Orphan Nuclear Receptors: The Exotics of Xenobiotics (2001)

nuclear receptor constitutive androstane receptor cytochrome P450 phenobarbital pregnenolone 16α-carbonitrile pregnane X receptor rifampicin steroid and xenobiotic receptor 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene retinoid X receptor Orphan nuclear receptors (NRs)1 include gene products that are structurally related to nuclear hormone receptors but lack known physiological ligands. It has become clear that orphan NRs represent a unique and pivotal resource to uncover new regulatory systems that impact both health and human diseases. In addition to production and clearance of endogenous hormones, mammals also confront numerous foreign chemicals (xenobiotics) such as ingested food, environmental pollutants, and carcinogens as well as prescription and non-prescription drugs. Such compounds may accumulate to toxic levels unless they are metabolized and eliminated, a process largely mediated by the supergene family of cytochrome P450 (CYP) enzymes. Expressed mainly in the liver and capable of recognizing an amazing diversity of xenobiotics, CYP enzymes catalyze the metabolic conversion of xenobiotics to polar derivatives that are more readily eliminated (for reviews, see Refs. 1Gonzalez F.J. Trends Pharmacol. Sci. 1992; 13: 346-352Abstract Full Text PDF PubMed Scopus (393) Google Scholar and 2Denison M.S. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Thus, CYP enzymes, especially members of the CYP 1–4 families, are crucial for xenobiotic detoxification and survival of organisms (for a review, see Ref. 3Waxman D.J. Arch. Biochem. Biophys. 1999; 369: 11-23Crossref PubMed Scopus (674) Google Scholar). Among CYP enzymes, the CYP3A and -2B isoenzymes are of particular medical significance. For example, the human CYP3A4 enzyme alone is involved in the metabolism of 50–60% of clinical drugs as well as neutraceuticals and herbal medicines (4Maurel P. Ioannides C. Cytochrome P450, Metabolic and Toxicological Aspects. CRC Press, Inc., Boca Raton, FL1996: 241-270Google Scholar). An additional 25–30% of these compounds are metabolized by the CYP2B isoenzymes. The combined metabolic versatility of CYP3A and -2B, coupled with their inducibility by xenobiotic substrates, constitutes a molecular basis for many clinical drug-drug interactions (4Maurel P. Ioannides C. Cytochrome P450, Metabolic and Toxicological Aspects. CRC Press, Inc., Boca Raton, FL1996: 241-270Google Scholar). Compounds that are strong CYP enzyme inducers such as glucocorticoids, phenobarbital (PB), or rifampicin (RIF) can dramatically affect the clearance of any co-consumed drug that is a substrate for the enzyme. Recently, St. Johns wort, a popular herbal remedy for depression, was found to trigger severe adverse drug-drug interactions with oral contraceptives, the HIV protease inhibitor indinavir, and the immunosuppressant cyclosporin as a consequence of activating the CYP3A system (Ref. 5Moore L.B. Goodwin B. Jones S.A. Wisely G.B. Serabjit-Singh C.J. Willson T.M. Collins J.L. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7500-7502Crossref PubMed Scopus (885) Google Scholar, and references therein). In 1992, the aryl hydrocarbon receptor was identified as a transcriptional sensor mediating the induction of CYP1A and -1B1 genes by dioxin and a variety of related polycyclic aromatic hydrocarbons (6Ema M. Sogawa K. Watanabe N. Chujoh Y. Matsushita N. Gotoh O. Funae Y. Fujii-Kuriyama Y. Biochem. Biophys. Res. Commun. 1992; 184: 246-253Crossref PubMed Scopus (371) Google Scholar). Aryl hydrocarbon receptor is a member of the basic helix-loop-helix family of transcription factors, and it was presumed that other basic helix-loop-helix proteins might control the CYP3A and -2B gene families. This turned out to be an incorrect assumption, and the molecular basis for induction of CYP3A and -2Bgenes remained largely unknown. A major conceptual challenge in defining the molecular mechanisms for xenobiotic response is thatCYP genes are induced by thousands of natural and synthetic compounds, and yet the inducibility of CYP enzymes shows clear target gene and species specificity. For example, the antibiotic RIF has been shown to be a specific CYP3A inducer in humans and rabbits, whereas pregnenolone 16α-carbonitrile (PCN), an anti-glucocorticoid, is a rodent-specific CYP3A inducer (Ref. 7Barwick J.L. Quattrochi L.C. Mills A.S. Potenza C. Tukey R.H. Guzelian P.S. Mol. Pharmacol. 1996; 50: 10-16PubMed Google Scholar, and references therein). Pharmacologic studies in cultured primary hepatocytes suggest that it is not the structure of CYP3A genes but rather species-specific cellular factor(s) that dictates the profile ofCYP3A inducibility (7Barwick J.L. Quattrochi L.C. Mills A.S. Potenza C. Tukey R.H. Guzelian P.S. Mol. Pharmacol. 1996; 50: 10-16PubMed Google Scholar). In the past 3–4 years, emerging evidence has increasingly pointed to a unique role for orphan NRs in the regulation of CYP genes by functioning as atypical pleotropic receptors for a remarkable diversity of xenobiotic compounds (Fig. 1). In 1998, a human orphan receptor SXR (steroid and xenobiotic receptor) and its rodent ortholog PXR (pregnane X receptor) were isolated as candidate xenobiotic receptors (xeno-sensors) postulated to regulate CYP3A gene expression (8Blumberg B. Sabbagh Jr., W. Juguilon H. Bolado Jr., J. van Meter C.M. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (828) Google Scholar, 9Kliewer 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. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar, 10Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow-Backman M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (801) Google Scholar) (for reviews, see Refs. 3Waxman D.J. Arch. Biochem. Biophys. 1999; 369: 11-23Crossref PubMed Scopus (674) Google Scholar, 11Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Crossref PubMed Scopus (290) Google Scholar, and 12Savas U. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1999; 56: 851-857Crossref PubMed Scopus (98) Google Scholar). Initially isolated as a homolog of the Xenopus BXR (benzoate X receptor) gene and predominantly expressed in the liver and intestine,SXR and PXR were found to be activated by a variety of xenobiotic compounds and steroids that were known to induce hepatic and intestinal CYP3A activity. Moreover, SXR and PXR have been shown to bind to the IR-6 and DR-3 xenobiotic response elements localized in the promoter regions of the human or rodent CYP3A genes (8Blumberg B. Sabbagh Jr., W. Juguilon H. Bolado Jr., J. van Meter C.M. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (828) Google Scholar, 9Kliewer 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. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar, 10Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow-Backman M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (801) Google Scholar). Recently, proof of the function identity of SXR/PXR has been established by targeted disruption of the mouse PXR locus, which abolishes the CYP3A xenobiotic response (13Xie 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. Nature. 2000; 406: 435-439Crossref PubMed Scopus (615) Google Scholar, 14Staudinger 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. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1182) Google Scholar). More importantly, replacement of PXR with its human homologSXR fully restores the xenobiotic response within the mouse liver but now with a humanized response profile (Fig.2) (13Xie 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. Nature. 2000; 406: 435-439Crossref PubMed Scopus (615) Google Scholar). Therefore, SXR and PXR function as species-specific xeno-sensors mediating the classic "adaptive hepatic response." It has long been postulated that evolutionary selection for the xenobiotic response was in part driven by the ability of one toxin to confer "metabolic immunity" to both itself and other co-consumed toxins by virtue of induction of common metabolic enzymes. If this hypothesis is correct, PXR null mice would fail to mount a hepatic response and thus remain sensitive to multidrug response. One common test of this idea is the ability of PCN pretreatment to protect mice from subsequent exposure to a second drug. Indeed, this key role for SXR/PXR in xenoprotection is confirmed by the failure of PCN-treated PXR null mice to protect against sedative xenotoxicants such as zoxazolamine and tribromoethanol (15Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3375-3380Crossref PubMed Scopus (694) Google Scholar) or the hepatotoxic bile acid lithocholic acid (14Staudinger 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. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1182) Google Scholar, 15Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3375-3380Crossref PubMed Scopus (694) Google Scholar). In contrast, hepatic expression of a constitutively activated SXR(VPSXR) transgene results in sustained up-regulation ofCYP3A gene expression and consequent protection against xenotoxicants (Fig. 3) (13Xie 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. Nature. 2000; 406: 435-439Crossref PubMed Scopus (615) Google Scholar).Figure 3Activation of SXR confers protection against xenobiotic toxicants. The zoxazolamine paralysis test in wild-type (WT) and VPSXR mice (the left andright mouse in B, respectively) is shown. The VPSXR mice, engineered to express an activated form of SXRtransgene in the liver, exhibit enhanced protection against paralytic zoxazolamine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to its ability to mediate CYP3A induction, SXR has been shown to induce the expression of the multiple drug resistance (MDR1) gene in response to several SXR agonists such as the chemotherapeutic agent Taxol (16Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (771) Google Scholar). MDR1 encodes a transporter that protects cells from toxicity by rapidly effluxing drugs. Therefore, SXR is implicated in both drug metabolism and clearance (Fig. 1). Constitutive androstane receptor (CAR) was initially isolated and shown to activate a DR-5 type of retinoid acid response element (βRARE) in a ligand-independent manner (17Baes M. Gulick T. Choi H.-S. Martinoli M.G. Simha D. Moore D.D. Mol. Cell. Biol. 1994; 14: 1544-1552Crossref PubMed Scopus (422) Google Scholar). The identity of CAR as a xenobiotic receptor was first hinted by the ability of selective androstane metabolites to inhibit its constitutive activity (18Forman B.M. Tzameli I. Choi H.S. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (440) Google Scholar). Its role in positive xenobiotic regulation was suggested when CAR was shown to activate the phenobarbital response element found in promoters of PB-inducible CYP2B genes (Ref. 19Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (659) Google Scholar, and references therein). Subsequently, this activation was found to be potentiated by PB and its derivatives such as TCPOBOP (19Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (659) Google Scholar). Moreover, disruption of the mouse CAR locus by homologous recombination resulted in loss of PB and TCPOBOP activation of theCYP2B10 gene (20Wei P. Zhang J. Egan-Hafley M. Liang S. Moore D.D. Nature. 2000; 407: 920-923Crossref PubMed Scopus (596) Google Scholar). Oddly, loss of CAR increases sensitivity to zoxazolamine-induced paralysis while decreasing sensitivity to cocaine-induced acute hepatic response (20Wei P. Zhang J. Egan-Hafley M. Liang S. Moore D.D. Nature. 2000; 407: 920-923Crossref PubMed Scopus (596) Google Scholar), furthering the complexity of the xenobiotic response. Similar to SXR and PXR, CAR also exhibits clear species-dependent ligand specificity. For example, TCPOBOP is an activating ligand for rodent CAR but fails to affect human CAR (21Moore 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. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar). 2W. Xie and R. M. Evans, unpublished observations. SXR/PXR and CAR were originally shown to regulate CYP3Aand -2B genes, respectively, presumably through distinct classes of drugs. Because the xenobiotic response elements in these two classes of CYP genes are different, it seemed reasonable to speculate that they were independently regulated. Surprisingly, several groups have recently demonstrated the existence of cross-talks between these receptors and their target CYP genes. Such reciprocal regulation is accomplished via adaptive recognition of each other's DNA response elements as diagrammed in Fig.4. The phenobarbital response element of the CYP2B promoter contains two imperfect DR-4 type NR binding sites that display measurable affinity for SXR, and subsequently SXR was found to regulate CYP2B both in cultured cells and in transgenic mice (22Xie W. Barwick J.L. Simon C.M. Pierce A. Safe S. Blumberg B. Guzelian P.S. Evans R.M. Genes Dev. 2000; 14: 3014-3023Crossref PubMed Scopus (466) Google Scholar). In a type of functional symmetry, CAR can activate CYP3A through previously defined SXR/PXR response elements (19Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (659) Google Scholar, 21Moore 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. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar, 22Xie W. Barwick J.L. Simon C.M. Pierce A. Safe S. Blumberg B. Guzelian P.S. Evans R.M. Genes Dev. 2000; 14: 3014-3023Crossref PubMed Scopus (466) Google Scholar, 23Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (364) Google Scholar). The in vivocross-talk between PXR and CAR could be more thoroughly explored via the generation of mice deficient in both PXR andCAR genes. Nevertheless, the cross-regulation ofCYP gene classes provides an explanation for the dual activation property of certain xenobiotics (24Strom S.C. Pisarov L.A. Dorko K. Thompson M.T. Schuetz J.D. Schuetz E.G. Methods Enzymol. 1996; 272: 388-400Crossref PubMed Google Scholar). However, not all drugs appear to be dual activators, which might correlate to their relative affinity for the receptors or reflect other more complex factors. Despite some limitations, this reciprocal regulation of CYPgenes by multiple xenobiotic receptors reveals the existence of a fail-safe metabolic safety net to protect against xenotoxicants and at the same time to increase the propensity for drug-drug interactions. Moreover, the overlap in their response element recognition establishes a molecular basis for a regulatory network of CYP gene expression that expands the function of an individual orphan receptor. The summarized work represents the beginning of an undoubtedly long road to understand the molecular complexity of the xenobiotic response. The identification and characterization of xenobiotic receptors such as SXR/PXR and CAR have created many future research opportunities described in the following paragraphs. Having demonstrated that SXR and PXR mediate CYP3A response, the presence of candidate DR-3 or IR-6 response elements in genes encoding additional phase I and phase II xenobiotic enzymes such as CYP2A, CYP2C, CYP2E, and UDP-glucuronosyltransferase raised the potential for a broader physiologic function (8Blumberg B. Sabbagh Jr., W. Juguilon H. Bolado Jr., J. van Meter C.M. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (828) Google Scholar). In addition, recent identification of MDR-1 (also known as P-glycoprotein) as a direct target of SXR implicates this receptor in drug efflux, which adds a new dimension to the action of these receptors (16Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (771) Google Scholar). It has become clear that many genes belonging to CYP 1–4 families can be transcriptionally activated by foreign chemicals through one or more NRs. TableI summarizes a list of P450 inducers, their responsive CYPs, the mediating NR, and the cognate response elements. Of note, many of the xenobiotic receptors also have endogenous ligands, indicating a broader physiologic role for these receptors. Interestingly, as a common heterodimerization partner for xenobiotic receptors, RXR seems to function as a master regulator for CYP gene expression because a liver-specific disruption of mouse RXRα gene causes decreased basal expression ofCYP3A, -2B, -4A, and -7A(25Wan Y.J. An D. Cai Y. Repa J.J. Hung-Po Chen T. Flores M. Postic C. Magnuson M.A. Chen J. Chien K.R. French S. Mangelsdorf D.J. Sucov H.M. Mol. Cell. Biol. 2000; 20: 4436-4444Crossref PubMed Scopus (205) Google Scholar). Accordingly, both CYP2B1/2 and CYP3A isoenzymes become elevated in rats treated with RXR-selective agonists (26Howell S.R. Shirley M.A. Ulm E.H. Drug Metab. Dispos. 1998; 26: 234-239PubMed Google Scholar).Table ICYP induction mediated by nuclear receptorsP450 inducersPrototypic responsive CYPsReceptorsNR-responsive elementsRepresentative endogenous ligandsPhenobarbitalh2B6CARDR4 (PBRE)1-aPhenobarbital response element.Androstane metabolites1-bInhibitory ligands.r2B1, 2B2DR3m2B10IR6Dexamethasoneh3A4SXR/PXRDR3Pregnenolone, corticosterone, bile acidsRifampicinr3A23IR6Hyperform1-hComponent of St. Johns wort.m3A11DR4Fibrate drugs4A1PPARα1-cPeroxisome proliferator-activated receptor.DR1Linoleic acid, arachidonic acid4A24A3Cholesterol7A1LXR1-dLiver X receptor.DR424(S)-HydroxycholesterolBile acids7A11-eRepression.FXR1-fFarnesoid X receptor.IR1Chenodeoxycholic acidThyroid hormoneP450 reductaseTR1-gThyroid hormone receptor.DR4Thyroid hormoneRetinoid acids2B, 3ARXRAll9-cis-Retinoid acid4A, 7A1-a Phenobarbital response element.1-b Inhibitory ligands.1-c Peroxisome proliferator-activated receptor.1-d Liver X receptor.1-e Repression.1-f Farnesoid X receptor.1-g Thyroid hormone receptor.1-h Component of St. Johns wort. Open table in a new tab For example, certain types of bile acids, known as potent ligands for farnesoid X receptor, have recently been shown to activate SXR and PXR in both cultured cells and mice (14Staudinger 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. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1182) Google Scholar, 15Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3375-3380Crossref PubMed Scopus (694) Google Scholar). A combination of knockout and transgenic mouse studies revealed that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer a resistance to toxic cholestatic bile acid lithocholic acid (14Staudinger 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. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1182) Google Scholar, 15Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3375-3380Crossref PubMed Scopus (694) Google Scholar). These observations not only establish a unique role for SXR/PXR-mediated xenobiotic response in the detoxification of bile acids but also provide a molecular mechanism for the clinical relief of cholestasis-associated pruritus by RIF or other CYP3Ainducers. As ligand-dependent transcription factors, these receptors can be used in conjunction with DNA array-based studies to characterize the network of target genes that comprise the genetic foundation of the xenobiotic response. Although long studied, the molecular basis of the interaction between external chemicals and the mammalian genome has not been well understood. NR-mediated xenobiotic regulation may represent the critical biochemical interface of man with his chemical environment. The combination of loss (gene knockout) and gain (transgene) of function provides a unique strategy to dissect the xenobiotic response through molecular, genomic, pharmacologic, and proteomic approaches. The humanized mouse system represents a major step toward generating a humanized rodent toxicologic model and thus provides an advanced way to explore the interface between the environment and the human genome. These xenobiotic receptors and genetically engineered animals, in conjunction with the completion of the human genome project, should greatly facilitate our understanding of the complexity of the xenobiotic response and its implication in pharmaceutical development including drug profiling, toxicity analysis, and drug-drug interaction. We thank our colleagues for their contribution to our studies described in this review. We thank Dr. Yanhong Shi for comments on the manuscript.