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Inhibitory Cross-talk between Estrogen Receptor (ER) and Constitutively Activated Androstane Receptor (CAR)

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m205239200

1083-351X

Gyesik Min, Hwajin Kim, Yangjin Bae, Larry N. Petz, Jongsook Kim Kemper,

Computational Drug Discovery Methods

Estrogen receptor (ER) activity can be modulated by the action of other nuclear receptors. To study whether ER activity is altered by orphan nuclear receptors that mediate the cellular response to xenobiotics, cross-talk between ER and constitutive androstane receptor (CAR), steroid and xenobiotic receptor, or peroxisome proliferator-activated receptor γ was examined in HepG2 cells. Of these receptors, CAR substantially inhibited ER-mediated transcriptional activity of the vitellogenin B1 promoter as well as a synthetic estrogen responsive element (ERE)-containing promoter. Treatment with an agonist of CAR, 1,4-bis-(2-(3,5-dichloropyridoxyl))benzene, potentiated CAR-mediated transcriptional repression. In contrast, an antagonist of CAR, androstenol, alleviated the repression effect. Although CAR interacted with the ER in solution, CAR did not interact with the ER bound to the ERE. CAR/retinoid X receptor bound to the ERE but with much lower affinity than ER. Incremental amounts of CAR elicited a progressive reduction of the ER activity induced by the p160 coactivator glucocorticoid receptor interacting protein 1 (GRIP-1). In turn, increasing amounts of GRIP-1 progressively reversed the depression of ER activity by CAR. An agonist or antagonist of CAR potentiated or alleviated, respectively, the CAR-mediated repression of the GRIP-1-enhanced ER activity, which is consistent with the ability of theses ligands to increase or decrease, respectively, the interaction of CAR with GRIP-1. A CAR mutant that did not interact with GRIP-1 did not inhibit ER-mediated transactivation. Our data demonstrate that xenobiotic nuclear receptor CAR antagonizes ER-mediated transcriptional activity by squelching limiting amounts of p160 coactivator and imply that xenobiotics may influence ER function of female reproductive physiology, cell differentiation, tumorigenesis, and lipid metabolism. Estrogen receptor (ER) activity can be modulated by the action of other nuclear receptors. To study whether ER activity is altered by orphan nuclear receptors that mediate the cellular response to xenobiotics, cross-talk between ER and constitutive androstane receptor (CAR), steroid and xenobiotic receptor, or peroxisome proliferator-activated receptor γ was examined in HepG2 cells. Of these receptors, CAR substantially inhibited ER-mediated transcriptional activity of the vitellogenin B1 promoter as well as a synthetic estrogen responsive element (ERE)-containing promoter. Treatment with an agonist of CAR, 1,4-bis-(2-(3,5-dichloropyridoxyl))benzene, potentiated CAR-mediated transcriptional repression. In contrast, an antagonist of CAR, androstenol, alleviated the repression effect. Although CAR interacted with the ER in solution, CAR did not interact with the ER bound to the ERE. CAR/retinoid X receptor bound to the ERE but with much lower affinity than ER. Incremental amounts of CAR elicited a progressive reduction of the ER activity induced by the p160 coactivator glucocorticoid receptor interacting protein 1 (GRIP-1). In turn, increasing amounts of GRIP-1 progressively reversed the depression of ER activity by CAR. An agonist or antagonist of CAR potentiated or alleviated, respectively, the CAR-mediated repression of the GRIP-1-enhanced ER activity, which is consistent with the ability of theses ligands to increase or decrease, respectively, the interaction of CAR with GRIP-1. A CAR mutant that did not interact with GRIP-1 did not inhibit ER-mediated transactivation. Our data demonstrate that xenobiotic nuclear receptor CAR antagonizes ER-mediated transcriptional activity by squelching limiting amounts of p160 coactivator and imply that xenobiotics may influence ER function of female reproductive physiology, cell differentiation, tumorigenesis, and lipid metabolism. estrogen receptor estrogen responsive element constitutively activated androstane receptor glucocorticoid receptor interacting protein steroid hormone receptor coactivator 1,4-bis-(2-(3,5-dichloropyridoxyl))benzene retinoid X receptor steroid and xenobiotic sensor receptor peroxisome proliferator-activated receptor ligand-binding domin glutathione S-transferase The hormone estrogen mediates diverse biological effects in the cell. Estrogen plays a fundamental role in development and maintenance of female reproductive organs and is involved in the initiation and progression of tumors in these organs. In addition, estrogen has been implicated in the control of gene regulation unrelated to cell growth and reproduction, such as lipid metabolism in the liver (1Archer T.K. Tam S.P. Deeley R.G. J. Biol. Chem. 1986; 261: 5067-5074Abstract Full Text PDF PubMed Google Scholar, 2Croston G.E. Milan L.B. Marchke K.B. Reichman M. Briggs M.R. Endocrinology. 1997; 138: 3779-3786Crossref PubMed Scopus (72) Google Scholar). Estrogen action is mediated by the nuclear receptor, estrogen receptor (ER),1 which is a ligand-dependent transcription factor and consists of distinct modular domains with distinct biological functions (3Evans R. Science. 1988; 240: 889-894Crossref PubMed Scopus (6292) Google Scholar, 4Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2841) Google Scholar). Ligand-bound ER either binds to the ERE or interacts indirectly to the DNA by tethering to other transcriptional factors in estrogen-responsive target genes (5Paech K. Webb P. Kuiper G. Nilsson S. Gustafsson J.-A. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2061) Google Scholar). Ligand binding induces a conformational change of the ER and recruits differential sets of coactivators or corepressors that determine biological activity by altering the magnitude of the transcriptional responses according to the physiological needs (6Katzenellenbogen B.S. Katzenellenbogen J.A. Breast Cancer Res. 2000; 2: 335-342Crossref PubMed Scopus (245) Google Scholar).There is extensive evidence that ER-mediated transcriptional activity is modulated by actions of other nuclear receptors and transcription factors. Thyroid hormone receptor and PPARα have been shown to bind the ERE with high affinity and inhibit gene expression, perhaps by competing with the ER for binding to the ERE (7Nunez S.B. Medin J.A. Braissant O. Kemp L. Wahli W. Ozato K. Segars J.H. Mol. Cell. Endo. 1997; 127: 27-40Crossref PubMed Scopus (90) Google Scholar, 8Glass O.K. Holloway J.M. Devary O.V. Rosenfeld M.G. Cell. 1988; 54: 313-323Abstract Full Text PDF PubMed Scopus (462) Google Scholar). The xenobiotic nuclear receptor, aromatic hydrocarbon receptor, has also been shown to modulate ER activity in human breast and hepatic cell lines (9Duan R. Porter W. Samudio I. Vyhlidal C. Kladde M. Safe S. Mol. Endocrinol. 1999; 13: 1511-1521Crossref PubMed Google Scholar, 10Ricci M.S. Tscano D.G. Mattingly C.J. Toscano J., WA. J. Biol. Chem. 1999; 274: 3430-3438Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Progesterone receptor isoforms A and B have been shown to act as potent repressors for ER activity by interfering with the interaction of ER with the transcriptional machinery (11Kraus W.L. Weis K.E. Katzenellenbogen B.S. Mol. Cell. Biol. 1995; 15: 1847-1857Crossref PubMed Scopus (173) Google Scholar). Rather than inhibiting ER activity, polypeptide growth factors such as epidermal growth factor and insulin-like growth factor have been shown to stimulate ER-mediated transcription in an E2-independent manner (12Smith C.L. Biol. Reprod. 1998; 58: 627-632Crossref PubMed Scopus (265) Google Scholar).Previous studies established that CAR influences steroid homeostasis through transcriptional regulation of CYP2B genes, which are steroid hydroxylases (13Wei P. Zhang J. Egan-Hafley M. Liang S. Moore D.D. Nature. 2000; 407: 920-923Crossref PubMed Scopus (582) Google Scholar). Unlike classical nuclear receptors, transcriptional activity of CAR is ligand-independent (14Baes M. Gulick T. Choi H.-S. Grazia M. Martinoli G. Simha D. Moore D.D. Mol. Cell. Biol. 1994; 14: 1544-1552Crossref PubMed Scopus (409) Google Scholar). This constitutive activity can be inhibited by the testosterone metabolites, androstenol and androstanol (15Forman B.M. Tzameli I. Choi H. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (433) Google Scholar). These androstanes antagonize ligand-independent transcription activation by decreasing the interaction of CAR with coactivators, such as GRIP-1 (16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) or SRC-1 (15Forman B.M. Tzameli I. Choi H. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (433) Google Scholar). CAR is sequestered in the cytoplasm, and treatment with agonists, such as TCPOBOP (17Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (355) Google Scholar) and phenobarbital, results in the translocation of CAR into the nucleus (18Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (484) Google Scholar), where it binds to its cognate recognition sites as a heterodimer with RXR. Some agonists of CAR also enhance transcriptional activity by promoting the interaction of CAR with the coactivator SRC-1 or GRIP-1 (15Forman B.M. Tzameli I. Choi H. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (433) Google Scholar, 16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition to the CYP2B gene, CAR has also been shown to be involved in regulation of genes involved in peroxisomal oxidation and the bilirubin UDP-glucuronosyl transferase gene and perhaps genes regulated by retinoic acid (19Sugatani J. Kojima H. Kakizaki S. Yoshinari K. Gong Q. Owens I. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (330) Google Scholar). The influence of CAR on the regulation of a broad spectrum of genes with multiple and diverse cellular functions suggests that the CAR-mediating signaling pathway is rather complex and thus that CAR may involve cross-regulation with other cellular signaling pathways.We have investigated the role of the xenobiotic orphan nuclear receptors, CAR, SXR, and PPARγ, in modulating ER activity. The liver is the major organ that metabolizes steroids and one of the target organs for estrogen action in the body. ERα as well as these liver-enriched orphan receptors and their heterodimeric partner RXR are all expressed in the liver (20Kliewer S.A. Lehmann J.M. Wilson T.M. Science. 1999; 284: 757-760Crossref PubMed Scopus (424) Google Scholar). Recent studies showed that the p160 coactivators, SRC-1, GRIP-1, and SRC-3, are expressed in the liver (21Hong H. Kohli K. Garbedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (493) Google Scholar,22Kim H.-J. Lee S.-K., Na, S.-Y. Choi H.-S. Lee J.W. Mol. Endocrinol. 1998; 12: 1038-1047Crossref PubMed Scopus (43) Google Scholar) and play a role in transcriptional activation mediated by the orphan receptors, CAR and SXR (16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Moore 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 (736) Google Scholar). Therefore, we examined whether biological cross-talk between the ER and these xenobiotic nuclear receptors occurred in the hepatic HepG2 cell line. We found that CAR significantly inhibited ER-mediated transactivation of the vitellogenin B1 promoter, as well as a synthetic ERE-driven promoter by a mechanism in which CAR squelches limiting amounts of p160 coactivators, such as SRC-1 and GRIP-1, which are essential for ER action.DISCUSSIONThe liver is a major target organ for estrogen action, so that the liver-abundant xenobiotic orphan nuclear receptors, CAR, or SXR, have the potential to modulate steroid hormone homeostasis (20Kliewer S.A. Lehmann J.M. Wilson T.M. Science. 1999; 284: 757-760Crossref PubMed Scopus (424) Google Scholar). On one level, these nuclear receptors may regulate estrogen action by virtue of their induction of the cytochrome P-450 drug metabolizing enzymes that alter hepatic metabolism of estrogens. On a second level, the receptors may more directly affect estrogen action by cross-regulation. These orphan receptors, for example, have been reported to interact with the p160 coactivator, SRC-1, which is also an essential transcriptional coactivator for ER action, so that the regulatory pathways of the two receptor systems have components in common (17Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (355) Google Scholar,23Moore 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 (736) Google Scholar). These observations led us to a hypothesis that the xenobiotic orphan nuclear receptors may influence the ER-mediated signaling pathway in hepatic cells. Our results demonstrated that SXR very modestly repressed the ER-mediated transactivation, whereas the PPARγ did not repress the ER-mediated transactivation in transfected human hepatic HepG2 cells. In contrast, cotransfection of increasing amounts of a CAR expression plasmid resulted in a substantial reduction of ER transactivation. Even without treatment with an agonistic ligand for CAR, TCPOBOP, CAR inhibited ER transcriptional activity, consistent with the constitutive activity of CAR (14Baes M. Gulick T. Choi H.-S. Grazia M. Martinoli G. Simha D. Moore D.D. Mol. Cell. Biol. 1994; 14: 1544-1552Crossref PubMed Scopus (409) Google Scholar). The inhibition of ER-mediated transactivation by CAR was increased by a CAR agonist, TCPOBOP (17Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (355) Google Scholar), and was decreased by a CAR antagonist, androstenol. These results demonstrate that there is regulatory cross-talk between CAR and the ER. The modulation of the effect by CAR ligands indicates that the cross-talk is related to the transcriptional activity of CAR.CAR-mediated Inhibitory Mechanism of Transcriptional Activity of the ERThree potential mechanisms by which CAR could repress ER-mediated transcriptional activity in the HepG2 cells are: 1) formation of an inactive complex with ER by interacting directly with the ER, 2) competition for ER binding to the ERE, and 3) squelching a coactivator of ER action, such as GRIP-1 or SRC-1. To determine whether CAR-mediated transcriptional repression of ER activity results from the inhibition of ER binding to the ERE site and/or the formation of the inactive heterodimer with ER by blocking the dimerization interface or coactivator interacting domain, we analyzed in vitro protein-DNA interaction studies using GST pull-down assays and gel mobility shift assay. Although CAR could interact with the ER in solution in GST pull-down assays, we were not able to detect an interaction of CAR with the ER when the ER was bound to the ERE. The first possibility of direct binding of CAR to the ER, therefore, is unlikely to explain the cross-regulation.CAR is promiscuous in binding to a variety of nuclear receptor-binding sites (17Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (355) Google Scholar, 31Paquet Y. Trottier E. Beaudet M.-J. Anderson A. J. Biol. Chem. 2000; 275: 38427-38436Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), so it was not too surprising that we found that transfected CAR/RXR from HepG2 nuclear extracts, as well as purified CAR/RXR, was able to bind to the consensus ERE. However, the binding affinity of CAR for the ERE was much less than that of the ER, so that a several hundred-fold excess of CAR did not affect binding of the ER to the ERE in vitro, whereas ER competed efficiently for CAR binding. Further, CAR ligands did not affect the binding of CAR to the ERE in contrast to their effects on inhibition of ER action (data not shown). It seems unlikely, therefore, that competition for binding to the ERE can explain the inhibition of ER action by CAR. The third possibility, the squelching of coactivators, seems the most likely possibility.ER works with many other non-DNA-binding proteins in the regulation of gene expression. These coregulators play crucial roles in the ER action in the cells. They influence the magnitude of transcriptional activation or repression and alter the dose-responsive profiles to the ligands depending on the natures of the ligand (6Katzenellenbogen B.S. Katzenellenbogen J.A. Breast Cancer Res. 2000; 2: 335-342Crossref PubMed Scopus (245) Google Scholar). The involvement of coregulators, coactivators such as SRC-1, GRIP-1, and corepressors, such as SMRT (silencing mediator forretinoid and thyroid hormone receptor), nuclear receptor corepressor-1, in transcriptional regulation by the ER is now well established (32Horwitz K. Jackson T. Bain D. Richer J. Takimoto G. Tung L. Mol. Endocrinol. 1996; 10: 1167-1177Crossref PubMed Scopus (834) Google Scholar, 33Robyr D. Wolffe A.P. Wahli W. Mol. Endocrinol. 2000; 14: 329-347Crossref PubMed Scopus (325) Google Scholar). Therefore, we tested the possibility that CAR interferes with the ER transactivation by squelching the ER coactivators, GRIP-1 and SRC-1. As shown recently (16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), the interaction of CAR with GRIP-1 and the transcriptional activity of CAR are increased and decreased by CAR agonists and antagonists, respectively. Similar modulation of the CAR-mediated inhibition of ER action by CAR ligands is consistent with competition for binding of these p160 coactivators between CAR and ER, resulting in squelching. Reversal of the CAR effect by increasing amounts of GRIP-1 further supports this possibility. The strongest evidence for this hypothesis is the loss in inhibition of ER action by CAR if the C-terminal 8 amino acids of CAR are deleted. This mutant retains the ability to bind to DNA (25Choi H.S. Chung M. Tzameli I. Simha D. Lee Y.K. Seol W. Moore D.D. J. Biol. Chem. 1997; 272: 23565-23571Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and is translocated into the nucleus as the wild type CAR is (18Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (484) Google Scholar). However, transcriptional activity and the interaction with GRIP1 is lost, which strongly suggests that competition for GRIP1, or possibly other coactivators recruited by GRIP1, is the basis for the inhibition of ER action by CAR.Physiological ImplicationsModulation of estrogen action by environmental compounds has been intensively studied recently. Much of this interest has focused on weakly estrogenic compounds present in the environment that can mediate increased estrogen action (34Tapiero H., Ba, G.N. Tew K.D. Biomed. Pharmacother. 2002; 56: 36-44Crossref PubMed Scopus (166) Google Scholar). However, a second mechanism for environmental modulation of estrogen action is suggested by this study in which cross-regulation is shown in human HepG2 cells between the ER and the xenobiotic orphan receptor CAR, which is activated by ingestion of xenobiotics. Estrogen has been shown to regulate transcriptional activity of hepatic genes, such as vitellogenin (28Keller H. Givel F. Perroud M. Wahli W. Mol. Endocrinol. 1995; 9: 794-804Crossref PubMed Google Scholar), apolipoprotein A-I (1Archer T.K. Tam S.P. Deeley R.G. J. Biol. Chem. 1986; 261: 5067-5074Abstract Full Text PDF PubMed Google Scholar, 35Lamon-Fava S. Ordovas J. Schaefer E.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2960-2965Crossref PubMed Scopus (53) Google Scholar), and low density lipoprotein receptor (2Croston G.E. Milan L.B. Marchke K.B. Reichman M. Briggs M.R. Endocrinology. 1997; 138: 3779-3786Crossref PubMed Scopus (72) Google Scholar). Therefore, we envision environmental agents that activate CAR may influence ER-mediated transcriptional activity of these genes by suppressing the ER activity in hepatic cells. Effects on the genes affecting lipid metabolism are interesting because functional cross-talk between xenobiotic metabolism and lipid metabolism has been implicated in recent studies (36Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. Mackenzie K. Latour A. Liu Y. Klaassen C. Brown K. Reinhard J. Wilson T. Koller B. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1114) Google Scholar). Because of their promiscuous binding to DNA and ligands, functional cross-talk between CAR and other xenobiotic orphan nuclear receptors, such as PXR, SXR, and PPARγ, in response to endogenous and exogenous chemicals has been documented (23Moore 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 (736) Google Scholar). This suggests that a network of regulation of cellular activity by xenobiotics may impinge on ER function in the liver. Such cross-regulation may occur in other tissues as well. For example, we found that in addition to ERα, xenobiotic nuclear receptors, CAR, SXR, PPARγ, and their heterodimeric partner RXR, are differentially expressed in five human breast cancer cell lines, and cross-regulation between the ER and the xenobiotic receptors occurs. 2J. K. Kemper, G. Min, and X. Xia, unpublished data. Thus, cross-talk studies from HepG2 cells as well as breast cancer cells imply the physiological relevance of receptor cross-talk between CAR and the ER in estrogen-responsive tissues such as female reproductive cells or hepatic cells. In conclusion, we demonstrate that in hepatic cells CAR inhibits ER signaling, which plays a crucial role in female reproductive physiology, cell growth, cell differentiation, and lipid metabolism in the cells. Such interactions may also occur in reproductive tissues. The hormone estrogen mediates diverse biological effects in the cell. Estrogen plays a fundamental role in development and maintenance of female reproductive organs and is involved in the initiation and progression of tumors in these organs. In addition, estrogen has been implicated in the control of gene regulation unrelated to cell growth and reproduction, such as lipid metabolism in the liver (1Archer T.K. Tam S.P. Deeley R.G. J. Biol. Chem. 1986; 261: 5067-5074Abstract Full Text PDF PubMed Google Scholar, 2Croston G.E. Milan L.B. Marchke K.B. Reichman M. Briggs M.R. Endocrinology. 1997; 138: 3779-3786Crossref PubMed Scopus (72) Google Scholar). Estrogen action is mediated by the nuclear receptor, estrogen receptor (ER),1 which is a ligand-dependent transcription factor and consists of distinct modular domains with distinct biological functions (3Evans R. Science. 1988; 240: 889-894Crossref PubMed Scopus (6292) Google Scholar, 4Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2841) Google Scholar). Ligand-bound ER either binds to the ERE or interacts indirectly to the DNA by tethering to other transcriptional factors in estrogen-responsive target genes (5Paech K. Webb P. Kuiper G. Nilsson S. Gustafsson J.-A. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2061) Google Scholar). Ligand binding induces a conformational change of the ER and recruits differential sets of coactivators or corepressors that determine biological activity by altering the magnitude of the transcriptional responses according to the physiological needs (6Katzenellenbogen B.S. Katzenellenbogen J.A. Breast Cancer Res. 2000; 2: 335-342Crossref PubMed Scopus (245) Google Scholar). There is extensive evidence that ER-mediated transcriptional activity is modulated by actions of other nuclear receptors and transcription factors. Thyroid hormone receptor and PPARα have been shown to bind the ERE with high affinity and inhibit gene expression, perhaps by competing with the ER for binding to the ERE (7Nunez S.B. Medin J.A. Braissant O. Kemp L. Wahli W. Ozato K. Segars J.H. Mol. Cell. Endo. 1997; 127: 27-40Crossref PubMed Scopus (90) Google Scholar, 8Glass O.K. Holloway J.M. Devary O.V. Rosenfeld M.G. Cell. 1988; 54: 313-323Abstract Full Text PDF PubMed Scopus (462) Google Scholar). The xenobiotic nuclear receptor, aromatic hydrocarbon receptor, has also been shown to modulate ER activity in human breast and hepatic cell lines (9Duan R. Porter W. Samudio I. Vyhlidal C. Kladde M. Safe S. Mol. Endocrinol. 1999; 13: 1511-1521Crossref PubMed Google Scholar, 10Ricci M.S. Tscano D.G. Mattingly C.J. Toscano J., WA. J. Biol. Chem. 1999; 274: 3430-3438Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Progesterone receptor isoforms A and B have been shown to act as potent repressors for ER activity by interfering with the interaction of ER with the transcriptional machinery (11Kraus W.L. Weis K.E. Katzenellenbogen B.S. Mol. Cell. Biol. 1995; 15: 1847-1857Crossref PubMed Scopus (173) Google Scholar). Rather than inhibiting ER activity, polypeptide growth factors such as epidermal growth factor and insulin-like growth factor have been shown to stimulate ER-mediated transcription in an E2-independent manner (12Smith C.L. Biol. Reprod. 1998; 58: 627-632Crossref PubMed Scopus (265) Google Scholar). Previous studies established that CAR influences steroid homeostasis through transcriptional regulation of CYP2B genes, which are steroid hydroxylases (13Wei P. Zhang J. Egan-Hafley M. Liang S. Moore D.D. Nature. 2000; 407: 920-923Crossref PubMed Scopus (582) Google Scholar). Unlike classical nuclear receptors, transcriptional activity of CAR is ligand-independent (14Baes M. Gulick T. Choi H.-S. Grazia M. Martinoli G. Simha D. Moore D.D. Mol. Cell. Biol. 1994; 14: 1544-1552Crossref PubMed Scopus (409) Google Scholar). This constitutive activity can be inhibited by the testosterone metabolites, androstenol and androstanol (15Forman B.M. Tzameli I. Choi H. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (433) Google Scholar). These androstanes antagonize ligand-independent transcription activation by decreasing the interaction of CAR with coactivators, such as GRIP-1 (16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) or SRC-1 (15Forman B.M. Tzameli I. Choi H. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (433) Google Scholar). CAR is sequestered in the cytoplasm, and treatment with agonists, such as TCPOBOP (17Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (355) Google Scholar) and phenobarbital, results in the translocation of CAR into the nucleus (18Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (484) Google Scholar), where it binds to its cognate recognition sites as a heterodimer with RXR. Some agonists of CAR also enhance transcriptional activity by promoting the interaction of CAR with the coactivator SRC-1 or GRIP-1 (15Forman B.M. Tzameli I. Choi H. Chen J. Simha D. Seol W. Evans R.M. Moore D.D. Nature. 1998; 395: 612-615Crossref PubMed Scopus (433) Google Scholar, 16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition to the CYP2B gene, CAR has also been shown to be involved in regulation of genes involved in peroxisomal oxidation and the bilirubin UDP-glucuronosyl transferase gene and perhaps genes regulated by retinoic acid (19Sugatani J. Kojima H. Kakizaki S. Yoshinari K. Gong Q. Owens I. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (330) Google Scholar). The influence of CAR on the regulation of a broad spectrum of genes with multiple and diverse cellular functions suggests that the CAR-mediating signaling pathway is rather complex and thus that CAR may involve cross-regulation with other cellular signaling pathways. We have investigated the role of the xenobiotic orphan nuclear receptors, CAR, SXR, and PPARγ, in modulating ER activity. The liver is the major organ that metabolizes steroids and one of the target organs for estrogen action in the body. ERα as well as these liver-enriched orphan receptors and their heterodimeric partner RXR are all expressed in the liver (20Kliewer S.A. Lehmann J.M. Wilson T.M. Science. 1999; 284: 757-760Crossref PubMed Scopus (424) Google Scholar). Recent studies showed that the p160 coactivators, SRC-1, GRIP-1, and SRC-3, are expressed in the liver (21Hong H. Kohli K. Garbedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (493) Google Scholar,22Kim H.-J. Lee S.-K., Na, S.-Y. Choi H.-S. Lee J.W. Mol. Endocrinol. 1998; 12: 1038-1047Crossref PubMed Scopus (43) Google Scholar) and play a role in transcriptional activation mediated by the orphan receptors, CAR and SXR (16Min G. Kemper J.K. Kemper B. J. Biol. Chem. 2002; 277: 25356-26363Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Moore L.B. Parks D.J. Jones S.A.