages/microglia and T cells to CNS inflammatory sites (Dong and Benveniste, 2001; Rebenko-Moll et al., 2006; Rostene et al., 2007). The actions of E2 on astrocytes are also relevant for the anti-inflammatory mechanisms of the hormone. Indeed, E2, in cultured astrocytes incubated with lipopolysaccharide (LPS), causes a decrease of NO level and of inflammatory markers, such as tumor necrosis factor (TNF)α, interleukin 6 (IL-6) and interferon gamma-inducible protein 10 (IP-10) (Kipp et al., 2007; Tenebaum et al., 2007; Cerciat et al., 2010). E2 also decreases the activation of NFκB induced by Aβ (1-40) and LPS in cultured astrocytes (Dodel et al., 1999). NFκB is a potent immediate-early transcriptional regulator of numerous pro-inflammatory genes and it is involved in the regulation of the expression of IL-6 and IP-10 in astrocytes as well as in other cell types (Arevalo et al., 2010; Azcoitia et al., 2010; Cerciat et al., 2010). The downregulation of the production of cytokines and chemokines, such as TNFα, IL-6 and IP-10, by reactive astrocytes may be involved in the neuroprotective mechanisms of E2, at least under chronic neurodegenerative conditions.

The anti-inflammatory effects of E2 involve genomic and membrane starting signals activated by ERs. A study using selective ERα and ERβ specific ligands suggests that both receptor subtypes are involved in the anti-inflammatory action of E2 on astrocytes in vitro (Lewis et al., 2008).

Aim of this part of the project is to evaluate the Ngb involvement in the well known anti-inflammatory effect of E2 in astrocytes. For this purpose, primary cortical astrocytes, isolated from P0 newborn mice, have been used as experimental model.

5.2 Results 5.2.1 17β-estradiol effect on neuroglobin protein levels in mouse primary cortical astrocytes

To assess the responsiveness of primary cortical astrocytes to E2, the expression of ERs was evaluated. In these cells ERα and ERβ were expressed (figure 5.1). Incubation of primary cortical astrocytes with E2 for 5 hours (figure 5.2a) caused a significant increase in Ngb levels with a maximum effect at 10 and 100 pM E2 concentrations, which decreased starting from 1 to 100 nM. At 24 hours of stimulation (figure 5.2b) the effect of E2 on Ngb protein level was still present although with a minor intensity.

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Figure 5.1 Characterization of ERs in mouse primary cortical astrocytes. ER subtype (ERα and ERβ) levels in non-stimulated cells compared to recombinant proteins (5 ng). The amount of protein was normalized to actin levels. Representative Western blots from five independent experiments.

Figure 5.2 Effect of E2 on Ngb protein levels in mouse primary cortical astrocytes. Dose-dependent analysis of E2 treatment (0.01-100 nM) on Ngb levels at 5 hours of stimulation (a) and at 24 hours (b) of stimulation. The amount of protein was normalized to actin levels. Representative Western blots from five independent experiments.

In line with these results 10 pM E2 and 5 hours of stimulation were used in the next experiments to stimulate primary cortical astrocytes.

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The pretreatment of astrocytes with E2 antagonist, ICI (1μM, 30 min before E2 treatment), completely prevented the E2 effect on Ngb levels (figure 5.3), suggesting an ER-mediated mechanism.

As primary astrocytes contain both ERα and ERβ subtypes (figure 5.1), cells were stimulated with either the specific ERα agonist PPT or the specific ERβ agonist DPN to discriminate the role of each ER isoform in the effect of E2 on Ngb levels. Only DPN mimicked the E2 effect on Ngb levels (figure 5.4a), whereas PPT was unable to increase Ngb levels at any tested concentration (figure 5.4b). This strongly suggests the involvement of ERβ in the E2-induced increase of Ngb protein levels.

Figure 5.3 Impact of ERs on Ngb protein levels in primary cortical astrocytes. Western blot analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM) and/or the ER inhibitor ICI (1 μM). ICI was administrated 30 min before E2. The amount of protein was normalized to actin levels. The figure represents a typical Western blot of five independent experiments.

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Figure 5.4 Impact of ERα and ERβ on Ngb protein levels in mouse primary cortical astrocytes. a, Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM) or the ERβ agonist DPN (0.01-100 nM). b, Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM) or the ERα agonist PPT (.0.1-100 nM). The amount of protein was normalized to actin levels. Representative Western blots from five independent experiments.

5.2.2 Effect of lipopolysaccharide on neuroglobin protein levels

First of all, the hypothesis that Ngb expression can be also affected by inflammatory stimuli, such LPS, was evaluated. LPS is commonly used as pro-inflammatory stimulus, inducing the activation of NFκB signaling and thus the synthesis and secretion of several cytokines (Arevalo et al., 2010; Azcoitia et al., 2010; Cerciat et al., 2010).

Compared with control, LPS (i.e., 500 ng/ml for 5 hours) was able to significantly increase Ngb protein levels, even though the effect was less strong than that of E2 (figure 5.5).

The co-treatment with E2 and LPS did not induce a significant difference in Ngb levels compared to the treatment with E2 alone, neither when E2 was administrated before LPS (i.e., E2 pretreatment 1 hour before LPS), nor when LPS was administrated before E2 (i.e., LPS pretreatment 1 hour before E2) (figure 5.5). Indeed, the increase in the levels of Ngb after

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the treatment with both E2 and LPS was similar to the increase after the treatment with E2 alone.

Figure 5.5 Impact of LPS and E2 on Ngb protein levels in mouse primary cortical astrocytes. Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM), LPS (500 ng/ml), E2 + LPS (E2 1 hour before LPS administration), or LPS + E2 (LPS 1 hour before E2 administration). The amount of protein was normalized to actin levels. Upper panel shows typical Western blot of five independent experiments. The results of the desnitometric analysis are represented in the bottom panel. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. (*) significant difference vs. vehicle, (°) vs. E2.

The pretreatment with the ER antagonist ICI prevented the E2 ability

to increase Ngb protein levels (figure 5.6), as also observed in figure 5.3, but did not affect the increase of Ngb mediated by LPS. When ICI was administrated before E2 and LPS co-treatment, only the LPS effect on Ngb increase was detectable (figure 5.6).

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Figure 5.6 Impact of LPS and ERs on Ngb protein levels in mouse primary cortical astrocytes. Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM), LPS (500 ng/ml), and/or ICI (1μM, 30 min before E2 or LPS administration). The amount of protein was normalized to actin levels. Upper panel shows typical Western blots of five independent experiments. The results of the desnitometric analysis are represented in the bottom panel. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. (*) significant difference vs. vehicle, (°) vs. E2.

Since it is known that E2 interferes with LPS-activated signals (i.e.,

NFκB pathway) (Dodel et al., 1999; Giraud et al., 2010) the role of cross-talk between each ER- and LPS-mediated signaling on Ngb protein levels was investigated.

The co-administration of the ERβ agonist, DPN (10 pM), with LPS indicated that LPS reduced the effect of DPN on Ngb induction, suggesting an antagonistic effect of LPS on ERβ signaling (figure 5.7a).

On the contrary, in presence of ERα agonist, PPT (10 pM), the effect of LPS on Ngb increase was barely detectable, indicating an impairing of LPS signaling by ERα (figure 5.7b), as already reported (Ghisletti et al., 2005; Vegeto et al., 2008).

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Figure 5.7 Impact of LPS and ERα or ERβ on Ngb protein levels in mouse primary cortical astrocytes. a, Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM), LPS (500 ng/ml), and/or DPN (10 pM). b, Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM), LPS (500 ng/ml), and/or PPT (10 pM). The amount of protein was normalized to actin levels. Left panels are typical Western blots of five independent experiments. Right panels show the results of the densitometric analysis. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. (*) significant difference vs. vehicle, (°) vs. E2, (§) vs. LPS. (#) vs. DPN.

After the pretreatment of astrocytes with the NFκB cell-permeable

inhibitor peptide, SN50 (10 μg/ml, 30 min before E2 and/or LPS), LPS was not able to increase Ngb levels, whereas the effect of E2 was unchanged (figure 5.8). Thus, SN50 treatment allowed to observe that LPS-activated NFκB was necessary for LPS-induced Ngb increase.

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Figure 5.8 Impact of LPS inhibitor on Ngb protein levels in mouse primary cortical astrocytes. Analysis of Ngb levels in cells stimulated for 5 hours with either vehicle, E2 (10 pM), LPS (500 ng/ml), and/or NFκB inhibitor SN50 (10 μg/ml, 30 min before E2 or LPS administration). The amount of protein was normalized to actin levels. Upper panel shows a typical Western blot of five independent experiments. Bottom panel shows the results of the densitometric analysis. Densitometric analysis (bottom panel) related to E2 and LPS treatment on Ngb levels. Data are means ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. (*) significant difference vs. vehicle, (°) vs. LPS. 5.2.3 Neuroglobin involvement in 17β-estradiol-mediated anti-inflammatory effects against lipopolysaccharide

The role played by increased levels of Ngb on E2 anti-inflammatory effects in primary cortical astrocytes was evaluated. As already reported (Cerciat et al., 2010), exposure of astrocyte cultures for 5 hours to 500 ng/ml LPS resulted in a significant increase in the mRNA levels of IL-6 and IP-10, and E2 10 pM treatment impaired the LPS-mediated increase of these pro-inflammatory molecules (figure 5.9).

Notably, the E2 anti-inflammatory effect against LPS required Ngb. Indeed, in cells transfected with siNgb the E2-induced decrease of IL-6 and IP-10 is completely prevented when co-administrated with LPS, showing no differences respect to the treatment with LPS alone. On the other hand,

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siNgb transfection did not affect the LPS ability to increase IL-6 and IP-10 mRNA levels (figure 5.9).

Figure 5.9 Effects of E2 and LPS on IL-6 and IP-10 mRNA in Ngb-silenced mouse primary cortical astrocytes. a, Analysis of Ngb protein levels in cell transfected with either MOCK (control) or Ngb small interference RNA (siNgb). The amount of protein was normalized to actin levels. A typical Western blot of five independent experiments is shown. Cells transfected with either MOCK (b, d) or siNgb (c, e) were stimulated for 5 hours with either vehicle, E2 (10 pm) and/or LPS (500ng/ml). The mRNA levels of IL-6 (b, c) and IP-10 (d, e) were measured on cell lysates by realtime RT-PCR. Data represent mean ± SD of five different experiments. Significant differences (p<0.001) were determined with ANOVA followed by Tukey-Kramer post-test. (*) significant difference vs. vehicle, (°) vs. LPS.

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5.3 Discussion

Our findings indicate that E2 induces Ngb upregulation also in primary astrocytes. The E2 effect on Ngb levels is already present after 5 hours of hormone treatment, and persists until 24 hours of stimulation. The effect is dose-dependent, with the maximum effect at 10 pM E2 concentration, two and three orders of magnitude lower than the effective dose in SK-N-BE cells or primary hippocampal neurons, respectively (i.e., 1 and 10 nM, respectively). Also in primary astrocytes the E2 dose-response curve results to be bell-shaped. As discussed before for the effect of E2 on Ngb in neurons, the lack of effect at higher concentrations could be considered the expression of a receptor downregulation phenomenon, by which the cells protect themselves against high hormone levels. Also, as previously mentioned, the two ERs can regulate each other their expression, affecting also in this way the cell response to the hormone (Galluzzo et al., 2009). Indeed, when cells are treated with DPN, which is the agonist of ERβ subtype only, the dose-response curve on Ngb level ends with a plateau, being ERα not activated.

ERα and ERβ are equally expressed in astrocytes and the ER pure antagonist ICI completely prevents the effect of E2 on Ngb protein level increase. In particular, the effect of E2 on Ngb levels specifically requires ERβ, confirming also in astrocytes the direct involvement of ERβ in Ngb modulation, as it was the case for human neuroblastoma SK-N-BE and mouse hippocampal neurons (see previous chapters).

The results reported here establish that Ngb is involved in the actions of E2 also in astrocytes. Indeed, Ngb is pivotal to mediate the E2 inhibitory effect on IL-6 and IP-10 synthesis, since siNgb treatment prevents the anti-inflammatory effect of E2; but at the same time siNgb does not affect LPS-mediated overexpression of inflammatory markers.

Acting also on the anti-inflammatory action of E2, Ngb can now be regarded as a key mediator of different E2 protective effects in the brain, including inflammatory mechanisms that often may represent the cause of principal neurodegenerative diseases and determine the prognostic consequences of these pathologies (Grammas, 2011; Li et al., 2011; Lim, 2011; Sastre et al., 2011; Scrivo et al., 2011). These findings, together with more recent works (DellaValle et al., 2010), indicate that Ngb likely plays a central role in different CNS disorders such as neurotrauma, infectious and autoimmune disease, acting directly in astrocytes. Together with the well known effects in neurons, the neuroprotective function of Ngb is now enriched of pivotal effects in astrocytes that are the most abundant cell

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population in CNS and represent the first contact with the blood brain barrier, pointing to a whole brain protective function of Ngb.

The mechanisms involved in the inhibition of the expression of inflammatory molecules by E2-induced Ngb needs to be further characterized. One possible mechanism is that Ngb can interact with critical sites involved in NFκB nuclear translocation, impairing, for instance, its dissociation from inhibitor of κB (IκB) proteins; Ngb may also interact with IκBs, inhibiting their degradation that is at the root of NFκB nuclear translocation; or Ngb may interfere with IκB kinases (IKKs) activity, which are important to phosphorylate IκB and promote their degradation.

Indeed, the transcriptional and/or translational control of Ngb expression is incompletely understood, as are the factors that regulate Ngb protein stability or turnover (Emara et al., 2009). For instance, some authors have been proposed that Ngb can be upregulated by hypoxic/ischemic-activated signals, but remains uncertain whether Ngb is actually controlled by the major regulator of cellular hypoxia response known as hypoxia-inducible factor-1 (HIF-1) (Sun et al., 2001). Using in silico techniques, Wystub and coworkers showed that conserved hypoxia-response elements, including HIF-1 consensus binding motifs, are absent from the upstream region of mouse and human Ngb (Wystub et al., 2004). However, the NGB promoter region does contain AP-1 and NFκB binding motifs, both of which have been reported to be activated by hypoxia or other insults, such as pro-inflammatory stimuli.

LPS, via NFκB signal, is able to increase Ngb levels. However, the role of this globin is not involved in the promotion of LPS effects, as observed for E2.

Interestingly, although both E2 and LPS are able to increase Ngb protein levels, a negative cross-talk between ERs and LPS-induced signal (i.e., NFκB) seems to be present. In fact, ERα-activated signals (which are not involved in E2-mediated Ngb upregulation) block NFκB-mediated Ngb increase, whereas on the other hand, also LPS impairs the ERβ-induced upregulation of Ngb protein levels. Therefore, the co-activation of ERα and ERβ is pivotal to regulate Ngb expression in presence of NFκB-activated signals in astrocytes.

These data open new avenues in the field of Ngb research in that the function played by this globin in CNS is larger than that has been thought previously, including also a protective role against neuroinflammation.

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6. CONCLUSION

Ngb received a great attention among scientific community due to its endogenous neuroprotective function. However, the mechanisms by which Ngb exerts this important role are still unclear.

Although it is well recognized that the overexpression of Ngb in transgenic animals reduces the size of cerebral infarct (Khan et al., 2006; Wang et al., 2008) and enhances cell survival after different brain injuries (Sun et al., 2003; Khan et al., 2006; Fordel et al., 2007a; Khan et al., 2008), the elucidation of the neuronal Ngb function(s) requires the identification of endogenous modulators of its expression and the molecular mechanisms responsible for Ngb expression and induction.

Indeed, this thesis was aimed to identify i) Ngb endogenous modulator(s), ii) the molecular mechanisms responsible of Ngb expression and induction, and iii) the role played by Ngb in neuroprotective signaling pathways.

In this thesis we have demonstrated that at physiological concentrations E2 strongly increases Ngb protein levels in human SK-N-BE cells and in mouse neurons via both ERβ-dependent genomic and membrane-initiated signals that specifically require p38 activation. The effect of E2 is specific, being not mimicked by either the male sex steroid hormone, DHT, or by the common precursor testosterone, or by IGF-1, another well-known neuroprotective hormone. These results represent the first evidence for steroid hormone modulation of globin expression in cells.

The E2 specificity on Ngb induction could raise the idea that Ngb expression is at the root of gender differences reported in male and female brain (Gillies and McArthur, 2010). However, ERs are expressed also in male brain. Testosterone freely enters the brain and could be converted to E2 by local aromatase (Simerly, 2005; Wilson and Davies, 2007; Forger, 2009; Tobet et al., 2009). Thus, E2 in particular could affects also male brain.

Moreover, we demonstrated that the well known neuroprotective effects elicited by E2 (McEwen, 2002; Marin et al., 2008; Morisette et al., 2008; Vasudevan and Pfaff, 2008; Gillies and McArthur, 2010) may, at least in part, be explained by an enhanced Ngb expression in neurons. Indeed, Ngb is essential to E2 protection against H2O2-induced toxicity by the inhibition of caspase-3 and PARP cleavage and by enhancing cell survival.

Intriguingly, we have detected that E2 not only affects Ngb expression, but also induces its reallocation in subcellular compartments, increasing Ngb levels into mitochondria. Indeed, we have confirmed that

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Ngb is present in the cell nucleus, mitochondria and cytosol, as reported also by other authors (Bentmann et al., 2005; Hundahl et al., 2010).

Several functions of Ngb have been proposed: (i) Ngb may exert a Mb-like role, enhancing O2 supply to the mitochondria of the metabolically active neurons; (ii) Ngb may scavenge damaging ROS or RNS, which are generated for example by the respiratory chain; (iii) Ngb may detoxify harmful excess of NO to nitrate at normoxia or produce NO for signaling functions from nitrite at hypoxia for the control of blood pressure; (iv) Ngb may be involved in a signal transduction pathway, e.g. by inhibiting the dissociation of GDP from G protein α; (v) Ngb may be part of a redox process that is instrumental in preventing apoptosis via reduction of cytochrome c (Burmester and Hankeln, 2009, and literature therein). All these functions have been supported by some experimental data or based on analogy with other globins.

In this study we have demonstrated that the major physiological role for Ngb in the mitochondria is the interception of the intrinsic pathway of apoptosis interfering directly with cytochrome c release, as previously suggested by in silico approaches (Fago et al., 2006; Bønding et al., 2008; Fago et al., 2008; Brittain et al., 2010a; Brittain et al., 2010b). In particular, E2 facilitates Ngb-cytochrome c interaction via ERβ activity. This specificity highlights the role of mitochondrial ERβ signaling in prevention of apoptosis as an essential mechanism of E2-mediated neuroprotection (Simpkins et al., 2008; Flynn et al., 2008; Yang et al., 2009, and literature therein).

Although first studies considered that Ngb has a neuron-specific localization (Laufs et al., 2004; Brunori and Vallone, 2006, and literature therein), we have confirmed that Ngb is expressed in both neurons and astrocytes, as observed by other authors (Chen et al., 2005; DellaValle et al., 2010; Dong et al., 2010). We have also found that E2, at physiological concentrations, strongly increases Ngb protein levels in mouse primary astrocytes through a mechanism involving ERβ. Thus, the ERβ specificity in Ngb induction is preserved also in this cell type.

Intriguingly, we have shown that Ngb induction occurs also after a pro-inflammatory stimulus such as LPS, with a mechanism requiring NFκB activation. This effect is in agreement with the presence of a NFκB consensus motif in Ngb promoter (Wystub et al., 2004), suggesting a direct action of NFκB on Ngb gene transcription.

Although both E2 and LPS are able to increase Ngb protein levels, we have detected a negative cross-talk between ERs and LPS signals. Indeed, ERα-activated signals (which are not involved in E2-mediated Ngb upregulation) inhibit LPS-mediated Ngb increase, whereas LPS impairs the

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ERβ-induced upregulation of Ngb protein levels. Therefore, the co-activation of ERα and ERβ is pivotal to regulate Ngb expression in presence of LPS-activated signals.

The increase in Ngb levels after LPS treatment is not involved in the promotion of LPS effects, as observed for E2. In contrast, Ngb is pivotal to mediate the E2 inhibitory effect on IL-6 and IP-10 synthesis, but, at the same time, does not affect LPS-mediated synthesis of inflammatory molecules. Therefore, Ngb is crucial also for E2-mediated anti-inflammatory effects. Thus, these results suggest that Ngb can be pinpointed as an additional regulatory protein of immune response, at least in astrocytes.

Intriguingly, we have observed that H2O2 treatment increases the interaction of Ngb with cytochrome c and LPS induces, via NFκB, an increase in Ngb protein levels. These effects exerted by two different insults may indicate that Ngb can act also as a compensatory protein able to counteract oxidative and inflammatory stimuli, as already observed during hypoxic conditions, where a Ngb role in sensing or responding to neuronal hypoxia has been suggested (Sun et al., 2001; Di Giulio et al., 2006; Li et al., 2006; Schmidt-Kastner et al., 2006). However, in both cases siNgb does not alter the effect of the insult (increase in cell death after H2O2 treatment, and increase in mRNA levels of inflammatory molecules after LPS treatment). This indicates that Ngb does not regulate with a feedback mechanism the effect of H2O2 and LPS.

In fact, the role of Ngb as compensatory protein suggested by other authors has been demonstrated after prolonged injuring stimuli (e.g., chronic hypoxia, MCAO/reperfusion) (Sun et al., 2001; Sun et al., 2003; Di Giulio et al., 2006), showing an increase of damage when Ngb was knocked-down (Sun et al., 2001; Sun et al., 2003). However one group has reported increased damage after a short-term oxidative insult (i.e., 6 hours) when Ngb knocking-down is prolonged for 48 hours before injury (Li et al., 2008). Thus, the cause why in our study there is no increased damage by H2O2 or LPS when cells are treated with siNgb may be related with the duration of Ngb silencing and to the single-dose administration of both H2O2 and LPS. Probably the exacerbation of the injury in absence of Ngb would be visible after more prolonged periods of Ngb silencing and/or after chronic neurodegenerative treatments.

In this scenario E2 function is to accelerate the protective cell response to external insults (De Marinis et al., 2011) by inducing a rapid and strong increase of endogenous Ngb, which in turn is pivotal to impair the trigger of the intrinsic apoptotic pathway in neurons and to reduce the inflammatory response of astrocytes. Further in vivo studies that will take into account the

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important role of E2 in Ngb induction are required to better understand how this new mechanism of Ngb endogenous modulation can be utilized in order to enhance the brain protective mechanisms.

In agreement with other authors (Yu et al., 2009, and literature therein), our findings suggest that the principal role played by Ngb in the brain could be the reduction of neuronal death by resetting the trigger level of apoptosis and by the inhibition of the expression of pro-inflammatory molecules, leading to the onset of physiological response to stress. E2 acts to accelerate Ngb neuroprotective effect by rapidly enhancing its protein levels in both neurons and astrocytes.

In addition, the possibility that other hormones and neurotransmitters may upregulate Ngb levels in the brain represents a potential new opportunity for the development of neuroprotective strategies and drugs against stroke, inflammation, neurodegenerative diseases (e.g., Alzhemer’s and Parkinson’s disease), excitotoxicity, and injuries related to oxygen or glucose deprivation.

Finally, the finding that Ngb is an E2-inducible nerve globin opens new avenues in the Ngb research field in that a strong re-evaluation of its anatomical and subcellular localization must be taken into account. In particular, recent ongoing studies that are carried out by Dr. Marco Fiocchetti, in the Cell and Animal Physiology laboratory of Roma Tre University, are demonstrating that Ngb is an E2-inducible protein also in non-neuronal tissues, suggesting that this novel globin have an important role in the pleiotropic effect of the most important female sex hormone.

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REFERENCES Acconcia, F., and Marino, M. (2011) The effects of 17β-estradiol in cancer

are mediated by estrogen receptor signaling at the plasma membrane. Front. Physiol. 2011 30, 1-8.

Acconcia, F., Ascenzi, P., Bocedi, A., Spisni, E., Tomasi, V., Trentalance, A., Visca, P., and Marino, M. (2005a) Palmitoylation-dependent estrogen receptor α membrane localization: regulation by 17β-estradiol. Mol. Biol. Cell 16, 231-237.

Acconcia, F., Ascenzi, P., Fabozzi, G., Visca, P., and Marino, M. (2004) S-palmitoylation modulates human estrogen receptor-alpha functions. Biochem. Biophys. Res. Commun. 316, 878-883.

Acconcia, F., Totta, P., Ogawa, S., Cardillo, I., Inoue, S., Leone, S., Trentalance, A., Muramatsu, M., and Marino, M. (2005b) Survival versus apoptotic 17β-estradiol effect: role of ERα- and ERβ- activated nongenomic signaling. J. Cell Physiol. 203, 193-201.

Alimirah, F., Chen, J., Basrawala, Z., Xin, H., and Choubey, D. (2006) DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation. FEBS Lett. 580, 2294-2300.

Antao, S.T., Duong, T.T., Aran, R., and Witting, P.K. (2010) Neuroglobin overexpression in cultured human neuronal cells protects against hydrogen peroxide insult via activating phosphoinositide-3 kinase and opening the mitochondrial K(ATP) channel. Antioxid. Redox Signal 13, 769-781.

Araújo, G.W., Beyer, C., and Arnold, S. (2008) Oestrogen influences on mitochondrial gene expression and respiratory chain activity in cortical and mesencephalic astrocytes. J. Neuroendocrinol. 20, 930-941.

Arevalo, M.A., Santos-Galindo, M., Bellini, M.J., Azcoitia, I., and Garcia-Segura, L.M. (2010) Actions of estrogens on glial cells: Implications for neuroprotection. Biochim. Biophys. Acta 1800, 1106-1112.

Arnold, S., and Beyer, C. (2009) Neuroprotection by estrogen in the brain: the mitochondrial compartment as presumed therapeutic target. J. Neurochem. 110, 1-11.

Arredondo-Peter, R., Hargrove, M.S., Moran, J.F., Sarath, G., and Klucas, R.V. (1998) Plant hemoglobins. Plant Physiol. 118, 1121-1125.

Ascenzi, P., Bellelli, A., Coletta, M., Colosimo, A., Falcioni, G., Giacometti, G. M., Ippoliti, R., Zolla, L., and Giardina, B. (2007) Multiple strategies for O2 transport: from simplicity to complexity. IUBMB Life 59, 600-616.

67

Ascenzi, P., Bocedi, A., and Marino, M. (2006) Structure-function relationship of estrogen receptor alpha and beta: impact on human health. Mol. Aspects Med. 27, 299-402.

Ascenzi, P., Polticelli, F., Marino, M., Santucci, R., and Coletta, M. (2011) Cardiolipin drives cytochrome c proapoptotic and antiapoptotic actions. IUBMB Life 63, 160-165.

Avivi, A., Gerlach, F., Joel, A., Reuss, S., Burmester, T., Nevo, E., and Hankeln, T. (2010) Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax. Proc. Natl. Acad. Sci. USA 107, 21570-21575.

Azcoitia, I., Santos-Galindo, M., Arevalo, M.A., and Garcia-Segura, L.M. (2010) Role of astroglia in the neuroplastic and neuroprotective actions of estradiol. Eur. J. Neurosci. 32, 1995-2002.

Behl, C. (2002) Oestrogen as a neuroprotective hormone. Nat. Rev. Neurosci. 3, 433-442.

Behl, C., Skutella, T., Lezoualc’h, F., Post, A., Widmann, M., Newton, C.J., and Holsboer, F. (1997) Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol. Pharmacol. 51, 535-541.

Behl, C., Widmann, M., Trapp, T., and Holsboer, F. (1995) 17-β Estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem. Biophys. Res. Commun. 216, 473-482.

Bentmann, A., Schmidt, M., Reuss, S., Wolfrum, U., Hankeln, T., and Burmester, T. (2005) Divergent distribution in vascular and avascular mammalian retinae links neuroglobin to cellular respiration. J. Biol. Chem. 280, 20660-20665.

Biagioli, M., Pinto, M., Cesselli, D., Zaninello, M., Lazarevic, D., Roncaglia, P., Simone, R., Vlachouli, C., Plessy, C., Bertin, N., Feltrami, A., Kobayashi, K., Gallo, V., Santoro, C., Ferrer, I., Rivella, S., Feltrami, C. A., Carninci, P., Raviola, E., and Gustincich, S. (2009) Unexpected expression of α- and β-globin in mesencephalic dopaminergic neurons and glial cells. Proc. Natl. Acad. Sci. USA 106, 15454-15459.

Bishop, J., and Simpkins, J.W. (1994) Estradiol treatment increases viability of glioma and neuroblastoma cells in vitro. Mol. Cell. Neurosci. 5, 303-308.

Bjornstrom, L., and Sjoberg, M. (2005) Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol. Endocrinol. 19, 833-842.

68

Bolognesi, M., Bordo, D., Rizzi, M., Tarricone, C., and Ascenzi, P. (1997) Nonvertebrate hemoglobins: Structural bases for reactivity. Prog. Biophys. Mol. Biol. 68, 29-68.

Bønding, S.H., Henty, K., Dingley, A.J., and Brittain, T. (2008) The binding of cytochrome c to neuroglobin: a docking and surface plasmon resonance study. Int. J. Biol. Macromol. 43, 295-299.

Borutaite, V., and Brown, G.C., (2007) Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state. J. Biol. Chem. 282, 31124-31130.

Brann, D.W., Dhandapani, K., Wakade, C., Mahesh, V.B., and Khan, M.M. (2007) Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids 72, 381-405.

Brittain, T., Skommer, J., Henty, K., Birch, N., and Raychaudhuri, S. (2010a) A role for human neuroglobin in apoptosis. IUBMB Life 62, 878-885.

Brittain, T., Skommer, J., Raychaudhuri, S., and Birch, N. (2010b) An antiapoptotic neuroprotective role for neuroglobin. Int. J. Mol. Sci. 11, 2306-2321.

Brunori, M., and Vallone, B. (2006) A globin for the brain. FASEB J. 20, 2192-2197.

Brunori, M., and Vallone, B. (2007) Neuroglobin, seven years after. Cell. Mol. Life Sci. 64, 1259-1268.

Brunori, M., Giuffrè, A., Nienhaus, K., Nienhaus, G.U., Scandurra, F.M. and Vallone, B. (2005) Neuroglobin, nitric oxide, and oxygen: functional pathways and conformational changes. Proc. Natl. Acad. Sci. USA 102, 8483-8488.

Bryan, N.S., Fernandez, B.O., Bauer, S.M., Garcia-Saura, M.F., Milsom, A.B., Rassaf, T., Maloney, R.E., Bharti, A., Rodriguez, J., and Feelisch, M. (2005) Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nature Chem. Biol. 1, 290-297.

Burmester, T., and Hankeln, T. (2004) Neuroglobin: a respiratory protein of the nervous system. News Physiol. Sci. 19, 110-113.

Burmester, T., and Hankeln, T. (2008) Neuroglobin and other nerve haemoglobins. In Dioxygen binding and sensing proteins. Bolognesi, M., di Prisco, G., and Verde, C. (eds.), Springer, Heidelberg, pp. 211-222.

Burmester, T., and Hankeln, T. (2009) What is the function of neuroglobin? J. Exp. Biol. 212, 1423-1428.

69

Burmester, T., Ebner, B., Weich, B., and Hankeln, T. (2002) Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues. Mol. Biol. Evol. 19, 416-421.

Burmester, T., Gerlach, F., and Hankeln, T. (2007). Regulation and role of neuroglobin and cytoglobin under hypoxia. Adv. Exp. Med. Biol. 618, 169-180.

Burmester, T., Haberkamp, M., Mitz, S., Roesner, A., Schmidt, M., Ebner, B., Gerlach, F., Fuchs, C. and Hankeln, T. (2004) Neuroglobin and cytoglobin: genes, proteins and evolution. IUBMB Life 56, 703-707.

Burmester, T., Weich, B., Reinhardt, S., and Hankeln, T. (2000) A vertebrate globin expressed in brain. Nature 407, 520-523.

Cerciat, M., Unkila, M., Garcia-Segura, L.M., and Arevalo, M.A. (2010) Selective estrogen receptor modulators decrease the production of interleukin-6 and interferon gamma-inducible protein-10 by astrocytes exposed to inflammatory challenge in vitro. Glia 58, 93-102.

Chen, X.Q., Qin, L.Y., Zhang, C.G., Yang, L.T., Gao, Z., Liu, S., Lau, L.T., Fung, Y.W., Greenberg, D.A., and Yu, A.C. (2005) Presence of neuroglobin in cultured astrocytes. Glia 50, 182-186.

Cheng, C.H., di Prisco, G., and Verde, C. (2009) The ‘‘icefish paradox’’: which is the task of neuroglobin in Antarctic hemoglobin-less icefish? IUBMB Life 61, 184-188.

Cyr, M., Calon, F., Morissette, M., and Di Paolo, T. (2002) Estrogenic modulation of brain activity: implications for schizophrenia and Parkinson’s disease. J. Psychiatry Neurosci. 27, 12-27.

Cyr, M., Calon, F., Morissette, M., Grandbois, M., Di Paolo, T., and Callier, S. (2000) Drugs with estrogen-like potency and brain activity: potential therapeutic application for the CNS. Curr. Pharm. Des. 6, 1287-1312.

Czlonkowska, A., Ciesielska, A., Gromadzka, G., and Kurkowska-Jastrzebska, I. (2006) Gender differences in neurological disease: role of estrogens and cytokines. Endocrine 29, 243-256.

De Marinis, E., Ascenzi, P., Pellegrini, M., Galluzzo, P., Bulzomi, P., Arevalo, M.A., Garcia-Segura, L.M., and Marino, M. (2010) 17β-estradiol-a new modulator of neuroglobin levels in neurons: role in neuroprotection against H2O2-induced toxicity. Neurosignals 18, 223-235.

De Marinis, E., Marino, M., and Ascenzi, P. (2011) Neuroglobin, estrogens, and neuroprotection. IUBMB Life 63, 140-145.

DellaValle, B., Hempel, C., Kurtzhals, J.A., and Penkowa, M. (2010) In vivo expression of neuroglobin in reactive astrocytes during

70

neuropathology in murine models of traumatic brain injury, cerebral malaria, and autoimmune encephalitis. Glia 58, 1220-1227.

Dewilde, S., Blaxter, M., Van Hauwaert, M.L., Vanfleteren, J., Esmans, E.L., Marden, M., Griffon, N., and Moens, L. (1996) Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata. J. Biol. Chem. 271, 19865-19870.

Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V., Aerts, T., Marden, M. C., Caubergs, R. and Moens, L. (2001) Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family. J. Biol. Chem. 276, 38949-38955.

Dhandapani, K.M., and Brann, D.W. (2002) Protective effects of estrogen and selective estrogen receptor modulators in the brain. Biol. Reprod. 67, 1379-1385.

Di Giulio, C., Bianchi, G., Cacchio, M., Artese, L., Piccirilli, M., Verratti, V., Valerio, R., and Iturriaga, R. (2006) Neuroglobin, a new oxygen binding protein is present in the carotid body and increases after chronic intermittent hypoxia. Adv. Exp. Med. Biol. 580, 15-19.

Dixon, B., and Pohajdak, B. (1992) Did the ancestral globin gene of plants and animals contain only two introns? Trends Biochem. Sci. 17, 486-488.

Dodel, R.C., Du, Y., Bales, K.R., Gao, F., and Paul, S.M. (1999) Sodium salicylate and 17beta-estradiol attenuate nuclear transcription factor NF-kappaB translocation in cultured rat astroglial cultures following exposure to amyloid A beta(1-40) and lipopolysaccharides. J. Neurochem. 73, 1453-1460.

Dong, Y., and Benveniste, E.N. (2001) Immune function of astrocytes. Glia 36, 180-190.

Dong, Y., Zhao, R., Chen, X.Q., and Yu, A.C. (2010) 14-3-3gamma and neuroglobin are new intrinsic protective factors for cerebral ischemia. Mol. Neurobiol. 41, 218-231.

Emara, M., Salloum, N., and Allalunis-Turner, J. (2009) Expression and hypoxic up-regulation of neuroglobin in human glioblastoma cells. Mol. Oncol. 3, 45-53.

Emara, M., Turner, A.R., and Allalunis-Turner, J. (2010) Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues. Cancer Cell Int. 10, 33.

Fago, A., Hundahl, C., Dewilde, S., Gilany, K., Moens, L. and Weber, R.E. (2004) Allosteric regulation and temperature dependence of oxygen binding in human neuroglobin and cytoglobin: molecular mechanisms and physiological significance. J. Biol. Chem. 279, 44417-44426.

71

Fago, A., Mathews, A.J., and Brittain, T. (2008) A role for neuroglobin: resetting the trigger level for apoptosis in neuronal and retinal cells. IUBMB Life 60, 398-401.

Fago, A., Mathews, A.J., Moens, L., Dewilde, S., and Brittain, T. (2006) The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c. FEBS Lett. 580, 4884-4888.

Fleming, G.F., Amato, J.M., Agresti, M., and Safa, A.R. (1992) Megestrol acetate reverses multidrug resistance and interacts with P-glycoprotein. Cancer Chemother. Pharmacol. 29, 445-449.

Flögel, U., Gödecke, A., Klotz, L.O., and Schrader, J. (2004) Role of myoglobin in the antioxidant defense of the heart. FASEB J. 18, 1156-1158.

Flögel, U., Merx, M.W., Gödecke, A., Decking, U.K. and Schrader, J. (2001) Myoglobin: a scavenger of bioactive NO. Proc. Natl. Acad. Sci. USA 98, 735-740.

Flynn, J.M., Dimitrijevich, S.D., Younes, M., Skliris, G., Murphy, L.C., and Cammarata, P.R. (2008) Role of wild-type estrogen receptor-beta in mitochondrial cytoprotection of cultured normal male and female human lens epithelial cells. Am. J. Physiol. Endocrinol. Metab. 295, E637-E647.

Fordel, E., Thijs, L., Martinet, W., Lenjou, M., Laufs, T., Van Bockstaele, D., Moens, L., and Dewilde, S. (2006) Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death. Neurosci. Lett. 20, 146-151.

Fordel, E., Thijs, L., Martinet, W., Schrijvers, D., Moens, L., and Dewilde, S. (2007b) Anoxia or oxygen and glucose deprivation in SH-SY5Y cells: a step closer to the unraveling of neuroglobin and cytoglobin functions. Gene 398, 114-122.

Fordel, E., Thijs, L., Moens, L., and Dewilde, S. (2007a) Neuroglobin and cytoglobin expression in mice: Evidence for a correlation with reactive oxygen species scavenging. FEBS J. 274, 1312-1317.

Forger, N.G. (2009) Control of cell number in the sexually dimorphic brain and spinal cord. J. Neuroendocrinol. 21, 393-399.

Fuchs, C., Burmester, T., and Hankeln, T. (2006) The amphibian globin gene repertoire as revealed by Xenopus genome sequence. Cytogenet. Genome Res. 112, 296-306.

Galluzzo, P., Ascenzi, P., Bulzomi, P., and Marino, M. (2008) The nutritional flavanone naringenin triggers antiestrogenic effects by regulating estrogen receptor α-palmitoylation. Endocrinology 149, 2567-2575.

72

Galluzzo, P., Caiazza, F., Moreno, S., and Marino, M. (2007) Role of ERβ palmitoylation in the inhibition of human colon cancer cell proliferation. Endocr. Relat. Cancer 14, 153-167.

Galluzzo, P., Rastelli, C., Bulzomi, P., Acconcia, F., Pallottini, V., and Marino, M. (2009) 17β-Estradiol regulates the first steps of skeletal muscle cell differentiation via ERβ-mediated signals. Am. J. Physiol. Cell Physiol. 297, C1249-C1262.

Garcia-Segura, L.M., Arevalo, M.A., and Azcoitia, I. (2010) Interactions of estradiol and insulin-like growth factor-I signalling in the nervous system: new advances. Prog. Brain Res. 181, 251-272.

Gardner, P.R., Gardner, A.M., Martin, L.A. and Salzman, A.L. (1998) Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc. Natl. Acad. Sci. USA 95, 10378-10383.

Geuens, E., Brouns, I., Flamez, D., Dewilde, S., Timmermans, J. P., and Moens, L. (2003) A globin in the nucleus! J. Biol. Chem. 278, 30417-30420.

Geuens, E., Dewilde, S., Hoogewijs, D., Pesce, A., Nienhaus, K., Nienhaus, G.U., Olson, J., Vanfleteren, J., Bolognesi, M., and Moens, L. (2004) Nerve globins in invertebrates. IUBMB Life 56, 653-656.

Ghisletti, S., Meda, C., Maggi, A., and Vegeto, E. (2005) 17beta-estradiol inhibits inflammatory gene expression by controlling NF-kappaB intracellular localization. Mol. Cell Biol. 25, 2957-2968.

Gillies, G.E., and McArthur, S. (2010) Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines. Pharmacol. Rev. 62, 155-198.

Giraud, S.N., Caron, C.M., Pham-Dinh, D., Kitabgi, P., and Nicot, A.B. (2010) Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes. Proc. Natl. Acad. Sci. U S A 107, 8416-8421.

Giuffrè, A., Moschetti, T., Vallone, B., and Brunori, M. (2008) Neuroglobin: enzymatic reduction and oxygen affinity. Biochem. Biophys. Res. Commun. 367, 893-898.

Go, M. (1981) Correlation of DNA exonic regions with protein structural units in haemoglobin. Nature 291, 90-92.

Goodman, M., Moore, G. W., and Matsuda, G. (1975) Darwinian evolution in the genealogy of haemoglobin. Nature 253, 603-608.

Gouras, G.K., Xu, H., Gross, R.S., Greenfield, J.P., Hai, B., Wang, R., and Greengard, P. (2000) Testosterone reduces neuronal secretion of Alzheimer’s beta amyloid peptides. Proc. Natl. Acad. Sci. USA 97, 1202-1205.

73

Grammas, P. (2011) Neurovascular dysfunction, inflammation and endothelial activation: implications for the pathogenesis of Alzheimer's disease. J Neuroinflammation 8, 26.

Green, P.S., and Simpkins J.W. (2000) Neuroprotective effects of estrogens: potential mechanisms of action. Int. J. Dev. Neurosci. 18, 347-358.

Hamdane, D., Kiger, L., Dewilde, S., Green, B.N., Pesce, A., Uzan, J., Burmester, T., Hankeln, T., Bolognesi, M., Moens, L. and Marden, M.C. (2004) Coupling of the heme and an internal disulfide bond in human neuroglobin. Micron. 35, 59-62.

Hamdane, D., Kiger, L., Dewilde, S., Green, B.N., Pesce, A., Uzan, J., Burmester, T., Hankeln, T., Bolognesi, M., Moens, L. and Marden, M.C. (2003) The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin. J. Biol. Chem. 278, 51713-51721.

Hankeln, T., Ebner, B., Fuchs, C., Gerlach, F., Haberkamp, M., Laufs, T. L., Roesner, A., Schmidt, M., Weich, B., Wystub, S., Saaler-Reinhardt, S., Reuss, S., Bolognesi, M., De Sanctis, D., Marden, M.C., Kiger, L., Moens, L., Dewilde, S., Nevo, E., Avivi, A., Weber, R.E., Fago, A., and Burmester, T. (2005) Neuroglobin and cytoglobin in search of their role in the vertebrate globin family. J. Inorg. Biochem. 99, 110-119.

Hankeln, T., Friedl, H., Ebersberger, I., Martin, J., and Schmidt, E.R. (1997) A variable intron distribution in globin genes of Chironomus: evidence for recent intron gain. Gene 205, 151-160.

Hankeln, T., Wystub, S., Laufs, T., Schmidt, M., Gerlach, F., Saaler- Reinhardt, S., Reuss, S., and Burmester, T. (2004) The cellular and subcellular localization of neuroglobin and cytoglobin-a clue to their function? IUBMB Life 56, 671-679.

Hansson, E., and Ronnback, L. (2003) Glial neuronal signaling in the central nervous system. FASEB J. 17, 341-348.

Hardison, R. (1999). The evolution of hemoglobin. Am. Scient. 87, 126-137.

Hardison, R.C. (1996) A brief history of hemoglobins: plant, animal, protist, and bacteria. Proc. Natl Acad. Sci. USA 93, 5675-5679.

Henderson, V.W. (1997) Estrogen, cognition, and a woman’s risk of Alzheimer’s disease. Am. J. Med. 103, 11S-18S.

Herold, S., Fago, A., Weber, R.E., Dewilde, S., and Moens, L. (2004) Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuroglobin reveal a potential role against oxidative stress. J. Biol. Chem. 279, 22841-22847.

74

Hou, S., Freitas, T., Larsen, R.W., Piatibratov, M., Sivozhelezov, V., Yamamoto, A., Meleshkevitch, E.A., Zimmer, M., Ordal, G.W. and Alam, M. (2001) Globin-coupled sensors: a class of heme-containing sensors in Archaea and Bacteria. Proc. Natl. Acad. Sci. USA 98, 9353-9358.

Hoy, J.A., and Hargrove, M.S. (2008) The structure and function of plant hemoglobins. Plant. Physiol. Biochem. 46, 371-379.

Hundahl, C., Fago, A., Dewilde, S., Moens, L., Hankeln, T., Burmester, T., and Weber, R.E. (2006a) Oxygen binding properties of non-mammalian nerve globins. FEBS J. 273, 1323-1329.

Hundahl, C., Kelsen, J., Ronn, L.C., Weber, R.E., Geuens, E., Hay-Schmidt, A., and Nyengaard, J.R. (2006b) Does neuroglobin protect neurons from ischemic insult? A quantitative investigation of neuroglobin expression following transient MCAO in spontaneously hypertensive rats. Brain Res. 1085, 19-27.

Hundahl, C.A., Allen, G.C., Hannibal, J., Kjaer, K., Rehfeld, J.F., Dewilde, S., Nyengaard, J.R., Kelsen, J., and Hay-Schmidt, A. (2010) Anatomical characterization of cytoglobin and neuroglobin mRNA and protein expression in the mouse brain. Brain Res. 1331, 58-73.

Jin, K., Mao, X.O., Xie, L., Khan, A.A., and Greenberg, D.A. (2008) Neuroglobin protects against nitric oxide toxicity. Neurosci. Lett. 430, 135-137.

Kakar, S., Hoffman, F.G., Storz, J.F., Fabian, M., and Hargrove, M.S., (2010) Structure and reactivity of hexacoordinate hemoglobins. Biophys. Chem. 152, 1-14.

Kast, M. (2001) The best supporting actors. Nature 412, 674-676. Khan, A.A., Mao, X.O., Banwait, S., DerMardirossian, C.M., Bokoch,

G.M., Jin, K., and Greenberg, D.A. (2008) Regulation of hypoxic neuronal death signaling by neuroglobin. FASEB J. 22, 1737-1747.

Khan, A.A., Mao, X.O., Banwait, S., Jin, K., and Greenberg, D.A. (2007) Neuroglobin attenuates beta-amyloid neurotoxicity in vitro and transgenic Alzheimer phenotype in vivo. Proc. Natl. Acad. Sci. USA 104, 19114-19119.

Khan, A.A., Wang, Y., Sun, Y., Mao, X.O., Xie, L., Miles, E., Graboski, J., Chen, S., Ellerby, L.M., Jin, K., and Greenberg, D.A. (2006) Neuroglobin-overexpressing transgenic mice are resistant to cerebral and myocardial ischemia. Proc. Natl. Acad. Sci. USA 103, 17944-17948.

Kiger, L., Uzan, J., Dewilde, S., Burmester, T., Hankeln, T., Moens, L., Hamdane, D., Baudin-Creuza, V., and Marden, M. (2004) Neuroglobin ligand binding kinetics. IUBMB Life 56, 709-719.

75

Kipp, M., Karakaya, S., Johann, S., Kampmann, E., Mey, J., and Beyer, C. (2007) Oestrogen and progesterone reduce lipopolysaccharide-induced expression of tumour necrosis factor-alpha and interleukin-18 in midbrain astrocytes. J. Neuroendocrinol. 19, 819-822.

Kompoliti, K. (2003) Estrogen and Parkinson’s disease. Front. Biosci. 8, s391-s400.

Kugelstadt, D., Haberkamp, M., Hankeln, T., and Burmester, T. (2004) Neuroglobin, cytoglobin and a novel, retina-specific globin from chicken. Biochem. Biophys. Res. Commun. 325, 719-725.

Kulkarni, J., Riedel, A., de Castella, A.R., Fitzgerald, P.B., Rolfe, T.J., Taffe, J., and Burger, H. (2002) A clinical trial of adjunctive oestrogen treatment in women with schizophrenia. Arch. Womens Ment. Health 5, 99-104.

Laflamme, N., Nappi, R.E., Drolet, G., Labrie, C., and Rivest, S. (1998) Expression and neuropeptidergic characterization of estrogen receptors (ERalpha and ERbeta) throughout the rat brain: anatomical evidence of distinct roles of each subtype. J. Neurobiol. 36, 357-378.

Laufs, T.L., Wystub, S., Reuss, S., Burmester, T., Saaler-Reinhardt, S., and Hankeln, T. (2004) Neuron-specific expression of neuroglobin in mammals. Neurosci. Lett. 362, 83-86.

Levin, E.R. (2002) Cellular functions of plasma membrane estrogen receptors. Steroids 67, 471-475.

Lewis, D.K., Johnson, A.B., Stohlgren, S., Harms, A., and Sohrabji, F. (2008) Effects of estrogen receptor agonists on regulation of the inflammatory response in astrocytes from young adult and middle-aged female rats. J. Neuroimmunol. 195, 47-59.

Li, F., Zhu, S., Wu, C., Yan, C., Liu, Y., and Shugan, L. (2011) Neuroinflammation and cell therapy for Parkinson's disease. Front. Biosci. 3, 1407-1420.

Li, R.C., Lee, S.K., Pouranfar, F., Brittian, K.R., Clair, H.B., Row, B.W., Wang, Y., and Gozal, D. (2006) Hypoxia differentially regulates the expression of neuroglobin and cytoglobin in rat brain. Brain Res. 1096, 173-179.

Li, R.C., Morris, M.W., Lee, S.K., Pouranfar, F., Wang, Y., and Gozal, D. (2008) Neuroglobin protects PC12 cells against oxidative stress. Brain Res. 1190, 159-166.

Lim, M. (2011) Treating inflammation in childhood neurodegenerative disorders. Dev. Med. Child Neurol. 53, 298-304.

Lipton, P. (1999) Ischemic cell death in brain neurons. Physiol. Rev. 79, 1431-1568.

76

Liu, J., Yu, Z., Guo, S., Lee, S.R., Xing, C., Zhang, C., Gao, Y., Nicholls, D.G., Lo, E.H., and Wang, X. (2009) Effects of neuroglobin overexpression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons. J. Neurosci. Res. 87,164-170.

Logsdon, J.M., Stoltzfus, A., and Doolittle, W.F. (1998) Molecular evolution: recent cases of spliceosomal intron gain? Curr. Biol. 8, R560-R563.

Makalowski, W., Zhang, J. and Boguski, M.S. (1996) Comparative analysis of 1196 orthologous mouse and human full length mRNA and protein sequences. Genome Res. 6, 846-857.

Marin, R., Diaz, M., Alonso, R., Sanz, A., Arevalo, M.A., and Garcia-Segura, L.M. (2009) Role of estrogen receptor α in membrane-initiated signaling in neural cells: Interaction with IGF-1 receptor. J. Steroid Biochem. Mol. Biol. 114, 2-7.

Marin, R., Ramirez, C., Morales, A., Gonzalez, M., Alonso, R., and Diaz, M. (2008) Modulation of Aβ-induced neurotoxicity by estrogen receptor α and other associated proteins in lipid rafts. Steroids 73, 992-996.

Marino, M., Acconcia, F., and Ascenzi, P. (2005) Estrogen receptor signalling: bases for drug actions. Curr. Drug Targets Immune Endocr. Metabol. Disord. 5, 305-314.

Marino, M., Acconcia, F., Bresciani, F., Weisz, A., and Trentalance, A. (2002) Distinct nongenomic signal transduction pathways controlled by 17β-estradiol regulate DNA synthesis and cyclin D1 gene transcription in HepG2 cells. Mol. Biol. Cell 13, 3720-3729.

Marino, M., and Ascenzi, P. (2006) Steroid hormone rapid signaling: the pivotal role of S-palmitoylation. IUBMB Life 58, 716-719.

Matthews, J., and Gustafsson, J.A. (2003) Estrogen signalling: a subtle balance between ERα and ERβ. Mol. Intervent 3, 281-292.

McEwen, B.S. (2002) Sex, stress and the hippocampus: allostasis, allostatic load and the aging process. Neurobiol. Aging 23, 921-939.

Mitz, S.A., Reuss, S., Folkow, L.P., Blix, A.S., Ramirez, J.M., Hankeln, T., and Burmester, T. (2009) When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain. Neurosci. 163, 552-560.

Moens, L., and Dewilde, S. (2000) Globins in the brain. Nature 407, 461-462.

Morissette, M., Le Saux, M., D’Astous, M., Jourdain, S., Al Sweidi, S., Morin, N., Estrada-Camarena, E., Mendez, P., Garcia-Segura, L.M., and Di Paolo, T. (2008) Contribution of estrogen receptors alpha and

77

beta to the effects of estradiol in the brain. J. Steroid Biochem. Mol. Biol. 108, 327-338.

Nguyen, T.V., Jayaraman, A., Quaglino, A., and Pike, C.J. (2010) Androgens selectively protect against apoptosis in hippocampal neurones. J. Neuroendocrinol. 22, 1013-1022.

Nguyen, T.V., Yao, M., and Pike, C.J. (2009) Dihydrotestosterone activates CREB signaling in cultured hippocampal neurons. Brain Res. 1298, 1-12.

Nicolis, S., Monzani, E., Ciaccio, C., Ascenzi, P., Moens, L., and Casella, L. (2007) Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger? Biochem. J. 407, 89-99.

Nienhaus, K., and Nienhaus, G.U. (2007) Searching for Neuroglobin’s role in the brain. IUBMB Life 59, 490-497.

Numakawa, Y., Matsumoto, T., Yokomaku, D., Taguchi, T., Niki, E., Hatanaka, H., Kunugi, H., and Numakawa, T. (2007) 17β-Estradiol protects cortical neurons against oxidative stress-induced cell death through reduction in the activity of mitogen-activated protein kinase and in the accumulation of intracellular calcium. Endocrinology 148, 627-637.

Oleksiewicz, U., Daskoulidou, N., Liloglou, T., Tasopoulou, K., Bryan, J., Gosney, J.R., Field, J.K., and Xinarianos, G. (2011). Neuroglobin and myoglobin in non-small cell lung cancer: Expression, regulation and prognosis. Lung Cancer. 2011 Jun 1. [Epub ahead of print]

Osterlund, M.K., Witt, M.R., Gustafsson, J.A. (2005) Estrogen action in mood and neurodegenerative disorders: estrogenic compounds with selective properties-the next generation of therapeutics. Endocrine 28, 235-242.

Ostojić, J., Sakaguchi, D.S., de Lathouder, Y., Hargrove, M.S., Trent, J.T. 3rd, Kwon, Y.H., Kardon, R.H., Kuehn, M.H., Betts, D.M., and Grozdanić, S. (2006) Neuroglobin and cytoglobin: oxygen-binding proteins in retinal neurons. Invest. Ophthalmol. Vis. Sci. 47, 1016-1023.

Parri, R., and Crunelli, V. (2003) An astrocyte bridge from synapse to blood flow. Nat. Neurosci. 6, 5-6.

Pesce, A., De Sanctis, D., Nardini, M., Dewilde, S., Moens, L., Hankeln, T., Burmester, T., Ascenzi, P., and Bolognesi, M. (2004) Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin. IUBMB Life 56, 657-664.

Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln, T., Burmester, T., and Bolognesi, M. (2003) Human brain neuroglobin

78

structure reveals a distinct mode of controlling oxygen affinity. Structure 11, 1087-1095.

Petersen, M.G., Dewilde, S., and Fago A. (2008) Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions. J. Inorg. Biochem. 102, 1777-1782.

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004) UCSF Chimera: a visualization system for exploratory research and analysis, J. Comput. Chem. 25, 1605-1612.

Pike, C.J., Carroll, J.C., Rosario, E.R., and Barron, A.M. (2009) Protective actions of sex steroid hormones in Alzheimer’s disease. Front. Neuroendocrinol. 30, 239-258.

Ramsden, M., Nyborg, A.C., Murphy, M.P., Chang, L., Stanczyk, F.Z., Golde, T.E., and Pike, C.J. (2003a) Androgens modulate beta-amyloid levels in male rat brain. J. Neurochem. 87, 1052-1055.

Ramsden, M., Shin, T.M., and Pike, C.J. (2003b) Androgens modulate neuronal vulnerability to kainate lesion. Neuroscience 122, 573-578.

Raychaudhuri, S., Skommer, J., Henty, K., Birch, N., and Brittain, T. (2010) Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death. Apoptosis 15, 401-411.

Rebenko-Moll, N.M., Liu, L., Cardona, A., and Ransohoff, R.M. (2006) Chemokines, mononuclear cells and the nervous system: heaven (or hell) is in the details. Curr. Opin. Immunol. 18, 683-689.

Reuss, S., Saaler-Reinhardt, S., Weich, B., Wystub, S., Reuss, M. H., Burmester, T., and Hankeln, T. (2002) Expression analysis of neuroglobin mRNA in rodent tissues. Neuroscience 115, 645-656.

Roesner, A., Fuchs, C., Hankeln, T., and Burmester, T. (2005) A globin gene of ancient evolutionary origin in lower vertebrates: evidence for two distinct globin gene families in animals. Mol. Biol. Evol. 22, 12-22.

Rosario, E.R., Chang, L., Stanczyk, F.Z., and Pike, C.J. (2004) Age-related testosterone depletion and the development of Alzheimer disease. JAMA 292, 1431-1432.

Rostene, W., Kitabgi, P., and Parsadaniantz, S.M. (2007) Chemokines: a new class of neuromodulator? Nat. Rev. Neurosci. 8, 895-903.

Ruffels, J., Griffin, M., and Dickenson, J.M. (2004) Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death. Eur. J. Pharmacol. 483, 163-173.

79

Sastre, M., Richardson, J.C., Gentleman, S.M., and Brooks, D.J. (2011) Inflammatory risk factors and pathologies associated with Alzheimer's disease. Curr. Alzheimer Res. 8, 132-1341.

Sawada, H., Ibi, M., Kihara, T., Urushitani, M., Akaike, A., and Shimohama, S. (1998) Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death. J. Neurosci. Res. 54, 707-719.

Schelshorn, D.W., Schneider, A., Kuschinsky, W., Weber, D., Kruger, C., Dittgen, T., Bourgers, H.F., Sabouri, F., Gassler, N., Bach, A., and Maurer, M.H. (2009) Expression of hemoglobin in rodent neurons. J. Cereb. Blood Flow Metab. 29, 585-595.

Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U., and Burmester, T. (2003) How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina. J. Biol. Chem. 278, 1932-1935.

Schmidt-Kastner, R., Haberkamp, M., Schmitz, C., Hankeln, T., and Burmester, T. (2006) Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture. Brain Res. 1103, 173-180.

Scrivo, R., Vasile, M., Bartosiewicz, I., and Valesini, G. (2011) Inflammation as “common soil” of the multifactorial diseases. Autoimmun. Rev. 10, 369-374.

Shao, G., Gong, K.R., Li, J., Xu, X.J., Gao, C.Y., Zeng, X.Z., Lu, G.W., and Huo, X. (2009) Antihypoxic effects of neuroglobin in hypoxia-preconditioned mice and SH-SY5Y cells. Neurosignals 17, 196-202.

Shulman, L.M., and Bhat, V. (2006) Gender disparities in Parkinson’s disease. Expert Rev. Neurother. 6, 407-416.

Simerly, R.B. (2005) Wired on hormones: endocrine regulation of hypothalamic development. Curr. Opin. Neurobiol. 15, 81-85.

Simpkins, J.W., Yang, S.H., Sarkar, S.N., and Pearce, V. (2008) Estrogen actions on mitochondria-physiological and pathological implications. Mol. Cell. Endocrinol. 290, 51-59.

Sinchak, K., Mills, R.H., Tao, L., LaPolt, P., Lu, J.K., and Micevych, P. (2003) Estrogen induces de novo progesterone synthesis in astrocytes. Dev. Neurosci. 25, 343-348.

Singer, C.A., Figueroa-Masot, X.A., Batchelor, R.H., and Dorsa, D.M. (1999) The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons. J. Neurosci. 19, 2455-2463.

Smagghe, B.J., Sarath, G., Ross, E., Hilbert, J-L. and Hargrove, M.S. (2006) Slow ligand binding kinetics dominate ferrous hexacoordinate

80

hemoglobin reactivities and reveal differences between plants and other species. Biochemistry 45, 561-570.

Sowa, A.W., Duff, S.M., Guy, P.A. and Hill, R.D. (1998) Altering hemoglobin levels changes energy status in maize cells under hypoxia. Proc. Natl. Acad. Sci. USA 95, 10317-10321.

Sun, Y., Jin, K., Mao, X.O., Zhu, Y., and Greenberg, D.A. (2001) Neuroglobin is upregulated by and protects neurons from hypoxic-ischemic injury. Proc. Natl. Acad. Sci. USA 98, 15306-15311.

Sun, Y., Jin, K., Peel, A., Mao, X.O., Xie, L., and Greenberg, D.A. (2003) Neuroglobin protects the brain from experimental stroke in vivo. Proc. Natl. Acad. Sci. USA 100, 3497-3500.

Suzuki, A., Sugihara, A., Uchida, K., Sato, T., Ohta, Y., Katsu, Y., Watanabe, H., and Iguchi, T. (2002) Developmental effects of perinatal exposure to bisphenol-A and diethylstilbestrol on reproductive organs in female mice. Reprod. Toxicol. 16, 107-116.

Suzuki, S., Brown, C.M., and Wise, P.M. (2009) Neuroprotective effects of estrogens following ischemic stroke. Front. Neuroendocrinol. 30, 201-211.

Swaab, D.F. (2004) Sexual differentiation of the human brain: relevance for gender identity, transsexualism and sexual orientation. Gynecol. Endocrinol. 19, 301-312.

Swaab, D.F. (2007) Sexual differentiation of the brain and behavior. Best Pract. Res. Clin. Endo. Metabol. 21, 431-444.

Tenenbaum, M., Azab, A.N., and Kaplanski, J. (2007) Effects of estrogen against LPS-induced inflammation and toxicity in primary rat glial and neuronal cultures, J. Endotoxin Res. 13, 158-166.

Tiwari-Woodruff, S., and Voskuhl, R.R. (2009) Neuroprotective and anti-inflammatory effects of estrogen receptor ligand treatment in mice. J. Neurol. Sci. 286, 81-85.

Tobet, S., Knoll, J.G., Hartshorn, C., Aurand, E., Stratton, M., Kumar, P., Searcy, B., and McClellan, K. (2009) Brain sex differences and hormone influences: a moving experience? J. Neuroendocrinol. 21, 387-392.

Totta, P., Acconcia, F., Leone, S., Cardillo, I., and Marino, M. (2004) Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involvement of estrogen receptor α and β signalling. IUBMB Life 56, 491-499.

Trent, J.T. 3rd, and Hargrove, M.S. (2002) A ubiquitously expressed human hexacoordinate hemoglobin. J. Biol. Chem. 277, 19538-19545.

81

Trent, J.T. 3rd,Watts, R.A., and Hargrove, MS. (2001) Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J. Biol. Chem. 276, 30106-30110.

Vallone, B., Nienhaus, K., Matthes, A., Brunori, M., and Nienhaus, G.U. (2004) The structure of carbonmonoxy neuroglobin reveals a hemesliding mechanism for control of ligand affinity. Proc. Natl. Acad. Sci. USA 101, 17351-17356.

Vandergon, T.L., and Riggs, A.F. (2008) Mini-hemoglobins from nemertean worms. Methods Enzymol. 436, 503-509.

Vasudevan, N., and Pfaff, D.W. (2008) Non-genomic actions of estrogens and their interaction with genomic actions in the brain. Front. Neuroendocrinol. 29, 238-257.

Vegeto, E., Benedusi, V., and Maggi, A. (2008) Estrogen anti-inflammatory activity in brain: a therapeutic opportunity for menopause and neurodegenerative diseases. Front. Neuroendocrinol. 29, 507-519.

Vinogradov, S.N., and Moens, L. (2008) Diversity of globin function: Enzymatic, transport, storage, and sensing. J. Biol. Chem. 283, 8773-8777.

Wajcman, H., and Kiger, L. (2002) Haemoglobin from microorganism to man: a single protein folding, a variety of functions. Comptes Rendus Biol. 325, 1159-1174.

Wajcman, H., Kiger, L., and Marden, M.C. (2009) Structure and function evolution in the superfamily of globins. C. R. Biol. 332, 273-282.

Wakasugi, K., and Morishima, I. (2005) Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity. Biochemistry 44, 2943-2948.

Wakasugi, K., Kitatsuji, C., and Morishima, I. (2005) Possible neuroprotective mechanism of human neuroglobin. Ann. NY Acad. Sci. 1053, 220-230.

Wakasugi, K., Nakano, T., and Morishima, I. (2003) Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor. J. Biol. Chem. 278, 36505-36512.

Wang, X., Dykens, J.A., Perez, E., Liu, R., Yang, S., Covey, D.F., and Simpkins, J.W. (2006) Neuroprotective effects of 17β-estradiol and nonfeminizing estrogens against H2O2 toxicity in human neuroblastoma SK-N-SH cells. Mol. Pharmacol. 70, 395-404.

Wang, X., Liu, J., Zhu, H., Tejima, E., Tsuji, K., Murata, Y., Atochin, D.N., Huang, P.L., Zhang, C., and Lo, E.H. (2008). Effects of neuroglobin overexpression on acute brain injury and long-term outcomes after focal cerebral ischemia. Stroke 39, 1869-1874.

82

Weber, R. E., and Fago (2004) Functional adaptation and its molecular basis in vertebrate hemoglobins, neuroglobins and cytoglobins. Respir. Physiol. Neurobiol. 144, 141-159.

Weber, R. E., and Vinogradov, S. N. (2001) Nonvertebrate hemoglobins: Functions and molecular adaptations. Physiol. Rev. 81, 569-628.

Williams, T. M., Zavanelli, M., Miller, M. A., Goldbeck, R. A., Morledge, M., Casper, D., Pabst, D. A., McLellan, W., Cantin, L. P., and Kliger, D. S. (2008) Running, swimming and diving modifies neuroprotecting globins in the mammalian brain. Proc. Biol. Sci. 275, 751-758.

Wilson, C.A., and Davies, D.C. (2007) The control of sexual differentiation of the reproductive system and brain. Reproduction 133, 331-359.

Wise, P.M., Dubal, D.B., Wilson, M.E., Rau, S.W., Böttner, M., and Rosewell, K.L. (2001) Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies. Brain Res. Brain Res. Rev. 37, 313-319.

Wittemberg, J. B., Bolognesi, M., Wittemberg, B. A., and Guertin, M. (2002) Truncated hemoglobins: a new family of hemoglobins widely distribuited in bacteria, unicellular eukaryotes, and plants. J. Biol. Chem. 277, 871-874.

Wittenberg, B. A., Briehl, R. W., and Wittenberg, J. B. (1965) Biochem. J. 96, 363-371.

Wittenberg, J. B. (1992) Functions of cytoplasmic hemoglobins and myohemerythrin. In Advances in Comparative and Environmental Physiology. pp. 59-85 Springer-Verlag Berlin, Heidelberg.

Wittenberg, J. B., and Wittenberg, B. A. (2003) Myoglobin function reassessed. J. Exp. Biol. 206, 2011-2020.

Wooten, G.F., Currie, L.J., Bovbjerg, V.E., Lee, J.K., and Patrie, J. (2004) Are men at greater risk for Parkinson’s disease than women? J. Neurol. Neurosurg. Psychiatry 75, 637-639.

Wystub, S., Ebner, B., Fuchs, C., Weich, B., Burmester, T., and Hankeln, T. (2004) Interspecies comparison of neuroglobin, cytoglobin and myoglobin: sequence evolution and candidate regulatory elements. Cytogenet. Genome Res. 105, 65-78.

Wystub, S., Laufs, T., Schmidt, M., Burmester, T., Maas, U., Saaler-Reinhardt, S., Hankeln, T., and Reuss, S. (2003) Localization of neuroglobinprotein in the mouse brain. Neurosci. Lett. 346, 114-116.

Yang, S.H., Sarkar, S.N., Liu, R., Perez, E.J., Wang, X., Wen, Y., Yan, L.J., and Simpkins, J.W. (2009) Estrogen receptor beta as a mitochondrial vulnerability factor. J. Biol. Chem. 284, 9540-9548.

Ye, S. Q., Zhou, X. Y., Lai, X. J., Zheng, L., and Chen, X. Q. (2009) Silencing neuroglobin enhances neuronal vulnerability to oxidative

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injury by down-regulating 14-3-3gamma. Acta Pharmacol. Sin. 30, 913-918.

Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) Global allostery model of hemoglobin: modulation of O2 affinity, cooperativity, and Bohr effect by heterotropic allosteric effectors. J. Biol. Chem. 277, 34508-34520.

Yu, Z., Fan, X., Lo, E.H., and Wang, X. (2009) Neuroprotective roles and mechanisms of neuroglobin. Neurol Res. 31, 122-127.

Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component. Biochim. Biophys. Acta 1041, 117-122.

Zhang, C., Wang, C., Deng, M., Li, L., Wang, H., Fan, M., Xu, W., Meng, F., Qian, L., and He, F. (2002) Full-length cDNA cloning of human neuroglobin and tissue expression of rat neuroglobin. Biochem. Biophys. Res. Commun. 290, 1411-1419.

Zhao, L., O’Neill, K., and Diaz Brinton R. (2005) Selective estrogen receptor modulators (SERMs) for the brain: current status and remaining challenges for developing NeuroSERMs. Brain Res. Brain Res. Rev. 49, 472-493.

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ACKNOWLEDGEMENTS

Looking back, I am surprised and at the same time very grateful for all I have received throughout these years. It has certainly shaped me as a person and has led me where I am now. All these years of PhD studies are full of such gifts.

I want first to thank my tutors Prof. Maria Marino and Prof. Paolo Ascenzi for all the hope they have put on me. They have always encouraged me to see science in its full depth. They have enlightened me through their wide knowledge and deep intuitions about where science should go and what is necessary to get there.

I also thank my tutors from Instituto Cajal, Prof. Luis Miguel Garcia-Segura and Maria Angeles Arevalo. The period I spent in Madrid enriched me of new methods, new scientific approaches and, most importantly, of very beautiful people.

Thanks to Dr. Filippo Acconcia for sharing his knowledge and for his precious advices with overwhelming generosity, and to Dr. Valentina Pallottini who is my first mentor and first taught me the beauty of physiology.

It was a pleasure to share doctoral studies and life with wonderful people who are very close friends now: Dr. Paola Galluzzo, Dr. Pamela Bulzomi, Dr. Marco Pellegrini and Dr. Marco Fiocchetti, who also strongly contributed to this thesis; Dr. Laura Trapani, Dr. Piergiorgio La Rosa, Dr. Marco Segatto and Dr. Alessandro Bolli. Thus, I thank them for their friendship and for sharing the glory and sadness of day-to-day research.

The months spent in Madrid would not have been as wonderful without my Spanish friends and colleagues from C-01 laboratory at Instituto Cajal, I thank for helping me to work better and for the wonderful friendship they offered me.

Special thanks go to my big family, especially to my father and my mother who supported me during all these years of studies.

Last but not least, big thanks to my irreplaceable and amazing friends, and to the man who gave me a lot of happiness and colorful life during these years. Thanks to Miriam and the “Tuesday group”, who rendered me able to build up my identity, as a woman and as a scientist.

“There is grandeur in this view of life that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved” (Charles Darwin, The Origin of Species).

1

A. MATERIALS AND METHODS

Reagents

E2, testosterone (T), 5α -androstan-17β -ol-3-one (dihydrotestosterone, DHT), naringenin (Nar), insulin-like growth factor 1 (IGF-1), bacterial endotoxin lipopolysaccharide (LPS) (Escherichia coli 026:B6), actinomycin (Act), cycloheximide (Cxm), Pen-Strep solution, H2O2, RPMI-1640 media without phenol red, Dulbecco’s modified Eagle medium (DMEM) without phenol red, charcoal- stripped fetal calf serum, the palmitoyl acyltransferase (PAT) inhibitor 2-bromohexadecanoid acid (2-Br-palmitate; 2Br), protease inhibitor cocktail, bovine serum albumin fraction V (BSA), mouse monoclonal anti-Flag®M2 antibody, and mouse monoclonal anti-β-actin (clone AC-74) antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Optimem, Hank’s buffer salt solution (HBSS 1×), Neurobasal medium, B27 serum-free supplement, and GlutaMAX-I were purchased from Gibco-BRL (Gaithersburg, MD, USA). The p38 inhibitor SB 203580 (SB), the AKT inhibitor, the NFκB inhibitor peptide SN50, and the IGF-1 receptor (IGF-1R) inhibitor picropodophyllin (PPP) were obtained from Calbiochem (San Diego, CA, USA). The E2 antagonist fulvestrant (ICI 182,780, ICI), the estrogen receptor (ER)α-selective agonist 4,4’,4’’-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), the ERβ-selective agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN), and the ERβ-selective antagonist (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC) were obtained from Tocris (Ballwin, MO, USA). Bradford protein assay was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Human recombinant ERα and ERβ were obtained by Pan-Vera (Madison, WI, USA). Anti-phospho-ERK1/2, anti-AKT, anti-ERα (MC20), anti-ERβ (H150), anti-caspase-3, antipoly(ADP-ribose)polymerase (PARP), anti-protein phosphatase 2A (PP2A), and anti-ERK1/2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-phospho-AKT, anti-phospho-p38, and anti-p38 antibodies were purchased from New England Biolabs (Beverly, MA, USA). Monoclonal anti-human Ngb (13C8) antibody was purchased from Abcam (Cambridge, UK). Anti-β-tubulin antibody was purchased from MP Biomedical (Solon, OH, USA). Polyclonal anti-cytochrome c and monoclonal anti-cytochrome c oxidase (COX-4) antibodies were purchased from Clontech Laboratories (Mountain View, CA, USA). Chemiluminescence reagent for Western blot ECL was obtained from GE Healthcare (Little Chalfont, UK). All the other products were from Sigma-Aldrich. Analytical or reagent grade products were used

2

without further purification. Cells Human neuroblastoma SK-N-BE cell line

Human SK-N-BE neuroblastoma cell line was routinely grown in air containing 5% CO2 in modified, phenol red-free, RPMI-1640 medium containing 10% (v/v) charcoal-stripped fetal calf serum, L -glutamine (2.0 mM), Pen-Strep solution (penicillin 100 U/ml, and streptomycin 100 mg/ml). Cells were passaged every 2 days. Cells were grown to approximately 70% confluence in 6-well plates before stimulation. Human cervix epithelioid carcinoma cell line

Human cervix epithelioid carcinoma cells (HeLa) were routinely grown in air containing 5% CO2 in modified, phenol red-free, DMEM medium, containing 10% (v/v) charcoal-stripped fetal calf serum, L-glutamine (2 mM), gentamicin (0.1 mg/ml), and penicillin (100 U/ml). Cells were passaged every 2 days and media changed every 2 days. Mouse primary hippocampal neurons

Hippocampal neurons were obtained from E18 mouse embryos after isolating the hippocampus in Ca2+- and Mg2+-free HBSS 1×. Mice were treated following the guidelines of the Council of Europe Convention ETS123, recently revised as indicated in the Directive 86/609/EEC. In addition, all protocols were approved by the Institutional Animal Care and Use Committee of CSIC-Cajal Institute (Madrid, Spain). Once 8-10 embryonic hippocampi were obtained, they were finely cut, washed twice in HBSS 1× buffer, and incubated in 0.1 mg/ml trypsin solution and 1 mg/ml DNAse (Roche Diagnostics GmbH, Mannheim, Germany) for 15 min at 37°C. Trypsin and DNAse were then eliminated by washing 3 times, with HBSS 1×, and the cut tissue was then triturated using a siliconized pipette. Cells were counted and plated in polylysine-coated (1 mg/ml) 6-well plates containing phenol red-free neurobasal medium supplemented with 2% (v/v) B27 serum-free supplement, 0.25% (v/v) GlutaMAX-I, and 1% (v/v) penicillin/streptomycin solution. Neurons were maintained under these conditions for 3 days at 5% CO2 and 37°C. Mouse primary cortical astrocytes

Astrocyte cultures were prepared by mechanical dissociation of the cerebral cortex from newborn (P0) C57 mice (Rubio et al., 2008). Mice were treated following the guidelines of the Council of Europe Convention ETS123, recently revised as indicated in the Directive 86/609/EEC. In addition, all protocols were approved by the Institutional Animal Care and

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Use Committee of CSIC-Cajal Institute (Madrid, Spain). The cortex was isolated under a dissecting microscope and cleaned of choroid plexus and meninges. Cell suspensions were filtered through a 100-lm nylon cell strainer into phenol red free DMEM containing 10% fetal calf serum and penicillin-streptomycin.

After centrifugation, cells were filtered through a 40 μm cell strainer and cultured in 75 cm2 tissue culture flasks at 37°C and 5% CO2. The medium was changed after 4 days in culture and subsequently two times a week for the entire culture period. Cellular confluence was observed 10 days after plating, producing around 4×106 cells per flask, showing a polygonal flat morphology.

Enriched astrocyte cultures were obtained after overnight shaking at 37°C a 250 rpm in a table top shaker (Thermo Forma, Marietta, OH) to minimize oligodendrocyte and microglia contamination. Astrocytes were removed from the flasks by incubation with 0.25% trypsin (type II-S; Sigma) and 0.04% EDTA, plated onto poly-L-lysine-coated six-well plates or coverslips at a density of 40,000 cells/cm2 in serum-free medium and used within 24 hours. Cell stimulation

Cells were simultaneously treated with vehicle (ethanol/PBS 1:10, v/v) and/or E2 (0.01-1000 nM), PPT (0.01-100 nM), DPN (0.01-100 nM), T (0.1-1000 nM), DHT (0.1-1000 nM), IGF-1 (100 ng/ml), LPS (500 ng/ml), and H2O2 (50 μM). When indicated, the anti-estrogen ICI (1 μM), the PAT inhibitor 2Br (10 μM), the AKT inhibitor (1 μM), the p38 inhibitor SB (5 μM), the ERβ inhibitor THC (1 μM), the IGF-1R inhibitor PPP (100 nM), the NFκB inhibitor SN50 (10 μg/ml), and the transcription inhibitor Act (1 μg/ml) were added 30 min before E2 or IGF-1 or LPS administration.

The translational inhibitor Cxm (10 μg/ml), was added 1hour before E2 administration.

Cell Viability

SK-N-BE cell lines were grown to 70% confluence in 6-well plates and stimulated either with vehicle or E2 (1 nM) or THC (1 μM). After 24 h of stimulation, cells were treated either with vehicle or with H2O2 50 μM for 24hours. After treatment, cells were harvested with trypsin, and counted with Coulter Model ZM electronic particle (Beckman, Palo Alto, CA, USA).

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Plasmid flag-Ngb

The pcDNA flag-Ngb (flag-Ngb) was obtained by subcloning the Ngb ORF from the NgbN1-pEGFP plasmid (Fordel et al., 2006) (kindly gifted by Prof. Sylvia Dewilde, University of Antwerp, Belgium) into the pcDNA-flag 3.1C. Transfection of flag-Ngb plasmid

HeLa cells were grown to ~70% confluence and then transfected with pcDNA-flag-Ngb plasmid using lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Six hours after transfection, the medium was changed and 24 hours after the cells were processed for confocal microscopy analysis.

DAPI staining

In some experiments, SK-N-BE cells were stimulated with E2 1 nM and/or 50 μM H2O2 for 24 hours. Cells were processed in chamber slides and rinsed with PBS, pH 7.4, followed by fixation in 100% (v/v) cold methanol for 15 min. The slides were cover-slipped using Prolong® Gold anti-fade reagent with 1 mg/ml DAPI (Invitrogen). Slides were viewed on an Olympus BX51 fluorescence microscope. Images were captured (40× magnification) with Leica DFC 420 camera (Leica Microsystems, Wetzlar, Germany). Confocal Microscopy Analysis

SK-N-BE cells and flag-Ngb transfected HeLa cells were stained with anti-Ngb (1:50) and anti-Flag®M2 (1:10,000) antibodies, respectively. Cells were processed in chamber slides and rinsed with PBS, pH 7.4, followed by fixation in formaldehyde 4% (v/v) for 1 hour, and permeabilization with cold acetone 95% for 3 min. Cells were rinsed in PBS and saturated with BSA 2% (w/v) for 1 hour and then incubated with primary antibody at 4°C o/n (anti-Ngb) or 1 hour at RT (anti-Flag®M2). After that cells were rinsed three times in PBS for 5 min and incubated with Alexa Fluor 488® and 578® donkey anti-mouse secondary antibodies (Invitrogen) (1:400). The slides were cover-slipped using Prolong® Gold anti-fade reagent. Confocal

5

analysis (63× magnification) was performed using LCS (Leica Microsystems).

Transfection of Short Interfering RNA

SK-N-BE cells and mouse primary cortical astrocytes, reaching 40-60% confluence, were transfected in a serum-free condition with either Stealth RNAi™ Ngb-silencing RNA or ERβ-silencing RNA (siNgb and siERβ; Invitrogen) according to the manufacturer’s instructions, using oligofectamine (Invitrogen) as the transfection reagent. The sequence used for Ngb oligonucleotides was:

5’-CGUGAUUGAUGCUGCAGUGACCAAU-3’ the sequence used for ERβ oligonucleotides was:

5’-GAAGAACUCUUUGCCCGGAAAUUUA-3’ The mismatch sequence used as a control for Ngb siRNA (siNgb) was:

5’-UGUGAUUUAUGGUGCAGUAACCAAC-3’ The mismatch sequence used as a control for ERβ siRNA (si ERβ) was:

5’-GAAUCAUUCCGUGCCAAGUAGAUUAAUUA-3’ Briefly, oligofectamine and oligonucleotides (400 and 200 pM for siNgb and siERβ, respectively) were mixed with Optimem. The mixture was incubated for 20 min at room temperature, diluted with Optimem, and added to the cell medium for 4 hours at 37°C. The medium was added to cells to reach the growing conditions (i.e., 10% (v/v) serum).

To evaluate the effective silencing of Ngb and ERβ, total proteins from cells transfected with MOCK (control), with scramble (mismatch sequence, data not shown), and with siNgb or siERβ oligonucleotides were extracted 48 hours after transfection, and Ngb and ERβ expression was tested by Western blot analysis using anti-Ngb and anti-ERβ antibodies.

Western Blot Assays

After treatments, cells were lysed and solubilized in 0.125 M Tris, pH 6.8, containing 10% (w/v) SDS, and protease inhibitor cocktail, and finally boiled for 2 min. Total proteins were quantified using the Bradford protein assay. Solubilized proteins (20 μg) were resolved by 7 or 15% SDS-PAGE at 100 V for 1 h at 25°C and then electrophoretically transferred to nitrocellulose for 45 min at 100 V and 4°C. The nitrocellulose was treated with 3% (w/v) BSA in 138 mM NaCl, 25 mM Tris, pH 8.0, and 0.1% (w/v) Tween-20 at 25°C for 1 hour and then probed overnight at 4°C with either

6

anti-Ngb (final dilution 1: 1000), anti-ERα MC-20 (final dilution 1:500), anti-ERβ H-150 (final dilution 1:3,000), anti-caspase-3 (final dilution 1:1000), anti-PARP (final dilution 1:500), anti-phospho-ERK1/2 (final dilution 1:200), anti-phospho-AKT (final dilution 1:1000), anti-phospho-p38 (final dilution 1:1000), anti-COX-4 (final dilution 1:1000) or anti-PP2A (final dilution 1:1000). The nitrocellulose was stripped by Restore Western Blot Stripping Buffer (Pierce Chemical, Rockford, IL, USA) for 10 min at room temperature and then probed with anti-β-tubulin (final dilution 1:1000) or anti-β-actin (final diluition 1:2500) to normalize total lysate. Moreover, the nitrocellulose incubated with either anti-phospho-ERK1/2, anti-phospho-AKT or anti-phospho-p38 was stripped and probed with anti-ERK1/2 (final dilution 1:200), anti-AKT (final dilution 1:100) and anti-p38 (final dilution 1:1000), respectively. To evidence ERα and ERβ levels, electrophoresis was performed in the presence of 5 ng of recombinant ERα and ERβ. Antibody reaction was visualized with ECL chemiluminescence.

Densitometric analyses were performed by ImageJ software for Microsoft Windows. The densitometry quantification of protein was normalized to tubulin or actin.

Quantitative Real-Time Polymerase Chain Reaction

After 5 h of treatment with LPS and/or E2, culture plates were briefly centrifuged and supernatants were removed. Cells were lysed and total RNA was extracted using the illustra RNAspin Mini RNA Isolation Kit (GE Healthcare, Buckinghamshire, UK) to measure the IP-10 and IL-6 mRNA expression levels.

First strand cDNA was prepared from 5 μg RNA using the RevertAidTM H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, Bath, UK) according to the supplied protocol. After reverse transcription (RT), the cDNA was diluted 1:10 and 5 μl were amplified by real-time PCR in 20 μl using SYBR Green master mix or TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a ABI Prism 7000 Sequence Detector (Applied Biosystems), with conventional Applied Biosystems cycling parameters (40 cycles of 95°C, 15 sec, 60°C, 1 min). Primer sequences were designed using Primer Express (Applied Biosystems) and were for IP-10, forward:

5’-CAGTGAGAATGAGGGCCATAGG-3’ and reverse:

5’-CGGATTCAGACATCTCTGCTCAT-3’;

7

for IL-6, forward: 5’-GAAACCGCTATGAAGTTCCTCTCTG-3’

and reverse: 5’-TGTTGGGAGTGGTATCCTCTGTGA-3’

Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was selected as control housekeeping gene. GADPH TaqMan probes and primers were the Assay-on-Demand gene expression products (Applied Biosystems). After amplification, a denaturing curve was performed to ensure the presence of unique amplification products. For visualizing and sequencing the PCR products, each mixture was electrophoresed in 2% (w/v) ethidium bromide-stained agarose gels. Then, bands were excised and cDNA was purified using the QIAquick PCR purification Kit (Qiagen, GmbH, Germany). 100 ng of each sample was sequenced (Automatic Sequencing Center, CSIC, Madrid, Spain) with the corresponding forward or reverse primer. The obtained sequence was aligned with the expected sequence of each transcript obtained from the GenBank. All reactions were performed in triplicate and the quantities of target gene expression were normalized to the corresponding GAPDH expression in test samples and plotted. Cell fractionation SK-N-BE cell fractionation was performed using ApoAlert™ Cell Fractionation kiy (Clontech) according to manufacturer’s instructions. After stimulation, cells were washed in ice-cold PBS, harvested by with trypsin (1%, v/v), resuspended with complete medium, and centrifuged at 600g for 5 min. Pellet was resuspended in Fractionation Buffer Mix containing DTT 1 mM and homogenized in a Dounce tissue grinder. Homogenate was centrifuged at 700g for 10 min. Pellet was resuspended in Sample buffer containing 0.125 M Tris, pH 6.8, and 10% (w/v) SDS (nuclear fraction); supernatant was centrifuged again at 10,000g for 25 min. Supernatant was collected (cytosolic fraction) and pellet resuspended in Fractionation Buffer Mix (mitochondrial fraction). Protein concentration of each fraction was determined using Bradford protein assay. Lysate of each fraction was then processed for Western Blot or used for immunoprecipitation assay.

8

True-blot co-immunoprecipitation

After stimulation, SK-N-BE cells were washed in ice-cold PBS, harvested by with trypsin (1%, v/v), and lysed in 50 μl lysis buffer 10 mM Tris, pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 10 mM NaCl, 1% (v/v) Triton X-100, and 1% (w/v) sodium cholate, containing protease inhibitor cocktail.

Cell lysates were clarified by centrifugation and immunoprecipitated with TrueBlot™ (eBioscience, San Diego, CA, USA) which preferentially detects the native disulfide form of mouse or rabbit IgG, reducing interference by the ~55 kDa heavy and ~23 kDa light chains of the immunoprecipitating antibody. Briefly, after stimulation equal amounts of soluble cell extracts were incubated with 10 μg of anti-cytochrome c. The lysates and antibodies were incubated at 4°C for 1 h, then 20 μl of anti-Mouse IgG Beads (eBioscience) were added and samples incubated for 1 hour on a rocking platform at 4°C. Samples were centrifuged at 10,000×g for 10 min, the supernatant was removed completely and beads (pelleted) were washed 3 times with 100 μl of lysis buffer. SDS-Reducing sample buffer (20 μl, containing 50 mM DTT) were added and samples were boiled at 100°C for 5 min. Proteins (pelleted and supernatant) were resolved using 15% SDS-PAGE at 100 V for 1 hour and then electrophoretically transferred to nitrocellulose for 45 min at 100 V at 4°C. The nitrocellulose was treated with 5% (w/v) non-fat dry milk in 150 mM NaCl, 50 mM Tris HCl (pH 8.0), 0.1% (w/v). Tween-20, and then probed at 4°C overnight with anti-Ngb antibody (1:1000). The antibody reaction was visualized with the ECL chemiluminescence reagent for Western blot. The nitrocellulose was stripped by Restore Western Blot Stripping Buffer for 10 min at room temperature and then probed with anti-cytochrome c antibody (final dilution 1:1000) to normalize the immunoprecipitate.

Statistical Analysis A statistical analysis was performed by using ANOVA followed by

Tukey-Kramer post-test with the GraphPad InStat3 software system for Windows. In all cases, p<0.05 was considered significant.

9

References Fordel, E., Thijs, L., Martinet, W., Lenjou, M., Laufs, T., Van Bockstaele,

D., Moens, L., and Dewilde, S. (2006) Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death. Neurosci. Lett. 410, 146-151.

Rubio, N., Gonzalez-Tirante, M., Arevalo, M.A., and Aranguez, I. (2008) Overexpression of GTP-binding proteins and GTPase activity in mouse astrocyte membranes in response to Theiler’s murine encephalomyelitis virus infection. J. Neurochem. 104, 100-112.

B. PEER REVIEWED PUBBLICATIONS FROM THE BEGINNING OF PhD FORMATION − De Marinis E.

, Casella L., Ciaccio C., Coletta M., Visca P., Ascenzi P. (2009) “Hypothesis. Catalytic peroxidation of nitrogen monoxide and peroxynitrite by heme-globins” IUBMB Life. 61, 62-73.

− Ascenzi P., De Marinis E.

, Visca P., Ciaccio C., Coletta M. (2009) “Peroxynitrite detoxification by ferryl Mycobacterium leprae truncated hemoglobin O” Biochem. Biophys. Res. Commun. 380, 392-396.

− di Masi A.*, De Marinis E.

*, Ascenzi P., Marino M. (2009) “Nuclear receptors CAR and PXR: molecular, functional, and biomedical aspects” Mol. Asp. Med. 30, 297-343 (* Equally contributed as a first author).

− Ascenzi P.*, De Marinis E.*

, di Masi A., Ciaccio C., Coletta M. (2009) “Peroxynitrite scavenging by ferryl sperm whale myoglobin and human hemoglobin” Biochem. Biophys. Res. Commun. 390, 27-31 (* Equally contributed as a first author).

− Gullotta F., De Marinis E.

, Ascenzi P., di Masi A. (2010) “Targeting the DNA Double Strand Breaks Repair for Cancer Therapy” Curr. Med. Chem. 17, 2017-2048.

− De Marinis E., Ascenzi P., Pellegrini M., Galluzzo P., Bulzomi P., Arevalo M.A., Garcia-Segura L.M., Marino M. (2010) “17β-estradiol - a new modulator of neuroglobin levels in neurons: role in neuroprotection against H2O2

-induced toxicity” Neurosignals 18: 223-235.

− De Marinis E.

, Marino M., Ascenzi P. (2011) “Neuroglobin, estrogens and neuroprotection” IUBMB Life 63: 140-145.

Hypothesis

Catalytic Peroxidation of Nitrogen Monoxide and Peroxynitriteby Globins

Elisabetta De Marinis1, Luigi Casella2, Chiara Ciaccio3, Massimo Coletta3,

Paolo Visca1,4 and Paolo Ascenzi1,41Dipartimento di Biologia e Laboratorio Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Roma, Italy2Dipartimento di Chimica Generale, Universita di Pavia, Pavia, Italy3Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita di Roma ‘Tor Vergata’, Roma, Italy4Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, Italy

Summary

Globins are generally considered as carriers of diatomic gas-eous ligands (e.g., O2 and NO) in metazoa. Recently, the(pseudo-)enzymatic activity of globins towards reactive nitrogenand oxygen species has been elucidated. In particular, some glo-bins (e.g., hemoglobin and myoglobin) catalyze the enzymaticscavenging of NO and peroxynitrite in the presence of H2O2.Indeed, H2O2 oxidizes some globins leading to the formation ofwater and of the heme-protein ferryl derivative, which, in turn,oxidizes NO and peroxynitrite leading to the formation of theglobin ferric species, NO2

2, and NO32. Here, we hypothesize that

NO, peroxynitrite, and H2O2 are co-substrates for the peroxi-dase activity of some globins, this catalytic activity was reportedin 1900 for the first time, even though the substrates have neverbeen identified firmly up to now. � 2008 IUBMB

IUBMB Life, 61(1): 62–73, 2009

Keywords globins; catalytic peroxidation; nitrogen monoxide; perox-

ynitrite; hydrogen peroxide; oxidative stress; nitrosative

stress.

Abbreviations EPO, eosinophil peroxidase; heme-Fe(III), ferric

heme-protein; heme-Fe(IV)¼¼O, ferryl heme-protein;

heme-Fe(III)-ONO, O-nitrito ferric heme-protein; Hb,

hemoglobin; Lb, leghemoglobin; Mb, myoglobin;

MPO, myeloperoxidase; Ngb, neuroglobin; trHbN,

truncated Hb N; trHbO, truncated Hb O.

The hemoglobin (Hb) superfamily includes several heme-

proteins, generally referred to as globins, which are found in all

kingdoms of living organisms (1, 2). Globin functions have

been the subject of active debate, in addition to dioxygen trans-

port and storage. Several functions have been proposed recently,

including control of nitrogen monoxide levels, O2 sensing, and

dehaloperoxidase activity (3–15).

Globins share physical, spectroscopic, and chemical similar-

ities with peroxidases (16, 17). In fact, as demonstrated first in

1900 (18), Hb reacts readily with hydrogen peroxide (H2O2). In

1923, the peroxidase activity of Hb has been reported (19), and

in 1938, the modulation of the peroxidase activity of Hb by

haptoglobin has been demonstrated (20). The reaction of myo-

globin (Mb) with H2O2, on the other hand, apparently was not

considered until 1952 (21), and the ability of Mb to catalyze

peroxide oxidation of substrates was not reported until 1955

(22). Upon reaction with H2O2, Mb and Hb form the cytotoxic

ferryl derivative (heme-Fe(IV)¼¼O), which is similar to com-

pound II formed by peroxidases (23, 24). Heme-Fe(IV)¼¼O is

able to oxidize a wide range of reducing substrates, such as

phenols and aromatic amines, even though substrate peroxida-

tion by Hb and Mb is far less efficient than that of peroxidases

(24, 25), ruling out the possibility that the potential peroxidase

activity of Hb and Mb is exerted on this class of substrates

under normal conditions.

Here, we hypothesize that the capability of some globins

(e.g., Hb and Mb) to form a compound II-like species under

oxidative stress may be actually exploited to avoid the building

up of NO and peroxynitrite,1 which can be then identified as

the ‘true’ substrates for the peroxidase activity of Hb and Mb.

Heme-proteins share the ability of detoxifying nitrogen reac-

tive species, for example, NO. Even though leukocyte peroxi-Address correspondence to: Paolo Ascenzi, Department of Biology

and Interdepartmental Laboratory for Electron Microscopy, University

Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy.

Tel: 139 06 5733 3200(2). Fax: 139 06 5733 6321. E-mail: ascenzi@

uniroma3.it

Received 22 July 2008; accepted 23 September 2008

1The term peroxynitrite is used in the text to refer generically toboth ONOO2 and its conjugated acid HOONO (38).

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.149

IUBMB Life, 61(1): 62–73, January 2009

dases are believed to play a dominant role in the consumption

of NO-derived oxidants at sites of inflammation (as a part of

host defenses against oxidative tissue injury), ferrous oxygen-

ated Hb and Mb (HbO2 and MbO2, respectively) indeed are

involved in the major pathway for NO removal from the vascu-

lar compartment and in the protection of mitochondrial respira-

tion (4, 6, 10, 12, 14, 26), respectively. Hereafter, we deal with

these reactions under aerobic and anaerobic conditions, discrim-

inating between reducing environmental conditions and oxida-

tive conditions.

Under aerobic and reducing conditions, the rapid and irre-

versible reaction of the ferrous oxygenated derivative of heme-

proteins (heme-Fe(II)-O2) with NO and peroxynitrite occurs.

This reaction gives rise to the ferric species (heme-Fe(III)) and

nitrate (NO32) as the final reaction products, displaying as a

reaction intermediate the heme-Fe(III)-peroxynitrite complex

(27, 28) (see Table 1). On the other hand, the reaction of heme-

Fe(II)-O2 with peroxynitrite gives rise to heme-Fe(IV)¼¼O, ni-

trite (NO22), O2, and H1; then, heme-Fe(IV)¼¼O may react with

a second peroxynitrite molecule, leading to the formation of the

heme-Fe(III) species and the peroxynitrite radical (ONOO�) as

the final reaction products (28) (see Table 2).

NO scavenging is also facilitated by the direct interaction of

ferrous nitrosylated heme-Fe(II) (heme-Fe(II)-NO) with O2, giv-

ing rise to heme-Fe(III) and NO32 as the final reaction products.

However, the intermediate(s) are different for reaction(s) cata-

lyzed by the hexa-coordinated human neuroglobin (Ngb) on one

side and by penta-coordinated globins, such as Mb (29–31) (see

Table 3). O2-mediated NO scavenging by ferrous nitrosylated

horse heart Mb and human Hb (Mb(II)-NO and Hb(II)-NO,

respectively) appears to occur with a reaction mechanism, in

which NO that is initially bound to heme-Fe(II) is displaced by

O2 but may stay trapped in a protein cavity(ies) close to the

heme. In the second step, ferrous oxygenated horse heart Mb

and human Hb (Mb(II)-O2 and Hb(II)-O2, respectively) react

with NO giving the transient heme-Fe(III)-peroxynitrite species

preceding the formation of the final products. The rate-limiting

step in catalysis appears to be NO dissociation from heme-

Fe(II)-NO (29). A slight rearrangement within the protein struc-

ture, taking place after formation of ferric human Ngb

[Ngb(III)] and possibly reflecting the penta-to-hexa-coordination

transition of the heme-Fe-atom, has been postulated to be the

rate- limiting step in O2-mediated NO scavenging (30).

NO and peroxynitrite detoxification by heme-Fe(II)-O2 and

O2-mediated NO scavenging by heme-Fe(II)-NO, indeed, all

reflect the superoxide character of the initial or transient heme-

Fe(II)-O2 species (i.e., heme-Fe(III)-O22) (32) (see Tables 1, 2,

and 3).

Table 1

NO scavenging by heme-Fe(II)-O2

Heme-protein kon (M21 s21) h (s21)

M. tuberculosis trHbNa 7.5 3 108 Fast

M. tuberculosis trHbOb 6.0 3 105 Fast

M. leprae trHbOc 2.1 3 106 3.4

E. coli flavoHbd ‡6.0 3 108 �2.0 3 102

Glycine max Lbe 8.2 3 107 Fast

Horse heart Mbf 4.4 3 107 [3.4 3 102

Murine Ngbg [7.0 3 107 �3.0 3 102

Human Hb 8.9 3 107f [5.8 3 101h

[3.3 3 101h

apH 5 7.5 and 23.08C. From (87).bpH 5 7.5 and 23.08C. From (88).cpH 5 7.3 and 20.08C. From (82).dpH 5 7.0 and 20.08C. From (36).epH 5 7.3 and 20.08C. From (89).fpH 5 7.0 and 20.08C. From (90).gpH 5 7.0 and 20.08C. From (91).hThe two values represent the decay rates for Fe(III)OONO a- and b-Hb subunits. pH 5 7.5 and 20.08C. From (27).

63PEROXIDATIVE ACTIVITY OF GLOBINS

Heme-Fe(II)-NO also facilitates peroxynitrite scavenging;

this reaction proceeds in two steps, a rapid conversion from

heme-Fe(II)-NO to the heme-Fe(III)-NO intermediate, which

then dissociates into NO and heme-Fe(III) (28) (see Table 4).

Preliminary results (33, 34) indicate that deoxygenated and fer-

rous carbonylated globins may also facilitate peroxynitrite

detoxification, giving rise to the heme-Fe(III) species.

All reactions depicted in Tables 1–4 are considered as

‘pseudo-enzymatic processes’ because they need a reductase

partner(s) to restore heme-Fe(II), which is absolutely necessary

for a new catalytic cycle. In particular, NADH-metHb and -

metMb reductases catalyze the conversion of heme-Fe(III) to

heme-Fe(II) in vivo. As a matter of fact, the enzymatic heme-

Fe(III) reduction is the rate-limiting step of the whole process,

this representing a severe limitation for the efficiency of these

mechanisms in vivo (4, 28, 35–43).

Under highly oxidative conditions, the redox equilibrium of

globins is shifted in favor of the heme-Fe(III) form, impairing

their role as O2 carriers. However, under these conditions, usu-

ally the high H2O2 concentration facilitates the oxidation of the

heme-Fe(III) of some globins (e.g., Hb and Mb), giving rise to

the formation of the compound II-like species heme-Fe(IV)¼¼O.

This highly oxidative form facilitates NO, peroxynitrite and

NO22 scavenging (see Tables 2, 5, and 6), because NO detoxifi-

cation by heme-Fe(IV)¼¼O leads to the formation of heme-

Fe(III) and NO22 (44–47) (see Table 5). The reactions of heme-

Fe(IV)¼¼O with peroxynitrite and NO22 generate ONOO� and

the nitrogen dioxide radical (�NO2), respectively, which could

contribute to tyrosine nitration and thus to the inactivation of

proteins (28, 48–51) (see Tables 2 and 6). The reaction of

heme-Fe(IV)¼¼O with NO (see Table 5) is significantly faster

than those of heme-Fe(IV)¼¼O with peroxynitrite and NO22 (see

Tables 2 and 6). These reactions, depicted in Tables 2, 5, and

6, do not require partner oxido-reductive enzyme(s), because

the system oscillates between the oxidation of the heme-Fe(III)

species to heme-Fe(IV)¼¼O by H2O2, and the heme-Fe(IV)¼¼O

reduction back to heme-Fe(III) by NO, peroxynitrite, and NO22

(28, 44–47). Interestingly, catalytic parameters for NO scaveng-

ing by heme-Fe(II)-O2 (43) and heme-Fe(IV)¼¼O (47) are

closely similar (see Tables 1 and 5) and high enough to indicate

that both reactions could occur efficiently in vivo.

In contrast with penta-coordinated globins (e.g., Hb and Mb)

(28, 44–51), heme-Fe(III) human Ngb apparently does not

generate the heme-Fe(IV)¼¼O form when exposed to H2O2 and

peroxynitrite, another feature of Ngb that may contribute to

neuronal survival after hypoxia and that may be related to

heme-Fe-atom hexa-coordination (28, 30, 31).

Beside globins, heme-Fe(IV)¼¼O peroxidases may facilitate

NO and NO22 detoxification (see Tables 5, 6, and 7). However,

in the case of mammalian peroxidases, such as myeloperoxidase

(MPO) and eosinophil peroxidase (EPO), the rate constants for

NO oxidation to NO22 are 2–3 orders of magnitude lower than

Table 2

Peroxynitrite scavenging by heme-Fe(II)-O2 and heme-Fe(IV)¼¼O

Heme-protein kon (M21 s21) hon (M

21 s21)

M. leprae trHbOa 4.8 3 104 1.3 3 104

Glycine max Lbb 5.5 3 104 2.1 3 104

Horse heart Mbc 5.4 3 104 2.2 3 104

Human Hbd 3.3 3 104 3.3 3 104

apH 7.3 and 20.08C. From (83).bpH 7.3 and 20.08C. From (89).cpH 7.5 and 20.08C. From (92).dpH 7.4 and 20.08C. From (93).

64 DE MARINIS ET AL.

those reported for the heme-Fe (IV)¼¼O derivative of globins,

whereas in the case of plant peroxidases, such as horseradish

peroxidase (HRP), the rate constant is only 10-fold slower than

for globins (47, 48, 52–59) (see Table 5). Further, the formation

of the heme-Fe(III)-ONO species is significantly faster in glo-

bins than in peroxidases (and possibly in catalase), where the

formation of the heme-Fe(III)-ONO species is the rate-limiting

step. Conversely, the dissociation of the heme-Fe(III)-ONO spe-

cies and the O-nitrito isomerization is significantly faster in per-

oxidases than in the heme-Fe(III) species of globins where

it represents instead the rate-limiting step (44–47, 52–55)

(see Table 5). On the other hand, the rate constant for NO22

scavenging by heme-Fe(IV)¼¼O MPO is similar to that observed

for heme-Fe(IV)¼¼O globin action (44–47, 53) (see Table 6).

Table 3

O2-mediated NO scavenging by heme-Fe(II)-NO

apH 7.5 and 25.08C. From (30).bpH 7.0 and 20.08C. From (94).cpH 7.2 and room temperature. From (95).


Table 4

Peroxynitrite scavenging by heme-Fe(II)-NO


M. leprae trHbOa [1.0 3 108 2.6 3 101

Glycine max Lbb 8.8 3 103 2.0

Horse heart Mbc 3.1 3 104 �1.2 3 101

Human Ngbd [1.3 3 105 1.2 3 1021

Human Hbe 6.1 3 103 �1.0

apH 7.3 and 20.08C. From (83).bpH 7.3 and 20.08C. From (46).cpH 7.5 and 20.08C. From (96).dpH 7.2 and 25.08C. From (30).epH 7.2 and 20.08C. From (97).

Table 5

NO scavenging by heme-Fe(IV)¼¼O


M. leprae trHbOa 7.8 3 106 2.1 3 101

Glycine max Lbb 1.8 3 106 [5.0 3 101

Horse heart Mbc 1.7 3 107 6.0

Human Hbd 2.4 3 107 4.8 3 1021

1.2 3 1021

Horseradish peroxidasee 1.0 3 106 Fast

Porcine eosinophyl peroxidasef 1.7 3 104 Fast

Bovine lactoperoxidasef 8.7 3 104 Fast

Human myeloperoxidaseg 8.0 3 103 Fast

apH 5 7.2 and 20.08C. From (47)bpH 5 7.0 and 20.08C. From (46).cpH 5 7.0 and 20.08C. From (44).dpH 5 7.0 and 20.08C. Biphasic kinetics of heme-Fe(III)-ONO decay (represented by values of h) has been attributed to a- and b-chains. From (45).epH 5 7.4 and 20.08C. From (52).fpH 5 7.0 and 25.08C. From (54).gpH 5 7.0 and 25.08C. From (53).


Both peroxidases and globins are able to perform the peroxy-

nitrite detoxification under oxidative stress conditions. In the

case of peroxidases, the reaction with peroxynitrite brings about

the fast formation of the compound II-like heme-Fe(IV)¼¼O

species (60), likely through the formation of a transient Fe(III)-

peroxynitrite complex, followed by its conversion to heme-

Fe(IV)¼¼O and �NO2 (61). This fast event is then followed by a

very slow reduction of heme-Fe(IV)¼¼O back to Fe(III), which

is driven by the oxidation of NO22 (in a redox equilibrium with

�NO2) to NO32 (61) (see Table 7). Although no kinetic parame-

ters are instead available for these reactions in catalase, a role

played by catalase in the detoxification of NO has been reported

(55).

In the case of globins, horse heart heme-Fe(III) Mb and

human heme-Fe(III) Hb catalyze the isomerization of peroxyni-

trite to NO32 (27, 62) (see Tables 1, 3, 5, and 7); in contrast,

peroxynitrite does not react with hexa-coordinated heme-Fe(III)

human Ngb, as reported for H2O2 (28, 30, 31).

Heme-Fe(III) species also facilitate NO scavenging through

the formation of the Fe(III)-NO complex, giving rise to heme-

Fe(II)-NO as the final reaction product. This reaction proceeds

in three steps: (i) reversible heme-Fe(III) nitrosylation (i.e.,

heme-Fe(III)-NO formation) followed by fast conversion to

heme-Fe(II)-NO1; (ii) H2O/OH2 catalyzed conversion of heme-

Fe(II)-NO1 to heme-Fe(II); and (iii) reversible heme-Fe(II)

nitrosylation by a second NO molecule (i.e., heme-Fe(II)-NO

formation). NO binding to heme-Fe(III) S. inaequivalvis HbI

and human Ngb(III) (30, 63) appears to be rate limiting,

whereas the conversion of heme-Fe(II)-NO1 to heme-Fe(II) is

the rate-limiting step for the reductive nitrosylation of heme-

Fe(III) Glycine max leghemoglobin (Lb(III)), sperm whale

Mb(III), human Hb(III), and human myeloperoxidase (46, 53,

64) (see Table 8).

Lastly, heme-based reactions involving peroxynitrite appear

to be facilitated by carbon dioxide (CO2). Indeed, peroxynitrite

may rapidly react with CO2 forming an adduct, believed to

be 1-carboxylato-2-nitrosodioxidane (ONOOC(O)O2). This

transient intermediate decays by homolysis of the O��O bond

giving rise to NO32 and CO2 as final products, trioxo-

carbonate(�12) (CO3�2) and �NO2 being the reaction intermedi-

ates. Note that CO3�2 and �NO2 are stronger oxidant species

than peroxynitrite (50, 65).

The comparison of globin and peroxidase action (see Tables

5, 6, 7, and 8) allows the following considerations. (i) The

detoxification activity of NO and peroxynitrite by the heme-

Fe(IV)¼¼O species of globins (occurring under oxidative condi-

tions) is higher than that of peroxidases. (ii) The NO22 detoxifi-

cation activity of mammalian peroxidases is higher than that

reported for plant peroxidases and globins. (iii) The heme-

Fe(III) derivative of peroxidases detoxifies peroxynitrite more

efficiently than the heme-Fe(IV)¼¼O species of globins.

Peroxidation of classical peroxidase substrates (e.g., phenols)

by globins occurs at a much slower rate, with respect to the

heme-enzymes (24, 48, 58). The different catalytic behavior of

globins and peroxidases for different substrates might be due to

the strong hydrogen bond present in peroxidases between the

proximal histidyl residue and a conserved aspartate residue

(45). Moreover, it may be also referred to the highly positive

charge present in the heme distal side of peroxidases (see

Fig. 1), which significantly lowers the pKa values of catalytic

His and Arg distal residues (66, 67). This idea is further

strengthened by the following: (i) the evidence that site-directed

mutants of horse heart Mb (Thr39Ile, Lys45Asp, Phe46Leu, and

Ile107Phe) and sperm whale Mb (Thr67Arg and Thr67Arg/

Ser92Asp) display a significant increase of the peroxidase activ-

ity (25, 68, 69), and (ii) site-directed mutants of cytochrome c

peroxidase (His175Gln, His175Glu, and His175Cys) and horse-

radish peroxidase (Arg38Leu, His42Glu, His42Gln) show a sub-

stantial decrease of the peroxidase activity (70–72).

The peroxidase activity of globins appears to be at the root

of the Mycobacterium leprae ability to persist in vivo in the

presence of reactive nitrogen and oxygen species. Indeed, dur-

ing infection, M. leprae is faced with the host macrophagic

environment, where low pH, low pO2, high pCO2, combined

with the toxic activity of reactive nitrogen and oxygen species

(including NO, superoxide (O2�2), and H2O2) contribute to limit

the growth of the bacilli in vivo (43, 47, 73–78).

The ability of M. leprae to persist in vivo in the presence of

reactive nitrogen and oxygen species implies the presence in

this elusive mycobacterium of (pseudo-)enzymatic detoxification

systems, including truncated hemoglobin O (trHbO) (43, 77–

84). M. leprae trHbO has been reported to facilitate NO and

peroxynitrite scavenging using O2, NO, and H2O2 as co-factors

(43, 47, 78, 82–84) (see Tables 1, 2, 4, 5, and 6). Interestingly,

kinetics of NO detoxification by the heme-Fe(IV)¼¼O derivative

Table 6

NO22 scavenging by heme-Fe(IV)¼¼O

Heme-protein kon (M21 s21)

M. leprae trHbOa 3.1 3 103

Glycine max Lbb 2.1 3 102

Horse heart Mbc 1.6 3 101

Human Hbd 7.5 3 102

Human myeloperoxidasee 5.5 3 102

apH 5 7.2 and 20.08C. From (47).bpH 5 7.0 and 20.08C. From (46).cpH 5 7.5 and 20.08C. From (44).dpH 5 7.0 and 20.08C. From (45).epH 5 7.0 and 15.08C. From (53).


of M. leprae, induced by H2O2, is faster than any other myco-

bacterial reactions involved in scavenging of reactive nitrogen

and oxygen species (47) (see Tables 1, 2, 4, 5, and 6). This

appears to be in agreement with the absence in M. leprae of a

specific reductase(s) converting heme-Fe(III) (obtained from the

reaction of heme-Fe(II)-O2 and heme-Fe(II)-NO with NO and

peroxynitrite) to heme-Fe(II), this enzymatic process being piv-

otal to start a new catalytic cycle (43, 47, 77, 78, 82–84).

Paradoxically, NO, peroxynitrite, and NO22 can serve as anti-

oxidants of the highly oxidizing heme-Fe(IV)¼¼O derivative of

globins, which could be responsible for the oxidative damage of

biological membranes (85) and inactivation of heme-based

enzymes (e.g., cytochrome c peroxidase) (86).

As a whole, peroxidases appear to be able to detoxify from

oxidative compounds (such as peroxynitrite) through the oxida-

tion of the resting heme-Fe(III) state to heme-Fe(IV)¼¼O under

normal oxidizing conditions. Whenever the environment

becomes highly oxidative massive oxidation of globins to

heme-Fe(IV)¼¼O takes place; this facilitates NO, peroxynitrite,

and NO22 detoxification, boosting the detoxification mechanism,

because NO, peroxynitrite, and NO22 can serve as antioxidants

of the highly oxidizing heme-Fe(IV)¼¼O species. Therefore,

under these highly oxidative conditions globins appear to facili-

tate NO, peroxynitrite, NO22, and H2O2 scavenging without

needing a reductase partner(s), which in such condition is

potentially devoid of reducing co-factors (e.g., NADH and

FADH2). Although the in vivo role of heme-Fe(IV)¼¼O globins

in scavenging reactive nitrogen species is still uncertain, NO,

peroxynitrite, and NO22 could be the ‘true’ substrates of globins

when acting as peroxidases, H2O2 being the co-substrate.

ACKNOWLEDGEMENTS

This work was partially supported by grants from the Ministry

for Education, University, and Research of Italy (Department of

Biology, University Roma Tre, Roma, Italy, ‘CLAR 2008’ to

Table 7

Peroxynitrite scavenging by heme-Fe(III)

kon (M21 s21) h (s21)

Horse heart Mb 2.9 3 104a [3.43102b

Human Hb 1.2 3 104a [5.83101c

[3.33101c

kon (5 K1 3 k2; M21 s21) h (s21)

Human myeloperoxidased 6.8 3 106 �0.1

Bovine lactoperoxidasee 3.3 3 105 nd

Horse radish peroxidasef 3.2 3 106 nd

apH 5 7.0 and 20.08C. From (98)bpH 5 7.0 and 20.08C. From (90).cThe two values represent the decay rates for Fe(III)OONO a- and b-Hb subunits. pH 5 7.5 and 20.08C. From (27).dpH 5 7.0 and 258C. From (61).epH 5 7.4 and 128C. From (60).fpH 5 6.8 and 258C. From (60).

nd, not determined.


Table

8

NO

scavengingbyhem

e-Fe(III)

Hem

e-protein

k on(M

21s2

1)

k off(s21)

h(s21)

l on(M

21s2

1)

l off(s21)

Glycine

max

Lb

1.4

3105a

3.0

a4.8

31024a

1.2

3108b

2.4

31025b

S.inaequivalvisHbI

3.2

3101c

\1.0

3103c

[6.0

31021c

1.6

3107d

nd

Sperm

whaleMb

1.9

3105e

1.4

3101e

\8.8

31024f

1.7

3107g

1.2

31024g

Mouse

Ngbh

nd

nd

nd

2.0

3105

2.0

31024

Human

Ngbi

2.1

3101

2.5

31023

[2.0

31021

nd

nd

2.9

2.5

31023

[5.0

31022

nd

nd

Human

Hbj

1.7

3103k

6.5

31021k

1.3

31023l

2.6

3107m

4.6

31025b

6.4

3103k

1.5k

1.3

31023l

2.6

3107m

2.2

31025b

Human

myeloperoxidasen

1.1

3106

1.1

3101

Slow

1.0

3105

4.6

apH

57.0

and20.08C

.From

(46).

bpH

57.0

and20.08C

.From

(99).

cpH

57.5

and20.08C

.From

(63).

dpH

57.0

and20.08C

.From

(100

).epH

56.5

and20.08C

.From

(101

).f pH\

8.3

and20.08C

.From

(64).

gpH

57.0

and20.08C

.From

(102

).hpH

57.0

and25.08C

.From

(103

).i pH

57.0

androom

temperature.Biphasic

kineticshas

beenattributedto

fastandslow

reactingform

.From

(30).

j Biphasic

kineticshas

beenattributedto

a-andb-chains.

kpH

57.0

and20.08C

.From

(104

).l pH

57.0

and20.08C

.From

(64).

mpH

57.0

and20.08C

.From

(105

).npH

57.0

and10.08C

.From

(53).

nd,notdetermined.


P.A.) and from the Ministry of Health of Italy (National Insti-

tute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’,

Roma, Italy, ‘Ricerca corrente 2007’ to P.A.).

REFERENCES1. Vinogradov, S. N., Hoogewijs, D., Bailly, X., Arredondo-Peter, R.,

Gough, J., Dewilde, S., Moens, L., and Vanfleteren, J. R. (2006) A

phylogenomic profile of globins. BMC Evol. Biol. 6, 31.

2. Vuletich, D. A. and Lecomte, J. T. (2006) A phylogenetic and struc-

tural analysis of truncated hemoglobins. J. Mol. Evol. 62, 196–210.

3. Poole, R. K. and Hughes, M. N. (2000) New functions for the ancient

globin family: bacterial responses to nitric oxide and nitrosative stress.

Mol. Microbiol. 36, 775–783.

4. Brunori, M. (2001) Nitric oxide moves myoglobin centre stage. TrendsBiochem. Sci. 26, 209–210.

5. Weber, R. E. and Vinogradov, S. N. (2001) Nonvertebrate hemoglo-

bins: functions and molecular adaptations. Physiol. Rev. 81, 569–628.

6. Godecke, A., Molojavyi, A., Heger, J., Flogel, U., Ding, Z., Jacoby,

C., and Schrader, J. (2003) Myoglobin protects the heart from induci-

ble nitric-oxide synthase (iNOS)-mediated nitrosative stress. J. Biol.

Chem. 278, 21761–21766.

7. Shikama, K. and Matsuoka, A. (2004) Structure-function relationships

in unusual nonvertebrate globins. Crit. Rev. Biochem. Mol. Biol. 39,

217–259.

8. Freitas, T. A., Saito, J. A., Hou, S., and Alam, M. (2005) Globin-

coupled sensors, protoglobins, and the last universal common ancestor.

J. Inorg. Biochem. 99, 23–33.

9. Gilles-Gonzalez, M. A. and Gonzalez, G. (2005) Heme-based sensors:

defining characteristics, recent developments, and regulatory hypothe-

ses. J. Inorg. Biochem. 99, 1–22.

10. Ascenzi, P., Bellelli, A., Coletta, M., Colosimo, A., Falcioni, G., Gia-

cometti, G. M., Ippoliti, R., Zolla, L., and Giardina, B. (2007) Multiple

strategies for O2 transport: from simplicity to complexity. IUBMB Life59, 600–616.

11. Nardini, M., Pesce, A., Milani, M., and Bolognesi, M. (2007) Protein

fold and structure in the truncated (2/2) globin family. Gene 398, 2–

11.

12. Wittenberg, J. B. (2007) On optima: the case of myoglobin-facilitated

oxygen diffusion. Gene 398, 156–161.

13. Bonamore, A. and Boffi, A. (2008) Flavohemoglobin: structure and

reactivity. IUBMB Life 60, 19–28.

14. Vinogradov, S. N. and Moens, L. (2008) Diversity of globin function:

enzymatic, transport, storage, and sensing. J Biol Chem. 283, 8773–

8777.

15. Wilson, M. T. and Reeder, B. J. (2008) Oxygen-binding haem proteins.

Exp Physiol. 93, 128–132.16. Keilin, D. and Hartree, E. F. (1951) Purification of horse-radish peroxi-

dase and comparison of its properties with those of catalase and meth-

aemoglobin. Biochem. J. 49, 88–104.

17. Willick, G. E., Schonbaum, G. R., and Kay, C. M. (1969) Circular

dichroism and absorption spectra of horse radish peroxidase and sperm

whale myoglobin in the Soret region. Biochemistry. 8, 3729–3734.

18. Kobert, R. (1900) Beitrage zur Kenntniss der Methamoglobine.

Pflugers Arch. Gesamte Physiol. Menschen Tiere. 82, 603–630.

19. Wu, H. (1923) Studies on hemoglobin: III. An ultra-micro-method for

the determination of hemoglobin as a peroxidase. J. Biochem. (Tokyo)

2, 189–194.

20. Polonovski, M. and Jayle, M. F. (1938) Existence dans le plasma

humain d’une substance activant l’action peroxydasique de l’hemo-

globine. C. R. Hebd. Seances Mem. Soc. Biol. Ses. Fil. 129, 457–460.

21. George, P. and Irvine, D. H. (1952) The reaction between metmyoglo-

bin and hydrogen peroxide. Biochem. J. 52, 511–517.

22. Keilin, D. and Hartree, E. F. (1955) Catalase, peroxidase and metmyo-

globin as catalysts of coupled peroxidatic reactions. Biochem. J. 60,310–325.

23. Yonetani, T. and Schleyer, H. (1967) Studies on cytochrome c peroxi-

dase. IX. The reaction of ferrimyoglobin with hydroperoxides and a

comparison of peroxidase-induced compounds of ferrimyoglobin and

cytochrome c peroxidase. J. Biol. Chem. 242, 1974–1979.

24. Everse, J., Everse, K. E., and Grisham, M. B., Eds (1991) Peroxidases

in Chemistry and Biology. Volumes I and II, CRC, Boca Raton.

Figure 1. Key residues of heme surroundings of horse heart Mb (PDB code: 2v1k) and horseradish peroxidase (PDB code: 1h58).


25. Redaelli, C., Monzani, E., Santagostini, L., Casella, L., Sanangelantoni,

A. M., Pierattelli, R., and Banci, L. (2002) Characterization and perox-

idase activity of a myoglobin mutant containing a distal arginine.

Chem.Bio.Chem. 3, 226–233.26. Abu-Soud, H. M. and Hazen, S. L. (2000) Nitric oxide is a physiologi-

cal substrate for mammalian peroxidases. J. Biol. Chem. 275, 37524–

37532.

27. Herold, S. (1999) Kinetic and spectroscopic characterization of an in-

termediate peroxynitrite complex in the nitrogen monoxide induced ox-

idation of oxyhemoglobin. FEBS Lett. 443, 81–84.

28. Herold, S. and Fago, A. (2005) Reactions of peroxynitrite with globin

proteins and their possible physiological role. Comp. Biochem. Physiol.A Mol. Integr. Physiol. 142, 124–129.

29. Fasano, M., Antonini, G., and Ascenzi, P. (2006) O2-mediated oxida-

tion of hemopexin-heme(II)-NO. Biochem. Biophys. Res. Commun.345, 704–712.

30. Herold, S., Fago, A., Weber, R. E., Dewilde, S., and Moens, L. (2004)

Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuro-

globin reveal a potential role against oxidative stress. J. Biol. Chem.279, 22841–22847.

31. Nicolis, S., Monzani, E., Ciaccio, C., Ascenzi, P., Moens, L., and

Casella, L. (2007) Reactivity and endogenous modification by nitrite

and hydrogen peroxide: does human neuroglobin act only as a scav-

enger? Biochem. J. 407, 89–99.

32. Momenteau, M. and Reed, C. (1994) Synthetic heme dioxygen com-

plexes. Chem. Rev. 94, 659–698.33. Exner, M. and Herold, S. (2000) Kinetic and mechanistic studies of

the peroxynitrite-mediated oxidation of oxymyoglobin and oxyhemo-

globin. Chem. Res. Toxicol. 13, 287–293.

34. Ascenzi, P., Ciaccio, C., and Coletta, M. (2007) Peroxynitrite-mediated

oxidation of ferrous carbonylated myoglobin is limited by carbon

monoxide dissociation. Biochem. Biophys. Res. Commun. 363, 931–

936.

35. Gow, A. J., Luchsinger, B. P., Pawloski, J. R., Singel, D. J., and Stam-

ler, J. S. (1999) The oxyhemoglobin reaction of nitric oxide. Proc.

Natl. Acad. Sci. USA 96, 9027–9032.

36. Gardner, A. M., Martin, L. A., Gardner, P. R., Dou, Y., and Olson, J.

S. (2000) Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin) The B10 tyrosine hydroxyl is

essential for dioxygen binding and catalysis. J. Biol. Chem. 275,

12581–12589.

37. Flogel, U., Merx, M. W., Godecke, A., Decking, U. K., and Schrader,

J. (2001) Myoglobin: a scavenger of bioactive NO. Proc. Natl. Acad.

Sci. USA 98, 735–740. Erratum in: Proc. Natl. Acad. Sci. USA 98,

4276.

38. Frauenfelder, H., McMahon, B. H., and Fenimore, P. W. (2003) Myo-

globin: the hydrogen atom of biology and a paradigm of complexity.

Proc. Natl. Acad. Sci. USA 100, 8615–8617.

39. Frey, A. D. and Kallio, P. T. (2003) Bacterial hemoglobins and flavo-

hemoglobins: versatile proteins and their impact on microbiology and

biotechnology. FEMS Microbiol. Rev. 27, 525–545.

40. Wu, G., Wainwright, L. M., and Poole, R. K. (2003) Microbial glo-

bins. Adv. Microb. Physiol. 47, 255–310.

41. Brunori, M., Bourgeois, D., and Vallone, B. (2004) The structural dy-

namics of myoglobin. J. Struct. Biol. 147, 223–234.

42. Frey, A. D. and Kallio, P. T. (2005) Nitric oxide detoxification - a

new era for bacterial globins in biotechnology? Trends Biotechnol. 23,

69–73.

43. Ascenzi, P. and Visca, P. (2008) Scavenging of reactive nitrogen spe-

cies by mycobacterial truncated hemoglobins. Methods Enzymol. 436,317–337.

44. Herold, S. and Rehmann, F. J. (2001) Kinetic and mechanistic studies

of the reactions of nitrogen monoxide and nitrite with ferryl myoglo-

bin. J. Biol. Inorg. Chem. 6, 543–555.

45. Herold, S. and Rehmann, F. J. (2003) Kinetics of the reactions of

nitrogen monoxide and nitrite with ferryl hemoglobin. Free Radic.

Biol. Med. 34, 531–545.

46. Herold, S. and Puppo, A. (2005) Kinetics and mechanistic studies of

the reactions of metleghemoglobin, ferrylleghemoglobin, and nitrosyl-

leghemoglobin with reactive nitrogen species. J. Biol. Inorg. Chem. 10,

946–957.

47. Ascenzi, P., De Marinis, E., Coletta, M., and Visca, P. (2008) H2O2

and �NO scavenging by Mycobacterium leprae truncated hemoglobin

O. Biochem. Biophys. Res. Commun. 373, 197–201.

48. Nicolis, S., Monzani, E., Roncone, R., Gianelli, L., and Casella, L.

(2004) Metmyoglobin-catalyzed exogenous and endogenous tyrosine

nitration by nitrite and hydrogen peroxide. Chem. Eur. J. 10, 2281–

2290.

49. Radi, R. (2004) Nitric oxide, oxidants, and protein tyrosine nitration.

Proc. Natl. Acad. Sci. USA. 101, 4003–4008.

50. Goldstein, S. and Merenyi, G. (2008) The chemistry of peroxynitrite:

implications for biological activity. Methods Enzymol. 436, 49–61.

51. Monzani, E., Nicolis, S., Roncone, R., Barbieri, M., Granata, A., and

Casella, L. (2008) Protein self-modification by heme-generated reactive

species. IUBMB Life. 60, 41–56.

52. Glover, R. E., Koshkin, V., Dunford, H. B., and Mason, R. P. (1999)

The reaction rates of NO with horseradish peroxidase compounds I and

II. Nitric Oxide. 3, 439–444.

53. Abu-Soud, H. M. and Hazen, S. L. (2000) Nitric oxide modulates

the catalytic activity of myeloperoxidase. J. Biol. Chem. 275, 5425–5430.

54. Abu-Soud, H. M., Khassawneh, M. Y., Sohn, J. T., Murray, P., Hax-

hiu, M. A., and Hazen, S. L. (2001) Peroxidases inhibit nitric oxide

(NO) dependent bronchodilation: development of a model describing

NO-peroxidase interactions. Biochemistry. 40, 11866–11875.

55. Brunelli, L., Yermilov, V., and Beckman, J. S. (2001) Modulation of

catalase peroxidatic and catalytic activity by nitric oxide. Free Radic.

Biol. Med. 30, 709–714.56. Burner, U., Furtmuller, P. G., Kettle, A. J., Koppenol, W. H., and

Obinger, C. (2000) Mechanism of reaction of myeloperoxidase with ni-

trite. J. Biol. Chem. 275, 20597–20601.

57. Bruck, T. B., Fielding, R. J., Symons, M. C. R., and Harvey, P. J.

(2001) Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

Eur. J. Biochem. 268, 3214–3222.

58. Monzani, E., Roncone, R., Galliano, M., Koppenol, W. H., and Case-

lla, L. (2004) Mechanistic insight into the peroxidase catalyzed nitra-

tion of tyrosine derivatives by nitrite and hydrogen peroxide. Eur. J.

Biochem. 271, 895–906.

59. van Dalen, C. J., Winterbourn, C. C., Kettle, A. J. (2006) Mechanism

of nitrite oxidation by eosinophil peroxidase: implications for oxidant

production and nitration by eosinophils. Biochem. J. 394, 707–713.

60. Floris, R., Piersma, S. R., Yang, G., Jones, P., and Wever, R. (1993)

Interaction of myeloperoxidase with peroxynitrite. A comparison with

lactoperoxidase, horseradish peroxidase and catalase. Eur. J. Biochem.

215, 767–775.

61. Furtmuller, P. G., Jantschko, W., Zederbauer, M., Schwanninger, M.,

Jakopitsch, C., Herold, S., Koppenol, W. H., and Obinger, C. (2005)

Peroxynitrite efficiently mediates the interconversion of redox inter-

mediates of myeloperoxidase. Biochem. Biophys. Res. Comm. 337,

944–954.

62. Herold, S., Kalinga, S., Matsui, T., and Watanabe, Y. (2004) Mecha-

nistic studies of the isomerization of peroxynitrite to nitrate catalyzed

by distal histidine metmyoglobin mutants. J. Am. Chem. Soc. 126,

6945–6955.

63. Boffi, A., Sarti, P., Amiconi, G., and Chiancone, E. (2002) The inter-

play between heme iron and protein sulfhydryls in the reaction

of dimeric Scapharca inaequivalvis hemoglobin with nitric oxide.

Biophys. Chem. 98, 209–216.


64. Hoshino, M., Maeda, M., Konishi, R., Seki, H., and Ford, P. C. (1996)

Studies on the reaction mechanism for reductive nitrosylation of

ferrihemoproteins in buffer solutions. J. Am. Chem. Soc. 118, 5702–

5707.

65. Ascenzi, P., Bocedi, A., Visca, P., Minetti, M., and Clementi, E.

(2006) Does CO2 modulate peroxynitrite specificity? IUBMB Life 58,

611–613.

66. Dunford, H. B. and Stillman, J. S. (1976) On the function and

mechanism of action of peroxidases. Coord. Chem. Rev. 19, 187–

251.

67. Welinder, K. G., Bjørnholm, B., and Dunford, H. B. (1995) Functions

of electrosctatic potentials and conserved distal and proximal His-Asp

H-bonding network in haem peroxidases. Biochem. Soc. Trans. 23,

257–262.

68. Wan, L., Twitchett, M. B., Eltis, L. D., Mauk, G., and Smith, M.

(1998) In vitro evolution of horse heart myoglobin to increase peroxi-

dase activity. Proc. Natl. Acad. Sci. USA 95, 12825–12831.

69. Roncone, R., Monzani, E., Murtas, M., Battaini, G., Pennati, A., San-

angelantoni, A. M., Zuccotti, S., Bolognesi, M., and Casella, L. (2004)

Engineering peroxidase activity in myoglobin: the haem cavity struc-

ture and peroxide activation in the T67R/S92D mutant and its deriva-

tive reconstituted with protohemin-L-histidine. Biochem. J. 377, 717–

724.

70. Choudhury, K., Sundaramoorthy, M., Hickman, A., Yonetani, T.,

Woehl, E., Dunn, M. F., and Poulos, T. L. (1994) Role of the proximal

ligand in peroxidase catalysis. Crystallographic, kinetic, and spectral

studies of cytochrome c peroxidase proximal ligand mutants. J. Biol.

Chem. 269, 20239–20249.

71. Rodriguez-Lopez, J. N., Smith, A. T., and Thorneley, R. N. F.

(1996) Role of arginine 38 in horse radish peroxidase. A critical

residue for substrate binding and catalysis. J. Biol. Chem. 271, 4023–

4030.

72. Tanaka, M., Ishimori, K., Mukai, M., Kitagawa, T., and Morishima, I.

(1997) Catalytic activities and structural properties of horseradish per-

oxidase distal His42?Glu or Gln mutant. Biochemistry. 36, 9889–

9898.

73. MacMicking, J., Xie, Q. W., and Nathan, C. (1997) Nitric oxide and

macrophage function. Annu. Rev. Immunol. 15, 323–350.74. Ratledge, C. and Dale, J., Eds (1999) Mycobacteria, Molecular Biology

and Virulence. Blackwell Science, Oxford.

75. Nathan, C. and Shiloh, M. U. (2000) Reactive oxygen and nitrogen

intermediates in the relationship between mammalian hosts and micro-

bial pathogens. Proc. Natl. Acad. Sci. USA 97, 8841–8848.

76. Cooper, A. M., Adams, L. B., Dalton, D. K., Appelberg, R., and

Ehlers, S. (2002) IFN-c and NO in mycobacterial disease: new jobs for

old hands. Trends Microbiol. 10, 221–226.

77. Visca, P., Fabozzi, G., Milani, M., Bolognesi, M., and Ascenzi, P.

(2002) Nitric oxide and Mycobacterium leprae pathogenicity. IUBMB

Life. 54, 95–99.78. Ascenzi, P., Bolognesi, M., Milani, M., Guertin, M., and Visca, P.

(2007) Mycobacterial truncated hemoglobins: from genes to functions.

Gene. 398, 42–51.79. Wittenberg, J. B., Bolognesi, M., Wittenberg, B. A., and Guertin, M.

(2002) Truncated hemoglobins: a new family of hemoglobins widely

distributed in bacteria, unicellular eukaryotes, and plants. J. Biol.

Chem. 277, 871–874.80. Milani, M., Pesce, A., Ouellet, H., Guertin, M., and Bolognesi, M.

(2003) Truncated hemoglobins and nitric oxide action. IUBMB Life.

55, 623–627.

81. Milani, M., Pesce, A., Nardini, M., Ouellet, H., Ouellet, Y., Dewilde,

S., Bocedi, A., Ascenzi, P., Guertin, M., Moens, L., Friedman, J. M.,

Wittenberg, J. B., and Bolognesi, M. (2005) Structural bases for heme

binding and diatomic ligand recognition in truncated hemoglobins.

J. Inorg. Biochem. 99, 97–109.

82. Ascenzi, P., Bocedi, A., Bolognesi, M., Fabozzi, G., Milani, M., and

Visca, P. (2006) Nitric oxide scavenging by Mycobacterium leprae

GlbO involves the formation of the ferric heme-bound peroxynitrite in-

termediate. Biochem. Biophys. Res. Commun. 339, 448–454.83. Ascenzi, P., Milani, M., and Visca, P. (2006) Peroxynitrite scavenging

by ferrous truncated hemoglobin GlbO from Mycobacterium leprae.

Biochem. Biophys. Res. Commun. 351, 528–533.

84. Fabozzi, G., Ascenzi, P., Di Renzi, S., and Visca, P. (2006) Truncated

hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide

toxicity. Microb. Pathog. 40, 211–220.

85. Rubbo, H., Radi, R., Anselmi, D., Kirk, M., Barnes, S., Butler, J.,

Eiserich, J. P., and Freeman, B. A. (2000) Nitric oxide reaction with

lipid peroxyl radicals spares a-tocopherol during lipid peroxidation.

Greater oxidant protection from the pair nitric oxide/a-tocopherol thana-tocopherol/ascorbate. J. Biol. Chem. 275, 10812–10818.

86. Brunori, M., Forte, E., Arese, M., Mastronicola, D., Giuffre, A., and

Sarti, P. (2006) Nitric oxide and the respiratory enzyme. Biochim. Bio-

phys. Acta 1757, 1144–1154.

87. Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B., Wit-

tenberg, J., and Guertin, M. (2002) Truncated hemoglobin HbN pro-

tects Mycobacterium bovis from nitric oxide. Proc. Natl. Acad. Sci.

USA 99, 5902–5907.

88. Ouellet, H., Juszczak, L., Dantsker, D., Samuni, U., Ouellet, Y. H.,

Savard, P. Y., Wittenberg, J. B., Wittenberg, B. A., Friedman, J. M.,

and Guertin, M. (2003) Reactions of Mycobacterium tuberculosis trun-

cated hemoglobin O with ligands reveal a novel ligand-inclusive

hydrogen bond network. Biochemistry 42, 5764–5774.

89. Herold, S. and Puppo, A. (2005) Oxyleghemoglobin scavenges nitro-

gen monoxide and peroxynitrite: a possible role in functioning nod-

ules? Biol. Inorg. Chem. 10, 935–945.90. Herold, S., Exner, M., and Nauser, T. (2001) Kinetic and mechanistic

studies of the NO�-mediated oxidation of oxymyoglobin and oxyhemo-

globin. Biochemistry 40, 3385–3395.

91. Brunori, M., Giuffre, A., Nienhaus, K., Nienhaus, G. U., Scandurra, F.

M., and Vallone, B. (2005) Neuroglobin, nitric oxide, and oxygen:

functional pathways and conformational changes. Proc. Natl. Acad.

Sci. USA 102, 8483–8488.

92. Herold, S., Exner, M., and Boccini, F. (2003) The mechanism of

the peroxynitrite-mediated oxidation of myoglobin in the absence

and presence of carbon dioxide. Chem. Res. Toxicol. 16, 390–

402.

93. Boccini, F. and Herold, S. (2004) Mechanistic studies of the oxida-

tion of oxyhemoglobin by peroxynitrite. Biochemistry. 43, 16393–

16404.

94. Møller, J. K. and Skibsted, L. H. (2004) Mechanism of nitrosylmyoglo-

bin autoxidation: temperature and oxygen pressure effects on the two

consecutive reactions. Chem. Eur. J. 10, 2291–2300.

95. Herold, S. and Rock, G. (2005) Mechanistic studies of the oxygen-

mediated oxidation of nitrosylhemoglobin. Biochemistry. 44, 6223–

6231.

96. Herold, S. and Boccini, F. (2006) NO� release from MbFe(II)NO and

HbFe(II)NO after oxidation by peroxynitrite. Inorg. Chem. 45, 6933–6943.

97. Herold, S. (2004) The outer-sphere oxidation of nitrosyliron(II)hemo-

globin by peroxynitrite leads to the release of nitrogen monoxide.

Inorg. Chem. 43, 3783–3785.98. Herold, S. and Shivashankar, K. (2003) Metmyoglobin and methemo-

globin catalyze the isomerization of peroxynitrite to nitrate. Biochemis-

try. 42, 14036–14046.

99. Rohlfs, R. J., Olson, J. S., and Gibson, Q. H. (1988) A comparison of

the geminate recombination kinetics of several monomeric heme pro-

teins. J. Biol. Chem. 263, 1803–1813.

100. Chiancone, E. and Gibson, Q. H. (1989) Ligand binding to the

dimeric hemoglobin from Scapharca inaequivalvis, a hemoglobin with


a novel mechanism for cooperativity. J. Biol. Chem. 264, 21062–

21065.

101. Hoshino, M., Oawa, K., Seki, H., and Ford, P. C. (1993) Photochemis-

try of nitric oxide adducts of water-soluble iron (III)porphyrin and fer-

rihemoproteins studied by nanosecond laser photolysis. J. Am. Chem.

Soc. 115, 9568–9575.

102. Moore, E. G. and Gibson, Q. H. (1976) Cooperativity in the dis-

sociation of nitric oxide from hemoglobin. J. Biol. Chem. 251, 2788–2794.

103. Van Doorslaer, S., Dewilde, S., Kiger, L., Nistor, S. V., Goovaerts, E.,

Marden, M. C., and Moens, L. (2003) Nitric oxide binding properties

of neuroglobin: a characterization by EPR and flash photolysis. J. Biol.

Chem. 278, 4919–4925.104. Sharma, V. S., Traylor, T. G., Gardiner, R., and Mizukami, H. (1987)

Reaction of nitric oxide with heme proteins and model compounds of

hemoglobin. Biochemistry 26, 3837–3843.

105. Cassoly, R. and Gibson, Q. (1975) Conformation, co-operativity and

ligand binding in human hemoglobin. J. Mol. Biol. 91, 301–313.


Biochemical and Biophysical Research Communications 380 (2009) 392–396

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate /ybbrc

Peroxynitrite detoxification by ferryl Mycobacterium leprae truncatedhemoglobin O

Paolo Ascenzi a,b,*, Elisabetta De Marinis a, Paolo Visca a,b, Chiara Ciaccio c,d, Massimo Coletta c,d

a Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italyb National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italyc Department of Experimental Medicine and Biochemical Sciences, University of Roma ‘Tor Vergata’, Via Montpellier 1, I-00133 Roma, Italyd Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems (CIRCMSB), Piazza Umberto I 1, I-70100 Bari, Italy

a r t i c l e i n f o

Article history:Received 15 January 2009Available online 23 January 2009

Keywords:Mycobacterium lepraeTruncated hemoglobin OPeroxynitriteHydrogen peroxideCarbon dioxideDetoxification of reactive nitrogen andoxygen species

0006-291X/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.bbrc.2009.01.088

Abbreviations: heme-Fe(III), ferric heme-protein;Fe(IV)] hemeprotein; heme-Fe(II), ferrous deoxygeFe(II)-NO, ferrous nitrosylated heme-protein; heme-heme-protein; Hb, hemoglobin; Lb, leghemoglobin;cated hemoglobin O; Ml-trHbO, Mycobacterium leprae

* Corresponding author. Address: Department of BLaboratory for Electron Microscopy, University Roma446, I-00146 Roma, Italy. Fax: +39 06 5733 6321.

E-mail address: [email protected] (P. Ascenzi).1 The recommended IUPAC nomenclature for peroxyn

and for peroxynitrous acid is hydrogen oxoperoxonitrused in the text to refer generically to both ONOO� and[14]).

a b s t r a c t

During infection, Mycobacterium leprae is faced with the host macrophagic environment limiting thegrowth of the bacilli. However, (pseudo-)enzymatic detoxification systems, including truncated hemo-globin O (Ml-trHbO), could allow this mycobacterium to persist in vivo. Here, kinetics of peroxynitrite(ONOOH/ONOO�) detoxification by ferryl Ml-trHbO (Ml-trHbOAFe(IV)@O), obtained by treatment withH2O2, is reported. Values of the second-order rate constant for peroxynitrite detoxification by Ml-trHbOAFe(IV)@O (i.e., of Ml-trHbOAFe(III) formation; kon), at pH 7.2 and 22.0 �C, are 1.5 � 104 M�1 s�1,and 2.2 � 104 M�1 s�1, in the absence of and presence of physiological levels of CO2 (�1.2 � 10�3 M),respectively. Values of kon increase on decreasing pH with a pKa value of 6.7, this suggests that ONOOHreacts preferentially with Ml-trHbOAFe(IV)@O. In turn, peroxynitrite acts as an antioxidant of Ml-trHbOAFe(IV)@O, which could be responsible for the oxidative damage of the mycobacterium. As awhole, Ml-trHbO can undertake within the same cycle H2O2 and peroxynitrite detoxification.

� 2009 Elsevier Inc. All rights reserved.

During infection, Mycobacterium leprae is faced with the host peroxocarbonate), which is a stronger nitrating agent than ONOOH� � ��
macrophagic environment, where low pH, low pO2, and high car-
bon dioxide (CO2) levels, combined with reactive nitrogen and oxy-gen species including peroxynitrite (ONOO�/ONOOH)1 andhydrogen peroxide (H2O2), contribute to limit the growth of the ba-cilli and to host tissue damage [1–10]. Peroxynitrite is more reactivethan its precursors nitrogen monoxide (�NO) and superoxide (O2

��),promoting oxidative tissue injury by different mechanisms. In fact,peroxynitrite reacts with bio-molecules either directly or afterhomolysis to nitrite radical (�NO2) and hydroxyl radical (�OH). Fur-thermore, one of the main targets of peroxynitrite is thought to beCO2, present in millimolar concentrations in most fluids and tissues,apparently forming an adduct whose composition is believed to beONOOC(O)O� (named 1-carboxylato-2-nitrosodioxidane or nitroso-

ll rights reserved.

heme-Fe(IV)@O, ferryl [oxo-nated heme-protein; heme-Fe(II)-O2, ferrous oxygenatedMb, myoglobin; trHbO, trun-

trHbO.iology and InterdepartmentalTre, Viale Guglielmo Marconi

itrite is oxoperoxonitrate (1�)ate. The term peroxynitrite isits conjugate acid ONOOH (see

and is homolyzed to trioxocarbonate( 1 ) (CO3 ) and NO2 [11–16].Note that leukocyte peroxidase catalyzes peroxynitrite conversionto hydrogen-peroxide-halide, representing an efficient antimicrobialagent. Thus, the respiratory burst of phagocytes serves as the pri-mary source of H2O2 for peroxidase-catalyzed reactions. In addition,microorganisms can generate H2O2, thus contributing to limit theirgrowth by the peroxidase system [1,17–19].

The ability of M. leprae to persist in vivo in the presence of reactivenitrogen and oxygen species implies the presence in this elusivemycobacterium of (pseudo-)enzymatic detoxification systems,including truncated hemoglobin O (Ml-trHbO) [7,9,20–25]. Ferrousoxygenated and nitrosylated Ml-trHbO (Ml-trHbOAFe(II)AO2 andMl-trHbOAFe(II)ANO, respectively) has been reported to facilitate�NO, O2, and peroxynitrite detoxification, moreover ferryl Ml-trHbO(Ml-trHbOAFe(IV)@O) acts as a �NO, nitrite (NO2

�), and H2O2 scaven-ger [9,20,21,23–25]. Peroxynitrite scavenging by ferrous oxygenatedand nitrosylated heme-proteins (heme-Fe(II)AO2 and heme-Fe(II)ANO, respectively), leading to the ferric heme-protein deriva-tive (heme-Fe(III)), needs a specific reductase(s) to restore theferrous species (heme-Fe(II)) in order to start a new catalytic cycle(see [26]). Since a reductase system(s) has been identified only forhemoglobin (Hb), flavohemoglobin, and myoglobin (Mb) (see [27–29]), alternative reaction mechanism(s) that does not need a partnerreductase(s) could be operative in vivo. Recently, �NO and NO2

�

have been reported to be detoxified by the ferryl derivative of

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P. Ascenzi et al. / Biochemical and Biophysical Research Communications 380 (2009) 392–396 393

heme-proteins (heme-Fe(IV)@O) leading to heme-Fe(III) and NO2�

and �NO2, respectively, then heme-Fe(III) can be oxidized to heme-Fe(IV)@O by H2O2. Therefore, �NO, NO2

�, and H2O2 facilitate theheme-Fe(IV)@O/heme-Fe(III) cycle (see [25,26,30–32]).

Here, kinetics of peroxynitrite detoxification by Ml-trHbOAFe(IV)@O, obtained by treatment with H2O2, are reported. In turn,peroxynitrite acts as an antioxidant of Ml-trHbOAFe(IV)@O leadingto Ml-trHbOAFe(III). Therefore, Ml-trHbO can undertake within thesame cycle not only �NO, NO2

�, and H2O2 scavenging [25], but alsoperoxynitrite detoxification (present study).

Materials and methods

Ml-trHbOAFe(III) was prepared as previously reported [33]. TheMl-trHbOAFe(III) concentration was determined by measuring theoptical absorbance at 409 nm (e409 nm = 1.15 � 105 M�1 cm�1) [20].The Ml-trHbOAFe(IV)@O stock solution was prepared by adding10–25 equivalents of H2O2 to a buffered Ml-trHbOAFe(III) solution(3.0 � 10�2 M phosphate buffer, pH 7.2), at 20.0 �C. After a reactiontime of 10–20 min, the Ml-trHbOAFe(IV)@O solution was stored onice and used within 1 h [25]. Before each experiment, theMl-trHbOAFe(IV)@O stock solution was diluted to the desired pHvalue (ranging between 6.2 and 8.1) with the appropriate4.0 � 10�1 M phosphate buffer solution. The Ml-trHbOAFe(IV)@Oconcentration was determined by measuring the optical absor-bance at 419 nm (e419 nm = 1.06 � 105 M�1 cm�1; pH 7.2 and20.0 �C) [25].

H2O2 (from Fluka GmbH, Buchs, Switzerland) was diluted withthe 5.0 � 10�2 M phosphate buffer solution (pH 7.2). The H2O2 con-centration was determined spectrophotometrically at 240 nm(e240 nm = 39.4 M�1 cm�1) [34].

Peroxynitrite was prepared from potassium superoxide (KO2)and �NO and from nitrous acid (HNO2) and H2O2 [35,36]. The per-oxynitrite stock solution was diluted with degassed 1.0 � 10�2 Msodium hydroxide (NaOH) to reach the desired concentration[9,21]. The peroxynitrite concentration was determined spectro-photometrically at 302 nm (e302 nm = 1.705 � 103 M�1 cm�1)[35,36]. Decomposed peroxynitrite was obtained by acidificationof the peroxynitrite solution [14].

All the other products (from Merck AG, Darmstadt, Germany, orSigma–Aldrich, St. Louis, MO, USA) were of analytical grade andused without purification.

The solutions of the experiments in the presence of CO2 wereprepared by adding the required amount of a 5.0 � 10�1 M NaHCO3

solution [9,21].Kinetics for peroxynitrite detoxification by Ml-trHbOAFe(IV)@O

was determined, in the absence and presence of CO2, by mixing theMl-trHbOAFe(IV)@O (final concentration, 2.7 � 10�6 M) solutionwith the peroxynitrite (final concentration, 2.0 � 10�5 M to4.0 � 10�4 M) solution, at pH values ranging between 6.2 and 8.1(final concentration, 2.0 � 10�1 M phosphate buffer) and 20.0 �C;no gaseous phase was present. Kinetics was monitored between360 nm and 460 nm.

The time course of peroxynitrite detoxification byMl-trHbOAFe(IV)@O, in the absence and presence of CO2, was fit-ted to a single exponential process according to the minimum reac-tion mechanism represented by Scheme 1 [37–39].

+ peroxynitrite + CO2

kon

Ml-trHbO-Fe(IV)=O + peroxynitrite Ml-trHbO-Fe(III)

Scheme 1.

Values of the pseudo-first-order rate constant for peroxynitrite-mediated Ml-trHbOAFe(IV)@O reduction (i.e., Ml-trHbOAFe(III)formation; k), in the absence and presence of CO2, were deter-mined according to Eq. (1) [40]:

½Ml-trHbOAFeðIVÞ@O�t ¼ ½Ml-trHbOAFeðIVÞ@O�i � e�k�t ð1Þ

Values of the second-order rate constant for peroxynitritedetoxification by Ml-trHbOAFe(IV)@O (i.e., Ml-trHbOAFe(III) for-mation; kon), in the absence and presence of CO2, were determinedaccording to Eq. (2) [40]:

k ¼ kon � ½peroxynitrite� ð2Þ

The pKa value describing the pH dependence of kon for peroxyni-trite-mediated detoxification of Ml-trHbOAFe(IV)@O in the ab-sence of CO2 was obtained, at 20.0 �C, according to Eq. (3) [41–43]:

kon ¼ klimðtopÞ � 10�pH� �.

10�pH þ 10�pKa� �

þ klimðbottomÞ ð3Þ

where klim(top) represents the asymptotic value of kon under condi-tions where pH� pKa, and klim(bottom) represents the asymptoticvalue of kon under conditions where pH� pKa.

In some cases, bovine liver catalase was added to theMl-trHbOAFe(IV)@O solution prior the reaction with peroxynitriteto destroy excess H2O2. According to literature [30,31], catalase didnot affect peroxynitrite scavenging by Ml-trHbOAFe(IV)@O, in theabsence and presence of CO2.

The results are given as mean values of at least four experi-ments plus or minus the corresponding standard deviation. Alldata were analyzed using the GraphPad Prism (GraphPad SoftwareInc., La Jolla, CA, USA) and MatLab (The Math Works Inc., Natick,MA, USA) programs.

Results and discussion

Mixing Ml-trHbOAFe(IV)@O and peroxynitrite solutions, in theabsence and presence of CO2, causes a shift of the optical absorp-tion maximum of the Soret band (i.e., kmax) from 419 nm (i.e.,Ml-trHbOAFe(IV)@O) to 409 nm (i.e., Ml-trHbOAFe(III)) and achange of the extinction coefficient from e419 nm = 1.06 �105 M�1 cm�1 (i.e., Ml-trHbOAFe(IV)@O) to e409 nm = 1.15 �105 M�1 cm�1 (i.e., Ml-trHbOAFe(III)), at pH 7.2 and 20.0 �C.

Under all the experimental conditions (i.e., 6.2 6 pH 6 8.1,2.0 � 10�5 M 6 [peroxynitrite]6 2.0 � 10�4 M, [CO2] = 0 M or1.2 � 10�3 M, and T = 20.0 �C), the time course for peroxynitritedetoxification by Ml-trHbOAFe(IV)@O corresponds to a monopha-sic process between 360 nm and 460 nm (see Scheme 1 and Fig. 1).Values of the pseudo-first-order rate constant for peroxynitrite-mediated Ml-trHbOAFe(IV)@O reduction (i.e., Ml-trHbOAFe(III)formation; k) are wavelength-independent at fixed pH and perox-ynitrite concentration, in the absence and presence of CO2. Plots ofk versus peroxynitrite concentration are linear (Fig. 1); at pH 7.2,the slope corresponds to kon = 1.5 � 104 M�1 s�1, in the absenceof CO2, and 2.2 � 104 M�1 s�1, in the presence of CO2 (Fig. 1 andTable 1). Under all the experimental conditions, the y-axis inter-cept of plots of k versus peroxynitrite concentration is very closeto 0 s�1 within the experimental error (Fig. 1), allowing to treatthe reaction as virtually irreversible. As expected, decomposed per-oxynitrite neither affects the spectroscopic properties nor inducesthe reduction of Ml-trHbOAFe(IV)@O.

As shown in Fig. 1 and Table 1, values of kon for peroxynitritedetoxification by Ml-trHbOAFe(IV)@O increase on decreasing pHfrom 8.1 to 6.2 (2.0 � 10�1 M phosphate buffer), in the absenceof CO2. The analysis of data according to Eq. (3) allowed to deter-mine the following parameters: pKa = 6.7 ± 0.1, klim(top) =(4.4 ± 0.1) � 104 M�1 s�1, and klim(bottom) = (6.4 ± 0.1) � 103 M�1

s�1, at 20.0 �C. The pKa value for peroxynitrite detoxification by

0.0 0.5 1.0 1.5 2.00.00

0.25

0.50

0.75

1.00

a

b

Time (s)

Nor

mal

ized

am

plitu

de

0.0 0.5 1.0 1.5 2.00.00

0.25

0.50

0.75

1.00

a

b

Time (s)

Nor

mal

ized

am

plitu

de

0 100 200 300 4000.0

2.5

5.0

7.5

10.0

[Peroxynitrite] ×106 (M)

k (s

-1)

6.0 6.5 7.0 7.5 8.00

1

2

3

4

pH

k on×

10-4

(M-1

s-1)

Fig. 1. Kinetics of peroxynitrite-mediated reduction of Ml-trHbOAFe(IV)@O, at pH 7.2 and 20.0 �C. (A) Normalized time courses for peroxynitrite-mediated reduction of Ml-trHbOAFe(IV)@O, in the absence of CO2. The time course analysis according to Eq. (1) allowed to determine the following values of k = 7.5 � 10�1 s�1 (trace a) and 3.1 s�1

(trace b). Values of k were obtained at [peroxynitrite] = 5.0 � 10�5 M (trace a) and 2.0 � 10�4 M (trace b). (B) Normalized time courses for peroxynitrite-mediated reduction ofMl-trHbOAFe(IV)@O, in the presence of CO2. The time course analysis according to Eq. (1) allowed to determine the following values of k = 1.1 s�1 (trace a) and 4.3 s�1 (traceb). Values of k were obtained at [peroxynitrite] = 5.0 � 10�5 M (trace a) and 2.0 � 10�4 M (trace b). (C) Dependence of k on the peroxynitrite concentration, in the absence andpresence of CO2 (circles and squares, respectively). The analysis of data according to Eq. (2) allowed to determine kon = 1.5 � 104 M�1 s�1, in the absence of CO2 (circles), and2.2 � 104 M�1 s�1, in the presence of CO2 (squares). (D) pH dependence of kon for peroxynitrite-mediated reduction of Ml-trHbOAFe(IV)@O, in the absence and presence ofCO2 (circles and squares, respectively). The analysis of data according to Eq. (3) allowed to determine klim(top) = (4.4 ± 0.1) � 104 M�1 s�1, klim(bottom) = (6.4 ± 0.1) � 103 M�1 s�1,and pKa = 6.7 ± 0.1 for peroxynitrite-mediated reduction of Ml-trHbOAFe(IV)@O in the absence of CO2 (circles). Values of kon for peroxynitrite-mediated reduction of Ml-trHbOAFe(IV)@O, in the presence of CO2 (squares) are grossly pH-independent, the average kon value is 2.1 � 104 M�1 s�1. Where not shown, standard deviation is smallerthan the symbol. The Ml-trHbOAFe(IV)@O concentration was 2.7 � 10�6 M. The CO2 concentration was 1.2 � 10�3 M. For details, see text.

Table 1Values of kon for peroxynitrite detoxification by Ml-trHbOAFe(IV)@O, in the absenceand presence of CO2, at 20.0 �C.a

pH kon (M�1 s�1)

[CO2] = 0 M [CO2] = 1.2 � 10�3 M

6.2 3.4 � 104 1.9 � 104

6.3 3.3 � 104 2.1 � 104

6.5 2.9 � 104 2.3 � 104

6.7 2.4 � 104 2.2 � 104

6.8 2.3 � 104 1.8 � 104

7.0 1.8 � 104 2.6 � 104

7.2 1.5 � 104 2.2 � 104

7.5 1.1 � 104 2.1 � 104

7.7 1.0 � 104 2.0 � 104

7.9 8.2 � 103 2.2 � 104

8.1 8.0 � 103 1.7 � 104

a 2.0 � 10�1 M phosphate buffer.

394 P. Ascenzi et al. / Biochemical and Biophysical Research Communications 380 (2009) 392–396

Ml-trHbOAFe(IV)@O in the absence of CO2 (=6.7 ± 0.1) correspondsto that reported for the ONOOH M ONOO� equilibrium (=6.5–6.8)(see [14]). Therefore, klim(top) should represent kon forMl-trHbOAFe(IV)@O reduction to Ml-trHbOAFe(III) by ONOOH atpH� pKa, while klim(bottom) should be referred to kon forMl-trHbOAFe(IV)@O reduction to Ml-trHbOAFe(III) by ONOO� atpH� pKa. In this respect, the reaction mechanisms proposed forheme-Fe(IV)@O reduction to heme-Fe(III) by ONOOH (i.e., atpH� pKa) [37–39,44] are represented by Schemes 2 and 3.

On the other hand, the reaction mechanism proposed for heme-Fe(IV)@O reduction to heme-Fe(III) by ONOO� (i.e., at pH� pKa)[37–39,44] is represented by Scheme 4.

Remarkably, klim(top) exceeds klim(bottom) by about one order ofmagnitude (i.e., klim(top)/klim(bottom) = 6.8), similarly to what ob-served for horse heart Mb [37]. In addition, values of kon for Ml-trHbOAFe(IV)@O reduction to Ml-trHbOAFe(III) are similar tothose reported for Glycine max leghemoglobin (Lb), horse heartMb, and human Hb (Table 2) [37–39].

As shown in Fig. 1 and Table 1, values of kon for peroxyni-trite detoxification by Ml-trHbOAFe(IV)@O in the presence ofCO2 do not show a clear pH dependence (the average kon valueis 2.1 � 104 M�1 s�1), as also reported for horse heartMb-Fe(IV)@O and human Hb-Fe(IV)@O [37,38]. The lack of apH effect finds the explanation on the basis of the reactionmechanism proposed for heme-Fe(IV)@O reduction to heme-Fe(III) by ONOOH/ONOO� in the presence of CO2 [37,38,44]and represented by Scheme 5.

Indeed, the reduction of heme-Fe(IV)@O to heme-Fe(III) occursupon the reaction with �NO2, which represents the rate-limitingstep of the whole process. Thus, on the basis of Scheme 5, the for-mation of �NO2 does not depend on the ONOOH M ONOO� equilib-rium (and thus on pH), but instead on the CO2 concentration[37,38,44].

Also in the presence of CO2, values of kon for Ml-trHbOAFe(IV)@Oreduction to Ml-trHbOAFe(III) are similar to those reported forGlycine max Lb, horse heart Mb, and human Hb (Table 2) [37–39].

Table 2Values of kinetic parameters for peroxynitrite detoxification by ferryl and ferrousoxygenated heme-proteins (in italics and bold, respectively; see Schemes 1 and 6,respectively).

Heme-protein [CO2] (M) hon (M�1 s�1) kon (M�1 s�1)

M. leprae trHbO 0a — 1.5 � 104a

1.2 � 10�3a — 2.2 � 104a

0b 4.8 � 104b 1.3 � 104b

1.2 � 10�3b 6.3 � 105b 1.7 � 104b

Glycine max Lbc 0 — 3.4 � 104

1.2 � 10�3 — 2.3 � 105

0 5.5 � 104 2.1 � 104

1.2 � 10�3 8.8 � 105 3.6 � 105

Horse heart Mb 0d — 1.9 � 104d

2.5 � 10�3e — 2.8 � 104e

0e 5.4 � 104e 2.2 � 104e

2.5 � 10�3e 3.1 � 105e 3.2 � 104e

Human Hbf 0 — 3.8 � 104

1.2 � 10�3 — 2.5 � 105

0 3.3 � 104 3.3 � 104

1.2 � 10�3 3.5 � 105 1.1 � 105

a pH 7.2 and 20.0 �C. Present study.b pH 7.3 and 20.0 �C. From [21].c pH 7.3 and 20.0 �C. From [39].d pH 7.5 and 20.0 �C. From [37].e pH 7.3 and 20.0 �C. From [37].f pH 7.4 and 20.0 �C. From [38].

NO3− + H+

ONOOH

•NO2 + •OH

heme-Fe(IV)=O + •NO2 heme-Fe(III)-OONO heme-Fe(III) + NO3−

Scheme 3.

± H+

ONOOH ONOO− + CO2 ONOOC(O)O

heme-Fe(IV)=O + •NO2 heme-Fe(II

Scheme

heme-Fe(IV)=O + ONOOH + H+ heme-Fe(III) + ONOO• + H2O

ONOO• •NO + O2

Scheme 2.

heme-Fe(IV)=O + ONOO− + H+ heme-Fe(III) + ONOO• + OH−

ONOO• •NO + O2

Scheme 4.


The heme-Fe(III)AOONO transient species (see Schemes 3 and5) has been previously demonstrated to be generated by the reac-tion of Ml-trHbOAFe(II)AO2 with �NO, which is then followed bythe decay of Ml-trHbOAFe(III)AOONO to Ml-trHbOAFe(III) andNO3

� [20].It is important to outline that values of kon for the peroxynitrite

detoxification by Ml-trHbOAFe(IV)@O, in the absence and presenceof CO2, determined here (see Scheme 1, Fig. 1, and Table 1) are inagreement with those reported in the literature [9,21] for the sec-ond step of peroxynitrite scavenging by Ml-trHbOAFe(II)AO2 (i.e.,values of kon given in Scheme 6; see Table 2), as reported for Glycinemax Lb, horse heart Mb, and human Hb (Table 2) [37–39].

This agreement reinforces the idea that we are actually measur-ing the rate constants for individual steps reported in Schemes 2–5;furthermore, the catalytic parameters for peroxynitrite detoxifica-tion by Ml-trHbOAFe(IV)@O in the absence and presence of CO2

are high enough to indicate that this reaction indeed could occurin vivo. However, in contrast to peroxynitrite scavenging byMl-trHbOAFe(II)AO2 (Scheme 6) [9,21], peroxynitrite detoxificationby Ml-trHbOAFe(IV)@O (Scheme 1) does not require partneroxido-reductive enzyme(s). Actually, Ml-trHbOAFe(III) oxidationto Ml-trHbOAFe(IV)@O is mediated by H2O2 [25], andMl-trHbOAFe(IV)@O reduction to Ml-trHbOAFe(III) is facilitatedby peroxynitrite (see Scheme 1, Fig. 1, and Tables 1 and 2), envisag-ing a short cycle between heme-Fe(IV)@O and heme-Fe(III) operatedthrough peroxynitrite without the necessity of a reductase(s). In thisframework, it becomes comprehensible why Ml-trHbOAFe(III)could not require a reductase system(s), which indeed has not yetbeen identified in this elusive mycobacterium [9,23].

NO3− + CO2

− ONOOC(O)O−

•NO2 + CO3•−

I)-OONO heme-Fe(III) + NO3−

5.

+ CO+ peroxynitrite + peroxynitrite 2 + CO2

hon kon

Ml-trHbO-Fe(II)-O2 + peroxynitrite Ml-trHbO-Fe(IV)=O + peroxynitrite Ml-trHbO-Fe(III)

reductase(s) + O2

Scheme 6.


As a whole, H2O2-induced Ml-trHbOAFe(IV)@O could be relevantfor M. leprae survival in vivo in the presence not only of �NO and NO2

�

[25] but also of peroxynitrite (present study), in the absence of asuitable reductase system(s) facilitating Ml-trHbOAFe(III) andMl-trHbOAFe(II) formation. Furthermore, as reported for �NO andNO2

� [25], peroxynitrite acts as an antioxidant (present study) pre-venting the Ml-trHbOAFe(IV)@O-mediated oxidation of myco-bacterial (macro)molecules such as membrane lipids (i.e., lipidperoxidation).

Acknowledgments

This work was partially supported by grants from the Ministryfor Education, University, and Research of Italy (Department ofBiology, University Roma Tre, Roma, Italy, ‘CLAR 2008’ to P.A.)and from the Ministry of Health of Italy (National Institute forInfectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, Italy, ‘Ric-erca corrente 2008’ to P.A.).

References

[1] S.J. Klebanoff, C.C. Shepard, Toxic effect of the peroxidase-hydrogenperoxidehalide antimicrobial system on Mycobacterium leprae, Infect. Immun.44 (1984) 534–536.

[2] W.R. Winrow, P.G. Winyard, C.J. Morris, D.R. Blake, Free radicals ininflammation: second messengers and mediators of tissue destruction, Br.Med. Bull. 49 (1993) 506–522.

[3] J. MacMicking, Q.W. Xie, C. Nathan, Nitric oxide and macrophage function,Annu. Rev. Immunol. 15 (1997) 323–350.

[4] C. Ratledge, J. Dale (Eds.), Mycobacteria Molecular Biology and Virulence,Blackwell Science, Oxford, 1999.

[5] C. Nathan, M.U. Shiloh, Reactive oxygen and nitrogen intermediates in therelationship between mammalian hosts and microbial pathogens, Proc. Natl.Acad. Sci. USA 97 (2000) 8841–8848.

[6] A.M. Cooper, L.B. Adams, D.K. Dalton, R. Appelberg, S. Ehlers, IFN-c and NO inmycobacterial disease: new jobs for old hands, Trends Microbiol. 10 (2002)221–226.

[7] P. Visca, G. Fabozzi, M. Milani, M. Bolognesi, P. Ascenzi, Nitric oxide andMycobacterium leprae pathogenicity, IUBMB Life 54 (2002) 95–99.

[8] D.M. Scollard, L.B. Adams, T.P. Gillis, J.L. Krahenbuhl, R.W. Truman, D.L.Williams, The continuing challenges of leprosy, Clin. Microbiol. Rev. 19 (2006)338–381.

[9] P. Ascenzi, P. Visca, Scavenging of reactive nitrogen species by mycobacterialtruncated hemoglobins, Methods Enzymol. 436 (2008) 317–337.

[10] D.M. Scollard, The biology of nerve injury in leprosy, Lepr. Rev. 79 (2008) 242–253.[11] S.V. Lymar, J.K. Hurst, Rapid reaction between peroxynitrite ion and carbon

dioxide: implications for biological activity, J. Am. Chem. Soc. 117 (1995)8867–8868.

[12] J.S. Beckman, W.H. Koppenol, Nitric oxide, superoxide, and peroxynitrite: thegood, the bad, and the ugly, Am. J. Physiol. 271 (1996) C1424–C1437.

[13] P. Ascenzi, A. Bocedi, P. Visca, M. Minetti, E. Clementi, Does CO2 modulateperoxynitrite specificity?, IUBMB Life 58 (2006) 611–613

[14] S. Goldstein, J. Lind, G. Merényi, Chemistry of peroxynitrites andperoxynitrates, Chem. Rev. 105 (2005) 2457–2470.

[15] S. Herold, A. Fago, Reactions of peroxynitrite with globin proteins and theirpossible physiological role, Comp. Biochem. Physiol. A Mol. Integr. Physiol. 142(2005) 124–129.

[16] S. Goldstein, G. Merényi, The chemistry of peroxynitrite: implications forbiological activity, Methods Enzymol. 436 (2008) 49–61.

[17] P.C. Andrews, N.I. Krinsky, A kinetic analysis of the interaction of humanmyeloperoxidase with hydrogen peroxide, chloride ions, and protons, J. Biol.Chem. 257 (1982) 13240–13245.

[18] H.B. Dunford, Heme Peroxidases, Wiley-VCH, New York, 1999.

[19] S.J. Klebanoff, Myeloperoxidase: friend and foe, J. Leukoc. Biol. 77 (2005) 598–625.[20] P. Ascenzi, A. Bocedi, M. Bolognesi, G. Fabozzi, M. Milani, P. Visca, Nitric oxide

scavenging by Mycobacterium leprae GlbO involves the formation of the ferricheme-bound peroxynitrite intermediate, Biochem. Biophys. Res. Commun. 339(2006) 450–456.

[21] P. Ascenzi, M. Milani, P. Visca, Peroxynitrite scavenging by ferrous truncatedhemoglobin GlbO from Mycobacterium leprae, Biochem. Biophys. Res.Commun. 351 (2006) 528–533.

[22] G. Fabozzi, P. Ascenzi, S. Di Renzi, P. Visca, Truncated hemoglobin GlbO fromMycobacterium leprae alleviates nitric oxide toxicity, Microb. Pathog. 40 (2006)211–220.

[23] P. Ascenzi, M. Bolognesi, M. Milani, M. Guertin, P. Visca, Mycobacterialtruncated hemoglobins: from genes to functions, Gene 398 (2007) 42–51.

[24] P. Ascenzi, M. Bolognesi, P. Visca, �NO dissociation represents the rate limitingstep for O2-mediated oxidation of ferrous nitrosylated Mycobacterium lepraetruncated hemoglobin O, Biochem. Biophys. Res. Commun. 357 (2007) 809–814.

[25] P. Ascenzi, E. De Marinis, M. Coletta, P. Visca, H2O2 and �NO scavenging byMycobacterium leprae truncated hemoglobin O, Biochem. Biophys. Res.Commun. 373 (2008) 197–201.

[26] E. De Marinis, L. Casella, C. Ciaccio, M. Coletta, P. Visca, P. Ascenzi, Catalyticperoxidation of nitrogen monoxide and peroxynitrite by globins, IUBMB Life61 (2009) 62–73.

[27] A.J. Gow, B.P. Luchsinger, J.R. Pawloski, D.J. Singel, J.S. Stamler, Theoxyhemoglobin reaction of nitric oxide, Proc. Natl. Acad. Sci. USA 96 (1999)9027–9032.

[28] M. Brunori, Nitric oxide moves myoglobin centre stage, Trends Biochem. Sci.26 (2001) 209–210.

[29] A. Bonamore, A. Boffi, Flavohemoglobin: structure and reactivity, IUBMB Life60 (2008) 19–28.

[30] S. Herold, F.J. Rehmann, Kinetic and mechanistic studies of the reactions ofnitrogen monoxide and nitrite with ferryl myoglobin, J. Biol. Inorg. Chem. 6(2001) 543–555.

[31] S. Herold, F.J. Rehmann, Kinetics of the reactions of nitrogen monoxide andnitrite with ferryl hemoglobin, Free Radic. Biol. Med. 34 (2003) 531–545.

[32] S. Herold, A. Puppo, Kinetics and mechanistic studies of the reactions ofmetleghemoglobin, ferrylleghemoglobin, and nitrosylleghemoglobin withreactive nitrogen species, J. Biol. Inorg. Chem. 10 (2005) 946–957.

[33] P. Visca, G. Fabozzi, A. Petrucca, C. Ciaccio, M. Coletta, G. De Sanctis, M. Milani,M. Bolognesi, P. Ascenzi, The truncated hemoglobin from Mycobacteriumleprae, Biochem. Biophys. Res. Commun. 294 (2002) 1064–1070.

[34] H. Østdal, B. Daneshvar, L.H. Skibsted, Reduction of ferrylmyoglobin by b-lactoglobulin, Free Radic. Res. 24 (1996) 429–438.

[35] D.S. Bohle, P.A. Glassbrenner, B. Hansert, Syntheses of puretetramethylammonium peroxynitrite, Methods Enzymol. 269 (1996) 302–311.

[36] W.H. Koppenol, R. Kissner, J.S. Beckman, Syntheses of peroxynitrite: to go withthe flow or on solid grounds?, Methods Enzymol 269 (1996) 296–302.

[37] S. Herold, M. Exner, F. Boccini, The mechanism of the peroxynitrite-mediatedoxidation of myoglobin in the absence and presence of carbon dioxide, Chem.Res. Toxicol. 16 (2003) 390–402.

[38] F. Boccini, S. Herold, Mechanistic studies of the oxidation of oxyhemoglobin byperoxynitrite, Biochemistry 43 (2004) 16393–16404.

[39] S. Herold, A. Puppo, Oxyleghemoglobin scavenges nitrogen monoxide andperoxynitrite: a possible role in functioning nodules?, J Biol. Inorg. Chem. 10(2005) 935–945.

[40] E. Antonini, M. Brunori, Hemoglobin and Myoglobin in their Reactions withLigands, North-Holland Publishing Co., Amsterdam, 1971.

[41] S. Pfeiffer, A.C. Gorren, K. Schmidt, E.R. Werner, B. Hansert, D.S. Bohle, B. Mayer,Metabolic fate of peroxynitrite in aqueous solutions. Reaction with nitricoxyde and pH-dependent decomposition to nitrite and oxygen in a 2:1stoichiometry, J. Biol. Chem. 272 (1997) 3465–3470.

[42] S. Herold, K. Shivashankar, Metmyoglobin and methemoglobin catalyzethe isomerization of peroxynitrite to nitrate, Biochemistry 42 (2003)14036–14046.

[43] S. Herold, S. Kalinga, T. Matsui, Y. Watanabe, Mechanistic studies of theisomerization of peroxynitrite to nitrate catalyzed by distal histidinemetmyoglobin mutants, J. Am. Chem. Soc. 126 (2004) 6945–6955.

[44] M. Exner, S. Herold, Kinetic and mechanistic studies of the peroxynitrite-mediated oxidation of oxymyoglobin and oxyhemoglobin, Chem. Res. Toxicol.13 (2000) 287–293.

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Review

Nuclear receptors CAR and PXR: Molecular, functional, andbiomedical aspects

Alessandra di Masi a,1, Elisabetta De Marinis a,1, Paolo Ascenzi a,b, Maria Marino a,*

a Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italyb National Institute for Infectious Diseases I.R.C.C.S. ‘‘Lazzaro Spallanzani”, Via Portuense 292, I-00149 Roma, Italy


Article history:Received 7 April 2009Accepted 28 April 2009

Keywords:XenosensorsXenobioticsConstitutive androstane receptorPregnane X receptorStructureFunction

a b s t r a c t

Nuclear receptors (NRs) are ligand-activated transcription factors sharing a common evo-lutionary history and having similar sequence features at the protein level. Selectiveligand(s) for some NRs is not known, therefore these NRs have been named ‘‘orphan recep-tors”. Whenever ligands have been recognized for any of the orphan receptor, it has beencategorized and grouped as ‘‘adopted” orphan receptor. This group includes the constitu-tive androstane receptor (CAR) and the pregnane X receptor (PXR). They function as sen-sors of toxic byproducts derived from endogenous metabolites and of exogenouschemicals, in order to enhance their elimination. This unique function of CAR and PXR setsthem apart from the steroid hormone receptors. The broad response profile has establishedthat CAR and PXR are xenobiotic sensors that coordinately regulate xenobiotic clearance inthe liver and intestine via induction of genes involved in drug and xenobiotic metabolism.In the past few years, research has revealed new and mostly unsuspected roles for CAR and

Abbreviations: 1,24,25(OH)2D3, 1,24,25-trihydroxyvitamin D3; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 25(OH)D3, 25-hydroxyvitamin D3; ABC, ATP-binding cassette; AhR, aryl hydrocarbon receptor; Aldh, aldehyde dehydrogenase; APAP, acetaminophen; AR, androgen receptor; ASC, apoptotic speckprotein; bp, base pair; CAR, constitutive androstane receptor; CCRP, CAR cytoplasmic retention protein; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; Colupulone, 3,5-dihydroxy-4,6,6-tris(3-methylbut-2-en-1-yl)-2-(2-methylpropanoyl)cyclo-hexa-2,4-dien-1-one; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; CPT, carnitine palmitoyltransferase; CYP, cytochrome P450;DBD, DNA-binding domain; dr, direct repeat; DSS, dextran sulphate sodium; E2, 17b-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; er,everted repeat; ERR, estrogen-related receptor; FoxO, insulin-responsive transcription factor; FTZ-F1, ‘‘fushi tarazu”-factor 1 receptor; FXR, farnesoid Xreceptor; G6Pase, glucose-6-phosphatase; GCNF, germ cell nuclear factor; GR, glucocorticoid receptor; GRIP, glucocorticoid receptor-interacting protein;GST, glutathione-S-transferase; GSTM, glutathione-S-transferase murine; H region, hinge region; hCAR, human CAR; HMGCS, 3-hydroxy-3-methylglutarate-CoA synthase; HNF4, hepatocyte nuclear factor 4; hPXR, human PXR; HRE, hormone response element; Hsp, heat shock protein; IBD, inflammatory boweldisease; kb, kilobase; LBD, ligand-binding domain; LPS, lipopolysaccharide; LXR, liver X receptor; MAPK, mitogen-activated protein kinase; mCAR, murineCAR; MDR, multidrug resistant protein; mPXR, murine PXR; MR, mineralocorticoid receptor; MRP, multidrug resistance-related protein; NAPQI, N-acetyl-p-benzo-quinone imine; NcoR, nuclear receptor co-repressor; NF-jB, nuclear factorjB; NGF1B, nerve growth factor inducible factor 1-B group; NLS, nuclearlocalization sequence; NR, nuclear receptor; OATP, organic anion transport protein; PB, phenobarbital; PBP, PPAR-binding protein; PBREM, phenobarbital-responsive enhancer module; PCN, pregnenolone 16a-carbonitrile; PEPCK, phosphoenolpyruvate carboxykinase; PGC, PPAR gamma co-activator; P-gp, P-glycoprotein; PKA, protein kinase A; PKC, protein kinase C; PP2A, phosphatase 2A; PPAR, peroxisome proliferator-activated receptor; Por, P450 oxidoreductase; PR, progesterone receptor; PXR, pregnane X receptor; RAR, retinoic acid receptor; RE, response element; RIP140, receptor-interacting protein140; RXR, retinoic acid X receptor; SCD, stearoyl-CoA desaturase; SF-1, steroidogenic factor-1; SH2, Src homology 2 domain; SHP, short heterodimerpartner; SMC, structural maintenance of chromosome; SMRT, silencing mediator of retinoid and thyroid receptors; SNP, single nucleotide polymorphism;SOD, superoxide dismutase; SR12813, [2-(3,5-di-tert-butyl-4-hydroxy-phenyl)-1-(diethoxy-phosphoryl)-vinyl]-phosphonic acid diethlyl ester; Src, aviansarcoma virus; SRC-1, steroid receptor co-regulator-1; SRC-1p1, steroid receptor co-regulator-1 peptide-1,ERHKILHRLLQEG; SRC-1p2, steroid receptor co-regulator-1 peptide-2, CPSSHSSLTERHKILHRLLQEGSPS; SRC-1p3, steroid receptor co-regulator-1 peptide-3,SLTERHKILHRLLQE; SREBP, sterol regulatoryelement-binding protein; SULT, sulfotransferase; T0901317, N-(2,2,2-trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}ben-zenesulfonamide; TCPOBOP, 3,5- dichloro-2-{4-[(3,5-dichloropyridin-2yl)oxy]phenoxy}pyridine; TH, thyroid hormone; THR, thyroid hormone receptor;TIF2, transcription intermediary factor 2; TIF2p, transcription intermediary factor 2 peptide AKENALLRYLLDKDDTKD; UGT, uridine diphosphate-glucuronyltransferase; VDR, vitamin D receptor; XREM, xenobiotic-responsive enhancer module.

0098-2997/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.mam.2009.04.002

* Corresponding author. Tel.: +39 06 57336345; fax: +39 06 57336321.E-mail address: [email protected] (M. Marino).

1 These authors contributed equally to this work.

Molecular Aspects of Medicine 30 (2009) 297–343


Molecular Aspects of Medicine

journal homepage: www.elsevier .com/locate /mam


Action mechanismsBio-medical aspects

PXR in modulating hormone, lipid, and energy homeostasis as well as cancer and liver ste-atosis. The purpose of this review is to highlight the structural and molecular bases of CARand PXR impact on human health, providing information on mechanisms through whichdiet, chemical exposure, and environment ultimately impact health and disease.

� 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2981.1. Who’s who. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2981.2. Evolutionary aspects of CAR and PXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

2. CAR and PXR structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3052.1. Gene organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

2.1.1. Human CAR gene: structure, polymorphisms and transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3052.1.2. Human PXR gene: structure, polymorphisms and transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

2.2. Protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3082.2.1. The primary structure of CAR and PXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3092.2.2. The architecture of CAR-DBD and PXR-DBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3092.2.3. The architecture of CAR-LBD and PXR-LBD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

3. CAR and PXR expression and activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3193.1. CAR and PXR expression patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3193.2. CAR and PXR ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3193.3. CAR and PXR activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

3.3.1. CAR activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3223.3.2. PXR activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

3.4. Activation of NRs: the case of ERs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3244. CAR and PXR functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

4.1. Detoxification by CAR and PXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3264.2. Role of CAR in bilirubin metabolism and heme biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3274.3. Role of CAR and PXR in bile acid homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3284.4. Role of CAR and PXR in steroid and thyroid hormone homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3294.5. Role of CAR and PXR in gluconeogenesis and lipid metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3304.6. Functional interplay between CAR/PXR and NRs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

5. From bench to bedside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3315.1. PXR-mediated drug–drug interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

5.1.1. Decreased drug efficacy by PXR-enhanced catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3315.1.2. Increased drug toxicity by PXR-mediated metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

5.2. PXR-mediated metabolic bone disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3325.3. PXR-mediated hepatic steatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3335.4. PXR role in inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3335.5. PXR role in cancer and chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3345.6. Miscellaneous implications of PXR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

5.6.1. PXR and antifibrogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3345.6.2. PXR and the oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

6. Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

1. Introduction

1.1. Who’s who

Living organisms are severely xenophobes in that they have an aversion to xenobiotics (from Greek, nemo1 ‘‘foreign” andbio1 ‘‘life”). This term was coined to cover all organic compounds that were foreign in the organism under study. The historyof xenobiotics is interwoven with the birth of organic chemistry. In the early 1800s, the prevailing belief was that the com-position of the human body, and indeed of all living things, was the result of a ‘‘vital force” or ‘‘internal flame” and that mere

298 A. di Masi et al. / Molecular Aspects of Medicine 30 (2009) 297–343


mortals were incapable of understanding these workings and would be particularly unable to synthesize constituent com-pounds of the human body (Murphy, 2001). Faced with this daunting concept, scientists were discouraged from thinkingabout the processes that dominated everyday digestion and nutrition. In 1828, Wöhler was able to shatter this myth ofsynthesizing urea, one of the compounds that he had examined in his studies on the urinary waste products, and sentthe following note to Berzelius ‘‘I must tell you that I can prepare urea without requiring a kidney of an animal, eitherman or dog” (Whöler and Frerichs, 1848). In modern-day terminology, this was a true ‘‘paradigm shift” and led to revisedthinking on what chemists could accomplish with regard to ‘‘organic” compounds. Wöhler test led to the first metabolismexperiments performed in 1841 by Ure who demonstrated the conjugation of benzoic acid with glycine in man (Ure, 1841;Keller, 1842). The transformation of compounds such as cinnamic acid (Erdmann and Marchand, 1842) and benzaldehyde(Whöler and Frerichs, 1848) to benzoic acid, further emphasized by the discovery of the conversion of benzene to phenol(Schultzen and Naunyn, 1867), indicated the ability of the body to oxidize ingested compounds. A rapid development ofchemical industries occurred soon after World War II, resulting in the manufacturing of a large number of chemical products.The increased use of fertilizers, insecticides, and herbicides led to a dramatic increase in world food production. This coupledwith improved medicine and pharmacological science helped to improve general public health rising life expectancy to anaverage of 75 years (Yu, 2001). Whereas many people of the world were enjoying the benefits of technological and economic

Fig. 1. Chemical structures of some CAR and PXR ligands.

A. di Masi et al. / Molecular Aspects of Medicine 30 (2009) 297–343 299


expansion and higher living standards, others perceived that these extraordinary developments were not without cost.Indeed, the impact of global environmental changes on human health has become a great concern. As early as the 1950sand 1960s, many urban dwellers and residents in the vicinity of industrial factories began to recognize undesirable changesin the environment (Yu, 2001).

Fig. 1 (continued)



Now it is clear that after a chemical pollutant gets into a mammalian organism, chemical reactions occur within the body,altering its reactivity and structural properties. The tome ‘‘Detoxication Mechanisms” by Williams (1947) heralded the birthof xenobiotic/drug metabolism as a distinct branch of science, providing a systematic approach based on the organic chem-istry classification. Williams also expanded on his concepts of the principal biochemical reactions whereby drugs and otherforeign compounds are metabolized in the body. Most importantly, he proposed that foreign compounds were metabolizedin two distinct phases: one including oxidation, reduction, and hydrolysis processes, and the other involving conjugationreactions.

In the early 1950s, biochemical research unveiled the mechanisms of a wide variety of transformation reactions. The pio-neering studies of James and Elizabeth Miller and Axelrod revealed the subcellular localization of metabolism reactions andbrought to the identification of enzymes responsible for catalytic transformations (Mueller and Miller, 1949; Axelrod, 1955).

A major class of catalytic oxidative transformations was initially characterized in 1955 by Hayashi and Mason. Since thesereactions need an oxidant (molecular oxygen) and a reductant (NAD(P)H), the enzymes catalyzing these processes were triv-ially named ‘‘mixed-function oxidases” (Hayashi et al., 1955; Mason et al., 1955).

The understanding of the biochemical nature of chemical transformations occurring in living organisms grew out fromearly studies on the liver pigments by Garfinkel and Klingenberg in 1958. These researchers observed in liver microsomesan unusual carbon monoxide-binding pigment with an absorbance maximum at 450 nm (Garfinkel, 1958; Klingenberg,1958). This pigment was ultimately characterized as a cytochrome by Omura and Sato (1962). The function of this uniquecytochrome (called P450; CYP) was initially revealed in 1963 by Estabrook, Cooper, and Rosenthal, while investigatingthe role of microsomes from the adrenal cortex in the catalytic conversion (i.e., hydroxylation) of 17-hydroxyprogesteroneto deoxycorticosterone (Estabrook et al., 1963). In the clinical setting, the discovery of the CYP polymorphism role in debr-isoquine and sparteine metabolism emphasized the importance of individual response to drugs (Thomas et al., 1976;Mahgoub et al., 1977; Eichelbaum et al., 1978). At present, 18 CYP families have been identified in mammals, among whichonly three are primarily responsible for most xenobiotic metabolism. Determining the molecular bases for the xenobiotic-mediated induction of CYP gene expression has become one of the most challenging dilemmas of modern toxicology(Dickins, 2004; Xu et al., 2005; Nakata et al., 2006; Plant, 2007; Graham and Lake, 2008).

Thus, the threat represented by xenobiotics for the organism is the target of a complex strategy. Metabolism of drugs andother xenobiotics in the liver is the body primary defence against accumulation of potentially toxic lipophilic compounds.This strategy comprises xenosensors, dedicated to the recognition of xenobiotics and dangerous endogenous molecules,as well as xenobiotic metabolizing and transporters systems. The first step of the detoxification mechanism is the xenobioticdetection by the constitutive androstane receptor (CAR) and/or the pregnane X receptor (PXR), both belonging to the nuclearreceptor (NR) super-family (Fuhr, 2000; Dixit et al., 2005; Chang and Waxman, 2006; Moreau et al., 2008), and/or by the arylhydrocarbon receptor (AhR), a member of the Per/ARNT/Sim (PAS) family of transcription factors (Francis et al., 2003).

CAR and PXR are activated by a variety of endogenous (i.e., steroids and bile acid salts), and exogenous ligands includingdrugs, insecticides, pesticides, and nutritional compounds (Fig. 1). Currently, it remains uncertain whether these receptorsdisplay a high ligand specificity or instead function as more generalized steroid/xenobiotic sensors. These xenosensors coor-dinate the expression of several genes encoding CYPs. Both CAR and PXR are able to elicit alterations in xeno- and endo-bioticmetabolism, regulating the expression of phases I and II metabolizing enzymes and phase III transporters (Fig. 2) (Maticet al., 2007; Plant, 2007; Timsit and Negishi, 2007; Tompkins and Wallace, 2007; Graham and Lake, 2008; Lim and Huang,2008; Ma and Lu, 2008; Moreau et al., 2008; Pascussi et al., 2008; Teng and Piquette-Miller, 2008; Köhle and Bock, 2009;Zhou et al., 2009). Although CAR and PXR play physiological roles (e.g., regulate cholesterol elimination pathways), no phys-iological ligands have been identified, therefore they still remain orphan NRs (Timsit and Negishi, 2007).

In contrast, AhR ligands are the hydrophobic environmental pollutants of polyhalogenated aromatic hydrocarbons,such as polychlorinated dibenzodioxins, dibenzofurans and coplanar biphenyls or polycyclic aromatic hydrocarbons

Fig. 2. Schematic illustration of phases I, II, and III of xenobiotic metabolizing systems. For details, see the text.



(e.g., benzo[a]pyrene, 3-methylcholanthrene, benzoflavones, and omeprazole) (Francis et al., 2003; Nguyen and Bradfield,2008; Gomez-Duran et al., 2009; Hahn et al., 2009).

Human CAR (hCAR), initially identified as the orphan receptor MB67 (Baes et al., 1994), was found as a heterodimer part-ner for the retinoic acid X receptor (RXR) (see Tompkins and Wallace, 2007; Graham and Lake, 2008; Lim and Huang, 2008;Ma and Lu, 2008; Moreau et al., 2008; Pascussi et al., 2008; Teng and Piquette-Miller, 2008; Köhle and Bock, 2009; Zhouet al., 2009). hCAR was able to bind to retinoic acid response elements (REs), but the extent of activation was much lowerthan that mediated by RXR. The isolation and characterization of mouse CAR (mCAR) was reported in 1997 (Choi et al.,1997); at that time, it was hypothesized to target a subset of retinoid acid REs, but its role in CYP regulation has not yet beenrealized (Stanley et al., 2006).

Meanwhile, separate lines of investigation were leading to the identification of mouse PXR (mPXR) that was identified in1998 by using an expressed sequence tag to screen a mouse liver library (Kliewer et al., 1998). mPXR was named ‘‘pregnaneX receptor” since it was shown to be activated by derivatives of dexamethasone and pregnenolone (Kliewer et al., 1998).

At approximately the same time, the human steroid X receptor was cloned in studies focused on the identification of theXenopus laevis benzoate X receptor homologues. The human steroid X receptor was shown to be related to CAR and to thevitamin D receptor (VDR) (Blumberg et al., 1998), and then it was established to be the human homologue of mPXR(Lehmann et al., 1998). A parallel computer modeling approach confirmed that the human steroid X receptor is thehuman homologue of mPXR (Bertilsson et al., 1998). According to the literature (Bertilsson et al., 1998), the human steroidX receptor has been renamed human PXR (hPXR).

One of the most intriguing hypotheses to justify the existence of NR xenosensors was built on the basis of studies dealingwith the broad implications of these receptors in steroid hormone homeostasis. It is intriguing to hypothesize that an ances-tral protein could act as the endogenous metabolite (endobiotics) sensor. Starting from this hypothetical receptor progenitor,evolution proceeded by ligand exploitation and serial genome expansions. If this hypothesis is true, the conceptual distancebetween specific endogenous hormones and exogenous environmental molecules is probably closer than previously thought(Buters et al., 1994; Zhai et al., 2007).

1.2. Evolutionary aspects of CAR and PXR

NRs form a super-family of ligand-activated transcription factors implicated in various physiological functions fromdevelopment to homoeostasis (Escriva et al., 2004; Ascenzi et al., 2006). NRs share a common evolutionary history as re-vealed by their conserved structure and by their high degree of sequence conservation (Escriva et al., 2004). The phylogeneticanalysis of the NR super-family brought to its sub-division into six sub-families of unequal size (Fig. 3): (i) the large sub-family I contains the thyroid hormone receptors (THRs), the retinoic acid receptors (RARs), VDR, the ecdysone receptor,the peroxisome proliferator-activated receptors (PPARs), as well as numerous orphan receptors including CAR and PXR;(ii) the sub-family II includes RXR, the chicken ovalbumin upstream promoter-transcription factor (COUP-TF), and thehepatocyte nuclear factor 4 (HNF4); (iii) the steroid receptor sub-family III includes the estrogen receptors (ERs), theestrogen-related receptors (ERRs), the glucocorticoid receptors (GRs), the mineralocorticoid receptor (MR), the progesteronereceptor (PR), as well as the androgen receptors (ARs); (iv) the sub-family IV contains the nerve growth factor inducible I-Bgroup of orphan receptors (NGF1B); (v) the sub-family V includes the steroidogenic factor-1 (SF-1) and the Drosophilamelanogaster ‘‘fushi tarazu” factor-1 receptor (FTZ-F1); and (vi) the small sub-family VI contains only the germ cell nuclearfactor-1 receptor (GCNF) (Laudet et al., 1999; Escriva et al., 2004; Ascenzi et al., 2006).

A hypothetical evolutionary path that might have been taken by the first NR in the early Metazoan is represented in Fig. 3.Since several sub-families were present in early Metazoan, it appears that the super-family underwent an ‘‘explosive expan-sion” during early Metazoan evolution. The diversification of the super-family followed two waves of gene duplication. Thefirst wave before the Protostome/Deuterostome split, during the emergence of Metazoan, led to the acquisition of the pres-ent six sub-families and the various groups of receptors within each sub-family. The second wave occurred after the arthro-pod/vertebrate split, specifically in vertebrates, producing the paralogous groups within each sub-family (see Laudet et al.,1999; Escriva et al., 2004; Ascenzi et al., 2006). In support of NR paralogous through genome or block duplications, mappingstudies have demonstrated the presence of extensive ‘‘paralogy groups” which include NRs on different chromosomes (Owenand Zelent, 2000).

The first NR arose as one unit consisting of the currently recognized DNA-binding domain (DBD) and ligand-binding do-main (LBD), based on the finding that the two domains existed together in lower Metazoan. However, such observation doesnot entirely dismiss the possibility that the two regions existed independently earlier in evolution, despite the fact thatstrong similarity to either domain has not yet been observed outside the Metazoan kingdom (Owen and Zelent, 2000).The ancestor DBD may have been similar to that of modern receptors, the LBD being present but not possessing a transac-tivation domain or the ability to dimerize (Owen and Zelent, 2000). The possibility that the VDR could be a chimera is inaccordance with its clustering into different families based on the phylogenetic trees compiled from either DBD or LBD se-quence (Laudet et al., 1999). The DBD of the VDR closely resembles that of FTZ-F1, while the LBD is closer to that of the RARgrouping. However, the more recently characterized CAR, PXR, and liver X receptor (LXR), along with several arthropodreceptors, cluster together with the VDR in phylogenetic trees based on both the DBD and LBD, thus throwing doubt onthe VDR being a chimera (Owen and Zelent, 2000).



Convergent evolution may be in part responsible for this phenomenon which is observed in other receptors such as GCNFand NGF1B. The possibility that each of these receptors diverged from a common chimeric ancestor still remains and a mech-anism for such an event, originally suggested by Laudet et al. (1999), is represented in Fig. 3. In constructing the evolutionary

Fig. 3. Evolutionary aspects. (A) Hypothetical model for the origins and evolutionary diversification of the NR super-family. Heterodimerization wasacquired early in the diversification of the family (arrow) and was either conserved (i.e., COUP, RXR, NGF1B, PPAR, RAR, THR, VDR, CAR, and PXR) or lost (i.e.,TR2, HNF4 and Rev-Erb). Dashed lines represent the possible origin of a chimeric VDR. The vertical dashed lines represent the time period of the firstMetazoan, the Protostome/Deuterostome split and the Cambrian explosion (modified from Owen and Zelent (2000)). (B) Phylogenetic relationship amongCAR and PXR was based on Neighbour-Joining tree, using the Mega 3.1 program. The segment indicates the 0.1% of the phylogenetic distance. For details, seethe text.



tree represented in Fig. 3, three assumptions have been made: (i), the earliest Metazoan possessed COUP-TF, RXR, and FTZ-F1, all of which are well conserved to the present day and thus are close to the NR ancestor of subfamilies II and V; (ii) theability to heterodimerize with RXR arose once and has diversified through sub-families I, II, and IV; and (iii) there is no cor-relation between the ligand recognized by a given receptor and its position in the family tree (see Laudet, 1997). The pres-ence of RXR in the earliest branches of the Metazoan enables the extrapolation that an archaic family II member gave rise tofamilies I and IV, which can also dimerize with RXR. It could be assumed from data in Cnidaria that a direct RXR ancestordeveloped the ability to bind the ligand 9-cis-retinoic acid with a subset diverging in RAR, thus acquiring the ability to bindall trans-retinoic acids (Owen and Zelent, 2000).

The gene comparison among human, chimpanzee, mouse, and rat suggests that the high degree of conservation withinmembers of the NR super-family may have arisen from negative evolution selection against changes in the protein aminoacid sequence (Clark et al., 2003; Zhang et al., 2004a). Indeed, the amino acid sequence identity between human and mouseortholog NR genes is typically greater than 95% and 85% in the DBD and in the LBD, respectively (Zhang et al., 2004a; Iyeret al., 2006). The only two exceptions of such conservation are CAR-LBD and PXR-LBD, both characterized by a high cross-species amino acid sequence difference, indicating divergence during the course of evolution (Fig. 3) (Jones et al., 2000;Moore et al., 2002; Zhang et al., 2004a; Krasowski et al., 2005). Indeed, CAR-LBD and PXR-LBD show considerable differencesin ligand recognition (Watkins et al., 2001, 2003a,b; Shan et al., 2004; Suino et al., 2004; Xu et al., 2004; Chrencik et al., 2005;Xue et al., 2007a,b; Teotico et al., 2008; Wang et al., 2008).

The percentage of identity among CAR and PXR expressed in several vertebrate species has been calculated by aligningtheir complete amino acidic sequences, and are summarized in Tables 1 and 2, respectively. While hCAR and mCAR shareapproximately 93.9% amino acid identity in their whole sequence (Table 1), only approximately 70% amino acid identityhas been observed in their LBDs (Escriva et al., 2002; Robinson-Rechavi et al., 2004). Moreover marked differences have beenobserved in their responses to xenobiotics. Indeed, clotrimazole is an efficacious deactivator of hCAR but has little or no ef-fect on the mCAR activity. Conversely, 3,5-dichloro-2-{4-[(3,5-dichloropyridin-2yl)oxy]phenoxy}pyridine (TCPOBOP) is a po-tent activator of mCAR but lacks any activity on hCAR. The divergence in the amino acid sequence across CAR orthologsundoubtedly contributes to cross-species differences in the physiological effects of xenobiotics (Moore et al., 2000a).

The analysis of the complete amino acid sequence of PXR of different species indicates an identity of 76.5% and 76%between hPXR and mouse and rat PXRs, respectively (Table 2). Similarly to CAR, the human, rabbit, mouse, and rat PXRsare all roughly equally divergent, sharing only approximately 80% amino acid identity in their LBDs (Moore et al., 2002;Zhang et al., 2004a). Indeed, although both the rabbit and human PXR are activated by rifampicin, there are marked differ-ences in their responsiveness to synthetic steroids such as dexamethasone and cyproterone, as indicated by the level of CYPs

Table 1Amino acid sequence identity of CAR from different species.a

NCBI code H.s. P.t. Ma.m. C.l. S.s. R.n. Mu.m. G.g. B.t. D.r.

Homo sapiens NP_005113 – 100.0 93.9 81.9 83.6 76.5 72.6 50.6 46.6 38.0Pan troglodytes NP_001129087.1 – 93.9 81.9 83.6 76.5 72.6 50.6 46.6 38.0Macaca mulatta NP_001028068 – 78.7 80.3 77.6 73.5 50.4 45.2 37.7Canis lupus XP_545770 – 81.3 73.5 71.2 47.0 48.4 36.0Sus scrofa NP_001033085 – 75.4 72.9 49.9 50.6 37.2Rattus norvegicus NP_075230 – 91.9 46.0 43.6 37.4Mus musculus NP_033933 – 45.0 43.0 36.0Gallus gallus NP_990033 – 29.0 42.0Bos taurus NP_001073236 – 21.1Danio rerio NP_001092087 –

a Amino acid sequences (from http://www.ncdi.nlm.nih.gov/entrez/query.fcg?CMD=search&DB=protein) were aligned by EMBOSS (from http://www.ebi.ac.uk/tools/emboss/align/index.html).

Table 2Amino acid sequence identity of PXR from different species.a

NCBI code H.s. P.t. Ma.m. C.l. S.s. R.n. Mu.m. G.g. B.t. D.r.

Homo sapiens NP_003880 – 90.3 89.8 38.5 82.8 76.0 76.5 43.1 79.3 46.7Pan troglodytes XP_001164074 – 90.1 39.3 76.4 69.8 70.2 39.7 72.9 44.6Macaca mulatta NP_001028054 – 39.5 79.4 72.4 72.6 41.9 75.0 46.7Canis lupus XP_535750 – 37.6 35.0 35.2 21.1 36.6 22.0Sus scrofa NP_001033094 – 74.1 75.3 46.1 84.8 46.3Rattus norvegicus NP_443212 – 96.5 43.7 73.4 46.2Mus musculus NP_035066 – 43.4 74.5 46.8Gallus gallus AAG18374.1 – 44.3 42.0Bos taurus NP_001096696 – 45.2Danio rerio NP_001092087 –

a Amino acid sequences (from http://www.ncdi.nlm.nih.gov/entrez/query.fcg?CMD=search&DB=protein) were aligned by EMBOSS (from http://www.ebi.ac.uk/tools/emboss/align/index.html).



gene expression in vivo (Jones et al., 2000). The PXR divergence represents an important component of cross-species differ-ences in the regulation of CYP3A expression by xenobiotics, and could correspond to either an adaptive response to differentenvironmental xenobiotic challenges or the different specificities of natural PXR ligands within vertebrate species. Despitetheir divergence, several lines of evidence suggest that PXRs are orthologs and not closely related members of sub-family 1Iof NRs (Jones et al., 2000).

The cross-species variation in CAR-LBD and PXR-LBD is even more striking when DNA sequences are compared; in fact,the ratio between the rate of non-synonymous (nucleotidic substitutions that result in amino acid replacement) and synon-ymous (nucleotidic substitutions that do not cause an amino acid replacement) nucleotide variation provides informationconcerning evolutionary selective forces acting on a given gene (Yang and Bielawski, 2000). The analysis of this parameterstrongly suggests that the natural selection has favored sequence diversity in CAR-LBD and PXR-LBD, possibly to adapt tocross-species differences in ligand recognition. CAR and PXR may thus represent unusual examples of NR genes that havechanged their ligand specificity across vertebrate species to adapt to cross-species differences in the recognition of exoge-nous and/or endogenous toxic compounds (Kliewer et al., 1998; Schuetz et al., 2001; Xie et al., 2001; Moore et al., 2002;Krasowski et al., 2005; Reschly and Krasowski, 2006).

The different CAR and PXR ligand recognition properties constitute an especially unusual finding in the NR super-family,representing the more extreme divergence of ligand-binding residues of any of the ligand-activated NRs in vertebrates(Krasowski et al., 2005; Reschly and Krasowski, 2006). Indeed, residues involved in ligand binding are strongly conservedin other NRs as demonstrated by VDR, an endocrine NR closely related to PXR; only four residues are not conserved acrossvertebrate species, ranging from sea lamprey to human (Reschly and Krasowski, 2006).

The origin and evolution of the NR1I sub-family (which currently includes VDR, CAR, and PXR) is still a matter of debate,although it appears probable that a single NR gene duplicated early in vertebrate evolution gave origin to VDR and PXR genes(Reschly and Krasowski, 2006). These two genes then diverged from each other, and additional duplications have resulted inmultiple VDR and PXR genes in some non-mammals species. Interestingly, distinct CAR and PXR genes appear to be solely foundin mammals (Reschly and Krasowski, 2006). Indeed, the chicken has only a single ‘‘xenobiotic-responsive” NR1I gene, currentlyclassified as CAR, the product of which has properties similar to both CAR and PXR (Fig. 3) (Handshin et al., 2004). A single‘‘xenobiotic-responsive” gene is also present in Danio renio (Fig. 3) and Xenopus tropicalis (Reschly and Krasowski, 2006). Alikely explanation is that an ancestral gene, similar to that of chicken CAR, duplicated just prior to or early in mammalian evo-lution. The two genes then diverged from each other to become the modern-day CAR and PXR genes found in all mammaliangenomes sequenced so far (including opossum, seal, dog, pig, mouse, rat, and rhesus monkey) (Reschly and Krasowski, 2006).

The different amino acid sequences of vertebrate CAR-LBD and PXR-LBD contrast with the fact that non-synonymous sub-stitutions are rare in hCAR and hPXR (Lamba et al., 2005). Interestingly, the hCAR and hPXR sequencing of 70 and 100 indi-viduals from three different ethnic groups, respectively, shows very low nucleotide diversity (lower than the genome-wideaverages for human genes) and no non-synonymous substitutions in the LBD coded by either gene (Zhang et al., 2001;Thompson et al., 2005). Overall, NR1I sub-family members show little variation in the LBD between human individuals.In addition, the nucleotide divergence between human and chimpanzee CAR and PXR is lower than the average of the nucle-otide divergence for other genes in the human genome (Ebersberger et al., 2002; Clark et al., 2003; Thompson et al., 2005).This suggests that CAR and PXR ligands, relevant at least in terms of influencing reproductive fitness, do not vary betweenhumans, and perhaps not even between humans and primates, but do vary between different animals (Ebersberger et al.,2002; Clark et al., 2003; Thompson et al., 2005).

2. CAR and PXR structures

2.1. Gene organization

A common paradigm for many genes, including those of NRs, is the generation of several splice variants determined bydifferential promoter usage, insertion or deletion of amino acid residues, shift of the reading frame, and/or introduction ofpremature termination codons (Lander et al., 2001; Venter, 2001). Approximately 80% of alternative splicing results in pro-tein isoforms that could vary in structure and functional properties (Modrek and Lee, 2002; Lamba et al., 2004a). In addition,single nucleotide polymorphisms (SNPs), which give origin to protein variants, could affect either constitutive (i.e., basal orligand independent) or inducible (i.e., ligand dependent) NR activity. Interestingly, it has been demonstrated that CAR andPXR variants play a role in the inter-individual variability of CYP genes expression, and may be involved in rare atypical re-sponses to drugs or altered sensitivity to carcinogens (Hustert et al., 2001; Arnold et al., 2004; Koyano et al., 2004; Ikedaet al., 2005; Mensah-Osman et al., 2007; Lin et al., 2009).

2.1.1. Human CAR gene: structure, polymorphisms and transcriptshCAR is the product of the NR1I3 gene, which is located on chromosomes 1, locus 1q23, and spans approximately

8.5 kilobases (kbs) (Thigpen, 2004). The NR1I3 gene is composed of 8545 base pairs (bps), and comprises 9 exons separatedby 8 intronic regions (Fig. 4) (Auerbach et al., 2003; Savkur et al., 2003).

Twenty-two unique hCAR splice variants, containing different combinations of splicing (e.g., deletions of exons 2, 4, 5, and7, partial deletion of exon 9, and insertion of 12 or 15 bps from introns 6 or 7), have been identified (Auerbach et al., 2003;



Savkur et al., 2003; Lamba et al., 2004a). Although some CAR mRNAs originate from a single splicing event, the majority ofthe CAR transcript isoforms originate from multiple alternative splice events in a variety of combinations (Table 3). SeveralCAR splice variants (i.e., isoforms-17, -18, -19, -20, and -21) are unlikely to encode functional proteins because they havepremature termination codons originating from nonsense mutations, and may be rapidly degraded (Lamba et al., 2004a).The other alternative CAR mRNAs could encode unique CAR proteins (Auerbach et al., 2003; Lamba et al., 2004a). In contrastto human liver where multiple mRNA transcripts have been observed, a single CAR mRNA has been detected in small intes-tine (isoform-6), heart (isoform-22), and prostate (isoform-10) (Lamba et al., 2004a).

In Fig. 4, the hCAR isoform-3 mRNA (NM_005122) and the corresponding isoform-3 protein (NP_005113) are repre-sented.2 The 348 amino acid long hCAR isoform-3 is composed of the DBD, encoded by exons 2 and 3, the hinge region(H region), encoded by exon 4, and the LBD, encoded by exons 4–8 and a 50 portion of exon 9 (Auerbach et al., 2003; Savkuret al., 2003; Lamba et al., 2004a).

Thirty SNPs have been found in the NR1I3 gene among the Japanese population (Ikeda et al., 2003, 2005). Among them, 16are located in the 50-flanking region (Ikeda et al., 2003). Moreover, one SNP is located in exon 3 (398T > G, responsible for thenon-synonymous amino acid substitution Val133Gly), one in exon 4 (540C > T, responsible for the silent amino acid substi-tution Pro180Pro), and one in exon 8 (1032G > A, responsible for the silent amino acid substitution Gln344Gln). Finally, sev-eral SNPs have been found in introns 1, 2, 4, 5, and 8 (Ikeda et al., 2003). Besides the SNP responsible for the amino acidsubstitution Val133Gly, three other polymorphisms localized in the CAR-LBD coding sequence have been detected(i.e., 737A > G, 923T > C, and 968A > G responsible for the amino acid substitutions His246Arg, Leu308Pro, and Asn323Ser,respectively) (Ikeda et al., 2005).

2.1.2. Human PXR gene: structure, polymorphisms and transcriptshPXR is the product of the NR1I2 gene, which is located on chromosome 3, locus 3q12–q13.3, and spans approximately 20

kbs (Hustert et al., 2001; Kliewer et al., 2002). The NR1I2 gene is composed of 38.002 bps, and comprises 10 exons separatedby 9 intronic regions (Fig. 5) (Hustert et al., 2001; Kliewer et al., 2002). The first two exons are used as alternative 50 ends ofhPXR transcripts both in the liver and in the small intestine. The transcript originating from exon 1 results in the synthesis ofthe isoform-1. The transcript originating from exon 2 results in the isoform-2 and is characterized by the extension of 39amino acid residues at the N-terminus (Table 4) (Bertilsson et al., 1998), whose role in hPXR activity is yet unknown. Theisoform-3, originating from an in-frame deletion of 111 bps of the 50 part of exon 5, represents a hPXR variant lacking 37amino acids in the LBD (Dotzlaw et al., 1999).

In Fig. 5, the hPXR isoform-1 mRNA (NM_003889) and the corresponding isoform-1 protein (NP_003880) are repre-sented.3 The 434 amino acid long hPXR isoform-1 is composed of the DBD, encoded by exons 3 and 4, the H region, encodedby exon 5, and the LBD, encoded by exons 5–10 (Hustert et al., 2001).

A total number of 28 variants of hPXR (including splicing variants and polymorphic variants) have been so far identified(Hustert et al., 2001). Six of these SNPs, observed among the Caucasians and Africans, result in missense mutations of thehPXR protein. In particular, three polymorphisms are located in exon 1 (52C > A, 79C > T, and 106G > A, responsible for

1 2 3 4 5 6 7 8 9

mRNA isoform-3

1381 base pairs

NM_005122

Gene, 1q23

8545 base pairs

NC_000001.9

1 2 3 4 5 6 7 8 9

171-3

10

1121

-1381

1-170

311-4

41

442-6

11

612-7

51

752-8

97

898-1

014

1015

-1120

DBD LBDH1-8

9-99

100-106

107-348Protein isoform-3

348 amino acids

NP_005113

1-170

1613

-1752

2234

-2364

4873

-5042

5265

-5404

6737

-6882

6978

-7094

7293

-7398

8285

-8545

Fig. 4. hCAR gene, mRNA, and protein organization. The hCAR gene, localized on chromosome 1, is composed of 9 exons and 8 introns. Twenty-two hCARisoforms, containing different combinations of splicing, have been so far identified. The isoform-3 is considered the wild-type one. This isoform encodes a348 amino acid long xenosensor. The DBD is encoded by exons 2 and 3, the H region is encoded by part of exon 4, and the LBD is encoded by exons 4–9(Auerbach et al., 2003; Savkur et al., 2003). For details, see the text.

2 The human CAR isoform-3 (see the NCBI database: http://www.ncbi.nlm.nih.gov) is generally accepted as the wild-type form of the receptor (Lamba et al.,2004a).

3 The human PXR isoform-1 (see the NCBI database: http://www.ncbi.nlm.nih.gov) is generally accepted as the wild-type form of the receptor (Hustert et al.,2001).



the amino acid substitutions Glu18Lys, Pro27Ser, and Gly36Arg, respectively), two are located in exon 4 (418G > A and488A > G, responsible for the amino acid substitutions Val140Met and Asp163Gly, respectively), and one in exon 8(1108G > A, responsible for the amino acid substitution Ala370Thr) (Hustert et al., 2001). The Glu18Lys, Pro27Ser, andGly36Arg substitutions are located at the N-terminus of hPXR, the Val140Met substitution is located in the H region, andthe Asp163Gly and Ala370Thr substitutions are located within the hPXR-LBD (Hustert et al., 2001). With the exception of

Table 3Isoforms of hCAR.a

Isoform Changes mRNA size (bp); NCBIdatabase code

Protein size (amino acids);NCBI database code

Isoform-1 Insertion of 27bp in exon 9 1408; NM_001077482 357; NP_001070950Isoform-2 Insertion of 8bp in exon 9 1393; NM_001077480 352; NP_001070948Isoform-3 Full length 1381; NM_005122 348; NP_005113Isoform-4 Partial deletion of exon 9 1279; NM_001077481 314; NP_001070949Isoform-5 Deletion of exon 7 1264; NM_001077471 309; NP_001070939Isoform-6 Insertion of 15bp, partial deletion of exon 9 1242; NM_001077469 340; NP_001070937Isoform-7 Insertion of 12bp, partial deletion of exon 9 1239; NM_001077478 339; NP_001070946Isoform-8 Deletion of exon 7, and partial deletion of exon 9 1110; NM_001077474 296; NP_001070942Isoform-9 Deletion of exon 2 and insertion of 15bp 1256; NM_001077472 324; NP_001070940Isoform-10 Deletion of exon 2 1241; NM_001077479 319; NP_001070947Isoform-11 Deletion of exon 2 and exon 7 1124; NM_001077470 280; NP_001070938Isoform-12 Deletion of exon 2, insertion of 15bp and 12bp, and partial deletion of exon 9 1114; NM_001077473 315; NP_001070941Isoform-13 Deletion of exon 2, insertion of 15bp, and partial deletion of exon 9 1102; NM_001077476 311; NP_001070944Isoform-14 Deletion of exon 2, and partial deletion of exon 9 1087; NM_001077477 306; NP_001070945Isoform-15 Deletion of exon 2, exon 7, and partial deletion of exon 9 970; NM_001077475 267; NP_001070943Isoform-16 Deletion of partial deletion of exon 4, insertion of 15bp, and exon 4 and exon 7 836; n.d. 360; n.d.Isoform-17 Partial deletion of exon 2 to exon 9 47; n.d. No protein producedIsoform-18 Deletion of exon 4, exon 5, and exon 7 726; n.d. No protein producedIsoform-19 Deletion of exon 4, exon 5, exon 7, and partial deletion of exon 9 620; n.d. No protein producedIsoform-20 Deletion of exon 2, exon 4, exon 5, exon 7, and partial deletion of exon 9 480; n.d. No protein producedIsoform-21 Deletion of exon 2, exon 4, exon 5, and exon 7 586; n.d. No protein producedIsoform-22 Deletion of exon 4 and exon 7 866; n.d. No protein produced

a The changes in the splicing events and in the exons structure are described. Moreover, the mRNA size (expressed as number of bps), and the relativeNCBI code, as well as the protein size (expressed as the number of amino acids), and the relative NBCI code, are indicated. In bold, the full-length isoform-3.

mRNA isoform-1

4446 base pairs

NM_003889

DBD LBDH1-38

39-130

131-143

144-434Protein isoform-1

434 amino acids

NP_003880

Gene, 3q12-q13.3

38002 base pairs

NC_000003.10

1-181

722

75-23

69

2674

6-269

64

2957

8-297

11

3105

6-312

43

3220

3-324

77

3449

6-346

38

3484

0-349

56

3524

3-353

48

3658

5-380

02

1 2 3 4 5 6 7 8 9 10

2894

-2999

1 3 4 5 6 7 8 9 10

1-181

7

2037

-2170

1818

-2036

2171

-2358

2359

-2633

2634

-2776

2777

-2893

3000

-4417

Fig. 5. hPXR gene, mRNA and protein organization. The hPXR gene, localized on chromosome 3, is composed of 10 exons and 9 introns. Three hPXRisoforms, containing different combinations of splicing, have been so far identified. The isoform-1 is considered the wild-type one. The isoform-1 encodes a434 amino acid long xenosensor. The DBD is encoded by exons 3 and 4, the H region is encoded by part of exon 5, and the LBD is encoded by exons 5–10(Hustert et al., 2001; Kliewer et al., 2002). For details, see the text.

Table 4Isoforms of hPXR.a

Isoform Changes mRNA size (bp); NCBI database code Protein size (amino acids); NCBI database code

Isoform-1 Originates from exon 1 4446; NM_003889 434; NP_003880Isoform-2 Originates from exon 2 2772; NM_022002 473; NP_071285Isoform-3 Deletion of 111bp in exon 5 4335; NM_033013 397; NP_148934

a The changes in the splicing events and in the exons structure are described. Moreover, the mRNA size (expressed as the number of bps), and the relativeNCBI code, as well as the protein size (expressed as tthe number of amino acids), and the relative NBCI code, are indicated. In bold, the full-length isoform-1.



Gly36Arg and Val140Met, all the amino acid substitutions affect residues that are conserved in human, rabbit, mouse, and ratPXRs (Moore et al., 2000a; Hustert et al., 2001). The most frequent hPXR polymorphism (Pro27Ser) occurs in 14.9% of Africanchromosomes. All the other xenosensor variants have an allelic frequency approximately lower than 3% and are specific foreither Caucasians of Africans (Hustert et al., 2001).

2.2. Protein structure

CAR and PXR, like all the members of the NR super-family, are modular proteins sharing common regions, including theN-terminal DBD, the H region, and the C-terminal LBD. These regions participate in the formation of independent but allos-terically interacting functional domains (Ribeiro et al., 1995; Kumar and Thompson, 1999; Olefsky, 2001; Mohan andHeyman, 2003; McEwan, 2004; Ascenzi et al., 2006).

| |CAR 1 MASREDEL---RNCVVCGDQATG 20 PXR 1 MEVRPKESWNHADFVHCEDTESVPGKPSVNADEEVGGPQICRVCGDKATG 50 | | | | |

| helix 1 | | | helix 2CAR 21 YHFNALTCEGCKGFFRRTVSKSIGPTCPF-AGSCEVSKTQRRHCPACRLQ 69 PXR 51 YHFNVMTCEGCKGFFRRAMKRNARLRCPFRKGACEITRKTRRQCQACRLR 100 | | | | |

| helix A | | CAR 70 KCLDAGMRKDMILSAEALALRRAKQAQRRAQQTPVQ------LSKEQEEL 113 PXR 101 KCLESGMKKEMIMSDEAVEERRALIKRKKSERTGTQPLGVQGLTEEQRMM 150 | | | | |

| | |α2’ | CAR 114 IRTLLGAHTRHMGTMFEQFVQFRPPAHLFIHHQ-PLPTLAP--------- 153 PXR 151 IRELMDAQMKTFDTTFSHFKNFRLPGVLSSGCELPESLQAPSREEAAKWS 200

α1 | α2 | | | |

| CAR 154 -----------------------------------VLPLVTHFADINTFM 168 PXR 201 QVRKDLCSLKVSLQLRGEDGSVWNYKPPADSGGKEIFSLLPHMADMSTYM 250

| β1 | β1’ | | α3 |

| | | | | CAR 169 VLQVIKFTKDLPVFRSLPIEDQISLLKGAAVEICHIVLNTTFCLQTQNFL 218 PXR 251 FKGIISFAKVISYFRDLPIEDQISLLKGAAFELCQLRFNTVFNAETGTWE 300

| α3’ | α4 | α5 | β2 β3 |

| | | | | CAR 219 CGPLRYTIEDGARVGFQVEFLELLFHFHGTLRKLQLQEPEYVLLAAMALF 268 PXR 301 CGRLSYCLEDTAG-GFQQLLLEPMLKFHYMLKKLQLHEEEYVLMQAISLF 349

β4 | α6 | α7 | | α8

| | | | | CAR 269 SPDRPGVTQRDEIDQLQEEMALTLQSYIKGQQRRPRDRFLYAKLLGLLAE 318 PXR 350 SPDRPGVLQHRVVDQLQEQFAITLKSYIECNRPQPAHRFLFLKIMAMLTE 399 | | α9 | | | α10

| | αX |CAR 319 LRSINEAYGYQIQHIQGLSAM-MPLLQEICS 348 PXR 400 LRSINAQHTQRLLRIQDIHPFATPLMQELFGITGS 434 | | | αAF |

T

Fig. 6. Amino acid sequence alignment of hCAR isoform-3 (NCBI code: NP_005113.1) and of hPXR isoform-2 (NCBI code: NP_071285.1). The xenosensorsequences were obtained from the NCBI database (http://www.ncbi.nlm-nih.gov). The amino acid sequence alignment was performed by EMBOSS (fromhttp://www.ebi.ac.uk/tools/emboss/align/index.html). Amino acid residues in green, azure, and yellow indicate conserved residues, conserved substitu-tions, and semi-conserved substitutions, respectively. Cys residues chelating zinc ions in DBDs are in bold and underlined. In hCAR-DBD, amino acidresidues forming a-helices are in black, bold, and boxed. In hCAR-LBD and hPXR-LBD, amino acid residues forming a-helices are in black and bold. In hCAR-LBD and hPXR-LBD, amino acid residues forming b-strands are in red and bold. The aX helix, present only in CAR, is also named a11; the aAF helix, presentin CAR and PXR, is also named a12. For every ten amino acid residues a vertical line has been drawn. For details, see the text.



2.2.1. The primary structure of CAR and PXRThe hCAR and hPXR sequences are closely related (Fig. 6) and appear to have arisen relatively recently from a common

progenitor (Handschin et al., 2000). Indeed, the amino acid identity and similarity of hCAR isoform-3 and hPXR isoform-1 is37.9% and 53.3%, respectively. However, their role in gene expression regulation is significantly different, hCAR and hPXRrecognizing different endogenous and exogenous ligands (see Sections 3 and 4).

Most of the amino acid residues involved in DNA binding are conserved or semi-conserved in hCAR and hPXR, the DBDbeing the most conserved region of NRs. In contrast the H region, connecting the DBD with the LBD, displays very low aminoacid identity and similarity; this may reflect the different subcellular distribution and the intracellular trafficking regulationof hCAR and hPXR. The LBD is moderately conserved in sequence and this reflects the different ligand-binding properties ofhCAR and hPXR. However, the LBD C-terminal boundary, corresponding to the transactivation region and involved in co-fac-tor binding, is variable in sequence (Fig. 6) (Ribeiro et al., 1995; Kumar and Thompson, 1999; Olefsky, 2001; Mohan and Hey-man, 2003; McEwan, 2004; Ascenzi et al., 2006).

Several natural mutants of hCAR and hPXR have been identified. His246Arg and Leu308Pro variants reduce the constitu-tive (i.e., ligand independent) transactivation activity of hCAR, this might be due to the conformational changes of thetransactivation region AF-2. Moreover, while the His246Arg variant is no longer activated by the agonistic 6-(4-chloro-phenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), the Leu308Pro variant retainsthe responsiveness to this ligand. The low affinity of CITCO for the hCAR His246Arg variant reflects the critical role of thisresidue in ligand recognition, in fact it is located in the a7 helix participating in the formation of the LBD ligand-bindingpocket. Moreover, the Val133Gly and Asn323Ser variants show levels of constitutive activity and responsiveness to CITCOsimilar to those of wild-type hCAR (Ikeda et al., 2005; Jyrkkarinne et al., 2005). Variants located at the N-terminus of thehPXR-DBD (i.e., Glu18Lys, Pro27Ser, and Gly36Arg) have no significant impact on the constitutive activity in comparisonto that of the wild-type xenosensor. Also all the hPXR-LBD variants, with the exception of Asp163Gly, show a significant con-stitutive activity. The Val140Met and Ala370Thr variants, located within the hPXR-LBD, or close to it, show a moderatethough dramatic effect on the ligand-dependent transcriptional activity (Hustert et al., 2001).

2.2.2. The architecture of CAR-DBD and PXR-DBDCAR-DBD and PXR-DBD are involved in receptor dimerization and in the binding of specific DNA sequences (Frank et al.,

2003; Watkins et al., 2003a; Suino et al., 2004; Xu et al., 2004; Xue et al., 2007a,b; Wang et al., 2008).The inspection of the molecular model of hCAR-DBD/hRXRa-DBD (Fig. 7) indicates that hCAR-DBD is folded into a glob-

ular shape, containing two a-helices perpendicular to one another (named helix 1, residues Glu29-Lys41, and helix 2,residues Pro64-Ala74), a connector loop (named T region, residues Arg77-Ala84) that lies perpendicular to the long axisof DNA, and a long a-helix (named helix A, residues Ala84-Arg97). The T region is interrupted by a helical turn (residuesLys78-Asp79-Met80). Both CAR-DBD and PXR-DBD contain eight conserved Cys residues found in two groups of four. Eachgroup is involved in the tetrahedral coordination of a single zinc ion (Figs. 6 and 7) (Frank et al., 2003).

The module 1 of hCAR-DBD is mainly involved in site-specific DNA binding due to its interaction with the sugar phos-phate backbone on the major DNA groove of the cognate RE. Minor hCAR-DBD:DNA contacts involve module 2. Moreover,both the T region and the A helix, composed of mostly charged amino acid residues, participate in DNA recognition(Fig. 7) (Frank et al., 2003).

Site-directed mutagenesis studies highlighted the role of residues Arg90 and Arg91 of hCAR-DBD to contact DNA, bothresidues being semi-conserved in hPXR-DBD (Arg121 and Arg122, respectively). Although Gln94, Arg97, and Arg98 are lo-cated at the DNA-binding site of the C-terminal extension of the hCAR-DBD, they play a minor role in DNA recognition. Notethat Gln94, Arg97, and Arg98 are replaced by Ile125, Lys128, and Lys129 in the hPXR-DBD (Fig. 7) (Frank et al., 2003).

hCAR-DBD and hRXRa-DBD heterodimerize in the ‘‘head-to-tail” orientation. The interaction between hCAR-DBD andhRXRa-DBD involves Tyr21, Asn24, and Asp79 of hCAR-DBD and Arg172 and Arg186 of hRXRa-DBD. In particular,hRXRa-DBD Arg172 makes a salt bridge with hCAR-DBD Asp79. Moreover, hRXRa-DBD Arg186 makes a hydrogen bond withthe hCAR-DBD Asn24 carbonyl oxygen atom, and a planar stacking interaction with hCAR-DBD Tyr21. Moreover, amino acidresidues surrounding the Arg186 residue of the hRXRa-DBD, notably Arg184, Asn185, and Gln188, form hydrogen bonds tothe DNA backbone and thereby support its proper position in the heterodimer interface (Fig. 7).

REs for CAR/RXR and PXR/RXR differ from one another by the number of neutral base pairs separating the direct oreverted DNA repeats (dr and er, respectively). CAR/RXR (both human and mouse) binds optimally both the dr-4-type andthe er-8-type REs formed by two hexameric-binding sites (i.e., AGTTCAnnnnAGTTCA and TGAACTnnnnnnnnAGTTCA, respec-tively). The dr-4-type RE is crucial for CYP2B6 regulation. Moreover, monomeric CAR binds the octameric sequenceAGAGTTCA containing two additional pyrimidine bases at the 50 position of the DNA-binding region. Although the affinityof DNA for monomeric CAR is lower than that for CAR/RXR, monomeric CAR:DNA binds co-activators (Frank et al., 2003).PXR/RXR (both human and mouse) binds efficiently both the dr3-type and the er6-type REs, formed by two hexameric-binding sites (i.e., AGTTCAnnnAGTTCA and TGAACTnnnnnnAGTTCA, respectively), which are crucial for CYP3A1 and CYP3A4regulation, respectively (Lehmann et al., 1998).

Heterodimerization and DNA binding highlight the importance of inter-half-site spacing. Indeed, the DBDs of NRs, whichare monomers in the absence of DNA and which generally bind weakly as monomers to a single half-site, bind with highaffinity and strong cooperativity to REs in which the appropriate half-sites are spaced correctly. Cooperative enhancementof NR/NR and NR/DNA interactions at the heterodimer interface brings the subunits in registration with the specific contact



surface of the half-site thus reinforcing the specific recognition. Remarkably, amino acid residues contributing to the hetero-dimer interface are relatively specific to a particular DBD, thereby ensuring a unique mode of heterodimerization on the cor-responding RE (Rastinejad et al., 1995).

2.2.3. The architecture of CAR-LBD and PXR-LBDThe three-dimensional structures of CAR-LBD and PXR-LBD (Table 5) share the canonical NR LBD a-helical sandwich fold

(Fig. 8) (see Holm et al., 1992; Holm and Sander, 1994, 1997; Kumar and Thompson, 1999; Ruff et al., 2000; Kumar et al.,2004; Carnahan and Redinbo, 2005; Orans et al., 2005; Ascenzi et al., 2006), displaying distinct regions crucial for ligandbinding, co-regulator recognition, and dimerization. The flexible ligand-binding pockets of CAR-LBD and PXR-LBD allow to

B

AModule 1 Module 2

G Y HT F

A N K T QQ A S RD L V RG T E HC C C C hCAR

G SZn2+ A Zn2+

F P9 RN C C KGFFRRTVSKSIGPTC C RLQKCLDAGMRKDMILSAEALALRRAKQAQRRA 99

helix 1 helix 2 helix A

G Y HT F

A N R K TK V T RD M I RG T E QC C C C hPXR

G AZn2+ Zn

2+

F P39 QI C C KGFFRRAMKRNARLRC C RLRKCLESGMKKEMIMSDEAVEERRALIKRKKS 130

KR

Q

A

E

G

V

R

TV

V

E

G

P

A

hRXRα-DBD hCAR-DBD

A

2

1

T

Fig. 7. hCAR-DBD and hPXR-DBD structures. (A) Schematic representation of hCAR-DBD and hPXR-DBD. Amino acid residues in green, azure, and yellowindicate conserved residues, conserved substitutions, and semi-conserved substitutions, respectively. Cys residues chelating zinc ions in DBDs are in boldand underlined. The zinc ions are highlighted by cyan disks. In hCAR-DBD, amino acid residues forming a-helices are in bold and boxed. (B) Three-dimensional model of hCAR-DBD/hRXRa-DBD bound to a DNA strand. Zinc ions are represented as cyan spheres. The DBD a-helices are shown in magentaand identified as indicated in panel A. The phosphate backbone of DNA is represented in orange. The nucleotide bases are shown in stick and coloredaccording to the atom type. The three-dimensional structure of hCAR-DBD isoform-3 has been modeled using the program Nest (Petrey et al., 2003) and thehVDR-DBD as the template (PDB code: 1kb2; Shaffer and Gewirth, 2002), which displays 73% similarity over ninety aligned residues. Docking of themodelled hCAR-DBD onto DNA has been performed by structural superimposition over the hRXR-DBD/hTHR-DBD heterodimer bound to the thyroidhormone DNA RE (PDB code: 2nll; Rastinejad et al., 1995). For details, see the text.



bind a wide range of structurally unrelated endogenous and exogenous ligands (see Sections 3 and 4). The CAR-LBD andPXR-LBD AF-2 region binds the Leu-Xxx-Xxx-Leu-Leu motifs of transcriptional co-activators, and the Ile/Leu-Xxx-Xxx-Ile/Val-Ile motifs present in co-repressors. However, different arrangements have been reported for CAR-LBD and PXR-LBD dimer-ization (Fig. 9) (Watkins et al., 2001, 2003a,b; Shan et al., 2004; Suino et al., 2004; Xu et al., 2004; Chrencik et al., 2005; Oranset al., 2005; Xue et al., 2007a,b; Teotico et al., 2008; Wang et al., 2008). Notably, the a10 helix contains a Leu-rich sequence thatseems to be required for xenosensor translocation from citosol to nucleus (see Section 3.3.1.) (Zelko et al., 2001).

2.2.3.1. The CAR-LBD structure. The hCAR-LBD and mCAR-LBD structures consist of eleven a-helices, two 310 helices(designed a2 and a20) between helices a1 and a3, and three b-strands. The 310 helices located between helices a1

Table 5X-ray three-dimensional structures of CAR and PXR.a

PDB code Receptor Ligand Co-activator Aggregation state References

1xnx mCAR-LBD 16,17-Androstane-3-ol Monomer Shan et al. (2004)1xv9 hCAR-LBD (5b)-Pregnane-3,20-dione SRC-1p1 Heterodimer Xu et al. (2004)

hRXRa-LBD Pentadecanoic acid SRC-1p11xvp hCAR-LBD CITCO SRC-1p1 Heterodimer Xu et al. (2004)

hRXRa-LBD Pentadecanoic acid SRC-1p11xls mCAR-LBD TCPOBOP TIF2p Heterodimer Suino et al. (2004)

hRXRa-LBD Retinoic acid TIF2p1ilg hPXR-LBD Monomer Watkins et al. (2001)1ilh hPXR-LBD SR12813 Monomer Watkins et al. (2001)1m13 hPXR-LBD Hyperforin Monomer Watkins et al. (2003b)1skx hPXR-LBD Rifampicin Monomer Chrencik et al. (2005)2qnv hPXR-LBD Colupulone Monomer Teotico et al. (2008)n.d.c. hPXR-LBD 17b-Estradiol Homodimer Xue et al. (2007b)3ctb hPXR-LBD SRC-1p2 Homodimer Wang et al. (2008)1nrl hPXR-LBD SR12813 SRC-1p2 Homodimer Watkins et al. (2003a)n.d. hPXR-LBD SR12813 SRC-1p2 Homodimer Wang et al. (2008)2o9i hPXR-LBD T0901317 SRC-1p3 Homodimer Xue et al. (2007a)

n.d., the atomic coordinates were not deposited at the Protein Data Bank.a From www.pdb.org.

Fig. 8. Three-dimensional structures of monomeric mCAR-LBD bound to 16,17-androstane-3-ol (PDB code: 1xnx), and of ligand-free monomeric hPXR-LBD(PDB code: 1ilg). The xenosensor structures are reciprocally rotated by 90� along the vertical axis. The LBD a-helices and b-strands are shown in magentaand green, respectively. The aX and aAF helices are shown in yellow. The mCAR-LBD ligand 16,17-androstane-3-ol is represented in spacefilling (white).Pictures have been generated using UCSF Chimera (Pettersen et al., 2004). For details, see the text.



and a3 are designed a20 and a200, and the a-helix located between helices a3 and a4 is named a30 (Fig. 8) (Shan et al., 2004;Suino et al., 2004; Xu et al., 2004). hCAR-LBD residues Leu336, Ser337, Ala338, and Met339 (corresponding to Leu346,Ser347, Ala348, and Met349 in mCAR-LBD) are located between a10 and the aAF helices and form the single turn helixnamed aX (Shan et al., 2004; Suino et al., 2004; Xu et al., 2004). In hCAR-LBD and mCAR-LBD, the conformation of the aX

Fig. 9. Three-dimensional structures of heterodimeric hRXRa-LBD/mCAR-LBD bound to TCPOBOP and TIF2p (PDB code: 1xls), and of homodimeric hPXR-LBD/hPXR-LBD bound to SR12813 and SRC-1p2 (PDB code: 1nrl). In heterodimeric hRXRa-LBD/mCAR-LBD: (i) the hRXRa-LBD a-helices and b-strands areshown in cyan and dark green, respectively; (ii) the mCAR-LBD a-helices and b-strands are shown in magenta and green, respectively; (iii) the hRXRa-LBDaAF helix is shown in blue; (iv) the mCAR-LBD aX and aAF helices are shown in yellow; (v) the hRXRa-LBD ligand retinoic acid and the mCAR-LBD ligandTCPOBOP are shown in spacefilling (white); and (vi) the TIF2p cofactor is represented in red. In homodimeric hPXR-LBD/hPXR-LBD: (i) the hPXR-LBD a-helices and b-strands are shown in magenta and green, respectively; (ii) the hPXR-LBD aAF helices are shown in yellow; (iii) the hPXR-LBD ligandsTCPOBOP are shown in spacefilling (white); and (iv) the SRC-1p2 cofactors are represented in red. For details, see Fig. 8 and the text.



helix contributes to the constitutive activity of CAR providing a rigid structure between helices a10 and aAF (Shan et al.,2004; Xu et al., 2004). In particular, the stabilization of the active hCAR-LBD conformation appears to be provided by theinteraction between the C-terminal carboxylate of the aAF helix and Lys195 from helix a4. Residues Leu336, Ser337,Ala338, and Met339 in the aX helix are identical across all the mammalian CAR-LBD orthologs (Sakai et al., 2004; Xuet al., 2004). Moreover, all the four residues that compose the barrier separating the ligand-binding pocket from the aAFhelix (i.e., Phe161, Asn165, Phe234, and Tyr326) are identical among the CAR orthologs (Xu et al., 2004).

The ligand-binding pocket of CAR-LBD is framed by helices a2, a3, a4, a5, a6, a7, and a10 and by b-strands 3 and 4(Fig. 8), the volume being about 600 Å3. Polar residues, present in the highly hydrophobic CAR-LBD ligand-binding pocket,neutralize each other by forming polar–polar interactions (e.g., the Arg156 � � � Asp238 salt bridge in mCAR-LBD) (Shan et al.,2004; Suino et al., 2004; Xu et al., 2004). In mCAR-LBD, the two 310 helices between helices a1 and a3 appear as a cap at thebottom of the structure contributing to the general hydrophobicity of the ligand-binding cavity. Moreover, this region hasbeen postulated to be an entry site for the ligand in mCAR-LBD (Shan et al., 2004).

CAR-LBD binds the second Leu-Xxx-Xxx-Leu-Leu a-helical motif of co-activators steriod receptor co-regulator-1peptide-1 (SRC-1p1) and transcription intermediary factor 2 (TIF2p) in a groove formed by helices a3, a4, and aAF(Fig. 9). Both co-activators adopt a two-turn a-helix structure with the three Leu residues oriented into the co-activator-binding site of CAR-LBD. Both ends of the co-activator helix are capped by hydrogen bonds, mediated by a charge clampformed by a conserved Glu from the aAF helix (at position 345 in hCAR-LBD and at position 355 in mCAR-LBD, respectively)and by a conserved Lys from the a3 helix (at position 177 in hCAR-LBD and at position 187 in mCAR-LBD, respectively).Moreover, the co-activator is bound by a second charge clamp formed by a conserved Arg from the a30 helix (at position183 in hCAR-LBD and at position 193 in mCAR-LBD) and by a conserved Glu from the a4 helix (at position 188 in hCAR-LBDand at position 198 in mCAR-LBD) (Suino et al., 2004; Xu et al., 2004).

The general architecture of the CAR-LBD/hRXRa-LBD back-to-back heterodimer is similar to that of other RXRa heterodi-meric structures in the active conformation (Fig. 9) (Bourguet et al., 2000; Gampe et al., 2000; Svensson et al., 2003; Suino et al.,2004; Xu et al., 2004). The large heterodimerization interface (about 1000 Å2) facilitates the association, playing a pivotal rolein CAR/RXR activation and signaling. In the CAR-LBD/hRXRa-LBD heterodimer, the CAR-LBD a10 helix packs parallel to thehRXRa-LBD a10 helix and contacts the hRXRa-LBD aAF helix (Suino et al., 2004; Xu et al., 2004). Moreover, the N-terminalend of hCAR-LBD a7 helix is close to the hRXRa-LBD aAF helix (Xu et al., 2004). However, the relatively short CAR-LBD aAFhelix limits the possibility of interaction with hRXRa-LBD (Suino et al., 2004; Xu et al., 2004).2.2.3.1.1. 16,17-Androstane-3-ol recognition by mCAR-LBD. The inverse agonist 16,17-androstane-3-ol nestles within thecenter of the mCAR-LBD hydrophobic ligand-binding pocket, being completely sequestered within the protein matrix(Fig. 10). The residues that interact with 16,17-androstane-3-ol come from helices a3, a5, a6, a7, aX, and the b2-strand.16,17-Androstane-3-ol makes no contacts with the aAF helix which is in a random conformation. The only polar interactionscritical for 16,17-androstane-3-ol recognition by mCAR-LBD occur between the 3a-hydroxyl moiety of the ligand andresidues Asn175 present in the a3 helix, and His213 present in the a5 helix. The Phe171Ala or Asn175Ala or His213Alamutations in mCAR-LBD determine a significant reduced transcriptional activity in response to 16,17-androstane-3-olbinding, without affecting the PXR constitutive activity (Shan et al., 2004).

The activity induced by 16,17-androstane-ol binding to mCAR-LBD appears to be associated with a ligand-induced kinkbetween helices a10 and aX. This disrupts the polar interaction(s) that locks the aAF helix in the basal transcriptionally ac-tive conformation (Shan et al., 2004).2.2.3.1.2. (5b)-Pregnane-3,20-dione and CITCO recognition by the hCAR-LBD/hRXRa-LBD heterodimer bound to SRC-1p1. ThehCAR-LBD/hRXRa-LBD heterodimer, bound to the 13 amino acid peptide from the second NR box of SRC-1 positioned in eachof the co-activator grooves and either the (5b)-pregnane-3,20-dione and pentadecanoic acid or the CITCO and pentadecanoicacid, is shown in Fig. 10 (Xu et al., 2004).

The steroidal agonist (5b)-pregnane-3,20-dione makes hydrophobic contacts with Phe161, Ile164, Leu206, Phe217,Tyr224, Phe234, and Leu242 in hCAR-LBD. In addition, the hydrogen bond between the ligand C21 ketone atom and the res-idue His203 orients the steroid in the ligand-binding pocket. Note that: (i) the Phe234Ala mutant impairs the constitutiveactivity; (ii) the Phe161Tyr mutant shows a slightly higher constitutive activity; and (iii) the Leu343Glu mutant induces adecrease in constitutive activity and in the ligand-binding affinity (Fig. 10) (Xu et al., 2004).

The selective agonist CITCO binds to hCAR-LBD in a range of possible conformations. The two binding modes of CITCO(Fig. 10) with the lowest energy have similar U-shaped conformations that make possible mostly hydrophobic interac-tions with hCAR-LBD. The p-chlorophenyl substituent of CITCO localizes into the pocket bounded by Phe161, Phe217,Val232, Phe234, Phe238, Leu239, Leu242, and Phe243, which is formed in part by helices a6 and a7. The oxime sidechain with the pendant dicholorophenyl fits into an adjacent hydrophobic pocket bounded by Phe132, Phe161, Ile164,Met168, Leu206, Phe217, Tyr224, Gly229, and Val232. Although no specific hydrogen bonds are observed between CITCOand the hCAR-LBD binding pocket, the imidazothiazole heterocycle of CITCO sits in a relatively polar region of the pocketformed by Asn165, Val199, Cys202, His203, and Tyr326 and makes weak electrostatic interactions with His203, Asn165,and Tyr326. CITCO does not form a direct contact with the aAF helix, indeed Phe161, Asn165, Phe234, and Tyr326 shieldthe ligand-binding pocket from the aAF helix. Moreover, the side chains of Leu336, Met340, Leu343, and Cys347 fromhelices aX and aAF pack against one face of the barrier, whereas the ligand-binding pocket lies on the other face. Inaddition, the hydrogen bond between Asn165 and Tyr326 participates in the maintenance of the structure rigidity(Xu et al., 2004).



Pentadecanoic acid binds to the hRXRa-LBD pocket as observed in other heterodimeric RXRa-structures. Pentadecanoicacid is completely buried within the bottom half of the receptor. The hRXRa-LBD L-shaped ligand-binding pocket is com-posed of residues from helices a3, a4, a5, a7, a10, and the b turn, the pocket volume being 470 Å3 (see Bourguet et al.,2000; Gampe et al., 2000; Svensson et al., 2003; Suino et al., 2004; Xu et al., 2004).2.2.3.1.3. TCPOBOP recognition by the mCAR-LBD/hRXRa-LBD heterodimer bound to TIF2p. The mCAR-LBD/hRXRa-LBD hetero-dimer active conformation binds the insecticide contaminant and most potent known member of the phenobarbital-like classof CYP-inducing agents 1,4-bis[2-(3,5-dichloro-pyridiloxy)]benzene (TCPOBOP) at the mCAR-LBD, and the retinoic acid at thehRXRa-LBD. Both receptors bind the 18 amino acid peptide from TIF2p in each of the co-activator grooves (Suino et al., 2004).

TCPOBOP is centrally located in the mCAR-LBD-binding pocket, adopting a C-shaped configuration (Fig. 10). The ligand isoriented with the A-ring toward the b-strand and the C-ring toward helices a10 and aAF. TCPOBOP forms an extensive net-work of hydrophobic interactions with the mCAR-LBD. In particular, the A-ring is sandwiched between Phe227 and Tyr234,whereas the C-ring is sandwiched between Phe244 and Tyr336. Moreover, TCPOBOP makes only two hydrogen bonds withthe mCAR-LBD, mediated through a water molecule that bridges the nitrogen atom of the pyridine rings with the side chainof Asn175 (Suino et al., 2004). In contrast with what reported for the mCAR-LBD:16,17-androstane-3-ol complex (Shan et al.,2004), and for the hCAR-LBD adducts with CITCO and (5b)-pregnane-3,20-dione (Xu et al., 2004), the TCPOBOP C-ring inter-acts directly with the Lys346, Lys353, and Thr350 residues. Moreover, in the mCAR-LBD bound to TCPOBOP, the C-terminalfree carboxylate group interacts with Lys205 from helix a4, which with Ser337, located in helix a10, contacts other carbonylgroups in the C-terminus of the aAF helix (Shan et al., 2004). This stabilizes the mCAR-LBD active conformation (Shan et al.,2004; Suino et al., 2004).

The Phe171Trp, Asn175Phe, Leu216Phe, Phe227Trp, Phe244Ala, Tyr336Ala, and Leu346Phe mutations determine a dimin-ished response to TCPOBOP, reflecting a critical role for ligand recognition by the mCAR-LBD. Moreover, the Leu346Phe mu-tant shows a significant increase in affinity for the TIF2p co-activator motif (Suino et al., 2004).

Fig. 10. Binding mode of 16,17-androstane-3-ol to mCAR-LBD (A; PDB code: 1xnx), of (5b)-pregnane-3,20-dione to hCAR-LBD (B; PDB code: 1xv9), of CITCOto hCAR-LBD (C; PDB code: 1xvp), and of TCPOBOP to mCAR-LBD (D; PDB code: 1xls). Note that CITCO may have multiple conformations. Amino acidresidues are shown in stick and colored according to the atom type. Ligands are in blue. For details, see Fig. 8 and the text.



Interestingly, TIF2p and TCPOBOP binding to hCAR-LBD/hRXRa-LBD and heterodimerization are allosterically coupled. In-deed, the affinity of TIF2p for hCAR-LBD/hRXRa-LBD is higher than that for monomeric hCAR-LBD. Moreover, TCPOBOP facil-itates TIF2p binding to hCAR-LBD/hRXRa-LBD and monomeric hCAR-LBD (Suino et al., 2004).

Retinoic acid binds to the hRXRa-LBD as reported in Section 2.2.3.1.2. (Bourguet et al., 2000; Gampe et al., 2000; Svenssonet al., 2003; Suino et al., 2004; Xu et al., 2004).

2.2.3.2. The PXR-LBD structure. hPXR-LBD consists of a three-layered a-helical sandwich (a1–a3/a4–a5–a8–a9/a7–a10)and a five-stranded antiparallel b-sheet (b1, b10, b2, b3, and b4) (Figs. 6, 8 and 9). This extended b-sheet is unique to PXR(Watkins et al., 2001, 2003a,b; Chrencik et al., 2005; Orans et al., 2005; Xue et al., 2007a,b; Teotico et al., 2008; Wanget al., 2008) since NR-LBDs typically contain only two- or three-stranded b-sheets (see Bourguet et al., 1995; Brzozowskiet al., 1997; Nolte et al., 1998; Lo et al., 1999; Shan et al., 2004; Suino et al., 2004; Xu et al., 2004; Orans et al., 2005;Renaud et al., 2005; Ascenzi et al., 2006). The PXR-LBD ends with a short helix (aAF) which is critical for the structuralorganization of the AF-2 region (i.e., for xenosensor activation) which is highly conserved in PXRs (see Reschly andKrasowski, 2006). The aAF is packed against the body of PXR-LBD in a position that appears critical to recruit transcrip-tional co-regulators (Watkins et al., 2001, 2003a,b; Chrencik et al., 2005; Orans et al., 2005; Xue et al., 2007a,b; Teoticoet al., 2008; Wang et al., 2008).

PXR-LBD deviates most significantly in the canonical structure of NR-LBDs at the bottom end of the receptor domain,this likely contributes to the xenosensor ability to bind chemically distinct ligands (see Fig. 1 and Sections 3 and 4). In-deed, PXR-LBD contains an insert of approximately 60 residues which is unique within members of the NR super-family(see Bourguet et al., 1995; Brzozowski et al., 1997; Nolte et al., 1998, Lo et al., 1999; Orans et al., 2005; Renaud et al.,2005; Ascenzi et al., 2006; Reschly and Krasowski, 2006). This insert (Val177-Pro228) contains the b1–b10 regions andthe novel a2 helix that folds along the underside of the expansive PXR ligand-binding pocket (Figs. 6, 8 and 9). In con-trast to hCAR-LBD, the hPXR-LBD ligand-binding pocket is characterized by three additional strands b1, b10, and b2, bypartial unwinding of helix a7, by displacement of helix a6, and by a different helix a20 geometry (see Figs. 6, 8 and 9)(Watkins et al., 2001, 2002, 2003a,b; Chrencik et al., 2005; Orans et al., 2005; Xue et al., 2007a,b; Teotico et al., 2008;Wang et al., 2008).

The ligand-binding cavity of the hPXR-LBD is largely hydrophobic and is lined by 28 amino acid residues. The bindingcavity volume is substantially larger than that of many other NRs (Kumar and Thompson, 1999; Ruff et al., 2000; Kumaret al., 2004; Chrencik et al., 2005) and is approximately double the size of the CAR-LBD cavity (Shan et al., 2004; Suinoet al., 2004; Xu et al., 2004), ranging from about 1200 Å3 in the absence of ligands (Watkins et al., 2001) to about 1600Å3 in the presence of rifampicin (Chrencik et al., 2005). Twenty cavity-lining residues are hydrophobic, four are polar(Ser208, Ser247, Cys284, and Gln285), and four are charged or potentially charged (Glu321, His327, His407, and Arg410).However, the salt bridge between Glu321 and Arg410 neutralizes their charged character, so that the inner surface of theligand-binding cavity is relatively uncharged and hydrophobic (Figs. 8 and 9) (Watkins et al., 2001; Orans et al., 2005;Xue et al., 2007a,b; Teotico et al., 2008; Wang et al., 2008).

The Ser192-Lys210 stretch, the flexible loop Ala229-Glu235, and the mobile hydrophobic loop Glu309-Glu321 (Fig. 8)are highly flexible and critical to recognize compounds of varying size and shape. Residues Ala229-Glu235 support theposition of the Ser192-Lys210 region, and their flexibility mirrors that of the longer region nearby. The Glu309-Glu321loop is linked to the ligand-binding cavity of hPXR by a non-solvent-accessible pore. These three regions cluster to-gether to create a flexible floor at the bottom of the hPXR-LBD ligand-binding cavity. Such a mobile floor is structurallyunique to the PXRs and appears critical to the promiscuous ligand-binding character of these xenosensors (Watkinset al., 2001, 2003a,b, Chrencik et al., 2005; Orans et al., 2005; Xue et al., 2007a,b; Teotico et al., 2008; Wang et al.,2008).

Residues Met243, Ser247, Gln325, Trp339, His446, and Phe459 are pivotal for ligand binding to hPXR. The mutation ofresidues Met243, Gln285, and His407 is at the root of the lesser degree of promiscuity of the mPXR (Moore et al., 2002).

hPXR-LBD a2 may function as a trapdoor, dropping out of the way so that ligands can enter the binding pocket. In somehPXR-LBD structures, a solvent-accessible channel of up to 3 Å wide and 9 Å long is present in the area adjacent to a2 (Wat-kins et al., 2001, 2003a,b; Orans et al., 2005).

In NR LBDs, the AF-2 region binds the Leu-Xxx-Xxx-Leu-Leu motifs in transcriptional co-activators and the Ile/Leu-Xxx-Xxx-Ile/Val-Ile motifs in co-repressors (McInerney et al., 1998; Hu and Lazar, 1999). The Leu residues in transcriptional co-activators pack via hydrophobic contacts against the surface of hPXR in a groove formed by a3, a4, and aAF. A charge clampinvolving PXR residues Lys259 and Glu427 stabilizes the weak helix dipole at the C- and N-terminus, respectively, of the Leu-Xxx-Xxx-Leu-Leu motif (Fig. 9) (Watkins et al., 2003a; Xue et al., 2007a, 2008).

Numerous single-site mutations have been introduced into the PXR-LBD with varying effects on transcriptional activity.Some of these mutations, including Ser247Trp, Trp299Ala, His407Asn, and Arg410Ala, lead to receptor variants thatexhibit an increased constitutive activity (Chrencik et al., 2005). Conversely, the mutation of charged residues (Asp205Ala,Glu321Ala, Arg410Asn, and Arg413Ala) appears to facilitate increased co-repressor or decreased co-activator binding,causing the partial or complete loss of the constitutive activity (see Orans et al., 2005).

The hPXR-LBD homodimer interaction is mediated largely by interdigitating aromatic residues from the b10 strand in eachmonomer (Fig. 9) (Watkins et al., 2003a; Xue et al., 2007a,b; Wang et al., 2008).



2.2.3.2.1. SR12813 recognition by hPXR-LBD. The high-affinity cholesterol-lowering drug SR12813 binds to hPXR-LBD in threedistinct modes (named positions 1, 2, and 3) (Fig. 11). Each orientation forms distinct interactions with residues that line theligand-binding cavity of hPXR. In particular: (i) position 1 makes van der Waals contacts with eight side chains and forms onehydrogen bond with Ser247; (ii) position 2 makes van der Waals contacts with seven side chains and forms one hydrogenbond with His407; and (iii) position 3 makes van der Waals contacts with six side chains, forms two hydrogen bonds with

Fig. 11. Binding mode of SR12813 (A; PDB code: 1ilh), hyperforin (B; PDB code: 1m13), T0901317 (C; PDB code: 2o9i), colupulone (D; PDB code: 2qnv), andrifampicin (E; PDB code: 1skx) to hPXR-LBD. For details, see Figs. 8 and 10 and the text.



Ser247 and Gln285, and forms a water-mediated hydrogen bond with Ser208. Of the 19 residues involved in contacting thedifferent orientations of SR12813, only Phe288 interacts with all three ligand conformations (Fig. 11). Two salt bridges, be-tween Glu321 and Arg410 and between Asp205 and Arg413, occur across the region of the hPXR-LBD ligand-binding pocketthat is closest to the receptor surface. As highlighted by site-directed mutagenesis, substitution of Asp205, Glu321, andArg413 to Ala reduces the constitutive (i.e., ligand independent) transcriptional activity of hPXR, whereas changingArg410 to Ala determines the increase in the hPXR constitutive activity. Lastly, the aAF helix of hPXR-LBD is in an active ori-entation and appears permissive to bind co-activators (Watkins et al., 2001).2.2.3.2.2. Hyperforin recognition by hPXR-LBD. Hyperforin, the psychoactive agent found in the herbal antidepressant St.John’s Wort, contacts a total of 12 amino acid side chains within hPXR-LBD (Fig. 11), inducing the volume increase of theligand-binding pocket by about 250 Å3 (Watkins et al., 2003b). This is generated by the movement of Leu209 and His407side chains and of the Cys207-Lys210 and Asp230-Glu235 main chain (Watkins et al., 2003b). A number of studies havedemonstrated that St. John’s Wort extracts are as effective as conventional medications for mild-to-moderate depression(Wheatley, 1997; Harrer et al., 1999; Schrader, 2000; Woelk, 2000; Szegedi et al., 2005). However, safety concerns have arisenover the concurrent use of St. John’s Wort with other medications after the publication of numerous case studies implicatingSt. John’s Wort extracts in pharmacokinetic drug interactions (Ruschitzka et al., 2000; Turtoon-weeks et al., 2001).

The hPXR-LBD:hyperforin complex exhibits peculiar structural features. A pseudo-helical region comprising residuesLys198-Lys210 begins the ordered portion of the structure after the Leu178-Trp199 disordered region. In addition, a mobilehydrophobic loop encompassing residues Leu309-Glu321 adopts a helical conformation (from Gln317 to Glu321) and is de-noted a6. The a6 helix in hPXR-LBD is distinct from a6 observed in other NRs (Bourguet et al., 1995; Brzozowski et al., 1997;Nolte et al., 1998; Williams and Sigler, 1998; Oberfield et al., 1999; Xu et al., 1999; Egea et al., 2000; Matias et al., 2000;Rochel et al., 2000; Orans et al., 2005; Renaud et al., 2005; Wagner et al., 2005; Ascenzi et al., 2006) as it is perpendicularto a7 (Watkins et al., 2003b). In other NRs, the a6 helix occupies the same space as the Lys198-Lys210 pseudo-helical regionin hPXR-LBD. Thus, the binding of hyperforin causes two loops adjacent to the ligand-binding pocket to adopt peculiar con-formations relative to hPXR-LBD structures (Watkins et al., 2003b).

As reported for other hPXR-LBD structures (Watkins et al., 2001, 2003a; Chrencik et al., 2005; Orans et al., 2005; Xue et al.,2007a,b; Teotico et al., 2008; Wang et al., 2008), hyperforin does not directly interact with the aAF helix, which is positionedin an active orientation permissive for co-activators binding. The activation of hPXR can be changed by introducing mutationof amino acid residues altering the size of the ligand-binding pocket (Watkins et al., 2003b).

The single-site mutations Trp299Met and His407Gln generate forms of hPXR that are less responsive to hyperforin rela-tive to wild-type hPXR. In contrast, the Phe288Ala mutant appears more responsive to hyperforin. These results indicate thatsmall changes within the hPXR-LBD-binding pocket can have large effects on the ligand-dependent activation of this recep-tor (Watkins et al., 2003b).2.2.3.2.3. Rifampicin recognition by hPXR-LBD. hPXR-LBD binds the macrolide antibiotic rifampicin (Fig. 11) (Chrencik et al.,2005), a drug widely used to treat tuberculosis and patients exposed to meningococcal disease and Haemophilus influenzaemeningitis (Vesely et al., 1998; Yogev and Guzman-Cottrill, 2005; Fraser et al., 2006; Prasad and Karlupia, 2007).

Rifampicin contacts 18 amino acid side residues (including Val211, Leu239, Leu308, and Arg410). This reflects thelarge size of the macrolide antibiotic that occupies regions of the hPXR-LBD-binding pocket not filled by smaller ligands(e.g., colupulone). Moreover, rifampicin forms hydrogen bonds with Ser247, Gln285 and His407. Surprisingly, bothGln285Ile and His407Gln mutations do not impact significantly drug binding to hPXR-LBD. However, the His407Glnmutation moderately increases the constitutive activity level of hPXR. Moreover, the Arg410Asn mutation reducesthe activation of hPXR by rifampicin. On the contrary, the Asp205Ala mutation improves the activation of hPXR by rif-ampicin, but eliminates the constitutive activity of the receptor. Furthermore, Ser247Trp and Trp299Ala mutations lar-gely eliminate hPXR response to rifampicin, without affecting the transcriptional activity. Indeed, Ser247 and Trp299 arepivotal for ligand recognition, forming hydrogen bonds or hydrophobic contacts. Lastly, residues Glu309-Glu321 adoptan a-helical structure upon rifampicin binding. These features in hPXR combine to generate a flexible and conformableligand-binding pocket that adjusts its shape to accommodate ligands of distinct size and structure (Chrencik et al.,2005).2.2.3.2.4. Colupulone recognition by hPXR-LBD. The three-dimensional structure of monomeric hPXR-LBD bound to the b-bit-ter acid colupulone is shown in Fig. 11 (Teotico et al., 2008). Colupulone, extracted from the flowers of the hops plant Humuluslupulus, has been reported to have antibacterial properties and to inhibit tumor cell proliferation (Mannering et al., 1993).

Colupulone binds to hPXR-LBD in a single orientation stabilized by van der Waals and hydrogen-bonding contacts involv-ing 13 residues. Note that residues Phe420 and Met425 belong to the aAF helix of the hPXR-LBD AF-2 region. A direct hydro-gen bond is formed between a colupulone hydroxyl and His407; moreover a second water-mediated hydrogen bond isobserved between another colupulone hydroxyl group and Gln285 (Teotico et al., 2008).2.2.3.2.5. 17b-Estradiol recognition by hPXR-LBD. The hPXR-LBD binds the sexual hormone 17b-estradiol (E2), the xenosensorhomodimerization being mediated by the extended b-sheet including b1 (Xue et al., 2007b).

E2 contacts eight amino acid residues, including Met243, Phe251, Leu411, Phe420, Met425, and Phe429, and forms hydro-gen bonds with Ser247 and Arg410. In particular, the 3-hydroxyl group on the steroid A-ring forms a hydrogen bond withSer247, whereas the 17b-hydroxyl group on the D-ring forms a hydrogen bond with Arg410. Moreover, Arg410 is stabilizedby a hydrogen bond with Ser208, which itself is close to the oxygen atom of the ligand 17b-hydroxyl group, and with the sidechain of Glu321 (Xue et al., 2007b).



Three single-site mutations, Ser208Ala, Cys284Ser, and Arg410Leu produce moderate changes in the constitutive activitylevels for hPXR. Moreover, the replacement of Ser247 with Ala significantly reduces the E2 affinity. The simultaneousreplacement of Met425 and Phe429 with Ala produces a form of hPXR that is completely unresponsive to ligands (Xueet al., 2007b).

The very low affinity of E2 for hPXR-LBD (EC50 = 22,000 nM) (see Sections 3 and 4) if compared with that for the estrogenreceptor-a (ERa; EC50 = 1 nM) reflects the different positioning of the hormone within the receptor ligand-binding pocket.Indeed, E2 binds closely adjacent to the aAF helix in PXR, leaving a notable 1000 Å3 of space unoccupied. In contrast, E2is more centrally located in the ERa pocket, which is oriented nearly perpendicular to that observed in the hPXR cleft, occu-pying almost all the space in the central pocket. Moreover, while hPXR-LBD forms only eight van der Waals interactions andtwo hydrogen bonds with E2, ERa forms 12 non-polar contacts and three hydrogen bonds (Xue et al., 2007b). The very lowaffinity of E2 for hPXR-LBD and the low level of the hormone in vivo (=1 nM) explain the negligible role of E2 on hPXR action(see Xue et al., 2007b).2.2.3.2.6. SR12813 and SRC-1p2 recognition by the hPXR homodimer. The hPXR-LBD homodimer binds SR12813 and a 25 ami-no acid residue fragment of the human steroid receptor co-activator-1 (SRC-1p2) containing one Leu-Xxx-Xxx-Leu-Leu motif(Fig. 9). SRC-1p2 was either non-covalently bound or fused to hPXR-LBD (Watkins et al., 2003a; Wang et al., 2008). A singlepolypeptide chain encompassing hPXR and SRC-1p2 tethered with a peptidyl linker was designed to promote intramolecularcomplex formation (Wang et al., 2008).

The SRC-1p2 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 region ofhPXR-LBD. The C-terminal sequence including the Leu-Xxx-Xxx-Leu-Leu motif forms an a-helix that binds to a groove on thesurface of hPXR-LBD composed of residues from a3, a4, and aAF. The Leu residues of the Leu-Xxx-Xxx-Leu-Leu motif areburied in this groove and pack adjacent to the body of hPXR-LBD. Two polar contacts occur between hPXR-LBD and theLeu-Xxx-Xxx-Leu-Leu a-helix: (i) between Lys259 of hPXR-LBD a3 and the carbonyl oxygen atom of the SRC-1p2 Leu694residue, and (ii) between Glu427 of hPXR-LBD aAF and the amine nitrogen atom of the SRC-1p2 Ile689 residue. Moreover,residues Ser682-Arg686 of SRC-1p2 form an additional turn of a-helix, kinked perpendicular to the Leu-Xxx-Xxx-Leu-Leua-helix and contacting a4 of hPXR-LBD. The SRC-1p2 Leu683 residue contacts both Glu270 and Ile273 on a4 of hPXR-LBD, and the SRC-1p2 His687 residue forms a hydrogen bond with the hPXR-LBD Lys277 residue. Moreover, the aAF helixof hPXR-LBD appears to have increased flexibility at its N-terminus, but it is more rigid at its C-terminus, which containsresidues that contact SRC-1p2 directly, including Glu427 (Fig. 9) (Watkins et al., 2003a).

SRC-1p2 binding to hPXR-LBD in complex with SR12813 increases the size of the binding cavity restructuring a2 in a no-vel conformation that opens a solvent-accessible channel gated by Leu209. This channel stretches along the loops joining a3to b10 and a2 to b1. While this channel is continuous and increases the volume of the ligand-binding cavity, the portion of thebinding cavity that contacts the ligand actually contracts around the singular orientation of SR12813, making this region ofthe binding cavity significantly smaller. In contrast with the structure of the hPXR-LBD:SR12813 complex, in which the li-gand is bound in multiple orientations (Watkins et al., 2001), the contracted binding pocket observed in the presence of SRC-1p2 binds SR12813 in a single orientation contacting the AF-2 region (Watkins et al., 2003a).

The SR12813 ligand makes hydrophobic contacts with eleven amino acid side chains that line the hPXR-LBD ligand-bind-ing cavity (Fig. 11). In addition, the ligand forms two hydrogen bonds with Ser247 and His407, and a p-stacking interactionbetween the O11 hydroxyl oxygen atom of SR12813 and the aromatic indole ring of Trp299. Nearly all amino acid sidechains, with the exception of Leu209, remain in relatively fixed positions in both the hPXR-LBD:SR12813:SRC-1p2 andhPXR-LBD:SR12813 complexes. In the presence of SRC-1p2, SR12813 makes van der Waals interactions with the aAF helixtriggering the ligand-dependent receptor activation (Watkins et al., 2003a).

Binding of SR12813 does not significantly affect the geometry of the SRC-1p2 cleft, including the AF-2 region in the fusedhPXR-LBD/SRC-1p2 protein (Wang et al., 2008). Moreover, the shape of the ligand-binding cavity in the tethered structures(Wang et al., 2008) closely resembles that of the non-covalent hPXR-LBD:SR12813:SRC-1p2 complex (Watkins et al., 2003a).

No significant changes at or near the SRC-1p2 cleft, including the AF-2 region, have been observed when homodimerichPXR-LBD bound to SRC-1p2 is compared to hPXR-LBD in complex with both SRC-1p2 and SR12813 (Watkins et al., 2001,2003a; Wang et al., 2008).

Lastly, the Ser192-Lys210 stretch is folded into a2 in the structures of hPXR-LBD bound to SR12813 and the SRC-1p2 co-activator peptide. Moreover, residues Glu309-Glu321 adopt an a-helical structure in complexes with SR12813 and SRC-1p2(Watkins et al., 2001, 2003a; Wang et al., 2008).2.2.3.2.7. T0901317 and SRC-1p3 recognition by the hPXR homodimer. The hPXR-LBD homodimer binds T0901317 in theabsence and presence of the second NR box of the human steroid receptor co-activator-1 (SRC-1p3) containing oneLeu-Xxx-Xxx-Leu-Leu motif. The NR box is associated with the AF-2 region of hPXR-LBD. The homodimer interaction ismediated largely by interdigitating aromatic residues from b10 in each monomer (Xue et al., 2007a).

T0901317 forms three polar and twelve van der Waals contacts with amino acid side chains that line the hPXR-LBD li-gand-binding pocket (Fig. 11). In particular, Tyr306 forms an edge-to-face with the benzyl ring of T0901317; moreover,the same benzyl ring forms parallel and edge-to-face aromatic stacking interactions with both Phe288 and Trp299. An aro-matic contact occurs also between His407 and Phe429, located on the aAF helix of the PXR-LBD AF-2 region. The two -CF3

groups of T0901317 form van der Waals contacts with five residues, including Met425, located on the hPXR-LBD aAF helix.The hPXR-LBD:T0901317 contacts involving residues belonging to the aAF helix help to stabilize the active conformation ofthe AF-2 region (Xue et al., 2007a). Only a small number of shifts in the positions of the amino acid residues that line the



ligand-binding pocket of hPXR-LBD were observed between the T0901317-bound and the apo structures (Watkins et al.,2001; Wang et al., 2008).

3. CAR and PXR expression and activation

3.1. CAR and PXR expression patterns

Due to their role in protecting living organisms from exogenous insults, the expression of xenosensors CAR and PXR at thepoints of xenobiotic entry and metabolism into the body is not unexpected. In fact, CAR is highly expressed in the liver and inthe epithelial cells of the small intestine villi; moreover, CAR is expressed at low levels in both mouse and human heart, skel-etal muscle, brain, kidney, and in human lung (Baes et al., 1994; Choi et al., 1997; Doherty and Charman, 2002; Swales andNegishi, 2004). However, the physiological role of CAR in these tissues has not been investigated thoroughly. Note that maleWistar-Kyoto rats contain a more elevated CAR protein levels compared to female rats (Yoshinari et al., 2001). The occur-rence of similar gender-related differences in other mammalian species is currently unknown.

PXR is primarily expressed in liver, intestine, and kidney (Kliewer et al., 1998). Expression in other tissues includes lung,stomach, peripheral blood monocytes, the blood–brain barrier, uterus, ovary, placenta, breast, osteoclasts, heart, adrenalgland, bone marrow, and specific regions of the brain (Bauer et al., 2004; Lamba et al., 2004a,b). This wide expression patterndesignates PXR as part of a protective system to prevent exposure of critical cells sensitive to aberrant levels of exogenous orendogenous compounds. Interestingly, PXR levels in the mouse liver and ovary increase approximately 50-fold during preg-nancy (Masuyama et al., 2001). These data suggest that PXR expression may be induced by pregnancy hormones and, fur-thermore, raise the intriguing possibility that PXR is involved in protecting the fetus and/or mother from eitherxenobiotics or high levels of endobiotics (Kliewer et al., 2002).

Although the PXR promoter has not yet been characterized, it has been reported that dexamethasone increases PXRexpression in human hepatocytes and rat hepatoma cells (Huss and Kasper, 2000; Pascussi et al., 2000). Since this effectis blocked by the GR antagonist RU486 (i.e., mifepristone), it appears that PXR expression is GR dependent (Kliewer et al.,2002; Matic et al., 2007).

3.2. CAR and PXR ligands

Historically, ligands of NRs have been identified in cell-based reporter assays, in which an expression plasmid encodingthe receptor of interest is co-introduced into cells with a reporter gene plasmid (Kliewer et al., 2002). This assay enabled theidentification of a variety of compounds that can activate both CAR and PXR, but this method appears somewhat misleadingwhen approaching CAR and PXR. As an example, the typical CAR activator phenobarbital (PB) does not bind directly to CAR,but activates nuclear translocation and successively CAR transcriptional activity (Moore et al., 2000a). However, ligand-bind-ing techniques indicate that CAR and PXR bind a structurally diverse panel of chemicals (Fig. 1) and the receptor ligand spec-ificity is species dependent (Table 6). CAR and PXR, in analogy with other ‘‘adopted” NRs that play a role in responsiveness todietary lipids (i.e., PPAR), oxysterols (i.e., LXR), and bile acids (i.e., farnesoid X receptor; FXR), have lower ligand affinity thanthe classical steroid hormone receptors (e.g., ERs) (Ascenzi et al., 2006; Tompkins and Wallace, 2007; Graham and Lake,2008; Lim and Huang, 2008; Ma and Lu, 2008; Moreau et al., 2008; Pascussi et al., 2008; Teng and Piquette-Miller, 2008;Köhle and Bock, 2009). Indeed, EC50 values for ligand binding to CAR and PXR are in the micromolar range compared withthe nanomolar range of classical steroid hormone receptors. This has led to a revision in thinking about the receptor-med-iated ligand recognition properties to accommodate the concept of low affinity/specificity but high capacity to induce cellsignaling (Tzameli and Moore, 2001).

The orphan nature of CAR generated a search for an endogenous ligand, especially in the light of the receptor apparentconstitutive activity (see Sections 2.2.2, 2.2.3 and 3.3). Two androstane metabolites, 5a-androst-16-en-3a-ol (androstenol)and 5a-androstan-3a-ol (androstanol) were identified as CAR ligands, but they repress the NR constitutive activity (Swalesand Negishi, 2004). Thus, an endogenous ligand with high affinity for CAR is still unknown and CAR should be considered anorphan receptor with many endogenous ligands, such as the above-mentioned androstane metabolites and 5b-pregnane-3,20-dione, and exogenous ligands, such as chlorpromazine, clotrimazole, o,p0-DDT, hydrocarbons (such as 2,3,30,40,50,6-hexa-chlorobiphenyl, CITCO, and TCPOBOP), methoxychlor, and retinoic acid (Tzameli et al., 2000; Sueyoshi and Negishi, 2001;Willson and Kliewer, 2002; Maglich et al., 2003; Timsit and Negishi, 2007). The ligand affinity for CAR varies across speciesand in some cases ligand binding to CAR still remains a matter of debate; TCPOBOP and CITCO are the only compoundsshown to bind specifically to mCAR and hCAR, respectively (Table 6).

PXR seems to be much more promiscuous than CAR (Moore et al., 2003). It is now established that PXR binds nearly allxenobiotics. These chemicals include prescription drugs such as dexamethasone, indinavir, paclitaxel, pregnenolone16a-carbonitrile (PCN), taxol, SR12813, and rifampicin. Notably, RU486, a GR and PR inhibitor, is also used for the medicaltermination of pregnancy (Christin-Maitre et al., 2000). The list of PXR ligands also includes pesticides (e.g., chlordane andtrans-nonachlor) and environmental contaminants (e.g., polychlorinated biphenols and nonylphenol) (Lemaire et al., 2006).However, the xenobiotic definition comprises not only the afore-mentioned anthropic-derived compounds (i.e., drugs andpollutants), but also natural occurring compounds. Like drugs and pollutants, natural compounds are able to activate



Table 6Ligand affinity for CAR and PXR.

Ligand Receptor EC50–IC50–Ki (nM) Notes References

16,17-Androstane-3-ol mCAR EC50 = 500 Shan et al. (2004)5a-Androstan-3a-ol hCAR EC50 = 1000–>10,000 Inverse agonist Moore et al. (2000a)

mCAR EC50 = 250–1500 Inverse agonist Moore et al. (2000a)Tzameli et al. (2000)Forman et al. (1998)

5a-Androst-16-en-3a-ol mCAR EC50 = 400–�5000 Inverse agonist Forman et al. (1998)Kawamoto et al. (2000)

CITCO hCAR EC50 = 25–304 Maglich et al. (2003)Huang et al. (2003)

Clotrimazole hCAR EC50 = 50–�1000 Inverse agonist Maglich et al. (2003)Huang et al. (2003)Moore et al. (2000a)

Di(2-ethylhexyl) phthalate hCAR EC50 = 211 DeKeyser et al. (2009)17b-Estradiol mCAR EC50 = 1000 Kawamoto et al. (2000)Estrone mCAR EC50 = 1000 Kawamoto et al. (2000)Meclizine hCAR EC50 = �500–1000 Inverse agonist Huang et al. (2003)

mCAR EC50 = 25 Huang et al. (2003)(5b)-Pregnane-3,20-dione hCAR EC50 = 670–3000 Moore et al. (2000a)

Maglich et al. (2003)mCAR EC50 = >10,000 Weak agonist Kawamoto et al. (2000)

�10,000 Inverse agonistProgesterone mCAR EC50 = �3000 Inverse agonist Kawamoto et al. (2000)TCPOBOP mCAR EC50 = 20–100 Suino et al. (2004)

Moore et al. (2000a)Tzameli et al. (2000)

Testosterone mCAR EC50 = �7000 Inverse agonist Kawamoto et al. (2000)Artemisinin hPXR EC50 = 5000–34,000 Burk et al. (2005)

Persson et al. (2006)Betamethasone hPXR EC50 = 20,000 Persson et al. (2006)Carbamazepine hPXR EC50 = 15,600 Persson et al. (2006)5b-Cholestan-3a,7a,12a-triol hPXR EC50 = 5000 Goodwin et al. (2003)

mPXR EC50 = 2500 Goodwin et al. (2003)CITCO hPXR EC50 = �3000 Maglich et al. (2003)Clotrimazole hPXR EC50 = 800–5000 Moore et al. (2000a)

Lehmann et al. (1998)Bertilsson et al. (1998)

mPXR EC50 = 1000 Moore et al. (2000a)Colupulone hPXR EC50 = 10 Teotico et al. (2008)Corticosterone hPXR EC50 = 30,000 Blumberg et al. (1998)

Kliewer et al. (1998)Coumestrol hPXR EC50 = 25,000 Blumberg et al. (1998)Dexamethasone hPXR EC50 = 5000–�10,000 Lehmann et al. (1998)

Persson et al. (2006)Moore et al. (2000a)

mPXR EC50 > 10,000 Moore et al. (2000a)Dexamethasone-t-butylacetate mPXR EC50 = 800 Kliewer et al. (1998)6,16a-Dimethylpregnenolone mPXR EC50 = 300 Kliewer et al. (1998)Ecteinascidin-743 hPXR IC50 = 3 Antagonist Synold et al. (2001)17b-Estradiol hPXR EC50 = 22,000–30,000 Xue et al. (2007a)

Blumberg et al. (1998)Ferutinine hPXR EC50 = 1800 Mnif et al. (2007)17-Hydroxy-pregnenolone hPXR EC50 > 10,000 Lehmann et al. (1998)

mPXR EC50 = 5000–20,000 Lehmann et al. (1998)Kliewer et al. (1998)

17-Hydroxy-progesterone hPXR EC50 > 10,000 Lehmann et al. (1998)mPXR EC50 = 5000–20,000 Lehmann et al. (1998)

3a-Hydroxy-5b-pregnane-3,20-dione-methansulphonate hPXR EC50 = 1000 Bertilsson et al. (1998)Hyperforin hPXR EC50 = 23 Moore et al. (2000b)

Ki = 27 Watkins et al. (2003b)Persson et al. (2006)

Indomethacin hPXR EC50 = 17,000 Persson et al. (2006)Lansoprazole hPXR EC50 = 3000 Persson et al. (2006)Lovastatin hPXR EC50 = 1000 Lehmann et al. (1998)Mono(2-ethylhexyl) phthalate hPXR EC50 = 8000 Hurst and Waxman (2004)Nifedipine hPXR EC50 = 4300 Bertilsson et al. (1998)Omeoprazole hPXR EC50 = 8600 Persson et al. (2006)Paclitaxel hPXR EC50 = 5000 Synold et al. (2001)Pantoprazole hPXR EC50 = 6800 Persson et al. (2006)PCN hPXR EC50 > 10,000 Moore et al. (2000a)

mPXR EC50 = 200–�700 Moore et al. (2000a)



metabolic pathways for detoxification. These compounds, produced by plants and fungi, face mammals only through dietaryintake (Galluzzo and Marino, 2006). Among diverse herbal derivatives, colupulone and hyperforin serve as PXR ligands (seeSections 2.2.3.2.2 and 2.2.3.2.4) together with the sesquiterpenoid ferutinine and the mycotoxin zearalenone which havebeen demonstrated to induce hPXR transcriptional activity (EC50 = 1800 nM and 1000 nM for ferutinine and zearalenone,respectively) (see Table 6) (Mnif et al., 2007). On the other hand, other plant-derived chemicals such as the flavonoids for-mononetin, genistein, apigenin, and kaempferol do not affect hPXR action (Mnif et al., 2007). Other PXR ligands includeendogenous steroids such as corticosterone, 17a-hydroxypregnenolone, 17a-hydroxyprogesterone, 5a-pregnane-3,20-dione, and progesterone as well as bile acids.

Notably, there are marked differences in the pharmacological activation profiles of PXR derived from different species. Infact, the mouse and rat PXRs are efficiently activated by PCN but not by rifampicin. On the contrary, rifampicin and SR12813,but not PCN, are specific for hPXR (Goodwin et al., 2002; Kliewer et al., 2002; Timsit and Negishi, 2007). Interestingly, someof these PXR ligands could also act as agonists of CAR (Schuetz et al., 1998; Masuyama et al., 2000; Blizard et al., 2001;Takeshita et al., 2001; Coumoul et al., 2002; Moore et al., 2000a; Landes et al., 2003; Makinen et al., 2003; Raucy, 2003;Tippin et al., 2003; Honkakoski et al., 2004; Lemaire et al., 2004; Kobayashi et al., 2005; Mani et al., 2005; Masuyamaet al., 2005; Timsit and Negishi, 2007; Zimber and Gespach, 2008).

Many of the diverse chemicals that activate CAR and PXR directly bind the receptor LBD (Kliewer et al., 2002). However,some of these ligands may also bind the LBD of other NRs such as ERs, FXR, LXR, AR, GR, VDR as well as MR, PR, and THR, thus,enlarging the body response to the potential toxicity of endobiotics and xenobiotics (Kretschmer and Baldwin, 2005; Ascenziet al., 2006; Galluzzo and Marino, 2006). However, CAR and PXR are capable to bind a remarkably diverse collection of chem-icals with molecular weights ranging from less than 250 kDa to more than 800 kDa. This promiscuity, unprecedented in theNR family, is warranted by the structural properties of CAR and PXR-LBDs (see Section 2.2.3). Moreover, in contrast to the

Table 6 (continued)

Ligand Receptor EC50–IC50–Ki (nM) Notes References

Kliewer et al. (1998)Pregnenolone mPXR EC50 = 5000–20,000 Kliewer et al. (1998)Progesterone mPXR EC50 = 5000–20,000 Kliewer et al. (1998)(5b)-Pregnane-3,20-dione hPXR EC50 > 10,000 Lehmann et al. (1998)

Lemaire et al. (2004)Kliewer et al. (1998)

mPXR EC50 = 5000–20,000 Lehmann et al. (1998)Kliewer et al. (1998)

Phenobarbital hPXR EC50 = 169,000–370,000 Lemaire et al. (2004)Persson et al. (2006)

Phenytoin hPXR EC50 = 8000 Persson et al. (2006)Primaquine hPXR EC50 = 13,600 Persson et al. (2006)Rabeprazole hPXR EC50 = 1500 Persson et al. (2006)Rifampicin hPXR EC50 = 200–3000 Chrencik et al. (2005)

Moore et al. (2000a)Lehmann et al. (1998)Persson et al. (2006)Lemaire et al. (2004)Bertilsson et al. (1998)Jones et al. (2000)Lehmann et al. (1998)

RU486 hPXR EC50 = 5500–10,000 Kliewer et al. (1998)Moore et al. (2000a)Lehmann et al. (1998)

mPXR EC50 = 1000–�20,000 Kliewer et al. (1998)Moore et al. (2000a)

(�)S20 hPXR ‘‘Weak” Mu et al. (2005)mPXR ‘‘Active” Mu et al. (2005)

(+)S20 hPXR ‘‘Active” Mu et al. (2005)mPXR ‘‘Weak” Mu et al. (2005)

Schisandrin hPXR EC50 = 2000 Mu et al. (2006)mPXR EC50 = 1250 Mu et al. (2006)

SR12813 hPXR EC50 = 120–200 Moore et al. (2000a)Ki = 41 Watkins et al. (2001)

mPXR EC50 = 4100 Moore et al. (2000a)T0901317 hPXR EC50 = 13 Xue et al. (2007b)TCPOBOP hPXR EC50 = 3900 Moore et al. (2000a)Troglitazone hPXR EC50 = �3000 Jones et al. (2000)

Persson et al. (2006)Warfarin hPXR EC50 = 49,500 Persson et al. (2006)Verapamil hPXR EC50 = 3200 Persson et al. (2006)Zearalenone hPXR EC50 = 1000 Mnif et al. (2007)



other NRs that bind few ligands with high affinity, PXR has evolved the ability to respond to a diverse set of low-affinity li-gands. The affinity of some ligands for CAR and PXR is reported in Table 6.

3.3. CAR and PXR activation

3.3.1. CAR activationIn mouse liver, CAR is cytoplasmic in the naïve state and translocates to the nucleus upon activation. An additional feature

recently uncovered for CAR activation includes its translocation to the membrane (Choi et al., 2005), which raises the pos-sibility of signaling components important in CAR activation located at the cell membrane (Swales and Negishi, 2004).

In contrast to AhR, which is triggered by direct ligand binding, nuclear translocation of CAR does not necessarily requirethe direct binding of ligands. In fact, PB and bilirubin have been shown to be CAR activators, but they do not appear to inter-act directly with the CAR-LBD (Moore et al., 2000a; Swales and Negishi, 2004) (Table 6). The potent PB-like activatorTCPOBOP binds directly to mCAR and translocates the receptor into the nucleus (Kawamoto et al., 1999). On the other hand,although hCAR does not appear to bind TCPOBOP, this xenosensor is translocated into the nucleus in the liver of humanizedmouse in which hCAR is over-expressed (Swales and Negishi, 2004). Therefore, activators can promote the nuclear translo-cation of CAR by a ligand-independent process, regardless of their receptor-binding ability.

Also de-phosphorylation is involved in regulating CAR translocation. In fact, okadaic acid, a protein phosphatase inhibitor,blocks the PB-induced target gene expression in rodents (Sidhu and Omiecinski, 1997) and prevents nuclear accumulation ofCAR (Honkakoski and Negishi, 1998). Co-overexpression of phosphatase 2A (PP2A) and mCAR enhances TCPOBOP-inducedmCAR nuclear translocation in hepatoma cells. Thus, de-phosphorylation of CAR constitutes a crucial signal for its releasefrom the cytosolic complex upon activator treatment; however, which kinase(s) phosphorylates CAR remains unknown(Timsit and Negishi, 2007). In addition, hCAR translocation occurs without a strong nuclear localization sequence signal(NLS) (Kawana et al., 2003) and is AF-2 independent (Zelko et al., 2001); however, the Leu-rich sequence localized withinthe a10 helix (see Section 2.2.3 and Fig. 6) is requested (Zelko et al., 2001; Squires et al., 2004; Swales and Negishi,2004). Notably, the Leu-rich sequence is also present in hPXR a10 helix (see Section 2.2.3 and Fig. 6). CAR is sequesteredin the cytosol in a multi-protein complex which includes a recently identified protein termed CAR cytoplasmic retention pro-tein (CCRP, Dnajc7 in the NCBI data base) and the heat shock chaperone Hsp90 (Kobayashi et al., 2003; Timsit and Negishi,2007). CCRP overexpression in human hepatoma cells, indeed, facilitates CAR accumulation in the cytosol; therefore, uponTCPOBOP stimulation, a more pronounced nuclear translocation of the xenoreceptor occurs. This highlights the role playedby CCRP in the CAR nuclear translocation mechanism (Timsit and Negishi, 2007).

In addition, the Hsp90:CAR complex also recruits PP2A which catalyzes mCAR Ser202 de-phosphorylation (Yoshinariet al., 2003). Note that Ser202Asp mutation, mimicking phosphorylation, abrogates the PB-induced nuclear accumulationof CAR; in contrast, the Ser202Ala mutation promotes CAR accumulation in the nucleus (Hosseinpour et al., 2006). Takentogether, these data revealed that the tight control of an otherwise constitutively active receptor is achieved by its cytosolicsequestration in vivo (Fig. 12).

Other proteins have been suggested to mediate CAR nuclear translocation. The p160 transcription factor GR-interactingprotein 1 (GRIP1) has been identified as a carrier assisting in CAR nuclear accumulation (Min et al., 2002a,b; Xia and Kemper,2005). Generally viewed as a NR co-regulator of the transcriptional activity, the overexpression of GRIP1 in the mouse liverenhances the PB-induced nuclear accumulation of CAR (Min et al., 2002a,b). However, questions remain as to the importanceof GRIP1 in CAR nuclear translocation, since attempts in other laboratories to recapitulate these effects have been unsuccess-ful (Hosseinpour et al., 2006; Timsit and Negishi, 2007). Interestingly, the PPAR-binding protein (PBP)/TRAP220/MED1 hasalso been suggested to play a role in CAR nuclear translocation. Using a mouse PBP knockout model, loss of PBP resultedin the abrogation of PB-induced CAR nuclear translocation, implicating PBP in the modulation of CAR activity (Jia et al.,2005). However, it remains to be determined, as for GRIP1, whether this is due to PBP either enhancing nuclear import orretaining CAR in the nucleus.

CAR nuclear translocation (i.e., activation) not necessarily leads to the increase of target gene transcription (Swales andNegishi, 2004). Translocation of CAR to the nucleus should be followed by CAR/RXRa association (i.e., heterodimerization),CAR-DBD binding to the RE, and recruitment of co-activators (e.g., apoptotic speck protein -2 (ASC-2), GRIP1/TIF2, PPAR gam-ma, co-activator-1 (PGC-1), structural maintenance of chromosome-1 (SMC-1), and SRC-1) (Kim et al., 1998; Muangmoonc-hai et al., 2001; Min et al., 2002a,b; Shiraki et al., 2003; Choi et al., 2005; Inoue et al., 2006). In this signaling paradigm,translocation of CAR into the nucleus serves as the first important step to regulate the xenosensor transcriptional activity(Timsit and Negishi, 2007).

Evidence is accumulating on the role of hormones and growth factors in regulating CAR activation and transcriptionalactivity. Glucocorticoids show stimulatory effects on CAR activity, this effect is mutual as CAR potentiates GR signaling. Thus,GR and CAR can synergize to induce target genes (Pascussi et al., 2001, 2003). The influence of glucocorticoids on the activityof CAR and PXR necessitates consideration in relation to physiological stress as a factor contributing to the response to xeno-biotics. Not only glucocorticoids, but also sex hormones might regulate CAR activity, this might explain the sexual dimor-phism in target gene induction by PB (Agrawal and Shapiro, 1996; Chang et al., 1997). Moreover, CAR has been shown torepress the ERa transcriptional activation by reducing the availability of co-activators (Min et al., 2002a,b). Furthermorewhile circulating thyroid hormone (TH) levels are regulated by CAR, TH does not appear to regulate CAR activity (Ganemet al., 1999). Hence, the cross-talk between THR and CAR signaling pathways is not mutual, in contrast to the cross-talk



observed between GR, ERa, and CAR (Timsit and Negishi, 2007). Lastly, the epidermal growth factor (EGF) represses the PB-induced CAR transcriptional activity (Kietzmann et al., 1999). The reduction in CAR transcriptional activity also appears to bemediated by the novel hepatotrophic growth factor, named ‘‘augmenter of liver regeneration” (Thasler et al., 2006).

3.3.2. PXR activationUnligated NRs may act as gene silencers. Indeed, some non-steroid NRs, such as RAR and THR, constitutively bind DNA

and silence target genes in an active manner by recruitment of co-repressors (Moehren et al., 2004). It has been suggestedthat also PXR may act as a gene silencer. Activation of PXR by ligands could result in the dissociation of co-repressors, such asthe silencing mediator of retinoid and thyroid receptors (SMRT) and of the nuclear receptor co-repressor (NcoR), allowing thebinding of the co-activators GRIP and SRC-1. While SMRT and NcoR stabilize chromatin and consequently repress transcrip-tion, SRC-1 and GRIP destabilize chromatin allowing the transcription machinery recruitment on DNA (Harmsen et al., 2007).

A completely different PXR-mediated signal transduction pathway raised from in vitro studies. In fact, recent results ofmouse liver section immunostaining suggest that mPXR may be located in the cytosol of untreated liver cells (Racz andBarsony, 1999). This indicates that PXR intracellular localization is similar to that of CAR (Squires et al., 2004). Nuclear trans-location of PXR appears to be regulated by a bipartite NLS located in the DBD (Kawana et al., 2003). Like CAR, mPXR appearsto be located in the cytosol in the resting state, and the AF-2 region appears to be involved in PCN-induced nucleartranslocation (Matic et al., 2007). Like CAR, PXR forms a protein complex with CCRP and Hsp90 which, in turn, increasethe cytosolic retention of PXR (Squires et al., 2004). Transient knockdown of CCRP by RNA-silencing technique attenuatesligand-induced PXR transcriptional activation. This outlines the importance of cytosolic sequestration in regulating thePXR transcriptional activity (Squires et al., 2004) (Fig. 13). Upon ligand binding to PXR, the xenosensor dissociates fromthe multi-protein complex and translocates to the nucleus to activate gene transcription as a heterodimer with RXRa(Goodwin et al., 1999; Frank et al., 2005). Increased transcription is mediated by recruitment of the p160 family co-activators(e.g., SRC-1 and GRIP), PBP, and PGC1-a (Ding and Staudinger, 2005a; Orans et al., 2005). On the other hand, the PXR tran-scriptional activity is inhibited by interactions with co-repressors including NCoR, receptor-interacting protein 140 (RIP140),short heterodimer partner (SHP), and SMRT (Orans et al., 2005).

The recognition of co-factors by PXR is governed by post-translational modifications of the receptor (i.e., phosphorylation/de-phosphorylation) and directly by the Leu-rich sequence of PXR (Matic et al., 2007). Protein phosphorylation plays animportant role in the regulation of NR functions, enabling integration of a variety of signals and rapid adaptation to environ-mental and physiological situations. hPXR can be phosphorylated by protein kinase A (PKA), resulting in strengthened inter-action with co-activators, such as SRC1 and PBP (Ding and Staudinger, 2005a). In contrast, the activity of PXR can berepressed by the activation of PKCa which alters the phosphorylation status of PXR and/or PXR-interacting proteins (i.e.,co-activators) (Ding and Staudinger, 2005a). In addition, okadaic acid, a protein phosphatase PP1/2A inhibitor, strongly

Fig. 12. Schematic illustration of the CAR activation. CAR resides in the cytosol in a multi-protein complex containing the CAR cytoplasmic retention protein(CCRP) and the heat shock protein 90 (Hsp90). After binding of the CAR ligand TCPOBOP, the protein phosphatase PP2A is recruited to the macromolecularcomplex. PP2A catalyzes the CAR Ser202 de-phosphorylation which, in turn, promotes CAR accumulation in the nucleus. Phenobarbital (PB) which is a CARactivator, but not a ligand, activates PP2A through a still unknown mechanism, thus promoting CAR translocation into the nucleus via a ligand-independentpathway. Translocation of CAR into the nucleus is followed by CAR/RXRa heterodimerization, CAR-DBD binding to the response element (RE) present in thetargets gene promoters, and the recruitment of co-activators. For details, see the text.



represses PXR transactivation (Ding and Staudinger, 2005b). Therefore, the (de-)phosphorylation state of PXR can modulateboth co-activator recruitment and xenosensor activation (Matic et al., 2007) (Fig. 13). This could reflect the ligand-indepen-dent activation of PXR.

Post-translational modifications of RXRa and of other co-factors may also impact on PXR activity. Phosphorylation of theRXRa Ser32 residue inhibits heterodimerization with PXR and several other NRs (Bruck et al., 2005; Mann et al., 2005; Maticet al., 2007). Furthermore, NR modification does not necessarily switch ‘‘on” or ‘‘off ” all subsequent transactivation events,but rather determines target gene selectivity for a given NR, as shown by phosphorylation of RXRa at Ser265 (Timsit andNegishi, 2007).

As a whole, nuclear translocation is an important step in the activation of CAR and PXR. However, these mechanisms havebeen assessed principally in mouse liver, thus their validity across the species remains to be elucidated.

3.4. Activation of NRs: the case of ERs

CAR and PXR, like all the members of the NR super-family, are modular proteins sharing common structural features (seeSection 2.2). Therefore, it is not surprising that NRs display common activation pathways. This point is highlighted by com-monalities of prototypical steroid hormone receptors ERs (both ERa and ERb) and CAR/PXR activation pathways.

CAR, PXR, and ERs (both ERa and ERb) activity is regulated in part by interaction with components of the cellular chap-erone machinery, such as Hsp90. In the classic model (Fig. 14), the unliganded ERs reside in the cytoplasm in a complex withHsp90 (Knoblauch and Garabedian, 1999). Ligand binding induces profound and rapid effects on the conformation of ERsallowing them to dimerize, producing both homodimers and heterodimers, and to translocate into the nucleus where spe-cific hormone REs present in DNA are recognized (Ascenzi et al., 2006). The ER–E2 complex can also function as a cytoplasmicsignaling molecule eliciting more rapid signal transduction pathways into the cells. Such extra-nuclear or non-genomic

Fig. 13. Schematic illustration of the PXR activation. Panel A: unligated PXR may act as a gene silencer. Activation of PXR by ligands (i.e., rifampicin andPCN) results in the dissociation of co-repressors and in the recruitment of co-activators, allowing gene transcription. Panel B: PXR resides in the cytosol in amulti-protein complex containing the CAR cytoplasmic retention protein (CCRP) and the heat shock protein 90 (Hsp90). Upon ligand binding (i.e., rifampicinand PCN) to PXR, the xenosensor dissociates from the multi-protein complex and translocates into the nucleus, thus activating gene transcription, as thePXR/RXRa heterodimer. PXR can be either phosphorylated by protein kinase A (PKA), resulting in strengthened interaction with co-activators, or de-phosphorylated by protein phosphatase PP2A, enhancing PXR transactivation. For details, see the text.



signaling pathways are independent of transcription. The recent finding that ERs also reside at the plasma membrane hasopened a new spectrum on E2 rapid signaling raising several new concerns (Marino and Ascenzi, 2008). Indeed, ERs arenow considered as very mobile proteins continuously shuttling between the membrane, the cytosol, and the nucleus. There-fore, ligand binding generates different and synergic signal transduction pathways, which provide plasticity for cell responseto E2 (Fig. 14) (Ascenzi et al., 2006).

Protein phosphorylation plays an important role in the regulation of NR functions, enabling integration of a variety of sig-nals and rapid adaptation to environmental and physiological situations. Phosphorylation of ERa is enhanced in response toE2 binding, but evidence for ligand-independent activation of these NRs through alternative signaling pathways has alsoemerged (see Weigel and Zhang, 1998; Ascenzi et al., 2006). Phosphorylation of ERb in response to E2 binding has not beenexamined in detail, although it has been shown to increase in response to activation of the mitogen-activated protein kinase(MAPK) pathway (see Lannigan, 2003).

ERa is phosphorylated at Ser104, Ser106, Ser118, Ser137, and Ser167, all located in the N-terminal transactivation domain(i.e., the A/B region), Ser236, located in the DBD (i.e., the C region), and Tyr537, located in the LBD (i.e., the E region). In re-sponse to E2 binding, ERa is predominately phosphorylated on Ser118 and to a lesser extent on Ser104 and Ser106. In re-sponse to activation of MAPK pathway, phosphorylation occurs on Ser118 and Ser167. Activation of PKA increases thephosphorylation of Ser236. Phosphorylation of Ser residues in the AF-1 domain (i.e., in the A/B region) appears to influencethe recruitment of co-activators, resulting in enhanced ER-mediated transcription. Ser236 phosphorylation does not appearto be involved in the activation of ERa-mediated transcription (see Lannigan, 2003; Smith and O’Malley, 2004). The phos-phorylated Tyr537 of ERa appears to play a role in the AF-2 function (see Lannigan, 2003) and represents the binding sitefor the Src-(avian sarcoma virus) homology domain (SH2) of the non-receptor tyrosine kinase Src (see Song et al., 2005).

4. CAR and PXR functions

Human and animals are exposed to potentially toxic chemicals from both endogenous and foreign sources including bilesalts, cholesterol and oxysterols, steroid hormones, bilirubin and fatty acids (endobiotics), as well as toxins, carcinogens, pol-lutants, drugs, dietary components, and herbal remedies (xenobiotics). To counter toxic insults and to maintain the homeo-static balance in important metabolic pathways, defense systems have been developed comprising enzymes and transportproteins capable of biotransformation reactions and subsequent elimination of endobiotics and xenobiotic metabolites.

Fig. 14. Schematic model illustrating the estrogen receptor a (ERa) activation. ERa localization at the plasma membrane is guaranteed by the post-translational addition of palmitic acid (PA), while in the cytosol ERa it forms complexes with heat shock proteins 90 or 70 (Hsp90 and Hsp70, respectively).17b-Estradiol (E2) binding induces ERa re-localization and activation of signaling proteins (i.e., extracellular regulated kinase, ERK, and AKT) whichphosphorylate ERa. This allows receptor homodimerization and translocation into the nucleus, where the transcription of target genes occurs. However, theactivation of epidermal growth factor receptor (EGFR) by EGF increases ERK and AKT activity, therefore ERa phosphorylation can occur also in a ligand-independent manner. For details, see the text.



Studies with knockout and transgenic mice for CAR and PXR have shown an overlapping set of target genes whose expres-sion is pivotal for the response to potentially harmful chemicals. CAR and PXR function as sensors of toxic byproducts ofendogenous metabolic compounds and of exogenous chemicals to facilitate their elimination. Both CAR and PXR are acti-vated by some of the same ligands (see Section 3), regulate a subset of common genes, and can signal through the sameREs (Moore et al., 2003; Swales and Negishi, 2004; Timsit and Negishi, 2007).

To further complicate this picture there is the evidence that, initially characterized as xenosensors, CAR and PXR also trig-ger pleiotropic effects on organ functions. Recent studies have shown the existence of cross-talk between xenosensors andother NRs or transcription factors controlling endogenous signaling pathways including lipid metabolism and maintenanceof glucose homeostasis (Moore et al., 2003; Lim and Huang, 2008). Such cross-talks could be at the root of alteration of phys-iological functions by xenobiotics and drugs provoking, among others, endocrine disruptions. Then, the cross-talk betweenCAR, PXR, and other NRs explains some clinical observations (Section 5). Therefore, an intriguing challenge is to differentiatethe function of these highly related NRs.

4.1. Detoxification by CAR and PXR

From Protozoa to mammals many mechanisms have been evolved to detoxify and/or employ the majority of xenobiotics,reducing their toxicity or, in some cases, making them available for body utilization. Versatile inducible metabolizing en-zymes and efflux transporters play a crucial role in these mechanisms that are a form of biotransformation and are consid-ered to be of ancient origin. After a toxicant or exogenous compound gets into a mammalian organism, chemical reactionsoccur within the body resulting in biotransformation(s). Although most of these reactions occur in the liver, xenosensor-dependent multi-enzymatic complexes are also expressed in gastrointestinal tract, lung, kidney, brain, and placenta (seeLamba et al., 2004a,b; Syme et al., 2004; Ganapathy and Prasad, 2005; Myllynen et al., 2005; Behravan and Piquette-Miller,2007; Pavek and Dvorak, 2008; Weier et al., 2008; Huls et al., 2009). These tissues metabolize not only many foreign chem-icals but also drugs and secondary products of metabolism. Biotransformations are thus critical processes in the body de-fense against the toxic effects of a wide variety of endobiotics and xenobiotics (Yu, 2001).

The biotransformation process(es) consists of phases I and II (Fig. 2). Major reactions in phase I include oxidation (e.g.,hydroxylation and deamination), reduction (e.g., addition of hydrogen atoms), and hydrolysis (e.g., splitting of ester andamide bonds). One of the most important characteristics of the phase I reactions is that a toxicant may acquire a functionalgroup, such as –OH, –NH2, –COOH, or –SH, to form a product called primary metabolite. Phase II reactions, on the other hand,are synthetic, or conjugation reactions, combining the toxicants or the primary metabolites directly with endogenous sub-stances (e.g., Gly, Cys, glutathione, sulfates, and glucuronic acid) (Yu, 2001).

The NADPH-cytochrome P450, commonly known as flavin mixed-function oxygenase system, is the most important en-zyme system involved in phase I oxidation reactions. Members of the CYP super-family (heme-dependent mono-oxygenases)are expressed within the liver, intestine and kidney, the primary organs for uptake, metabolism, and excretion of xenobiotics(Michalets, 1998; Yu, 2001; Kliewer et al., 2002). The CYP microsomal enzymes represent a supergene family of heme pro-teins that catalyze the metabolic conversion to more polar derivatives of an amazing diversity of foreign chemicals as well asendogenous substrates (e.g., steroid hormones) (Meyer, 1996; Xu et al., 2005). CYP isoforms are involved selectively in the

Table 7Nomenclature for the main families of Cytochrome P450 involved in xenobiotic metabolism.

Family Main characteristics Members Names

CYP1 Found in liver and extra-hepatic tissues; importantin drug metabolism, steroid metabolism (mainlyestrogens), and heme metabolism; induced bypolycyclic aromatic hydrocarbons, aromatic aminesand nitrosamines

3 sub-families CYP1A1, CYP1A2, CYP1B13 genes1 pseudogene

CYP2 Found in liver; important in the metabolism ofmany classes of drugs; induced by ethanol andacetone

13 sub-families CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8,CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1,CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1

16 genes16 pseudogenes

CYP3 Found in fetal and adult liver, in gastrointestinaltract, in kidney, and in placenta; important in themetabolism of majority of drugs and steroids andmany hydrophobic substrates; induced mainly byglucocorticoids and phenobarbital

1 sub-family CYP3A4, CYP3A5, CYP3A7, CYP3A434 genes2 pseudogenes

CYP4 Found in liver and kidney; important in metabolismof free fatty acids, in the catabolization ofleukotrienes and prostanoids, in the synthesis ofbioactive metabolites from arachidonic acid, and inxenobiotic metabolism

6 sub-families CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3,CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2,CYP4X1, CYP4Z1

11 genes10 pseudogenes

CYP7 Found in liver and brain, important in the synthesisof bile acids; in neurosteroid metabolism, and sexhormone synthesis

2 sub-families CYP7A1, CYP7B12 genes



metabolism of endogenous chemicals and of xenobiotics (Matic et al., 2007; Plant, 2007; Zhou et al., 2009). Fifty-seven CYPgenes are present in humans, members of families CYP1, CYP2, CYP3, CYP4, and CYP7 playing crucial roles in xenobioticmetabolism (Nakata et al., 2006; Matic et al., 2007; Plant, 2007; Zhou et al., 2009) (Table 7). The CYP3A sub-family representsperhaps the most relevant group. Indeed, CYP3A enzymes are the most abundant CYPs in human liver, comprising 30–50% ofCYPs, and hence representing the bulk of the CYP enzymes that a chemical is likely to be exposed to. Moreover, the adaptiveactive site of CYP3A enzymes is the prerequisite of ligand-binding promiscuity, indeed up 60% of therapeutics in use todayare subject to phase I metabolism as substrates of CYP3A enzymes (Cholerton et al., 1992; Matic et al., 2007; Plant, 2007;Zhou et al., 2009). Once the xenobiotic challenge is resolved, the CYP3A concentration returns to normal levels. Inductionof CYP3A is also at the root of drug–drug interactions (Section 5.) (Kliewer et al., 2002).

Contrary to the CYPs, mainly localized in the smooth endoplasmic reticulum, phase II enzymes are located in the cyto-plasmic matrix (Yu, 2001). The phase II metabolizing or conjugating enzymes belong to many super-families including sul-fotransferase (SULT), UDP-glucoronosyltransferase (UGT), NAD(P)H-quinone oxidoreductase, NAD(P)H-menadionereductase, epoxide hydrolases, glutathione-S-transferase (GST), and N-acetyltransferase (Xu et al., 2005). Increased hydro-philicity of xenobiotics, obtained by conjugation reactions catalyzed by phase II enzymes, generally enhances their excretionin the bile and/or the urine. However, xenobiotic conjugation by phase II enzymes could result in the biosynthesis of toxicmetabolites (Cholerton et al., 1992; Schilter et al., 1993; Hinson and Forkert, 1995; Chen et al., 2000; Kong et al., 2000;Rushmore and Kong, 2002; Matic et al., 2007; Plant, 2007; Zhou et al., 2009).

The biotransformation of lipophilic compounds by the two phases of reactions often results in the production of a morestable, water-soluble, metabolite that is more readily excretable via the transporter-mediated elimination pathway or phaseIII.

Phase III transporters, including P-glycoprotein (P-gp; also known as multidrug resistant protein-1, MDR1), multidrugresistance-associated proteins (MRPs), and the organic anion transporting polypeptide 2 (OATP2) play crucial roles in drugabsorption, distribution, and excretion (Brinkmann and Eichelbaum, 2001; Kerb et al., 2001; Dean et al., 2002; Kim, 2003;Mizuno et al., 2003; Staudinger et al., 2003; Matic et al., 2007; Plant, 2007; Zhou et al., 2009). ATP-dependent P-gp and MRPsfacilitate substrate (e.g., amino acids, metal ions, sugars, lipids, and xenobiotics) transport across the membrane, for this rea-son they are known as ATP-binding cassette transporters (i.e., ABC). In human, 46 ATP-binding cassette transporters havebeen identified (Dean et al., 2001; Mizuno et al., 2003; Xu et al., 2005). The OATP2 is a member of the OATP family that medi-ates sodium- and ATP-independent transport of a variety of endogenous and exogenous compounds, including conjugatedand unconjugated bilirubin, conjugated steroids, neutral compounds, THs, and bile salts (Xu et al., 2005; Matic et al.,2007; Plant, 2007; Timsit and Negishi, 2007; Zhou et al., 2009).

An important feature for regulation of phases I and II enzymes, as well as of phase III transporters, is that they can beinduced to higher levels of expression following exposure to specific substrates, as well as to structurally unrelated com-pounds (Yu, 2001). As a whole, the regulation of gene expression of various phase I, phase II drug metabolizing enzymes,and phase III transporters has potential impact on the metabolism, elimination, pharmacokinetics, pharmacodynamics, tox-icokinetics, toxicodynamics, and drug–drug interactions, protecting the human body against exposure to health-impairingxenobiotics (Xu et al., 2005; Plant, 2007; Timsit and Negishi, 2007; Zhou et al., 2009) (Fig. 2).

CAR coordinates the regulation of multiple hepatic genes (Table 8) resulting, in most cases, in metabolic detoxification byCYPs and transferases within the hepatocyte, followed by the net clearance of xenobiotics from the blood by transporters suchas OATP2. CAR hepatoprotective action includes blockage of the induction by PB of certain genes, which were only identifiedby their induction in CAR-null mice. For example, CAR prevents the induction of CYP4A, the major microsomal lipid peroxi-dase, by PB-dependent induction of superoxide dismutase-3 (SOD); therefore, CYP4A and SOD3 together may suppress oxi-dative stress (Swales and Negishi, 2004). Moreover, sleep induced by PB was prolonged in CAR-null mice. Similarly, the musclerelaxant zoxazolamine resulted in paralysis in wild-type mice that was avoided by pre-treatment with CAR activators, but wasprolonged in CAR knockout mice (Wei et al., 2000). Thus, the CAR-coordinated induction of metabolic activity increases theelimination of these drugs in a protective manner. This CAR-mediated defense system against chemical toxicity is applicableto endogenous and exogenous compounds and may have evolved to deal with both of them (Swales and Negishi, 2004).

PXR serves as a master transcriptional regulator of CYP3A isozymes (Quattrochi and Guzelian, 2001; Kliewer et al., 2002).Indeed, PXR and CYP3A result co-expressed in several tissues; moreover PXR is activated by CYP3A inducers. The discoverythat PXR is activated by PCN provided the original link to CYP3A (Kliewer et al., 1998). CYP3A is dysregulated in PXR-nullmice which do not have any overt phenotype under standard laboratory conditions (Xie et al., 2000a; Staudinger et al.,2001); however, their response to xenobiotics is severely compromised (Kliewer et al., 2002).

Besides regulating members of the CYP families, PXR is involved in other aspects of xenobiotic metabolism (Table 8), reg-ulating carboxylesterases, alcohol dehydrogenase, GST, UGT, SULT, and transporters such as P-gp, several MRPs, and OATP2(Maglich et al., 2002; Rosenfeld et al., 2003).

4.2. Role of CAR in bilirubin metabolism and heme biosynthesis

Bilirubin, an oxidative end product of heme catabolism, is one of the most toxic natural breakdown products in the body.Accumulation is associated with jaundice that can chronically lead to neurotoxicity and eventually fatal encephalopathy(Swales and Negishi, 2004). Glucuronidation by uridine diphosphate-glucuronyl transferase (UGT)1A1 is the major detoxi-fication pathway of bilirubin, and the conjugate is secreted across the bile canicular membrane of hepatocytes into the bile



by an active transporter MRP 2 (Swales and Negishi, 2004). For a long time it has been known that PB could decrease elevatedbilirubin levels. This is now revealed to be due to the ability of CAR to promote the bilirubin excretion by inducing the biliarytransporters UGT1A1, OATP2, MRP2, and GST A1 (Sugatani et al., 2001; Huang et al., 2003). An additional biliary transporterMRP3, with overlapping substrate specificity to MRP2, is also PB inducible, but PB induction of MRP3 appears to be CAR inde-pendent (Xiong et al., 2002). Thus, the role of CAR in regulating these MRPs is not clear.

The induction of 5-aminolevulinic acid synthase 1 by PB seems to indicate that CAR may also coordinate the induction ofheme biosynthesis by increasing CYP levels. However, induction studies with PB and TCPOBOP in CAR-null mice indicate thatthe regulation of 5-aminolevulinic acid synthase 1 is CAR independent (Maglich et al., 2002; Ueda et al., 2002).

4.3. Role of CAR and PXR in bile acid homeostasis

Bile acids, which are produced by the liver, are essential for the elimination of excess of cholesterol from the body and thesolubilization, absorption, and transport of dietary lipids in the intestine. Bile acid homeostasis is tightly regulated because

Table 8Some of the CAR and PXR target genes involved in the phases I, II and III metabolism.

Nuclar receptor Target gene Organism Effect on target gene

Phase ICAR, PXR Aldh1A1 Mouse "CAR, PXR Aldh1A7 Mouse "CAR CYP1A1 Mouse "PXR CYP1A1 Mouse ;PXR CYP1A1 Human "PXR CYP1A2 Human "PXR CYP1A6 Human "CAR CYP2A4 Mouse "CAR CYP2A6 Human "CAR, PXR CYP2B1 Rat "CAR, PXR CYP2B2 Rat "CAR, PXR CYP2B6 Human "CAR, PXR CYP2B10 Mouse, human "CAR CYP2C6 Rat "CAR CYP2C7 Rat "PXR CYP2C8 Human "CAR, PXR CYP2C9 Human "CAR, PXR CYP2C19 Human "CAR, PXR CYP3A1 Rat "PXR CYP3A2 Rat "CAR, PXR CYP3A4 Human "CAR, PXR CYP3A11 Mouse, human "PXR CYP3A13 Mouse "PXR CYP3A23 Rat "PXR CYP3A44 Mouse "PXR CYP7A1 Human ;PXR CYP11A1 Human "PXR CYP11B1 Human "PXR CYP11B2 Human "CAR, PXR Por Mouse "

Phase IICAR, PXR GSTA1 Mouse, rat "CAR, PXR GSTA2 Mouse, rat "CAR GSTA3 Mouse, rat "PXR GSTA4 Mouse "CAR, PXR Gstm1 Mouse, rat "CAR, PXR Gstm2 Mouse "PXR Sult2A1 Mouse, human "CAR, PXR UGT1A1 Mouse, human "PXR UGT1A3 Human "PXR UGT1A4 Human "CAR UGT2B1 Rat "

Phase IIICAR, PXR MDR1A Mouse, human "PXR MDR1B Mouse "CAR MRP1 Mouse "CAR, PXR MRP2 Mouse, rat, human "CAR, PXR MRP3 Mouse, human "CAR MRP4 Mouse "CAR, PXR OATP2 Mouse, rat "



bile acids are detergents that can be extremely toxic if their levels become elevated. In addition, bile secretion is an impor-tant pathway for the elimination of large hydrophobic endobiotics and xenobiotic metabolites, including many high molec-ular weight conjugates. The major components of bile are cholesterol, lecithin, bile pigments, bile acids, and bicarbonate ions(Nguyen and Bouscarel, 2008).

Even if shown only in mouse, CAR induces enzymes and transporters involved in bile acids elimination, namely CYP3A11,SULT2A1, and the transporter MRP3 (Zhang et al., 2004b). Hence the defensive role of CAR contributes to maintain normalcholesterol levels indirectly through regulation of bile acid homeostasis. This CAR role occurs in the absence of the key bileacid sensors FXR (Kliewer et al., 1998; Makishima et al., 1999; Wang et al., 1999) and PXR (Guo et al., 2003). In fact, in FXR/PXR double knockout mice, cholic acid elevated the levels of CAR and CYP2B mRNA, and pre-treatment with PB or TCPOBOPinduced a collection of target genes, incorporating those involved in protection against bilirubin toxicity, which reduced ser-um bile acid and bilirubin concentration (Guo et al., 2003).

PXR has been recently found to play a major role in protection from bile acid toxicity. The role of PXR in bile acid homeo-stasis comes from in vivo observations showing that PXR activation is protective against hepatotoxic bile acid and that accu-mulation of bile acid precursors leads to PXR activation (Ma et al., 2008). These studies revealed the role of PXR in regulatingthe conversion of cholesterol into bile acids and detoxifying oxidized cholesterol (‘‘oxysterols”). Accordingly, high mortalityassociated with hepatitis and hepatocellular injury occurs in PXR-null mice fed with a cholesterol- and cholic acid-enricheddiet (Sonoda et al., 2005). Cholic acid was included in the diet to block cholesterol catabolism and increase intestinal cho-lesterol absorption. In addition to liver injury, the PXR target genes CYP3A11 and OATP2 were also induced in wild-type mice,but not in PXR-null mice. Thus, PXR displays a protective role against cholesterol toxicity (Ma et al., 2008).

PXR activation regulates the levels of murine CYP3A11/human CYP3A4, SULT, and OATP2, which may promote the metab-olism and transport of bile acids (Timsit and Negishi, 2007). Indeed, OATP2, localized on the basolateral membrane of thehepatocyte, is involved in the cellular uptake of bile acids. Its induction by PXR would presumably increase the uptake ofbile acids from the sinusoidal blood into the hepatocyte where the detoxification pathways, such as CYP-mediated hydrox-ylation and sulfation could take place (Timsit and Negishi, 2007). PXR activation was proposed to decrease the bile acid syn-thesis via down-regulation of CYP7A1 and to accelerate bile acid metabolism and elimination through up-regulation ofmetabolic enzymes and transporters (Nguyen and Bouscarel, 2008).

As whole, CAR, PXR, and FXR appear to cooperate for protection against hepatic bile acid toxicity.

4.4. Role of CAR and PXR in steroid and thyroid hormone homeostasis

The first endogenous modifiers of CAR activity identified were the steroids androstanol and androstenol, which inhibit theactivity of the receptor by dissociating the interaction between CAR and SRC-1 (Forman et al., 1998). Similarly, progesteroneand testosterone repress the constitutive activity of CAR (Swales and Negishi, 2004). In contrast, pharmacological levels of E2and estrone as well as the progesterone metabolite pregnane-3,20-dione can activate rodent and human CAR, respectively(Swales and Negishi, 2004).

As CYP2B, a major target for CAR activation, metabolizes both estrogen and androgen and CAR-regulated UGT1A1 glucu-ronidates estrogens, induction of these enzymes due to CAR activation by xenobiotics and endobiotics could increase steroidhormone catabolism (Sugatani et al., 2001; Kawamoto et al., 2000; Swales and Negishi, 2004). CAR may augment steroid sul-fation in the presence of PB by transactivating the 30-phosphoadenosine 50-phosphosulfate synthase 2 gene (Ueda et al.,2002). Thus, xenobiotics, through CAR, may influence the different stages of steroid hormone homeostasis by inducingthe CYPs and transferases involved in the metabolism of estrogens and their precursor steroids (Swales and Negishi, 2004).

Preclinical studies support the concept of PXR as a potential endocrine disrupting factor that might have broad implica-tions in steroid hormone homeostasis and drug–hormone interactions. Activation of PXR markedly increases plasma concen-trations of corticosterone and aldosterone, and their increase was associated with the enhanced expression of adrenalsteroidogenic enzymes, including CYP11A1, CYP11B1, CYP11B2, and 3b-hydroxysteroid dehydrogenase (Zhai et al., 2007).The PXR target geneCYP3A4 also contributes to the metabolism of steroid hormones, exhibiting a significant role in cortisoland testosterone metabolism (Buters et al., 1994). Cortisol and testosterone 6b-hydroxylase activities were also used as bio-markers for CYP3A4 induction or inhibition (Fayer et al., 2001; Konishi et al., 2004). Among the major human hepatic P450s,CYP3A4 most efficiently catalyzes 6b- and 16a-hydroxylation of progesterone (Niwa et al., 1998). In addition, CYP3A4catalyzes 2-, 4-, and 16-hydroxylation of E2 (Badawi et al., 2001). In a CYP3A4-transgenic mouse line expressing both humanand murine CYP3A, females were found to be deficient in lactation, leading to a markedly lower pup survival. This impairedlactation phenotype was associated with significantly reduced serum E2 levels in CYP3A4-transgenic mice, suggesting thatCYP3A4 could play an important role in E2 homeostasis (Yu et al., 2005). This could be of relevance in administrating drugsthat are PXR activators to pregnant and lactating women. Of note, rifampicin is contraindicated in pregnancy except in thepresence of a severe disease untreatable with other drugs, such as tuberculosis, because of teratogenicity found in animalstudies and case reports of malformation, death, and haemorrhage in infants whose mothers were exposed to this macrolideantibiotic (Ma et al., 2008). The role of PXR in the homeostasis of steroid hormones, especially sex hormones, could providean important clue as to the mechanism by which rifampicin compromises pregnancy (Ma et al., 2008).

TH has a well-established role in liver regeneration and energy usage. Levels of TH are controlled by a balance of its syn-thesis, metabolism, and secretion. Thyroid-stimulating hormone enhances synthesis of inactive 3-5-30-50-tetraiodothyronine(T4) in the thyroid gland which is subsequently converted to various forms of TH by deiodinases in the peripheral target



tissues, such as liver and kidney. Chronic treatment with PB is known to promote thyroid hypertrophy in humans and rats(Konno et al., 2008). PB-induced CAR activation decreases the serum level of total T4, however serum T3 levels were notmeasured (Maglich et al., 2004; Qatanani et al., 2005). Chronic treatment with the CAR activator phenytoin decreases theserum levels of total T4, promoting thyroid hypertrophy (Curran and DeGroot, 1991). Since TH is known to be sulfated orglucuronidated for its clearance and excretion, it was hypothesized that phase II enzymes (i.e., UGT1A1 and SULT1A1) aredetermining factors responsible for T4 decrease (Konno et al., 2008). However, it remains elusive whether UGT1A1 andSULT1A1 are the major enzymes conjugating TH and how these enzymes more effectively conjugate T4 over T3 (Konnoet al., 2008).

4.5. Role of CAR and PXR in gluconeogenesis and lipid metabolism

Hepatic gluconeogenesis is tightly controlled by insulin and glucagon and plays a major role for survival during fasting orstarvation. Genes involved in gluconeogenesis include notably glucose-6-phosphatase (G6Pase) and phosphoenolpyruvatecarboxykinase (PEPCK). G6Pase is the critical enzyme that controls the serum level of glucose by catalyzing the de-phosphory-lation of glucose-6-phosphate generated from both gluconeogenesis and glycogenolysis. PEPCK catalyzes the formation ofphosphoenolpyruvate from oxaloacetate, with the release of carbon dioxide and GDP. In the liver, gluconeogenesis is con-trolled positively by glucocorticoids, cAMP, and glucagon, and negatively by insulin and glucose (Konno et al., 2008).

Functional links between insulin- and xenobiotic-mediated pathways occur (Tien and Negishi, 2006). It has long beenknown that treatment with drugs which are now known as activators of CAR and PXR represses hepatic gluconeogenic en-zymes and genes (Argaud, 1991; Ueda et al., 2002; Kodama et al., 2004). PB has been shown to decrease plasma glucose indiabetic patients (Lahtela et al., 1985). Treatment with the mPXR activator PCN decreases blood glucose levels in fastingwild-type but not in PXR-null mice. Although the exact mechanism remains unclear, most of the data obtained so far stronglysuggest that CAR and PXR activation represses the gluconeogenic pathway by interfering with transcription factors orco-factors involved in the transcriptional regulation of these gluconeogenic enzymes. Regulation of gluconeogenesis medi-ated by the fork-head insulin-responsive transcription factor 1 (FoxO1) can also be influenced by CAR. Similarly to insulin,CAR was shown to down-regulate FoxO1 binding to insulin response sequences, thus attenuating the ability of FoxO1 tostimulate gluconeogenic genes such as PEPCK (Kodama et al., 2004). This provides a likely explanation of how PB treatmentimproves insulin sensitivity in non-insulin-dependent/Type II diabetes (Lahtela et al., 1985). The direct interaction of CARand PXR with FoxO1 appears to be the underlying mechanism(s) repressing the G6Pase and PEPCK1 genes in response toxenobiotics. As a result, although the molecular mechanism(s) differs from that of insulin, CAR and PXR cross-talk withFoxO1 to repress gluconeogenesis. Moreover, CAR is associated to PPAR-c co-activator 1a (PGC-1a), a transcriptionalco-factor induced by fasting which regulates energy metabolism (Timsit and Negishi, 2007). PXR also represses transcrip-tional activity of FoxO1 on insulin-responsive element (Kodama et al., 2004), acting as a negative transcriptional regulatorof genes involved in glucose metabolism.

Hepatic lipid metabolism plays a major role in survival during fasting and/or prolonged exercise. When blood glucose lev-els are low, the liver increases fatty acid oxidation and ketogenesis to provide extra-hepatic tissues with ketone bodiesthrough b-oxidation and ketogenesis. At the same time, the liver decreases lipogenesis to attenuate hepatic storage of tri-glycerides. Under these conditions, carnitine palmitoyltransferase 1A (CPT1A) and mitochondrial 3-hydroxy-3-methylglut-arate-CoA synthase 2 (HMGCS2), which are the key enzymes in b-oxidation and ketogenesis, respectively, are up-regulated(Hegardt, 1999; Louet et al., 2001), while stearoyl-CoA desaturase 1 (SCD1), which is the key enzyme for the synthesis ofunsaturated fatty acids, is up-regulated by glucose and fructose (Dobrzyn and Ntambi, 2005). In the absence of insulin,FoxA2, a winged helix/fork-head transcription factor, stimulates CPT1A and HMGCS2 expression (Wolfrum et al., 2003). Insu-lin represses these two genes by inactivating the FoxA2 through the AKT-dependent signal pathway (Konno et al., 2008).Insulin also increases the transcription of SCD1, in part, by activating the sterol regulatory element-binding protein (SREBP)(Martini and Pallottini, 2007).

Drug-activating PXR acts like insulin and represses hepatic energy metabolism by increasing triglyceride synthesis and bydecreasing b-oxidation and ketogenesis, although the molecular mechanism differs from that of insulin (Konno et al., 2008).Since fatty acid b-oxidation produces and supplies chemical energy such as ATP and NADH to gluconeogenesis in the liver,the repression of b-oxidation by PXR could lead to down-regulation of gluconeogenesis. Consistent with this conclusion, in-creased hepatic deposit of triglycerides and the concomitant up-regulation of the free fatty acid transporter genes in consti-tutively active PXR transgenic mice have been reported (Konno et al., 2008).

4.6. Functional interplay between CAR/PXR and NRs

CAR and PXR were originally shown to regulate CYP2B and CYP3A genes, respectively. However, several groups have nowdemonstrated that PXR can regulate CYP2B genes in cell-based reporter assays and in vivo (Xie et al., 2000b). Conversely, CARregulates CYP3A expression (Sueyoshi et al., 1999; Tzameli et al., 2000; Xie et al., 2000b; Wei et al., 2000; Goodwin et al.,2001). Recent studies indicate that the overlap in PXR and CAR target genes extends well beyond the CYP2B and CYP3A genes.In the liver, CAR and PXR co-regulate members of the CYP2C, GST, SULT, and UGT families and the canalicular MRP2 trans-porter (Gerbal-Chaloin et al., 2001, 2002; Kast et al., 2002; Maglich et al., 2002). These findings suggest a functional redun-dancy in the CAR and PXR signaling pathways. Nevertheless, there are differences in the degree to which specific genes are



activated by either CAR or PXR agonists (Maglich et al., 2002), which undoubtedly contribute to the distinct pharmacologiesof these xenobiotics.

CAR and PXR are regulated by several of the same chemicals, including xenobiotics and natural steroids (Moore et al.,2000a). However, studies in which CAR and PXR were tested against collections of xenobiotics and natural steroids incell-based reporter assays revealed that CAR is much less promiscuous in its interactions with chemicals than PXR (Mooreet al., 2000a, 2002). Molecular modeling of the CAR-LBD based on the PXR crystal structures suggests that CAR is likely tohave a much smaller ligand-binding pocket than PXR (see Section 2.2.3). Nevertheless, CAR may be activated indirectlyby a variety of chemicals that promote its translocation into the nucleus. Thus, CAR and PXR may play complementary rolesin sensing potentially harmful chemicals that either alter the phosphorylation status of the cell or enter the nucleus.

An extensive cross-talk between AhR, CAR and PXR has been reported. For example, AhR appears to be a target gene ofPXR (Maglich et al., 2004), and CAR could be regulated by AhR (Patel et al., 2007). AhR-controlled CYPs are CYP1A1, CYP1A2,and CYP1B1 which are involved in both bioactivation of the carcinogen benzopyrene and in its first-pass detoxification in theintestinal epithelium (Köhle and Bock, 2009). Although clusters of functional REs have been characterized in the enhancerregion of human CYP1A1 (Ueda et al., 2006), CYP1A1 and CYP1A2 appear to be indirectly upregulated by PXR ligands dueto the induction of AhR by PXR (Maglich et al., 2004). Evidence is accumulating that uptake and export conjugate transportersare also co-regulated by the nuclear receptors PXR/CAR and AhR (Köhle and Bock, 2009).

In conclusion, members of CYP families 1 to 4 appear to be differentially induced by NRs whereas receptor control appearsto have converged in the case of phase II enzymes. Tight coupling between phases I and II enzymes may attenuate the riskposed by accumulation of toxic intermediates of the metabolic pathways such as bile acids, leading to efficient enterohepaticcirculation of bile acids and their conjugates (Köhle and Bock, 2009). Coupling may also have evolved in the case of dietaryphytochemicals, compounds to which the mammalian organisms are exposed from millions of years. On the other hand, cou-pling seems to be loose for newly developed drugs (Köhle and Bock, 2009). In the latter case, particular care has to be takento investigate potential generation of toxic intermediates.

FXR mediates the feedback inhibition of bile acid regulation. The interplay between FXR and PXR is mediated by bile acidswhich serve as FXR and PXR ligands at physiologically relevant concentrations. Two functional FXR-responsive elements inthe RE of CYP3A4 have been reported. One of them is known to bind PXR. As reported before, transcriptional activation of PXRby bile acids increases CYP3A and OATP2 transcription, whereas in contrast it represses CYP7A1. Thus, bile acids, which aresubstrates of CYP3A4 and activators for both FXR and PXR, can initiate a feedback mechanism to protect hepatocytes fromtoxicity of bile acids (Lim and Huang, 2008).

Liver X receptors (LXRa and LXRb) are NRs that coordinate carbohydrate and lipid metabolism by binding oxysterols.Other LXR agonists, such as 24(S),25-epoxycholesterol and T0901317, are shown to induce CYP3A4 and CYP2B6 mRNAexpression through the activation of PXR (Duniec-Dmuchowski et al., 2007). The synthetic LXR agonist, T0901317, is alsoa high-affinity ligand for PXR. It stimulates LXR activity and PXR target genes. Thus, many of the biological gene ordinaryassociated with LXR may be mediated by PXR, not by LXR (Mitro et al., 2007; Lim and Huang, 2008). First characterizedas xenosensors, recent reports have enlarged the list of ligands/activators of CAR and PXR. Thus, these receptors exert pro-found effects on bile acids, lipids, hormones, and xenobiotics regulatory networks including induction or down-regulation ofvarious enzymes and transporters involved in phases I, II, and III xenobiotic metabolizing/transporting systems.

5. From bench to bedside4

5.1. PXR-mediated drug–drug interactions

The most common clinical implication for the activation of PXR is the occurrence of drug–drug interactions especially inpatients affected by tuberculosis, cancer, AIDS, cardiovascular diseases, and diabetes. The clinical consequences of drug–druginteractions are generally a decreased therapeutic efficacy and, occasionally, an increased drug toxicity. Therefore, under-standing the mechanism(s) involved in drug–drug interactions represents an important goal for the improvement of drugsafety. The identification of PXR sheds light on molecular mechanisms involved in drug–drug interactions, which occur whenone PXR ligand induces the expression of enzymes or transporters affecting the metabolism of another co-administered drug(Lazarou et al., 1998; Ma et al., 2008).

5.1.1. Decreased drug efficacy by PXR-enhanced catabolismIn most cases, PXR facilitates drug elimination by increased metabolism. Remarkably, rifampicin, a human PXR ligand

used at a high dose and long term for tuberculosis treatment, is associated with PXR-mediated drug–drug interactions(Niemi et al., 2003). Rifampicin interacts with over 100 drugs, most notably drugs that are CYP3A substrates, including oralcontraceptives, pre-anesthetic midazolam, and anti-HIV protease inhibitors (Ivanovic et al., 2008; Ma et al., 2008). Drug–drug interactions between rifampicin and oral contraceptives were first reported in the early 1970s. In tuberculosis patientsunder chemotherapy with rifampicin, a significant high incidence of unwanted pregnancies and menstrual cycle disorderswas noted in female patients using oral contraceptives (Ma et al., 2008). Drug–drug interactions were also found with

4 Most of the data concerning clinical implications of CAR and PXR refer to PXR.



midazolam, a fast-acting benzodiazepine used for short minor surgical procedures such as dental extraction. In fact,midazolam is ineffective in patients treated with rifampicin (Backman et al., 1996). Drug–drug interactions between rifam-picin and anti-HIV protease inhibitors in tuberculosis and HIV co-infected patients can result in the loss of HIV suppression.As a whole, rifampicin activates PXR and up-regulates the PXR target gene CYP3A4, resulting in increased metabolic clearanceof oral contraceptives, midazolam, and anti-HIV protease inhibitors, leading to decreased efficacy (Ivanovic et al., 2008; Maet al., 2008).

The herbal medicine St. John’s Wort is used for centuries, dating from the early Greeks, for medicinal purposes in thetreatment of mental disorders and nerve pain. Today, St. John’s Wort is widely used for depression, anxiety, and sleep dis-orders (Ma et al., 2008). However, the alarm regarding St. John’s Wort was raised when a life-threatening adverse drug reac-tion was reported in two heart transplant patients treated with cyclosporine, one who had self-administered St. John’s Wortand the other who was prescribed by a psychiatrist (Ruschitzka et al., 2000). Cyclosporine is an immunosuppressant used inorgan transplant patients to reduce the risk of organ rejection. However, in organ transplant patients using St. John’s Wort,the organ transplant failed. Further studies revealed that St. John’s Wort contains multiple PXR ligands, most notablyhyperforin, which was found to activate PXR with EC50 = 23 nM (Moore et al., 2000b). Thus, St. John’s Wort activates PXR,up-regulates CYP3A expression, and accelerates cyclosporine metabolism. St. John’s Wort–cyclosporine interaction leadsto a marked decrease of cyclosporine blood levels (Murakami et al., 2006). Remarkably, the feeding of herbal derivatives(e.g., St. John’s Wort and colupulone) together with prescribed medications increases the risk of potentially dangerousdrug–herb interactions due to changes in CYP450 expression profiles following hPXR activation (Burka, 2003).

5.1.2. Increased drug toxicity by PXR-mediated metabolismAltered metabolism might be harmful for some drugs because of the production and accumulation of toxic metabolites.

Acetaminophen (APAP), widely used as an analgesic for relief of fever and headaches, is metabolized primarily in the liver,where its major metabolites include metabolically inactive sulfate and glucuronide conjugates (Ma et al., 2008). However, aminor metabolic pathway involves the hepatic CYP, which is responsible for the generation of the putative toxic alkylatingmetabolite N-acetyl-p-benzo-quinone imine (NAPQI). At recommended doses, NAPQI is quickly detoxificated through con-jugation with glutathione to produce a non-toxic derivative. However, under conditions of CYP induction, the risk of APAPtoxicity increases due to excess hepatic NAPQI. Accordingly, APAP hepatotoxicity occurs upon treatment of humans with iso-niazid (a CYP2E1 inducer) and of mice with 3-methylcholanthrene (a CYP1A2 inducer) (Szymanska et al., 1992; Crippin,1993) or PCN (a CYP3A11 inducer) (Guo et al., 2004). PCN pre-treatment of PXR-null mice does not induce APAP hepatotox-icity, suggesting that PXR plays a critical role in APAP bio-activation (Guo et al., 2004).

5.2. PXR-mediated metabolic bone disorders

Vitamin D promotes bone formation and mineralization and is essential in skeleton development. Vitamin D deficiencyleads to bone softening diseases, such as rickets in children and osteomalacia in adults. In mammals, two major forms ofvitamin D exist, vitamin D2 and vitamin D3. In humans, vitamin D3 is more effective than vitamin D2 while vitamin D2is more effective than vitamin D3 in rats. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the physiologically active form of vita-min D in humans, is synthesized from vitamin D3 by hepatic CYP27A1 and CYP2R1, and renal CYP27B1. 1,25(OH)2D3 medi-ates its biological effects by binding to the VDR. The NR activation in the intestine, bone, and kidney leads to the maintenanceof calcium and phosphorus levels in the blood and to the maintenance of bone content (Ma et al., 2008).

Renal CYP24 is well known to be the major enzyme contributing to the metabolism of 1,25(OH)2D3 to the inactive form1,24,25-trihydroxyvitamin D3 (1,24,25(OH)2D3), which decreases the formation of 1,25(OH)2D3 from 25-hydroxyvitaminD3 (25(OH)D3) (Ma et al., 2008). Recently, drugs such as PXR ligands have been reported to modulate the expression ofthe PXR target gene CYP24 both in vitro and in vivo, altering the homeostasis of 1,25(OH)2D3 (Pascussi et al., 2005). More-over, the PXR target gene CYP3A4 catalyzes 23- and 24-hydroxylation of 1,25(OH)2D3, resulting in the production of biolog-ically inactive metabolites (Xu et al., 2006).

Whatever the effect of CYP24 and CYP3A4 on 1,25(OH)2D3 homeostasis, the role of PXR in metabolic bone disorders inhumans remains unclear. In fact, although a significant proportion of the world’s population has been treated with the PXRligand rifampicin over the past 40 years, rifampicin-mediated osteomalacia was not reported to any significant degree (Maet al., 2008). Moreover, no significant change in 1,25(OH)2D3 plasma levels during short- or long-term treatment with rif-ampicin was observed (Ma et al., 2008).

Overall, the role of PXR in vitamin D homeostasis and metabolic bone disorders is not conclusive. Indeed, the role of CARin drug-induced osteomalacia should also be considered, since CAR ligands can also regulate metabolism and affect vitaminD homeostasis. Moreover, CAR ligands, such as phenytoin and PB, are much more frequently associated with osteomalaciathan the PXR ligand rifampicin (Ma et al., 2008).

Vitamin K2, appearing to protect against the fracture risk and bone loss at the spine, is a weak PXR ligand(Landes et al., 2003). Indeed, vitamin K2 supplementation increases bone density in vivo, and it is used clinicallyin the management of osteoporosis. In vitro, vitamin K2 is able to induce bone markers in primary osteocytesisolated from wild-type mouse calvaria but not in cells isolated from PXR-null mice (Tabb et al., 2003). Note thatthe osteoblastgenic transcription factor MSX2, a PXR target gene, mediates the osteoprotective action of vitamin K2(Igarashi et al., 2007).



5.3. PXR-mediated hepatic steatosis

The abnormal retention of lipids within the cells results in steatosis, which reflects an impairment of the normal pro-cesses of synthesis and breakdown of triglycerides. As the liver is the primary organ of lipid metabolism, steatosis most oftenoccurs in this tissue. While patients with hepatic steatosis have few or no symptoms, infrequently they may complain offatigue, malaise, and dull right upper quadrant abdominal discomfort. However, the danger of hepatic steatosis is the resultof the sequelae, such as liver fibrosis, cirrhosis, and carcinoma. In humans, hepatic steatosis is commonly associated withalcohol abuse or metabolic syndrome (diabetes, hypertension, and dyslipidemia), but may also be caused by drugs and cer-tain toxins (Lee et al., 2008).

Compared to the wild-type mice, the ‘‘humanized” hPXR transgenic mice, which express the activated hPXR in the liver,had a marked increase in liver weight and in triglyceride accumulation (Lee et al., 2008). Treating ‘‘humanized” hPXR trans-genic mice with rifampicin (an hPXR agonist) for 5 weeks induced a significant liver triglyceride accumulation. The lipo-genic effect of PXR was independent of the activation of the dedicated lipogenic transcriptional factors such as sterolregulatory element-binding proteins (SREBPs) and their primary lipogenic target enzymes (i.e., fatty acid synthase, FAS,and acetyl-CoA carboxylase) (Collot-Teixeira et al., 2007; Koonen et al., 2007; Lee et al., 2008; Ma et al., 2008). Instead,PXR-mediated lipid accumulation likely results from increased hepatic free fatty acid uptake by activating the fatty acidtransporter CD36. Activation of CD36 and induction of the accessory lipogenic enzymes such as stearoyl-CoA desaturaseand fatty acid elongase were observed both in the untreated and in the rifampicin-treated ‘‘humanized” hPXR transgenicmice (Lee et al., 2008). CD36, a multi-ligand scavenger receptor present on the surface of a number of cell types, may con-tribute to hepatic steatosis by facilitating the high-affinity uptake of fatty acids from the circulation (Lee et al., 2008). CD36levels in the liver are correlated with hepatic triglyceride storage and secretion, suggesting that CD36 may play a causativerole in the pathogenesis of hepatic steatosis (Koonen et al., 2007). Moreover, PXR may promote hepatic steatosis byincreasing the expression of CD36 directly or indirectly through the PXR-mediated activation of PPARc (Lee et al., 2008).PXR-mediated gene regulation and lipid accumulation are required for the hepatic regenerative response to liver resection,and it was suggested that PXR is essential for normal progression of liver regeneration by modulating lipid homeostasis(Dai et al., 2008).

Although there are very few clinical reports concerning drug-induced hepatic steatosis by PXR ligands, such as dexameth-asone, clotrimazole, and rifampicin, direct attention should be addressed to the safety of these drugs (Lee et al., 2008; Maet al., 2008). However, no evidence indicating rifampicin-induced hepatic steatosis has been reported, although this drugis used by a large number of tuberculosis patients since 1970s (Morere et al., 1975; Ma et al., 2008). Surprisingly, fatty liverswere noted in rats given a high dose of rifampicin in preclinical studies (Truhaut et al., 1978; Piriou et al., 1987). The phys-iological and pharmacological relevance of this finding to humans is questionable since supra-pharmacological doses wereused in rats; in fact, rifampicin is a human specific PXR ligand, having virtually no effect on rat PXR at human-equivalentdoses (Kliewer et al., 2002). Overall, the role of human PXR in lipid metabolism and hepatic steatosis warrants furtherinvestigation.

5.4. PXR role in inflammatory bowel disease

The intestinal wall has crucial functions in host-environment interactions in the context of nutrient intake and process-ing. It operates in contact with an extremely populated bacterial colony and has to cope with drastic changes in its envi-ronment, sometimes hostile and harmful (Wahli, 2008). Maintenance of key functions during the lifespan of organismsrequires sophisticated sensing and response processes, including detoxification, inflammation and its control, efficient re-pair involving suppression of apoptosis and controlled cell proliferation, and mechanisms of rapid renewal of the intestinallining. These tasks mobilize several NRs as well as inflammatory/immune and proliferation/differentiation signaling path-ways that are interconnected and finely tuned, but sometimes compete with each other with consequences for the asso-ciated signaling partners (Wahli, 2008). Dysfunctions of this homeostasis may result in inflammatory and auto-immunebowel diseases and cancer. Inflammatory bowel disease (IBD) refers to a chronic inflammatory condition of the digestivetract occurring as one of the two major types, ulcerative colitis, and Crohn’s disease. Ulcerative colitis is limited to the co-lon while Crohn’s disease most commonly affects the small intestine and/or the colon, but can involve any part of the gas-trointestinal tract from the mouth to the anus. However, the etiology of IBD is unknown. To date, genetic, infectious,immunologic, and psychological factors have all been implicated in influencing the development of IBD (Ma et al.,2008; Wahli, 2008).

Recently PXR has been identified as a gene strongly associated with the susceptibility to IBD in humans (Dring et al.,2006). In patients with IBD, a decreased expression of PXR and of PXR target genes have been observed (Langmann et al.,2004; Martinez et al., 2007). In the dextran sulphate sodium (DSS)-induced IBD mouse acute colitis model, treatment withthe PXR ligand PCN protected against DSS-induced colitis compared with vehicle-treated mice, as defined by bodyweight loss, diarrhea, rectal bleeding, colon length, and histology. However, this treatment did not decrease the severityof DSS-induced colitis in PXR-null mice indicating a role for PXR in protection against IBD (Shah et al., 2007). It has recentlybeen reported that hepatic SCD1 is down-regulated in mice with DSS-induced colitis, this leading to elevated levels of pro-inflammatory saturated fatty acids and reduced levels of anti-inflammatory unsaturated fatty acids (Chen et al., 2008). Itshould be noted that SCD1 is up-regulated in mice by PXR activation (Zhou et al., 2006a); thus, PXR activation should be



expected to ameliorate the symptoms of DSS-induced colitis in mice having low levels of expression of SCD1 throughincreased production of unsaturated fatty acids (Ma et al., 2008). Interestingly, budesonide, a glucocorticoid derivativefrequently used as an anti-inflammatory drug for IBD, has been recently identified as a PXR ligand (Maier et al., 2007).

Rifaximin, used for the treatment of travelers’ diarrhea, is beneficial in the treatment of active ulcerative colitis,mild-to-moderate Crohn’s disease as well as prevention of postoperative recurrence of Crohn’s disease, despite the differ-ences in dose and duration (Laustsen and Wimmett, 2004; Shafran and Johnson, 2005; Guslandi et al., 2006). The mechanismcontributing to the beneficial effects of rifaximin in chronic gastrointestinal disorders is not fully understood. By using PXR-humanized, PXR-null, and wild-type mice, rifaximin was identified as a gut-specific human PXR activator (Ma et al., 2007).

Bacterial lipopolysaccharide (LPS), a highly pro-inflammatory component of the Gram-negative bacteria, elicits an arrayof host responses that are mediated by the nuclear transcription factor kappa (NF-jB), which includes an increased produc-tion of pro-inflammatory cytokines that contribute to host defence mechanisms (Wahli, 2008). In PXR-null mice, the en-hanced expression of NF-jB target genes and the increased small intestinal inflammation was observed, suggesting afunctional interaction between the PXR and the NF-jB pathways. Furthermore, the activation of NF-jB inhibits the PXR func-tion, thereby causing a reduced expression of its target genes, whilst inhibition of NF-jB increases the PXR activity and targetgenes expression (Zhou et al., 2006b; Wahli, 2008). This mutual negative cross-talk between NF-jB and PXR explains theimmunosuppressive effects of PXR ligands such as rifampicin, which reduces NF-jB activity, and sheds light on the de-creased expression of PXR target genes such as CYPs in inflammation and infection when NF-jB activity is high. Thus,PXR can function as a negative mediator of inflammation and immunity linking drug and xenobiotic metabolism to immuneresponses. This provides an insight for treating infectious diseases and may have direct clinical implications for the treat-ment of immunocompromised patients (Xie and Tian, 2006; Zhou et al., 2006b; Wahli, 2008).

5.5. PXR role in cancer and chemotherapy

Resistance to chemotherapeutic agents is the major clinical problem and cause of failure in the chemotherapy of humancancer. Understanding the molecular basis of chemoresistance will be valuable for developing more effective chemotherapy.Several molecular targets have been shown to be related to chemoresistance, which include efflux transporters, phases I andII detoxication enzymes, and DNA-repair enzymes. Most of these chemoresistance-related enzymes are encoded by PXR tar-get genes, such as P-GP, MRPs, CYP3A, UGT, and GST (Harmsen et al., 2007).

Some chemotherapeutic agents, such as cyclophosphamide, tamoxifen, and taxol, have been identified as hPXR ligands(Ma et al., 2008). Remarkably, activation of PXR induces a battery of enzymes and transporters that accelerate the metabo-lism and the elimination of chemotherapeutic agents, contributing to resistance to chemotherapy in breast cancer (Dotzlawet al., 1999), in prostate cancer (Chen et al., 2007), in endometrial cancer (Masuyama et al., 2007), and in osteosarcoma(Mensah-Osman et al., 2007).

5.6. Miscellaneous implications of PXR

5.6.1. PXR and antifibrogenesisPXR was recently proposed as a potential target for antifibrotic therapy. In fact, the extent of fibrosis caused by carbon

tetrachloride is significantly inhibited by the PXR ligand PCN in chronically treated rats. Moreover, the short-term treatmentof hepatic stellate cells with the PXR ligand rifampicin inhibits the expression of fibrosis-related genes whereas the long-term treatment reduces the proliferation and the trans-differentiation of hepatic stellate cells. Both rifampicin effects arePXR dependent (Marek et al., 2005; Haughton et al., 2006).

5.6.2. PXR and the oxidative stressA heightened sensitivity to oxidative toxicant paraquat was noted both in PXR humanized and in wild-type mice upon

PXR activation. Consistent with this in vivo study, cell lines with activated human PXR were also sensitive to paraquat,and an increased production of reactive oxygen species was observed. These data suggest that PXR activation is a risk factorfor oxidative stress caused by an imbalance between the production of reactive oxygen species and detoxification of thereactive intermediates (Gong et al., 2006). However, further studies are necessary to highlight the role of PXR in oxidativestress, due to its importance in drug toxicity and human disease.

6. Concluding remarks and future directions

CAR (also called MB67) was originally described in 1994 as a new orphan member of the NR super-family predominantlyexpressed in the liver (Baes et al., 1994). Remarkably, an important role of CAR in the complex network of proteins involvedin the response to retinoic acid and its metabolites was proposed. At the start of the new century the interest for this receptorwas very low (just 27 articles cited in PubMed data base from 1994 to January 2000) to grow up exponentially in the recentyears (481 articles cited in PubMed data base from 2000 to the end of 2008; already there are 28 articles cited in the firsttrimester of 2009). PXR (initially called steroid X receptor) was originally described in 1998 as a xenobiotic sensor (Bertilssonet al., 1998; Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998), catching immediately the attention of



researchers (18 articles cited in PubMed data base from 1998 to January 2000) and still stimulating research (757 articlescited in PubMed data base from 2000 to the end of 2008; already there are 60 articles cited in the first trimester of 2009).

The bulk of these investigations contributes to define the role of PXR as a key mediator of an elaborate network of genesinvolved in the detoxification and clearance of endobiotics and xenobiotics (Zhou et al., 2009). Although several interspeciesdifferences between rodents and humans have been observed, the data generated from the PXR-null and PXR-humanizedmouse lines served as valuable in vivo models for investigations on PXR (Kliewer et al., 2002). The role of CAR, however,is still ambiguous probably due to the lack of good models. CAR has the potential to impact numerous signaling pathwaysvia the genes that it modulates directly and by its interference with other NR signaling pathways (Swales and Negishi, 2004).This creates a unique integrative mechanism to modulate the metabolism of not only xenobiotics but also endogenously pro-duced steroids and dietary factors. Unlike its closest relative PXR, the function of which relies solely on ligand binding, CARfunctions ligand independently and can be regulated by both direct ligand binding and indirect activation processes (Lim andHuang, 2008). In light of this, the elucidation of the mechanistic aspects of CAR activation, nuclear translocation and tran-scriptional activity is extremely important to divulge the pathways involved and the evolutionary drive that separatedCAR from PXR.

However, this is just the beginning of the story. Several new avenues of research have been opened in the recent yearsthat have revealed new and mostly unsuspected roles for CAR and PXR in energy and hormone homeostasis, inflammation,bone homeostasis, and cancer (Ma et al., 2008; Zhou et al., 2009). These results suggest that CAR and PXR possess a numberof important functions in the body that remain to be fully explored. Moreover, a field in rapid expansion is the study of cross-talk between other NRs (e.g., FXR, LXR, PPAR, ER, and GR). This recently discovered interplay appears to be important for theregulation of diverse target genes (Lim and Huang, 2008).

The extreme flexibility and versatility of NRs open the prospect of regulating their transcriptional activity by ligands,post-translational modifications, partners, co-receptors, and promoter context. While NRs activate batteries of genes, co-activators activate batteries of NRs and transcription factors. In addition, co-activators exist as multi-protein complexes,are subjected to transcriptional regulation, post-translational modification, and controlled degradation, and exhibit poly-morphisms, which are expected to influence the activity of their partners. We are therefore far from having a wide and clearview of the tangle of regulatory networks in which the signaling pathways controlling xenobiotic/drug metabolism and dis-position are embedded even if CAR and PXR suggest new mechanisms through which diet, chemical exposure, and environ-ment ultimately impact human health and diseases (Ma et al., 2008; Zhou et al., 2009).

The studies described in this review substantiate both structural and functional differences between CAR and PXR. Futurestudies will continue to enlarge our knowledge of the differences between these receptors. For example, crystal structureswill allow a detailed comparison of ligand-binding pockets of CAR and PXR and will aid in the understanding of the uniquestructure/function relationships inherent in each LBD. Also, microarray experiments will allow an unbiased view of the dif-ferential effects of these two receptors on gene expression. These experiments should be performed not just in liver, but inother tissues where CAR and PXR are expressed such as intestine and kidney. Overall, the data will ultimately lead to a betterunderstanding of the distinct physiological role of these two receptors.

Acknowledgements

The authors wish to thank Dr. Alessandro Bolli, Dr. Fabio Polticelli, and Dr. Daniele Salvi (University Roma Tre, Roma,Italy) for helpful discussions. This work was partially supported by grants from the Ministry for Health of Italy (NationalInstitute for Infectious Diseases I.R.C.C.S. ‘‘Lazzaro Spallanzani”, Roma, Italy, Ricerca corrente 2008 to P.A.) and from the Min-istry of Education, University, and Research of Italy (CLAR 2008 to P.A.).

References

Agrawal, A.K., Shapiro, B.H., 1996. Phenobarbital induction of hepatic CYP2B1 and CYP2B2: pretranscriptional and post-transcriptional effects of gender,adult age, and phenobarbital dose. Mol. Pharmacol. 49, 523–531.

Argaud, D., 1991. Inhibition of gluconeogenesis in isolated rat hepatocytes after chronic treatment with phenobarbital. Biochem. J. 280, 663–669.

Arnold, K.A., Eichelbaum, M., Burk, O., 2004. Alternative splicing affects the function and tissue-specific expression of the human constitutive androstanereceptor. Nucl. Recept. 2, 1.

Ascenzi, P., Bocedi, A., Marino, M., 2006. Structure-function relationship of estrogen receptor alpha and beta: impact on human health. Mol. Aspects Med. 27,299–402.

Auerbach, S.S., Ramsden, R., Stoner, M.A., Verlinde, C., Hassett, C., Omiecinski, C.J., 2003. Alternatively spliced isoforms of the human constitutive androstanereceptor. Nucl. Acid. Res. 31, 3194–3207.

Axelrod, J., 1955. The enzymatic deamination of amphetamine (bezedrine). J. Biol. Chem. 214, 753–763.Backman, J.T., Olkkola, K.T., Neuvonen, P.J., 1996. Rifampin drastically reduces plasma concentrations and effects of oral midazolam. Clin. Pharmacol. Ther.

59, 7–13.Badawi, A.F., Cavalieri, E.L., Rogan, E.G., 2001. Role of human cytochrome P450 1A1, IA2, 1B1, and 3A4 in the 2-, 4-, and 16 alpha-hydroxylation of 17 beta-

estradiol. Metabolism 50, 1001–1003.Baes, M., Gulick, T., Choi, H.S., Martinoli, M.G., Simha, D., Moore, D.D., 1994. A new orphan member of the nuclear hormone receptor superfamily that

interacts with a subset of retinoic acid response elements. Mol. Cell. Biol. 14, 1544–1552.Bauer, B., Hartz, A.M., Fricker, G., Miller, D.S., 2004. Pregnane X receptor up-regulation of P-glycoprotein expression and transport function at the blood–

brain barrier. Mol. Pharmacol. 66, 413–419.Behravan, J., Piquette-Miller, M., 2007. Drug transport across the placenta, role of the ABC drug efflux transporters. Expert Opin. Drug Metab. Toxicol. 3, 819–

830.



Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Backman, M., Ohlsson, R., Postlind, H., Blomquist, P., Berkenstam, A., 1998.Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. USA 95, 12208–12213.

Blizard, D., Sueyoshi, T., Negishi, M., Dehal, S.S., Kupfer, D., 2001. Mechanism of induction of cytochrome p450 enzymes by the proestrogenic endocrinedisruptor pesticide-methoxychlor: interactions of methoxychlor metabolites with the constitutive androstane receptor system. Drug Metab. Dispos. 29,781–785.

Blumberg, B., Sabbagh, W., Juguilon, H., Bolado, J., van Meter, C.M., Ono, E.S., Evans, R.M., 1998. SXR, a novel steroid and xenobiotic-sensing nuclear receptor.Genes Dev. 12, 3195–3205.

Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H., Moras, D., 1995. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-alpha. Nature 375, 377–382.

Bourguet, W., Germain, P., Gronemeyer, H., 2000. Nuclear receptor ligand-binding domains: three-dimensional structures, molecular interactions andpharmacological implications. Trends Pharmacol. Sci. 21, 381–388.

Brinkmann, U., Eichelbaum, M., 2001. Polymorphisms in the ABC drug transporter gene MDR1. Pharmacogenom. J. 1, 59–64.Bruck, N., Bastien, J., Bour, G., Tarrade, A., Plassat, J.L., Bauer, A., Adam-Stitah, S., Rochette-Egly, C., 2005. Phosphorylation of the Retinoid X Receptor at the

Omega loop, modulates the expression of retinoic-acid-target genes with a promoter context specificity. Cell. Signal. 17, 1229–1239.Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard, R.E., Bonn, T., Engstrom, O., Ohman, L., Greene, G.L., Gustafsson, J.Å., Carlquist, M., 1997. Molecular basis

of agonism and antagonism in the oestrogen receptor. Nature 389, 753–758.Burk, O., Arnold, K.A., Nussler, A.K., Schaeffeler, E., Efimova, E., Avery, B.A., Avery, M.A., Fromm, M.F., Eichelbaum, M., 2005. Antimalarial artemisinin drugs

induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor. Mol. Pharmacol.67, 1954–1965.

Buters, J.T.M., Korzekwa, K.R., Kunze, K.L., Omata, Y., Hardwick, J.P., Gonzalez, F.J., 1994. cDNA-directed expression of human cytochrome-p450 cyp3a4 usingbaculovirus. Drug Metab. Dispos. 22, 688–692.

Carnahan, V.E., Redinbo, M.R., 2005. Structure and function of the human nuclear xenobiotic receptor PXR. Curr. Drug Metab. 6, 357–367.Chang, T.K., Waxman, D.J., 2006. Synthetic drugs and natural products as modulators of constitutive androstane receptor (CAR) and pregnane X receptor

(PXR). Drug Metab. Rev. 38, 51–73.Chang, T.K.H., Anderson, M.D., Bandiera, S.M., Bellward, G.D., 1997. Effect of ovariectomy and androgen on phenobarbital induction of hepatic CYP2B1 and

CYP2B2 in Sprague-Dawley rats. Drug Metab. Dispos. 25, 994–1000.Chen, C., Yu, R., Owuor, E.D., Kong, A.N., 2000. Activation of antioxidant-response element (ARE), mitogen-activated protein kinases (MAPKs) and caspases

by major green tea polyphenol components during cell survival and death. Arch. Pharm. Res. 23, 605–612.Chen, Y., Tang, Y., Wang, M.T., Zeng, S., Nie, D., 2007. Human pregnane X receptor and resistance to chemotherapy in prostate cancer. Cancer Res. 67, 10361–

10367.Chen, C., Shah, Y.M., Morimura, K., Krausz, K.W., Miyazaki, M., Richardson, T.A., Morgan, E.T., Ntambi, J.M., Idle, J.R., Gonzalez, F.J., 2008. Metabolomics

reveals that hepatic stearoyl-CoA desaturase 1 downregulation exacerbates inflammation and acute colitis. Cell Metab. 7, 135–147.Choi, H.S., Chung, M., Tzameli, I., Simha, D., Lee, Y.K., Seol, W., Moore, D.D., 1997. Differential transactivation by two isoforms of the orphan nuclear hormone

receptor CAR. J. Biol. Chem. 272, 23565–23571.Choi, E., Lee, S., Yeom, S.Y., Kim, G.H., Lee, J.W., Kim, S.W., 2005. Characterization of activating signal cointegrator-2 as a novel transcriptional coactivator of

the xenobiotic nuclear receptor constitutive androstane receptor. Mol. Endocrinol. 19, 1711–1719.Cholerton, S., Daly, A.K., Idle, J.R., 1992. The role of individual human cytochromes P450 in drug metabolism and clinical response. Trends Pharmacol. Sci. 13,

434–439.Chrencik, J.E., Orans, J., Moore, L.B., Xue, Y., Peng, L., Collins, J.L., Wisely, G.B., Lambert, M.H., Kliewer, S.A., Redinbo, M.R., 2005. Structural disorder in the

complex of human pregnane X receptor and the macrolide antibiotic rifampicin. Mol. Endocrinol. 19, 1125–1134.Christin-Maitre, S., Bouchard, P., Spitz, I.M., 2000. Medical termination of pregnancy. New. Engl. J. Med. 342, 946–956.Clark, A.G., Glanowski, S., Nielsen, R., Thomas, P.D., Kejariwal, A., Todd, M.A., Tanenbaum, D.M., Civello, D., Lu, F., Murphy, B., Ferriera, S., Wang, G., Zheng, X.,

White, T.J., Sninsky, J.J., Adams, M.D., Cargill, M., 2003. Inferring nonneutral evolution from human-chimp-mouse orthologous gene trios. Science 302,1960–1963.

Collot-Teixeira, S., Martin, J., McDermott-Roe, C., Poston, R., McGregor, J.L., 2007. CD36 and Macrophages in Atherosclerosis. Cardiovasc. Res. 75, 468–477.Coumoul, X., Diry, M., Barouki, R., 2002. PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides. Biochem. Pharmacol. 64,

1513–1519.Crippin, J.S., 1993. Acetaminophen hepatotoxicity – potentiation by isoniazid. Am. J. Gastroenterol. 88, 590–592.Curran, P.G., DeGroot, L.J., 1991. The effect of hepatic enzyme inducing drugs on thyroid hormones and the thyroid gland. Endocrinol. Rev. 12, 135–150.Dai, G., He, L., Bu, P., Wan, Y.J., 2008. Pregnane X receptor is essential for normal progression of liver regeneration. Hepatology 47, 1277–1287.Dean, M., Rzhetsky, A., Allikmets, R., 2001. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res. 11, 1156–1166.Dean Jr., C.E., Benjamin, S.A., Chubb, L.S., Tessari, J.D., Keefe, T.J., 2002. Nonadditive hepatic tumor promoting effects by a mixture of two structurally

different polychlorinated biphenyls in female rat livers. Toxicol. Sci. 66, 54–61.DeKeyser, J.G., Stagliano, M.C., Auerbach, S.S., Prabu, K.S., Jones, A.D., Omiecinski, C.J., 2009. Di(2-ethylhexyl) phthalate is a highly potent agonist for the

human constitutive androstane receptor splice variant, CAR2. Mol. Pharmacol. 75, 1005–1013.Dickins, M., 2004. Induction of cytochromes P450. Curr. Top Med. Chem. 4, 1745–1766.Ding, X., Staudinger, J.L., 2005a. Induction of drug metabolism by forskolin: the role of the pregnane X receptor and the protein kinase a signal transduction

pathway. J. Pharmacol. Exp. Ther. 312, 849–856.Ding, X., Staudinger, J.L., 2005b. Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C. Biochem. Pharmacol. 69, 867–

873.Dixit, S.G., Tirona, R.G., Kim, R.B., 2005. Beyond CAR and PXR. Curr. Drug Metab. 6, 385–397.Dobrzyn, A., Ntambi, J.M., 2005. The role of stearoyl-CoA desaturase in the control of metabolism. Prostaglandins Leukot Essent Fatty Acids 73, 35–41.Doherty, M.M., Charman, W.N., 2002. The mucosa of the small intestine: how clinically relevant as an organ of drug metabolism? Clin. Pharmacokinetics 41,

235–253.Dotzlaw, H., Leygue, E., Watson, P., Murphy, L.C., 1999. The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast

tissue. Clin. Cancer Res. 5, 2103–2107.Dring, M.M., Goulding, C.A., Trimble, V.I., Keegan, D., Ryan, A.W., Brophy, K.M., Smyth, C.M., Keeling, P.W., O’Donoghue, D., O’Sullivan, M., O’Morain, C.,

Mahmud, N., Wikstrom, A.C., Kelleher, D., McManus, R., 2006. The pregnane X receptor locus is associated with susceptibility to inflammatory boweldisease. Gastroenterology 130, 341–348.

Duniec-Dmuchowski, Z., Ellis, E., Strom, S.C., Kocarek, T.A., 2007. Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists. Biochem.Pharmacol. 74, 1535–1540.

Ebersberger, I., Metzler, D., Schwarz, C., Pääbo, S., 2002. Genomewide comparison of DNA sequences between humans and chimpanzees. Am. J. Hum. Genet.70, 1490–1497.

Egea, P.F., Klaholz, B.P., Moras, D., 2000. Ligand–protein interactions in nuclear receptors of hormones. FEBS Lett. 476, 62–67.Eichelbaum, M., Spannbrucker, N., Dengler, H.J., 1978. In: Gorrod, J.W. (Ed.), A Probably Genetic Defect of the Metabolism of Sparteine. Biological Oxidation

of Nitrogen. Elsevier, Amsterdam, pp. 113–118.Erdmann, O.L., Marchand, R.F., 1842. Metabolism of cinnamic acid to hippuric acid in animals. Annu. Chem. Pharm. 44, 344.Escriva, H., Manzon, L., Youson, J., Laudet, V., 2002. Analysis of lamprey and hagfish genes reveals a complex history of gene duplications during early

vertebrate evolution. Mol. Biol. Evol. 19, 1440–1450.



Escriva, H., Bertrand, S., Laudet, V., 2004. The evolution of the nuclear receptor superfamily. Essays Biochem. 40, 11–26.Estabrook, R.W., Cooper, D.Y., Rosenthal, S., 1963. The light-reversible carbon monoxide inhibition of the steroid C-21 hydroxylation system of the adrenal

cortex. Biochem. Z. 338, 741–755.Fayer, J.L., Petullo, D.M., Ring, B.J., Wrighton, S.A., Ruterbories, K.J., 2001. A novel testosterone 6 beta-hydroxylase activity assay for the study of CYP3A-

mediated metabolism, inhibition, and induction in vitro. J. Pharmacol. Toxicol. Methods 46, 117–123.Forman, B.M., Tzameli, I., Choi, H.S., Chen, J., Simha, D., Seol, W., Evans, R.M., Moore, D.D., 1998. Androstane metabolites bind to and deactivate the nuclear

receptor CAR-beta. Nature 395, 612–615.Francis, G.A., Fayard, E., Picard, F., Auwerx, J., 2003. Nuclear receptors and the control of metabolism. Annu. Rev. Physiol. 65, 261–311.Frank, C., Gonzalez, M.M., Oinonen, C., Dunlop, T.W., Carlberg, C., 2003. Characterization of DNA complexes formed by the nuclear receptor constitutive

androstane receptor. J. Biol. Chem. 278, 43299–43310.Frank, C., Makkonen, H., Dunlop, T.W., Matilainen, M., Vaisanen, S., Carlberg, C., 2005. Identification of pregnane X receptor binding sites in the regulatory

regions of genes involved in bile acid homeostasis. J. Mol. Biol. 346, 505–519.Fraser, A., Gafter-Gvili, A., Paul, M., Leibovici, L., 2006. Antibiotics for preventing meningococcal infections. Cochrane Database Syst. Rev. 18, CD004785.Fuhr, U., 2000. Induction of drug metabolising enzymes: pharmacokinetic and toxicological consequences in humans. Clin. Pharmacokinet. 38, 493–504.Galluzzo, P., Marino, M., 2006. Nutritional flavonoids impact on nuclear and extranuclear estrogen receptor activities. Genes Nutr. 1, 161–176.Gampe Jr., R.T., Montana, V.G., Lambert, M.H., Miller, A.B., Bledsoe, R.K., Milburn, M.V., Kliewer, S.A., Willson, T.M., Xu, H.E., 2000. Asymmetry in the PPARc/

RXRa crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol. Cell. 5, 545–555.Ganapathy, V., Prasad, P.D., 2005. Role of transporters in placental transfer of drugs. Toxicol. Appl. Pharmacol. 207, 381–387.Ganem, L.G., Trottier, E., Anderson, A., Jefcoate, C.R., 1999. Phenobarbital induction of CYP2B1/2 in primary hepatocytes: endocrine regulation and evidence

for a single pathway for multiple inducers. Toxicol. Appl. Pharmacol. 155, 32–42.Garfinkel, D., 1958. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch. Biochem. Biophys. 77,

493–509.Gerbal-Chaloin, S., Pascussi, J.M., Pichard-Garcia, L., Daujat, M., Waechter, F., Fabre, J.M., Carrere, N., Maurel, P., 2001. Induction of CYP2C genes in human

hepatocytes in primary culture. Drug Metab. Dispos. 29, 242–251.Gerbal-Chaloin, S., Daujat, M., Pascussi, J.M., Pichard-Garcia, L., Vilarem, M.J., Maurel, P.J., 2002. Transcriptional regulation of CYP2C9 gene. Role of

glucocorticoid receptor and constitutive androstane receptor. J. Biol. Chem. 277, 209–217.Gomez-Duran, A., Carvajal-Gonzalez, J.M., Mulero-Navarro, S., Santiago-Josefat, B., Puga, A., Fernandez-Salguero, P.M., 2009. Fitting a xenobiotic receptor

into cell homeostasis: how the dioxin receptor interacts with TGFbeta signaling. Biochem. Pharmacol. 77, 700–712.Gong, H., Singh, S.V., Singh, S.P., Mu, Y., Lee, J.H., Saini, S.P., Toma, D., Ren, S., Kagan, V.E., Day, B.W., Zimniak, P., Xie, W., 2006. Orphan nuclear receptor

pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells. Mol. Endocrinol. 20, 279–290.Goodwin, B., Hodgson, E., Liddle, C., 1999. The orphan human Pregnane X Receptor mediates the transcriptional activation of CYP3A4 by rifampicin through

a distal enhancer module. Mol. Pharmacol. 56, 1329–1339.Goodwin, B., Moore, L.B., Stoltz, C.M., McKee, D.D., Kliewer, S.A., 2001. Regulation of the human CYP2B6 gene by the nuclear pregnane X receptor. Mol.

Pharmacol. 60, 427–431.Goodwin, B., Redinbo, M.R., Kliewer, S.A., 2002. Regulation of cyp3a gene transcription by the pregnane x receptor. Annu. Rev. Pharmacol. Toxicol. 42, 1–23.Goodwin, B., Gauthier, K.C., Umetani, M., Watson, M.A., Lochansky, M.I., Collins, J.L., Leitersdorf, E., Mangelsdorf, D.J., Kliewer, S.A., Repa, J.J., 2003.

Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor. Proc. Natl. Acad. Sci. USA 100, 223–228.Graham, M.J., Lake, B.G., 2008. Induction of drug metabolism: species differences and toxicological relevance. Toxicology 254, 184–191.Guo, G.L., Lambert, G., Negishi, M., Ward, J.M., Brewer Jr., H.B., Kliewer, S.A., Gonzalez, F.J., Sinal, C.J., 2003. Complementary roles of farnesoid X receptor,

pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity. J. Biol. Chem. 278, 45062–45071.Guo, G.L., Moffit, J.S., Nicol, C.J., Ward, J.M., Aleksunes, L.A., Slitt, A.L., Kliewer, S.A., Manautou, J.E., Gonzalez, F.J., 2004. Enhanced acetaminophen toxicity by

activation of the pregnane X receptor. Toxicol. Sci. 82, 374–380.Guslandi, M., Petrone, M.C., Testoni, P.A., 2006. Rifaximin for active ulcerative colitis. Inflamm. Bowel Dis. 12, 335.Hahn, M.E., Allan, L.L., Sherr, D.H., 2009. Regulation of constitutive and inducible AHR signaling: complex interactions involving the AHR repressor. Biochem.

Pharmacol. 77, 485–497.Handschin, C., Podvinec, M., Meyer, U.A., 2000. CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X

receptor (PXR) and constitutive androstane receptor (CAR). Proc. Natl. Acad. Sci. USA 97, 10769–10774.Harmsen, S., Meijerman, I., Beijnen, J.H., Schellens, J.H., 2007. The role of nuclear receptors in pharmacokinetic drug–drug interactions in oncology. Cancer

Treat. Rev. 33, 369–380.Harrer, G., Hubner, W., Prodzuwent, I., 1999. Effectiveness and tolerance of the hypericum extract LI 160 compared to maprotiline: a multicenter double-

blind study. J. Geriatr. Psychiat. Neurol. 7, 524–528.Haughton, E.L., Tucker, S.J., Marek, C.J., Durward, E., Leel, V., Bascal, Z., Monaghan, T., Koruth, M., Collie-Duguid, E., Mann, D.A., Trim, J.E., Wright, M.C., 2006.

Pregnane X receptor activators inhibit human hepatic stellate cell transdifferentiation in vitro. Gastroenterology 131, 194–209.Hayashi, O., Katagiri, M., Rothberg, S., 1955. Mechanism of the pyrocatechase reaction. J. Am. Chem. Soc. 77, 5450–5451.Hegardt, F.G., 1999. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a control enzyme in ketogenesis. Biochem. J. 338, 569–582.Hinson, J.A., Forkert, P.G., 1995. Phase II enzymes and bioactivation. Can. J. Physiol. Pharmacol. 73, 1407–1413.Holm, L., Sander, C., 1994. The FSSP database of structurally aligned protein fold families. Nucl. Acid. Res. 22, 3600–3609.Holm, L., Sander, C., 1997. Dali/FSSP classification of threedimensional protein folds. Nucl. Acid. Res. 25, 231–234.Holm, L., Ouzounis, C., Sander, C., Tuparev, G., Vriend, G., 1992. A database of protein structure families with common folding motifs. Protein Sci. 1, 1691–

1698.Honkakoski, P., Negishi, M., 1998. Protein serine/threonine phosphatase inhibitors suppress phenobarbitalinduced Cyp2b10 gene transcription in mouse

primary hepatocytes. Biochem. J. 330, 889–895.Honkakoski, P., Palvimo, J.J., Penttila, L., Vepsalainen, J., Auriola, S., 2004. Effects of triaryl phosphates on mouse and human nuclear receptors. Biochem.

Pharmacol. 67, 97–106.Hosseinpour, F., Moore, R., Negishi, M., Sueyoshi, T., 2006. Serine 202 regulates the nuclear translocation of constitutive active/androstane receptor. Mol.

Pharmacol. 69, 1095–1102.Hu, X., Lazar, M.A., 1999. The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402, 93–96.Huang, W., Zhang, J., Chua, S.S., Qatanani, M., Han, Y., Granata, R., Moore, D.D., 2003. Induction of bilirubin clearance by the constitutive androstane receptor

(CAR). Proc. Natl. Acad. Sci. USA 100, 4156–4161.Huls, M., Russel, F.G., Masereeuw, R., 2009. The role of ATP binding cassette transporters in tissue defense and organ regeneration. J. Pharmacol. Exp. Ther.

328, 3–9.Hurst, C.H., Waxman, D.J., 2004. Environmental phthalate monoesters activate pregnane X receptor-mediated transcription. Toxicol. Appl. Pharmacol. 199,

266–274.Huss, J.M., Kasper, C.B., 2000. Two-stage glucocorticoid induction of CYP3A23 through both the glucocorticoid and pregnane X receptors. Mol. Pharmacol.

58, 48–57.Hustert, E., Zibat, A., Presecan-Siedel, E., Eiselt, R., Mueller, R., Fuss, C., Brehm, I., Brinkmann, U., Eichelbaum, M., Wojnowski, L., Burk, O., 2001. Natural

protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4. Drug Metab. Dispos. 29, 1454–1459.Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H., Kato, S., 2007. Vitamin K induces osteoblast differentiation through pregnane X receptor-

mediated transcriptional control of the Msx2 gene. Mol. Cell. Biol. 27, 7947–7954.



Ikeda, S., Kurose, K., Ozawa, S., Sai, K., Hasegawa, R., Komamura, K., Ueno, K., Kamakura, S., Kitakaze, M., Tomoike, H., Nakajima, T., Matsumoto, K., Saito, H.,Goto, Y., Kimura, H., Katoh, M., Sugai, K., Minami, N., Shirao, K., Tamura, T., Yamamoto, N., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., Saito, Y., Sawada, J.,2003. Twenty-six novel single nucleotide polymorphisms and their frequencies of the NR1I3 (CAR) gene in a Japanese population. Drug Metab.Pharmacokinet. 18, 413–418.

Ikeda, S., Kurose, K., Jinno, H., Sai, K., Ozawa, S., Hasegawa, R., Komamura, K., Kotake, T., Morishita, H., Kamakura, S., Kitakaze, M., Tomoike, H., Tamura, T.,Yamamoto, N., Kunitoh, H., Yamada, Y., Ohe, Y., Shimada, Y., Shirao, K., Kubota, K., Minami, H., Ohtsu, A., Yoshida, T., Saijo, N., Saito, Y., Sawada, J., 2005.Functional analysis of four naturally occurring variants of human constitutive androstane receptor. Mol. Genet. Metab. 86, 314–319.

Inoue, K., Borchers, C.H., Negishi, M., 2006. Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 geneby xenobiotics. Biochem. J. 398, 125–133.

Ivanovic, J., Nicastri, E., Ascenzi, P., Bellagamba, R., De Marinis, E., Notari, S., Pucillo, L.P., Tozzi, V., Ippolito, G., Narciso, P., 2008. Therapeutic drug monitoringin the management of HIV-infected patients. Curr. Med. Chem. 15, 1925–1939.

Iyer, M., Reschly, E.J., Krasowski, M.D., 2006. Functional evolution of the pregnane X receptor. Expert Opin. Drug Metab. Toxicol. 2, 381–397.Jia, Y., Guo, G.L., Surapureddi, S., Sarkar, J., Qi, C., Guo, D., Xia, J., Kashireddi, P., Yu, S., Cho, Y.W., Rao, M.S., Kemper, B., Ge, K., Gonzalez, F.J., Reddy, J.K., 2005.

Transcription coactivator peroxisome proliferatoractivated receptor-binding protein/mediator 1 deficiency abrogates acetaminophen hepatotoxicity.Proc. Natl. Acad. Sci. USA 102, 12531–12536.

Jones, S.A., Moore, L.B., Shenk, J.L., Wisely, G.B., Hamilton, G.A., McKee, D.D., Tomkinson, N.C., LeCluyse, E.L., Lambert, M.H., Willson, T.M., Kliewer, S.A.,Moore, J.T., 2000. The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol. Endocrinol. 14, 27–39.

Jyrkkarinne, J., Windshugel, B., Makinen, J., Ylisirnio, M., Perakyla, M., Poso, A., Sippl, W., Honkakoski, P., 2005. Amino acids important for ligand specificity ofthe human constitutive androstane receptor. J. Biol. Chem. 280, 5960–5971.

Kast, H.R., Goodwin, B., Tarr, P.T., Jones, S.A., Anisfeld, A.M., Stoltz, C.M., Tontonoz, P., Kliewer, S., Willson, T.M., Edwards, P.A., 2002. Regulation of multidrugresistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstanereceptor. J. Biol. Chem. 277, 2908–2915.

Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K., Negishi, M., 1999. Phenobarbital-responsive nuclear translocation of the receptor CAR ininduction of the CYP2B gene. Mol. Cell. Biol. 19, 6318–6322.

Kawamoto, T., Kakizaki, S., Yoshinari, K., Negishi, M., 2000. Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in inductionof the mouse Cyp2b10 gene. Mol. Endocrinol. 14, 1897–1905.

Kawana, K., Ikuta, T., Kobayashi, Y., Gotoh, O., Takeda, K., Kawajiri, K., 2003. Molecular mechanism of nuclear translocation of an orphan nuclear receptor,SXR. Mol. Pharmacol. 63, 524–531.

Keller, W., 1842. On the conversion of benzoic acid into hippuric acid. Annu. Chem. Pharm. 125, 9.Kerb, R., Hoffmeyer, S., Brinkmann, U., 2001. ABC drug transporters: hereditary polymorphisms and pharmacological impact in MDR1, MRP1 and MRP2.

Pharmacogenomics 2, 51–64.Kietzmann, T., Hirsch-Ernst, K.I., Kahl, G.F., Jungermann, K., 1999. Mimicry in primary rat hepatocyte cultures of the in vivo perivenous induction by

phenobarbital of cytochrome P-450 2B1 mRNA: role of epidermal growth factor and perivenous oxygen tension. Mol. Pharmacol. 56, 46–53.Kim, R.B., 2003. Organic anion-transporting polypeptide (OATP) transporter family and drug disposition. Eur. J. Clin. Invest. 33, 1–5.Kim, H.J., Lee, S.K., Na, S.Y., Choi, H.S., Lee, J.W., 1998. Molecular cloning of xSRC-3, a novel transcription coactivator from Xenopus, that is related to AIB1, p/

CIP, and TIF2. Mol. Endocrinol. 512, 1038–1047.Kliewer, S.A., Moore, J.T., Wade, L., Staudinger, J.L., Watson, M.A., Jones, S.A., McKee, D.D., Oliver, B.B., Willson, T.M., Zetterstrom, R.H., Perlmann, T., Lehmann,

J.M., 1998. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 73–82.Kliewer, S.A., Goodwin, B., Willson, T.M., 2002. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocrinol. Rev. 23, 687–702.Klingenberg, M., 1958. Pigments of rat liver microsomes. Arch. Biochem. Biophys. 75, 376–386.Knoblauch, R., Garabedian, M.J., 1999. Role for Hsp90-associated cochaperone p23 in estrogen receptor signal transduction. Mol. Cell. Biol. 19, 3748–3759.Kobayashi, K., Sueyoshi, T., Inoue, K., Moore, R., Negishi, M., 2003. Cytoplasmic accumulation of the nuclear receptor CAR by a tetratricopeptide repeat

protein in HepG2 cells. Mol. Pharmacol. 64, 1069–1075.Kobayashi, K., Yamanaka, Y., Iwazaki, N., Nakajo, I., Hosokawa, M., Negishi, M., Chiba, K., 2005. Identification of HMG–CoA reductase inhibitors as activators

for human, mouse and rat constitutive androstane receptor. Drug Metab. Dispos. 33, 924–929.Kodama, S., Koike, C., Negishi, M., Yamamoto, Y., 2004. Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-

metabolizing and gluconeogenic enzymes. Mol. Cell. Biol. 24, 7931–7940.Köhle, C., Bock, K.W., 2009. Coordinate regulation of human drug-metabolizing enzymes, and conjugate transporters by the Ah receptor, pregnane X

receptor and constitutive androstane receptor. Biochem. Pharmacol. 77, 689–699.Kong, A.N., Yu, R., Chen, C., Mandlekar, S., Primiano, T., 2000. Signal transduction events elicited by natural products: role of MAPK and caspase pathways in

homeostatic response and induction of apoptosis. Arch. Pharm. Res. 23, 1–16.Konishi, H., Tanaka, K., Minouchi, T., Yamaji, A., 2004. Urinary 6 beta-hydroxycortisol/17-hydroxycorticosteroids ratio as a measure of hepatic CYP3A4

capacity after enzyme induction. Annu. Clin. Biochem. 41, 335–337.Konno, Y., Negishi, M., Kodama, S., 2008. The roles of nuclear receptors CAR and PXR in hepatic energy metabolism. Drug Metab. Pharmacokinet. 23, 8–13.Koonen, D.P.Y., Jacobs, R.L., Febbraio, M., Young, M.E., Soltys, C.L.M., Ong, H., Vance, D.E., Dyck, J.R.B., 2007. Increased hepatic CD36 expression contributes to

dyslipidemia associated with diet-induced obesity. Diabetes 56, 2863–2871.Koyano, S., Kurose, K., Saito, Y., Ozawa, S., Hasegawa, R., Komamura, K., Ueno, K., Kamakura, S., Kitakaze, M., Nakajima, T., Matsumoto, K., Akasawa, A., Saito,

H., Sawada, J., 2004. Functional characterization of four naturally occurring variants of human pregnane X receptor (PXR): one variant causes dramaticloss of both DNA binding activity and the transactivation of the CYP3A4 promoter/enhancer region. Drug Metab. Dispos. 32, 149–154.

Krasowski, M.D., Yasuda, K., Hagey, L.R., Schuetz, E.G., 2005. Evolution of the pregnane x receptor: adaptation to cross-species differences in biliary bile salts.Mol. Endocrinol. 19, 1720–1739.

Kretschmer, X.C., Baldwin, W.S., 2005. CAR and PXR: xenosensors of endocrine disrupters? Chem. Biol. Interact. 155, 111–128.Kumar, R., Thompson, E.B., 1999. The structure of the nuclear hormone receptors. Steroids 64, 310–319.Kumar, R., Johnson, B.H., Thompson, E.B., 2004. In: McEwan, I.J. (Ed.), Essay in Biochemistry: The Nuclear Receptor Superfamily. Portland Press, London, pp.

27–39.Lahtela, J.T., Arranto, A.J., Sotaniemi, E.A., 1985. Enzyme inducers improve insulin sensitivity in non-insulindependent diabetic subjects. Diabetes 34, 911–

916.Lamba, J.K., Lamba, V., Yasuda, K., Lin, Y.S., Assem, M., Thompson, E., Strom, S., Schuetz, E., 2004a. Expression of constitutive androstane receptor splice

variants in human tissues and their functional consequences. J. Pharmacol. Exp. Ther. 311, 811–821.Lamba, V., Yasuda, K., Lamba, J.K., Assem, M., Davila, J., Strom, S., Schuetz, E., 2004b. PXR (NR1I2): splice variants in human tissues, including brain, and

identification of neurosteroids and nicotine as PXR activators. Toxicol. Appl. Pharmacol. 199, 251–265.Lamba, J., Lamba, V., Schuetz, E., 2005. Genetic variants of PXR (NR1I2) and CAR (NR1I3) and their implications in drug metabolism and pharmacogenetics.

Curr. Drug Metab. 6, 369–383.Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., Funke, R., Gage, D., Harris, K., Heaford,

A., Howland, J., Kann, L., Lehoczky, J., LeVine, R., McEwan, P., McKernan, K., Meldrim, J., Mesirov, J.P., Miranda, C., Morris, W., Naylor, J., Raymond, C.,Rosetti, M., Santos, R., Sheridan, A., Sougnez, C., Stange-Thomann, N., Stojanovic, N., Subramanian, A., Wyman, D., Rogers, J., Sulston, J., Ainscough, R.,Beck, S., Bentley, D., Burton, J., Clee, C., Carter, N., Coulson, A., Deadman, R., Deloukas, P., Dunham, A., Dunham, I., Durbin, R., French, L., Grafham, D.,Gregory, S., Hubbard, T., Humphray, S., Hunt, A., Jones, M., Lloyd, C., McMurray, A., Matthews, L., Mercer, S., Milne, S., Mullikin, J.C., Mungall, A., Plumb, R.,Ross, M., Shownkeen, R., Sims, S., Waterston, R.H., Wilson, R.K., Hillier, L.W., McPherson, J.D., Marra, M.A., Mardis, E.R., Fulton, L.A., Chinwalla, A.T., Pepin,



K.H., Gish, W.R., Chissoe, S.L., Wendl, M.C., Delehaunty, K.D., Miner, T.L., Delehaunty, A., Kramer, J.B., Cook, L.L., Fulton, R.S., Johnson, D.L., Minx, P.J.,Clifton, S.W., Hawkins, T., Branscomb, E., Predki, P., Richardson, P., Wenning, S., Slezak, T., Doggett, N., Cheng, J.F., Olsen, A., Lucas, S., Elkin, C.,Uberbacher, E., Frazier, M., Gibbs, R.A., Muzny, D.M., Scherer, S.E., Bouck, J.B., Sodergren, E.J., Worley, K.C., Rives, C.M., Gorrell, J.H., Metzker, M.L., Naylor,S.L., Kucherlapati, R.S., Nelson, D.L., Weinstock, G.M., Sakaki, Y., Fujiyama, A., Hattori, M., Yada, T., Toyoda, A., Itoh, T., Kawagoe, C., Watanabe, H., Totoki,Y., Taylor, T., Weissenbach, J., Heilig, R., Saurin, W., Artiguenave, F., Brottier, P., Bruls, T., Pelletier, E., Robert, C., Wincker, P., Rosenthal, A., Platzer, M.,Nyakatura, G., Taudien, S., Rump, A., Yang, H.M., Yu, J., Wang, J., Huang, G.Y., Gu, J., Hood, L., Rowen, L., Madan, A., Qin, S.Z., Davis, R.W., Federspiel, N.A.,Abola, A.P., Proctor, M.J., Myers, R.M., Schmutz, J., Dickson, M., Grimwood, J., Cox, D.R., Olson, M.V., Kaul, R., Shimizu, N., Kawasaki, K., Minoshima, S.,Evans, G.A., Athanasiou, M., Schultz, R., Roe, B.A., Chen, F., Pan, H.Q., Ramser, J., Lehrach, H., Reinhardt, R., McCombie, W.R., de la Bastide, M., Dedhia, N.,Blocker, H., Hornischer, K., Nordsiek, G., Agarwala, R., Aravind, L., Bailey, J.A., Bateman, A., Batzoglou, S., Birney, E., Bork, P., Brown, D.G., Burge, C.B.,Cerutti, L., Chen, H.C., Church, D., Clamp, M., Copley, R.R., Doerks, T., Eddy, S.R., Eichler, E.E., Furey, T.S., Galagan, J., Gilbert, J.G.R., Harmon, C., Hayashizaki,Y., Haussler, D., Hermjakob, H., Hokamp, K., Jang, W.H., Johnson, L.S., Jones, T.A., Kasif, S., Kaspryzk, A., Kennedy, S., Kent, W.J., Kitts, P., Koonin, E.V., Korf,I., Kulp, D., Lancet, D., Lowe, T.M., McLysaght, A., Mikkelsen, T., Moran, J.V., Mulder, N., Pollara, V.J., Ponting, C.P., Schuler, G., Schultz, J.R., Slater, G., Smit,A.F.A., Stupka, E., Szustakowki, J., Thierry-Mieg, D., Thierry-Mieg, J., Wagner, L., Wallis, J., Wheeler, R., Williams, A., Wolf, Y.I., Wolfe, K.H., Yang, S.P., Yeh,R.F., Collins, F., Guyer, M.S., Peterson, J., Felsenfeld, A., Wetterstrand, K.A., Patrinos, A., Morgan, M.J.Int Human Genome Sequencing, C., 2001. Initialsequencing and analysis of the human genome. Nature 409, 860–921.

Landes, N., Birringer, M., Brigelius-Flohe, R., 2003. Homologous metabolic and gene activating routes for vitamins E and K. Mol. Aspects Med. 24, 337–344.

Langmann, T., Moehle, C., Mauerer, R., Scharl, M., Liebisch, G., Zahn, A., Stremmel, W., Schmitz, G., 2004. Loss of detoxification in inflammatory boweldisease: dysregulation of pregnane X receptor target genes. Gastroenterology 127, 26–40.

Lannigan, D.A., 2003. Estrogen receptor phosphorylation. Steroids 68, 1–9.Laudet, V., 1997. Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. J. Mol. Endocrinol. 19, 207–226.Laudet, V., Auwerx, J., Gustafsson, J.Å., Wahli, W., 1999. A unified nomenclature system for the nuclear receptor superfamily. Cell 97, 161–163.Laustsen, G., Wimmett, L., 2004. Drug approval highlights: FDA update. Nurse Pract. 30, 14–29.Lazarou, J., Pomeranz, B.H., Corey, P.N., 1998. Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. JAMA 279,

1200–1205.Lee, J.H., Zhou, J., Xie, W., 2008. PXR and LXR in hepatic steatosis: a new dog and an old dog with new tricks. Mol. Pharm. 5, 60–66.Lehmann, J.M., McKee, D.D., Watson, M.A., Willson, T.M., Moore, J.T., Kliewer, S.A., 1998. The human orphan nuclear receptor PXR is activated by compounds

that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102, 1016–1023.Lemaire, G., de Sousa, G., Rahmani, R., 2004. A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides. Biochem.

Pharmacol. 68, 2347–2358.Lemaire, G., Mnif, W., Pascussi, J.M., Pillon, A., Rabenoelina, F., Fenet, H., Gomez, E., Casellas, C., Nicolas, J.C., Cavailles, V., Duchesne, M.J., Balaguer, P., 2006.

Identification of new human PXR ligands among pesticides using a stable reporter cell system. Toxicol. Sci. 91, 501–509.Lim, Y.P., Huang, J.D., 2008. Interplay of pregnane X receptor with other nuclear receptors on gene regulation. Drug Metab. Pharmacokinet. 23, 14–21.Lin, Y., Yasuda, K., Assem, M., Cline, C., Barber, J., Li, C.W., Kholodovych, V., Ai, N., Chen, J.D., Welsh, W.J., Ekins, S., Schuetz, E., 2009. The major human PXR

splice variant, PXR.2, exhibits significantly diminished ligand-activated transcriptional regulation. Drug Metab. Dispos. 37, 1295–1304.Lo, J.C., Schwitzgebel, V.M., Tyrrell, J.B., Fitzgerald, P.A., Kaplan, S.L., Conte, F.A., Grumbach, M.M., 1999. Normal female infants born of mothers with classic

congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J. Clin. Endocrinol. Metab. 84, 930–936.Louet, J.F., LeMay, C., Pegorier, J.P., Decaux, J.F., Girard, J., 2001. Regulation of liver carnitine palmitoyltransferase I gene expression by hormones and fatty

acids. Biochem. Soc. Trans. 29, 310–316.Ma, Q., Lu, A.Y., 2008. The challenges of dealing with promiscuous drug-metabolizing enzymes, receptors and transporters. Curr. Drug Metab. 9, 374–383.Ma, X., Shah, Y.M., Guo, G.L., Wang, T., Krausz, K.W., Idle, J.R., Gonzalez, F.J., 2007. Rifaximin is a gut-specific human pregnane X receptor activator. J.

Pharmacol. Exp. Ther. 322, 391–398.Ma, X., Idle, J.R., Gonzalez, F.J., 2008. The pregnane X receptor: from bench to bedside. Expert Opin. Drug Metab. Toxicol. 4, 895–908.Maglich, J.M., Stoltz, C.M., Goodwin, B., Hawkins-Brown, D., Moore, J.T., Kliewer, S.A., 2002. Nuclear pregnane x receptor and constitutive androstane

receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol. Pharmacol. 62, 638–646.Maglich, J.M., Parks, D.J., Moore, L.B., Collins, J.L., Goodwin, B., Billin, A.N., Stoltz, C.A., Kliewer, S.A., Lambert, M.H., Willson, T.M., Moore, J.T., 2003.

Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J. Biol. Chem. 278,17277–17283.

Maglich, J.M., Watson, J., McMillen, P.J., Goodwin, B., Willson, T.M., Moore, J.T., 2004. The nuclear receptor CAR is a regulator of thyroid hormone metabolismduring caloric restriction. J. Biol. Chem. 279, 19832–19838.

Mahgoub, A., Dring, L.G., Idle, J.R., Lancaster, R., Smith, R.L., 1977. Polymorphic hydroxylation of debrisoquine in man. Lancet 2, 584–586.Maier, A., Zimmermann, C., Beglinger, C., Drewe, J., Gutmann, H., 2007. Effects of budesonide on P-glycoprotein expression in intestinal cell lines. Br. J.

Pharmacol. 150, 361–368.Makinen, J., Reinisalo, M., Niemi, K., Viitala, P., Jyrkkarinne, J., Chung, H., Pelkonen, O., Honkakoski, P., 2003. Dual action of oestrogens on the mouse

constitutive androstane receptor. Biochem. J. 376, 465–472.Makishima, M., Okamoto, A.Y., Repa, J.J., Tu, H., Learned, R.M., Luk, A., Hull, M.V., Lustig, K.D., Mangelsdorf, D.J., Shan, B., 1999. Identification of a nuclear

receptor for bile acids. Science 284, 1362–1365.Mani, S., Huang, H., Sundarababu, S., Liu, W., Kalpana, G., Smith, A.B., Horwitz, S.B., 2005. Activation of the steroid and xenobiotic receptor (human pregnane

X receptor) by nontaxane microtubule-stabilizing agents. Clin. Cancer Res. 11, 6359–6369.Mannering, G.J., Shoeman, J.A., Nutter, L.M., 1993. Effects of the Hops Component, Colupulone, on the Induction of P4503A and the Replication of Human

Tumor Cells. Smith Gordon, London.Mann, K.K., Padovani, A.M., Guo, Q., Colosimo, A.L., Lee, H.Y., Kurie, J.M., Miller Jr., W.H., 2005. Arsenic trioxide inhibits nuclear receptor function via SEK1/

JNK-mediated RXRalpha phosphorylation. J. Clin. Invest. 115, 2924–2933.Marek, C.J., Tucker, S.J., Konstantinou, D.K., Elrick, L.J., Haefner, D., Sigalas, C., Murray, G.I., Goodwin, B., Wright, M.C., 2005. Pregnenolone-16alpha-

carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms. Biochem. J. 387, 601–608.Marino, M., Ascenzi, P., 2008. Membrane association of estrogen receptor a and b influences 17b-estradiol-mediated cancer cell proliferation. Steroids 73,

853–858.Martinez, A., Marquez, A., Mendoza, J., Taxonera, C., Fernandez-Arquero, M., Diaz-Rubio, M., de la Concha, E.G., Urcelay, E., 2007. Role of the PXR gene locus

in inflammatory bowel diseases. Inflamm. Bowel Dis. 13, 1484–1487.Martini, C., Pallottini, V., 2007. Cholesterol: from feeding to gene regulation. Genes Nutr. 2, 181–193.Mason, H.S., Fowlks, L., Peterson, E., 1955. Oxygen transfer and electron transport by the phenolase complex. J. Am. Chem. Soc. 77, 2851–2914.Masuyama, H., Hiramatsu, Y., Kunitomi, M., Kudo, T., MacDonald, P.N., 2000. Endocrine disrupting chemicals, phthalic acid and nonylphenol, activate

Pregnane X receptor-mediated transcription. Mol. Endocrinol. 14, 421–428.Masuyama, H., Hiramatsu, Y., Mizutani, Y., Inoshita, H., Kudo, T., 2001. The expression of pregnane X receptor and its target gene, cytochrome P450 3A1, in

perinatal mouse. Mol. Cell. Endocrinol. 172, 47–56.Masuyama, H., Suwaki, N., Tateishi, Y., Nakatsukasa, H., Segawa, T., Hiramatsu, Y., 2005. The pregnane X receptor regulates gene expression in a ligand- and

promoter-selective fashion. Mol. Endocrinol. 19, 1170–1180.Masuyama, H., Nakatsukasa, H., Takamoto, N., Hiramatsu, Y., 2007. Down-regulation of pregnane X receptor contributes to cell growth inhibition and

apoptosis by anticancer agents in endometrial cancer cells. Mol. Pharmacol. 72, 1045–1053.



Matias, P.M., Donner, P., Coelho, R., Thomaz, M., Peixoto, C., Macedo, S., Otto, N., Joschko, S., Scholz, P., Wegg, A., Bäsler, S., Schäfer, M., Egner, U., Carrondo,M.A., 2000. Structural evidence for ligand specificity in the binding domain of the human androgen receptor. Implications for pathogenic genemutations. J. Biol. Chem. 275, 26164–26171.

Matic, M., Mahns, A., Tsoli, M., Corradin, A., Polly, P., Robertson, G.R., 2007. Pregnane X receptor: promiscuous regulator of detoxification pathways. Int. J.Biochem. Cell. Biol. 39, 478–483.

McEwan, I.J. (Ed.), 2004. Essay in Biochemistry: The Nuclear Receptor Superfamily. Portland Press, London.McInerney, E.M., Rose, D.W., Flynn, S.E., Westin, S., Mullen, T.M., Krones, A., Inostroza, J., Torchia, J., Nolte, R.T., Assa-Munt, N., Milburn, M.V., Glass, C.K.,

Rosenfeld, M.G., 1998. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev. 12, 3357–3368.Mensah-Osman, E.J., Thomas, D.G., Tabb, M.M., Larios, J.M., Hughes, D.P., Giordano, T.J., Lizyness, M.L., Rae, J.M., Blumberg, B., Hollenberg, P.F., Baker, L.H.,

2007. Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines. Cancer 109, 957–965.Meyer, U.A., 1996. Overview of enzymes of drug metabolism. J. Pharmacokinet. Biopharm. 24, 449–459.Michalets, E.L., 1998. Update: clinically significant cytochrome P-450 drug interactions. Pharmacotherapy 18, 84–112.Min, G., Kemper, J.K., Kemper, B., 2002a. Glucocorticoid receptor-interacting protein 1 mediates ligandindependent nuclear translocation and activation of

constitutive androstane receptor in vivo. J. Biol. Chem. 277, 26356–26363.Min, G., Kim, H., Bae, Y., Petz, L., Kemper, J.K., 2002b. Inhibitory cross-talk between estrogen receptor (ER) and constitutively activated androstane receptor

(CAR). CAR inhibits ER-mediated signaling pathway by squelching p160 coactivators. J. Biol. Chem. 277, 34626–34633.Mitro, N., Vargas, L., Romeo, R., Koder, A., Saez, E., 2007. T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR. FEBS Lett. 581, 1721–

1726.Mizuno, N., Niwa, T., Yotsumoto, Y., Sugiyama, Y., 2003. Impact of drug transporter studies on drug discovery and development. Pharmacol. Rev. 55, 425–

461.Mnif, W., Pascussi, J.M., Pillon, A., Escande, A., Bartegi, A., Nicolas, J.C., Cavailles, V., Duchesne, M.J., Balaguer, P., 2007. Estrogens and antiestrogens activate

hPXR. Toxicol. Lett. 170, 19–29.Modrek, B., Lee, C., 2002. A genomic view of alternative splicing. Nat. Genet. 30, 13–19.Moehren, U., Ekey, M., Baniahmad, A., 2004. Gene repression by nuclear hormone receptors. In: The Nuclear Receptor Superfamily. Portland Press, Oxford,

pp. 89–105.Mohan, R., Heyman, R.A., 2003. Orphan nuclear receptor modulators. Curr. Top. Med. Chem. 3, 163747.Moore, L.B., Parks, D.J., Jones, S.A., Bledsoe, R.K., Consler, T.G., Stimmel, J.B., Goodwin, B., Liddle, C., Blanchard, S.G., Willson, T.M., Collins, J.L., Kliewer, S.A.,

2000a. Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J. Biol. Chem. 275,15122–15127.

Moore, L.B., Goodwin, B., Jones, S.A., Wisely, G.B., Serabjit-Singh, C.J., Willson, T.M., Collins, J.L., Kliewer, S.A., 2000b. St. John’s wort induces hepatic drugmetabolism through activation of the pregnane X receptor. Proc. Natl. Acad. Sci. USA 97, 7500–7502.

Moore, L.B., Maglich, J.M., McKee, D.D., Wisely, B., Willson, T.M., Kliewer, S.A., Lambert, M.H., Moore, J.T., 2002. Pregnane X receptor (PXR), constitutiveandrostane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Mol. Endocrinol. 16,977–986.

Moore, J.T., Moore, L.B., Maglich, J.M., Kliewer, S.A., 2003. Functional and structural comparison of PXR and CAR. Biochim. Biophys. Acta 1619, 235–238.

Moreau, A., Vilarem, M.J., Maurel, P., Pascussi, J.M., 2008. Xenoreceptors CAR and PXR activation and consequences on lipid metabolism, glucosehomeostasis, and inflammatory response. Mol. Pharm. 5, 35–41.

Morere, P., Nouvet, G., Stain, J.P., Paillot, B., Metayer, J., Hemet, J., 1975. Information obtained by liver biopsy in 100 tuberculous patients. Sem. Hop. 51,2095–2102.

Muangmoonchai, R., Smirlis, D., Wong, S.C., Edwards, M., Phillips, I.R., Shephard, E.A., 2001. Xenobiotic induction of cytochrome P450 2B1 (CYP2B1) ismediated by the orphan nuclear receptor constitutive androstane receptor (CAR) and requires steroid co-activator 1 (SRC-1) and the transcription factorSp1. Biochem. J. 355, 71–78.

Mueller, G.C., Miller, J.A., 1949. The reductive cleavage of 4-dimethylamino-azo-benzene by rat liver: the intracellular distribution of the enzyme systemand its requirement for triphosphopyridine nucleotide. J. Biol. Chem. 180, 1125.

Mu, Y., Stephenson, C.R., Kendall, C., Saini, S.P., Toma, D., Ren, S., Cai, H., Strom, S.C., Day, B.W., Wipf, P., Xie, W., 2005. A pregnane X receptor agonist withunique species-dependent stereoselectivity and its implications in drug development. Mol. Pharmacol. 68, 403–413.

Mu, Y., Zhang, J., Zhang, S., Zhou, H.H., Toma, D., Ren, S., Huang, L., Yaramus, M., Baum, A., Venkataramanan, R., Xie, W., 2006. Traditional Chinese medicinesWu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) activate pregnane X receptor and increase warfarin clearance in rats. J.Pharmacol. Exp. Ther. 316, 1369–1377.

Murakami, Y., Tanaka, T., Murakami, H., Tsujimoto, M., Ohtani, H., Sawada, Y., 2006. Pharmacokinetic modelling of the interaction between St. John’s wortand ciclosporin A. Br. J. Clin. Pharmacol. 61, 671–676.

Murphy, P.J., 2001. Xenobiotic metabolism: a look from the past to the future. Drug Metab. Dispos. 29, 779–780.Myllynen, P., Pasanen, M., Pelkonen, O., 2005. Human placenta: a human organ for developmental toxicology research and biomonitoring. Placenta 26, 361–

371.Nakata, K., Tanaka, Y., Nakano, T., Adachi, T., Tanaka, H., Kaminuma, T., Ishikawa, T., 2006. Nuclear receptor-mediated transcriptional regulation in Phase I, II,

and III xenobiotic metabolizing systems. Drug Metab. Pharmacokinet. 21, 437–457.Nguyen, A., Bouscarel, B., 2008. Bile acids and signal transduction: Role in glucose homeostasis. Cell Signal. 20, 2180–2197.Nguyen, L.P., Bradfield, C.A., 2008. The search for endogenous activators of the aryl hydrocarbon receptor. Chem. Res. Toxicol. 21, 102–116.Niemi, M., Backman, J.T., Fromm, M.F., Neuvonen, P.J., Kivistö, K.T., 2003. Pharmacokinetic interactions with rifampicin: clinical relevance. Clin.

Pharmacokinet. 42, 819–850.Niwa, T., Yabusaki, Y., Honma, K., Matsuo, N., Tatsuta, K., Ishibashi, F., Katagiri, M., 1998. Contribution of human hepatic cytochrome P450 isoforms to

regioselective hydroxylation of steroid hormones. Xenobiotica 28, 539–547.Nolte, R.T., Wisely, G.B., Westin, S., Cobb, J.E., Lambert, M.H., Kurokawa, R., Rosenfeld, M.G., Willson, T.M., Glass, C.K., Milburn, M.V., 1998. Ligand binding and

co-activator assembly of the peroxisome proliferator-activated receptor-c. Nature 395, 137–143.Oberfield, J.L., Collins, J.L., Holmes, C.P., Goreham, D.M., Cooper, J.P., Cobb, J.E., Lenhard, J.M., Hull-Ryde, E.A., Mohr, C.P., Blanchard, S.G., Parks, D.J., Moore,

L.B., Lehmann, J.M., Plunket, K., Miller, A.B., Milburn, M.V., Kliewer, S.A., Willson, T.M., 1999. A peroxisome proliferator-activated receptor gamma ligandinhibits adipocyte differentiation. Proc. Natl. Acad. Sci. USA 96, 6102–6106.

Olefsky, J.M., 2001. Nuclear receptor minireview series. J. Biol. Chem. 276, 36863–36864.Omura, T., Sato, R., 1962. A new cytochrome in liver microsomes. J. Biol. Chem. 237, 1375–1376.Orans, J., Teotico, D.G., Redinbo, M.R., 2005. The nuclear xenobiotic receptor pregnane X receptor: recent insights and new challenges. Mol. Endocrinol. 19,

2891–2900.Owen, G.I., Zelent, A., 2000. Origins and evolutionary diversification of the nuclear receptor superfamily. Cell. Mol. Life Sci. 57, 809–827.Pascussi, J.M., Drocourt, L., Fabre, J.M., Maurel, P., Vilarem, M.J., 2000. Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression

in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators. Mol. Pharmacol. 58, 361–372.Pascussi, J.M., Drocourt, L., Gerbal-Chaloin, S., Fabre, J.M., Maurel, P., Vilarem, M.J., 2001. Dual effect of dexamethasone on CYP3A4 gene expression in human

hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor. Eur. J. Biochem. 268, 6346–6358.Pascussi, J.M., Gerbal-Chaloin, S., Drocourt, L., Maurel, P., Vilarem, M.J., 2003. The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of

nuclear and steroid receptors. Biochim. Biophys. Acta 1619, 243–253.



Pascussi, J.M., Robert, A., Nguyen, M., Walrant-Debray, O., Garabedian, M., Martin, P., Pineau, T., Saric, J., Navarro, F., Maurel, P., Vilarem, M.J., 2005. Possibleinvolvement of pregnane X receptor-enhanced CYP24 expression in drug-induced osteomalacia. J. Clin. Invest. 115, 177–186.

Pascussi, J.M., Gerbal-Chaloin, S., Duret, C., Daujat-Chavanieu, M., Vilarem, M.J., Maurel, P., 2008. The tangle of nuclear receptors that controls xenobioticmetabolism and transport: crosstalk and consequences. Annu. Rev. Pharmacol. Toxicol. 48, 1–32.

Patel, R.D., Hollingshead, B.D., Omiecinski, C.J., Perdew, G.H., 2007. Aryl hydrocarbon receptor activation regulates constitutive androstane levels in murineand human liver. Hepatology 46, 209–218.

Pavek, P., Dvorak, Z., 2008. Xenobiotic-induced transcriptional regulation of xenobiotic metabolizing enzymes of the cytochrome P450 superfamily inhuman extrahepatic tissues. Curr. Drug Metab. 9, 129–143.

Persson, K.P., Ekehed, S., Otter, C., Lutz, E.S., McPheat, J., Masimirembwa, C.M., Andersson, T.B., 2006. Evaluation of human liver slices and reporter geneassays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans. Pharm. Res. 23, 56–69.

Petrey, D., Xiang, Z., Tang, C.L., Xie, L., Gimpelev, M., Mitros, T., Soto, C.S., Goldsmith-Fischman, S., Kernytsky, A., Schlessinger, A., Koh, I.Y., Alexov, E., Honig,B., 2003. Using multiple structure alignments, fast model building, and energetic analysis in fold recognition and homology modeling. Proteins 53, 430–435.

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., 2004. UCSF Chimera–a visualization system for exploratoryresearch and analysis. Comput. Chem. 25, 1605–1612.

Piriou, A., Maissiat, R., Jacqueson, A., Warnet, J.M., Claude, J.R., 1987. Ultrastructural changes in the parenchymal liver cells of rats treated with high doses ofrifampicin. Br. J. Exp. Pathol. 68, 201–207.

Plant, N., 2007. The human cytochrome P450 sub-family: transcriptional regulation, inter-individual variation and interaction networks. Biochim. Biophys.Acta 1770, 478–488.

Prasad, K., Karlupia, N., 2007. Prevention of bacterial meningitis: an overview of Cochrane systematic reviews. Respir. Med. 101, 2037–2043.Qatanani, M., Zhang, J., Moore, D.D., 2005. Role of the constitutive androstane receptor in xenobiotic-induced thyroid hormone metabolism. Endocrinology

146, 995–1002.Quattrochi, L.C., Guzelian, P.S., 2001. Cyp3A regulation: from pharmacology to nuclear receptors. Drug Metab. Dispos. 29, 615–622.Racz, A., Barsony, J., 1999. Hormone-dependent translocation of vitamin D receptors is linked to transactivation. J. Biol. Chem. 274, 19352–19360.Rastinejad, F., Perlmann, T., Evans, R.M., Sigler, P.B., 1995. Structural determinants of nuclear receptor assembly on dna direct repeats. Nature 375, 203–211.Raucy, J.L., 2003. Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products. Drug Metab. Dispos. 31, 533–539.Renaud, J., Bischoff, S.F., Buhl, T., Floersheim, P., Fournier, B., Geiser, M., Halleux, C., Kallen, J., Keller, H.J., Ramage, P., 2005. Selective estrogen receptor

modulators with conformationally restricted side chains. Synthesis and structure-activity relationship of ERa-selective tetrahydroisoquinoline ligands.J. Med. Chem. 48, 364–379.

Reschly, E.J., Krasowski, M.D., 2006. Evolution and function of the NR1I nuclear hormone receptor subfamily (VDR, PXR, and CAR) with respect tometabolism of xenobiotics and endogenous compounds. Curr. Drug Metab. 7, 349–365.

Ribeiro, R.C., Kushner, P.J., Baxter, J.D., 1995. The nuclear hormone receptor gene superfamily. Annu Rev. Med. 46, 443–453.Robinson-Rechavi, M., Boussau, B., Laudet, V., 2004. Phylogenetic dating and characterization of gene duplications in vertebrates: the cartilaginous fish

reference. Mol. Biol. Evol. 21, 580–586.Rochel, N., Wurtz, J.M., Mitschler, A., Klaholz, B., Moras, D., 2000. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol.

Cell. 5, 173–179.Rosenfeld, J.M., Vargas Jr., R., Xie, W., Evans, R.M., 2003. Genetic profiling defines the xenobiotic gene network controlled by the nuclear receptor pregnane X

receptor. Mol. Endocrinol. 17, 1268–1282.Ruff, M., Gangloff, M., Wurtz, J.M., Moras, D., 2000. Estrogen receptor transcription and transactivation: structure–function relationship in DNA- and ligand-

binding domains of estrogen receptors. Breast Cancer Res. 2, 353–359.Ruschitzka, F., Meier, P., Turina, M., Luscher, T., Noll, G., 2000. Acute heart transplant rejection due to Saint John’s Wort. Lancet 355, 548–549.Rushmore, T.H., Kong, A.N., 2002. Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Curr. Drug Metab. 3,

481–490.Sakai, H., Iwata, H., Kim, E.Y., Tanabe, S., Baba, N., 2004. Identification of constitutive androstane receptor cDNA in northern fur seal (Callorhinus ursinus).

Mar. Environ. Res. 58, 107–111.Savkur, R.S., Wu, Y., Bramlett, K.S., Wang, M., Yao, S., Perkins, D., Totten, M., Searfoss 3rd, G., Ryan, T.P., Su, E.W., Burris, T.P., 2003. Alternative splicing within

the ligand binding domain of the human constitutive androstane receptor. Mol. Genet. Metab. 80, 216–226.Schilter, B., Turesky, R.J., Juillerat, M., Honegger, P., Guigoz, Y., 1993. Phase I and phase II xenobiotic reactions and metabolism of the food-borne carcinogen

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in aggregating liver cell cultures. Biochem. Pharmacol. 45, 1087–1096.Schrader, E., 2000. Equivalence of St. John’s wort extract (Ze 117) and fluoxetine: a randomized, controlled study in mild-moderate depression. Int. Clin.

Psychopharmacol. 15, 61–68.Schuetz, E.G., Brimer, C., Schuetz, J.D., 1998. Environmental xenobiotics and the antihormones cyproterone acetate and spironolactone use the nuclear

hormone pregnenolone X receptor to activate the CYP3A23 hormone response element. Mol. Pharmacol. 54, 1113–1117.Schuetz, E.G., Strom, S., Yasuda, K., Lecureur, V., Assem, M., Brimer, C., Lamba, J., Kim, R.B., Ramachandran, V., Komoroski, B.J., Venkataramanan, R., Cai, H.,

Sinal, C.J., Gonzalez, F.J., Schuetz, J.D., 2001. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors,transporters, and cytochrome P450. J. Biol. Chem. 276, 39411–39418.

Schultzen, O., Naunyn, B., 1867. The behavior of benzene-derived hydrocarbons in the animal organism duBois-Reymond’s. Arch. Anat. Physiol., 349.Shaffer, P.L., Gewirth, D.T., 2002. Structural basis of VDR–DNA interactions on direct repeat response elements. EMBO J. 21, 2242–2252.Shafran, I., Johnson, L.K., 2005. An open-label evaluation of rifaximin in the treatment of active Crohn’s disease. Curr. Med. Res. Opin. 21, 1165–

1169.Shah, Y.M., Ma, X., Morimura, K., Kim, I., Gonzalez, F.J., 2007. Pregnane X receptor activation ameliorates DSS-induced inflammatory bowel disease via

inhibition of NF-kappaB target gene expression. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1114–1122.Shan, L., Vincent, J., Brunzelle, J.S., Dussault, I., Lin, M., Ianculescu, I., Sherman, M.A., Forman, B.M., Fernandez, E.J., 2004. Structure of the murine constitutive

androstane receptor complexed to androstenol: a molecular basis for inverse agonism. Mol. Cell. 16, 907–917.Shiraki, T., Sakai, N., Kanaya, E., Jingami, H., 2003. Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by

peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state. J. Biol. Chem.278, 11344–11350.

Sidhu, J.S., Omiecinski, C.J., 1997. An okadaic acid-sensitive pathway involved in the phenobarbital-mediated induction of CYP2B gene expression in primaryrat hepatocyte cultures. J. Pharmacol. Exp. Ther. 282, 1122–1129.

Smith, C.L., O’Malley, B.W., 2004. Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocrinol. Rev. 25, 45–71.

Song, R.X.D., Zhang, Z., Santen, R.J., 2005. Estrogen rapid action via protein complex formation involving ERa and Src. Trends Endocrinol. Metab. 16, 347–353.

Sonoda, J., Chong, L.W., Downes, M., Barish, G.D., Coulter, S., Liddle, C., Lee, C.H., Evans, R.M., 2005. Pregnane X receptor prevents hepatorenal toxicity fromcholesterol metabolites. Proc. Natl. Acad. Sci. USA 102, 2198–2203.

Squires, E.J., Sueyoshi, T., Negishi, M., 2004. Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver. J.Biol. Chem. 279, 49307–49314.

Stanley, L.A., Horsburgh, B.C., Ross, J., Scheer, N., Wolf, C.R., 2006. PXR and CAR: nuclear receptors which play a pivotal role in drug disposition and chemicaltoxicity. Drug Metab. Rev. 38, 515–597.



Staudinger, J.L., Goodwin, B., Jones, S.A., Hawkins-Brown, D., MacKenzie, K.I., LaTour, A., Liu, Y., Klaassen, C.D., Brown, K.K., Reinhard, J., Willson, T.M., Koller,B.H., Kliewer, S.A., 2001. The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proc. Natl. Acad. Sci. USA 98, 3369–3374.

Staudinger, J.L., Madan, A., Carol, K.M., Parkinson, A., 2003. Regulation of drug transporter gene expression by nuclear receptors. Drug Metab. Dispos. 31,523–527.

Sueyoshi, T., Negishi, M., 2001. Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu. Rev. Pharmacol. Toxicol. 41, 123–143.

Sueyoshi, T., Kawamoto, T., Zelko, I., Honkakoski, P., Negishi, M., 1999. The repressed nuclear receptor CAR responds to phenobarbital in activating thehuman CYP2B6 gene. J. Biol. Chem. 274, 6043–6046.

Sugatani, J., Kojima, H., Ueda, A., Kakizaki, S., Yoshinari, K., Gong, Q.H., Owens, I.S., Negishi, M., Sueyoshi, T., 2001. The phenobarbital response enhancermodule in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology 33, 1232–1238.

Suino, K., Peng, L., Reynolds, R., Li, Y., Cha, J.Y., Repa, J.J., Kliewer, S.A., Xu, H.E., 2004. The nuclear xenobiotic receptor CAR: structural determinants ofconstitutive activation and heterodimerization. Mol. Cell. 16, 893–905.

Svensson, S., Ostberg, T., Jacobsson, M., Norström, C., Stefansson, K., Hallén, D., Johansson, I.C., Zachrisson, K., Ogg, D., Jendeberg, L., 2003. Crystal structure ofthe heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation. EMBO J. 22, 4625–4633.

Swales, K., Negishi, M., 2004. CAR, driving into the future. Mol. Endocrinol. 18, 1589–1598.Syme, M.R., Paxton, J.W., Keelan, J.A., 2004. Drug transfer and metabolism by the human placenta. Clin. Pharmacokinet. 43, 487–514.Synold, T.W., Dussault, I., Forman, B.M., 2001. The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat. Med. 7, 584–590.Szegedi, A., Kohnen, R., Dienel, A., Kieser, M., 2005. Acute treatment of moderate to severe depression with hypericum extracts WS 5570 (St. John’s wort):

randomised controlled double blind non-inferiority trial versus paroxetine. BMJ 330, 503.Szymanska, J.A., Swietlicka, E.A., Piotrowski, J.K., Skrzypinska-Gawrysiak, M., Sporny, S., 1992. Effects of 3-methylcholanthrene or diethyl maleate on the

hepatotoxicity of acetaminophen. J. Appl. Toxicol. 12, 415–419.Tabb, M.M., Sun, A., Zhou, C., Grun, F., Errandi, J., Romero, K., Pham, H., Inoue, S., Mallick, S., Lin, M., Forman, B.M., Blumberg, B., 2003. Vitamin K2 regulation

of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR. J. Biol. Chem. 278, 43919–43927.Takeshita, A., Koibuchi, N., Oka, J., Taguchi, M., Shishiba, Y., Ozawa, Y., 2001. Bisphenol-A, an environmental estrogen, activates the human orphan nuclear

receptor, steroid and xenobiotic receptor-mediated transcription. Eur. J. Endocrinol. 145, 513–517.Teng, S., Piquette-Miller, M., 2008. Regulation of transporters by nuclear hormone receptors: implications during inflammation. Mol. Pharm. 5, 67–76.Teotico, D.G., Bischof, J.J., Peng, L., Kliewer, S.A., Redinbo, M.R., 2008. Structural basis of human PXR activation by the hops constituent colupulone. Mol.

Pharmacol. 74, 1512–1520.Thasler, W.E., Dayoub, R., Muhlbauer, M., Hellerbrand, C., Singer, T., Grabe, A., Jauch, K.W., Schlitt, H.J., Weiss, T.S., 2006. Repression of cytochrome P450

activity in human hepatocytes in vitro by a novel hepatotrophic factor, augmenter of liver regeneration. J. Pharmacol. Exp. Ther. 316, 822–829.Thigpen, K.G., 2004. International sequencing consortium. Environ. Health Perspect. 112, 406.Thomas, P.E., Lu, A.Y., Ryan, D., West, S.B., Kawalek, J., Levin, W., 1976. Multiple forms of rat liver cytochrome P-450. J. Biol. Chem. 251, 1385–1391.Thompson, E.E., Kuttab-Boulos, H., Krasowski, M.D., Di Rienzo, A., 2005. Functional constraints on the constitutive androstane receptor inferred from human

sequence variation and cross-species comparisons. Hum. Genom. 2, 168–178.Tien, E.S., Negishi, M., 2006. Nuclear receptors CAR and PXR in the regulation of hepatic metabolism. Xenobiotica 36, 1152–1163.Timsit, Y.E., Negishi, M., 2007. CAR and PXR: the xenobiotic-sensing receptors. Steroids 72, 231–246.Tippin, T.K., Hamilton, G., Moore, L., Beaudet, E.J., Jolley, S., Brodie, T.A., Andrews, R.C., Becherer, J.D., McDougald, D.L., Gaul, M.D., Hoivik, D.J., Mellon-

Kusibab, K., Lehmann, J., Kliewer, S., Novick, S., Laethem, R., Zhao, Z., LeCluyse, E.L., 2003. CYP3A induction by N-hydroxyformamide tumor necrosisfactor-alpha converting enzyme/matrix metalloproteinase inhibitors use of a pregname X receptor activation assay and primary hepatocyte culture forassessing induction potential in humans. Drug Metab. Dispos. 31, 870–877.

Tompkins, L.M., Wallace, A.D., 2007. Mechanisms of cytochrome P450 induction. J. Biochem. Mol. Toxicol. 21, 176–181.Truhaut, R., Claude, J.R., Warnet, J.M., Jacqueson, A., Piriou, A., 1978. Liver steatogenic power of high doses of rifampicin in rats. C.R. Acad. Sci. Hebd. Seances

Acad. Sci. D. 286, 493–497.Turtoon-weeks, S., Barone, G., Gurley, B., Ketel, B., Lightfoot, M., Abul-Ezz, S., 2001. St. John’s wort: a hidden risk for transplant patients. Prog. Transplant. 11,

116–120.Tzameli, I., Moore, D.D., 2001. Role reversal: new insights from new ligands for the xenobiotic receptor CAR. Trends Endocrinol. Metab. 12, 7–10.Tzameli, I., Pissios, P., Schuetz, E.G., Moore, D.D., 2000. The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the

nuclear receptor CAR. Mol. Cell. Biol. 20, 2951–2958.Ueda, A., Hamadeh, H.K., Webb, H.K., Yamamoto, Y., Sueyoshi, T., Afshari, C.A., Lehmann, J.M., Negishi, M., 2002. Diverse roles of the nuclear orphan receptor

CAR in regulating hepatic genes in response to phenobarbital. Mol. Pharmacol. 61, 1–6.Ueda, R., Iketaki, H., Nagata, K., Kimura, S., Gonzalez, F.J., Kusano, K., Yoshimura, T., Yamazoe, Y., 2006. A common regulatory region functions bidirectionally

in transcriptional activation of the human CYP1A1 and CYP1A2 genes. Mol. Pharmacol. 69, 1924–1930.Ure, A., 1841. On gouty concretions; with a new method of treatment. Pharm. J. Transact. 1, 24.Venter, J.C., 2001. The sequence of the human genome. Science 292, 1838 (p. 1304).Vesely, J.J., Pien, F.D., Pien, B.C., 1998. Rifampin, a useful drug for nonmycobacterial infections. Pharmacotherapy 18, 345–357.Wagner, M., Halilbasic, E., Marschall, H.U., Zollner, G., Fickert, P., Langner, C., Zatloukal, K., Denk, H., Trauner, M., 2005. CAR and PXR agonists stimulate

hepatic bile acid and bilirubin detoxification and elimination pathways in mice. Hepatology 42, 420–430.Wahli, W., 2008. A gut feeling of the PXR, PPAR and NF-kappaB connection. J. Intern. Med. 263, 613–619.Wang, H., Chen, J., Hollister, K., Sowers, L.C., Forman, B.M., 1999. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol. Cell. 3, 543–553.Wang, W., Prosise, W.W., Chen, J., Taremi, S.S., Le, H.V., Madison, V., Cui, X., Thomas, A., Cheng, K.C., Lesburg, C.A., 2008. Construction and characterization of

a fully active PXR/SRC-1 tethered protein with increased stability. Protein Eng. Des. Sel. 21, 425–433.Watkins, R.E., Wisely, G.B., Moore, L.B., Collins, J.L., Lambert, M.H., Williams, S.P., Willson, T.M., Kliewer, S.A., Redinbo, M.R., 2001. The human nuclear

xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 292, 2329–2333.Watkins, R.E., Noble, S.M., Redinbo, M.R., 2002. Structural insights into the promiscuity and function of the human pregnane X receptor. Curr. Opin. Drug

Discov. Devel. 5, 150–158.Watkins, R.E., Davis-Searles, P.R., Lambert, M.H., Redinbo, M.R., 2003a. Coactivator binding promotes the specific interaction between ligand and the

pregnane X receptor. J. Mol. Biol. 331, 815–828.Watkins, R.E., Maglich, J.M., Moore, L.B., Wisely, G.B., Noble, S.M., Davis-Searles, P.R., Lambert, M.H., Kliewer, S.A., Redinbo, M.R., 2003b. 2.1 A crystal

structure of human PXR in complex with the St. John’s wort compound hyperforin. Biochemistry 42, 1430–1438.Weier, N., He, S.M., Li, X.T., Wang, L.L., Zhou, S.F., 2008. Placental drug disposition and its clinical implications. Curr. Drug Metab. 9, 106–121.Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., Moore, D.D., 2000. The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature

407, 920–923.Weigel, N.L., Zhang, Y., 1998. Ligand-independent activation of steroid hormone receptors. J. Mol. Med. 76, 469–479.Wheatley, D., 1997. LI 160, an extract of St. John’s wort, versus amitriptyline in mildly to moderately depressed outpatients-a controlled 6-week clinical

trial. Pharmacopsychiatry 30, 77–80.Whöler, F., Frerichs, F.T., 1848. Concerning the modifications which particular organic materials undergo in their transition to the urine. Annu. Chem. Pharm.

63, 335.Williams, R.T., 1947. Detoxication Mechanisms. John Wiley and Sons, Inc, New York.



Williams, S.P., Sigler, P.B., 1998. Atomic structure of progesterone complexed with its receptor. Nature 393, 392–396.Willson, T.M., Kliewer, S.A., 2002. PXR, CAR and drug metabolism. Nat. Rev. Drug Discov. 1, 259–266.Woelk, H., 2000. Comparison of St. John’s wort and imipramine for treating depression: randomised controlled trial. BMJ 321, 536–539.Wolfrum, C., Asilmaz, E., Luca, E., Friedman, J.M., Stoffel, M., 2003. FoxA2 regulates lipid metabolism and ketogenesis in the liver during fasting and in

diabetes. Nature 432, 1027–1032.Xia, J., Kemper, B., 2005. Structural determinants of constitutive androstane receptor required for its glucocorticoid receptor interacting protein-1-mediated

nuclear accumulation. J. Biol. Chem. 280, 7285–7293.Xie, W., Tian, Y., 2006. Xenobiotic receptor meets NF-kB, a collision in the small bowel. Cell. Metab. 4, 177–178.Xie, W., Barwick, J.L., Downes, M., Blumberg, B., Simon, C.M., Nelson, M.C., Neuschwander-Tetri, B.A., Brunt, E.M., Guzelian, P.S., Evans, R.M., 2000a.

Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406, 435–439.Xie, W., Barwick, J.L., Simon, C.M., Pierce, A.M., Safe, S., Blumberg, B., Guzelian, P.S., Evans, R.M., 2000b. Reciprocal activation of xenobiotic response genes by

nuclear receptors SXR/PXR and CAR. Genes. Dev. 14, 3014–3023.Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C.M., Nelson, M.C., Ong, E.S., Waxman, D.J., Evans, R.M., 2001. An essential role for nuclear receptors SXR/PXR

in detoxification of cholestatic bile acids. Proc. Natl. Acad. Sci. USA 98, 3375–3380.Xiong, H., Yoshinari, K., Brouwer, K.L., Negishi, M., 2002. Role of constitutive androstane receptor in the in vivo induction of Mrp3 and CYP2B1/2 by

phenobarbital. Drug Metab. Dispos. 30, 918–923.Xue, Y., Chao, E., Zuercher, W.J., Willson, T.M., Collins, J.L., Redinbo, M.R., 2007a. Crystal structure of the PXR-T1317 complex provides a scaffold to examine

the potential for receptor antagonism. Bioorg. Med. Chem. 15, 2156–2166.Xue, Y., Moore, L.B., Orans, J., Peng, L., Bencharit, S., Kliewer, S.A., Redinbo, M.R., 2007b. Crystal structure of the pregnane X receptor-estradiol complex

provides insights into endobiotic recognition. Mol. Endocrinol. 21, 1028–1038.Xu, L., Glass, C.K., Rosenfeld, M.G., 1999. Coactivator and corepressor complexes in nuclear receptor function. Curr. Opin. Genet. Dev. 9, 140–147.Xu, R.X., Lambert, M.H., Wisely, B.B., Warren, E.N., Weinert, E.E., Waitt, G.M., Williams, J.D., Collins, J.L., Moore, L.B., Willson, T.M., Moore, J.T., 2004. A

structural basis for constitutive activity in the human CAR/RXRalpha heterodimer. Mol. Cell. 16, 919–928.Xu, C., Li, C.Y., Kong, A.N., 2005. Induction of phase I, II and III drug metabolism/transport by xenobiotics. Arch. Pharm. Res. 28, 249–268.Xu, D.X., Chen, Y.H., Wang, J.P., Sun, M.F., Wang, H., Wei, L.Z., Wei, W., 2005. Perinatal lipopolysaccharide exposure downregulates pregnane X receptor and

Cyp3a11 expression in fetal mouse liver. Toxicol. Sci. 87, 38–45.Xu, Y., Hashizume, T., Shuhart, M.C., Davis, C.L., Nelson, W.L., Sakaki, T., Kalhorn, T.F., Watkins, P.B., Schuetz, E.G., Thummel, K.E., 2006. Intestinal and hepatic

CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): implications for drug-induced osteomalacia. Mol. Pharmacol. 69, 56–65.Yang, Z., Bielawski, J.P., 2000. Statistical methods for detecting molecular adaptation. Trends Ecol. Evol. 15, 496–503.Yogev, R., Guzman-Cottrill, J., 2005. Bacterial meningitis in children: critical review of current concepts. Drugs 65, 1097–1112.Yoshinari, K., Sueyoshi, T., Moore, R., Negishi, M., 2001. Nuclear receptor CAR as a regulatory factor for the sexually dimorphic induction of CYB2B1 gene by

phenobarbital in rat livers. Mol. Pharmacol. 59, 278–284.Yoshinari, K., Kobayashi, K., Moore, R., Kawamoto, T., Negishi, M., 2003. Identification of the nuclear receptor CAR:HSP90 complex in mouse liver and

recruitment of protein phosphatase 2A in response to phenobarbital. FEBS Lett. 548, 17–20.Yu, M.H., 2001. Metabolism of environmental chemicals. In: Lewis Publishers (Ed.), Impacts of environmental toxicants on living systems. Boca Raton.Yu, A.M., Fukamachi, K., Krausz, K.W., Cheung, C., Gonzalez, F.J., 2005. Potential role for human cytochrome P450 3A4 in estradiol homeostasis.

Endocrinology 146, 2911–2919.Zelko, I., Sueyoshi, T., Kawamoto, T., Moore, R., Negishi, M., 2001. The peptide near the C terminus regulates receptor CAR nuclear translocation induced by

xenochemicals in mouse liver. Mol. Cell. Biol. 21, 2838–2846.Zhai, Y., Pai, H.V., Zhou, J., Amico, J.A., Vollmer, R.R., Xie, W., 2007. Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid

homeostasis. Mol. Endocrinol. 21, 138–147.Zhang, J., Kuehl, P., Green, E.D., Touchman, J.W., Watkins, P.B., Daly, A., Hall, S.D., Maurel, P., Relling, M., Brimer, C., Yasuda, K., Wrighton, S.A., Hancock, M.,

Kim, R.B., Strom, S., Thummel, K., Russell, C.G., Hudson Jr., J.R., Schuetz, E.G., Boguski, M.S., 2001. The human pregnane X receptor: genomic structure andidentification and functional characterization of natural allelic variants. Pharmacogenetics 11, 555–572.

Zhang, Z., Burch, P.E., Cooney, A.J., Lanz, R.B., Pereira, F.A., Wu, J., Gibbs, R.A., Weinstock, G., Wheeler, D.A., 2004a. Genomic analysis of the nuclear receptorfamily: new insights into structure, regulation, and evolution from the rat genome. Genome Res. 14, 580–590.

Zhang, J., Huang, W., Qatanani, M., Evans, R.M., Moore, D.D., 2004b. The constitutive androstane receptor and pregnane X receptor function coordinately toprevent bile acid-induced hepatotoxicity. J. Biol. Chem. 279, 49517–49522.

Zhou, J., Zhai, Y., Mu, Y., Gong, H., Uppal, H., Toma, D., Ren, S., Evans, R.M., Xie, W., 2006a. A novel pregnane X receptor-mediated and sterol regulatoryelement-binding protein-independent lipogenic pathway. J. Biol. Chem. 281, 15013–15020.

Zhou, C., Tabb, M.M., Nelson, E.L., Grun, F., Verma, S., Sadatrafiei, A., Lin, M., Mallick, S., Forman, B.M., Thummel, K.E., Blumberg, B., 2006b. Mutual repressionbetween steroid and xenobiotic receptor and NF-kappaB signaling pathways links xenobiotic metabolism and inflammation. J. Clin. Invest. 116, 2280–2289.

Zhou, C., Verma, S., Blumberg, B., 2009. The steroid and xenobiotic receptor (SXR), beyond xenobiotic metabolism. Nucl. Recept. Signal. 7, 1–21.Zimber, A., Gespach, C., 2008. Bile acids and derivatives, their nuclear receptors FXR, PXR and ligands: role in health and disease and their therapeutic

potential. Anticancer Agents Med. Chem. 8, 540–563.


Biochemical and Biophysical Research Communications 390 (2009) 27–31


Biochemical and Biophysical Research Communications

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Peroxynitrite scavenging by ferryl sperm whale myoglobinand human hemoglobin

Paolo Ascenzi a,b,*,1, Elisabetta De Marinis a,1, Alessandra di Masi a, Chiara Ciaccio c,d, Massimo Coletta c,d

a Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italyb National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, I-00149 Roma, Italyc Department of Experimental Medicine and Biochemical Sciences, University of Roma ‘Tor Vergata’, Via Montpellier 1, I-00133 Roma, Italyd Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, I-70121 Bari, Italy


Article history:Received 8 September 2009Available online 17 September 2009

Keywords:Ferryl myoglobin reductionFerryl hemoglobin reductionFerric myoglobin formationFerric hemoglobin formationPeroxynitrite scavengingEffect of CO2

Kinetics

0006-291X/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.bbrc.2009.09.050

Abbreviations: Fe(III), ferric heme-protein; Fe(IV)@protein; Fe(II)ANO, ferrous nitrosylated heme-proteiated heme-protein; Hb, hemoglobin; Lb, leghemoglotruncated HbO

* Corresponding author. Address: Department of BiLaboratory for Electron Microscopy, University Roma446, I-00146 Roma, Italy. Fax: +39 06 5733 6321.

E-mail address: [email protected] (P. Ascenzi).1 These Authors contributed equally to this study.

a b s t r a c t

Globins protect from the oxidative and nitrosative cell damage. Here, kinetics of peroxynitrite scavengingby ferryl sperm whale myoglobin (MbAFe(IV)@O) and human hemoglobin (HbAFe(IV)@O), between pH5.8 and 8.3 at 20.0 �C, are reported. In the absence of CO2, values of the second-order rate constant forperoxynitrite scavenging by MbAFe(IV)@O and HbAFe(IV)@O (i.e., for MbAFe(III) and HbAFe(III) forma-tion; kon) are 4.6 � 104 M�1 s�1 and 3.3 � 104 M�1 s�1, respectively, at pH 7.1. Values of kon increase ondecreasing pH with pKa values of 6.9 and 6.7, this suggests that the ONOOH species reacts preferentiallywith MbAFe(IV)@O and HbAFe(IV)@O. In the presence of CO2 (=1.2 � 10�3 M), values of kon for peroxyni-trite scavenging by MbAFe(IV)@O and HbAFe(IV)@O are essentially pH-independent, the average kon

values are 7.1 � 104 M�1 s�1 and 1.2 � 105 M�1 s�1, respectively. As a whole, MbAFe(IV)@O andHbAFe(IV)@O, obtained by treatment with H2O2, undertake within the same cycle H2O2 and peroxynitritedetoxification.

� 2009 Elsevier Inc. All rights reserved.

Peroxynitrite is implicated in several physiological and patho-logical events, including cell signaling, drug metabolism, microbialpathogenesis, atherosclerosis, inflammation, and neurodegenera-tive disorders. It reacts with various bio-molecules including pro-teins, lipids, and DNA by either direct reaction with a targetmolecule or immediately after homolysis to �NO2 and hydroxylradical (�OH) or after reaction with CO2 and homolysis to CO3

��

and �NO2 [1–11].Besides their role in O2 transport and storage, globins also cat-

alyze several reactions aimed to scavenge toxic reactive nitrogenand oxygen species. These reactions play an important physiolog-ical role in the defense against nitrosative and oxidative stress[7,12–16]. Peroxynitrite scavenging has been reported to be facili-tated by the ferrous oxygenated (heme–Fe(II)–O2), ferrous nitrosy-lated (heme–Fe(II)–NO), and ferric (heme–Fe(III)) derivatives ofheme-proteins [7,15,17–29].

ll rights reserved.

O, ferryl [oxo-Fe(IV)] heme-n; Fe(II)AO2, ferrous oxygen-bin; Mb, myoglobin; trHbO,

ology and InterdepartmentalTre, Viale Guglielmo Marconi

Here, a detailed kinetic study of peroxynitrite scavenging by theferryl derivative of sperm whale Mb (MbAFe(IV)@O) and humanHb (HbAFe(IV)@O) is reported. MbAFe(IV)@O and HbAFe(IV)@O,obtained by treatment with hydrogen peroxide (H2O2), catalyzeperoxynitrite scavenging. In turn, peroxynitrite acts as an antioxi-dant of MbAFe(IV)@O and HbAFe(IV)@O and could prevent celldamage. Therefore, Mb and Hb appear to be involved in bothH2O2 and peroxynitrite scavenging.

Materials

Ferric sperm whale Mb (MbAFe(III)) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Ferrous oxygenated sperm whale Mb(MbAFe(II)AO2) was prepared by adding few grains of sodiumdithionite to the MbAFe(III) solution, then the solution wasdesalted by passing it throughout a G25 Sephadex gel filtration col-umn equilibrated in air with 1.0 � 10�1 M phosphate buffer, at pH7.2 and 20 �C [30]. Ferrous oxygenated human Hb (HbAFe(II)AO2)was prepared from blood samples according to literature [30]. Fer-ric human Hb (HbAFe(III)) was prepared by adding a few grains ofsodium ferricyanide to the HbAFe(II)AO2 solution [30]. Spermwhale MbAFe(IV)@O and human HbAFe(IV)@O were prepared byadding 7–15 equivalents of H2O2 to the MbAFe(III) and HbAFe(III)solutions (5.0 � 10�2 M phosphate buffer, pH 7.2) at 20.0 �C. After areaction time of few minutes, the MbAFe(IV)@O and HbAFe(IV)@O

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kon

heme-Fe(IV)=O + peroxynitrite heme-Fe(III)

Scheme 1.


solutions were stored on ice and used within 1 h. The heme-pro-tein concentration was determined spectrophotometrically with evalues listed in Supplementary Table 1.

The solutions of the experiments in the presence of CO2 wereprepared by adding the required amount of a 5.0 � 10�1 M NaHCO3

solution [15,19,21,22,24,25,29].H2O2 (from Fluka GmbH, Buchs, Switzerland) was diluted with

the 5.0 � 10�2 M phosphate buffer solution (pH 7.2); the H2O2 con-centration was determined spectrophotometrically at 240 nm(e240nm = 3.94 � 101 M�1 cm�1) [31].

Peroxynitrite was prepared from potassium superoxide (KO2)and �NO and from nitrous acid (HNO2) and H2O2 [32,33]. The per-oxynitrite stock solution was diluted with degassed 1.0 � 10�2 Msodium hydroxide (NaOH) to reach the desired concentration.The peroxynitrite concentration was determined spectrophoto-metrically at 302 nm (e302nm = 1.67 � 103 M�1 cm�1) [34].

All the other products (from Merck AG, Darmstadt, Germany, orSigma–Aldrich, St. Louis, MO, USA) were of analytical grade andused without purification.

Methods

Kinetics of peroxynitrite scavenging by sperm whale MbAFe-(IV)@O and human HbAFe(IV)@O were determined, in the absenceand presence of CO2, by rapid mixing the MbAFe(IV)@O andHbAFe(IV)@O solutions (final concentration, 3.2 � 10�6 and2.9 � 10�6 M, respectively) with the peroxynitrite solution (finalconcentration, 2.0 � 10�5 to 4.0 � 10�4 M), at pH values rangingbetween 5.8 and 8.3 (final concentration, 2.0 � 10�1 M phosphatebuffer) and 20.0 �C; no gaseous phase was present. Kinetics wasmonitored between 360 and 460 nm [15,19,21,23–29].

The time course of peroxynitrite scavenging by MbAFe(IV)@Oand HbAFe(IV)@O, in the absence and presence of CO2, was fittedto a single exponential process according to the minimum reactionmechanism represented by Scheme 1 [29].

Values of the pseudo-first-order rate constant k for peroxynitritescavenging by MbAFe(IV)@O and HbAFe(IV)@O, in the absence andpresence of CO2, were determined according to Eq. (1) [29]:

½FeðIVÞ@O�t ¼ ½FeðIVÞ@O�i � e�k�t ð1Þ

Values of kon, in the absence and presence of CO2, were deter-mined according to Eq. (2) [29]:

k ¼ kon � ½peroxynitrite� þ a ð2Þ

where a is the value of k in the absence of peroxynitrite.The pKa values describing the pH-dependence of kon for perox-

ynitrite scavenging by MbAFe(IV)@O and HbAFe(IV)@O, in the ab-sence of CO2, were obtained, at 20.0 �C, according to Eq. (3) [29]:

kon ¼ ððklimðtopÞ � klimðbottomÞÞ � 10�pHÞ=ð10�pH þ 10�pKaÞþ klimðbottomÞ ð3Þ

where klim(top) and klim(bottom) represent the asymptotic values of kon

under conditions where pH �pKa and pH �pKa, respectively.

hon

heme-Fe(II)-O2 + peroxynitrite heme-Fe(

Scheme

In some cases, catalase was added to the MbAFe(IV)@O andHbAFe(IV)@O solutions prior to reaction with peroxynitrite to de-stroy excess H2O2. According to literature [29,35,36], catalase doesnot affect peroxynitrite scavenging by MbAFe(IV)@O andHbAFe(IV)@O, in the absence and presence of CO2.

Kinetics of peroxynitrite scavenging by sperm whale MbAFe(II)AO2 and human HbAFe(II)AO2 were determined, in the absence andpresence of CO2, by rapid mixing the MbAFe(II)AO2 and HbAFe-(II)AO2 solutions (final concentration, 3.4 � 10�6 and 3.3 �10�6 M, respectively) with the peroxynitrite solution (final concen-tration, 2.0 � 10�5 to 4.0 � 10�4 M), at pH 7.1 (final concentration,2.0 � 10�1 M phosphate buffer) and 20.0 �C; no gaseous phase waspresent. Kinetics was monitored between 360 and 460 nm [19,22,24,25].

The time course of peroxynitrite scavenging by sperm whaleMbAFe(II)AO2 and human HbAFe(II)AO2, in the absence and pres-ence of CO2, was fitted to two consecutive mono-exponential pro-cesses according to the minimum reaction mechanism representedby Scheme 2 [19,22,24,25].

Values of the pseudo-first-order rate constants h and k for per-oxynitrite scavenging by MbAFe(II)AO2 and HbAFe(II)AO2, in theabsence and presence of CO2, were determined according to Eqs.(4a–c) [37]:

½FeðIIÞAO2�t ¼ ½FeðIIÞAO2�i � e�h�t ð4aÞ

½FeðIVÞ@O�t ¼ ½FeðIIÞAO2�i � ðh� ððe�h�t=ðk� hÞÞþ ðe�k�t=ðh� kÞÞÞÞ ð4bÞ

½FeðIIIÞ�t ¼ ½FeðIIÞAO2�i � ½FeðIIÞAO2�t þ ½FeðIVÞ@O�t ð4cÞ

Values of hon and kon, in the absence and presence of CO2, weredetermined according to Eqs. (5a) and (5b) [29]:

h ¼ hon � ½peroxynitrite� þ a ð5aÞk ¼ kon � ½peroxynitrite� þ a ð5bÞ

where a is the value of h or k in the absence of peroxynitrite.The results are given as mean values of at least four experi-

ments plus or minus the corresponding standard deviation. Alldata were analyzed using the MatLab program (The Math WorksInc., Natick, MA, USA).

Results and discussion

Mixing of sperm whale MbAFe(IV)@O or human HbAFe(IV)@Owith peroxynitrite solutions, in the absence and presence of CO2,leads to the formation of MbAFe(III) and HbAFe(III), respectively.Under all the experimental conditions, the time course of perox-ynitrite scavenging by MbAFe(IV)@O and HbAFe(IV)@O corre-sponds to a monophasic process (Scheme 1). Moreover, values ofk for peroxynitrite scavenging by MbAFe(IV)@O and HbAFe(IV)@Oare wavelength-independent under pseudo-first order conditionsat fixed peroxynitrite concentration and pH (data not shown).

Plots of k versus [peroxynitrite] are linear, the slope corre-sponds to kon (Figs. 1 and 2). In the absence of CO2, the y-axis inter-cept of plots of k versus [peroxynitrite] (i.e., a; see Eq. (2))corresponds to a ffi 0 s�1 (Figs. 1 and 2 and Supplementary Tables2 and 3). On the other hand, in the presence of CO2 (Figs. 1 and 2and Supplementary Tables 2 and 3), the y-axis intercept of plots

kon

IV)=O + peroxynitrite heme-Fe(III)

2.

0 100 200 300 4000

10

20

30

40A

[Peroxynitrite]×106 (M)


k (s

-1)

0 100 200 300 4000

10

20

30

40B

k (s

-1)

5.5 6.0 6.5 7.0 7.5 8.0 8.50.0

2.5

5.0

7.5

10.0C

pH

k on×

10-4

(M

-1s-1

)

Fig. 1. Kinetics of peroxynitrite scavenging by sperm whale MbAFe(IV)@O, at 20.0 �C.(A) Dependence of k on the peroxynitrite concentration, in the absence of CO2, at pH5.8, 7.1, and 8.3 (circles, squares, and triangles, respectively). The analysis of dataaccording to Eq. (2) allowed to determine kon = 8.1 � 104 M�1 s�1 (circles),4.6 � 104 M�1 s�1 (squares), and 2.0 � 104 M�1 s�1 (triangles). (B) Dependence of kon the peroxynitrite concentration, in the presence of CO2, at pH 6.1, 7.1, and 7.9(circles, squares, and triangles, respectively). The analysis of data according to Eq. (2)allowed to determine kon = 8.1 � 104 M�1 s�1 and a = 2.1 s�1 (circles), kon = 7.4 �104 M�1 s�1 and a = 2.8 s�1 (squares), and kon = 6.2 � 104 M�1 s�1 and a = 2.4 s�1

(triangles). (C) pH-dependence of kon in the absence of CO2. The analysis of dataaccording to Eq. (3) allowed to determine pKa = 6.9 ± 0.1, klim(top) = (8.4 ± 0.2) �104 M�1 s�1, and klim(bottom) = (1.8 ± 0.1) � 104 M�1 s�1. Where not shown, standarddeviation is smaller than the symbol. The MbAFe(IV)@O concentration was3.2 � 10�6 M. The CO2 concentration was 1.2 � 10�3 M. For details, see text.

0 100 200 300 4000

10

20

30

40A



k (s

-1)

0 100 200 300 4000

25

50

75B

k(s

-1)

5.5 6.0 6.5 7.0 7.5 8.0 8.50.0

2.5

5.0

7.5

10.0C

pH

k on×

10-4

(M

-1s-1

)

Fig. 2. Kinetics of peroxynitrite scavenging by human HbAFe(IV)@O, at 20.0 �C. (A)Dependence of k on the peroxynitrite concentration, in the absence of CO2, at pH 5.9,7.1, and 8.2 (circles, squares, and triangles, respectively). The analysis of dataaccording to Eq. (2) allowed to determine kon = 7.7 � 104 M�1 s�1 (circles),3.3 � 104 M�1 s�1 (squares), and 1.3 � 104 M�1 s�1 (triangles). (B) Dependence of kon the peroxynitrite concentration, in the presence of CO2, at pH 5.9, 7.1, and 8.2(circles, squares, and triangles, respectively). The analysis of data according to Eq. (2)allowed to determine kon = 1.3 � 105 M�1 s�1 and a = 4.1 s�1 (circles), kon = 1.9 �105 M�1 s�1 and a = 5.1 s�1 (squares), and kon = 9.1 � 104 M�1 s�1 and a = 4.7 s�1

(triangles). (C) pH-dependence of kon in the absence of CO2. The analysis of dataaccording to Eq. (3) allowed to determine pKa = 6.7 ± 0.2, klim(top) = (8.7 ± 0.9) �104 M�1 s�1, and klim(bottom) = (1.1 ± 0.1) � 104 M�1 s�1. The HbAFe(IV)@O concen-tration was 2.9 � 10�6 M. The Co2 concentration was 1.2 � 10�3 M. For details, seetext and Fig. 1.


of k versus [peroxynitrite] shows values of a ranging between 2.1and 6.2 s�1 at different pH values. Since peroxynitrite scavengingby MbAFe(IV)@O and HbAFe(IV)@O is not likely to be a reversibleprocess, 2.1 P a P 6.2 s�1 may be indicative of a reaction mecha-nism more complex than that reported in Scheme 1 [19,22,24,25].

As shown in Figs. 1 and 2 and Supplementary Tables 2 and 3,values of kon for peroxynitrite scavenging by MbAFe(IV)@O andHbAFe(IV)@O increase on decreasing pH from 8.3 to 5.8, in the ab-sence of CO2; the analysis of data according to Eq. (3) allowed todetermine values of pKa = 6.9 and 6.7, respectively. The pKa valuesfor peroxynitrite scavenging by MbAFe(IV)@O (=6.9) andHbAFe(IV)@O (=6.7), in the absence of CO2, are similar to those re-ported for: (i) peroxynitrite detoxification by ferryl Mycobacteriumleprae truncated HbO (=6.7; trHbOAFe(IV)@O) [29], and (ii) the

peroxynitrous acid/peroxynitrite (i.e., ONOOH/ONOO) equilibrium(=6.5–6.8) [10,34]. This suggests that peroxynitrous acid is the spe-cies that reacts preferentially with heme–Fe(IV)@O. According toEq. (3), klim(top) and klim(bottom) could represent the second-orderrate constants for MbAFe(IV)@O- and HbAFe(IV)@O-mediatedscavenging of peroxynitrous acid at pH� pKa and of peroxynitriteat pH� pKa, respectively. In agreement with: (i) kinetics of perox-ynitrite scavenging by M. leprae trHbOAFe(IV)@O [29], and (ii) ki-netic simulations concerning peroxynitrite scavenging by horseheart MbAFe(IV)@O [19], klim(top) values for peroxynitrite scaveng-ing by MbAFe(IV)@O and HbAFe(IV)@O exceed those of klim(bottom)

(i.e., klim(top)/klim(bottom) = 4.7 and 7.9, respectively). Accordingly,the reaction of HbAFe(IV)@O with ONOOH shows a lower activa-

Table 1Values of kinetic parameters for peroxynitrite scavenging by ferryl and ferrousoxygenated heme-proteins (in italics and bold, respectively; see Schemes 1 and 2,respectively).

Heme-protein [CO2] (M) hon (M�1 s�1) kon (M�1 s�1)

Mycobacterium leprae trHbO 0a — 1.5 � 104a

1.2 � 10�3a — 2.2 � 104a

0 4.8 � 104b 1.3 � 104b

1.2 � 10�3b 6.3 � 105b 1.7 � 104b

Glycine max Lbc 0 — 3.4 � 104

1.2 � 10�3 — 2.3 � 105

0 5.5 � 104 2.1 � 104

1.2 � 10�3 8.8 � 105 3.6 � 105

Sperm whale Mbd 0 — 4.6 � 104

1.2 � 10�3 — 7.4 � 104

0 7.3 � 104 3.8 � 104

1.2 � 10�3 6.8 � 104 4.6 � 104

Horse heart Mb 0e — 1.9 � 104e

1.2 � 10�3e — 2.6 � 104e

0f 5.4 � 104f 2.2 � 104f

1.2 � 10�3f 4.1 � 105f 3.2 � 104f

Human Hbd 0 — 3.3 � 104

1.2 � 10�3 — 1.9 � 105

0 2.9 � 104 1.7 � 1041.2 � 10�3 2.1 � 105 1.6 � 105

a pH 7.2 and 20.0 �C. From [29].b pH 7.3 and 20.0 �C. From [25].c pH 7.3 and 20.0 �C. From [24].d pH 7.1 and 20.0 �C. Present study.e pH 7.5 and 20.0 �C. From [19].f pH 7.3 and 20.0 �C. From [19].


tion barrier (by about 5.1 kJ mol�1) with respect to that withONOO�. In the case of MbAFe(IV)@O, the reactivity difference be-tween ONOOH and ONOO� is much lower (amounting to about3.7 kJ mol�1).

In the presence of CO2, values of kon for peroxynitrite scaveng-ing by MbAFe(IV)@O and HbAFe(IV)@O are pH-independent (theaverage kon values are 7.1 � 104 M�1 s�1 and 1.2 � 105 M�1 s�1,respectively; Figs. 1 and 2, and Supplementary Tables 2 and 3),as reported for M. leprae trHbO–Fe(IV)@O, Glycine max leghemo-globin–Fe(IV)@O (Lb–Fe(IV)@O), horse heart MbAFe(IV)@O, andhuman HbAFe(IV)@O [19,22,24,29]. This agrees with the reactionmechanism proposed for peroxynitrite scavenging by heme–Fe(IV)@O in the presence of CO2 involving the transient highlyreactive species �NO2. The formation of �NO2, possibly representingthe rate-limiting step of the whole process, does not depend onthe ONOOH M ONOO� + H+ equilibrium (and thus on pH), but in-stead on the CO2 concentration [19,22,24,29]. Also values of a forperoxynitrite scavenging by MbAFe(IV)@O and HbAFe(IV)@O inthe presence of CO2 are pH-independent (the average a valuesare 2.9 and 4.9 s�1, respectively) (see Supplementary Tables 2and 3).

To support the kinetic mechanism of peroxynitrite scavengingby MbAFe(IV)@O and HbAFe(IV)@O (Scheme 1), kinetics of perox-ynitrite detoxification by sperm whale MbAFe(II)AO2 and humanHbAFe(II)AO2 were investigated. Mixing of MbAFe(II)AO2 orHbAFe(II)AO2 with peroxynitrite solutions, in the absence andpresence of CO2, leads to the formation of MbAFe(III) and HbAFe(III), respectively, via the transient formation of MbAFe(IV)@Oand HbAFe(IV)@O, respectively. Under all the experimental condi-tions, the time course for peroxynitrite scavenging by MbAFe(II)AO2 and HbAFe(II)AO2 corresponds to a biphasic process (Scheme2). Moreover, values of h and k for peroxynitrite scavenging byMbAFe(III) and HbAFe(III) are wavelength-independent underpseudo-first order conditions at fixed peroxynitrite concentration(data not shown).

Plots of h and k versus [peroxynitrite] are linear, the slope cor-responds to hon and kon (see Eqs. (5a) and (5b)) (SupplementaryFigs. 1 and 2). In the absence of CO2, the y-axis intercept of plotsof h and k versus [peroxynitrite] corresponds to a ffi 0 s�1 (Supple-mentary Figs. 1 and 2). On the other hand, in the presence of CO2

(Supplementary Figs. 1 and 2), the y-axis intercept of plots of hand k versus [peroxynitrite] display values of a ranging between4.7 s�1 and 1.2 � 101 s�1. Since peroxynitrite scavenging byMbAFe(II)AO2 and HbAFe(II)AO2 is not likely to be a reversibleprocess, 4.7 s�1 P a P 1.2 � 101 s�1 may be indicative of a reactionmechanism more complex than that reported in Scheme 2[19,22,24,25].

Values of kon for peroxynitrite scavenging by MbAFe(IV)@O andMbAFe(II)AO2, and by HbAFe(IV)@O and HbAFe(II)AO2 match eachother (Table 1), according to Schemes 1 and 2. Moreover, values ofhon and kon for the peroxynitrite scavenging by HbAFe(IV)@O andHbAFe(II)AO2 are in agreement with those reported previously, inthe absence and presence of CO2 [22] (see Table 1).

Values of kon for the peroxynitrite scavenging by sperm whaleMb and human Hb derivatives are grossly similar to those reportedfor M. leprae trHbO, Glycine max Lb, and horse heart Mb action (Ta-ble 1) [19,22,24,29], indicating that the reactions depicted inSchemes 1 and 2 do not appear to reflect the different geometryof the heme-distal pocket. In fact, sperm whale Mb, horse heartMb, and human Hb display the classical histidyl-based heme-distalpocket; the ligand entry to and exit from the heme-distal site oc-curs via the so-called ‘E7-gate’ [38–40]. On the other hand, theheme-distal region of M. leprae trHbO is completely different, in-deed the HisE7 residue present in sperm whale Mb and humanHb chains is replaced by Ala [15,41]. Moreover, cavity systemspresent in the protein matrix appear to facilitate ligand entry to

and exit from the M. leprae trHbO heme-distal pocket, the so-called‘E7-gate’ being inoperative [15,41].

Conclusions

The catalytic parameters for peroxynitrite-mediated reductionof heme–Fe(IV)@O (present study) and heme–Fe(II)AO2 are similar(Table 1) and high enough to indicate that both reactions couldoccur in vivo [7,15]. Peroxynitrite scavenging by heme–Fe(IV)@O,obtained by treatment with H2O2, could be relevant under anaero-bic and oxidative conditions, as occurs in ischemia-reperfusioninjury and other cardiovascular pathological situations [3,7,10].In turn, peroxynitrite can act as a scavenger of the highly oxidizingheme-Fe(IV)@O species, which could be responsible for the oxida-tive cell damage [42]. Therefore, heme-globins can undertakewithin the same cycle H2O2 and peroxynitrite detoxification.

Acknowledgments

This work was partially supported by grants from the Ministryfor Education, University, and Research of Italy (Department ofBiology, University Roma Tre, Roma, Italy, ‘CLAR 2009’ to P.A.)and from the Ministry of Welfare of Italy (National Institute forInfectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, Italy,‘Ricerca corrente 2009’ to P.A.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2009.09.050.

References

[1] J.S. Beckman, W.H. Koppenol, Nitric oxide, superoxide, and peroxynitrite: thegood, the bad, and the ugly, Am. J. Physiol. 271 (1996) C1424–C1437.

[2] C. Ducrocq, B. Blanchard, B. Pignatelli, H. Oshima, Peroxinitrite: an endogenousoxidizing and nitrating agent, Cell. Mol. Life Sci. 55 (1999) 1068–1077.

[3] R.S. Ronson, M. Nakamura, J. Vinten-Johansen, The cardiovascular effects andimplications of peroxynitrite, Cardiov. Res. 44 (1999) 47–59.

http://dx.doi.org/10.1016/j.bbrc.2009.09.050


[4] L.J. Ignarro, Nitric oxide as a unique signaling molecule in the vascular system:a historical overview, J. Physiol. Pharmacol. 53 (2002) 503–514.

[5] E. Clementi, E. Nisoli, Nitric oxide and mitochondrial biogenesis: a key to long-term regulation of cellular metabolism, Comp. Biochem. Physiol. 142 (2005)102–110.

[6] A. Denicola, R. Radi, Peroxynitrite and drug-dependent toxicity, Toxicology 208(2005) 273–288.

[7] S. Herold, A. Fago, Reactions of peroxynitrite with globin proteins and theirpossible physiological role, Comp. Biochem. Physiol. A Mol. Integr. Physiol. 142(2005) 124–129.

[8] M.H. Zaki, T. Akuta, T. Akaike, Nitric oxide-induced nitrative stress involved inmicrobial pathogenesis, J. Pharmacol. Sci. 98 (2005) 117–129.

[9] A. Olmos, R.M. Giner, S. Máñez, Drugs modulating the biological effects ofperoxynitrite and related nitrogen species, Med. Res. Rev. 27 (2007) 1–64.

[10] S. Goldstein, G. Merényi, The chemistry of peroxynitrite: implications forbiological activity, Methods Enzymol. 436 (2008) 49–61.

[11] L. Liaudet, G. Vassalli, P. Pacher, Role of peroxynitrite in the redox regulation ofcell signal transduction pathways, Front. Biosci. 14 (2009) 4809–4814.

[12] M. Brunori, Nitric oxide moves myoglobin centre stage, Trends Biochem. Sci.26 (2001) 209–210.

[13] J.B. Wittenberg, B.A. Wittenberg, Myoglobin function reassessed, J. Exp. Biol.206 (2003) 2011–2020.

[14] M. Angelo, A. Hausladen, D.J. Singel, J.S. Stamler, Interactions of NO withhemoglobin: from microbes to man, Methods Enzymol. 436 (2008)131–168.

[15] P. Ascenzi, P. Visca, Scavenging of reactive nitrogen species by mycobacterialtruncated hemoglobins, Methods Enzymol. 436 (2008) 317–337.

[16] D.A. Greenberg, K. Jin, A.A. Khan, Neuroglobin: an endogenousneuroprotectant, Curr. Opin. Pharmacol. 8 (2008) 20–24.

[17] S. Herold, T. Matsui, Y. Watanabe, Peroxynitrite isomerization catalyzed byhis64 myoglobin mutants, J. Am. Chem. Soc. 123 (2001) 4085–4086.

[18] S. Herold, K. Shivashankar, M. Mehl, Myoglobin scavenges peroxynitritewithout being significantly nitrated, Biochemistry 41 (2002) 13460–13472.

[19] S. Herold, M. Exner, F. Boccini, The mechanism of the peroxynitrite-mediatedoxidation of myoglobin in the absence and presence of carbon dioxide, Chem.Res. Toxicol. 16 (2003) 390–402.

[20] N. Romero, R. Radi, E. Linares, O. Augusto, C.D. Detweiler, R.P. Mason, A.Denicola, Reaction of human hemoglobin with peroxynitrite. Isomerization tonitrate and secondary formation of protein radicals, J. Biol. Chem. 278 (2003)44049–44057.

[21] S. Herold, The outer-sphere oxidation of nitrosyliron(II)hemoglobin byperoxynitrite leads to the release of nitrogen monoxide, Inorg. Chem. 43(2004) 3783–3785.

[22] F. Boccini, S. Herold, Mechanistic studies of the oxidation of oxyhemoglobin byperoxynitrite, Biochemistry 43 (2004) 16393–16404.

[23] S. Herold, A. Fago, R.E. Weber, S. Dewilde, L. Moens, Reactivity studies of theFe(III) and Fe(II)NO forms of human neuroglobin reveal a potential role againstoxidative stress, J. Biol. Chem. 279 (2004) 22841–22847.

[24] S. Herold, A. Puppo, Oxyleghemoglobin scavenges nitrogen monoxide andperoxynitrite: a possible role in functioning nodules?, J Biol. Inorg. Chem. 10(2005) 935–945.

[25] P. Ascenzi, M. Milani, P. Visca, Peroxynitrite scavenging by ferrous truncatedhemoglobin GlbO from Mycobacterium leprae, Biochem. Biophys. Res.Commun. 351 (2006) 528–533.

[26] S. Herold, F. Boccini, NO� release from MbFe(II)NO and HbFe(II)NO afteroxidation by peroxynitrite, Inorg. Chem. 45 (2006) 6933–6943.

[27] P. Ascenzi, M. Fasano, Abacavir modulates peroxynitrite-mediated oxidation offerrous nitrosylated human serum heme–albumin, Biochem. Biophys. Res.Commun. 353 (2007) 469–474.

[28] P. Ascenzi, A. Bocedi, G. Antonini, M. Bolognesi, M. Fasano, Reductivenitrosylation and peroxynitrite-mediated oxidation of heme–hemopexin,FEBS J. 274 (2007) 551–562.

[29] P. Ascenzi, E. De Marinis, P. Visca, C. Ciaccio, M. Coletta, Peroxynitritedetoxification by ferryl Mycobacterium leprae truncated hemoglobin O,Biochem. Biophys. Res. Commun. 380 (2009) 392–396.

[30] E. Antonini, M. Brunori, Hemoglobin and Myoglobin in their Reactions withLigands, 1971, North-Holland Publishing Co., Amsterdam, London.

[31] H. Østdal, B. Daneshvar, L.H. Skibsted, Reduction of ferrylmyoglobin byb-lactoglobulin, Free Radic. Res. 24 (1996) 429–438.

[32] D.S. Bohle, P.A. Glassbrenner, B. Hansert, Syntheses of pure tetramethyl-ammonium peroxynitrite, Methods Enzymol. 269 (1996) 302–311.

[33] W.H. Koppenol, R. Kissner, J.S. Beckman, Syntheses of peroxynitrite: to go withthe flow or on solid grounds?, Methods Enzymol 269 (1996) 296–302.

[34] S. Goldstein, J. Lind, G. Merényi, Chemistry of peroxynitrites andperoxynitrates, Chem. Rev. 105 (2005) 2457–2470.

[35] S. Herold, F.J. Rehmann, Kinetic and mechanistic studies of the reactions ofnitrogen monoxide and nitrite with ferryl myoglobin, J. Biol. Inorg. Chem. 6(2001) 543–555.

[36] S. Herold, F.J. Rehmann, Kinetics of the reactions of nitrogen monoxide andnitrite with ferryl hemoglobin, Free Radic. Biol. Med. 34 (2003) 531–545.

[37] H. Bateman, Solution of a system of differential equations occurring in thetheory of radioactive transformations, Proc. Cambridge Philos. Soc. 15 (1910)423–427.

[38] M.F. Perutz, Myoglobin and haemoglobin: role of distal residues in reactionswith haem ligands, Trends Biochem. Sci. 14 (1989) 42–44.

[39] M. Bolognesi, D. Bordo, M. Rizzi, C. Tarricone, P. Ascenzi, Nonvertebratehemoglobins: structural bases for reactivity, Prog. Biophys. Mol. Biol. 68(1997) 29–68.

[40] P. Ascenzi, A. Bellelli, M. Coletta, A. Colosimo, G. Falcioni, G.M. Giacometti, R.Ippoliti, L. Zolla, B. Giardina, Multiple strategies for O2 transport: fromsimplicity to complexity, IUBMB Life 59 (2007) 600–616.

[41] P. Visca, G. Fabozzi, M. Milani, M. Bolognesi, P. Ascenzi, Nitric oxide andMycobacterium leprae pathogenicity, IUBMB Life 54 (2002) 95–99.

[42] A.I. Alayash, R.P. Patel, R.E. Cashon, Redox reactions of hemoglobin andmyoglobin: biological and toxicological implications, Antioxid. Redox Signal. 3(2001) 313–327.

Current Medicinal Chemistry, 2010, 17, ????-???? 1

0929-8673/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

Targeting the DNA Double Strand Breaks Repair for Cancer Therapy

Francesca Gullotta1, Elisabetta De Marinis

1, Paolo Ascenzi

1,2 and Alessandra di Masi*

,1

1Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy

2National Institute for Infectious Diseases I.R.C.C.S. “Lazzaro Spallanzani”, Via Portuense 292, I-00149 Roma, Italy

Abstract: Among several types of DNA lesions, the DNA double strand breaks (DSBs) are one of the most deleterious and harmful. Mammalian cells mount a coordinated response to DSBs with the aim of appropriately repair the DNA damage. Indeed, failure of the DNA damage response (DDR) can lead to the development of cancer-prone genetic diseases. The identification and development of drugs targeting proteins involved in the DDR is even more investigated, as it gives the possibility to specifically target cancer cells. Indeed, the administration of DNA repair inhibitors could be combined with chemo- and radiotherapy, thus improving the eradication of tumor cells. Here, we provide an overview about DSBs damage response, focusing on the role of the DSBs repair mechanisms, of chromatin modifications, and of the cancer susceptibility gene BRCA1 which plays a multifunctional role in controlling genome integrity. Moreover, the most investigated DSBs enzyme inhibitors tested as potential therapeutic agents for anti-cancer therapy are reported.

Keywords: DNA damage response, DNA double strand breaks, DNA repair, cancer susceptibility, inhibitors of DNA repair, cancer therapy.

1. INTRODUCTION

The integrity of an organism genome is of primary im-portance for genome stability. Therefore, the DNA damage induced by endogenous and exogenous factors, including reactive nitrogen and oxygen species, radiations, and car-cinogens, is continuously repaired to avoid genetic altera-tions. The DNA repair is coordinated through cell cycle checkpoints in order to properly remove the DNA damage. Checkpoint proteins play a pivotal role ensuring that each cell cycle phase is completed before progression to the sub-sequent one. Moreover, in the presence of DNA damage, the activation of checkpoint proteins pauses the cell cycle giving to the cell the necessary time to repair the damage before starting DNA replication and mitosis [1-4].

The critical role played by the DNA repair mechanisms

in the maintenance of the genome stability is evidenced by

the fact that many enzymes involved in these pathways have

been conserved throughout evolution [5]. The DNA damage

response (DDR) is strongly linked to cancer in both homo-

zygous and heterozygous individuals. Indeed germline muta-

tions in several genes involved in DDR are at the basis of

cancer-predisposing syndromes, and are associated with in-

herited chromosome instability [6,7]. Aneuploidy is a char-

acteristic of cancer, with greater than 90% of all solid tumors

in humans carrying an aberrant karyotype. The mitotic

checkpoint plays a pivotal role in the maintenance of genome

stability, disruption of mitosis often leading to defects in

chromosome segregation and to the production of genetically

unstable aneuploid cells [8]. Indeed, several studies indicate

that aneuploidy can promote both tumor cell growth and

tumor cell death, the cellular outcome depending on the ex-

tent of mitotic checkpoint silencing and of aneuploidy in-

duced. Partial loss of the mitotic checkpoint results in low

levels of aneuploidy and promotes tumor initiation and pro-

gression. On the contrary, the complete loss of the mitotic

*Address correspondence to this author at the Department of Biology, Uni-

versity Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy; Tel:

+39-06-57333494; Fax: +39-06-57336321; E-mail: [email protected]

checkpoint induces high levels of aneuploidy and lethality [9]. Thus differential checkpoint silencing has a profound outcome on cell fate; however, how this condition contrib-utes to tumorigenesis is not yet clear [8]. Many human inher-ited disorders (e.g., Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and ligase 4 syndrome (LIG4), all characterized by homozygous autosomal mutations in genes involved in DNA damage sensing and/or repairing, display genome instability and cancer susceptibility [3,10-14].

Carriers of mutations in genes involved in DDR also show an increased risk to develop malignancies with respect to the normal population [15-18]. In particular, heterozygous cells expressing gene mutations are generally characterized by haplo-insufficiency that allows the development of tu-mors due to a defect in the proper removal of DNA lesions. By the time the tumor develops, the second allele of the re-pair gene is inactivated in almost all cases, either by loss of heterozygosity or by gene conversion of the wild-type to the mutant allele [19].

Variations in DDR in the normal population may influ-ence cancer susceptibility and outcomes [5]. In fact, besides germline mutations that negate gene function, variants like polymorphisms of DDR genes, although having a less dra-matic functional impact, may bear upon cancer risk and out-come for patients [5]. Polymorphic changes are present throughout the human genome and may account for the high variability in the susceptibility to many common diseases (e.g., diabetes, cardiovascular disorders, and cancer) [20]. Indeed, polymorphisms in DDR genes could confer subop-timal DNA repair capacity, thus leading to accumulation of mutations and hence to a predisposition to cancer [5].

The identification and characterization of DDR defects in cancer has opened up opportunities for cancer therapy. In-deed, as these defects are specific to tumor cells, the devel-opment of new anti-cancer agents targeting DNA repair pathways may give the possibility to develop therapies able to selectively kill cancerous cells and reducing toxic effects in normal tissues [19]. For common solid tumors, radiother-apy and chemotherapy are the two main treatments currently

2 Current Medicinal Chemistry, 2010 Vol. 17, No. 1 Gullotta et al.

available to improve outcomes in cancer patients [5]. The cytotoxicity of many of these agents is directly related to their ability to induce DNA damage. The ability of cancer cells to recognize this damage and to initiate DDR is an im-portant mechanism for therapeutic resistance that negatively impacts upon therapeutic efficacy. Pharmacological inhibi-tion of DDR, therefore, has the potentiality to enhance cyto-toxicity of a diverse range of anti-cancer agents. The use of inhibitors of DDR pathways appears also to provide an inter-esting opportunity to target the genetic differences that exist between normal and tumor tissues [5].

Here, we report a critical analysis of DSBs repair path-ways, with particular attention to chromatin modifications, to the role of the breast cancer associated protein 1 (BRCA1), generally considered as a molecular model, and to cancer therapeutic strategies based on the inhibition of the DSBs repair pathways.

2. DSBs DAMAGE SENSING AND REPAIR

Eukaryotic cells have evolved specialized multi-component macro-molecular systems to sense, respond to, and repair DNA damage [21-24]. The range of different DNA lesions or adducts induced by DNA-damaging agents is broad, and hence there is a necessity for multiple DNA repair pathways to exist in cells. These pathways are in-volved in the repair of: (i) alkyl adducts by O6-alkylguanine DNA alkyltransferase; (ii) base damage and DNA single strand breaks (SSBs) by base excision repair (BER); (iii) DNA double strand breaks (DSBs) by homologous recombi-nation (HR) and non-homologous end joining (NHEJ); (iv) bulky adducts by nucleotide excision repair (NER); (v) cross-links by DNA inter-strand cross-link repair; and (vi) mismatches and insertion/deletion loops by DNA mismatch repair (MMR). While each damaged lesion is repaired by the lesion-specific DNA repair pathway, there are some overlaps and interactions between the pathways involved in the repair of specific lesions. In Table 1 an overview of some of the DNA damaging agents, the types of lesion induced, and the DNA repair pathways activated has been reported.

Among several types of lesion, the DSB is one of the most deleterious and harmful, as chromosomal breakage may result in an extreme loss of genetic integrity [14,24,25]. DSBs are more common than was once expected and are estimated to occur at levels of 10 per cell per day in mam-mals [26]. DSBs are generated not only by environmental factors, such as ionizing radiation (IR) and chemical geno-toxic compounds, but also by endogenous factors. In particu-lar, products of oxidative metabolism (e.g., ˙OH and ˙H) and physiological processes (e.g., DNA replication, meiotic re-combination, and immunoglobulin V(D)J gene rearrange-

ments), represent the most important sources of DSBs [14,25,27].

DSB originates by a break in the sugar phosphate back-bone of each of the two strands in duplex DNA. If the breaks in each strand are sufficiently far apart so that the strands remain attached through base pairing, they will be repaired as separate single stranded breaks. On the contrary, if the single breaks in each strand close together, the ends will dis-sociate from one another, resulting in a DSB [28]. Because the DNA ends can become physically detached, either persis-tent DSBs or inaccurate repair can have profound effects. In particular, the incorrect rejoining of broken ends of DNA can produce aberrant chromosomal rearrangements (e.g., translo-cations, insertions, duplications, and deletions) enhancing genome instability, whereas unrepaired DSBs can cause cell death [6,29-31]. Large chromosomal deletions can lead to the loss of tumor suppressor genes or to the activation of proto-oncogenes, resulting in carcinogenesis [29,32,33].

The harmful effects of just one DSB underline the impor-tance of a sensitive damage detection system and of a rapid response. Cells must be capable of sense the lesion(s) in a selective and extremely sensitive manner, and must dis-criminate between a real lesion and a common intermediate of DNA metabolism. After sensing the DNA lesion, the sig-nal that detects the break is rapidly amplified in order to in-duce the global cell functions that are involved in the dam-age response (including cell cycle control, transcriptional regulation, and/or post-translational modifications of repair proteins, and, if necessary, apoptosis) (Fig. (1)) [21-24].

2.1. The DSBs Repair Mechanisms

When DNA is damaged by IR, many DNA damage-signaling proteins are recruited to the damaged loci forming discrete nuclear IR-induced foci (IRIF) [34-36] (Fig. (2)). The hierarchy and timing of protein recruitment (e.g., the Ataxia telangiectasia mutated protein (ATM), the mediator of DNA-damage checkpoint 1 protein (MDC1), the meiotic recombination 11 protein (MRE11), the Nijmegen breakage syndrome mutated protein (NBN), and RAD50) to form IRIFs are thought to be critical for checkpoint response and DDR [36-38]. Indeed, the hierarchy and timing of IRIF for-mation provides the order of the molecular events ensuing DNA damage detection and signal transduction [36,39,40] (Fig. (2)).

In mammalian cells, one of the main players in IRIF for-mation is the H2A histone family member X (H2AX). In response to DNA damage, H2AX is phosphorylated ( -H2AX) at the Ser139 residue by ATM, a member of the phosphoinositide-3-kinase (PI3K)-related protein kinase (PIKK) family. The epigenetic signal generated by -H2AX

Table 1. Overview of Some of the DNA Damaging Agents, the Types of Lesion Induced, and the DNA Repair Pathways Activated

Damaging agent Reactive nitrogen and oxy-

gen species, IR Alkylating agents Replication inhibitors Topoisomerase inhibitors

Type of damage induced SSB, DSB Cross links, DSB, base

modifications DSB SSB, DSB

Repair pathway BER, NER, NHEJ, HR BER, NER, NHEJ, HR HR BER, NER NHEJ, HR

Targeting DSBs Repair for Cancer Therapy Current Medicinal Chemistry, 2010 Vol. 17, No. 1 3

Fig. (1). Induction of DSBs by IR and carcinogens. The DSBs damage response includes a number of sensing and signaling proteins that

coordinate the cell cycle checkpoint activation, the gene transcription, and the DNA repair to allow for the proper DSB rejoining and cell

survival. In the presence of heavily damaged or dysregulated cells, the apoptotic programmed cell death pathway and the long-term cell cycle

arrest (known as senescence) are activated. Lack of proper DNA repair or inaccurate DSBs repair may result in cell death or survival of cell

with mis-repaired DNA, which may then lead to tumorigenesis.

is then recognized by sensor proteins [41-43]. Indeed, -H2AX marks the chromatin region at or near the DSB site and serves as a platform for the recruitment of DNA check-point signaling and BRCT-containing repair proteins, includ-ing the p53 binding protein 1 (53BP1), the breast cancer 1 protein (BRCA1), MDC1, and NBN [7,34,37,44 51]. The association of the MRE11/RAD50/NBN (MRN) complex to the DSBs represents the first event in the DSBs response [39,52-55]. Suddenly after the localization of the MRN com-plex at the damaged site, ATM undergoes to auto-phosphorylation at the Ser1981 residue, with the consequent dissociation of ATM dimers and ATM kinase activation [56]. Once activated, ATM can phosphorylate several sub-strates, many of them involved in cell cycle checkpoints (e.g. p53, the checkpoint homolog 1 protein (CHK1), the check-point homolog 2 protein (CHK2), the cell division cycle 25 homolog A protein (CDC25A), the structural maintenance of chromosomes 1 protein (SMC1), and BRCA1) [56,57]. This determines the execution of DSBs responses, including cell cycle arrest, DNA repair and induction of a transcriptional program [7,58]. Depending on the severity of the DNA dam-age and on the cell type involved, cells may undergo apopto-sis instead of attempting to repair the DNA damage (gener-ally during interphase, before entering mitosis). In particular, after exposure to IR, lymphocytes and mouse embryonic

stem cells undergo apoptosis, whereas fibroblasts do not [59]. It is well known that tumors differ in their sensitivity to IR [60,61]. Low doses of IR can induce interphase death of some tumors, especially of hematopoietic origin. In this case, morphology and biochemistry of dying cells resemble apop-tosis [60]. Cells that do not undergo death in interphase may become arrested in G1/S and/or in G2/M, but if the cellular machinery is not able to repair the injury during the arrest, the DNA damage can lead to different death-associated con-sequences. Tumor cells with impaired or lost checkpoint functions are unable to maintain this arrest, and thus enter mitosis prematurely in the presence of unrepaired DNA. The fate of such cells depends on several factors. Indeed, tumor cells can either die by apoptosis after one or even repeated mitotic cycles or undergo mitotic catastrophe due to prema-ture mitosis [62]. The mitotic catastrophe associated with giant multinucleated cells and uncondensed chromosomes, is the most prominent mode of cell death after IR in solid tu-mors and may occur at long intervals after exposure depend-ing on the proliferation rate and DNA repair capacity of irra-diated cells [62].

Tumorigenic abnormalities that deregulate DNA replica-tion determine DNA damage and checkpoint activation, compromising the efficiency of the DDR pathways. This causes deregulated cell proliferation, survival, increased ge-


nomic instability and tumor progression [63]. Indeed, defi-ciencies of -H2AX or NBN have been shown to increase the accumulation of chromosome aberrations [64,65].

Two distinct DSBs repair pathways, namely the HR and the NHEJ, have been identified both in mammalian cells and in yeast to preserve the integrity of DNA after DSBs induc-tion [3,23,29,66,67] (Fig. (2)). Each pathway consists of a different array of repair enzymes and related factors. HR uses a DNA template, as can be found on a sister chromatid during the late S and the G2 cell cycle phases. On the other hand, NHEJ brings the DNA termini in a protein-DNA com-plex and joins them without the need for homology, repre-senting the major DSB repair pathway during the G1 cell cycle phase. Therefore, the choice of the repair pathway (ei-ther HR or NHEJ) is linked to the cell cycle phase. However, both pathways may be also activated simultaneously during the S and G2 phases, thus repairing DNA lesions coopera-tively [23,68,69].

The HR repair mechanism utilizes extensive homology to faithfully restore the sequence at the break site by means of

processes involving proteins of the RAD52 epistasis group [70-72]. In human cells, the main steps in HR are mediated by the single-strand binding replication protein A (RPA) [73,74], which stabilizes the single strand DNA (ssDNA) helped by mediator factors involved in the promotion or sta-bilization of the RAD51 nucleoprotein filament (Fig. (2)). Once RAD51 is correctly loaded, a joint molecule, resulting from the strand invasion of the RAD51 nucleoprotein fila-ment into the sister chromatid, is formed. Then, a second strand invasion happens, in which the dislocated filament searches for homology in the duplex DNA with the broken single-strand end. The joint molecule intermediate is known as the Holliday junction (HJ), a DNA structure consisting of two duplex crossed strands forming a heteroduplex DNA molecule [75]. In the last step of the process, helicase and endonucleolytic activities are required for branch migration and HJ strand nicking [7,76].

The NHEJ repair mechanism restores broken ends with little or no requirement for sequence homology. As a result of the DSB, the DNA broken ends can be incompatible and

Fig. (2). DSBs response mechanisms. After the IR-induced DSBs damage, the MRN complex is rapidly recruited at the damaged site, follo-

wed by the localization of the activated ATM. In turn, ATM kinase phosphorylates the H2AX histone and NBN, and the epigenetic signal

generated by -H2AX is recognized by sensor proteins (e.g., BRCA1, MDC1, 53BP1, and SMC1). After sensing, the signal must be rapidly

amplified in order to induce the proper cell response, including cell cycle control, DSBs repair, and, if necessary, apoptosis. The DSBs repair

machinery is recruited to the lesion in relation to the cell cycle stage: in the S and G2 phases the presence of the replicated DNA allows the

repair mainly through the HR pathway, whereas in the G1 phase cells undergo repair predominantly through NHEJ repair pathway. For de-

tails, see text (modified from [7]).


not complementary. The overall process involves the DNA-dependent protein kinase (DNA-PK) holo-enzyme, a mem-ber of the PIKK protein family, consisting of the DNA end-binding heterodimer Ku70/Ku80 and the catalytic subunit (DNA-PKcs) [69,77-80] (Fig. (2)). Upon activation of the DNA-PK, nucleases and polymerases specific for the DNA end processing (e.g., Artemis and Cernunnos) are recruited to fill in the gaps and remove flaps prior rejoining [81-86]. After the DNA ends have been processed, the complex formed by the X-ray repair cross complementing protein 4 (XRCC4) and the DNA ligase IV completes the ligation step [87 90]. Cell lines defective in any of these genes are gener-ally highly IR sensitive and show marked deficiencies in DSBs repair [3,91-93].

2.2. Role of Chromatin Modifications in the DSBs Response

Eukaryotic DNA is packaged around nucleosomes made of a histone octamer, forming chromatin. Chromatin modifi-cation represents a crucial aspect of the DSBs detection and response [94]. Indeed, highly localized changes, as expan-sion and contraction in chromatin structure, are necessary to allow the recruitment of repair proteins to DNA damage sites [94-96].

Regional changes of the higher-order chromatin structure are promoted by at least two distinct classes of enzymes, namely chromatin-remodeling and histone modifying en-zymes. Chromatin-remodeling enzymes, including INO80, SWI/SNF, RSC, Tip60, SWR1, and RAD54, show ATPase activity and catalyze nucleosome mobility [97-104]. On the other hand, histone modifying enzymes add chemical groups (i.e., methyl, phosphate, acetyl, and ubiquitin) to the N- and/or C-terminal regions of the histone tails. Such modifica-tions may represent a “histone code” acting as surface mark-ers that can be recognized by non-histone proteins altering the state of chromatin. This leads to a variety of downstream events (i.e., transcriptional activation, cell cycle checkpoints control, and DNA repair) [105].

Chromatin-remodeling and histone-modifying enzymes are able to change the chromatin topology after cell exposure to DNA damaging-agents (e.g., IR) [106]. In this respect, histone acetylation facilitates both protein recruitment and chromatin structure relaxation. Indeed, acetylation of the Lys residue located at the N-terminal tails of histones H3 and H4 abolishes the positive charge allowing the relaxation of the higher-order chromatin structure [107]. Both acetyltrans-ferases (HATs) and histone deacetylases (HDACs) regulate the acetylation status of histones which can be modified in response to the DNA-induced damage [108]. In mammalian cells, Tip60 is responsible for acetylation of the N-terminal tail of H4, a step which seems to facilitate the loading of ATM signaling mediator proteins, such as 53BP1, BRCA1, and RAD51 [109,110]. Interestingly, a dominant negative form of Tip60 histone acetylase is associated with a DSBs repair defect, and siRNA-mediated Tip60 depletion reduces markedly ATM autophosphorylation of Ser1981 as well as ATM kinase activity [98]. Furthermore, ATM acetylation seems to be Tip60-dependent. Indeed, Tip60-deficient cells show a degree of radiation sensitivity very similar to that of ATM-mutated cells [111]. This points to a link between his-

tone acetylation and the DDR pathway governed by ATM. Consistently, Tip60 is constitutively bound to ATM, its ac-tivity increasing after DNA damage [111]. HAT activation, ATM acetylation, and Tip60 recruitment to IRIF occur in an ATM kinase-independent fashion. These findings, together with the observation that in Tip60-deficient cells ATM kinase activity is reduced, suggest that acetylation is an es-sential upstream event for ATM activation [111].

Members of the HAT Tip60 core complex NuA4, such as the transactivation-transformation domain-associated protein (TRRAP; also named PAF400), interact with the MRN com-plex and play a role in DSBs repair [112]. Notably, TRRAP

-/-

murine cells show a defective HR repair mechanism due to the impairment of both Tip60 recruitment and H4 acetylation at the DSB sites [109,113]. The recruitment of RAD51 and BRCA1 is also compromised in TRRAP-deficient cells. The restoration of a correct recruitment of RAD51 and the execu-tion of HR is achieved by treating TRRAP-deficient cells with hypotonic shock and relaxing chromatin agents [109,113].

On the other hand, the recruitment of chromatin-remodeling proteins may be also a downstream step in the DNA damage signaling pathways. In fact, though the local-ization of DDR proteins, such as MDC1 and NBN, is unaf-fected by chromatin conformation [109], chromatin remodel-ing at a single DSB in yeast depends on the activity of the Mre11/Rad50/Xrs2 complex (the yeast ortholog of the mammalian MRN complex) [114]. After the initial sensing of the lesion mediated by the MRN complex, the response to the DSBs may branch out the chromatin relaxation-independent and the chromatin relaxation-dependent path-ways [113]. Although a number of human genetic syndromes characterized by mutations in genes involved in chromatin remodeling have been described, the sensitivity of patients cells to DNA-damaging agents remains to be investigated [115,116].

2.3. Role of BRCA1 in the Genome Stability Maintenance

The BRCA1 gene was cloned and mapped to chromo-some 17q21 through its link to breast and ovarian cancer [117]. Mutations in one or both BRCA1 alleles occurs in 40-50% of germline cancers and in 10% of sporadic breast can-cers [117]. Moreover, secondary tumors (e.g., pancreatic and melanoma) commonly arise at later stages in breast cancer patients [118,119]. These evidences led to the notion that BRCA1 represents a cancer-susceptibility gene. Defining BRCA1 functions in DDR is essential to understand its role in tumor suppression, and to develop new anti-cancer thera-peutic approaches able to specifically target BRCA1-mutated cancer cells.

BRCA1 plays a multifunctional role in controlling ge-nome integrity. Indeed, cells lacking intact BRCA1 manifest a mitotic-checkpoint defect [120,121]. BRCA1 interacts with several nuclear polypeptides, including RAD51, thus having a key role in DSBs repair and in HR [46,120,122]. Remarka-bly, embrionic stem cells lacking BRCA1 are defective in transcription-coupled repair of oxidative damage [123,124].

BRCA1 encodes a 1863 amino acids protein with a mo-lecular mass of 220 kDa [117,125,126]. The BRCA1 protein


is characterized by a multi-domain structure comprising (i) the N-terminal RING finger (a specialized zinc-finger) [117], (ii) three nuclear localization signals in the central region [127], and (iii) a tandem of two C-terminal BRCA1 carboxy-terminal (BRCT) domains, named BRCT1 and BRCT2 [128-130].

BRCA1 is the highest profile BRCT superfamily mem-ber, from which the BRCT motif was first described [129-131]. The BRCT domains constitute a class of phosphopro-tein binding modules involved both in the cellular response to the DNA damage and in the cell cycle control [132]. The high degree of conservation among several species highlights the importance of these domains in cellular metabolism. The BRCT repeat structure provides a flexible framework for protein-protein and protein-ligand interactions that are cru-cial in DNA damage signaling complexes. Therefore, this protein domain appear particularly attractive as scaffolding elements at the heart of large multi-protein complexes [132]. BRCT repeats are characterized by conserved clusters of hydrophobic residues forming the BRCT:BRCT contact re-gion. This hydrophobic region recognizes proteins and pep-tides containing the pSer-Xxx-Xxx-Phe recognition motif (where pSer indicates the phosphorylated Ser residue and Xxx indicates any residue) [132]. Several mutations in BRCA1, linked to an enhanced risk of breast and ovarian cancer, are localized at the interface between the two BRCT repeats, thus indicating that their correct packing is essential for BRCA1 function and tumor suppression [133].

The loss of BRCA1 induces a pleiotropic phenotype dis-playing not only defects in DNA-repair processes, but also alterations in a number of diverse cellular processes [134,135]. Indeed, several proteins have been reported to interact with BRCA1 at multiple sites [135,136], including the BRCA1 associated RING domain 1 (BARD1) [135,137], importin (a subunit of the nuclear transport signal receptor) [127,135], and the ubiquitin hydrolase BRCA1 associated protein 1 (BAP1) [135,138]. Furthermore, the tumor-suppressor p53 [135,139], the tumor suppressor retinoblas-toma protein (RB) [135,140], the transcriptional repressor CTBP interacting protein (CtIP) [135,141,142], and the RNA polymerase II holoenzyme complex [135,143,144] bind the C-terminal region of BRCA1, supporting its role in both cell cycle control and transcriptional regulation [135].

Interestingly, the tandem BRCT C-terminal domains of BRCA1 bind histone deacetylase-1 and -2 (HDAC1 and HDAC2, respectively), the BTB and CNC homology 1 pro-tein (BACH1), a member of the DEAH family of DNA heli-cases [135,145,146], and the BRG1 subunit of the SWI/SNF-related complex [135,147]. This demonstrates a role of BRCA1 in chromatin remodeling, and thus in the regulation of DNA replication, transcription, and repair mechanisms [135].

Among several functions, BRCA1 is involved in the

DNA damage signaling and acts as a checkpoint mediator.

Indeed, BRCA1 is a target for ATM and Ataxia telangiecta-

sia and RAD3-related (ATR) kinase (a member of the PIKK

proteins family). Note that ATM and ATR signaling path-

ways are activated by IR- and ultraviolet (UV)-induced dam-

age, respectively [148]. The BRCA1 phosphorylation by

ATM and ATR facilitates the interaction of these kinases

with their substrates at the damaged site, by mediating the

spatio-temporal assembly of damage-specific multiprotein

complexes [135,149]. Moreover, BRCA1 is involved in the

DSBs repair. After the induction of DSB, BRCA1 is stably

associated with the MRN complex [150], this being neces-

sary for BRCA1-mediated ubiquitylation events at the DSB

sites [151]. Moreover, BRCA1 is able to bind the -H2AX

histone by the BRCT tandem domains; consistently, cell

lines deficient for -H2AX are compromised for BRCA1-mediated ubiquitylation at the DSB sites [151].

BRCA1 associates with p53 thus enhancing the p53-

dependent transcription from the cyclin-dependent kinase 1A

(CDKN1A) and the BCL2-associated X protein (BAX) pro-

moters [135,152,153]. Furthermore, BRCA1 affects the ex-

pression of the cyclin-dependent kinase inhibitor CDKN1A

in a p53-independent manner [135,152], thus representing a

modulator of the G1/S checkpoint. Since BRCA1 is required

for the S- and G2/M-phase checkpoint arrest in response to

DSBs [135,154], cells lacking intact BRCA1 manifest a mi-

totic-checkpoint defect and are more likely than their normal

counterparts to become aneuploid [135,155,156]. Finally,

BRCA1 is able to enhance the expression of the xeroderma

pigmentosum-complementation group C (XPC) protein, the

damage-specific DNA-binding protein 2 (DDB2), and the

growth-arrest and DNA-damage inducible protein 45

(GADD45), independently of p53. However, it is currently

unclear whether this role of BRCA1 is a direct or an indirect

consequence of DNA repair and checkpoint signaling, even

if it has been demonstrated that BRCA1 can associate with

the RNA polymerase II holoenzyme complex via the RNA helicase A [135,149].

The identification of mutations in the breast cancer sus-

ceptibility gene BRCA1 has provided the opportunity to help

in the identification of women who are at high risk of devel-

oping breast and ovarian cancer [157]. All known BRCA1

mutations are collected at the Breast Cancer Information

Core [158]. Truncating and missense mutations are two of

the predominant types of BRCA1 mutations so far identified.

However, while most of the truncating mutations have been

found to be cancer-associated, missense variants are more

difficult to classify [159]. At present, about three-hundreds

mutations have been found in the whole BRCA1 gene

[160,161]; remarkably one-hundred missense mutations have

been detected in the BRCT domains through breast and ovar-

ian cancer screening programs [157]. However, there is suf-

ficient pedigree data to relate only few of them to breast and

ovarian cancer [157]. In situations in which clinical and ge-

netic data are not sufficient to classify BRCA1 variants,

structural and functional studies are used to assess specific

biochemical properties of the mutated protein in order to

classify the mutation(s) as deleterious or neutral [162-166].

Moreover, cancer-associated missense mutations of BRCA1

have been found to exhibit loss of function with respect to

the transcriptional activity, while neutral variants display an

activity similar to that of the wild-type protein [164,167]. Of

particular interest is the significance of breast cancer risk

associated with unclassified variants, where genetic pene-

trance studies are not available to assess their effects [161,166]. A set of twenty-five BRCT missense variants


Table 2. Functional Characteristics of Some of the Cancer-Related Mutations Located within the BRCT Domains of BRCA1 Iden-

tified so Far (n.a., not Available Data)

Genetic classification Functional classification pSer-Xxx-Xxx-Phe binding ability References

Mutations within the BRCT1 domain (amino acids 1642-1736)

Ser1655Phe Cancer-related n.a. [157]

Val1665Met Cancer-related Trascriptionally active n.a. [168]

Ala1669Ser Cancer-related Trascriptionally active n.a. [168,319]

Thr1685Ile Cancer-related Not trascriptionally active n.a. [161,164,167]

Val1688del Cancer-related Not trascriptionally active n.a. [168]

Asp1692Asn Cancer-related Trascriptionally active +/ [157,168]

Phe1695Leu Cancer-related n.a. + [157]

Val1696Leu Cancer-related n.a. [157]

Cys1697Arg Cancer-related Not trascriptionally active [157,168]

Arg1699Gln Cancer-related Trascriptionally active [168,320]

Arg1699Leu Cancer-related Not trascriptionally active n.a. [321]

Arg1699Ser Cancer-related Trascriptionally active n.a. [168]

Arg1699Trp Cancer-related Trascriptionally active [157,161,168]

Gly1706Glu Cancer-related Not trascriptionally active [157,161,164,167]

Ala1708His Cancer-related Not trascriptionally active [157,168]

Ala1708Glu Cancer-related Not trascriptionally active n.a. [168]

Ser1715Asn Cancer-related Not trascriptionally active n.a. [168]

Ser1715Arg Cancer-related Not trascriptionally active [157,161,168]

Trp1718Cys Cancer-related n.a. [157]

Thr1720Ala Cancer-related Trascriptionally active + [157,161,321,322]

Asp1733Gly Cancer-related Trascriptionally active n.a. [168]

Mutations within the BRCT linker region (amino acids 1737-1755)

Gly1738His Cancer-related Not trascriptionally active [157,168]

Asp1739Gly Cancer-related Trascriptionally active [157,321]

Pro1749Arg Cancer-related n.a. +/ [157]

Arg1751Gln Cancer-related n.a. [157]

Ala1752Pro Cancer-related Trascriptionally active +/ [157,321]

Mutations within the BRCT2 domain (amino acids 1756-1855)

Leu1764Pro Cancer-related Not trascriptionally active n.a. [161,164,167]

Ile1766Ser Cancer-related n.a. [157]

Met1775Arg Cancer-related Not trascriptionally active [117,157]

Gly1788Val Cancer-related Not trascriptionally active [161,157]

Val1809Phe Cancer-related n.a. [157]

Trp1837Arg Not trascriptionally active [157,168]

Trp1837Gly Cancer-related n.a. [157]

Trp1837X Cancer-related Not trascriptionally active n.a. [168]


have been analyzed by pull-down assay to test the pSer bind-ing capacity [157], demonstrating that the structural integrity of the BRCT tandem domains is required for phospho-protein recognition. Remarkably, the missense variants that highly destabilized the BRCA1 fold are unable to bind the pSer-Xxx-Xxx-Phe peptide [157]. Moreover, mutations of any amino acid residues located within the Phe binding pocket impair specific phospho-protein recognition.

In Table 2, some of the functionally characterized cancer-related missense and nonsense mutations located within ei-ther the BRCT1, or the BRCT2, or the BRCT linker region of BRCA1 are summarized. These mutations may either pre-vent the correct folding of BRCA1 or destabilize the BRCT tandem domains folding, altering the BRCT domains func-tion [157,168,169].

3. TARGETING THE DSBs REPAIR PATHWAY:

IMPLICATIONS FOR CANCER THERAPY

The cytotoxicity of most chemotherapeutic drugs, as well as of IR, is related to their ability to cause DNA damage. Very different DNA lesions and adducts are induced by chemotherapeutic drugs and radiations, DNA repair reflect-ing the multiple cellular mechanisms. Since a tumor cell is characterized by a defect in a specific DNA repair pathway [170-172], targeting the other pathways involved in the dam-age removal should have a much greater impact on the sur-vival of the tumor cells than on the survival of normal cells [173]. Therefore, the development of anti-cancer agents spe-cifically targeting DNA repair pathways in cancer cells may give the possibility to develop therapies able to kill only can-cerous cells reducing the overall cytotoxic effects [19,172,174-178]. These drugs, playing per se a relevant role in cancer therapy [176], may be also used in combination with other chemotherapeutic and radiotherapeutic protocols [175]. The aim of radiation therapy is to maximize radiation dose to the tumor to achieve local control and minimize the dose to normal tissues. This concept, defined as “therapeutic ratio”, is relevant when designing trials using inhibitors of DSBs repair as adjuncts to radiotherapy. Since radiotherapy is usually fractionated into daily treatments to improve cellu-lar and DSBs repair in normal cells relative to tumor cells, it will be important to measure the DNA repair in both malig-nant and normal tissues to develop optimized protocols within the context of a “molecular therapeutic ratio” [179-181].

As a whole, several therapeutic protocols based on the inhibition of DSBs repair pathways have been developed; drugs specifically impairing repair mechanisms are actually under evaluation in several clinical trials. Results obtained provide convincing evidence that DNA repair proteins are viable drug targets. However, several crucial clinical issues need to be addressed to evaluate the potential of these agents. Table 3 reports the most investigated enzyme inhibitors of DSBs repair and the clinical trials based on these molecules administrated in mono- and/or combined-therapies.

3.1. ATM, ATR, and DNA-PK

Three DNA repair-related kinases, ATM, ATR, and DNA-PK, have been targeted for the development of novel anti-cancer agents [182].

As mentioned above, ATM and ATR protein kinases play a crucial role in cellular DDR, regulating DNA repair and cell cycle checkpoints [174] (see paragraph 2.1.); the key role played by these two proteins in the DDR is highlighted by the severe phenotypes caused by homozygous mutations in the codifying genes, e.g. the cancer-prone-diseases AT and Seckel syndrome (SS), respectively.

ATM and ATR inhibition abolishes the cell cycle check-point functions [183-186] sensitizing cells to anti-cancer radiotherapy and chemotherapy [187,188]. The rationale behind the clinical use of ATM inhibitors assumes that ATM signaling is dysfunctional in tumor cells, ATM inhibition inducing a hypersensitivity to agents causing DSBs [189]. However, most of ATM inhibitors, such as caffeine, LY294002, and wortmannin are not specific since they in-hibit a number of members of the PI3K family, thus causing side-effects [5,190-193].

Wortmannin binds to the ATP binding site of PI3K, so that one face of wortmannin packs against the N-terminal lobe, and the other face packs against the C-terminal lobe (PDB ID: 1E7U) [194]. Moreover, the primary amine of the active site Lys833 attacks wortmannin at the furan ring [195]. The resulting covalent complex causes a large con-formational rearrangement in the active site and irreversibly inhibits the enzyme [194]. Recently, Ku-55933 has been reported to inhibit selectively ATM, impairing the phos-phorylation of several substrates (including SMC1, p53, NBN, and H2AX) and sensitizing tumor cells to IR, to the topoisomerase I inhibitor camptothecin, and to the topoi-somerase II inhibitors amsacrine, doxorubicin, and etoposide [187,196].

Recently, Schisandrin B, an active component of Fructus schisandrae, has been reported to be an highly specific ATR protein kinase inhibitor, able to impair the DNA damage repair [197] and to enhance the anti-cancer effects of the topoisomerase II inhibitor doxorubicin [198].

Given its crucial role in the DSBs repair, the NHEJ ma-chinery represents an important target for adjuvant treat-ments aimed to radio-sensitizing tumor cells and improving radiotherapy efficacy [5,173,175,196]. In this respect, the discovery that inhibition of the DNA-PK, a crucial compo-nent of the NHEJ pathway, renders cells acutely sensitive to IR and topoisomerase II inhibitors, stimulated efforts to syn-thesize DNA-PK inhibitors as potential therapeutics. Cur-rently, a number of potent and selective DNA-PK inhibitors are available, including IC 87102, IC 87361, NU7026, NU7441, OK-1035, SU11752, salvicine, and vanillin [5,173,175,177,196,199-205]. Preclinical trials using these inhibitors are in progress [203,205-207]. In particular, salvicine has been administrated in mono-therapy in a phase II clinical trial [196].

3.2. Chromatin Modifications

Chromatin modifications play a critical role in DSBs re-pair, representing an important target for anti-cancer therapy [19]. In particular, the use of HDAC inhibitors (HDACIs) represents a promising novel strategy in cancer treatment [208,209]. Indeed, the inhibition of HDACs activity en-hances the sensitivity to IR both in vitro [210,211] and in vivo [212-215].












HDACIs target tumor cells via the re-activation of si-lenced checkpoints and tumor suppressor genes [216]. How-ever, because the HDACs downstream targets are involved in several cellular processes, the exact mechanism of cell killing is not well understood [19]. HDACIs, such as suberoylanilide hydroxamic acid (SAHA, also known as vorinostat) and thricostatin A (TSA), enhance and stabilize -H2AX foci formation after IR exposure [214,217-219].

This suggests a mechanism of increased cell killing by modulating the DSBs repair [19]. A very high number of phase I, II, and III clinical oncological trials testing SAHA efficacy both in mono- and in combined-therapy with other agents are in progress or have been completed [207]. The electron density map of both the HDAC7-SAHA and HDAC7-TSA complexes reveal poor electron density for the capping group but strong and clear electron density for the hydroxamate moiety of the inhibitors. The hydroxamate moiety of both SAHA and TSA inhibitors forms hydrogen bonds with the side chains of two active site residues (i.e., His669 and His670) mimicking the interactions of the water molecule in the active site of apo-HDAC7. The hydroxamate group of both SAHA and TSA forms a monodentate com-plex with the catalytic zinc ion involving only the hydroxyl oxygen rather than both the hydroxyl and the carbonyl oxy-gen of the inhibitor. Upon inhibitor binding, the side chain of some amino acids, which are at the periphery of the pocket and mostly solvent-exposed, change conformation to ac-commodate the inhibitor (e.g., Thr625, Asp626, Phe679, and Leu810) (HDAC7-SAHA, PDB ID: 3C0Z; HDAC7-TSA, PDB ID: 3C10) [220].

PCI-24781 (also named CRA-02478), currently tested in phase I/II clinical trials [207,221], sensitizes human tumor cells to IR [222], increases apoptosis, inhibits the formation of IR-induced RAD51 foci formation, and leads to down-regulation of RAD51 expression [223]. Currently, there is an ongoing clinical trial testing the combination of the valproic acid HDACI with the radiotherapeutic treatment [207,224]. In recent years, even more specific and selective HDACIs are becoming available [225,226].

In the light of the pivotal role of the H2AX histone in the DNA damage signaling, this protein has been proposed as a target for anti-cancer therapy. Indeed, peptide inhibitors of H2AX phosphorylation increase tumor cell sensitivity to IR [227].

3.3. CHK1 and CHK2

A large number of chemically diverse CHK1 and CHK2 kinase inhibitors have been reported in the literature; cur-rently, several compounds are under development for clinical use [228].

CHK1 and CHK2 are Ser/Thr protein kinases which function as key regulators of the DDR pathways, limiting cell cycle progression in the presence of DNA damage [229,230]. Both biochemical and genetic evidences suggest that CHK1, an important downstream kinase of the PI3Ks, is a key mediator of both G1/S and G2/M checkpoints. In p53-deficient cells, which are incapable to sustain the G1/S checkpoint arrest, loss of CHK1 function sensitizes cells to the DNA damage [189]. CHK1 has been recently identified as the target of the staurosporine analog UCN-01, which

causes the abrogation of the G2/M checkpoint activation in response to IR in p53

-/- cells [231]. Thus, targeting the

CHK1-related G1/S and G2/M checkpoint activation would possibly lead to preferential sensitization of p53 mutant can-cer cells to genotoxic agents [189,232]. UCN-01 potentiates the cytotoxicity of a variety of anti-cancer agents, including IR, cisplatin, and camptothecin [233-235]; this encouraged the investigation of UCN-01 in combination therapy for can-cer treatment [207,234-241]. Compared with staurosporine, UCN-01 is a more selective CHK1 inhibitor but less potent against the cyclin-dependent kinase 1 (CDK1) [242]. Hydro-phobic and hydrogen-bonding interactions occur in the CHK1:UCN-01 complex, highlighting the high en-zyme:inhibitor structural complementarity. In particular, a hydroxyl group of the lactam moiety of UCN-01 interacts with the CHK1 ATP-binding pocket (PDB ID:1NVQ) [243].

Three UCN-01 structurally-related compounds, namely Gö6976, SB-218078, and isogranulatimide, inhibit CHK1 activity [244-247]; however their selectivity towards CHK1 and CHK2 is openly debated [189]. Although SB-218078 and UCN-01 bind at the same pocket of CHK1, both the CHK1:SB-218078 and the CHK1:UCN-01 complexes differ in the conformation of residues Glu91, Phe93, Glu134, Ser147, and Asp148. Remarkably, although different hydro-gen bonds stabilize the CHK1:SB-218078 and CHK1:UCN-01 complexes (PDB ID: 1NVS) [243], the IC50 values are similar [248].

Clinical studies aimed at the evaluation of the therapeutic potential of several CHK1 and CHK2 inhibitors (i.e., PD-321852, AZD7762, PF-00477736, XL-844 (also named EXEL-9844), IC-83 (also named LY2603618), and CEP-3891) have been conducted or are in progress [232,249-252]. In particular, CEP-3891 has been reported as a potent and selective CHK1 inhibitor [253,254].

Two other important classes of CHK1 inhibitors are ureas and indolinones, both showing potent inhibitory activ-ity towards CHK1 [255,256].

N-aryl-N’-pyrazinylureas have been discovered to be a

new class of CHK1 inhibitors, able to abrogate the G2/M checkpoint [255,257 260]. Starting from the lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl) urea, the R

4 position of the urea phenyl ring was modified

with several chemical structures, thus synthesizing a variety of N-aryl-N

’-pyrazinylureas [260]. Each compound so ob-

tained was analyzed to verify possible improvements of physical properties such as polarity and solubility, while keeping similar potency. In 15 of 41 compounds the enzy-matic activity reported was less than 20 nM (IC50: 3 - >10,000). The compound N-(2-chloro-4-(3-(5-cyanopyrazin-2-yl)ureido)-5-methoxyphenyl)-2-(dimethylamino)acetamide, carrying a highly hydrophobic tertiary aliphatic amine, shows the highest affinity (IC50 = 3 nM). The compound N-(2-chloro-4-(3-(5-cyanopyrazin-2-yl)ureido)-5-methoxyphenyl) isonicotinamide shows very good results throughout the en-zymatic and cellular assays, as it abrogates doxorubicin-induced cell cycle arrest (IC50 = 1,700 nM) and enhanced doxorubicin cytotoxicity (IC50 = 440 nM), while displaying no single agent activity [260].

Among indolinones, analogs of 3-ethylidene-1,3-dihydro-indol-2-one were synthesized and tested in vitro for


their inhibitory activities. The most promising compound is the analog 6-(4-hydroxy-3-methoxyphenyl)-3-(1H-pyrrolo [2,3-b]pyridin-3-ylmethylene)-1,3-dihydro-indol-2-one which possess potent inhibitory and cytotoxic activities [256]. This compound shows also potent anti-proliferative activity in the presence of doxorubicin (EC50 = 180 nM) [256]. To under-stand how these CHK1 inhibitors interact with the enzyme, the X-ray co-crystal structure of the complex formed by the potent inhibitor (Z)-3-((1H-pyrrol-2-yl)methylene)-6-(4-hydroxy-3-methoxyphenyl)indolin-2-one and CHK1 was determined. The inhibitor 3’-OMe group is hydrogen bonded to Lys38, and the inhibitor 4’-OH functionality forms a hy-drogen bond with Glu55. Furthermore, the hinge region of the inhibitor forms two additional hydrogen bonds with Glu85 and Cys87 (PDB ID: 2AYP) [256].

Because CHK2 can mediate G1/S and G2/M phase arrest and can regulate the apoptotic response [170], it has been hypothesized that CHK2 inhibition may sensitizes p53-deficient tumor cells to genotoxic agents while transiently protecting normal cells through blocking their apoptotic re-sponse [246]. Several selective inhibitors of CHK2 have been reported, including CEP-6367 [253], a synthetic ana-logue of the marine sponge metabolite hymenialdisine [261], and a series of 2-arylbenzimidazoles [262]. In particular, 2-(3-(3,4-dichlorophenoxy)phenyl)-1H-benzo[d]imidazole-5-carboxamide is an ATP-competitive inhibitor of CHK2 that protects human CD4

+ and CD8

+ T-cells from IR-induced

apoptosis [262].

In conclusion, CHK1 and CHK2 inhibitors might be suit-able to selectively treat tumors with a p53-mutant back-ground, which occurs in almost half of the malignant tumors [232].

3.4. Strategies for Targeting the BRCA Pathway

Although there are many examples of DDR loss in cancer cells, the loss of BRCA1 is of particular interest due to its correlation with breast and ovarian cancer. Tumors arising in BRCA1 mutation carriers have generally lost the wild-type allele, thus being unable to express a functional BRCA1 pro-tein. Therefore, BRCA-tumors are characterized by dysfunc-tional BRCA1-dependent events, leading to a tumor-specific dysfunction in the DSB repair by HR. The essential role of BRCA1 in the DDR, both as mediator of HR repair and in cell cycle control, highlights strong differences in the ability to repair DSBs by HR between normal and tumoral cells. Therefore, BRCA1 may be considered as a primary target in the development of new therapeutic strategies.

The identification of small molecules that disrupt protein-protein interactions in a potent, specifc and reproducible manner represent a significant challenge in cancer therapy. Indeed, inhibitors impairing the binding of the BRCT do-mains of BRCA1 to phosphorylated proteins (e.g., Abraxas, BACH1, and CtIP) implicated in DDR may be useful to sen-sitise tumors to chemotherapeutic agents [263,264]. Syn-thetic tetrapeptides sharing the pSer-Xxx-Xxx-Phe motif derived from the C-terminal region of Abraxas, BACH1, and CtIP have been reported to bind BRCT domains; pSer-Pro-Thr-Phe shows the highest affinity (IC50 = 1.4 10

4 nM)

[263]. Moreover, non-peptidic compounds have been re-ported to inhibit the interaction of BRCT with the phos-

phorylated peptide Ser-Arg-Ser-Thr-pSer-Pro-Thr-Phe-Asn-Lys derived from the C-terminus of BACH1; the highest potency was shown by NCGC00094849-01 (IC50 ~ 6 10

3

nM) [264]. However, none of these small-molecules inhibi-tors have been tested in clinical trials. Currently, no other specific BRCA1 inhibitors have been discovered.

Two classes of drugs are considered fundamental in the treatment of BRCA-associated tumors, i.e. the inhibitors of poly(ADP-ribose) polymerase (PARP) and the platinum-based DNA cross-linking agents. The PARP enzyme is a key protein in the SSB repair by the BER pathway. In the ab-sence of PARP activity, these lesions are thought to be con-verted into DSBs. On the contrary, cisplatin and carboplatin mostly induce interstrand and intrastrand cross-links, the processing of these lesions resulting in intermediates whose resolution depends on intact HR.

3.4.1. The Synthetic Lethality Model

A synthetic lethal interaction between two genes occurs when a mutation in a single gene is compatible with viabil-ity, but mutations in both genes lead to death [265,266]. Syn-thetic lethality has been proposed as a potential therapeutic approach for cancer treatment, especially when a defect in a tumor suppressor gene is known [267]. Exploiting this con-cept, the DNA repair enzyme PARP has been suggested to be a synthetic lethal partner of both BRCA1 and BRCA2, highlighting the possibility to develop a highly selective anti-cancer therapy [268,269].

PARP1 and PARP2 are members of the PARP protein superfamily, catalyzing the polymerization (“poly ADP ribosylation”) of ADP-ribose units from NAD

+ molecules on

target proteins [270-272]. PARP1 is involved in the BER pathway, by detecting and binding to SSBs [273]. The PARP1-SSB complex formation results in the poly ADP-ribosylation of PARP1 and of other proteins such as the X-ray repair cross complementing protein 1 (XRCC1) [274]. While the poly ADP-ribosylation of XRCC1 is thought to initiate BER, the poly ADP-ribosylation of the histones H1 and H2B most probably aids the access of repair proteins to DNA by enabling the relaxation of the chromatin structure. It is also possible that the poly ADP-ribosylation of proteins at the DNA damage site provides a local source of energy to enable repair, in the form of ATP [275]. When PARP1 is deficient or absent in cells, BER efficiency is greatly reduced [276]. Indeed, inhibition of PARP1 may lead to decreased BER repair and increased production of SSBs throughout the cell cycle [277, 278]. In this context, during the S phase, an accumulation of SSBs induces the formation of DNA repli-cation fork arrest, giving rise to DSBs which requires HR for repair. Inhibition of PARP1 in a cell incapable of HR (due to mutations in BRCA1 or BRCA2) forces the cell to repair the damaged DNA by means of error-prone repair pathways like NHEJ and single-strand annealing [269]. The inhibition of PARP1 by RNA interference and of both PARP1 and PARP2 by low molecular weight inhibitors is able to selec-tively kill BRCA1 and BRCA2 homozygous mutated cells [268,269]. Since both wild-type and heterozygous BRCA1 and BRCA2 cells show minimal effects after inhibitor treat-ment [268,269], this therapeutic strategy seems to be quite specific for homozygous mutated cells only [172]. Therefore, inhibition of PARP should cause synthetic lethality along


with defects in essential components of the DNA repair pathway, as it would cause the persistence of DNA lesions.

Recently, some low molecular weight inhibitors of PARP1, based on nicotinamide analogs, have been character-ized [279,280]. In the context of cancer treatment, PARP inhibitors were initially proposed as chemo-sensitizers. In fact, PARP1 inhibitors can sensitize tumor cells to cytotoxic therapies based on IR treatment and temozolomide, topoi-somerase I inhibitors, and platinum administration [172,207,281-288]. In particular, the PARP1 inhibitor AG014699 has been tested in monotherapy (phase II study), and co-administered with either temozolomide or topotecan (preclinical, phase I and II studies) [289-291]. Remarkably, the demonstration of PARP inhibitor selectivity towards BRCA homozygous mutated cells has now led to clinical trials testing these agents in the treatment of BRCA-associated cancers [292]. Phase II clinical trials testing the KU-0059436 (also named Olaparib or AZD2281) PARP-1 inhibitor are in progress [207,292,293]. Phase I and II clini-cal trials aimed to test ABT-888 efficacy in cancer treatment, both in monotherapy and in combination with several agents, have been carried out or are in progress [207].

Recently, a novel series of 2-phenyl-2H-indazole-7-carboxamides as PARP1 and PARP2 inhibitors has been developed and optimised to improve enzyme and cellular activity. The compound MK-4827, which displays excellent PARP1 and PARP2 inhibition (IC50 = 3.8 and 2.1 nM, re-spectively), is currently tested in phase I clinical trials [294].

A number of other clinical oncology trials testing PARP inhibitors, such as BSI-201, CEP-9722, GPI 21016, and INO-1001 (per se or in combined therapy) are planned or in progress [207,241,285,295].

3.4.2. The Topoisomerase II Inhibitors

During replication, the unwinding of DNA may cause the formation of tangling structures, such as supercoils or catenanes. The major role of topoisomerases is to prevent the DNA tangling, by catalyzing and guiding the unknotting of DNA, with the creation of transient breaks in the nucleic acid [296]. In particular, topoisomerase II produces transient DSBs, passes another unbroken DNA strand through it, and then re-anneals the cutted strand in an ATP-dependent man-ner [296].

BRCA1 and BRCA2 homozygous mutated cells display increased sensitivity to the topoisomerase II inhibitor etoposide [297], a chemotherapeutic agent commonly used for the treatment of multiple tumor types [298]. Etoposide blocks the re-ligation step by binding covalently the enzyme and creating a “frozen cleavable complex” [299]. The sensi-tivity of the BRCA1- and BRCA2-mutated cells to etoposide is lost after pre-treatment with aclarubicin, a compound in-hibiting the topoisomerase II-dependent formation of the DSBs [297]. This suggests that the sensitivity of BRCA ho-mozygous mutated cells is dependent on the etoposide-induced DSBs, suggesting a specific role of this anti-cancer agent in BRCA-mutated tumors [19].

3.4.3. Cross-Linking and Alkylating Agents

Since BRCA1- and BRCA2-mutated cells are character-ized by defects of the DSBs HR repair mechanism, it is

not

surprising that preclinical models demonstrated that these

cells show a particular sensitivity towards DNA cross-linking

agents (e.g., cisplatin, carboplatin, melphalan, mito-

mycin-C and nimustine) [300-304]. These agents cause ge-nomic lesions that lead to a collapse of the DNA replication forks, which subsequently require the DSBs repair by HR [303,305].

BRCA1 appears a major target of cisplatin and perhaps

of other chemotherapeutic DNA damaging agents. Accord-

ingly, cell lines carrying a BRCA1 mutation are more sensi-

tive to cisplatin treatment than wild-type cells [306]. Re-

cently, it has been reported that the treatment of thirty pa-

tients with the neo-adjuvants epirubicin, cisplatin, and infu-

sional fluoracil results in the complete pathologic response in

almost twelve patients [307].

A clinical trial aimed to asses the efficacy of carboplatin

in the treatment of tumors in BRCA1 and BRCA2 heterozy-

gote patients is underway [303]. Indeed, carboplatin is syn-

ergistic with cetiximab in the treatment of basal-like breast

cancer cell lines [308].

Although breast and ovarian BRCA1-mutated cancers

may become resistant to platinum therapy, phase III clinical

trials are currently investigating platinums in the neo-

adjuvant setting and in the treatment of metastatic triple-

negative breast cancer [309].

The alkylating agent trabectedin (also named YondelisTM

or ET 743), an anti-tumor agent isolated from Ecteinascidia

turbinate, has been selected for clinical investigations due to

its potent cytotoxic activity against a variety of tumor cell

lines [310] and human tumor xenografts in vivo in combina-

tion with cisplatin [311]. Currently, trabectedin is undergo-

ing phase I, II, and III clinical oncological trials, both in

monotherapy and in combination with other agents, with

promising results for the treatment of soft tissue sarcomas,

and breast and ovarian cancers [207,312-314]. The current

model that explains the trabectedin mechanism of action

suggests the following sequence of four events: (i) trabecte-

din binds covalently to the DNA minor groove and the ad-

duct is recognized by the NER system; (ii) the recruited ex-

cision repair cross-complementing rodent repair deficiency-

complementation group 5 protein (ERCC5) binds to the

DNA and interacts with the minor groove-bound drug; (iii)

the excision repair cross-complementing rodent repair defi-

ciency-complementation group 1 protein (ERCC1):

xeroderma pigmentosum-complementation group F (XPF)

complex localizes on the DNA and, together with proteins of

other DNA repair pathways that try to correct the DNA le-

sions, are hijacked at the sites of damage, creating stronger

cytotoxic complexes; (iv) during the S phase of the cell cy-

cle, these complexes give rise to DSBs that need to be re-

paired by HR [315]. This implies that HR-proficient cells

can repair the damage, whereas those defective in one or

more HR proteins (like the breast and ovarian BRCA-

associated cancer cells) are sensitive to the trabectidin action

[315]. Thus, patients exhibiting an intact NER pathway but

deficient in the HR repair mechanism, result more sensitive

to the trabectedin treatment [315,316].


4. CONCLUSION AND PERSPECTIVES

Significant advances have been made recently in the elu-cidation of the molecular mechanisms of the DSBs damage response to IR, carcinogens, and environmental factors. The importance of a robust DNA-damage surveillance network is evidenced by the fact that defects in signaling and repair of DNA damage are linked to the development of inherited chromosome instability syndromes and cancer onset. Moreo-ver, epidemiological studies evidenced that also heterozy-gous carriers of both polymorphisms and mutations within DDR genes are characterized by a high cancer risk. The pos-sibility to specifically interfere with the DSBs repair mecha-nisms for the development of novel and highly specific therapies may have a significant clinical impact.

For the future there is a need to develop strategies for a rapid identification of the defect in DSBs sensing and/or repair mechanisms responsible for cancer development, in order to identify the proper pathway-specific therapeutic strategy. Indeed, by using anti-cancer agents specifically toxic for cancer cells, it could be possible to develop specific therapies able to kill tumoral cells, reducing the overall cito-toxic effects.

The use of DSBs inhibitors within clinical radiotherapy is expected to improve patient outcomes by exploiting preci-sion-guided radiotherapy. In order to maximize the therapeu-tic effect of DSBs inhibitors with a clinical course of radio-therapy, it is important to establish a rational scheduling re-gime that accounts for inhibitor pharmacodynamics, provid-ing optimal radiosensitization [317]. To optimize the “thera-peutic ratio” it will be also important to determine whether the inhibitor should be given throughout the entire course of radiotherapy or rather only during the latter weeks of radia-tion treatment, with the intent of eradicating any remaining radioresistant populations of tumors cells [317]. However, IR, alkylating agents and topoisomerase II inhibitors may induce secondary tumors several years after exposure, due to the mutagenic DNA damage that they induced to normal cells. Therefore, addition of a DNA repair inhibitor to such therapies may enhance the risk of mutagenic damage, and hence the risk of secondary tumors. This implies that addi-tional clinical studies need to be performed in order to evalu-ate the long-term effect of combined therapy using already tested, and novel inhibitors of the DSBs response pathway.

Important questions remain about the best biomarkers to use in clinical trials using DNA repair inhibitors. The opti-mal timing of the biomarkers is also unknown when com-bined with radiotherapy as it depends on the pharmacology of the agent, the dose of radiotherapy, and the accessibility of tumor and normal tissues during a clinical trial [317].

Remarkably, a particular attention should be focused on possible gender differences in radiotherapeutic and che-motherapeutic treatments. Indeed, the lower DNA repair capacity in females may influence DDR in tumor cells after exposure to cytotoxic cancer therapy, and determine the ac-cumulation of mutations in normal cells [318].

Lastly, since both homozygous and heterozygous carriers of mutations within genes involved in DNA sensing, repair-ing, and in the cell cycle control, need precaution regarding any diagnostic and therapeutic exposure to IR, the adoption

of appropriate radiodiagnostic and radiotherapeu-tic/chemotherapeutic protocols results of significant impor-tance.

ACKNOWLEDGEMENTS

Authors wish to thank Dr. Antonio Antoccia, Prof. Caterina Tanzarella, and Prof. Maria Marino for helpful dis-cussions. This work was partially supported by grants from the Italian Ministry of Education, University and Research (CLAR 2009 to A.d.M. and to P.A.) and from the Italian Ministry of Health (National Institute for Infectious Diseases I.R.C.C.S. ”Lazzaro Spallanzani”, Roma, Italy, Ricerca cor-rente 2008 to P.A.).

ABBREVIATIONS

53BP1 = p53 binding protein 1

AT = Ataxia telangiectasia

ATM = Ataxia telangiectasia mutated protein

ATR = Ataxia telangiectasia and RAD3-related pro-tein

BACH1 = BTB and CNC homology 1 protein

BAP1 = BRCA1 associated protein 1

BARD1 = BRCA1 associated RING domain 1

BAX = BCL2-associated protein

BER = base excision repair

BRCA = breast cancer associated protein

BRCA1 = breast cancer associated protein 1

BRCA2 = breast cancer associated protein 2

BRCT = BRCA1 carboxy-terminal domain

CDC25A = cell division cycle 25 homolog A protein

CDK1 = cyclin-dependent kinase 1

CDKN1A = cyclin-dependent kinase inhibitor 1A

CHK1 = checkpoint homolog kinase 1 protein

CHK2 = checkpoint homolog kinase 2 protein

CtIP = CTBP interacting protein

DDB2 = damage-specific DNA-binding 2 protein

DDR = DNA damage response

DSB = DNA double strand break

DNA-PK = DNA-dependent protein kinase

DNA- = DNA-dependent protein kinase catalytic

PKcs subunit

ERCC1 = excision repair cross-complementing rodent repair deficiency- complementation group 1 protein

ERCC5 = excision repair cross-complementing rodent repair deficiency- complementation group 5 protein

GADD45 = growth-arrest and DNA-damage inducible 45 protein


H2AX = H2A histone family member X

-H2AX = H2A histone family member X phosphory-lated on Ser139

HAT = histone acetyltransferases

HDAC = histone deacetylase

HDACI = HDAC inhibitor

HJ = Holliday junction

HR = homologous recombination

IR = ionizing radiation

IRIF = ionizing radiation induced foci

LIG4 = ligase 4

MDC1 = mediator of DNA-damage checkpoint 1 pro-tein

MMR = DNA mismatch repair

MRE11 = meiotic recombination 11 protein

MRN = MRE11/RAD50/NBN complex

NBS = Nijmegen breakage syndrome

NBN = Nijmegen breakage syndrome mutated pro-tein

NHEJ = non-homologous end-joining

NER = nucleotide excision repair

pSer = phosphorylated serine

PARP = poly(ADP-ribose)polymerase

PI3K = phosphoinositide-3-kinase

PIKK = phosphoinositide-3-kinase related kinase

PTEN = phosphatase and tensin homolog

RB = retinoblastoma protein

RPA = replication protein A

SAHA = suberoylanilide hydroxamic acid

SSB = DNA single strand break

SMC1 = structural maintenance of chromosomes 1 protein

SS = Seckel syndrome

ssDNA = single strand DNA

TRRAP = transactivation-transformation domain-associated protein

TSA = Thricostatin A

UV = ultraviolet

XPC = xeroderma pigmentosum-complementation group C protein

XPF = xeroderma pigmentosum-complementation group F protein

XRCC1 = X-ray repair cross complementing 1 protein

XRCC4 = X-ray repair cross complementing 4 protein

REFERENCES

[1] Humpal, S.E.; Robinson, D.A.; Krebs, J.E. Marks to stop the clock:

histone modifications and checkpoint regulation in the DNA dam-

age response. Biochem. Cell Biol., 2009, 87, 243-53.

[2] Kaufmann, W.K.; Paules, R.S. DNA damage and cell cycle check-

points. FASEB J., 1996, 10, 238-47.

[3] O'Driscoll, M.; Jeggo, P.A. The role of double-strand break repair -

insights from human genetics. Nat. Rev. Genet., 2006, 7, 45-54.

[4] Sancar, A.; Lindsey-Boltz, L.A.; Unsal-Kacmaz, K.; Linn, S. Mo-

lecular mechanisms of mammalian DNA repair and the DNA dam-

age checkpoints. Annu. Rev. Biochem., 2004, 73, 39-85.

[5] Madhusudan, S.; Middleton, M.R. The emerging role of DNA

repair proteins as predictive, prognostic and therapeutic targets in

cancer. Cancer Treat. Rev., 2005, 31, 603-17.

[6] Hoeijmakers, J.H. Genome maintenance mechanisms for prevent-

ing cancer. Nature, 2001, 411, 366-74.

[7] di Masi, A.; Beradinelli, F.; Antoccia, A. In Genetic Inheritance

Patterns; Ren Kimura, Ed.; Nova Science Publishers: Hauppauge

NY, 2008; Vol. 21-73.

[8] Bannon, J.H.; Mc Gee, M.M. Understanding the role of aneuploidy

in tumorigenesis. Biochem. Soc. Trans., 2009, 37, 910-3.

[9] Weaver, B.A.; Cleveland, D.W. Aneuploidy: instigator and inhibi-

tor of tumorigenesis. Cancer Res., 2007, 67, 10103-5.

[10] O'Driscoll, M.; Cerosaletti, K.M.; Girard, P.M.; Dai, Y.; Stumm,

M.; Kysela, B.; Hirsch, B.; Gennery, A.; Palmer, S.E.; Seidel, J.;

Gatti, R.A.; Varon, R.; Oettinger, M.A.; Neitzel, H.; Jeggo, P.A.;

Concannon, P. DNA ligase IV mutations identified in patients ex-

hibiting developmental delay and immunodeficiency. Mol. Cell,

2001, 8, 1175-85.

[11] Ben Omran, T.I.; Cerosaletti, K.; Concannon, P.; Weitzman, S.;

Nezarati, M.M. A patient with mutations in DNA Ligase IV: clini-

cal features and overlap with Nijmegen breakage syndrome. Am. J.

Med. Genet. A, 2005, 137A, 283-7.

[12] Altieri, F.; Grillo, C.; Maceroni, M.; Chichiarelli, S. DNA damage

and repair: from molecular mechanisms to health implications. An-

tioxid. Redox. Signal., 2008, 10, 891-937.

[13] Chistiakov, D.A.; Voronova, N.V.; Chistiakov, P.A. Genetic varia-

tions in DNA repair genes, radiosensitivity to cancer and suscepti-

bility to acute tissue reactions in radiotherapy-treated cancer pa-

tients. Acta Oncol., 2008, 47, 809-24.

[14] O'Driscoll, M.; Jeggo, P.A. The role of the DNA damage response

pathways in brain development and microcephaly: insight from

human disorders. DNA Repair (Amst), 2008, 7, 1039-50.

[15] Bender, C.F.; Sikes, M.L.; Sullivan, R.; Huye, L.E.; Le Beau,

M.M.; Roth, D.B.; Mirzoeva, O.K.; Oltz, E.M.; Petrini, J.H. Cancer

predisposition and hematopoietic failure in Rad50(S/S) mice.

Genes Dev., 2002, 16, 2237-51.

[16] Ahmed, M.; Rahman, N. ATM and breast cancer susceptibility.

Oncogene, 2006, 25, 5906-11.

[17] Ralhan, R.; Kaur, J.; Kreienberg, R.; Wiesmuller, L. Links between

DNA double strand break repair and breast cancer: accumulating

evidence from both familial and nonfamilial cases. Cancer Lett.,

2007, 248, 1-17.

[18] di Masi, A.; Antoccia, A. NBS1 heterozygosity and cancer risk.

Curr. Genom., 2008, 9, 275-81.

[19] Powell, S.N.; Bindra, R.S. Targeting the DNA damage response for

cancer therapy. DNA Repair (Amst), 2009, 8, 1153-65.

[20] Schmith, V.D.; Campbell, D.A.; Sehgal, S.; Anderson, W.H.;

Burns, D.K.; Middleton, L.T.; Roses, A.D. Pharmacogenetics and

disease genetics of complex diseases. Cell Mol. Life Sci., 2003, 60,

1636-46.

[21] Jeggo, P.A.; Lobrich, M. Contribution of DNA repair and cell cycle

checkpoint arrest to the maintenance of genomic stability. DNA

Repair (Amst), 2006, 5, 1192-8.

[22] Shrivastav, M.; De Haro, L.P.; Nickoloff, J.A. Regulation of DNA

double-strand break repair pathway choice. Cell Res., 2008, 18,

134-47.

[23] Bernstein, K.A.; Rothstein, R. At loose ends: resecting a double-

strand break. Cell, 2009, 137, 807-10.

[24] Pardo, B.; Gomez-Gonzalez, B.; Aguilera, A. DNA repair in

mammalian cells: DNA double-strand break repair: how to fix a

broken relationship. Cell Mol. Life Sci., 2009, 66, 1039-56.

[25] Jeggo, P.A.; Lobrich, M. DNA double-strand breaks: their cellular

and clinical impact? Oncogene, 2007, 26, 7717-9.

[26] Shin, D.S.; Chahwan, C.; Huffman, J.L.; Tainer, J.A. Structure and

function of the double-strand break repair machinery. DNA Repair

(Amst), 2004, 3, 863-73.


[27] Mahaney, B.L.; Meek, K.; Lees-Miller, S.P. Repair of ionizing

radiation-induced DNA double-strand breaks by non-homologous

end-joining. Biochem. J., 2009, 417, 639-50.

[28] Ward, J.F. The complexity of DNA damage: relevance to biologi-

cal consequences. Int. J. Radiat. Biol., 1994, 66, 427-32.

[29] van Gent, D.C.; Hoeijmakers, J.H.; Kanaar, R. Chromosomal sta-

bility and the DNA double-stranded break connection. Nat. Rev.

Genet., 2001, 2, 196-206.

[30] Povirk, L.F. Biochemical mechanisms of chromosomal transloca-

tions resulting from DNA double-strand breaks. DNA Repair

(Amst), 2006, 5, 1199-212.

[31] Natarajan, A.T.; Palitti, F. DNA repair and chromosomal altera-

tions. Mutat. Res., 2008, 657, 3-7.

[32] Pierce, A.J.; Stark, J.M.; Araujo, F.D.; Moynahan, M.E.; Berwick,

M.; Jasin, M. Double-strand breaks and tumorigenesis. Trends Cell

Biol., 2001, 11, S52-S59.

[33] Sakata, K.; Someya, M.; Matsumoto, Y.; Hareyama, M. Ability to

repair DNA double-strand breaks related to cancer susceptibility

and radiosensitivity. Radiat. Med., 2007, 25, 433-8.

[34] Paull, T.T.; Rogakou, E.P.; Yamazaki, V.; Kirchgessner, C.U.;

Gellert, M.; Bonner, W.M. A critical role for histone H2AX in re-

cruitment of repair factors to nuclear foci after DNA damage. Curr.

Biol., 2000, 10, 886-95.

[35] Rouse, J.; Jackson, S.P. Interfaces between the detection, signaling,

and repair of DNA damage. Science, 2002, 297, 547-51.

[36] Kobayashi, J.; Iwabuchi, K.; Miyagawa, K.; Sonoda, E.; Suzuki,

K.; Takata, M.; Tauchi, H. Current topics in DNA double-strand

break repair. J. Radiat. Res. (Tokyo), 2008, 49, 93-103.

[37] Stewart, G.S.; Wang, B.; Bignell, C.R.; Taylor, A.M.; Elledge, S.J.

MDC1 is a mediator of the mammalian DNA damage checkpoint.

Nature, 2003, 421, 961-6.

[38] Rai, R.; Dai, H.; Multani, A.S.; Li, K.; Chin, K.; Gray, J.; Lahad,

J.P.; Liang, J.; Mills, G.B.; Meric-Bernstam, F.; Lin, S.Y. BRIT1

regulates early DNA damage response, chromosomal integrity, and

cancer. Cancer Cell, 2006, 10, 145-57.

[39] Petrini, J.H.; Stracker, T.H. The cellular response to DNA double-

strand breaks: defining the sensors and mediators. Trends Cell

Biol., 2003, 13, 458-62.

[40] Kuo, L.J.; Yang, L.X. Gamma-H2AX - a novel biomarker for DNA

double-strand breaks. In Vivo, 2008, 22, 305-9.

[41] Rogakou, E.P.; Boon, C.; Redon, C.; Bonner, W.M. Megabase

chromatin domains involved in DNA double-strand breaks in vivo.

J. Cell Biol., 1999, 146, 905-16.

[42] Shroff, R.; Arbel-Eden, A.; Pilch, D.; Ira, G.; Bonner, W.M.;

Petrini, J.H.; Haber, J.E.; Lichten, M. Distribution and dynamics of

chromatin modification induced by a defined DNA double-strand

break. Curr. Biol., 2004, 14, 1703-11.

[43] Berkovich, E.; Monnat, R.J., Jr.; Kastan, M.B. Roles of ATM and

NBS1 in chromatin structure modulation and DNA double-strand

break repair. Nat. Cell Biol., 2007, 9, 683-90.

[44] Scully, R.; Chen, J.; Ochs, R.L.; Keegan, K.; Hoekstra, M.; Feun-

teun, J.; Livingston, D.M. Dynamic changes of BRCA1 subnuclear

location and phosphorylation state are initiated by DNA damage.

Cell, 1997, 90, 425-35.

[45] Carney, J.P.; Maser, R.S.; Olivares, H.; Davis, E.M.; Le Beau, M.;

Yates, J.R., III; Hays, L.; Morgan, W.F.; Petrini, J.H. The

hMre11/hRad50 protein complex and Nijmegen breakage syn-

drome: linkage of double-strand break repair to the cellular DNA

damage response. Cell, 1998, 93, 477-86.

[46] Zhong, Q.; Chen, C.F.; Li, S.; Chen, Y.; Wang, C.C.; Xiao, J.;

Chen, P.L.; Sharp, Z.D.; Lee, W.H. Association of BRCA1 with

the hRad50-hMre11-p95 complex and the DNA damage response.

Science, 1999, 285, 747-50.

[47] Rappold, I.; Iwabuchi, K.; Date, T.; Chen, J. Tumor suppressor p53

binding protein 1 (53BP1) is involved in DNA damage-signaling

pathways. J. Cell Biol., 2001, 153, 613-20.

[48] Celeste, A.; Petersen, S.; Romanienko, P.J.; Fernandez-Capetillo,

O.; Chen, H.T.; Sedelnikova, O.A.; Reina-San-Martin, B.; Cop-

pola, V.; Meffre, E.; Difilippantonio, M.J.; Redon, C.; Pilch, D.R.;

Olaru, A.; Eckhaus, M.; Camerini-Otero, R.D.; Tessarollo, L.; Li-

vak, F.; Manova, K.; Bonner, W.M.; Nussenzweig, M.C.;

Nussenzweig, A. Genomic instability in mice lacking histone

H2AX. Science, 2002, 296, 922-7.

[49] Goldberg, M.; Stucki, M.; Falck, J.; D'Amours, D.; Rahman, D.;

Pappin, D.; Bartek, J.; Jackson, S.P. MDC1 is required for the in-

tra-S-phase DNA damage checkpoint. Nature, 2003, 421, 952-6.

[50] van den, B.M.; Bree, R.T.; Lowndes, N.F. The MRN complex:

coordinating and mediating the response to broken chromosomes.

EMBO Rep., 2003, 4, 844-9.

[51] Xu, X.; Stern, D.F. NFBD1/MDC1 regulates ionizing radiation-

induced focus formation by DNA checkpoint signaling and repair

factors. FASEB J., 2003, 17, 1842-8.

[52] Lisby, M.; Rothstein, R. DNA damage checkpoint and repair cen-

ters. Curr. Opin. Cell Biol., 2004, 16, 328-34.

[53] Helmink, B.A.; Bredemeyer, A.L.; Lee, B.S.; Huang, C.Y.;

Sharma, G.G.; Walker, L.M.; Bednarski, J.J.; Lee, W.L.; Pandita,

T.K.; Bassing, C.H.; Sleckman, B.P. MRN complex function in the

repair of chromosomal Rag-mediated DNA double-strand breaks.

J. Exp. Med., 2009, 206, 669-79.

[54] Porter-Goff, M.E.; Rhind, N. The role of MRN in the S-phase DNA

damage checkpoint is independent of its Ctp1-dependent roles in

double-strand break repair and checkpoint signaling. Mol. Biol.

Cell, 2009, 20, 2096-107.

[55] Shikazono, N.; Noguchi, M.; Fujii, K.; Urushibara, A.; Yokoya, A.

The yield, processing, and biological consequences of clustered

DNA damage induced by ionizing radiation. J. Radiat. Res. (To-

kyo), 2009, 50, 27-36.

[56] Bakkenist, C.J.; Kastan, M.B. DNA damage activates ATM

through intermolecular autophosphorylation and dimer dissocia-

tion. Nature, 2003, 421, 499-506.

[57] Kitagawa, R.; Bakkenist, C.J.; McKinnon, P.J.; Kastan, M.B.

Phosphorylation of SMC1 is a critical downstream event in the

ATM-NBS1-BRCA1 pathway. Genes Dev., 2004, 18, 1423-38.

[58] Lowndes, N.F.; Murguia, J.R. Sensing and responding to DNA

damage. Curr. Opin. Genet. Dev., 2000, 10, 17-25.

[59] Sionov, R.V.; Haupt, Y. The cellular response to p53: the decision

between life and death. Oncogene, 1999, 18, 6145-57.

[60] Vakifahmetoglu, H.; Olsson, M.; Zhivotovsky, B. Death through a

tragedy: mitotic catastrophe. Cell Death Differ., 2008, 15, 1153-62.

[61] Frosina, G. DNA repair and resistance of gliomas to chemotherapy

and radiotherapy. Mol. Cancer Res., 2009, 7, 989-99.

[62] Chang, B.D.; Watanabe, K.; Broude, E.V.; Fang, J.; Poole, J.C.;

Kalinichenko, T.V.; Roninson, I.B. Effects of p21Waf1/Cip1/Sdi1

on cellular gene expression: implications for carcinogenesis, senes-

cence, and age-related diseases. Proc. Natl. Acad. Sci. U.S.A.,

2000, 97, 4291-6.

[63] Bartkova, J.; Horejsi, Z.; Koed, K.; Kramer, A.; Tort, F.; Zieger,

K.; Guldberg, P.; Sehested, M.; Nesland, J.M.; Lukas, C.; Orntoft,

T.; Lukas, J.; Bartek, J. DNA damage response as a candidate anti-

cancer barrier in early human tumorigenesis. Nature, 2005, 434,

864-70.

[64] Celeste, A.; Difilippantonio, S.; Difilippantonio, M.J.; Fernandez-

Capetillo, O.; Pilch, D.R.; Sedelnikova, O.A.; Eckhaus, M.; Ried,

T.; Bonner, W.M.; Nussenzweig, A. H2AX haploinsufficiency

modifies genomic stability and tumor susceptibility. Cell, 2003,

114, 371-83.

[65] Difilippantonio, S.; Celeste, A.; Fernandez-Capetillo, O.; Chen,

H.T.; Reina, S.M.; Van Laethem, F.; Yang, Y.P.; Petukhova, G.V.;

Eckhaus, M.; Feigenbaum, L.; Manova, K.; Kruhlak, M.; Camerini-

Otero, R.D.; Sharan, S.; Nussenzweig, M.; Nussenzweig, A. Role

of Nbs1 in the activation of the Atm kinase revealed in humanized

mouse models. Nat. Cell Biol., 2005, 7, 675-85.

[66] Kanaar, R.; Hoeijmakers, J.H.; van Gent, D.C. Molecular mecha-

nisms of DNA double strand break repair. Trends Cell Biol., 1998,

8, 483-9.

[67] Lieber, M.R.; Ma, Y.; Pannicke, U.; Schwarz, K. Mechanism and

regulation of human non-homologous DNA end-joining. Nat. Rev.

Mol. Cell Biol., 2003, 4, 712-20.

[68] Pierce, A.J.; Jasin, M. NHEJ deficiency and disease. Mol. Cell,

2001, 8, 1160-1.

[69] Rothkamm, K.; Kruger, I.; Thompson, L.H.; Lobrich, M. Pathways

of DNA double-strand break repair during the mammalian cell cy-

cle. Mol. Cell Biol., 2003, 23, 5706-15.

[70] Thompson, L.H.; Schild, D. The contribution of homologous re-

combination in preserving genome integrity in mammalian cells.

Biochimie, 1999, 81, 87-105.

[71] Haber, J.E. Partners and pathwaysrepairing a double-strand break.

Trends Genet., 2000, 16, 259-64.


[72] Thompson, L.H.; Schild, D. Homologous recombinational repair of

DNA ensures mammalian chromosome stability. Mutat. Res., 2001,

477, 131-53.

[73] Golub, E.I.; Gupta, R.C.; Haaf, T.; Wold, M.S.; Radding, C.M.

Interaction of human rad51 recombination protein with single-

stranded DNA binding protein, RPA. Nucleic Acids Res., 1998, 26,

5388-93.

[74] Sigurdsson, S.; Trujillo, K.; Song, B.; Stratton, S.; Sung, P. Basis

for avid homologous DNA strand exchange by human Rad51 and

RPA. J. Biol. Chem., 2001, 276, 8798-806.

[75] Wyman, C.; Kanaar, R. Homologous recombination: down to the

wire. Curr. Biol., 2004, 14, R629-R631.

[76] Johnson, R.D.; Jasin, M. Sister chromatid gene conversion is a

prominent double-strand break repair pathway in mammalian cells.

EMBO J., 2000, 19, 3398-407.

[77] Dvir, A.; Peterson, S.R.; Knuth, M.W.; Lu, H.; Dynan, W.S. Ku

autoantigen is the regulatory component of a template-associated

protein kinase that phosphorylates RNA polymerase II. Proc. Natl.

Acad. Sci. U.S.A., 1992, 89, 11920-4.

[78] Smith, G.C.; Jackson, S.P. The DNA-dependent protein kinase.

Genes Dev., 1999, 13, 916-34.

[79] Jackson, S.P. Sensing and repairing DNA double-strand breaks.

Carcinogenesis, 2002, 23, 687-96.

[80] Weterings, E.; van Gent, D.C. The mechanism of non-homologous

end-joining: a synopsis of synapsis. DNA Repair (Amst), 2004, 3,

1425-35.

[81] Moshous, D.; Callebaut, I.; de Chasseval, R.; Corneo, B.; Cavaz-

zana-Calvo, M.; Le Deist, F.; Tezcan, I.; Sanal, O.; Bertrand, Y.;

Philippe, N.; Fischer, A.; de Villartay, J.P. Artemis, a novel DNA

double-strand break repair/V(D)J recombination protein, is mutated

in human severe combined immune deficiency. Cell, 2001, 105,

177-86.

[82] Riballo, E.; Kuhne, M.; Rief, N.; Doherty, A.; Smith, G.C.; Recio,

M.J.; Reis, C.; Dahm, K.; Fricke, A.; Krempler, A.; Parker, A.R.;

Jackson, S.P.; Gennery, A.; Jeggo, P.A.; Lobrich, M. A pathway of

double-strand break rejoining dependent upon ATM, Artemis, and

proteins locating to gamma-H2AX foci. Mol. Cell, 2004, 16, 715-

24.

[83] Zhang, X.; Succi, J.; Feng, Z.; Prithivirajsingh, S.; Story, M.D.;

Legerski, R.J. Artemis is a phosphorylation target of ATM and

ATR and is involved in the G2/M DNA damage checkpoint re-

sponse. Mol. Cell Biol., 2004, 24, 9207-20.

[84] Ahnesorg, P.; Smith, P.; Jackson, S.P. XLF interacts with the

XRCC4-DNA ligase IV complex to promote DNA nonhomologous

end-joining. Cell, 2006, 124, 301-13.

[85] Buck, D.; Malivert, L.; de Chasseval, R.; Barraud, A.; Fondaneche,

M.C.; Sanal, O.; Plebani, A.; Stephan, J.L.; Hufnagel, M.; Le Deist,

F.; Fischer, A.; Durandy, A.; de Villartay, J.P.; Revy, P. Cernun-

nos, a novel nonhomologous end-joining factor, is mutated in hu-

man immunodeficiency with microcephaly. Cell, 2006, 124, 287-

99.

[86] Callebaut, I.; Malivert, L.; Fischer, A.; Mornon, J.P.; Revy, P.; de

Villartay, J.P. Cernunnos interacts with the XRCC4 x DNA-ligase

IV complex and is homologous to the yeast nonhomologous end-

joining factor Nej1. J. Biol. Chem., 2006, 281, 13857-60.

[87] Critchlow, S.E.; Bowater, R.P.; Jackson, S.P. Mammalian DNA

double-strand break repair protein XRCC4 interacts with DNA li-

gase IV. Curr. Biol., 1997, 7, 588-98.

[88] Grawunder, U.; Wilm, M.; Wu, X.; Kulesza, P.; Wilson, T.E.;

Mann, M.; Lieber, M.R. Activity of DNA ligase IV stimulated by

complex formation with XRCC4 protein in mammalian cells. Na-

ture, 1997, 388, 492-5.

[89] Chen, L.; Trujillo, K.; Sung, P.; Tomkinson, A.E. Interactions of

the DNA ligase IV-XRCC4 complex with DNA ends and the

DNA-dependent protein kinase. J. Biol. Chem., 2000, 275, 26196-

205.

[90] Frank, K.M.; Sharpless, N.E.; Gao, Y.; Sekiguchi, J.M.; Ferguson,

D.O.; Zhu, C.; Manis, J.P.; Horner, J.; DePinho, R.A.; Alt, F.W.

DNA ligase IV deficiency in mice leads to defective neurogenesis

and embryonic lethality via the p53 pathway. Mol. Cell, 2000, 5,

993-1002.

[91] Kurimasa, A.; Kumano, S.; Boubnov, N.V.; Story, M.D.; Tung,

C.S.; Peterson, S.R.; Chen, D.J. Requirement for the kinase activity

of human DNA-dependent protein kinase catalytic subunit in DNA

strand break rejoining. Mol. Cell Biol., 1999, 19, 3877-84.

[92] Riballo, E.; Critchlow, S.E.; Teo, S.H.; Doherty, A.J.; Priestley, A.;

Broughton, B.; Kysela, B.; Beamish, H.; Plowman, N.; Arlett, C.F.;

Lehmann, A.R.; Jackson, S.P.; Jeggo, P.A. Identification of a de-

fect in DNA ligase IV in a radiosensitive leukaemia patient. Curr.

Biol., 1999, 9, 699-702.

[93] Wachsberger, P.R.; Li, W.H.; Guo, M.; Chen, D.; Cheong, N.;

Ling, C.C.; Li, G.; Iliakis, G. Rejoining of DNA double-strand

breaks in Ku80-deficient mouse fibroblasts. Radiat. Res., 1999,

151, 398-407.

[94] Kouzarides, T. Chromatin modifications and their function. Cell,

2007, 128, 693-705.

[95] Strahl, B.D.; Allis, C.D. The language of covalent histone modifi-

cations. Nature, 2000, 403, 41-5.

[96] Taverna, S.D.; Li, H.; Ruthenburg, A.J.; Allis, C.D.; Patel, D.J.

How chromatin-binding modules interpret histone modifications:

lessons from professional pocket pickers. Nat. Struct. Mol. Biol.,

2007, 14, 1025-40.

[97] Cairns, B.R.; Lorch, Y.; Li, Y.; Zhang, M.; Lacomis, L.; Erd-

jument-Bromage, H.; Tempst, P.; Du, J.; Laurent, B.; Kornberg,

R.D. RSC, an essential, abundant chromatin-remodeling complex.

Cell, 1996, 87, 1249-60.

[98] Ikura, T.; Ogryzko, V.V.; Grigoriev, M.; Groisman, R.; Wang, J.;

Horikoshi, M.; Scully, R.; Qin, J.; Nakatani, Y. Involvement of the

TIP60 histone acetylase complex in DNA repair and apoptosis.

Cell, 2000, 102, 463-73.

[99] Symington, L.S. Role of RAD52 epistasis group genes in homolo-

gous recombination and double-strand break repair. Microbiol.

Mol. Biol. Rev., 2002, 66, 630-70.

[100] Cai, S.; Han, H.J.; Kohwi-Shigematsu, T. Tissue-specific nuclear

architecture and gene expression regulated by SATB1. Nat. Genet.,

2003, 34, 42-51.

[101] Mizuguchi, G.; Shen, X.; Landry, J.; Wu, W.H.; Sen, S.; Wu, C.

ATP-driven exchange of histone H2AZ variant catalyzed by SWR1

chromatin remodeling complex. Science, 2004, 303, 343-8.

[102] Mohrmann, L.; Verrijzer, C.P. Composition and functional speci-

ficity of SWI2/SNF2 class chromatin remodeling complexes. Bio-

chim. Biophys. Acta, 2005, 1681, 59-73.

[103] Thoma, N.H.; Czyzewski, B.K.; Alexeev, A.A.; Mazin, A.V.;

Kowalczykowski, S.C.; Pavletich, N.P. Structure of the

SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54.

Nat. Struct. Mol. Biol., 2005, 12, 350-6.

[104] Osley, M.A.; Shen, X. Altering nucleosomes during DNA double-

strand break repair in yeast. Trends Genet., 2006, 22, 671-7.

[105] Spotswood, H.T.; Turner, B.M. An increasingly complex code. J.

Clin. Invest, 2002, 110, 577-82.

[106] Fernandez-Capetillo, O.; Nussenzweig, A. Linking histone deacety-

lation with the repair of DNA breaks. Proc. Natl. Acad. Sci. U. S.

A, 2004, 101, 1427-8.

[107] Narlikar, G.J.; Fan, H.Y.; Kingston, R.E. Cooperation between

complexes that regulate chromatin structure and transcription. Cell,

2002, 108, 475-87.

[108] Costelloe, T.; Fitzgerald, J.; Murphy, N.J.; Flaus, A.; Lowndes,

N.F. Chromatin modulation and the DNA damage response. Exp.

Cell Res., 2006, 312, 2677-86.

[109] Murr, R.; Loizou, J.I.; Yang, Y.G.; Cuenin, C.; Li, H.; Wang, Z.Q.;

Herceg, Z. Histone acetylation by Trrap-Tip60 modulates loading

of repair proteins and repair of DNA double-strand breaks. Nat.

Cell Biol., 2006, 8, 91-9.

[110] Squatrito, M.; Gorrini, C.; Amati, B. Tip60 in DNA damage re-

sponse and growth control: many tricks in one HAT. Trends Cell

Biol., 2006, 16, 433-42.

[111] Sun, Y.; Jiang, X.; Chen, S.; Fernandes, N.; Price, B.D. A role for

the Tip60 histone acetyltransferase in the acetylation and activation

of ATM. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 13182-7.

[112] Robert, F.; Hardy, S.; Nagy, Z.; Baldeyron, C.; Murr, R.; Dery, U.;

Masson, J.Y.; Papadopoulo, D.; Herceg, Z.; Tora, L. The transcrip-

tional histone acetyltransferase cofactor TRRAP associates with the

MRN repair complex and plays a role in DNA double-strand break

repair. Mol. Cell Biol., 2006, 26, 402-12.

[113] Downey, M.; Durocher, D. Chromatin and DNA repair: the bene-

fits of relaxation. Nat. Cell Biol., 2006, 8, 9-10.

[114] Tsukuda, T.; Fleming, A.B.; Nickoloff, J.A.; Osley, M.A. Chroma-

tin remodelling at a DNA double-strand break site in Saccharomy-


ces cerevisiae. Nature, 2005, 438, 379-83.

[115] Ausio, J.; Levin, D.B.; De Amorim, G.V.; Bakker, S.; Macleod,

P.M. Syndromes of disordered chromatin remodeling. Clin. Genet.,

2003, 64, 83-95.

[116] Cho, B.P. Dynamic conformational heterogeneities of carcinogen-

DNA adducts and their mutagenic relevance. J. Environ. Sci.

Health C. Environ. Carcinog. Ecotoxicol. Rev., 2004, 22, 57-90.

[117] Miki, Y.; Swensen, J.; Shattuck-Eidens, D.; Futreal, P.A.; Harsh-

man, K.; Tavtigian, S.; Liu, Q.; Cochran, C.; Bennett, L.M.; Ding,

W.; . A strong candidate for the breast and ovarian cancer suscepti-

bility gene BRCA1. Science, 1994, 266, 66-71.

[118] Liede, A.; Karlan, B.Y.; Narod, S.A. Cancer risks for male carriers

of germline mutations in BRCA1 or BRCA2: A review of the lit-

erature. J. Clin. Oncol., 2004, 22, 735-42.

[119] Hemminki, K.; Bermejo, J.L. Effects of screening for breast cancer

on its age-incidence relationships and familial risk. Int. J. Cancer,

2005, 117, 145-9.

[120] Scully, R.; Chen, J.J.; Plug, A.; Xiao, Y.H.; Weaver, D.; Feunteun,

J.; Ashley, T.; Livingston, D.M. Association of BRCA1 with

Rad51 in mitotic and meiotic cells. Cell, 1997, 88, 265-75.

[121] Wang, Y.; Cortez, D.; Yazdi, P.; Neff, N.; Elledge, S.J.; Qin, J.

BASC, a super complex of BRCA1-associated proteins involved in

the recognition and repair of aberrant DNA structures. Genes Dev.,

2000, 14, 927-39.

[122] Baumann, P.; West, S.C. Role of the human RAD51 protein in

homologous recombination and double-stranded break repair.

Trends Biochem. Sci., 1998, 23, 247-51.

[123] Gowen, L.C.; Avrutskaya, A.V.; Latour, A.M.; Koller, B.H.;

Leadon, S.A. BRCA1 required for transcription-coupled repair of

oxidative DNA damage (Retracted article. See vol 300, pg 1657,

June 13 2003). Science, 1998, 281, 1009-12.

[124] Abbott, D.W.; Thompson, M.E.; Robinson-Benion, C.; Tomlinson,

G.; Jensen, R.A.; Holt, J.T. BRCA1 expression restores radiation

resistance in BRCA1-defective cancer cells through enhancement

of transcription-coupled DNA repair. J. Biol. Chem., 1999, 274,

18808-12.

[125] Chen, Y.; Chen, C.F.; Riley, D.J.; Allred, D.C.; Chen, P.L.; Von

Hoff, D.; Osborne, C.K.; Lee, W.H. Aberrant subcellular localiza-

tion of BRCA1 in breast cancer. Science, 1995, 270, 789-91.

[126] Scully, R.; Ganesan, S.; Brown, M.; De Caprio, J.A.; Cannistra,

S.A.; Feunteun, J.; Schnitt, S.; Livingston, D.M. Location of

BRCA1 in human breast and ovarian cancer cells. Science, 1996,

272, 123-6.

[127] Chen, C.F.; Li, S.; Chen, Y.; Chen, P.L.; Sharp, Z.D.; Lee, W.H.

The nuclear localization sequences of the BRCA1 protein interact

with the importin-alpha subunit of the nuclear transport signal re-

ceptor. J. Biol. Chem., 1996, 271, 32863-8.

[128] Koonin, E.V.; Tatusov, R.L.; Rudd, K.E. Protein sequence com-

parison at genome scale. Methods Enzymol., 1996, 266, 295-322.

[129] Bork, P.; Hofmann, K.; Bucher, P.; Neuwald, A.F.; Altschul, S.F.;

Koonin, E.V. A superfamily of conserved domains in DNA dam-

age-responsive cell cycle checkpoint proteins. FASEB J., 1997, 11,

68-76.

[130] Callebaut, I.; Mornon, J.P. From BRCA1 to RAP1: a widespread

BRCT module closely associated with DNA repair. FEBS Lett.,

1997, 400, 25-30.

[131] Koonin, E.V.; Altschul, S.F.; Bork, P. BRCA1 protein products ...

Functional motifs. Nat. Genet., 1996, 13, 266-8.

[132] Glover, J.N.; Williams, R.S.; Lee, M.S. Interactions between

BRCT repeats and phosphoproteins: tangled up in two. Trends Bio-

chem. Sci., 2004, 29, 579-85.

[133] Williams, R.S.; Lee, M.S.; Hau, D.D.; Glover, J.N. Structural basis

of phosphopeptide recognition by the BRCT domain of BRCA1.

Nat. Struct. Mol. Biol., 2004, 11, 519-25.

[134] Scully, R.; Puget, N.; Vlasakova, K. DNA polymerase stalling,

sister chromatid recombination and the BRCA genes. Oncogene,

2000, 19, 6176-83.

[135] Boulton, S.J. Cellular functions of the BRCA tumour-suppressor

proteins. Biochem. Soc. Trans., 2006, 34, 633-45.

[136] Deng, C.X.; Brodie, S.G. Roles of BRCA1 and its interacting pro-

teins. Bioessays, 2000, 22, 728-37.

[137] Wu, L.C.; Wang, Z.W.; Tsan, J.T.; Spillman, M.A.; Phung, A.; Xu,

X.L.; Yang, M.C.; Hwang, L.Y.; Bowcock, A.M.; Baer, R. Identi-

fication of a RING protein that can interact in vivo with the BRCA1

gene product. Nat. Genet., 1996, 14, 430-40.

[138] Jensen, D.E.; Proctor, M.; Marquis, S.T.; Gardner, H.P.; Ha, S.I.;

Chodosh, L.A.; Ishov, A.M.; Tommerup, N.; Vissing, H.; Sekido,

Y.; Minna, J.; Borodovsky, A.; Schultz, D.C.; Wilkinson, K.D.;

Maul, G.G.; Barlev, N.; Berger, S.L.; Prendergast, G.C.; Rauscher,

F.J., III BAP1: a novel ubiquitin hydrolase which binds to the

BRCA1 RING finger and enhances BRCA1-mediated cell growth

suppression. Oncogene, 1998, 16, 1097-112.

[139] Chai, Y.L.; Cui, J.; Shao, N.; Shyam, E.; Reddy, P.; Rao, V.N. The

second BRCT domain of BRCA1 proteins interacts with p53 and

stimulates transcription from the p21WAF1/CIP1 promoter. Onco-

gene, 1999, 18, 263-8.

[140] Aprelikova, O.N.; Fang, B.S.; Meissner, E.G.; Cotter, S.; Camp-

bell, M.; Kuthiala, A.; Bessho, M.; Jensen, R.A.; Liu, E.T. BRCA1-

associated growth arrest is RB-dependent. Proc. Natl. Acad. Sci.

U.S.A., 1999, 96, 11866-71.

[141] Wong, A.K.; Ormonde, P.A.; Pero, R.; Chen, Y.; Lian, L.; Salada,

G.; Berry, S.; Lawrence, Q.; Dayananth, P.; Ha, P.; Tavtigian, S.V.;

Teng, D.H.; Bartel, P.L. Characterization of a carboxy-terminal

BRCA1 interacting protein. Oncogene, 1998, 17, 2279-85.

[142] Yu, X.; Wu, L.C.; Bowcock, A.M.; Aronheim, A.; Baer, R. The C-

terminal (BRCT) domains of BRCA1 interact in vivo with CtIP, a

protein implicated in the CtBP pathway of transcriptional repres-

sion. J. Biol. Chem., 1998, 273, 25388-92.

[143] Scully, R.; Anderson, S.F.; Chao, D.M.; Wei, W.; Ye, L.; Young,

R.A.; Livingston, D.M.; Parvin, J.D. BRCA1 is a component of the

RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. U. S. A,

1997, 94, 5605-10.

[144] Anderson, S.F.; Schlegel, B.P.; Nakajima, T.; Wolpin, E.S.; Parvin,

J.D. BRCA1 protein is linked to the RNA polymerase II holoen-

zyme complex via RNA helicase A. Nat. Genet., 1998, 19, 254-6.

[145] Yarden, R.I.;Brody, L.C. BRCA1 interacts with components of the

histone deacetylase complex. Proc. Natl. Acad. Sci. U.S.A., 1999,

96, 4983-8.

[146] Cantor, S.B.; Bell, D.W.; Ganesan, S.; Kass, E.M.; Drapkin, R.;

Grossman, S.; Wahrer, D.C.; Sgroi, D.C.; Lane, W.S.; Haber, D.A.;

Livingston, D.M. BACH1, a novel helicase-like protein, interacts

directly with BRCA1 and contributes to its DNA repair function.

Cell, 2001, 105, 149-60.

[147] Bochar, D.A.; Wang, L.; Beniya, H.; Kinev, A.; Xue, Y.; Lane,

W.S.; Wang, W.; Kashanchi, F.; Shiekhattar, R. BRCA1 is associ-

ated with a human SWI/SNF-related complex: linking chromatin

remodeling to breast cancer. Cell, 2000, 102, 257-65.

[148] Hurley, P.J.; Bunz, F. ATM and ATR: components of an integrated

circuit. Cell Cycle, 2007, 6, 414-7.

[149] Hartman, A.R.;Ford, J.M. BRCA1 induces DNA damage recogni-

tion factors and enhances nucleotide excision repair. Nat. Genet.,

2002, 32, 180-4.

[150] Greenberg, R.A.; Sobhian, B.; Pathania, S.; Cantor, S.B.; Nakatani,

Y.; Livingston, D.M. Multifactorial contributions to an acute DNA

damage response by BRCA1/BARD1-containing complexes.

Genes Dev., 2006, 20, 34-46.

[151] Polanowska, J.; Martin, J.S.; Garcia-Muse, T.; Petalcorin, M.I.;

Boulton, S.J. A conserved pathway to activate BRCA1-dependent

ubiquitylation at DNA damage sites. EMBO J., 2006, 25, 2178-88.

[152] Somasundaram, K.; Zhang, H.; Zeng, Y.X.; Houvras, Y.; Peng, Y.;

Zhang, H.; Wu, G.S.; Licht, J.D.; Weber, B.L.; El Deiry, W.S. Ar-

rest of the cell cycle by the tumour-suppressor BRCA1 requires the

CDK-inhibitor p21WAF1/CiP1. Nature, 1997, 389, 187-90.

[153] Ouchi, T.; Monteiro, A.N.; August, A.; Aaronson, S.A.; Hanafusa,

H. BRCA1 regulates p53-dependent gene expression. Proc. Natl.

Acad. Sci. U. S. A, 1998, 95, 2302-6.

[154] Xu, X.; Qiao, W.; Linke, S.P.; Cao, L.; Li, W.M.; Furth, P.A.;

Harris, C.C.; Deng, C.X. Genetic interactions between tumor sup-

pressors Brca1 and p53 in apoptosis, cell cycle and tumorigenesis.

Nat. Genet., 2001, 28, 266-71.

[155] Xu, X.; Wagner, K.U.; Larson, D.; Weaver, Z.; Li, C.; Ried, T.;

Hennighausen, L.; Wynshaw-Boris, A.; Deng, C.X. Conditional

mutation of Brca1 in mammary epithelial cells results in blunted

ductal morphogenesis and tumour formation. Nat. Genet., 1999, 22,

37-43.

[156] Xu, X.; Weaver, Z.; Linke, S.P.; Li, C.; Gotay, J.; Wang, X.W.;

Harris, C.C.; Ried, T.; Deng, C.X. Centrosome amplification and a

defective G2-M cell cycle checkpoint induce genetic instability in


BRCA1 exon 11 isoform-deficient cells. Mol. Cell, 1999, 3, 389-

95.

[157] Glover, J.N. Insights into the molecular basis of human hereditary

breast cancer from studies of the BRCA1 BRCT domain. Fam.

Cancer, 2006, 5, 89-93.

[158] Breast Cancer Information Core. Available at

http://research.nhgri.nih.gov/bic/. Accessed on October 2009.

[159] Goldgar, D.E.; Easton, D.F.; Deffenbaugh, A.M.; Monteiro, A.N.;

Tavtigian, S.V.; Couch, F.J. Integrated evaluation of DNA se-

quence variants of unknown clinical significance: application to

BRCA1 and BRCA2. Am. J. Hum. Genet., 2004, 75, 535-44.

[160] Szabo, C.I.; Schutte, M.; Broeks, A.; Houwing-Duistermaat, J.J.;

Thorstenson, Y.R.; Durocher, F.; Oldenburg, R.A.; Wasielewski,

M.; Odefrey, F.; Thompson, D.; Floore, A.N.; Kraan, J.; Klijn,

J.G.; van den Ouweland, A.M.; Wagner, T.M.; Devilee, P.; Simard,

J.; 't Veer, L.J.; Goldgar, D.E.; Meijers-Heijboer, H. Are ATM mu-

tations 7271T-->G and IVS10-6T-->G really high-risk breast can-

cer-susceptibility alleles? Cancer Res., 2004, 64, 840-3.

[161] Easton, D.F.; Deffenbaugh, A.M.; Pruss, D.; Frye, C.; Wenstrup,

R.J.; Allen-Brady, K.; Tavtigian, S.V.; Monteiro, A.N.; Iversen,

E.S.; Couch, F.J.; Goldgar, D.E. A systematic genetic assessment

of 1,433 sequence variants of unknown clinical significance in the

BRCA1 and BRCA2 breast cancer-predisposition genes. Am. J.

Hum. Genet., 2007, 81, 873-83.

[162] Williams, R.S.;Glover, J.N. Structural consequences of a cancer-

causing BRCA1-BRCT missense mutation. J. Biol. Chem., 2003,

278, 2630-5.

[163] Mirkovic, N.; Marti-Renom, M.A.; Weber, B.L.; Sali, A.; Mon-

teiro, A.N. Structure-based assessment of missense mutations in

human BRCA1: implications for breast and ovarian cancer

predisposition. Cancer Res., 2004, 64, 3790-7.

[164] Carvalho, M.A.; Marsillac, S.M.; Karchin, R.; Manoukian, S.;

Grist, S.; Swaby, R.F.; Urmenyi, T.P.; Rondinelli, E.; Silva, R.;

Gayol, L.; Baumbach, L.; Sutphen, R.; Pickard-Brzosowicz, J.L.;

Nathanson, K.L.; Sali, A.; Goldgar, D.; Couch, F.J.; Radice, P.;

Monteiro, A.N. Determination of cancer risk associated with germ

line BRCA1 missense variants by functional analysis. Cancer Res.,

2007, 67, 1494-501.

[165] Karchin, R.; Monteiro, A.N.; Tavtigian, S.V.; Carvalho, M.A.; Sali,

A. Functional impact of missense variants in BRCA1 predicted by

supervised learning. PLoS Comput. Biol., 2007, 3, e26.

[166] Domchek, S.; Weber, B.L. Genetic variants of uncertain signifi-

cance: flies in the ointment. J. Clin. Oncol., 2008, 26, 16-7.

[167] Carvalho, M.A.; Couch, F.J.; Monteiro, A.N. Functional assays for

BRCA1 and BRCA2. Int. J. Biochem. Cell Biol., 2007, 39, 298-

310.

[168] Vallon-Christersson, J.; Cayanan, C.; Haraldsson, K.; Loman, N.;

Bergthorsson, J.T.; Brondum-Nielsen, K.; Gerdes, A.M.; Moller,

P.; Kristoffersson, U.; Olsson, H.; Borg, A.; Monteiro, A.N. Func-

tional analysis of BRCA1 C-terminal missense mutations identified

in breast and ovarian cancer families. Hum. Mol. Genet., 2001, 10,

353-60.

[169] Williams, R.S.; Bernstein, N.; Lee, M.S.; Rakovszky, M.L.; Cui,

D.; Green, R.; Weinfeld, M.; Glover, J.N. Structural basis for phos-

phorylation-dependent signaling in the DNA-damage response.

Biochem. Cell Biol., 2005, 83, 721-7.

[170] Zhou, B.B.; Bartek, J. Targeting the checkpoint kinases: chemosen-

sitization versus chemoprotection. Nat. Rev. Cancer, 2004, 4, 216-

25.

[171] Longley, D.B.; Johnston, P.G. Molecular mechanisms of drug

resistance. J. Pathol., 2005, 205, 275-92.

[172] Martin, S.A.; Lord, C.J.; Ashworth, A. DNA repair deficiency as a

therapeutic target in cancer. Curr. Opin. Genet. Dev., 2008, 18, 80-

6.

[173] Madhusudan, S.; Hickson, I.D. DNA repair inhibition: a selective

tumour targeting strategy. Trends Mol. Med., 2005, 11, 503-11.

[174] Belzile, J.P.; Choudhury, S.A.; Cournoyer, D.; Chow, T.Y. Target-

ing DNA repair proteins: a promising avenue for cancer gene ther-

apy. Curr. Gene Ther., 2006, 6, 111-23.

[175] Ding, J.; Miao, Z.H.; Meng, L.H.; Geng, M.Y. Emerging cancer

therapeutic opportunities target DNA-repair systems. Trends

Pharmacol. Sci., 2006, 27, 338-44.

[176] O'Connor, M.J.; Martin, N.M.; Smith, G.C. Targeted cancer thera-

pies based on the inhibition of DNA strand break repair. Oncogene,

2007, 26, 7816-24.

[177] Kelley, M.R.; Fishel, M.L. DNA repair proteins as molecular tar-

gets for cancer therapeutics. Anticancer Agents Med. Chem., 2008,

8, 417-25.

[178] Kinsella, T.J. Understanding DNA damage response and DNA

repair pathways: applications to more targeted cancer therapeutics.

Semin. Oncol., 2009, 36, S42-S51.

[179] Olive, P.L.; Vikse, C.M.; Durand, R.E. Hypoxic fractions measured

in murine tumors and normal tissues using the comet assay. Int. J.

Radiat. Oncol. Biol. Phys., 1994, 29, 487-91.

[180] Choudhury, A.; Cuddihy, A.; Bristow, R.G. Radiation and new

molecular agents part I: targeting ATM-ATR checkpoints, DNA

repair, and the proteasome. Semin. Radiat. Oncol., 2006, 16, 51-8.

[181] Bristow, R.G.; Ozcelik, H.; Jalali, F.; Chan, N.; Vesprini, D. Ho-

mologous recombination and prostate cancer: a model for novel

DNA repair targets and therapies. Radiother. Oncol., 2007, 83,

220-30.

[182] Hopkins, A.L.; Groom, C.R. The druggable genome. Nat. Rev.

Drug Discov., 2002, 1, 727-30.

[183] Abraham, R.T. Cell cycle checkpoint signaling through the ATM

and ATR kinases. Genes Dev., 2001, 15, 2177-96.

[184] Shiloh, Y. ATM and ATR: networking cellular responses to DNA

damage. Curr. Opin. Genet. Dev., 2001, 11, 71-7.

[185] Brown, E.J.; Baltimore, D. Essential and dispensable roles of ATR

in cell cycle arrest and genome maintenance. Genes Dev., 2003, 17,

615-28.

[186] Matsuoka, S.; Ballif, B.A.; Smogorzewska, A.; McDonald, E.R.,

III; Hurov, K.E.; Luo, J.; Bakalarski, C.E.; Zhao, Z.; Solimini, N.;

Lerenthal, Y.; Shiloh, Y.; Gygi, S.P.; Elledge, S.J. ATM and ATR

substrate analysis reveals extensive protein networks responsive to

DNA damage. Science, 2007, 316, 1160-6.

[187] Hickson, I.; Zhao, Y.; Richardson, C.J.; Green, S.J.; Martin, N.M.;

Orr, A.I.; Reaper, P.M.; Jackson, S.P.; Curtin, N.J.; Smith, G.C.

Identification and characterization of a novel and specific inhibitor

of the ataxia-telangiectasia mutated kinase ATM. Cancer Res.,

2004, 64, 9152-9.

[188] Rainey, M.D.; Charlton, M.E.; Stanton, R.V.; Kastan, M.B. Tran-

sient inhibition of ATM kinase is sufficient to enhance cellular sen-

sitivity to ionizing radiation. Cancer Res., 2008, 68, 7466-74.

[189] Lord, C.J.; Garrett, M.D.; Ashworth, A. Targeting the double-

strand DNA break repair pathway as a therapeutic strategy. Clin.

Cancer Res., 2006, 12, 4463-8.

[190] Sarkaria, J.N.; Tibbetts, R.S.; Busby, E.C.; Kennedy, A.P.; Hill,

D.E.; Abraham, R.T. Inhibition of phosphoinositide 3-kinase re-

lated kinases by the radiosensitizing agent wortmannin. Cancer

Res., 1998, 58, 4375-82.

[191] Izzard, R.A.; Jackson, S.P.; Smith, G.C. Competitive and noncom-

petitive inhibition of the DNA-dependent protein kinase. Cancer

Res., 1999, 59, 2581-6.

[192] Block, W.D.; Merkle, D.; Meek, K.; Lees-Miller, S.P. Selective

inhibition of the DNA-dependent protein kinase (DNA-PK) by the

radiosensitizing agent caffeine. Nucleic Acids Res., 2004, 32, 1967-

72.

[193] Kong, D.; Yamori, T. Phosphatidylinositol 3-kinase inhibitors:

promising drug candidates for cancer therapy. Cancer Sci., 2008,

99, 1734-40.

[194] Walker, E.H.; Pacold, M.E.; Perisic, O.; Stephens, L.; Hawkins,

P.T.; Wymann, M.P.; Williams, R.L. Structural determinants of

phosphoinositide 3-kinase inhibition by wortmannin, LY294002,

quercetin, myricetin, and staurosporine. Mol. Cell, 2000, 6, 909-19.

[195] Wymann, M.P.; Bulgarelli-Leva, G.; Zvelebil, M.J.; Pirola, L.;

Vanhaesebroeck, B.; Waterfield, M.D.; Panayotou, G. Wortmannin

inactivates phosphoinositide 3-kinase by covalent modification of

Lys-802, a residue involved in the phosphate transfer reaction. Mol.

Cell Biol., 1996, 16, 1722-33.

[196] Sanchez-Perez, I. DNA repair inhibitors in cancer treatment. Clin.

Transl. Oncol., 2006, 8, 642-6.

[197] Nishida, H.; Tatewaki, N.; Nakajima, Y.; Magara, T.; Ko, K.M.;

Hamamori, Y.; Konishi, T. Inhibition of ATR protein kinase activ-

ity by schisandrin B in DNA damage response. Nucleic Acids Res.,

2009, 37, 5678-89.

[198] Li, L.; Lu, Q.; Shen, Y.; Hu, X. Schisandrin B enhances doxorubi-

cin-induced apoptosis of cancer cells but not normal cells. Bio-

chem. Pharmacol., 2006, 71, 584-95.


[199] Hollick, J.J.; Golding, B.T.; Hardcastle, I.R.; Martin, N.;

Richardson, C.; Rigoreau, L.J.; Smith, G.C.; Griffin, R.J. 2,6-

disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-

Dependent protein kinase (DNA-PK). Bioorg. Med. Chem. Lett.,

2003, 13, 3083-6.

[200] Ismail, I.H.; Martensson, S.; Moshinsky, D.; Rice, A.; Tang, C.;

Howlett, A.; McMahon, G.; Hammarsten, O. SU11752 inhibits the

DNA-dependent protein kinase and DNA double-strand break re-

pair resulting in ionizing radiation sensitization. Oncogene, 2004,

23, 873-82.

[201] Leahy, J.J.; Golding, B.T.; Griffin, R.J.; Hardcastle, I.R.;

Richardson, C.; Rigoreau, L.; Smith, G.C. Identification of a highly

potent and selective DNA-dependent protein kinase (DNA-PK) in-

hibitor (NU7441) by screening of chromenone libraries. Bioorg.

Med. Chem. Lett., 2004, 14, 6083-7.

[202] Willmore, E.; de Caux, S.; Sunter, N.J.; Tilby, M.J.; Jackson, G.H.;

Austin, C.A.; Durkacz, B.W. A novel DNA-dependent protein

kinase inhibitor, NU7026, potentiates the cytotoxicity of topoi-

somerase II poisons used in the treatment of leukemia. Blood,

2004, 103, 4659-65.

[203] Shinohara, E.T.; Geng, L.; Tan, J.; Chen, H.; Shir, Y.; Edwards, E.;

Halbrook, J.; Kesicki, E.A.; Kashishian, A.; Hallahan, D.E. DNA-

dependent protein kinase is a molecular target for the development

of noncytotoxic radiation-sensitizing drugs. Cancer Res., 2005, 65,

4987-92.

[204] Salles, B.; Calsou, P.; Frit, P.; Muller, C. The DNA repair complex

DNA-PK, a pharmacological target in cancer chemotherapy and

radiotherapy. Pathol. Biol. (Paris), 2006, 54, 185-93.

[205] Zhao, Y.; Thomas, H.D.; Batey, M.A.; Cowell, I.G.; Richardson,

C.J.; Griffin, R.J.; Calvert, A.H.; Newell, D.R.; Smith, G.C.;

Curtin, N.J. Preclinical evaluation of a potent novel DNA-

dependent protein kinase inhibitor NU7441. Cancer Res., 2006, 66,

5354-62.

[206] Nutley, B.P.; Smith, N.F.; Hayes, A.; Kelland, L.R.; Brunton, L.;

Golding, B.T.; Smith, G.C.; Martin, N.M.; Workman, P.; Raynaud,

F.I. Preclinical pharmacokinetics and metabolism of a novel proto-

type DNA-PK inhibitor NU7026. Br. J. Cancer, 2005, 93, 1011-8.

[207] ClinicalTrials.gov. Available at http://www.clinicaltrials.gov. Ac-

cessed on October 2009.

[208] Carey, N.; La Thangue, N.B. Histone deacetylase inhibitors: gath-

ering pace. Curr. Opin. Pharmacol., 2006, 6, 369-75.

[209] Mann, B.S.; Johnson, J.R.; Cohen, M.H.; Justice, R.; Pazdur, R.

FDA approval summary: vorinostat for treatment of advanced pri-

mary cutaneous T-cell lymphoma. Oncologist, 2007, 12, 1247-52.

[210] Prasad, K.N.; Sinha, P.K.; Ramanujam, M.; Sakamoto, A. Sodium

ascorbate potentiates the growth inhibitory effect of certain agents

on neuroblastoma cells in culture. Proc. Natl. Acad. Sci. U. S. A,

1979, 76, 829-32.

[211] Nackerdien, Z.; Michie, J.; Bohm, L. Chromatin decondensed by

acetylation shows an elevated radiation response. Radiat. Res.,

1989, 117, 234-44.

[212] Camphausen, K.; Scott, T.; Sproull, M.; Tofilon, P.J. Enhancement

of xenograft tumor radiosensitivity by the histone deacetylase in-

hibitor MS-275 and correlation with histone hyperacetylation. Clin.

Cancer Res., 2004, 10, 6066-71.

[213] Camphausen, K.; Cerna, D.; Scott, T.; Sproull, M.; Burgan, W.E.;

Cerra, M.A.; Fine, H.; Tofilon, P.J. Enhancement of in vitro and in

vivo tumor cell radiosensitivity by valproic acid. Int. J. Cancer,

2005, 114, 380-6.

[214] Geng, L.; Cuneo, K.C.; Fu, A.; Tu, T.; Atadja, P.W.; Hallahan,

D.E. Histone deacetylase (HDAC) inhibitor LBH589 increases du-

ration of gamma-H2AX foci and confines HDAC4 to the cyto-

plasm in irradiated non-small cell lung cancer. Cancer Res., 2006,

66, 11298-304.

[215] Cuneo, K.C.; Fu, A.; Osusky, K.; Huamani, J.; Hallahan, D.E.;

Geng, L. Histone deacetylase inhibitor NVP-LAQ824 sensitizes

human nonsmall cell lung cancer to the cytotoxic effects of ioniz-

ing radiation. Anti-Cancer Drugs, 2007, 18, 793-800.

[216] Johnstone, R.W.; Licht, J.D. Histone deacetylase inhibitors in can-

cer therapy: is transcription the primary target? Cancer Cell, 2003,

4, 13-18.

[217] Karagiannis, T.C.; Harikrishnan, K.N.; El Osta, A. The histone

deacetylase inhibitor, Trichostatin A, enhances radiation sensitivity

and accumulation of gammaH2A.X. Cancer Biol. Ther., 2005, 4,

787-93.

[218] Munshi, A.; Tanaka, T.; Hobbs, M.L.; Tucker, S.L.; Richon, V.M.;

Meyn, R.E. Vorinostat, a histone deacetylase inhibitor, enhances

the response of human tumor cells to ionizing radiation through

prolongation of gamma-H2AX foci. Mol. Cancer Ther., 2006, 5,

1967-74.

[219] Zhang, Y.; Adachi, M.; Zou, H.; Hareyama, M.; Imai, K.; Shino-

mura, Y. Histone deacetylase inhibitors enhance phosphorylation

of histone H2AX after ionizing radiation. Int. J. Radiat. Oncol.

Biol. Phys., 2006, 65, 859-66.

[220] Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.; Shuen, M.;

Loppnau, P.; Mazitschek, R.; Kwiatkowski, N.P.; Lewis, T.A.;

Maglathin, R.L.; McLean, T.H.; Bochkarev, A.; Plotnikov, A.N.;

Vedadi, M.; Arrowsmith, C.H. Human HDAC7 harbors a class IIa

histone deacetylase-specific zinc binding motif and cryptic deace-

tylase activity. J. Biol. Chem., 2008, 283, 11355-63.

[221] Pharmacyclis. Available at http://www.pharmacyclics.com. Ac-

cessed on October 2009.

[222] Banuelos, C.A.; Banath, J.P.; MacPhail, S.H.; Zhao, J.; Reitsema,

T.; Olive, P.L. Radiosensitization by the histone deacetylase inhibi-

tor PCI-24781. Clin. Cancer Res., 2007, 13, 6816-26.

[223] Adimoolam, S.; Sirisawad, M.; Chen, J.; Thiemann, P.; Ford, J.M.;

Buggy, J.J. HDAC inhibitor PCI-24781 decreases RAD51 expres-

sion and inhibits homologous recombination. Proc. Natl. Acad. Sci.

U.S.A., 2007, 104, 19482-7.

[224] Camphausen, K.;Tofilon, P.J. Inhibition of histone deacetylation: a

strategy for tumor radiosensitization. J. Clin. Oncol., 2007, 25,

4051-6.

[225] Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner,

E.; Buggy, J.J. A novel histone deacetylase 8 (HDAC8)-specific

inhibitor PCI-34051 induces apoptosis in T-cell lymphomas. Leu-

kemia, 2008, 22, 1026-34.

[226] Chou, C.J.; Herman, D.; Gottesfeld, J.M. Pimelic diphenylamide

106 is a slow, tight-binding inhibitor of class I histone deacetylases.

J. Biol. Chem., 2008, 283, 35402-9.

[227] Kao, J.; Milano, M.T.; Javaheri, A.; Garofalo, M.C.; Chmura, S.J.;

Weichselbaum, R.R.; Kron, S.J. gamma-H2AX as a therapeutic

target for improving the efficacy of radiation therapy. Curr. Cancer

Drug Targets, 2006, 6, 197-205.

[228] Janetka, J.W.; Ashwell, S. Checkpoint kinase inhibitors: a review

of the patent literature. Expert. Opin. Ther. Pat., 2009, 19, 165-97.

[229] Chen, Y.;Poon, R.Y. The multiple checkpoint functions of CHK1

and CHK2 in maintenance of genome stability. Front Biosci., 2008,

13, 5016-29.

[230] Reinhardt, H.C.; Yaffe, M.B. Kinases that control the cell cycle in

response to DNA damage: Chk1, Chk2, and MK2. Curr. Opin. Cell

Biol., 2009, 21, 245-55.

[231] Busby, E.C.; Leistritz, D.F.; Abraham, R.T.; Karnitz, L.M.; Sark-

aria, J.N. The radiosensitizing agent 7-hydroxystaurosporine

(UCN-01) inhibits the DNA damage checkpoint kinase hChk1.

Cancer Res., 2000, 60, 2108-12.

[232] Liang, Y.; Lin, S.Y.; Brunicardi, F.C.; Goss, J.; Li, K. DNA dam-

age response pathways in tumor suppression and cancer treatment.

World J. Surg., 2009, 33, 661-6.

[233] Bunch, R.T.;Eastman, A. Enhancement of cisplatin-induced cyto-

toxicity by 7-hydroxystaurosporine (UCN-01), a new G2-

checkpoint inhibitor. Clin. Cancer Res., 1996, 2, 791-7.

[234] Wang, Q.; Fan, S.; Eastman, A.; Worland, P.J.; Sausville, E.A.;

O'Connor, P.M. UCN-01: a potent abrogator of G2 checkpoint

function in cancer cells with disrupted p53. J. Natl. Cancer Inst.,

1996, 88, 956-65.

[235] Shao, R.G.; Cao, C.X.; Shimizu, T.; O'Connor, P.M.; Kohn, K.W.;

Pommier, Y. Abrogation of an S-phase checkpoint and potentiation

of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01)

in human cancer cell lines, possibly influenced by p53 function.

Cancer Res., 1997, 57, 4029-35.

[236] Akinaga, S.; Nomura, K.; Gomi, K.; Okabe, M. Enhancement of

antitumor activity of mitomycin C in vitro and in vivo by UCN-01,

a selective inhibitor of protein kinase C. Cancer Chemother. Phar-

macol., 1993, 32, 183-9.

[237] Hsueh, C.T.; Kelsen, D.; Schwartz, G.K. UCN-01 suppresses

thymidylate synthase gene expression and enhances 5-fluorouracil-

induced apoptosis in a sequence-dependent manner. Clin. Cancer

Res., 1998, 4, 2201-6.


[238] Hirose, Y.; Berger, M.S.; Pieper, R.O. Abrogation of the Chk1-

mediated G(2) checkpoint pathway potentiates temozolomide-

induced toxicity in a p53-independent manner in human glioblas-

toma cells. Cancer Res., 2001, 61, 5843-9.

[239] Shi, Z.; Azuma, A.; Sampath, D.; Li, Y.X.; Huang, P.; Plunkett, W.

S-Phase arrest by nucleoside analogues and abrogation of survival

without cell cycle progression by 7-hydroxystaurosporine. Cancer

Res., 2001, 61, 1065-72.

[240] Tse, A.N.; Carvajal, R.; Schwartz, G.K. Targeting checkpoint

kinase 1 in cancer therapeutics. Clin. Cancer Res., 2007, 13, 1955-

60.

[241] Hosoya, N.;Miyagawa, K. Clinical importance of DNA repair

inhibitors in cancer therapy. Magazine Europ. Med. Oncol.

(MEMO), 2009, 2, 9-14.

[242] Jackson, J.R.; Gilmartin, A.; Imburgia, C.; Winkler, J.D.; Marshall,

L.A.; Roshak, A. An indolocarbazole inhibitor of human check-

point kinase (Chk1) abrogates cell cycle arrest caused by DNA

damage. Cancer Res., 2000, 60, 566-72.

[243] Zhao, B.; Bower, M.J.; McDevitt, P.J.; Zhao, H.; Davis, S.T.; Jo-

hanson, K.O.; Green, S.M.; Concha, N.O.; Zhou, B.B. Structural

basis for Chk1 inhibition by UCN-01. J. Biol. Chem., 2002, 277,

46609-15.

[244] Kohn, E.A.; Yoo, C.J.; Eastman, A. The protein kinase C inhibitor

Go6976 is a potent inhibitor of DNA damage-induced S and G2

cell cycle checkpoints. Cancer Res., 2003, 63, 31-5.

[245] Jiang, X.; Zhao, B.; Britton, R.; Lim, L.Y.; Leong, D.; Sanghera,

J.S.; Zhou, B.B.; Piers, E.; Andersen, R.J.; Roberge, M. Inhibition

of Chk1 by the G2 DNA damage checkpoint inhibitor isogranu-

latimide. Mol. Cancer Ther., 2004, 3, 1221-7.

[246] Kawabe, T. G2 checkpoint abrogators as anticancer drugs. Mol.

Cancer Ther., 2004, 3, 513-9.

[247] Collins, I.; Garrett, M.D. Targeting the cell division cycle in can-

cer: CDK and cell cycle checkpoint kinase inhibitors. Curr. Opin.

Pharmacol., 2005, 5, 366-73.

[248] Zhou, B.B.; Sausville, E.A. Drug discovery targeting Chk1 and

Chk2 kinases. Prog. Cell Cycle Res., 2003, 5, 413-21.

[249] Parsels, L.A.; Parsels, J.D.; Booth, R.J.; Denny, W.A.; Kraker,

A.J.; Maybaum, J. The small-molecule chk1 inhibitor, PD-321852,

causes synergistic depletion of chk1 protein and clonogenic death

when combined with gemcitabine in colorectal and pancreatic tu-

mor cells. Proc. Amer. Assoc. Cancer Res., 2006, 47, 1154.

[250] Ashwell, S.; Janetka, J.W.; Zabludoff, S. Keeping checkpoint

kinases in line: new selective inhibitors in clinical trials. Expert.

Opin. Investig. Drugs, 2008, 17, 1331-40.

[251] Zabludoff, S.D.; Deng, C.; Grondine, M.R.; Sheehy, A.M.; Ash-

well, S.; Caleb, B.L.; Green, S.; Haye, H.R.; Horn, C.L.; Janetka,

J.W.; Liu, D.; Mouchet, E.; Ready, S.; Rosenthal, J.L.; Queva, C.;

Schwartz, G.K.; Taylor, K.J.; Tse, A.N.; Walker, G.E.; White,

A.M. AZD7762, a novel checkpoint kinase inhibitor, drives check-

point abrogation and potentiates DNA-targeted therapies. Mol.

Cancer Ther., 2008, 7, 2955-66.

[252] Parsels, L.A.; Morgan, M.A.; Tanska, D.M.; Parsels, J.D.; Palmer,

B.D.; Booth, R.J.; Denny, W.A.; Canman, C.E.; Kraker, A.J.; Law-

rence, T.S.; Maybaum, J. Gemcitabine sensitization by checkpoint

kinase 1 inhibition correlates with inhibition of a Rad51 DNA

damage response in pancreatic cancer cells. Mol. Cancer Ther.,

2009, 8, 45-54.

[253] Sorensen, C.S.; Syljuasen, R.G.; Falck, J.; Schroeder, T.; Ronn-

strand, L.; Khanna, K.K.; Zhou, B.B.; Bartek, J.; Lukas, J. Chk1

regulates the S phase checkpoint by coupling the physiological

turnover and ionizing radiation-induced accelerated proteolysis of

Cdc25A. Cancer Cell, 2003, 3, 247-58.

[254] Syljuasen, R.G.; Sorensen, C.S.; Nylandsted, J.; Lukas, C.; Lukas,

J.; Bartek, J. Inhibition of Chk1 by CEP-3891 accelerates mitotic

nuclear fragmentation in response to ionizing Radiation. Cancer

Res., 2004, 64, 9035-40.

[255] Wang, G.T.; Li, G.; Mantei, R.A.; Chen, Z.; Kovar, P.; Gu, W.;

Xiao, Z.; Zhang, H.; Sham, H.L.; Sowin, T.; Rosenberg, S.H.; Lin,

N.H. 1-(5-Chloro-2-alkoxyphenyl)-3-(5-cyanopyrazin-2-yl)ureas

[correction of cyanopyrazi] as potent and selective inhibitors of

Chk1 kinase: synthesis, preliminary SAR, and biological activities.

J. Med. Chem., 2005, 48, 3118-21.

[256] Lin, N.H.; Xia, P.; Kovar, P.; Park, C.; Chen, Z.; Zhang, H.;

Rosenberg, S.H.; Sham, H.L. Synthesis and biological evaluation

of 3-ethylidene-1,3-dihydro-indol-2-ones as novel checkpoint 1 in-

hibitors. Bioorg. Med. Chem. Lett., 2006, 16, 421-6.

[257] Keegan, K.; Kesicki, E.A.; Gaudino, J.J.; Cook, A.W.; Cowen,

S.D.; Gurgess, L.E.WO 02/070494, 2002.

[258] Boyle, R.G.; Imogai, H.J.; Cherry D.WO 03/101444, 2003.

[259] Li, G.; Li, Q.; Li, T.; Lin, N.H.; Mantei, R.A.; Sham, H.L.; Wang,

G.T.U.S. 034,038, 2004.

[260] Li, G.; Hasvold, L.A.; Tao, Z.F.; Wang, G.T.; Gwaltney, S.L.;

Patel, J.; Kovar, P.; Credo, R.B.; Chen, Z.; Zhang, H.; Park, C.;

Sham, H.L.; Sowin, T.; Rosenberg, S.H.; Lin, N.H. Synthesis and

biological evaluation of 1-(2,4,5-trisubstituted phenyl)-3-(5-

cyanopyrazin-2-yl)ureas as potent Chk1 kinase inhibitors. Bioorg.

Med. Chem. Lett., 2006, 16, 2293-8.

[261] Sharma, V.;Tepe, J.J. Potent inhibition of checkpoint kinase activ-

ity by a hymenialdisine-derived indoloazepine. Bioorg. Med.

Chem. Lett., 2004, 14, 4319-21.

[262] Arienti, K.L.; Brunmark, A.; Axe, F.U.; McClure, K.; Lee, A.;

Blevitt, J.; Neff, D.K.; Huang, L.; Crawford, S.; Pandit, C.R.;

Karlsson, L.; Breitenbucher, J.G. Checkpoint kinase inhibitors:

SAR and radioprotective properties of a series of 2-

arylbenzimidazoles. J. Med. Chem., 2005, 48, 1873-85.

[263] Lokesh, G.L.; Muralidhara, B.K.; Negi, S.S.; Natarajan, A. Ther-

modynamics of phosphopeptide tethering to BRCT: the structural

minima for inhibitor design. J. Am. Chem. Soc., 2007, 129, 10658-

9.

[264] Simeonov, A.; Yasgar, A.; Jadhav, A.; Lokesh, G.L.; Klumpp, C.;

Michael, S.; Austin, C.P.; Natarajan, A.; Inglese, J. Dual-

fluorophore quantitative high-throughput screen for inhibitors of

BRCT-phosphoprotein interaction. Anal. Biochem., 2008, 375, 60-

70.

[265] Hartwell, L.H.; Szankasi, P.; Roberts, C.J.; Murray, A.W.; Friend,

S.H. Integrating genetic approaches into the discovery of anticancer

drugs. Science, 1997, 278, 1064-8.

[266] Garber, K. Running interference: pace picks up on synthetic lethal-

ity research. J. Natl. Cancer Inst., 2004, 96, 982-3.

[267] Kaelin, W.G., Jr. The concept of synthetic lethality in the context

of anticancer therapy. Nat. Rev. Cancer, 2005, 5, 689-98.

[268] Bryant, H.E.; Schultz, N.; Thomas, H.D.; Parker, K.M.; Flower, D.;

Lopez, E.; Kyle, S.; Meuth, M.; Curtin, N.J.; Helleday, T. Specific

killing of BRCA2-deficient tumours with inhibitors of poly(ADP-

ribose) polymerase. Nature, 2005, 434, 913-7.

[269] Farmer, H.; McCabe, N.; Lord, C.J.; Tutt, A.N.; Johnson, D.A.;

Richardson, T.B.; Santarosa, M.; Dillon, K.J.; Hickson, I.; Knights,

C.; Martin, N.M.; Jackson, S.P.; Smith, G.C.; Ashworth, A. Target-

ing the DNA repair defect in BRCA mutant cells as a therapeutic

strategy. Nature, 2005, 434, 917-21.

[270] Ame, J.C.; Spenlehauer, C.; de Murcia, G. The PARP superfamily.

Bioessays, 2004, 26, 882-93.

[271] Otto, H.; Reche, P.A.; Bazan, F.; Dittmar, K.; Haag, F.; Koch-

Nolte, F. In silico characterization of the family of PARP-like

poly(ADP-ribosyl)transferases (pARTs). BMC. Genomics, 2005, 6,

139.

[272] Gagne, J.P.; Hendzel, M.J.; Droit, A.; Poirier, G.G. The expanding

role of poly(ADP-ribose) metabolism: current challenges and new

perspectives. Curr. Opin. Cell Biol., 2006, 18, 145-51.

[273] Hassa, P.O.;Hottiger, M.O. The diverse biological roles of mam-

malian PARPS, a small but powerful family of poly-ADP-ribose

polymerases. Front. Biosci., 2008, 13, 3046-82.

[274] Dantzer, F.; Ame, J.C.; Schreiber, V.; Nakamura, J.; Menissier-de

Murcia, J.; de Murcia, G. Poly(ADP-ribose) polymerase-1 activa-

tion during DNA damage and repair. Methods Enzymol., 2006, 409,

493-510.

[275] Zaremba, T.;Curtin, N.J. PARP inhibitor development for systemic

cancer targeting. Anticancer Agents Med. Chem., 2007, 7, 515-23.

[276] Dantzer, F.; de La, R.G.; Menissier-de Murcia, J.; Hostomsky, Z.;

de Murcia, G.; Schreiber, V. Base excision repair is impaired in

mammalian cells lacking Poly(ADP-ribose) polymerase-1. Bio-

chemistry, 2000, 39, 7559-69.

[277] Peralta-Leal, A.; Rodriguez, M.I.; Oliver, F.J. Poly(ADP-

ribose)polymerase-1 (PARP-1) in carcinogenesis: Potential role of

PARP inhibitors in cancer treatment. Clin. Transl.Oncol., 2008, 10,

318-23.

[278] Woodhouse, B.C.; Dianova, I.I.; Parsons, J.L.; Dianov, G.L.

Poly(ADP-ribose) polymerase-1 modulates DNA repair capacity


and prevents formation of DNA double strand breaks. Dna Repair,

2008, 7, 932-40.

[279] Virag, L.; Szabo, C. The therapeutic potential of poly(ADP-ribose)

polymerase inhibitors. Pharmacol. Rev., 2002, 54, 375-429.

[280] Southan, G.J.; Szabo, C. Poly(ADP-ribose) polymerase inhibitors.

Curr. Med. Chem., 2003, 10, 321-40.

[281] Miknyoczki, S.J.; Jones-Bolin, S.; Pritchard, S.; Hunter, K.; Zhao,

H.; Wan, W.; Ator, M.; Bihovsky, R.; Hudkins, R.; Chatterjee, S.;

Klein-Szanto, A.; Dionne, C.; Ruggeri, B. Chemopotentiation of

temozolomide, irinotecan, and cisplatin activity by CEP-6800, a

poly(ADP-ribose) polymerase inhibitor. Mol. Cancer Ther., 2003,

2, 371-82.

[282] Calabrese, C.R.; Almassy, R.; Barton, S.; Batey, M.A.; Calvert,

A.H.; Canan-Koch, S.; Durkacz, B.W.; Hostomsky, Z.; Kumpf,

R.A.; Kyle, S.; Li, J.; Maegley, K.; Newell, D.R.; Notarianni, E.;

Stratford, I.J.; Skalitzky, D.; Thomas, H.D.; Wang, L.Z.; Webber,

S.E.; Williams, K.J.; Curtin, N.J. Anticancer chemosensitization

and radiosensitization by the novel poly(ADP-ribose) polymerase-1

inhibitor AG14361. J. Natl. Cancer Inst., 2004, 96, 56-67.

[283] Curtin, N.J. PARP inhibitors for cancer therapy. Expert Rev. Mol.

Med., 2005, 7, 1-20.

[284] Miknyoczki, S.; Chang, H.; Grobelny, J.; Pritchard, S.; Worrell, C.;

McGann, N.; Ator, M.; Husten, J.; Deibold, J.; Hudkins, R.; Zulli,

A.; Parchment, R.; Ruggeri, B. The selective poly(ADP-ribose) po-

lymerase-1(2) inhibitor, CEP-8983, increases the sensitivity of

chemoresistant tumor cells to temozolomide and irinotecan but

does not potentiate myelotoxicity. Mol. Cancer Ther., 2007, 6,

2290-302.

[285] Ratnam, K.; Low, J.A. Current development of clinical inhibitors

of poly(ADP-ribose) polymerase in oncology. Clin. Cancer Res.,

2007, 13, 1383-8.

[286] Ashworth, A. Drug resistance caused by reversion mutation. Can-

cer Res., 2008, 68, 10021-3.

[287] Tan, D.S.; Marchio, C.; Reis-Filho, J.S. Hereditary breast cancer:

from molecular pathology to tailored therapies. J. Clin. Pathol.,

2008, 61, 1073-82.

[288] Chalmers, A.J. The potential role and application of PARP inhibi-

tors in cancer treatment. Br. Med. Bull., 2009, 89, 23-40.

[289] Plummer, E.R.; Middleton, M.R.; Jones, C.; Olsen, A.; Hickson, I.;

McHugh, P.; Margison, G.P.; McGown, G.; Thorncroft, M.; Wat-

son, A.J.; Boddy, A.V.; Calvert, A.H.; Harris, A.L.; Newell, D.R.;

Curtin, N.J. Temozolomide pharmacodynamics in patients with

metastatic melanoma: dna damage and activity of repair enzymes

O6-alkylguanine alkyltransferase and poly(ADP-ribose) polym-

erase-1. Clin. Cancer Res., 2005, 11, 3402-9.

[290] Plummer, E.R. Inhibition of poly(ADP-ribose) polymerase in can-

cer. Curr. Opin. Pharmacol., 2006, 6, 364-8.

[291] Plummer, R.; Jones, C.; Middleton, M.; Wilson, R.; Evans, J.;

Olsen, A.; Curtin, N.; Boddy, A.; McHugh, P.; Newell, D.; Harris,

A.; Johnson, P.; Steinfeldt, H.; Dewji, R.; Wang, D.; Robson, L.;

Calvert, H. Phase I study of the poly(ADP-ribose) polymerase in-

hibitor, AG014699, in combination with temozolomide in patients

with advanced solid tumors. Clin. Cancer Res., 2008, 14, 7917-23.

[292] de Bono, J.; Boss, D.; Fong, P.; Roelvink, M.; Yap, T.; Tutt, A.;

Mortimer, P.; O'Connor, M.; Schellens, J.H.; Kaye, S. First in hu-

man phase I pharmacokinetic (PK) and pharmacodynamic (PD)

study of KU-0059436 (KU), a small molecule inhibitor of poly

ADP-RIBOSE polymerase (PARP) in cancer patients (P), including

BRCA 1/2 mutation carriers. Ann. Oncol., 2007, 18, 26-7.

[293] Tuma, R.S. Combining carefully selected drug, patient genetics

may lead to total tumor death. J. Natl. Cancer Inst., 2007, 99,

1505-6, 1509.

[294] Jones, P.; Altamura, S.; Boueres, J.; Ferrigno, F.; Fonsi, M.;

Giomini, C.; Lamartina, S.; Monteagudo, E.; Ontoria, J.M.; Orsale,

M.V.; Palumbi, M.C.; Pesci, S.; Roscilli, G.; Scarpelli, R.; Schultz-

Fademrecht, C.; Toniatti, C.; Rowley, M. Discovery of 2-{4-[(3S)-

piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a

novel oral poly(ADP-ribose)polymerase (PARP) inhibitor effica-

cious in BRCA-1 and -2 mutant tumors. J. Med. Chem., 2009, 52,

7170-85.

[295] Drew, Y.; Plummer, R. PARP inhibitors in cancer therapy: two

modes of attack on the cancer cell widening the clinical applica-

tions. Drug Resist. Updat., 2009, 12, 153-6.

[296] Champoux, J.J. DNA topoisomerases: structure, function, and

mechanism. Annu. Rev. Biochem., 2001, 70, 369-413.

[297] Treszezamsky, A.D.; Kachnic, L.A.; Feng, Z.H.; Zhang, J.R.; To-

kadjian, C.; Powell, S.N. BRCA1- and BRCA2-deficient cells are

sensitive to etoposide-induced DNA double-strand breaks via

topoisomerase II. Cancer Res., 2007, 67, 7078-81.

[298] Chemo.care. Available at www.chemocare.com/bio/etoposide.asp.

Accessed on October 2009.

[299] Chen, G.L.; Yang, L.; Rowe, T.C.; Halligan, B.D.; Tewey, K.M.;

Liu, L.F. Nonintercalative antitumor drugs interfere with the break-

age-reunion reaction of mammalian DNA topoisomerase II. J. Biol.

Chem., 1984, 259, 13560-6.

[300] Bhattacharyya, A.; Ear, U.S.; Koller, B.H.; Weichselbaum, R.R.;

Bishop, D.K. The breast cancer susceptibility gene BRCA1 is re-

quired for subnuclear assembly of Rad51 and survival following

treatment with the DNA cross-linking agent cisplatin. J. Biol.

Chem., 2000, 275, 23899-903.

[301] Howlett, N.G.; Taniguchi, T.; Olson, S.; Cox, B.; Waisfisz, Q.;

Die-Smulders, C.; Persky, N.; Grompe, M.; Joenje, H.; Pals, G.;

Ikeda, H.; Fox, E.A.; D'Andrea, A.D. Biallelic inactivation of

BRCA2 in Fanconi anemia. Science, 2002, 297, 606-9.

[302] Kraakman-van der Zwet, M.; Overkamp, W.J.; van Lange, R.E.;

Essers, J.; Duijn-Goedhart, A.; Wiggers, I.; Swaminathan, S.; van

Buul, P.P.; Errami, A.; Tan, R.T.; Jaspers, N.G.; Sharan, S.K.; Ka-

naar, R.; Zdzienicka, M.Z. Brca2 (XRCC11) deficiency results in

radioresistant DNA synthesis and a higher frequency of spontane-

ous deletions. Mol. Cell Biol., 2002, 22, 669-79.

[303] Tutt, A.N.; Lord, C.J.; McCabe, N.; Farmer, H.; Turner, N.; Martin,

N.M.; Jackson, S.P.; Smith, G.C.; Ashworth, A. Exploiting the

DNA repair defect in BRCA mutant cells in the design of new

therapeutic strategies for cancer. Cold Spring Harb. Symp. Quant.

Biol., 2005, 70, 139-48.

[304] Evers, B.; Schut, E.; van der, B.E.; Braumuller, T.M.; Egan, D.A.;

Holstege, H.; Edser, P.; Adams, D.J.; Wade-Martins, R.;

Bouwman, P.; Jonkers, J. A high-throughput pharmaceutical screen

identifies compounds with specific toxicity against BRCA2-

deficient tumors. Clin. Cancer Res., 2010, 16, 99-108.

[305] Tutt, A.; Bertwistle, D.; Valentine, J.; Gabriel, A.; Swift, S.; Ross,

G.; Griffin, C.; Thacker, J.; Ashworth, A. Mutation in Brca2 stimu-

lates error-prone homology-directed repair of DNA double-strand

breaks occurring between repeated sequences. EMBO J., 2001, 20,

4704-16.

[306] Tassone, P.; Tagliaferri, P.; Perricelli, A.; Blotta, S.; Quaresima,

B.; Martelli, M.L.; Goel, A.; Barbieri, V.; Costanzo, F.; Boland,

C.R.; Venuta, S. BRCA1 expression modulates chemosensitivity of

BRCA1-defective HCC1937 human breast cancer cells. Br. J. Can-

cer, 2003, 88, 1285-91.

[307] Torrisi, R.; Balduzzi, A.; Ghisini, R.; Rocca, A.; Bottiglieri, L.;

Giovanardi, F.; Veronesi, P.; Luini, A.; Orlando, L.; Viale, G.;

Goldhirsch, A.; Colleoni, M. Tailored preoperative treatment of lo-

cally advanced triple negative (hormone receptor negative and

HER2 negative) breast cancer with epirubicin, cisplatin, and infu-

sional fluorouracil followed by weekly paclitaxel. Cancer Che-

mother. Pharmacol., 2008, 62, 667-72.

[308] Hoadley, K.A.; Weigman, V.J.; Fan, C.; Sawyer, L.R.; He, X.;

Troester, M.A.; Sartor, C.I.; Rieger-House, T.; Bernard, P.S.;

Carey, L.A.; Perou, C.M. EGFR associated expression profiles vary

with breast tumor subtype. BMC. Genom., 2007, 8, 258.

[309] Chia, S.K.; Speers, C.H.; D'yachkova, Y.; Kang, A.; Malfair-

Taylor, S.; Barnett, J.; Coldman, A.; Gelmon, K.A.; O'Reilly, S.E.;

Olivotto, I.A. The impact of new chemotherapeutic and agents on

survival in a population-based of women with metastatic breast

cancer hormone cohort. Cancer, 2007, 110, 973-9.

[310] D'Incalci, M.; Erba, E.; Damia, G.; Galliera, E.; Carrassa, L.; Mar-

chini, S.; Mantovani, R.; Tognon, G.; Fruscio, R.; Jimeno, J.; Fair-

cloth, G.T. Unique features of the mode of action of ET-743. On-

cologist, 2002, 7, 210-6.

[311] D'Incalci, M.; Colombo, T.; Ubezio, P.; Nicoletti, I.; Giavazzi, R.;

Erba, E.; Ferrarese, L.; Meco, D.; Riccardi, R.; Sessa, C.; Cavallini,

E.; Jimeno, J.; Faircloth, G.T. The combination of yondelis and

cisplatin is synergistic against human tumor xenografts. Eur. J.

Cancer, 2003, 39, 1920-6.

[312] Demetri, G.D. ET-743: the US experience in sarcomas of soft

tissues. Anticancer Drugs, 2002, 13 Suppl 1, S7-S9.

[313] D'Incalci, M.; Jimeno, J. Preclinical and clinical results with the


natural marine product ET-743. Expert Opin. Investig. Drugs,

2003, 12, 1843-53.

[314] Le Cesne, A.; Blay, J.Y.; Judson, I.; van Oosterom, A.; Verweij, J.;

Radford, J.; Lorigan, P.; Rodenhuis, S.; Ray-Coquard, I.; Bonvalot,

S.; Collin, F.; Jimeno, J.; Di Paola, E.; Van Glabbeke, M.; Nielsen,

O.S. Phase II study of ET-743 in advanced soft tissue sarcomas: a

European Organisation for the Research and Treatment of Cancer

(EORTC) soft tissue and bone sarcoma group trial. J. Clin. Oncol.,

2005, 23, 576-84.

[315] Herrero, A.B.; Martin-Castellanos, C.; Marco, E.; Gago, F.; Mo-

reno, S. Cross-talk between nucleotide excision and homologous

recombination DNA repair pathways in the mechanism of action of

antitumor trabectedin. Cancer Res., 2006, 66, 8155-62.

[316] Soares, D.G.; Escargueil, A.E.; Poindessous, V.; Sarasin, A.; de

Gramont, A.; Bonatto, D.; Henriques, J.A.P.; Larsen, A.K. Replica-

tion and homologous recombination repair regulate DNA double-

strand break formation by the antitumor alkylator ecteinascidin

743. Proc. Natl. Acad. Sci. U.S.A., 2007, 104, 13062-7.

[317] Liu, S.K.; Olive, P.L.; Bristow, R.G. Biomarkers for DNA DSB

inhibitors and radiotherapy clinical trials. Cancer Metastasis Rev.,

2008, 27, 445-58.

[318] Gorbunova, V.;Seluanov, A. Making ends meet in old age: DSB

repair and aging. Mech. Ageing Dev., 2005, 126, 621-8.

[319] Judkins, T.; Hendrickson, B.C.; Deffenbaugh, A.M.; Scholl, T.

Single nucleotide polymorphisms in clinical genetic testing: the

characterization of the clinical significance of genetic variants and

their application in clinical research for BRCA1. Mutat. Res., 2005,

573, 168-79.

[320] Chenevix-Trench, G.; Healey, S.; Lakhani, S.; Waring, P.; Cum-

mings, M.; Brinkworth, R.; Deffenbaugh, A.M.; Burbidge, L.A.;

Pruss, D.; Judkins, T.; Scholl, T.; Bekessy, A.; Marsh, A.; Love-

lock, P.; Wong, M.; Tesoriero, A.; Renard, H.; Southey, M.; Hop-

per, J.L.; Yannoukakos, K.; Brown, M.; Easton, D.; Tavtigian,

S.V.; Goldgar, D.; Spurdle, A.B. Genetic and histopathologic

evaluation of BRCA1 and BRCA2 DNA sequence variants of un-

known clinical significance. Cancer Res., 2006, 66, 2019-27.

[321] Huyton, T.; Bates, P.A.; Zhang, X.; Sternberg, M.J.; Freemont, P.S.

The BRCA1 C-terminal domain: structure and function. Mutat.

Res., 2000, 460, 319-32.

[322] Phelan, C.M.; Dapic, V.; Tice, B.; Favis, R.; Kwan, E.; Barany, F.;

Manoukian, S.; Radice, P.; van der Luijt, R.B.; van Nesselrooij,

B.P.; Chenevix-Trench, G.; kConFab; Caldes, T.; de la, H.M.;

Lindquist, S.; Tavtigian, S.V.; Goldgar, D.; Borg, A.; Narod, S.A.;

Monteiro, A.N. Classification of BRCA1 missense variants of un-

known clinical significance. J. Med. Genet., 2005, 42, 138-46.

[323] Selleck. Available at http://www.selleckchem.com/. Accessed on

October 2009.

[324] Daniel, R.A.; Rozanska, A.L.; Thomas, H.D.; Mulligan, E.A.;

Drew, Y.; Castelbuono, D.J.; Hostomsky, Z.; Plummer, E.R.;

Boddy, A.; Tweddle, D.A.; Curtin, N.J.; Clifford, S.C. Inhibition of

poly (ADP-ribose) polymerase-1 enhances temozolomide and topo-

tecan activity against childhood neuroblastoma. Clin. Cancer Res.,

2009, 15, 1241-9.

[325] Mante, S.; Minneman, K.P. Caffeine Inhibits Forskolin-Stimulated

Cyclic-Amp Accumulation in Rat-Brain. Eur. J. Pharmacol., 1990,

175, 203-5.

[326] Cortez, D. Caffeine inhibits checkpoint responses without inhibit-

ing the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-

related (ATR) protein kinases. J. Biol. Chem., 2003, 278, 39-45.

[327] Sarkaria, J.N.; Busby, E.C.; Tibbetts, R.S.; Roos, P.; Taya, Y.;

Karnitz, L.M.; Abraham, R.T. Inhibition of ATM and ATR kinase

activities by the radiosensitizing agent, caffeine. Cancer Res.,

1999, 59, 4375-82.

[328] Foukas, L.C.; Daniele, N.; Ktori, C.; Anderson, K.E.; Jensen, J.;

Shepherd, P.R. Direct effects of caffeine and theophylline on p110

delta and other phosphoinositide 3-kinases - Differential effects on

lipid kinase and protein kinase activities. J. Biol. Chem., 2002, 277,

37124-30.

[329] National Cancer Institute. Available at http:// www.cancer.gov.


[330] Sorensen, C.S.; Syljuasen, R.G.; Lukas, J.; Bartek, J. ATR, claspin

and the Rad9-Rad1-Hus1 complex regulate Chk1 and Cdc25A in

the absence of DNA damage. Cell Cycle, 2004, 3, 941-5.

[331] Kashishian, A.; Douangpanya, H.; Clark, D.; Schlachter, S.T.;

Eary, C.T.; Schiro, J.G.; Huang, H.; Burgess, L.E.; Kesicki, E.A.;

Halbrook, J. DNA-dependent protein kinase inhibitors as drug can-

didates for the treatment of cancer. Mol. Cancer Ther., 2003, 2,

1257-64.

[332] Shinohara, K.; Master, V.; Carroll, P.R.; Mcleod, D.G.; Beyer,

D.C. Intermediate results of ferromagnetic thermal ablation for ra-

diation failure local recurrence of prostate cancer. J. Urol., 2004,

171, 217.

[333] Szabo, G.; Soos, P.; Mandera, S.; Heger, U.; Flechtenmacher, C.;

Bahrle, S.; Seres, L.; Cziraki, A.; Gries, A.; Zsengeller, Z.; Vahl,

C.F.; Hagl, S.; Szabo, C. INO-1001 a novel poly(ADP-ribose) po-

lymerase (PARP) inhibitor improves cardiac and pulmonary func-

tion after crystalloid cardioplegia and extracorporal circulation.

Shock, 2004, 21, 426-32.

[334] Morrow, D.A.; Brickman, C.M.; Murphy, S.A.; Baran, K.;

Krakover, R.; Dauerman, H.; Kumar, S.; Slomowitz, N.; Grip, L.;

McCabe, C.H.; Salzman, A.L. A randomized, placebo-controlled

trial to evaluate the tolerability, safety, pharmacokinetics, and

pharmacodynamics of a potent inhibitor of poly(ADP-ribose) po-

lymerase (INO-1001) in patients with ST-elevation myocardial in-

farction undergoing primary percutaneous coronary intervention:

results of the TIMI 37 trial. J. Thromb. Thrombolysis., 2009, 27,

359-64.

[335] Santa Cruz Biotechnology. Available at http://www.scbt.com.


[336] Fong, P.C.; Spicer, J.; Reade, S.; Reid, A.; Vidal, L.; Schellens,

J.H.; Tutt, A.; Harris, P.A.; Kaye, S.; De Bono, J.S. Phase I phar-

macokinetic (PK) and pharmacodynamic (PD) evaluation of a

small molecule inhibitor of poly ADP-ribose polymerase (PARP),

KU-0059436 (Ku) in patients (p) with advanced tumours. J. Clin.

Oncol., 2006, 24, 126S.

[337] Dungey, F.A.; Loser, D.A.; Chalmers, A.J. Replication-Dependent

Radiosensitization of Human Glioma Cells by Inhibition of

Poly(Adp-Ribose) Polymerase: Mechanisms and Therapeutic Po-

tential. Int. J. Radiat. Oncol. Biol. Phys., 2008, 72, 1188-97.

[338] Take, Y.; Kumano, M.; Hamano, Y.; Fukatsu, H.; Teraoka, H.;

Nishimura, S.; Okuyama, A. Ok-1035, A Selective Inhibitor of

Dna-Dependent Protein-Kinase. Biochem. Biophys. Res. Commun.,

1995, 215, 41-7.

[339] Stockley, M.; Clegg, W.; Fontana, G.; Golding, B.T.; Martin, N.;

Rigoreau, L.J.M.; Smith, G.C.M.; Griffin, R.J. Synthesis, crystal

structure determination, and biological properties of the DNA-

dependent protein kinase (DNA-PK) inhibitor 3-cyano-6-hydra-

zonomethyl-5-(4-pyridyl)pyrid-[1H]-2-one (OK-1035). Bioorg.

Med. Chem. Lett., 2001, 11, 2837-41.

[340] Blasina, A.; Hallin, J.; Chen, E.H.; Arango, M.E.; Kraynov, E.;

Register, J.; Grant, S.; Ninkovic, S.; Chen, P.; Nichols, T.; O'Con-

nor, P.; Anderes, K. Breaching the DNA damage checkpoint via

PF-00477736, a novel small-molecule inhibitor of checkpoint

kinase 1. Mol. Cancer Ther., 2008, 7, 2394-04.

[341] Peixoto, P.;Lansiaux, A. Histone-deacetylases inhibitors: from TSA

to SAHA. Bull. Cancer, 2006, 93, 27-36.

[342] Tocris Bioscence. Available at http://www.tocris.com. Accessed on

October 2009.

[343] Woo, S.H.; Frechette, S.; Abou, K.E.; Bouchain, G.; Vaisburg, A.;

Bernstein, N.; Moradei, O.; Leit, S.; Allan, M.; Fournel, M.;

Trachy-Bourget, M.C.; Li, Z.; Besterman, J.M.; Delorme, D. Struc-

turally simple trichostatin A-like straight chain hydroxamates as

potent histone deacetylase inhibitors. J. Med. Chem., 2002, 45,

2877-85.

[344] Hu, E.D.; Dul, E.; Sung, C.M.; Chen, Z.X.; Kirkpatrick, R.; Zhang,

G.F.; Johanson, K.; Liu, R.G.; Lago, A.; Hofmann, G.; Macarron,

R.; los Frailes, M.; Perez, P.; Krawiec, J.; Winkler, J.; Jaye, M.

Identification of novel isoform-selective inhibitors within class I

histone deacetylases. J. Pharmacol. Exp. Ther., 2003, 307, 720-8.

[345] Graves, P.R.; Yu, L.J.; Schwarz, J.K.; Gales, J.; Sausville, E.A.;

O'Connor, P.M.; Piwnica-Worms, H. The Chk1 protein kinase and

the Cdc25C regulatory pathways are targets of the anticancer agent

UCN-01. J. Biol. Chem., 2000, 275, 5600-5.

[346] Sato, S.; Fujita, N.; Tsuruo, T. Interference with PDK1-Akt sur-

vival signaling pathway by UCN-01 (7-hydroxystaurosporine). On-

cogene, 2002, 21, 1727-38.

[347] Yu, Q.; La Rose, J.; Zhang, H.L.; Takemura, H.; Kohn, K.W.;

Pommier, Y. UCN-01 inhibits p53 up-regulation and abrogates


gamma-radiation-induced G(2)-M checkpoint independently of p53

by targeting both of the checkpoint kinases, Chk2 and Chk1. Can-

cer Res., 2002, 62, 5743-8.

[348] Eikel, D.; Lampen, A.; Nau, H. Teratogenic effects mediated by

inhibition of histone deacetylases: Evidence from quantitative

structure activity relationships of 20 valproic acid derivatives.

Chem. Res. Toxicol., 2006, 19, 272-8.

[349] Durant, S.;Karran, P. Vanillins - a novel family of DNA-PK inhibi-

tors. Nucleic Acids Res., 2003, 31, 5501-12.

[350] Nakanishi, S.; Kakita, S.; Takahashi, I.; Kawahara, K.; Tsukuda,

E.; Sano, T.; Yamada, K.; Yoshida, M.; Kase, H.; Matsuda, Y.; Ha-

shimoto, Y.; Nonomura, Y. Wortmannin, A microbial product in-

hibitor of myosin light chain kinase. J. Biol. Chem., 1992, 267,

2157-63.

[351] Stoyanova, S.; BulgarelliLeva, G.; Kirsch, C.; Hanck, T.; Klinger,

R.; Wetzker, R.; Wymann, M.P. Lipid kinase and protein kinase ac-

tivities of G-protein-coupled phosphoinositide 3-kinase gamma:

Structure-activity analysis and interactions with wortmannin. Bio-

chem. J., 1997, 324, 489-95.

[352] Ihle, N.T.; Williams, R.; Chow, S.; Chew, W.; Berggren, M.I.;

Paine-Murrieta, G.; Minion, D.J.; Halter, R.J.; Wipf, P.; Abraham,

R.; Kirkpatrick, L.; Powis, G. Molecular pharmacology and anti-

tumor activity of PX-866, a novel inhibitor of phosphoinositide-3-

kinase signaling. Mol. Cancer Ther., 2004, 3, 763-72.

[353] Ihle, N.T.; Paine-Murrieta, G.; Berggren, M.I.; Baker, A.; Tate,

W.R.; Wipf, P.; Abraham, R.T.; Kirkpatrick, D.L.; Powis, G. The

phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resis-

tance to the epidermal growth factor receptor inhibitor gefitinib in

A-549 human non-small cell lung cancer xenografts. Mol. Cancer

Ther., 2005, 4, 1349-57.

[354] Knight, Z.A.; Gonzalez, B.; Feldman, M.E.; Zunder, E.R.; Golden-

berg, D.D.; Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth,

B.; Balla, T.; Weiss, W.A.; Williams, R.L.; Shokat, K.M. A phar-

macological map of the PI3-K family defines a role for p110alpha

in insulin signaling. Cell, 2006, 125, 733-47.

[355] Matthews, D.J.; Yakes, F.M.; Chen, J.; Tadano, M.; Bornheim, L.;

Clary, D.O.; Tai, A.; Wagner, J.M.; Miller, N.; Kim, Y.D.; Robert-

son, S.; Murray, L.; Karnitz, L.M. Pharmacological abrogation of

S-phase checkpoint enhances the anti-tumor activity of gemcitabine

in vivo. Cell Cycle, 2007, 6, 104-10.

Table 3. Chemical 2D structure, activity, and clinical trials of some of the most investigated enzyme inhibitors of the DSBs repair (n.a., not available data).

Inhibitor Structure Target IC50 - Ki

(nM) Ref.

Mono- or combined- ther-

apy

Clinical study

stage Ref.

2-(3-(3,4-dichlorophenoxy)phenyl)-

1H-benzo[d]imidazole-5-

carboxamide NH

NH2N

OO

Cl

Cl

CHK2

CHK1

IC50 = 15

n.a. [262]

6-(4-hydroxy-3-methoxyphenyl)-3-

(1H-pyrrolo[2,3-b]pyridin-3-

ylmethylene)-1,3-dihydro-indol-2-

one NH

O

HO

OCH3

NH

N

CHK1

CHK2

IC50 = 4

IC50 = 22,679 [256] Doxorubicin Preclinical [240,256]

ABT-888 NH

N

NH

O NH2

H3C

PARP IC50 = 10 [317]

Monotherapy

IR

Carboplatin

Cyclophosphamide

Doxorubicin

Irinotecan

Paclitaxel

Temozolomide

Topotecan

Phase I

Phase I

Phase I

Phase I/II

Phase I/II

Phase I

Phase I

Phase I/II

Phase I

[207,241]

AG014699

NH

HN

O

NHCH3

F

PARP Ki < 5 [323]

Monotherapy

Temolozomide

Topotecan

Phase II

Preclinical/

Phase I/II

Preclinical

[207,241,

289,324]

(Table 3). Contd…..


(nM) Ref.


apy

Clinical study

stage Ref.

AZD7762

FS

NH

NH2

O

O

HN

NH

CHK1

CHK2

IC50 = 5

IC50 < 10 [176,251]

Gemcitabine

Irinotecan

Phase I

Phase I [207]

BSI-201

I

NO2

OH2N

PARP1 n.a.

Monotherapy

Carboplatin/Paclitaxel

Gemcitabine

Gemcitabine/Carboplatin

Irinotecan

Temozolomide

Topotecan

Phase I/II

Phase I/II

Phase I

Phase II/III

Phase I/II

Phase I/II

Phase I

[207,241]

Caffeine

N

N N

N

O

H3C

O CH3

CH3

DNA-PK

PI3K

ATM

ATR

CHK1

IC50 = 200 - > 10,000,000

n.a.

p110 : IC50 = 10,000

p110 : IC50 = 3,000

IC50 = 200,000

IC50 = 1,100,000

IC50 > 10,000,000

[192,246, 325-328]

Cisplatinum and high-dose cytosine arabinoside

Cisplatinum, highdose cyta-rabine (ARA-C)

Phase III

Phase I/II

[329]

CEP-3891 Structure not disclosed

(Cephalon Inc.)

CHK1

CHK2

IC50 = 4

IC50 = 300,000 [246,330] IR Preclinical [240,254]


(Cephalon Inc.)

CHK2

CHK1

IC50 = 20

IC50 = 300 [246,330]


(Cephalon Inc.) PARP n.a.

Monotherapy

Temozolomide Phase I [207, 295]



(nM) Ref.


apy

Clinical study

stage Ref.

Gö6976

N N

HN

CH3

O

CN

PKC CHK1

CHK2

IC50 = 0.2

IC50 = 50 - 100

IC50 = 10,000

[244,246]

GPI 21016 Structure not disclosed

(MGI Pharma) PARP n.a. Temozolomide Phase I planned [295]

Hymenialdisine

NH

Br

NH

N

HN

O

H2N

O

CHK2

PKC

CHK1

IC50 = 42

n.a.

PKC (h): IC50 = 700

PKC II(h): IC50 = 1200

IC50 = 1,950

[261]

IC-83 Structure not disclosed

(Eli Lilly & CO.)

CHK1

CHK2

n.a.

n.a. Pemetrexeb Phase I [207]

IC 87102

O

N

OH O

OH

DNA-PK

PI3K

IC50 = 35

n.a.

p110 : IC50 = 2700

p110 : IC50 = 400

p110 : IC50 = 1800

p110 : IC50 = 5000

[331] IR Preclinical [332]



(nM) Ref.


apy

Clinical study

stage Ref.

IC 87361

O

N

OH

O

O

DNA-PK

PI3K

IC50 = 34

n.a.

p110 : IC50 = 3800

p110 : IC50 = 1700

p110 : IC50 = 2800

p110 : IC50 = 7900

[331] Monotherapy

IR

Preclinical

Preclinical [207,332]

INO-1001 Structure not disclosed

(Inotek Pharmaceuticals) PARP IC50 = 3 - < 10 [333,334] Temozolomide Phase I [207,241]

Isogranulatimide

NH

NN

HN

O O

CHK1

CHK2

DNA-PK

IC50 = 100

IC50 = 3,000

IC50 = 10,000

[245,335]

KU-0059436

N

NH

O

F

O

N

N

O

PARP2

PARP1

IC50 = 1 - 2

IC50 = 2 - 5 [323,336]

Monotherapy

Bevacizumab

Carboplatin

Carboplatin and paclitaxel

Cisplatin

Dacarbazine

Doxorubicin

Gemcitabine

Irinotecan

Paclitaxel

Topotecan

Phase I/II

Phase I

Phase I

Phase I

Phase I

Phase I

Phase I/II

Phase I

Phase I

Phase I/II

Phase I

[207,241,

337]

KU-55933 S

SO N

O

O

ATM

DNA-PK

PI3K

ATR

IC50 = 13

IC50 = 2,500

IC50 = 16,600

IC50 > 100,000

[187]



(nM) Ref.


apy

Clinical study

stage Ref.

MK4827

O NH2

N

N NH

PARP2

PARP1

IC50 = 2.1

IC50 = 3.8 [294] Monotheraphy Phase I [207,295]

N-(2-chloro-4-(3-(5-cyanopyrazin-2-

yl)ureido)-5-

methoxyphenyl)isonicotinamide

Cl

OCH3

NH

NH

O

N

N CNHN

O

N

CHK1

CHK2

IC50 = 8

n.a. [260] Doxorubicin Preclinical [240,255]

NCGC00094849-01

OHO

N

O OH

O

HO

HO

O

O

N

OHO

O

OH

BRCA1 IC50 ~ 6,000 [264]

NU7026

O N

O

O

DNA-PK

PI3K

ATM

ATR

IC50 = 230

n.a.

p110 : IC50 = 13,000

IC50 > 100,000

IC50 > 100,000

[199,201] Monotherapy Preclinical [206]

NU7441

S

O N

O

O

DNA-PK

PI3K

ATM

ATR

IC50 = 14

n.a.

p110 : IC50 = 5,000

IC50 > 100,000

IC50 > 100,000

[201]

Monotherapy

IR

Doxorubicin

Etoposide

Preclinical

Preclinical

Preclinical

Preclinical

[205]

OK-1035

NH

O

NC

CHNNH2

N

DNA-PK IC50 = 8,000 - 100,000 [200,338,

339]



(nM) Ref.


apy

Clinical study

stage Ref.

PCI-24781

O

N

CH3

H3C

O

HN

O

NH

O

OH

HDAC

n.a.

HDAC1: Ki = 7 [323]

Monotherapy

Doxorubicin

Phase I/II

Phase I/II [207,221]

PD-321852

N

NH

O

O

Cl

ClH3CHN

HO

CHK1

CHK2

IC50 = 5

n.a. [252] Gemcitabine Preclinical [240,249]

PF-00477736

NH2

O

NH

NH

HNO N

N

N

CH3

CHK1

CHK2

CDK1

Ki = 0.49 - 0.75

Ki = 47 - 75

Ki = 9,900

[176,340] Gemcitabine Phase I [207]

SAHA NH

NHOH

O

O

HDAC

IC50 = 2,500 - 7,500 HDAC1: IC50 = 10

HDAC7: IC50 = 113

[323,341]

Monotherapy IR

Alvocidib AMG 655

Azacitidine Bevacizumab

Bevacizumab, carboplatin, and paclitaxel

Bevacizumab and irinotecan Bevacizumab and temo-

zolomide Bexarotene

Bicalutamide Bortezomib

Capecitabine Carboplatin

Carboplatin and etoposide Cisplatin

Cladribine Cyclophosphamide

Cytarabine

Phase I/II/III Phase I/II

Phase I Phase I

Phase I/II Phase I/II

Phase I

Phase I

Phase I/II

Phase I Phase II

Phase I/II/III Phase I


Phase I Phase I/II

Phase I/II

[207]



(nM) Ref.


apy

Clinical study

stage Ref.

Dasatinib Decitabine

Dexamethasone Docetaxel

Doxorubicin Erlonitib Etoposide Filgrastim

Fludarabine Fluoracil

Gemcitabine Gemtuzumab ozogamicin

Goserelin Idarubicin Ifosfamide

Imatinib mesylate Irinotecan

Isotretinoin Lenalidomide Leucovorin Leuprolide Metotrexate

MK0683 Niacinamide

Niacinamide and etoposide NPI-0052

Oxaliplatin Paclitaxel

Paclitaxel and carboplatin Pegaspargase Pegfilgrastim

Pegylated Liposomal Doxorubicin (PLD)

Pemetrexed and cisplatin Prednisone Rituximab

Rituximab, cyclophos-phamide, etoposide, and

prednisone Sorafenib

Sorafenib and LBH589 Tamoxifen

Temozolomide Thiotepa

Topotecan Trastuzumab

Velcade Vincristine Vinorelbine

Phase I/II Phase I Phase I Phase II Phase I

Phase I/II Phase I/II Phase I/II Phase I/II Phase I/II Phase I/II Phase I/II Phase I/II Phase II

Phase I/II Phase I/II Phase I Phase I

Phase I/II Phase I/II Phase I/II Phase II Phase I Phase I Phase I Phase I Phase I Phase I

Phase I/II Phase I/II Phase I


Phase I/II Phase I/II Phase I/II Phase I

Phase I Phase II

Phase I/II n.a.


Phase I/II Phase I

n.a.



(nM) Ref.


apy

Clinical study

stage Ref.

Salvicine H3C

HO

H3C CH3OH

O

O

CH3

CH3

DNA-PK n.a. Monotherapy Phase II [196]

SB-218078

N

HN

N

O

O

O

CHK1

CDC2

PKC

CHK2

IC50 = 15

IC50 = 250

IC50 = 1000

n.a.

[242,246, 248,342]

SU11752 HN

S

O

O

NH

NH

OH

O

CH3

H3C

O

DNA-PK

PI3K IC50 = 130

n.a.

p110 : IC50 = 1,100

[200]

TSA N

H3C

CH3

O

CH3 CH3

NHOH

O

HDAC

IC50 = 5

HDAC1: IC50 = 70 - 300

HDAC3: IC50 = 100 - 300

HDAC7: IC50 = 0.3

HDAC8: IC50 = 100 - 300

[220,323,

343,344]



(nM) Ref.


apy

Clinical study

stage Ref.

UCN-01 N N

HN

O

CH3

OCH3

NHCH3

O OH

PKC

CHK1

PDK1 CHK2

IC50 = 4 - 30

PKC : IC50 = 1 IC50 = 5 - 11

IC50 = 33 IC50 = 10 - > 1,000

[231,242,

245,246, 248,262,

345-347]

Monotherapy

IR Carboplatin

Cisplatin Cytarabine (Ara-c)

Fludarabin Fluoracil

Gemcitabine Irinotecan

Leucovorin Mitomycin C

Perifosine Prednisone

Temozolomide Topoisomerase I poisons

Topotecan

Phase I/II

Phase I/II Phase I

Phase I Phase I


Phase I Phase I

Phase I Phase I/II

Phase I Phase I


Phase I/II

[207,234-

241]

Valproic acid OH

O

H3C

CH3

HDAC IC50 = 398,000 [348]

Monotherapy IR

Adriamycin, cyclophos-

phamide and an-dandvindesine

All-trans retinoic acid (ATRA)

Azacitidine Azacytidine and retinoic

acid Bevacizumab

Carboplatin Cyclophpspamide

Cytarabine (Ara-C) Dasatinib

Decitabine Doxorubicin

Erlotinib Epirubicin

Etoposide Fludarabine

Fluoracil Hydralazine

Karenitecin Lapatinib

Lenalidomide Sorafenib

Sunitinib Temozolomide

Teophyllin Zidovudine

Phase I/II Preclinical/

Phase I/II Phase II

Phase I/II

Phase I/II Phase II

Phase I/II

Phase I/II Phase I

Phase II Phase I

Phase I/II Phase II

Phase I Phase I

Phase I Phase II

Phase I Phase II/III

Phase I/II Phase I

Phase I/II Phase I

Phase I Phase I/II

Phase I/II n.a

[207,224]



(nM) Ref.


apy

Clinical study

stage Ref.

Vanillin

HO

OCH3

H

O

DNA-PK IC50 = 1,500 [349]

Wortmannin O

O

OO

H3CO O

O CH3

CH3O

CH3

PI3K

DNA-PK

ATM

ATR

n.a.

class IA PI3K: IC50 = 4.2

p110 : IC50 = 4 - 38

p110 : IC50 = 1 - 45

p110 : IC50 = 2 - 14

p110 : IC50 = 4 - 70

IC50 = 16 - 150

IC50 = 150

IC50 = 1,800

[190,191,

193,194,

200,331,

350-354]

XL-844 Structure not disclosed (Exelixis) CHK2

CHK1

Ki = 0.07 - 0.2

Ki = 2.2 [176,355] Gemcitabine Phase I [241]

(Z)-3-((1H-pyrrol-2-yl)methylene)-

6-(4-hydroxy-3-

methoxyphenyl)indolin-2-one NH

O

HO

OCH3

NH

CHK1

CHK2

IC50 = 7

IC50 = 21,730 [256]

Critical Review

Neuroglobin, Estrogens, and Neuroprotection

Elisabetta De Marinis1, Maria Marino1 and Paolo Ascenzi1,2*1Department of Biology, University Roma Tre, Roma, Italy2Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Roma, Italy

Summary

Globins have been found in glial cells and neurons of inverte-brates and vertebrates. The first nerve globin has been recognizedin the nerve cord of the polychaete annelid Aphrodite aculeata in1872. In some invertebrates, the nerve globin reaches a millimolarconcentration which is likely sufficient to sustain the aerobic me-tabolism and thus the excitability of the nervous system. In 2000,the first vertebrate nerve globin, named neuroglobin (Ngb), hasbeen identified in neuronal tissues of mice and humans. In contrastto invertebrate nerve globins, the concentration of Ngb, the proto-type of vertebrate nerve globins, is low (lM), reaching a maximumof 100 lM in retina cells. Therefore, Ngb appears unlikely to actprimarily as an O2 buffer and to facilitate O2 diffusion to the mito-chondria. Indeed, Ngb has been hypothesized to catalyze the for-mation/decomposition of reactive nitrogen and/or oxygen speciesand to be part of intracellular signaling pathways enhancing cellsurvival. Here, we report that neuronal Ngb levels are stronglyinduced by the steroid hormone 17b-estradiol. Furthermore, Ngbparticipates to mechanisms involved in 17b-estradiol-induced pro-tective effects against H2O2-induced neurotoxicity. � 2011 IUBMB

IUBMBLife, 63(3): 140–145, 2011

Keywords nerve globins; neuroglobin; neuroprotection; estrogen re-

ceptor; 17b-estradiol; H2O2 neurotoxicity; apoptosis.

Abbreviations Cygb, cytoglobin; ER, estrogen receptor; E2, 17b-estradiol; Hb, hemoglobin, Mb, myoglobin; mini-Hb,

mini-hemoglobin; Ngb, neuroglobin; trHb, truncated

hemoglobin.

INTRODUCTION

Globins occur in all kingdoms of living organisms and dis-

play a variety of functions, including not only O2 transport and

storage but also (pseudo-)enzymatic properties (1–10). Despite

the large variability in their primary and quaternary structures,

globins display a well-conserved tertiary structure (the ‘‘globin

fold’’) consisting of 140–160 amino acids typically organized in

a three-on-three a-helical sandwich (1, 8, 11). The tertiary struc-

tures of truncated hemoglobins and mini-hemoglobins are sube-

ditings of the three-on-three a-helical sandwich, highlighting the

striking structural plasticity of the globin fold (2, 12, 13).

Globin-like molecules are dissolved in the circulating body

fluids of invertebrates or localized into the cells. Three catego-

ries can be distinguished within the intracellular, mainly cyto-

plasmatic, globins: (i) globins located in circulating erythrocyte-

like cell types (e.g., hemoglobin, Hb), (ii) tissue globins (e.g.,

cytoglobin, Cygb, myoglobin, Mb, and neuroglobin, Ngb), and

(iii) globins of unicellular organisms (2–4, 6, 14–18).

Several intracellular globins have been found in the inverte-

brate and vertebrate nervous system possibly sustaining the con-

sume of large amounts of metabolic energy, which requires a

continuous supply of O2 (19, 20). The role of globins in the

brain seems to be pivotal to support nervous system functions

during temporary periods of hypoxia, which may follow envi-

ronmental or pathologic insults, therefore avoiding serious dam-

ages to the nervous system (21–25).

Invertebrate nerve globins have been observed in several

Phyla such as Annelida, Arthropoda, Echiura, Mollusca, Nemer-

tea, and Nematoda (15, 26), the first nerve globin having been

recognized in the nerve cord of the polychaete annelid Aphro-

dite aculeata in 1872 (27). In some invertebrates, nerve globins

reach a millimolar local concentration, which is likely sufficient

to facilitate O2 diffusion and storage (13, 19, 20, 28).

Only in 2000, the first vertebrate nerve globin, named neuro-

globin (Ngb), has been identified in neuronal tissues of mice

and humans (14). More recently, Cygb, globin E, globin X, and

a- and b-chains of Hb have been reported to be expressed in

the vertebrate nervous system (16, 29–34). In contrast to their

invertebrate counterparts, the concentration of Ngb and Cygb is

relatively low (lM) (35); however, Ngb has been found at rela-

tively high concentration in neurons of the hypothalamus (36)

This article is dedicated to Prof. Austin F. Riggs who explored Peru

Andes and pioneered invertebrate nerve globins.

Received 5 January 2011; accepted 5 January 2011

Address correspondence to: Paolo Ascenzi, Interdepartmental Labo-

ratory for Electron Microscopy, University Roma Tre, Via della Vasca

Navale 79, I-00146 Roma, Italy. Tel: 139-06-57333200(2). Fax: 139-

06-57336321. E-mail: [email protected]

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.426

IUBMB Life, 63(3): 140–145, March 2011

reaching very high levels (100 lM) in retina rod cells (37, 38).

Although the concentration of globin E, globin X, and a- and

b-chains of Hb in neural tissues is unknown, it is likely to be

very low (29–34). The relatively low concentration of vertebrate

nerve globins suggests that they are not simply involved in O2

storage and supply to mitochondria but could display (pseudo-

)enzymatic functions and could be part of signaling pathways

(16, 29–31, 34, 35, 39–44).

NEUROGLOBIN, AN HEXA-COORDINATEDGLOBIN IN THE BRAIN

Ngb is a highly conserved protein with an evolutionary rate

that is about threefold slower than that of Mb and Hb. Thus,

Ngb has remained largely unchanged during evolution, pointing

to an important role of this protein; phylogenetic studies have

indicated an ancient origin for Ngb, dating back 800 mya (16,

17).

Ngb is a monomeric, �150-amino acid-long heme protein

displaying less than 25% sequence identity to conventional ver-

tebrate Hbs or Mbs (14, 45). Ngb displays the classical three-

on-three globin fold adapted to host the heme hexa-coordinated

structure of the HisF8-Fe-HisE7 type in both deoxygenated fer-

rous and ferric forms and an elongated protein matrix cavity/

tunnel held to facilitate ligand diffusion from the bulk to the

heme and vice versa (45, 46). Therefore, ligand binding to the

heme-Fe atom of Ngb needs the formation of the transient

penta-coordinated HisF8-Fe species; the reversible intramolecu-

lar hexa-to-penta-coordination transition of the heme-Fe atom

modulates exogenous ligand-binding properties of Ngb (47–49).

Ngb binds several ligands, including diatomic gaseous

ligands (e.g., O2, NO, and CO), and displays (pseudo-)enzy-

matic properties (e.g., O2-mediated NO detoxification). The P50

value for O2 binding to Ngb has been reported to range between

2 and 10 torr depending on pH, temperature, and the redox state

of the cell (14, 42, 45–47, 50–60). However, the hexacoordina-

tion of the heme-Fe atom impairs Ngb-mediated peroxynitrite

detoxification and the formation of the H2O2-mediated ferryl

species (42, 54).

Ngb, the most investigated vertebrate nerve globin (43, 49),

is expressed not only in neurons of the central and peripheral

nervous systems but also in the gastrointestinal tract and in en-

docrine organs (14, 36, 39, 43, 49, 61–67). Recently, Ngb has

also been detected in quiescent astrocytes of the healthy seal

brain (43) and in human glioblastoma cell lines (67). Ngb has

been hypothesized: (i) to act as an O2 buffer, (ii) to facilitate

O2 diffusion to the mitochondria, (iii) to catalyze the formation

and the decomposition of reactive nitrogen and/or oxygen spe-

cies, and (iv) to be part of intracellular signaling pathways by

inhibiting the dissociation of GDP from Ga proteins and trigger-

ing the release of the Gbc complex, and by reducing cyto-

chrome c. Although it is unlikely that Ngb has so many distinct

roles, there is no doubt that Ngb displays a protective func-

tion(s) in the brain (14, 35, 41–44, 54, 56, 59, 60, 68–77).

NEUROPROTECTIVE EFFECTS OF NEUROGLOBIN

Despite the cellular mechanisms remain poorly defined and

still controversial, many in vivo studies performed in Ngb-over-

expressing transgenic mice, in primary neurons, and in cultured

cell lines sustain the neuroprotective role played by Ngb (35,

41, 44, 72, 76, 78, 79). In Ngb-overexpressing transgenic ani-

mals, the size of cerebral infarct is drastically reduced (79, 80),

and the cell survival is enhanced under conditions of Alzheimer

disease, anoxia, and oxygen and glucose deprivation (69, 73,

78, 79). Similarly, intracerebral administration of Ngb-express-

ing adeno-associated virus vector reduces the infarct size in rats

after focal cerebral ischemia, with opposite effects observed fol-

lowing knockdown of endogenous Ngb in rat brains (78). More-

over, the neuroprotective role of endogenous Ngb has been sup-

ported by its knockdown in vitro, which renders cortical neuro-

nal cultures more susceptible to hypoxia (72) decreasing

viability of neuroblastoma cells under oxidative stress (81).

However, some questions have been raised concerning the

capacity of Ngb to provide general protection to neurons in vivo

(82).

Contradictions in data derived from in vivo experiments

almost certainly arise, at least in major part, from differences in

the nature, severity, and duration of challenge used in the vari-

ous studies. Any proposed mechanism of action of Ngb, in neu-

rons, thus must consider not only its capacity to provide a level

of protection to many cell types in the brain but also account

for its very nonuniform distribution in the brain.

However, all the neuroprotective effects associated to Ngb

are correlated with its ability to prevent the neuronal apoptotic

death. It has been proposed that the neuroprotective effect of

Ngb is the result of the reduction of ferric cytochrome c by fer-

rous Ngb, thereby preventing cytochrome c-induced apoptosis

(35, 41). This suggests that the physiological role of Ngb could

be to reset the trigger level for the postmitochondrial execution

of apoptosis (44, 77).

17b-ESTRADIOL AND NEUROPROTECTION

Besides the female reproductive tract, the brain is an impor-

tant target for estrogen actions. Indeed, the low estrogen levels

present in postmenopausal women have been associated to diffi-

culties in memory as well as deficits in fine motor coordination,

reduction in reaction times, feelings of depression and anxiety,

and Alzheimer’s disease (83–86). Remarkably, estrogens play a

pivotal role also in the male brain because 17b-estradiol (E2,

the most efficient within estrogens) is synthesized locally from

steroid precursors, including circulating testosterone, by P450-

aromatase enzyme (87–89).

As an example, E2 neuroprotection against b-amyloid toxic-

ity and glutamate-mediated oxidative stress (90) occurs via its

nuclear receptors (i.e., ERa and ERb), which are widely distrib-

uted in the brain of males as well as females (89, 91–95). ER-

mediated cellular responses to E2 have been loosely grouped

into two interconnected categories: genomic and rapid extranu-

141NEUROGLOBIN, ESTROGENS, AND NEUROPROTECTION

clear signals (96), which involve the rapid stimulation of the

Src-protein tyrosine kinase and mitogen-activated protein kinase

(MAPK) pathways (95).

Although there are contradictory reports on the relative con-

tributions of ERa and ERb to the neuroprotective effects of

estrogens in most disease models (93, 97), evidence is emerging

that both ERs have protective capacity, but they operate via dif-

ferent mechanisms and possibly in different time frames. For

example, in ischemic brain injury and experimental autoimmune

encephalomyelitis, ERa is induced early, whereas ERb is

induced later (98, 99). Considerable attention is now focused on

which ER signaling pathway controls various E2-induced pro-

tective mechanisms ranging from antiapoptotic, neurotrophic,

and neurogenic actions. These mechanisms suppress neuroin-

flammation, which accompanies the progression, if not initia-

tion, of so many pathological brain conditions, including Par-

kinson’s disease, Alzheimer’s disease, stroke, and multiple scle-

rosis (85, 89, 100–103).

Notably, several of the above reported E2 protective effects in

brain overlap that described for Ngb raising the intriguing possibil-

ity that E2 could act as an possible endogenous Ngb modulator.

NEUROGLOBIN LEVELS ARE UPREGULATEDBY 17b-ESTRADIOL VIA ERb

The hypothesis that E2 could modulate endogenous Ngb lev-

els has been verified in both human neuroblastoma cell line and

mouse primary hippocampal neurons. E2 increases up to 300%

Ngb levels in a dose-dependent manner (with the maximum

effect at E2 physiological concentration, i.e., 1–10 nM). The E2

effect was rapid (1 h), persistent (24 h), and specific, being not

mimicked by either the male sex steroid hormone dihydrotestos-

terone or by the common precursor testosterone.

These results represent the first evidence for steroid hormone

modulation of globin levels in cells (104). Recently, it has been

reported that Hb is specifically expressed in neurons, its expres-

sion being upregulated by erythropoietin and accompanied by

enhanced brain oxygenation under physiologic and hypoxic con-

ditions (33). At present, the relationship between Hb and Ngb in

neurons is still unclear. Although Hb and Ngb are expressed in

the same nerve cells, Ngb levels are not increased by erythro-

poietin (33). It is, therefore, unlikely that they have a tightly

linked function, for example, in facilitated oxygen transport;

however, Hb and Ngb could fulfill independent tasks in neurons.

It is now well known that E2 has numerous effects on the

male brain throughout the lifespan, beginning during gestation

and continuing on into senescence (89, 105). Although it is pos-

sible that the experimental models we used (i.e., neuroblastoma

cell line and embryonic mouse hippocampal neurons) do not

express P450-aromatase causing the inability of testosterone to

modulate Ngb levels, the reported results firmly indicate Ngb as

a new E2 target that should be added to the variety of E2-spe-

cific actions in the brain (104).

Human neuroblastoma cell line and mouse primary hippo-

campal neurons express different levels of both ER isoforms.

However, the effect of E2 on Ngb levels specifically requires

all ERb activities. In fact, ERb extranuclear and genomic sig-

nals crosstalk each other to guarantee both the rapid and the

persistent E2 effects. In particular, the rapid (15 min) and per-

sistent (24 h) ERb-mediated p38 activation, a MAPK family

component, is required for E2-induced Ngb increase. Thus,

although Ngb promoter does not contain any canonical ERE, it

is not surprising that the transcription inhibitor actinomycin

completely prevents the increase of the E2-induced Ngb levels.

However, the sequence analysis of Ngb promoter indicates that

several noncanonical half ERE sites are present along with re-

sponsive element for other transcription factors (104). These

results suggest that the E2-induced Ngb transcription could be

mediated by tethered interactions of ER with other transcription

factors to activate gene expression (i.e., indirect genomic mech-

anism) (96). Consequently, the integration between extranuclear

and genomic ERb-mediated events is required to provide plas-

ticity for this neuronal response to E2.

NEUROGLOBIN IS PART OF E2-INDUCED SIGNALSPROTECTING NEURONS FROM APOPTOSIS

The E2 effect in enhancing Ngb levels prompted us to

explore the role played by Ngb in E2-induced neuroprotection.

Exposure to H2O2 (50 lM) induces neuroblastoma cell death,

which is accompanied by a dramatic increase in caspase-3 acti-

vation. The cell pretreatment with physiological E2 concentra-

tion decreases cell death and reduced caspase-3 activation trig-

gers by exposure to H2O2 (104). This E2 effect against H2O2

toxicity requires ERb and Ngb being prevented by the ERb in-

hibitor (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol

and by knocking out Ngb using small interfering RNA (104).

Although this observation does not solve the problem of the

antiapoptotic mechanism of neuroprotection by Ngb, it has been

recently shown that the elevation of human Ngb expression in neu-

rons prior to insult with H2O2 enhances cell viability and results in

a significant decrease in oxidative stress and an increased intracel-

lular ATP concentration (106). In addition, a linkage of Ngb to the

oxidative metabolism has been proposed (24). These data are

strongly suggestive of the involvement of Ngb in the E2-induced

attenuated ATP depletion. Furthermore, in the presence of Ngb,

the initially released cytochrome c may be sequestered by the Ngb

(77) and the apoptotic cascade avoided (35, 44, 79).

CONCLUSIONS AND PERSPECTIVES

Although it is well recognized that the overexpression of

Ngb in transgenic animals reduces the size of cerebral infarct

(79, 80) and enhances cell survival after different brain injuries

(69, 73, 78, 79), the elucidation of the neuronal Ngb function(s)

requires that its expression is regulated by endogenous

modulator(s). Strikingly, we found that E2 stimulates Ngb levels

in human and mouse neurons via ERb-dependent rapid and

genomic signals that require p38 activation (Fig. 1).

Thus, the well-known neuroprotective effects elicited by E2


(85, 89, 100–102) may, at least in part, be explained by an

enhanced Ngb expression in neurons.

In the next future, different strategies (e.g., E2-induced Ngb

localization, association between Ngb and ERs, and E2-induced

ERs-mediated Ngb promoter activity) will substantiate Ngb

action in neurons, our laboratories being active in this field.

However, from now, Ngb can be regarded as part of signals

activated by E2 to exert neuroprotective effects definitely vali-

dating the role played by Ngb as an antiapoptotic neuroprotec-

tive globin.

Finally, the finding that Ngb is an E2-inducible nerve globin

opens new avenues in the Ngb research field in that a strong

reevaluation of its anatomical and subcellular localization is

necessary. In addition, the possibility that other hormones and

neurotransmitters may regulate Ngb levels in brain and in other

tissues provides a potential new opportunity for the develop-

ment of neuroprotective drugs.

ACKNOWLEDGEMENTS

This work was partly supported by a grant from Italian Ministry

of Health (Strategico 2008 to M.M.).

REFERENCES1. Bolognesi, M., Bordo, D., Rizzi, M., Tarricone, C., and Ascenzi, P.

(1997) Nonvertebrate hemoglobins: Structural bases for reactivity.

Prog. Biophys. Mol. Biol. 68, 29–68

2. Wittemberg, J. B., Bolognesi, M., Wittemberg, B. A., and Guertin, M.

(2002) Truncated hemoglobins: a new family of hemoglobins widely

distribuited in bacteria, unicellular eukaryotes, and plants. J. Biol.

Chem. 277, 871–874.

3. Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K.

(2002) Global allostery model of hemoglobin: modulation of O2 affin-

ity, cooperativity, and Bohr effect by heterotropic allosteric effectors.

J. Biol. Chem. 277, 34508–34520.4. Wittenberg, J. B., and Wittenberg, B. A. (2003) Myoglobin function

reassessed. J. Exp. Biol. 206, 2011–2020.5. Ascenzi, P., Bolognesi, M., Milani, M., Guertin, M., and Visca, P.

(2007) Mycobacterial truncated hemoglobins: from genes to functions.

Gene 398, 42–51.

6. Ascenzi, P., Bellelli, A., Coletta, M., Colosimo, A., Falcioni, G., Gia-

cometti, G. M., Ippoliti, R., Zolla, L., and Giardina, B. (2007) Multiple

strategies for O2 transport: from simplicity to complexity. IUBMB Life

59, 600–616.

7. Bonamore, A., and Boffi, A. (2008) Flavohemoglobin: structure and

reactivity. IUBMB Life 60, 19–28.

8. Hoy, J. A., and Hargrove, M. S. (2008) The structure and function of

plant hemoglobins. Plant. Physiol. Biochem. 46, 371–379.9. Pullan, S. T., Monk, C. E., Lee, L., and Poole, R. K. (2008) Microbial

responses to nitric oxide and nitrosative stress: Growth, ‘‘omic’’, and

physiological methods. Methods Enzymol. 437, 499–519.10. Vinogradov, S. N., and Moens, L. (2008) Diversity of globin function:

Enzymatic, transport, storage, and sensing. J. Biol. Chem. 283, 8773–

8777.

11. Wajcman, H., Kiger, L., and Marden, M. C. (2009) Structure and

function evolution in the superfamily of globins. C. R. Biol. 332, 273–

282.

12. Nardini, M., Pesce, A., Milani, M., and Bolognesi, M. (2007) Protein

fold and structure in the truncated (2/2) globin family. Gene 398, 2–11.

13. Vandergon, T. L., and Riggs, A. F. (2008) Mini-hemoglobins from

nemertean worms. Methods Enzymol. 436, 503–509.14. Burmester, T., Weich, B., Reinhardt, S., and Hankeln, T. (2000) A ver-

tebrate globin expressed in brain. Nature 407, 520–523.

15. Weber, R. E., and Vinogradov, S. N. (2001) Nonvertebrate hemoglo-

bins: Functions and molecular adaptations. Physiol. Rev. 81, 569–628.16. Burmester, T., Ebner, B., Weich, B., and Hankeln, T. (2002) Cytoglo-

bin: A novel globin type ubiquitously expressed in vertebrate tissues.

Mol. Biol. Evol. 19, 416–421.17. Burmester, T., Haberkamp, M., Mitz, S., Roesner, A., Schmidt, M.,

Ebner, B., Gerlach, F., Fuchs, C., and Hankeln, T. (2004). Neuroglobin

and cytoglobin: genes, proteins and evolution. IUBMB Life 56, 703–707.

18. Egawa, T., and Yeh, S. R. (2005) Structural and functional properties

of hemoglobins from unicellular organisms as revealed by resonance

Raman spectroscopy. J. Inorg. Biochem. 99, 72–96.19. Geuens, E., Dewilde, S., Hoogewijs. D., Pesce, A., Nienhaus, K.,

Nienhaus, G. U., Olson, J., Vanfleteren, J., Bolognesi, M., and Moens,

L. (2004) Nerve globins in invertebrates. IUBMB Life 56, 653–656.

20. Burmester, T., and Hankeln, T. (2008) Neuroglobin and other nerve hae-

moglobins. In Dioxygen binding and sensing proteins. Bolognesi, M., di

Prisco, G., and Verde, C. (eds.)., Springer, Heidelberg, pp. 211–222.

21. Burmester, T., Gerlach, F., and Hankeln, T. (2007). Regulation and

role of neuroglobin and cytoglobin under hypoxia. Adv. Exp. Med.

Biol. 618, 169–180.

22. Williams, T. M., Zavanelli, M., Miller, M. A., Goldbeck, R. A., Mor-

ledge, M., Casper, D., Pabst, D. A., McLellan, W., Cantin, L. P., and

Kliger, D. S. (2008) Running, swimming and diving modifies neuropro-

tecting globins in the mammalian brain. Proc. Biol. Sci. 275, 751–758.

23. Cheng, C. H., di Prisco, G., and Verde, C. (2009) The ‘‘icefish para-

dox’’: which is the task of neuroglobin in Antarctic hemoglobin-less

icefish? IUBMB Life 61, 184–188.

24. Mitz, S. A., Reuss, S., Folkow, L. P., Blix, A. S., Ramirez, J. M., Han-

keln, T., and Burmester, T. (2009) When the brain goes diving: glial

oxidative metabolism may confer hypoxia tolerance to the seal brain.

Neuroscience 163, 552–560.

25. Avivi, A., Gerlach, F., Joel, A., Reuss, S., Burmester, T., Nevo, E.,

and Hankeln, T. (2010) Neuroglobin, cytoglobin, and myoglobin con-

Figure 1. E2-induced Ngb participates in the mechanisms

involved in hormone-based protective effects against H2O2-

induced neurotoxicity.


tribute to hypoxia adaptation of the subterranean mole rat Spalax.


26. Vinogradov, S. N., Hoogewijs, D., Bailly, X., Arredondo-Peter, R.,

Gough, J., Dewilde, S., Moens L, and Vanfleteren, J. R. (2006) A phy-

logenomic profile of globins. BMC Evol. Biol. 6, 31.

27. Lankester, E. R. (1872) A contribution to the knowledge of haemoglo-

bin. Proc. Royal Soc. London 21, 70–81.

28. Hundahl, C., Fago, A., Dewilde, S., Moens, L., Hankeln, T.,

Burmester, T., and Weber, R. E. (2006) Oxygen binding properties of

non-mammalian nerve globins. FEBS J. 273, 1323–1329.

29. Kugelstadt, D., Haberkamp, M., Hankeln, T., and Burmester, T. (2004)

Neuroglobin, cytoglobin and a novel, retina-specific globin from

chicken. Biochem. Biophys. Res. Commun. 325, 719–725.

30. Roesner, A., Fuchs, C., Hankeln, T., and Burmester, T. (2005) A globin

gene of ancient evolutionary origin in lower vertebrates: evidence for

two distinct globin gene families in animals. Mol. Biol. Evol. 22, 12–22.

31. Fuchs, C., Burmester, T., and Hankeln, T. (2006) The amphibian glo-

bin gene repertoire as revealed by Xenopus genome sequence. Cytoge-

net. Genome Res. 112, 296–306.

32. Biagioli, M., Pinto, M., Cesselli, D., Zaninello, M., Lazarevic, D., Ron-

caglia, P., Simone, R., Vlachouli, C., Plessy, C., Bertin, N., Feltrami,

A., Kobayashi, K., Gallo, V., Santoro, C., Ferrer, I., Rivella, S., Fel-

trami, C. A., Carninci, P., Raviola, E., and Gustincich, S. (2009) Unex-

pected expression of a- and b-globin in mesencephalic dopaminergic

neurons and glial cells. Proc. Natl. Acad. Sci. USA 106, 15454–15459.

33. Schelshorn, D. W., Schneider, A., Kuschinsky, W., Weber, D., Kruger,

C., Dittgen, T., Burgers, H. F., Sabouri, F., Gassler, N., Bach, A., and

Maurer, M. H. (2009) Expression of hemoglobin in rodent neurons. J.

Cereb. Blood Flow Metab. 29, 585–595.

34. Richter, F., Meurers, B. H., Zhu, C., Medvedeva, V. P., and Chesselet,

M. F. (2009) Neurons express hemoglobin a- and b-chains in rat and

human brains. J. Comp. Neurol. 515, 538–547.

35. Fago, A., Mathews, A. J., and Brittain, T. (2008) A Role for neuroglo-

bin: resetting the trigger level for apoptosis in neuronal and retinal

cells. IUBMB Life 60, 398–401.

36. Hundahl, C. A., Allen, G. C., Hannibal, J., Kjaer, K., Rehfeld, J. F.,

Dewilde, S., Nyengaard, J. R., Kelsen, J., and Hay-Schmidt, A. (2010)

Anatomical characterization of cytoglobin and neuroglobin mRNA and

protein expression in the mouse brain. Brain Res. 1331, 58–73.

37. Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U., and

Burmester, T. (2003) How does the eye breathe? Evidence for neuro-

globin-mediated oxygen supply in the mammalian retina. J. Biol.

Chem. 278, 1932–1935.

38. Bentmann, A., Schmidt, M., Reuss, S., Wolfrum, U., Hankeln, T., and

Burmester, T. (2005) Divergent distribution in vascular and a-vascular

mammalian retinae links neuroglobin to cellular respiration. J. Biol.Chem. 280, 20660–20665.

39. Trent, J. T. III, and Hargrove, M. S. (2002) A ubiquitously expressed

human hexacoordinate hemoglobin. J. Biol. Chem. 277, 19538–19545.

40. Geuens, E., Brouns, I., Flamez, D., Dewilde, S., Timmermans, J. P.,

and Moens L. (2003) A globin in the nucleus! J. Biol. Chem. 278,

30417–30420.

41. Fago, A., Mathews, A. J., Moens, L., Dewilde, S., and Brittain, T.

(2006) The reaction of neuroglobin with potential redox protein part-

ners cytochrome b5 and cytochrome c. FEBS Lett. 580, 4884–4888.

42. Nicolis, S., Monzani, E., Ciaccio, C., Ascenzi, P., Moens, L., and

Casella, L. (2007) Reactivity and endogenous modification by nitrite

and hydrogen peroxide: does human neuroglobin act only as a scav-

enger? Biochem. J. 407, 89–99.

43. Burmester, T, and Hankeln, T. (2009) What is the function of neuro-

globin? J. Exper. Biol. 212, 1423–1428.

44. Raychaudhuri, S., Skommer, J., Henty, K., Birch, N., and Brittain, T.

(2010) Neuroglobin protects nerve cells from apoptosis by inhibiting

the intrinsic pathway of cell death. Apoptosis 15, 401–411.

45. Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln,

T., Burmester, T., and Bolognesi, M. (2003) Human brain neuroglobin

structure reveals a distinct mode of controlling oxygen affinity. Struc-

ture 11, 1087–1095.

46. Vallone, B., Nienhaus, K., Matthes, A., Brunori, M., and Nienhaus, G.

U. (2004) The structure of carbonmonoxy neuroglobin reveals a heme-

sliding mechanism for control of ligand affinity. Proc. Natl. Acad. Sci.

USA 101, 17351–17356.

47. Kiger, L., Uzan, J., Dewilde, S., Burmester, T., Hankeln, T., Moens,

L., Hamdane, D., Baudin-Creuza, V., and Marden, M. (2004) Neuro-

globin ligand binding kinetics. IUBMB Life 56, 709–719.

48. Pesce, A., De Sanctis, D., Nardini, M., Dewilde, S., Moens, L., Hankeln,

T., Burmester, T., Ascenzi, P., and Bolognesi, M. (2004) Reversible

hexa- to penta-coordination of the heme Fe atom modulates ligand bind-

ing properties of neuroglobin and cytoglobin. IUBMB Life 56, 657–664.

49. Brunori, M., and Vallone, B. (2006) A globin for the brain. FASEB J.

20, 2192–2197.

50. Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V.,

Aerts, T., Marden, M. C., Caubergs, R., and Moens, L. (2001) Biochemi-

cal characterization and ligand binding properties of neuroglobin, a novel

member of the globin family. J. Biol. Chem. 276, 38949–38955.

51. Trent, J. T. III, Watts, R. A., and Hargrove, M. S. (2001) Human neu-

roglobin, a hexacoordinate hemoglobin that reversibly binds oxygen. J.Biol. Chem. 276, 30106–30110.

52. Hamdane, D., Kiger, L., Dewilde, S., Green, B. N., Pesce, A.,

Uzan, J., Burmester, T., Hankeln, T., Bolognesi, M., Moens, L.,

and Marden, M. C. (2003) The redox state of the cell regulates the

ligand binding affinity of human neuroglobin and cytoglobin. J. Biol.Chem. 278, 51713–51721.

53. Fago, A., Hundahl, C., Malte, H., and Weber, R. E. (2004) Functional

properties of neuroglobin and cytoglobin: insights into the ancestral

physiological roles of globins. IUBMB Life 56, 689–696.

54. Herold, S., Fago, A., Weber, R. E., Dewilde, S., and Moens, L. (2004)

Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuro-

globin reveal a potential role against oxidative stress. J. Biol. Chem.279, 22841–22847.

55. Trandafir, F., van Doorslaer, S., Dewilde, S., and Moens, L. (2004)

Temperature dependence of NO binding modes in human neuroglobin.

Biochim. Biophys. Acta 1702, 153–161.

56. Brunori, M., Giuffre, A., Nienhaus, K., Nienhaus, G. U., Scandurra, F.

M., and Vallone B. (2005) Neuroglobin, nitric oxide, and oxygen:

functional pathways and conformational changes. Proc. Natl. Acad.

Sci. USA 102, 8483–8488.

57. Fago, A., Mathews, A. J., Dewilde, S., Moens, L., and Brittain, T.

(2006) The reactions of neuroglobin with CO: Evidence for two forms

of the ferrous protein. J. Inorg. Biochem. 100, 1339–1343.

58. Brittain, T., Yosaatmadja, Y., and Henty, K. (2008) The interaction of

human neuroglobin with hydrogen sulphide. IUBMB Life 60, 135–138.

59. Giuffre, A., Moschetti, T., Vallone, B., and Brunori, M. (2008) Neuro-

globin: enzymatic reduction and oxygen affinity. Biochem. Biophys.

Res. Commun. 367, 893–898.

60. Kakar, S., Hoffman, F. G., Storz, J. F., Fabian, M., and Hargrove, M.

S. (2010) Structure and reactivity of hexacoordinate hemoglobins. Bio-

phys. Chem. 152, 1–14.

61. Moens, L., and Dewilde, S. (2000) Globins in the brain. Nature 407,

461–462.

62. Reuss, S., Saaler-Reinhardt, S., Weich, B., Wystub, S., Reuss, M. H.,

Burmester, T., and Hankeln, T. (2002) Expression analysis of neuro-

globin mRNA in rodent tissues. Neuroscience 115, 645–656.

63. Wystub, S., Laufs, T., Schmidt, M., Burmester, T., Maas, U., Saaler-

Reinhardt, S., Hankeln, T., and Reuss, S. (2003) Localization of neuro-

globin protein in the mouse brain. Neurosci. Lett. 346, 114–116.

64. Burmester, T., and Hankeln, T. (2004) Neuroglobin: A respiratory pro-

tein of the nervous system. News Physiol. Sci. 19, 110–113.


65. Hankeln, T., Wystub, S., Laufs, T., Schmidt, M., Gerlach, F., Saaler-

Reinhardt, S., Reuss, S., and Burmester, T. (2004) The cellular and

subcellular localization of neuroglobin and cytoglobin–a clue to their

function? IUBMB Life 56, 671–679.

66. Hankeln, T., Ebner, B., Fuchs, C., Gerlach, F., Haberkamp, M., Laufs, T.

L., Roesner, A., Schmidt, M., Weich, B., Wystub, S., Saaler-Reinhardt,

S., Reuss, S., Bolognesi, M., De Sanctis, D., Marden, M. C., Kiger, L.,

Moens, L., Dewilde, S., Nevo, E., Avivi, A., Weber, R. E., Fago, A.,

and Burmester, T. (2005) Neuroglobin and cytoglobin in search of their

role in the vertebrate globin family. J. Inorg. Biochem. 99, 110–119.67. Emara, M., Turner, A. R., and Allalunis-Turner, J. (2010) Hypoxic

regulation of cytoglobin and neuroglobin expression in human normal

and tumor tissues. Cancer Cell Int. 10, 33–49.68. Ostojic, J., Sakaguchi, D. S., de Lathouder, Y., Hargrove, M. S., Trent,

J. T. 3rd, Kwon, Y. H., Kardon, R. H., Kuehn, M. H., Betts, D. M., and

Grozdanic, S. (2006) Neuroglobin and cytoglobin: oxygen-binding pro-

teins in retinal neurons. Invest. Ophthalmol. Vis. Sci. 47, 1016–1023.69. Fordel, E., Thijs, L., Moens, L., and Dewilde, S. (2007) Neuroglobin

and cytoglobin expression in mice: Evidence for a correlation with re-

active oxygen species scavenging. FEBS J. 274, 1312–1317.70. Nienhaus, K., and Nienhaus, G. U. (2007) Searching for neuroglobin’s

role in the brain. IUBMB Life 59, 490–497.

71. Greenberg, D. A., Jin, K., and Khan, A. A. (2008) Neuroglobin: an en-

dogenous neuroprotectant. Curr. Opin. Pharmacol. 8, 20–24.72. Jin, K., Mao, X. O., Xie, L., Khan, A. A., and Greenberg, D. A.

(2008) Neuroglobin protects against nitric oxide toxicity. Neurosci.

Lett. 430, 135–137.73. Khan, A. A., Mao, X. O., Banwait, S., DerMardirossian, C. M., Bokoch,

G. M., Jin, K., and Greenberg, D. A. (2008) Regulation of hypoxic neu-

ronal death signaling by neuroglobin. FASEB J. 22, 1737–1747.74. Petersen, M. G., Dewilde, S., and Fago, A. (2008) Reactions of ferrous

neuroglobin and cytoglobin with nitrite under anaerobic conditions. J.Inorg. Biochem. 102, 1777–1782.

75. Watanabe, S., and Wakasugi, K. (2008) Neuroprotective function of

human neuroglobin is correlated with its guanine nucleotide dissocia-

tion inhibitor activity. Biochem. Biophys. Res. Commun. 369, 695–700.

76. Yu, Z., Fan, X., Lo, E. H., and Wang, X. (2009) Neuroprotective roles

and mechanisms of neuroglobin. Neurol. Res. 31, 122–127.

77. Brittain, T., Skommer, J., Raychaudhuri, S., and Birch, N. (2010) An

antiapoptotic neuroprotective role for neuroglobin. Int. J. Mol. Sci. 11,

2306–2231.

78. Sun, Y., Jin. K., Peel, A., Mao, X. O., and Xie, L. (2003) Greenberg,

D. A. Neuroglobin protects the brain from experimental stroke in vivo.


79. Khan, A. A., Wang, Y., Sun, Y., Mao, X. O., Xie, L., Miles, E., Gra-

boski, J., Chen, S., Ellerby, L. M., Jin, K., Greenberg, D. A. (2006) Neu-

roglobin-overexpressing transgenic mice are resistant to cerebral and

myocardial ischemia. Proc. Natl. Acad. Sci. USA 103, 17944–17948.

80. Wang, X., Liu, J., Zhu, H., Tejima, E., Tsuji, K., Murata, Y., Atochin,

D. N., Huang, P. L., Zhang, C., and Lo, E. H. (2008). Effects of neu-

roglobin overexpression on acute brain injury and long-term outcomes

after focal cerebral ischemia. Stroke 39, 1869–1874.

81. Ye, S. Q., Zhou, X. Y., Lai, X. J., Zheng, L., and Chen, X. Q. (2009)

Silencing neuroglobin enhances neuronal vulnerability to oxidative injury

by down-regulating 14–3-3gamma. Acta Pharmacol. Sin. 30, 913–918.

82. Hundahl, C., Kelsen, J., Ronn, L. C., Weber, R. E., Geuens, E., Hay-

Schmidt, A., and Nyengaard, J. R. (2006) Does neuroglobin protect

neurons from ischemic insult? A quantitative investigation of neuroglo-

bin expression following transient MCAO in spontaneously hyperten-

sive rats. Brain Res. 1085, 19–27.

83. Behl, C., Skutella, T., Lezoualc’h, F., Post, A., Widmann, M., Newton, C.

J., and Holsboer, F. (1997) Neuroprotection against oxidative stress by

estrogens: structure-activity relationship. Mol. Pharmacol. 51, 535–541.

84. McEwen, B. S., and Alves, S. E. (1999) Estrogen actions in the central

nervous system. Endocr. Rev. 20, 279–307.

85. McEwen, B. S. (2002) Sex, stress and the hippocampus: Allostasis,

allostatic load and the aging process. Neurobiol. Aging 23, 921–939.

86. Brinton, R. D. (2004) Impact of estrogen therapy on Alzheimer’s dis-

ease: A fork in the road? CNS Drugs 18, 405–422.87. Sharpe, R. M. (1998) The roles of oestrogen in the male. Trends Endo-

crinol. Metab. 9, 371–377.88. Jones, M. E., Boon, W. C., Proietto, J., and Simpson, E. R. (2006) Of

mice and men: the evolving phenotype of aromatase deficiency. Trends

Endocrinol. Metab. 17, 55–64.89. Gillies, G. E., and McArthur, S. (2010) Estrogen actions in the brain

and the basis for differential action in men and women: A case for

sex-specific medicines. Pharmacol. Rev. 62, 155–198.90. Fitzpatrick, J. L., Mize, A. L., Wade, C. B., Harris, J. A., Shapiro, R. A.,

and Dorsa, D. M. (2002) Estrogen-mediated neuroprotection against

beta-amyloid toxicity requires expression of estrogen receptor a or b and

activation of the MAPK pathway. J. Neurochem. 82, 674–682.91. Toran-Allerand, C. D. (2005) Estrogen and the brain: beyond ER-a,

ER-b, and 17b-estradiol. Ann. NY. Acad. Sci. 1052, 136–144.92. Balthazart, J., and Ball, G. F. (2006) Is brain estradiol a hormone or a

neurotransmitter? Trends Neurosci. 29, 241–249.93. Brann, D. W., Dhandapani, K., Wakade, C., Mahesh, V. B., and Khan,

M. M. (2007) Neurotrophic and neuroprotective actions of estrogen:

basic mechanisms and clinical implications. Steroids 72, 381–405.94. Micevych, P. and Dominguez, R. (2009) Membrane estradiol signaling

in the brain. Front. Neuroendocrinol. 30, 315–327.95. Singer, C. A., Figueroa-Masot, X. A., Batchelor, R. H., and Dorsa, D.

M. (1999) The mitogen-activated protein kinase pathway mediates

estrogen neuroprotection after glutamate toxicity in primary cortical

neurons. J. Neurosci. 19, 2455–2463.

96. Ascenzi, P., Bocedi, A., and Marino, M. (2006) Structure-function

relationship of estrogen receptor a and b: Impact on human health.

Mol. Aspects Med. 27, 299–402.

97. Suzuki, S., Brown, C. M., and Wise, P. M. (2009) Neuroprotective effects of

estrogens following ischemic stroke. Front. Neuroendocrinol. 30, 201–211.

98. Suzuki, A., Sugihara, A., Uchida, K., Sato, T., Ohta, Y., Katsu, Y.,

Watanabe, H., and Iguchi, T. (2002) Developmental effects of perinatal

exposure to bisphenol-A and diethylstilbestrol on reproductive organs

in female mice. Reprod. Toxicol. 16, 107–116.

99. Tiwari-Woodruff, S., and Voskuhl, R. R. (2009) Neuroprotective and

anti-inflammatory effects of estrogen receptor ligand treatment in

mice. J. Neurol. Sci. 286, 81–85.

100. Marin, R., Ramirez, C., Morales, A., Gonzalez, M., Alonso, R., and Diaz,

M. (2008) Modulation of Ab-induced neurotoxicity by estrogen receptor aand other associated proteins in lipid rafts. Steroids 73, 992–996.

101. Morissette, M., Le Saux, M., D’Astous, M., Jourdain, S., Al Sweidi, S.,

Morin, N., Estrada-Camarena, E., Mendez, P., Garcia-Segura, L. M., and Di

Paolo, T. (2008) Contribution of estrogen receptors a and b to the effects of

estradiol in the brain. J. Steroid Biochem. Mol. Biol. 108, 327–338.

102. Vasudevan, N., and Pfaff, D. W. (2008) Non-genomic actions of estro-

gens and their interaction with genomic actions in the brain. Front.Neuroendocrinol. 29, 238–257.

103. Marin, R., Diaz, M., Alonso, R., Sanz, A., Arevalo, M. A., and Gar-

cia-Segura, L. M. (2009) Role of estrogen receptor a in membrane-ini-

tiated signaling in neural cells: Interaction with IGF-1 receptor. J. Ste-

roid Biochem. Mol. Biol. 114, 2–7.

104. De Marinis, E., Ascenzi, P., Pellegrini, M., Galluzzo, P., Bulzomi, P.,

et al. (2011) 17b-Estradiol, a new modulator of neuroglobin levels in neu-

rons: effects against H2O2-induced neurotoxicity.Neurosignals, in press.

105. Swaab, D. F. (2007) Sexual differentiation of the brain and behavior.

Best Pract. Res. Clin. Endocrinol. Metab. 21, 431–444

106. Antao, S. T., Duong, T. T., Aran, R., and Witting, P. K. (2010) Neuro-

globin overexpression in cultured human neuronal cells protects

against hydrogen peroxide insult via activating phosphoinositide-3 ki-

nase and opening the mitochondrial K(ATP) channel. Antioxid. Redox

Signal. 13, 769–781


Fax +41 61 306 12 34E-Mail [email protected]

Original Paper

Neurosignals 2010;18:223–235 DOI: 10.1159/000323906

17 � -Estradiol – A New Modulator of Neuroglobin Levels in Neurons: Role in Neuroprotection againstH 2 O 2 -Induced Toxicity

Elisabetta De Marinis a Paolo Ascenzi a, b Marco Pellegrini a Paola Galluzzo a Pamela Bulzomi a Maria Angeles Arevalo c Luis Miguel Garcia-Segura c Maria Marino a

a Department of Biology and b Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Rome , Italy; c Instituto Cajal, CSIC, Madrid , Spain

these data suggest that Ngb is part of the E 2 signaling mech-anism that is activated to exert protective effects against H 2 O 2 -induced neurotoxicity.

Copyright © 2011 S. Karger AG, Basel

Introduction

Neuroglobin (Ngb), the third member of the globin family [1] , is a monomeric hexa-coordinated heme pro-tein of 17 kDa expressed not only in neurons of the cen-tral and peripheral nervous systems, but also in the gas-trointestinal tract and in endocrine organs [1–18] . Re-cently, Ngb has also been detected in human glioblastoma cell lines [17] and in quiescent astrocytes of the healthy seal brain [15] . Although Ngb occurs at relatively low con-centrations ( � M ) in a wide range of tissues, Ngb is found at relatively high concentrations in highly metabolically active cells and certain specialized cells, such as neurons of the hypothalamus, and particularly in retinal rod cells where its concentration has been estimated to be up to 100 � M [18–21] . Ngb binds several ligands, including di-atomic gaseous ligands, and displays (pseudo-)enzymatic properties [1, 3, 6, 10, 22–33] . The P 50 value for O 2 binding

Key Words Neuroglobin � Estrogen receptor � H 2 O 2 neurotoxicity � Neuroprotection � Apoptosis

Abstract Although discovered in 2000, neuroglobin (Ngb) functions are still uncertain. A contribution to the role played by Ngb in neurons could certainly derive from the identification of Ngb endogenous modulators. Here, we evaluate the possi-bility that Ngb could be regulated by 17 � -estradiol (E 2 ) sig-naling in both SK-N-BE human neuroblastoma cell line and mouse hippocampal neurons. 1 n M E 2 rapidly induced a 300% increase in Ngb levels in both models. The E 2 effect was specific, being not induced by testosterone or dihy-drotestosterone. The E 2 -induced Ngb increase requires es-trogen receptor (ER) � , but not ER � , as evaluated by the mi-metic effect of ER � -specific agonist DPN and by the block-age of E 2 effect in ER � -silenced SK-N-BE cells. Furthermore, both rapid (15 min) ER � -dependent activation of p38/MAPK and transcriptional ER � activity were required for the estro-genic regulation of Ngb. Finally, E 2 exerted a protective ef-fect against H 2 O 2 -induced neuroblastoma cell death which was completely prevented in Ngb-silenced cells. Overall,

Received: November 17, 2010 Accepted: December 28, 2010 Published online: February 18, 2011

Maria Marino Department of Biology, University Roma Tre Viale Guglielmo Marconi 446 IT–00146 Rome (Italy) Tel. +39 06 57 336 345, E-Mail m.marino @ uniroma3.it

© 2011 S. Karger AG, Basel1424–862X/10/0184–0223$26.00/0

Accessible online at:www.karger.com/nsg

De Marinis /Ascenzi /Pellegrini /Galluzzo /Bulzomi /Arevalo /Garcia-Segura /Marino

Neurosignals 2010;18:223–235224

to Ngb has been reported to range between 2 and 10 Torr depending on pH, temperature, and the redox state of the cell [1, 22–24, 32] .

Although discovered in 2000 [1] , the cell function(s) of Ngb is still controversial. Indeed, the O 2 supply by Ngb to the mitochondria of the metabolically active neurons and retinal rod cells is highly debated [1, 15, 19, 33, 34] . In vi-tro, Ngb has been reported to scavenge nitrogen monox-ide (NO) in the presence of high O 2 levels [25, 28, 35] ; however, at low O 2 conditions Ngb may react with NO 2 – resulting in the formation of NO [36] . Therefore, the pro-tective role of Ngb against NO in vivo is controversial [32, 33, 37] . Although in vitro Ngb does not react with hydro-gen peroxide (H 2 O 2 ) [25, 30] and the Ngb-NO 2 adduct reacts with H 2 O 2 facilitating the nitration of aromatic substrates [30] , the correlation between reactive oxygen species formation/decomposition and Ngb expression in vivo is debated [15, 38] . Moreover, Ngb has been reported to interact with several proteins (see [ 15] ). In particular, Ngb binding to the G � protein inhibits GDP dissociation, thereby protecting cells from apoptosis [39, 40] . Recently, in silico simulations indicate Ngb capability to reduce cy-tochrome c released from mitochondria suggesting its protective role against programmed cell death [19, 41–43] .

Although Ngb properties are highly debated [12, 15, 19, 33, 44] , it is unlikely that Ngb has so many distinct roles [15] ; nevertheless, there is no doubt that Ngb is ben-eficial to neurons [15] . In vivo experiments, using trans-genic rodents, have shown that increased levels of Ngb significantly protect both heart and brain tissues from hypoxic insult, whereas decreased Ngb levels lead to an exacerbation of tissue death [45–47] . In this way, Ngb could protect neurons from hypoxic insult by modulating the activation of the apoptotic cascade [19, 41] . A signifi-cant contribution to highlight the role played by Ngb in neuroprotection could derive from the identification of Ngb endogenous modulator(s) (e.g., hormones and neu-rotransmitters), but, as far as we know, no Ngb involve-ment in the hormone signal transduction pathways has been identified yet.

Female sex steroid hormones could represent good candidates as Ngb modulators. Indeed, in addition to their well-established role in reproductive organs, es-trogens affect areas of the brain that are not primarily involved in reproduction [48] . Growing evidence do-cuments profound effects of estrogens on learning, mem-ory, and mood as well as neurodevelopmental and neurodegenerative processes [49, 50] . Although most studies have been conducted on females, there is mount-ing recognition that estrogens play important roles in the

male brain, where they can be generated from circulating testosterone by local aromatase or synthesized de novo by neurons and glia [51] . Several sources of evidence con-firm that estrogens serve as neurotrophic and neuropro-tective agents. Notably, 17 � -estradiol (E2) attenuates the toxicity of the amyloid- � peptide and glutamate in a hip-pocampal cell line [52] . In addition, estrogen therapy in post-menopausal women is associated with decreasedincidence and enhanced recovery from ischemic stroke [51] . The protective effects of estrogens have been widely reported in different types of neuronal cells against a va-riety of insults, including H 2 O 2 [53, 54] , serum depriva-tion [55] , oxygen-glucose deprivation [56] , and iron [57] . Due to myriad and often tissue-specific estrogen effects, the precise molecular events that mediate these protec-tive actions are not fully understood. Here, we evaluate the possibility that Ngb could be part of 17 � -estradiol-induced signals and effects in neuronal cells.

Materials and Methods

Reagents E 2 , testosterone (T), 5 � -androstan-17 � -ol-3-one (dihydrotes-

tosterone, DHT), naringenin (Nar), insulin-like growth factor 1 (IGF-1), actinomycin D (Act), cycloheximide (Cxm), Pen-Strep solution, H 2 O 2 , RPMI-1640 media without phenol red, and char-coal-stripped fetal calf serum, the palmitoyl acyltransferase (PAT) inhibitor 2-bromohexadecanoid acid (2-Br-palmitate; 2-Br), the protease inhibitor cocktail, and the bovine serum albu-min fraction V (BSA) were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

Optimem, Hank’s buffer salt solution (HBSS 1 ! ), Neurobasal medium, B27 serum-free supplement, and GlutaMAX-I were pur-chased from Gibco-BRL (Gaithersburg, Md., USA). The p38 in-hibitor SB 203 580 (SB), the AKT inhibitor, and the IGF-1 receptor (IGF-1R) inhibitor picropodophyllin (PPP) were obtained from Calbiochem (San Diego, Calif., USA). The E 2 antagonist fulves-trant (ICI 182,780, ICI), the estrogen receptor (ER) � -selectiveagonist 4,4 � ,4 � � -(4-propyl-[ 1 H]-pyrazole-1,3,5-triyl)trisphenol (PPT), the ER � -selective agonist 2,3-bis(4-hydroxyphenyl)pro-pionitrile (DPN), and the ER � -selective antagonist (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC) were ob-tained from Tocris (Ballwin, Mo., USA). Bradford protein assay was obtained from Bio-Rad Laboratories (Hercules, Calif., USA). The human recombinant ER � and ER � were obtained by Pan-Vera (Madison, Wisc., USA). The anti-phospho-ERK1/2, anti-AKT, anti-ER � (MC20), anti-ER � (H150), anti-caspase-3, anti-poly(ADP-ribose)polymerase (PARP), and anti-ERK1/2 antibod-ies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The polyclonal anti-phospho-AKT, anti-phospho-p38, and anti-p38 antibodies were purchased from New England Biolabs (Beverly, Mass., USA). The monoclonal anti-human Ngb (13C8) was purchased from Abcam (Cambridge, UK). The anti- � -tubulin was purchased from MP Biomedical (Solon, Ohio, USA). The chemiluminescence reagent for Western blot ECL was

17 � -Estradiol Signaling and Neuroglobin Neurosignals 2010;18:223–235 225

obtained from GE Healthcare (Little Chalfont, UK). All the other products were from Sigma-Aldrich. Analytical or reagent grade products were used without further purification.

Cells The human SK-N-BE neuroblastoma cell line was routinely

grown in air containing 5% CO 2 in modified, phenol red-free, RPMI-1640 medium containing 10% (v/v) charcoal-stripped fetal calf serum, L -glutamine (2.0 m M ), Pen-Strep solution (penicillin 100 U/ml, and streptomycin 100 mg/ml). Cells were passaged ev-ery 2 days. Cells were grown to approximately 70% confluence in 6-well plates before stimulation.

Hippocampal neurons were obtained from E18 mouse embry-os after isolating the hippocampus in Ca 2+ - and Mg 2+ -free HBSS 1 ! . Mice were treated following the guidelines of the Council of Europe Convention ETS123, recently revised as indicated in the Directive 86/609/EEC. In addition, all protocols were approved by the Institutional Animal Care and Use Committee of CSIC-Cajal Institute (Madrid, Spain). Once 8–10 embryonic hippocampi were obtained, they were finely cut, washed twice in HBSS 1 ! buffer, and incubated in 0.1 mg/ml trypsin solution and 1 mg/ml DNAse (Roche Diagnostics GmbH, Mannheim, Germany) for 15 min at 37 ° C. Trypsin and DNAse were then eliminated by wash-ing 3 times, with HBSS 1 ! , and the cut tissue was then triturated using a siliconized pipette. Cells were counted and plated in poly-lysine-coated (1 mg/ml) 6-well plates containing phenol red-free neurobasal medium supplemented with 2% (v/v) B27 serum-free supplement, 0.25% (v/v) GlutaMAX-I, and 1% (v/v) penicillin/streptomycin solution. Neurons were maintained under these conditions for 3 days at 5% CO 2 and 37 ° C.

Cells were simultaneously treated with vehicle (ethanol/PBS1: 10, v/v) and/or E 2 (0.1–1,000 n M ), PPT (0.1–100 n M ), DPN (0.1–100 n M ), T (0.1–1,000 n M ), DHT (0.1–1,000 n M ), IGF-1 (100 ng/ml), and H 2 O 2 (50 � M ). When indicated, the anti-estrogen ICI(1 � M ), the PAT inhibitor 2-Br (10 � M ), the AKT inhibitor (1 � M ), the p38 inhibitor SB (5 � M ), the ER � inhibitor THC (1 � M ), the IGF-1R inhibitor PPP (100 n M ), and the transcription inhibitor Act (1 � g/ml) were added 30 min before E 2 or IGF-1 administra-tion. The translational inhibitor Cxm (10 � g/ml), was added 1 h before E 2 administration.

Cell Viability SK-N-BE cell lines were grown to 70% confluence in 6-well

plates and stimulated either with vehicle or E 2 (1 n M ) or THC(1 � M ). After 24 h of stimulation, cells were treated either with vehicle or with H 2 O 2 50 � M for 24 h. After treatment, cells were harvested with trypsin, and counted with Beckman Coulter Mod-el ZM electronic particle (Palo Alto, Calif., USA).

Transfection of Short Interfering RNA SK-N-BE cells, reaching 40–60% confluence, were transfected

in a serum-free condition with either Stealth RNAi TM Ngb-silenc-ing RNA or ER � -silencing RNA (siRNA; Invitrogen) according to the manufacturer’s instructions, using oligofectamine (Invit-rogen) as the transfection reagent. The sequence used for Ngb oligonucleotides was 5 � -CGUGAUUGAUGCUGCAGUGACC-AAU-3 � ; the sequence used for ER � oligonucleotides was 5 � -GAAGAACUCUUUGCCCGGAAAUUUA-3 � . The mismatchsequences used as a control were 5 � -UGUGAUUUAUGGUGC-AGUAACCAAC-3 � and 5 � -GAAUCAUUCCGUGC CA AG UA G-

A UUA-3 � for Ngb and ER � si-RNA, respectively. Briefly, oligo-fectamine and oligonucleotides (400 and 200 p M for Ngb siRNA and ER � siRNA, respectively) were mixed with Optimem. The mixture was incubated for 20 min at room temperature, diluted with Optimem, and added to the cell medium for 4 h at 37 ° C. The medium was added to cells to reach the growing conditions (i.e., 10% (v/v) serum).

To evaluate the effective silencing of Ngb and ER � , total pro-teins from cells transfected with MOCK (control), with scramble (mismatch sequence, data not shown), and with Ngb or ER � oli-gonucleotides were extracted 48 h after transfection, and Ngb and ER � expression was tested by Western blot analysis using anti-Ngb and anti-ER � antibodies.

Western Blot Assays After stimulation, the SK-N-BE cell line and hippocampal

neurons were lysed and solubilized in 0.125 M Tris, pH 6.8, con-taining 10% (w/v) SDS and the protease inhibitor cocktail, then the cell lysates were boiled for 2 min. Total proteins were quanti-fied using the Bradford protein assay. Solubilized proteins (20 � g) were resolved by 7 or 15% SDS-PAGE at 100 V for 1 h at 25 ° C and then electrophoretically transferred to nitrocellulose for 45 min at 100 V and 4 ° C. The nitrocellulose was treated with 3% (w/v) BSA in 138 m M NaCl, 25 m M Tris, pH 8.0, at 25 ° C for 1 h andthen probed overnight at 4 ° C either with anti-Ngb (final dilution 1: 1,000) or anti-ER � MC-20 (final dilution 1: 500) or anti-ER � H-150 (final dilution 1: 3,000) or anti-caspase-3 (final dilution1: 1,000) or anti-PARP (final dilution 1: 500) or anti-phospho-ERK1/2 (final dilution 1: 200) or anti-phospho-AKT (final dilu-tion 1: 1,000) or anti-phospho-p38 (final dilution 1: 1,000). The ni-trocellulose was stripped by Restore Western Blot Stripping Buf-fer (Pierce Chemical, Rockford, Ill., USA) for 10 min at room temperature and then probed with anti- � -tubulin (final dilution1: 1,000) to normalize total lysate. Moreover, the nitrocellulose in-cubated with either anti-phospho-ERK1/2 or anti-phospho-AKT or anti-phospho-p38 was stripped and probed with anti-ERK1/2 (final dilution 1: 200), anti-AKT (final dilution 1: 100) and anti-p38 (final dilution 1: 1,000), respectively. To evidence ER � and ER � levels, electrophoresis was performed in the presence of 5 ng of recombinant ER � and ER � . Antibody reaction was visualized with chemiluminescence Western blot detection reagent.

Densitometric analyses were performed by ImageJ software for Windows. The densitometry quantification of protein was normalized to tubulin.

Statistical Analysis A statistical analysis was performed by using ANOVA fol-

lowed by Tukey-Kramer post-test with the GraphPad InStat3 soft-ware system for Windows. In all cases, p ! 0.05 was considered significant.

Results

E 2 Specifically Increases Ngb Levels in Neurons Figure 1 shows that E 2 stimulation induced a time- and

dose-dependent increase in Ngb levels in SK-N-BE cells ( fig. 1 A, B). The E 2 (10 n M ) effect on Ngb levels started 30


Neurosignals 2010;18:223–235226

min after stimulation being significant 1 h after stimula-tion and remained constant 24 h after hormone stimula-tion. The E 2 dose-response curve was bell-shaped with a maximum effect at physiological E 2 concentrations (i.e., 1–10 n M ; 24 h stimulation).

In contrast, the male sex steroid hormone DHT and the common precursor of E 2 and DHT, T, did not modify Ngb levels at any tested concentration (data not shown), suggesting the specificity of the E 2 effect. These data were confirmed in freshly isolated mouse hippocampal neu-rons ( fig. 1 C, D). Indeed, in these primary neurons, the E 2 effect was rapid (1 h), persistent (24 h) ( fig. 1 C), and specific in that neither DHT nor T were able to increase Ngb levels at any tested concentration (data not shown). In addition, even in mouse hippocampal neurons, 10 n M E 2 increased Ngb levels which remained significantly higher than control cells even at higher E 2 concentrations ( fig. 1 D). In line with the slight differences found in the E 2 concentration to obtain the maximum effect in both

cell types, 1 and 10 n M were used in the consecutive ex-periments to stimulate the SK-N-BE cell line and hippo-campal neurons, respectively.

ER � was necessary for E 2 -induced increase in Ngb lev-els. The pretreatment of SK-N-BE cells with the pure E 2 antagonist, ICI, completely prevented the E 2 effect on Ngb levels ( fig. 2 A), suggesting an ER-mediated mecha-nism. As SK-N-BE cells contain high ER � and low ER � levels ( fig. 2 B), cells were stimulated with either the spe-cific ER � agonist PPT or the specific ER � agonist DPN to discriminate the role of each ER isoform in the E 2 -in-duced Ngb level increase. Only 1 and 10 n M DPN mim-icked the E 2 effect on Ngb levels ( fig. 2 C), whereas PPT was unable to increase Ngb levels, at any concentration investigated ( fig. 2 D). This result was confirmed by cell pretreatment with the specific ER � inhibitor THC, which completely prevented the E 2 effect ( fig. 2 C). Furthermore, the decrease of ER � protein level by ER � SiRNA transfec-tion caused an impairment of the E 2 ability to increase

0

1

2

3

4a,b

a,b,c

c c0

0.5

1.0

1.5

2.0 a,b a,ba,b a,b

D

0

0.5

1.0

1.5a,b,c

a,b,c,da,b,c,d

E2 concentration (nM)



0

0

0 0 0.1 1 10 100 1,000

B E2 concentration (nM)0 0.1 1 10 100 1,000

1 4 6 24

1 4 6 24 0 10.1 10 100 1,000

Ngb

Tubulin

Ngb

Tubulin

00 11 2 4 24 0.10.5 10 100 1,000

Ngb

Tubulin

Ngb

Tubulin

0.5

1.0

1.5a a a a

E2 stimulation time (h)

E2 time (h)

E2 time (h)

C

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

0 0.5 1 2 4 24E2 stimulation time (h)A

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Fig. 1. Effect of 17 � -estradiol (E 2 ) on Ngb protein levels in SK-N-BE human neuro-blastoma cell line ( A , B ) and in mouse hip-pocampal primary neurons ( C , D ). A , C Time-course analysis of E 2 treatment (10 n M ) on Ngb levels. B , D E 2 dose-dependent (0.1–1,000 n M ) effect on Ngb levels (24 h of stimulation). The amount of protein was normalized by comparison with tubulin levels. The data are typical Western blots of five independent experiments (top pan-els); densitometric analysis related to E 2 dose- and time-dependent experiments (bottom panels). Data are means 8 SD of five different experiments. p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test. A , C a significant vs. 0 h and b vs. 0.5 h; B , D a vs. 0, b vs. 0.1, c vs. 1, and d vs. 10 n M .


A B

0

0.5

1.0

1.5

2.0

2.5a,c

a,b

a,c

b,c,d b

C

0

0.5

1.0

1.5

1 10 100

a

D

0

0.5

1.0

1.5 a

b

a,b,c

a,c,d a,c,d

FE

Ngb

Ngb

Tubulin

Tubulin

Ngb

Tubulin

Vehi

cle

E 2

Vehi

cle

E 2 Vehi

cle

E 2

Vehi

cle

Vehicle

E 2

E2 0.01 0.1 101Vehicle E2

Vehi

cle

E 2Ve

hicl

e

E 2 THC

THC

THC

+ E

2

THC+E2

E 2 +

ICI

ICI

Ngb

Tubulin

Ngb

1 10 100

DPN (nM)

DPNconcentration

(nM)

Tubulin

Tubulin

SK-N

-BE

Reco

mbi

nant

prot

eins

ER�

ER�

Tubulin

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

1 10 100

PPT (nM)

PPTconcentration

(nM)

MOCK si-ER�

ER�

1 100.01 0.01

Nar (μM)

Nar concentration (μM)

10 1001Vehicle E2

Fig. 2. Impact of estrogen receptors (ER) � and � on Ngb protein expression in SK-N-BE human neuroblastoma cell line. A West-ern blot analysis of Ngb levels in cells stimulated for 24 h with either vehicle or E 2 (1 n M ) and/or the ER inhibitor ICI 182,870 (ICI; 1 � M ). B ER isoform levels in non-stimulated cells compared to recombinant proteins (5 ng). C Analysis of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (1 n M ), the ER � agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN; 1–100 n M ) or the ER � -selective antagonist (R,R)-5,11-diethyl-5,6,11,12-tetrahy-dro-2,8-chrysenediol (THC; 1 � M ). D Analysis of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (1 n M ) or the ER � agonist 4,4 � ,4 � � -(4-propyl [ 1 H]-pyrazole-1,3,5-triyl)trisphenol (PPT; 1–100 n M ). E Analysis of Ngb and ER � levels in cells trans-

fected with either MOCK (control) or ER � small interference mRNA (si-ER � ) in the absence or presence of E 2 (1 n M ). F Analy-sis of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (1 n M ) or naringenin (Nar; 0.01–10 � M ). The amount of proteins was normalized by comparison with tubulin levels. The data are typical Western blots of four independent experiments; densito-metric analyses related to DPN, PPT, and Nar dose-dependent experiments ( C , D and F bottom panels). Data are means 8 SD of four different experiments. p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test. C , D a significant vs. vehicle, b vs. E 2 , c vs. 1, and d vs. 10 n M . F a significant vs. vehicle, b vs. E 2 , c vs. 0.01, and d vs. 0.1 � M .


Neurosignals 2010;18:223–235228

Ngb levels ( fig. 2 E). A further confirmation of the ER � involvement in the effect of E 2 effects derives from the results obtained using the flavonoid Nar ( fig. 2 F). Indeed, we previously reported that this flavonoid is a partial an-tagonist of E 2 in the presence of ER � [58] and an E 2 mi-metic in the presence of ER � [59] . Figure 2 F shows that, like E 2 , 0.1 � M Nar was sufficient to increase Ngb levels. This effect persisted at Nar high concentrations (i.e., 1 and 10 � M ). ER � was also necessary for the E 2 -induced Ngb increase in mouse primary neurons ( fig. 3 ). In these cells, containing a similar amount of ER � and ER �

( fig. 3 A), ICI prevented the E 2 effect ( fig. 3 B), DPN mim-icked the E 2 effect ( fig. 3 C), whereas PPT was unable to increase Ngb levels ( fig. 3 D). Cell pretreatment with the specific ER � inhibitor THC further confirmed these re-sults ( fig. 3 E).

Mechanism Involved in the E 2 -Induced Increase of Ngb Levels in Neurons ERs are ligand-activated transcription factors which

possess both transcriptional and extranuclear activities [48] . Thus, we evaluated the impact on the E 2 -induced

A B

C0.1 1 10 100

D E

Ngb

Tubulin

Ngb

Tubulin

Ngb

Tubulin

Ngb

Tubulin

Vehi

cle

E 2

Vehicle E2

E 2 +

ICI

ICI

Vehi

cle

E 2 THC

+ E

2

THC

DPNconcentration

(nM)

Reco

mbi

nant

prot

eins

Hip

poca

mpa

lne

uron

s

Tubulin

ER�

ER�

Tubulin

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Ngb

leve

l/tu

bulin

leve

l(a

rbitr

ary

units

)

Vehi

cle

E 2 10.1 10 100

DPN (nM)

PPTconcentration

(nM)

0.1 1 10 100Vehicle E2

Vehi

cle

E 2 10.1 10 100

PPT (nM)

0

0.5

1.0

1.5

2.0

2.5

0

0.5

1.0

1.5

2.0

2.5 *** * * *

Fig. 3. Impact of estrogen receptors (ER) � and � on Ngb protein expression in mouse hippocampal primary neurons. A ER isoform levels in non-stimulated cells compared to recombinant proteins (5 ng). B Western blot analyses of Ngb levels in cells stimulated for 24 h with either vehicle or E 2 (10 n M ) and/or the ER inhibitor ICI 182,870 (ICI; 1 � M ). C Analyses of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (10 n M ) or the ER � agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN; 0.1–100 n M ). D Analyses of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (10 n M ) or the ER � agonist 4,4 � ,4 � � -(4-propyl [ 1 H]-pyrazole-1,3,5-tri-

yl)trisphenol (PPT; 0.1–100 n M ). E Analyses of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (10 n M ) and/or the ER � -selective antagonist (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC; 1 � M ). The amount of proteins was normal-ized by comparison with tubulin levels. Typical blots of five inde-pendent experiments are shown; densitometric analyses related to DPN and PPT dose-dependent experiments ( C , D bottom panels). Data are means 8 SD of five different experiments. * p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test with respect to vehicle-treated samples.


Ngb levels of the transcription inhibitor Act, the transla-tion inhibitor Cxm, and the ER membrane localization inhibitor 2-Br [60] . Although the Ngb promoter sequence analysis (accession No. 12,581 from Transcriptional Reg-ulatory Element Database http://rulai.cshl.edu/cgibin/TRED/tred.cgi?process=home) indicates that no canon-ical estrogen-responsive element (ERE) is present, SK-N-BE cell pretreatment for 24 h with Act completely pre-vented the E 2 effect on Ngb levels ( fig. 4 A). Similarly, Cxm ( fig. 4 B) and 2-Br ( fig. 4 C) impaired the increase of Ngb levels induced by E 2 , suggesting that both transcrip-tional and extranuclear mechanisms contribute to E 2 ef-fects. This prompted us to evaluate which signal trans-duction cascade was activated by E 2 in neurons. After 15 min of stimulation with 1 n M E 2 , an increase of AKT and p38 phosphorylation in SK-N-BE cells was observed ( fig. 5 A, B). The E 2 -induced activation of these kinases was still present 1 h after E 2 stimulation ( fig. 5 A, B), but only the E 2 -induced p38 phosphorylation persisted 24 h after hormone stimulation (data not shown). By 30 min after 1 n M E 2 stimulation, the ERK1/2 phosphorylation status decreased ( fig. 5 A, B), in agreement with previous data obtained in cortical neurons [61] . The cell pretreat-ment with either AKT or p38 or ERK1/2 inhibitors sug-gested that neither AKT nor ERK1/2 are involved in the E 2 -induced increase of Ngb levels (data not shown),

whereas p38 activation was required for both rapid (i.e., 1 h) and long term (i.e., 24 h) E 2 effects on Ngb levels ( fig. 5 C, D). Similarly, only p38 inhibitor prevents an E 2 -induced Ngb level increase in hippocampal neurons (data not shown). Notably, the SK-N-BE cell transfection with ER � SiRNA reduced both ER � levels and the E 2 ability to induce p38 phosphorylation ( fig. 5 E). On the other hand, ER � SiRNA did not impair the E 2 -induced AKT activation (data not shown), suggesting that ER � could be the molecular mediator of AKT activation in these cells.

Since it has been reported that several actions of E 2 in the nervous system involve cross-talk between ER � and the IGF-1 receptor [62] , we evaluated the possibility that the ER � -dependent E 2 -induced p38 phosphorylation and the Ngb-increased levels are dependent on the ER � -IGF-1 receptor cross-talk. IGF-1 is more efficient than E 2 to ac-tivate AKT phosphorylation and cell pretreatment with PPP, the IGF-1 receptor inhibitor, strongly prevented both IGF-1 and E 2 effects on AKT activation ( fig. 5 F), confirm-ing that cross-talk between the IGF-1 receptor and ERs is important for AKT activation. However, IGF-1 was un-able to increase p38 phosphorylation and PPP did not pre-vent E 2 -induced p38 activation ( fig. 5 F). In addition, IGF-1 did not modify Ngb levels in SK-N-BE cells ( fig. 5 G) fur-ther sustaining the high specificity of the E 2 effect.

Ngb

Tubulin

Ngb

Tubulin

Ngb

Tubulin

Vehi

cle

E 2 E 2 +

Act

Act Ve

hicl

e

E 2 E 2 +

Cxm

Cxm

A

Ngb

leve

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ary

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)

Vehicle E2 Act E2 + Act Vehicle E2 Cxm E2 + Cxm Vehicle E2 2-Br E2 + 2-Br0

0.5

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ary

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ary

units

)

0

0.5

1.0

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* * *

ºº º

Vehi

cle

E 2 2-Br

E 2 +

2-B

r

Fig. 4. Mechanisms underlying E 2 effects on Ngb protein expres-sion in SK-N-BE human neuroblastoma cells. A Western blot analyses of Ngb levels in cells stimulated for 24 h with either ve-hicle, E 2 (1 n M ) and/or the transcription inhibitor actinomycin D (Act, 1 � g/ml). B Analyses of Ngb levels in cells stimulated for24 h with either vehicle, E 2 (1 n M ) and/or the translation inhibitor cycloheximide (Cxm, 10 � g/ml). C Analyses of Ngb levels in cells stimulated for 24 h with either vehicle, E 2 (1 n M ) and/or the pal-

mitoylacyl transferase inhibitor 2-bromohexadecanoic acid (2-Br; 10 � M ). The amount of proteins was normalized by comparison with tubulin levels. Data are representative Western blots of inde-pendent experiments ( A – C top panels); densitometric analyses ( A – C bottom panels). Data are means 8 SD of four different ex-periments. p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test with respect to ( * ) vehicle- or (°) E 2 -treat-ed samples.


Neurosignals 2010;18:223–235230

AB

D

NgbNgb

p38

p38-P

Tubulin

Ngb

Tubulin

Ngb

Tubulin

Tubulin

p38

p38-P

Tubulin

AKT-P

p38-P

p38

Tubulin

AKT-P

AKT

AKT

PBS

E 2 PBS

E 2

Vehi

cle

IGF-

1

E 2

Vehi

cle

IGF-

1

IGF-

1 +

PPP

E 2 +

PPP

E 2

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cle

E 2 1

h

E 2 +

SB

SB Vehi

cle

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SB

E 2 2

4 h

SB

Prot

ein

leve

ls/t

ubul

in le

vel

(arb

itrar

y un

its)

0 15 30 60

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0 15 30 60 0 15 30 60

MOCK si-ER�

ER�

E2 time (min)

0

0.5

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AKT-P p38-P ERK1/2-P

ERK1/2-P

ERK1/2

E2 stimulation time (min)

aa

a

a aa

a,ba,b,c

F

C

E

G

Fig. 5. Rapid signal transduction pathways activated by E 2 and impact of E 2 -dependent rapid signal inhibitors on Ngb protein levels in SK-N-BE human neuroblastoma cell line. A Time-course analyses of phosphorylated (P) and unphosphorylated AKT, p38, and ERK1/2 in cells stimulated for 0, 15, 30, and 60 min with E 2 (1 n M ). B Densitometric analysis related to E 2 -induced AKT, p38, and ERK1/2 phosphorylation experiments. Data are means 8 SD of four different experiments. * p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test: a significant vs. 0, b vs. 15, and c vs. 30 min. C , D Analyses of Ngb levels in cells stimulated for 1 h ( C ) or 24 h ( D ) with either vehicle, E 2 (1 n M ) and/or the p38 inhibitor SB-203580 (SB; 5 � M ). E Analysis of Ngb, ER � , and phosphorylated (P) and unphosphorylated p38 levels in cells

transfected with either MOCK (control) or ER � small interfer-ence mRNA (si-ER � ) in the absence or presence of E 2 (1 n M ). The amount of proteins was normalized by comparison with tubulin levels. Data are representative Western blots of three independent experiments. F Western blot analyses of phosphorylated (P) and unphosphorylated AKT and p38 levels in cells treated for 24 h with either vehicle, E 2 (1 n M ) or IGF-1 (100 ng/ml). When indi-cated, cells were pretreated with the IGF-1 receptor inhibitor pic-ropodophyllin (PPP; 100 � M ). G Analyses of Ngb protein levels in cells treated for 24 h with either vehicle, E 2 (1 n M ) or IGF-1 (100 ng/ml). The amount of proteins was normalized by comparison with tubulin levels. Data are representative Western blots of three independent experiments.


Fig. 6. E 2 effect on SK-N-BE human neuroblastoma cell line viabil-ity. A Cells were grown in the presence of either vehicle or E 2 (1 n M ) and counted at the indicated time. Data are means 8 SD of five independent experiments carried out in duplicate. B Cells were grown for 24 h in the presence of either vehicle, E 2 (1 n M ) or E 2 in the presence of the ER � -selective antagonist (R,R)-5,11-di-ethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC; 1 � M ). After 24 h, cells were stimulated with H 2 O 2 50 � M (24 h of stimulation), and counted. Data are means 8 SD of five independent experi-ments carried out in duplicate. p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test: a significant vs. Ve-hicle – H 2 O 2 , b vs. vehicle + H 2 O 2 , c vs. E 2 – H 2 O 2 , d vs. E 2 + H 2 O 2 , and e vs. THC + E 2 – H 2 O 2 . C Western blot analyses of caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage were performed on cells stimulated with either the vehicle or pre-treated with E 2 (1 n M ) for 24 h in the presence or absence of THC pretreatment, and then treated with H 2 O 2 50 � M (24 h of stimula-tion). Staurosporin (2 � M for 24 h) was used as positive control of caspase activation. The amount of proteins was normalized by comparison with tubulin level. Data are representative Western

blots of three independent experiments. D Western blot analysis of Ngb protein levels in cells transfected with either MOCK (control) or with Ngb small interference mRNA (si-Ngb) in the absence or presence of E 2 (1 n M ). The amount of proteins was normalized by comparison with tubulin levels. Data are representative Western blots of three independent experiments. Cells transfected with ei-ther MOCK (empty bars) or si-Ngb (filled bars) were grown for 24 h in the presence of either the vehicle or E 2 (1 n M ), stimulated with H 2 O 2 50 � M (24 h of stimulation), and counted. Data are means 8 SD of three independent experiments carried out in duplicate. p ! 0.001 was calculated with ANOVA followed by Tukey-Kramer post-test: a significant vs. vehicle – H 2 O 2 , b vs. vehicle + H 2 O 2 , c vs. E 2 + H 2 O 2 , d vs. vehicle – H 2 O 2 si-Ngb, and e vs. E 2 – H 2 O 2 si-Ngb. E Western blot analyses of caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage were performed on cells transfected with either MOCK or si-Ngb and stimulated with either vehicle or 50 � M H 2 O 2 in the presence or absence of E 2 (1 n M ) (24 h pretreatment). The amount of proteins was normal-ized by comparison with tubulin level. Data are representative Western blots of three independent experiments.

0 4 6 24 30 480

50

100

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Stimulation time (h)A

ab

a,c,d,e

B C

D

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num

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E 2 +

TH

C

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C

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Stau

rosp

orin

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– H2O2

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cle

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Vehicle

Vehicle

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THC + E 2

Vehicle

Vehi

cle

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E 2 Vehi

cle

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E 2 +

H2O

2

H2O

2

Vehi

cle

E 2 E 2 +

H2O

2

H2O

2

– H2O2 + H2O2

+ H2O2

– H2O2 + H2O2

MOCK si-Ngb

MOCK si-Ngb

Ngb


Neurosignals 2010;18:223–235232

Ngb Involvement in E 2 -Induced Protection against H 2 O 2 -Induced Oxidative Stress Finally we evaluated the role played by increased lev-

els of Ngb on E 2 effects in SK-N-BE cells. E 2 stimulation did not modify SK-N-BE cell number ( fig. 6 A) but, as ex-pected [61] , reduced by 50% the H 2 O 2 -induced decrease in cell number ( fig. 6 B) as well as the increase of the 17-kDa active caspase-3 subunit and the cleavage of the cas-pase-3 substrate PARP ( fig. 6 C). Staurosporin (2 � M for 24 h) was used as positive control of caspase activation ( fig. 6 C). Notably, the E 2 -protective effect against H 2 O 2 -induced neuron toxicity seems to require ER � , since the cell pretreatment with the specific ER � inhibitor THC completely prevented E 2 effects ( fig. 6 B, C). In line with this result, E 2 was unable to counteract the H 2 O 2 -in-duced decrease in cell number ( fig. 6 D) and the activa-tion of the pro-apoptotic cascade (i.e., caspase-3 activa-tion and PARP cleavage) ( fig. 6 E) in Ngb-silenced SK-N-BE cells.

Discussion

The aim of this paper was to identify a possible endog-enous modulator of Ngb. Thus, we investigated the effect of E 2 , a well-known neurotrophic and neuroprotective hormone [52–57, 63–66] , on Ngb expression. Our results indicated that E 2 increases Ngb levels of about 300% in both the human neuroblastoma cell line and mouse pri-mary hippocampal neurons. Although it has been ob-tained with a qualitative technique (i.e., Western blot), this effect is conspicuous in that the well-known E 2 effect on cyclin D1 expression, playing a relevant role in E 2 -in-duced cell proliferation, is only of about 50–70% [67] . In addition, the E 2 -induced Ngb increase is rapid (1 h), per-sistent (24 h), and specific, being not mimicked by either the male sex steroid hormone DHT or by the common precursor T or by IGF-1, another well-known neuropro-tective hormone. These results represent the first evi-dence for steroid hormone modulation of globin levels in cells. Recently, it has been reported that hemoglobin is specifically expressed in neurons, its expression being upregulated by erythropoietin and accompanied by en-hanced brain oxygenation under physiologic and hypox-ic conditions [68] . At the present, the relationship be-tween hemoglobin and Ngb in neurons is still unclear. Although hemoglobin � -chains and Ngb are expressedin the same nerve cells, Ngb levels are not increased by erythropoietin [68] . It is therefore unlikely that they have a tightly linked function, e.g. in facilitated oxygen trans-

port; however, hemoglobin and Ngb could fulfill inde-pendent tasks in neurons.

It is now well known that sex steroid hormones have numerous effects on the brain throughout the lifespan, beginning during gestation and continuing on into se-nescence [69] . However, the inability of Ngb to react with androgens renders Ngb a new E 2 target that should be added to the variety of E 2 -specific actions on the brain which include mood, locomotor activity, pain sensitivity, vulnerability to epilepsy, attentional mechanisms, and cognition [64] .

The E 2 effect on Ngb levels is rapid and dose-depen-dent with the maximum effect at E 2 physiological con-centration (i.e., 1–10 n M ). Notably, the E 2 dose-response curve results in being bell-shaped. This is typical for E 2 [70] , whose effects are mediated by two receptor isoforms. Accordingly, the plant-derived flavonoid Nar, which par-tially blocks the rapid activities of ER � [58] , increased Ngb levels with a plateau at 1 � M concentration. This re-sult suggests a functional antagonism between the activ-ities of ER � and ER � in neurons, as has been reported in other cell types [71] .

Although human neuroblastoma cell line and mouse primary hippocampal neurons express different levels of both ER isoforms, the effect of E 2 on Ngb levels specifi-cally requires all ER � activities. In fact, ER � extranucle-ar and genomic signals cross-talk each other to guarantee both the rapid (1 h) and the persistent (24 h) E 2 effects. In particular, the rapid (15 min) and persistent (24 h) ER � -mediated p38 activation is required for E 2 -induced Ngb increase. The E 2 -dependent activation of p38, a mitogen-activated protein kinase (MAPK) family component, seems to represent a conserved pathway in ER � -based E 2 rapid signals. Indeed, the E 2 -induced ER � -mediated ac-tivation of the p38/MAPK occurs in ER � -transfected HeLa cells and in ER � -containing rat myoblasts and co-lon adenocarcinoma cells [70, 72, 73] . This signaling pathway transduces different E 2 effects depending on the cell context. In fact, p38 activation is required for E 2 -in-duced apoptosis of cancer cells [72, 73] , for E 2 -induced gene transcription [73] , and for E 2 -induced protection against oxidative stress in neuroblastoma cell line (pres-ent results) and in rat myoblasts [Marino, unpubl. re-sults]. Thus, although Ngb promoter does not contain any canonical ERE, it is not surprising that the transcrip-tion inhibitor Act completely prevents the increase of the E 2 -induced Ngb levels. The sequence analysis (accession No. 12,581 from Transcriptional Regulatory ElementDatabase http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home) indicates that several non-canonical half


ERE sites are present in the Ngb promoter along with a responsive element for other transcription factors. These results suggest that the E 2 -induced Ngb transcription could be mediated by tethered interactions of ER with other transcription factors to activate gene expression (i.e., indirect genomic mechanism) [48] . Thus, the inte-gration between extranuclear and genomic events may be required to provide plasticity for the neuronal response to E 2 . The hormone rapidly induces AKT activation and ERK dephosphorylation in neuroblastoma cells. These effects are still detected in ER � -containing but ER � -si-lenced cells. Although these kinases are not directly in-volved in an E 2 -induced Ngb increase, they could con-tribute to the E 2 effects in neurons, since AKT activation has been associated with the increase of the anti-apoptotic protein Bcl-2 and to the E 2 -induced cell sur-vival [72] , while E 2 protected cortical neurons against ox-idative stress by reducing H 2 O 2 -induced activation of ERK1/2 [61] .

The main result reported here is that Ngb is part of the E 2 response to H 2 O 2 -induced toxicity. In fact, exposure to 50 � M H 2 O 2 induces neuroblastoma cell death (about 50%) which is accompanied by a dramatic increase in cas-pase-3 activation. The cell pretreatment with E 2 (1 n M ) decreases cell death and reduces caspase-3 activation triggered by exposure to H 2 O 2 in good accordance with the literature [52–54, 61, 74] . This E 2 effect against H 2 O 2 toxicity is completely prevented by treatment with THC, ER � inhibitor, and by knocking out Ngb using small in-terfering RNA.

Exposure to H 2 O 2 induces a robust increase of reactive oxygen species in cells (followed by oxidation of lipids, proteins, and DNA), intracellular calcium increase, glu-tathione depletion, mitochondria dysfunction, and cas-pase-3 activation followed by apoptotic cell death [74] . It has been demonstrated that E 2 exerts protective effects on several of these cellular events including potent attenua-tion of lipid peroxidation, attenuated ATP depletion, al-

leviated intracellular calcium elevation, ablated mito-chondrial calcium loading (with the subsequent mito-chondrial membrane potential maintenance), reduced caspase-3 activation, and enhanced cell survival ( [74] and present results). At present, it is difficult to discriminate the role played by Ngb in each of these E 2 -induced cellu-lar outcomes because it is not yet clear if they require ER � or ER � or both receptors. Moreover, most of the E 2 -pro-tective effects have been obtained at pharmacological E 2 concentrations (0.1–10 � M ) [74] . However, it has recently shown that the elevation of human Ngb expression in neurons prior to insult with H 2 O 2 enhances cell viability and results in a significant decrease in oxidative stress and an increased intracellular ATP concentration [75] . In addition, a linkage of Ngb to oxidative metabolism has been proposed [76] . These data are strongly suggestive of the involvement of Ngb in E 2 -induced attenuated ATP depletion. Furthermore, in the presence of Ngb the ini-tially released cytochrome c may be sequestered by the Ngb [42, 43] and the apoptotic cascade avoided [19] .

In the future, different strategies (e.g., E 2 -induced Ngb localization, association between Ngb and ERs, and E 2 -induced ER-mediated Ngb promoter activity) will sub-stantiate Ngb action in neurons, our laboratories being active in this field. However, from now, Ngb can be re-garded as part of signals activated by E 2 to exert neuro-protective effects definitely validating the role played by Ngb as an anti-apoptotic neuroprotective globin.

Acknowledgements

The generous gift of SK-N-BE human neuroblastoma cell line from Dr. Roberto Rivabene (Institute of Public Health, ISS, Rome, Italy) and of staurosporin from Dr. Antonio Antoccia (University Roma Tre) are gratefully acknowledged. This work was supported by grants from University Roma Tre (CLAR 2009 to P.A.) and the Italian Ministry of Health (Strategico 2008 to M.M).

References

1 Burmester T, Weich B, Reinhardt S, Hankeln T: A vertebrate globin expressed in brain. Nature 2000; 407: 520–523.

2 Moens L, Dewilde S: Globins in the brain. Nature 2000; 407: 461–462.

3 Trent JT 3rd, Watts RA, Hargrove MS: Hu-man neuroglobin, a hexacoordinate hemo-globin that reversibly binds oxygen. J Biol Chem 2001; 276: 30106–30110.

4 Pesce A, Bolognesi M, Bocedi A, Ascenzi P, Dewilde S, Moens L, Hankeln T, Burmester T: Neuroglobin and cytoglobin. Fresh blood for the vertebrate globin family. EMBO Rep 2002; 3: 1146–1151.

5 Reuss S, Saaler-Reinhardt S, Weich B, Wys-tub S, Reuss MH, Burmester T, Hankeln T: Expression analysis of neuroglobin mRNA in rodent tissues. Neurosci 2002; 115: 645–656.

6 Pesce A, Dewilde S, Nardini M, Moens L, As-cenzi P, Hankeln T, Burmester T, Bolognesi M: Human brain neuroglobin structure re-veals a distinct mode of controlling oxygen affinity. Structure 2003; 11: 1087–1095.

7 Wystub S, Laufs T, Schmidt M, BurmesterT, Maas U, Saaler-Reinhardt S, Hankeln T, Reuss S: Localization of neuroglobin protein in the mouse brain. Neurosci Lett 2003; 346: 114–116.


Neurosignals 2010;18:223–235234

8 Burmester T, Hankeln T: Neuroglobin: a re-spiratory protein of the nervous system. News Physiol Sci 2004; 19: 110–113.

9 Hankeln T, Wystub S, Laufs T, Schmidt M, Gerlach F, Saaler-Reinhardt S, Reuss S, Bur-mester T: The cellular and subcellular local-ization of neuroglobin and cytoglobin – a clue to their function? IUBMB Life 2004; 56: 671–679.

10 Vallone B, Nienhaus K, Matthes A, Brunori M, Nienhaus GU: The structure of carbon-monoxy neuroglobin reveals a heme-sliding mechanism for control of ligand affinity. Proc Natl Acad Sci USA 2004; 101: 17351–17356.

11 Hankeln T, Ebner B, Fuchs C, Gerlach F, Haberkamp M, Laufs TL, Roesner A, Schmidt M, Weich B, Wystub S, Saaler-Rein-hardt S, Reuss S, Bolognesi M, De Sanctis D, Marden MC, Kiger L, Moens L, Dewilde S, Nevo E, Avivi A, Weber RE, Fago A, Bur-mester T: Neuroglobin and cytoglobin in search of their role in the vertebrate globin family. J Inorg Biochem 2005; 99: 110–119.

12 Brunori M, Vallone B: A globin for the brain. FASEB J 2006; 20: 2192–2197.

13 Nienhaus K, Nienhaus GU: Searching for neuroglobin’s role in the brain. IUBMB Life 2007; 59: 490–497.

14 Greenberg DA, Jin K, Khan AA: Neuroglo-bin: an endogenous neuroprotectant. Curr Opin Pharmacol 2008; 8: 20–24.

15 Burmester T, Hankeln T: What is the func-tion of neuroglobin? J Exp Biol 2009; 212: 1423–1428.

16 Yu Z, Fan X, Lo EH, Wang X: Neuroprotec-tive roles and mechanisms of neuroglobin. Neurol Res 2009; 31: 122–127.

17 Emara M, Turner AR, Allalunis-Turner J: Hypoxic regulation of cytoglobin and neu-roglobin expression in human normal and tumor tissues. Cancer Cell Int 2010; 10: 33–49.

18 Hundahl CA, Allen GC, Hannibal J, Kjaer K, Rehfeld JF, Dewilde S, Nyengaard JR, Kelsen J, Hay-Schmidt A: Anatomical characteriza-tion of cytoglobin and neuroglobin mRNA and protein expression in the mouse brain. Brain Res 2010; 1331: 58–73.

19 Fago A, Mathews AJ, Brittain T: A role for neuroglobin: resetting the trigger level for apoptosis in neuronal and retinal cells. IUBMB Life 2008; 60: 398–401.

20 Schmidt M, Giessl A, Laufs T, Hankeln T, Wolfrum U, Burmester T: How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina. J Biol Chem 2003; 278: 1932–1935.

21 Bentmann A, Schmidt M, Reuss S, Wolfrum U, Hankeln T, Burmester T: Divergent distri-bution in vascular and avascular mammali-an retinae links neuroglobin to cellular res-piration. J Biol Chem 2005; 280: 20660–20665.

22 Dewilde S, Kiger L, Burmester T, Hankeln T, Baudin-Creuza V, Aerts T, Marden MC, Caubergs R, Moens L: Biochemical charac-terization and ligand binding properties of neuroglobin, a novel member of the globin family. J Biol Chem 2001; 276: 38949–38955.

23 Hamdane D, Kiger L, Dewilde S, Green BN, Pesce A, Uzan J, Burmester T, Hankeln T, Bolognesi M, Moens L, Marden MC: The re-dox state of the cell regulates the ligand bind-ing affinity of human neuroglobin and cyto-globin. J Biol Chem 2003; 278: 51713–51721.

24 Fago A, Hundahl C, Malte H, Weber RE: Functional properties of neuroglobin and cytoglobin: insights into the ancestral physi-ological roles of globins. IUBMB Life 2004; 56: 689–696.

25 Herold S, Fago A, Weber RE, Dewilde S, Moens L: Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuroglobin re-veal a potential role against oxidative stress. J Biol Chem 2004; 279: 22841–22847.

26 Kiger L, Uzan J, Dewilde S, Burmester T, Hankeln T, Moens L, Hamdane D, Baudin-Creuza V, Marden M: Neuroglobin ligand binding kinetics. IUBMB Life 2004; 56: 709–719.

27 Trandafir F, van Doorslaer S, Dewilde S, Moens L: Temperature dependence of NO binding modes in human neuroglobin. Bio-chim Biophys Acta 2004; 1702: 153–161.

28 Brunori M, Giuffré A, Nienhaus K, Nienhaus GU, Scandurra FM, Vallone B: Neuroglobin, nitric oxide, and oxygen: functional path-ways and conformational changes. Proc Natl Acad Sci USA 2005; 102: 8483–8488.

29 Fago A, Mathews AJ, Dewilde S, Moens L, Brittain T: The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein. J Inorg Biochem 2006; 100: 1339–1343.

30 Nicolis S, Monzani E, Ciaccio C, Ascenzi P, Moens L, Casella L: Reactivity and endoge-nous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger? Biochem J 2007; 407: 89–99.

31 Brittain T, Yosaatmadja Y, Henty K: The in-teraction of human neuroglobin with hydro-gen sulphide. IUBMB Life 2008; 60: 135–138.

32 Giuffrè A, Moschetti T, Vallone B, Brunori M: Neuroglobin: enzymatic reduction and oxygen affinity. Biochem Biophys Res Com-mun 2008; 367: 893–898.

33 Kakar S, Hoffman FG, Storz JF, Fabian M, Hargrove MS: Structure and reactivity of hexacoordinate hemoglobins. Biophys Chem 2010; 152: 1–14.

34 Ostojić J, Sakaguchi DS, de Lathouder Y, Hargrove MS, Trent JT 3rd, Kwon YH, Kar-don RH, Kuehn MH, Betts DM, Grozdanić S: Neuroglobin and cytoglobin: oxygen-bind-ing proteins in retinal neurons. Invest Oph-thalmol Vis Sci 2006; 47: 1016–1023.

35 Herold S, Fago A: Reactions of peroxynitrite with globin proteins and their possible phys-iological role. Comp Biochem Physiol A Mol Integr Physiol 2005; 142: 124–129.

36 Petersen MG, Dewilde S, Fago A: Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions. J Inorg Biochem 2008; 102: 1777–1782.

37 Jin K, Mao XO, Xie L, Khan AA, Greenberg DA: Neuroglobin protects against nitric ox-ide toxicity. Neurosci Lett 2008; 430: 135–137.

38 Fordel E, Thijs L, Moens L, Dewilde S: Neu-roglobin and cytoglobin expression in mice. Evidence for a correlation with reactive oxy-gen species scavenging. FEBS J 2007; 274: 1312–1317.

39 Khan AA, Mao XO, Banwait S, DerMardi-rossian CM, Bokoch GM, Jin K, Greenberg DA: Regulation of hypoxic neuronal death signaling by neuroglobin. FASEB J 2008; 22: 1737–1747.

40 Watanabe S, Wakasugi K: Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation in-hibitor activity. Biochem Biophys Res Com-mun 2008; 369: 695–700.

41 Fago A, Mathews AJ, Moens L, Dewilde S, Brittain T: The reaction of neuroglobin with potential redox protein partners cytochrome b 5 and cytochrome c . FEBS Lett 2006; 580: 4884–4888.

42 Brittain T, Skommer J, Raychaudhuri S, Birch N: An antiapoptotic neuroprotective role for neuroglobin. Int J Mol Sci 2010; 11: 2306–2231.

43 Raychaudhuri S, Skommer J, Henty K, Birch N, Brittain T: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrin-sic pathway of cell death. Apoptosis 2010; 15: 401–411.

44 Pesce A, De Sanctis D, Nardini M, Dewilde S, Moens L, Hankeln T, Burmester T, Ascen-zi P, Bolognesi M: Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin. IUBMB Life 2004; 56: 657–664.

45 Sun Y, Jin K, Peel A, Mao XO, Xie L, Green-berg DA: Neuroglobin protects the brain from experimental stroke in vivo. Proc Natl Acad Sci USA 2003; 100: 3497–3500.

46 Khan AA, Wang Y, Sun Y, Mao XO, Xie L, Miles E, Graboski J, Chen S, Ellerby LM,Jin K, Greenberg DA: Neuroglobin-overex-pressing transgenic mice are resistant to ce-rebral and myocardial ischemia. Proc Natl Acad Sci USA 2006; 103: 17944–17948.

47 Jin K, Mao Y, Mao X, Xie L, Greenberg DA: Neuroglobin expression in ischemic stroke. Stroke 2010; 41: 557–559.

48 Ascenzi P, Bocedi A, Marino M: Structure-function relationship of estrogen receptor � and � : impact on human health. Mol Aspects Med 2006; 27: 299–402.

49 Manthey D, Behl C: From structural bio-chemistry to expression profiling: neuropro-tective activities of estrogen. Neuroscience 2006; 138: 845–850.


50 Correia SC, Santos RX, Cardoso S, Carvalho C, Santos MS, Oliveira CR, Moreira PI: Ef-fects of estrogen in the brain: is it a neuropro-tective agent in Alzheimer’s disease? Curr Aging Sci 2010; 3: 113–126.

51 Gillies GE, McArthur S: Estrogen actions in the brain and the basis for differential action in men and women: a case for sex-specific medicines. Pharmacol Rev 2010; 62: 155–198.

52 Behl C, Skutella T, Lezoualc’h F, Post A, Widmann M, Newton CJ, Holsboer F: Neu-roprotection against oxidative stress by es-trogens: structure-activity relationship. Mol Pharmacol 1997; 51: 535–541.

53 Sawada H, Ibi M, Kihara T, Urushitani M, Akaike A, Shimohama S: Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death. J Neurosci Res 1998; 54: 707–719.

54 Green PS, Simpkins JW: Neuroprotective ef-fects of estrogens: potential mechanisms of action. Int J Dev Neurosci 2000; 18: 347–358.

55 Bae YH, Hwang JY, Kim YH, Koh JY: Anti-oxidative neuroprotection by estrogens in mouse cortical cultures. J Korean Med Sci 2000; 15: 327–336.

56 Wilson ME, Dubal DB, Wise PM: Estradiol protects against injury-induced cell death in cortical explant cultures: a role for estrogen receptors. Brain Res 2000; 873: 235–242.

57 Blum-Degen D, Haas M, Pohli S, Harth R, Romer W, Oettel M, Riederer P, Gotz ME: Scavestrogens protect IMR 32 cells from ox-idative stress-induced cell death. Toxicol Appl Pharmacol 1998; 152: 49–55.

58 Galluzzo P, Ascenzi P, Bulzomi P, Marino M: The nutritional f lavanone naringenin trig-gers antiestrogenic effects by regulating es-trogen receptor � -palmitoylation. Endocri-nology 2008; 149: 2567–2575.

59 Totta P, Acconcia F, Leone S, Cardillo I, Ma-rino M: Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involve-ment of estrogen receptor � and � signalling. IUBMB Life 2004; 56: 491–499.

60 Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V, Trentalance A, Visca P, Marino M: Palmitoylation-dependent estrogen receptor � membrane localization: regulation by 17 � -estradiol. Mol Biol Cell 2005; 16: 231–237.

61 Numakawa Y, Matsumoto T, Yokomaku D, Taguchi T, Niki E, Hatanaka H, Kunugi H, Numakawa T: 17 � -Estradiol protects corti-cal neurons against oxidative stress-induced cell death through reduction in the activity of mitogen-activated protein kinase and in the accumulation of intracellular calcium. Endocrinology 2007; 148: 627–637.

62 Marin R, Diaz M, Alonso R, Sanz A, Arevalo MA, Garcia-Segura LM: Role of estrogen re-ceptor � in membrane-initiated signaling in neural cells: interaction with IGF-1 receptor. J Steroid Biochem Mol Biol 2009; 114: 2–7.

63 Regan RF, Guo Y: Estrogens attenuate neu-ronal injury due to hemoglobin, chemical hypoxia and excitatory amino acids in mu-rine cortical cultures. Brain Res 1997; 764: 133–140.

64 McEwen BS: Sex, stress and the hippocam-pus: allostasis, allostatic load and the aging process. Neurobiol Aging 2002; 23: 921–939.

65 Marin R, Ramirez C, Morales A, Gonzalez M, Alonso R, Diaz M: Modulation of A � -in-duced neurotoxicity by estrogen receptor � and other associated proteins in lipid rafts. Steroids 2008; 73: 992–996.

66 Vasudevan N, Pfaff DW: Non-genomic ac-tions of estrogens and their interaction with genomic actions in the brain. Front Neuro-endocrinol 2008; 29: 238–257.

67 Marino M, Acconcia F, Bresciani F, Weisz A, Trentalance A: Distinct nongenomic signal transduction pathways controlled by 17 � -estradiol regulate DNA synthesis and cyclin D 1 gene transcription in HepG2 cells. Mol Biol Cell 2002; 13: 3720–3729.

68 Schelshorn DW, Schneider A, Kuschinsky W, Weber D, Kruger C, Dittgen T, Bourgers HF, Sabouri F, Gassler N, Bach A, Maurer MH: Expression of hemoglobin in rodent neurons. J Cereb Blood Flow Metab 2009; 29: 585–595.

69 Swaab DF: Sexual differentiation of the brain and behavior. Best Pract Res Clin Endo Me-tabol 2007; 21: 431–444.

70 Galluzzo P, Rastelli C, Bulzomi P, Acconcia F, Pallottini V, Marino M: 17 � -Estradiol reg-ulates the first steps of skeletal muscle cell differentiation via ER � -mediated signals. Am J Physiol Cell Physiol 2009; 297:C1249–C1262.

71 Matthews J, Gustafsson JA: Estrogen signal-ling: a subtle balance between ER � and ER � . Mol Intervent 2003; 3: 281–292.

72 Acconcia F, Totta P, Ogawa S, Cardillo I,Inoue S, Leone S, Trentalance A, Muramatsu M, Marino M: Survival versus apoptotic 17 � -estradiol effect: role of ER � - and ER � -activated nongenomic signaling. J Cell Physiol 2005; 203: 193–201.

73 Galluzzo P, Caiazza F, Moreno S, Marino M: Role of ER � palmitoylation in the inhibition of human colon cancer cell proliferation. En-docr Relat Cancer 2007; 14: 153–167.

74 Wang X, Dykens JA, Perez E, Liu R, Yang S, Covey DF, Simpkins JW: Neuroprotective ef-fects of 17 � -estradiol and nonfeminizing es-trogens against H 2 O 2 toxicity in human neu-roblastoma SK-N-SH cells. Mol Pharmacol 2006; 70: 395–404.

75 Antao ST, Duong TT, Aran R, Witting PK: Neuroglobin overexpression in cultured hu-man neuronal cells protects against hydro-gen peroxide insult via activating phos-phoinositide-3 kinase and opening the mito-chondrial K(ATP) channel. Antioxid Redox Signal 2010; 13: 769–781.

76 Mitz SA, Reuss S, Folkow LP, Blix AS, Ramirez JM, Hankeln T, Burmester T: When the brain goes diving: glial oxidative metab-olism may confer hypoxia tolerance to the seal brain. Neurosci 2009; 163: 552–560.

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