The Farnesoid X Receptor Controls Gene Expression in a Ligand- and Promoter-selective Fashion

Farnesoid X receptor (FXR) is a nuclear receptor for bile acids. Ligand activated-FXR regulates transcription of genes to allow feedback control of bile acid synthesis and secretion. There are five major bile acids in humans. We have previously demonstrated that lithocholate acts as an FXR antagonist, and here we show that the other four bile acids, chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA), and ursodeoxycholate (UDCA), act as selective FXR agonists in a gene-specific fashion. In an in vitro coactivator association assay, CDCA fully activated FXR, whereas CA partially activated FXR and DCA and UDCA had negligible activities. Similar results were also obtained from a glutathione S-transferase pull-down assay in which only CDCA and the synthetic FXR agonist GW4064 significantly increased the interaction of SRC-1 with FXR. In FXR transactivation assays with a bile salt export pump (BSEP) promoter-driven luciferase construct, bile acids showed distinct abilities to activate the BSEP promoter: CDCA, DCA, CA, and UDCA increased luciferase activity by 25-, 20-, 18-, and 8-fold, respectively. Consistently, CDCA increased BSEP mRNA by 750-fold in HepG2 cells, whereas DCA, CA, and UDCA induced BSEP mRNA by 250-, 75-, and 15-fold, respectively. Despite the partial induction of BSEP mRNA, CA, DCA, and UDCA effectively repressed expression of cholesterol 7α-hydroxylase, another FXR target. We further showed that all four bile acids significantly increased FXR protein, suggesting the existence of an auto-regulatory loop in FXR signaling pathways. In conclusion, these results suggest that the binding of each bile acid results in a different FXR conformations, which in turn differentially regulates expression of individual FXR targets. Farnesoid X receptor (FXR) is a nuclear receptor for bile acids. Ligand activated-FXR regulates transcription of genes to allow feedback control of bile acid synthesis and secretion. There are five major bile acids in humans. We have previously demonstrated that lithocholate acts as an FXR antagonist, and here we show that the other four bile acids, chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA), and ursodeoxycholate (UDCA), act as selective FXR agonists in a gene-specific fashion. In an in vitro coactivator association assay, CDCA fully activated FXR, whereas CA partially activated FXR and DCA and UDCA had negligible activities. Similar results were also obtained from a glutathione S-transferase pull-down assay in which only CDCA and the synthetic FXR agonist GW4064 significantly increased the interaction of SRC-1 with FXR. In FXR transactivation assays with a bile salt export pump (BSEP) promoter-driven luciferase construct, bile acids showed distinct abilities to activate the BSEP promoter: CDCA, DCA, CA, and UDCA increased luciferase activity by 25-, 20-, 18-, and 8-fold, respectively. Consistently, CDCA increased BSEP mRNA by 750-fold in HepG2 cells, whereas DCA, CA, and UDCA induced BSEP mRNA by 250-, 75-, and 15-fold, respectively. Despite the partial induction of BSEP mRNA, CA, DCA, and UDCA effectively repressed expression of cholesterol 7α-hydroxylase, another FXR target. We further showed that all four bile acids significantly increased FXR protein, suggesting the existence of an auto-regulatory loop in FXR signaling pathways. In conclusion, these results suggest that the binding of each bile acid results in a different FXR conformations, which in turn differentially regulates expression of individual FXR targets. Bile acids are the end products of cholesterol catabolism. The five major bile acids in humans are chenodeoxycholate (CDCA), 1The abbreviations used are: CDCA, chenodeoxycholate; FXR, farnesoid X receptor; BSEP, bile salt export pump; Cyp7a, cholesterol 7α-hydroxylase; DCA, deoxycholate; CA, cholate; UDCA, ursodeoxycholate; LCA, lithocholate; SRC-1, steroid receptor coactivator protein-1; FBS, fetal bovine serum; CS, charcoal-stripped; DMEM, Dulbecco’s modified Eagle’s medium. GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SHP, small heterodimer partner. deoxycholate (DCA), cholate (CA), ursodeoxycholate (UDCA), and lithocholate (LCA) (1Hofmann A.F. Arch. Intern. Med. 1999; 159: 2647-2658Crossref PubMed Scopus (679) Google Scholar). In addition to their critical roles in lipid and vitamin absorption, bile acids are ligands for the nuclear receptor FXR and regulate expression of genes whose products are critically important for bile acid and cholesterol homeostasis (2Makishima M. 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Edwards P.A. J. Lipid Res. 2002; 43: 2037-2041Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and kininogen (12Zhao A. Lew J.L. Huang L. Yu J. Zhang T. Hrywna Y. Thompson J.R. Pedro Nd N. Blevins R.A. Pelaez F. Wright S.D. Cui J. J. Biol. Chem. 2003; 21: 21Google Scholar). FXR represses expression of cholesterol 7α-hydroxylase (Cyp7a) (11Mak P.A. Kast-Woelbern H.R. Anisfeld A.M. Edwards P.A. J. Lipid Res. 2002; 43: 2037-2041Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 13Chiang J.Y. Kimmel R. Weinberger C. Stroup D. J. Biol. Chem. 2000; 275: 10918-10924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 14Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar, 15Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar), sterol 12 α-hydroxylase (16Zhang M. Chiang J.Y. J. Biol. Chem. 2001; 276: 41690-41699Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), the Na+/taurocholate co-transporting polypeptide (17Denson L.A. Sturm E. Echevarria W. Zimmerman T.L. Makishima M. Mangelsdorf D.J. Karpen S.J. Gastroenterology. 2001; 121: 140-147Abstract Full Text PDF PubMed Scopus (370) Google Scholar), and apolipoprotein A-I (18Claudel T. Sturm E. Duez H. Torra I.P. Sirvent A. Kosykh V. Fruchart J.C. Dallongeville J. Hum D.W. Kuipers F. Staels B. J. Clin. Invest. 2002; 109: 961-971Crossref PubMed Scopus (287) Google Scholar). A few functional distinct classes of FXR ligands have been identified. We have previously shown that LCA is an FXR antagonist and that guggulsterones are SBARMs (selective bile acid receptor modulators) (6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Cui J. Huang L. Zhao A. Lew J.L. Yu J. Sahoo S. Meinke P.T. Royo I. Pelaez F. Wright S.D. J. Biol. Chem. 2003; 278: 10214-10220Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). GW4064 is a synthetic FXR agonist (20Maloney P.R. Parks D.J. Haffner C.D. Fivush A.M. Chandra G. Plunket K.D. Creech K.L. Moore L.B. Wilson J.G. Lewis M.C. Jones S.A. Willson T.M. J. Med. Chem. 2000; 43: 2971-2974Crossref PubMed Scopus (458) Google Scholar). Bile acids such as CDCA, DCA, and CA have been previously shown to be FXR agonists that regulate expression of FXR targets (2Makishima 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. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2164) Google Scholar, 3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1843) Google Scholar, 4Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Mol. Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). In this study, we systematically characterized the FXR agonist activities of these bile acids using a series of cell-free and cell-based assays including coactivator recruitment, GST pull-down assay, FXR transactivation, and quantitative real-time PCR for examining the expression of FXR targets. Our results indicate that CDCA is a full FXR agonist that effectively regulates expression of FXR targets, whereas DCA, CA, and UDCA are partial agonists in FXR transactivation assays and regulate expression of FXR targets in a gene-selective fashion. These three bile acids partially induced BSEP mRNA but effectively repressed Cyp7a and strongly increased FXR protein expression. This study shows for the first time that distinct bile acids have unique properties as FXR agonists and reveals an auto-regulatory loop in FXR signaling pathways. Reagents—The following reagents were obtained from Invitrogen: DMEM and Opti-MEM I; regular fetal bovine serum (FBS) and charcoal-stripped FBS (CS-FBS); and TRIzol reagents. l-[35S]Methionine (1000 Ci/mmol) was obtained from Amersham Biosciences. The TnT T7 QuickCoupled transcription/translation kit and reagents for β-galactosidase and luciferase assays were from Promega (Madison, WI). Fu-GENE 6 transfection reagent was obtained from Roche Diagnostics. Bile acids were obtained from Steraloids, Inc. (Newport, RI). GW4064 was synthesized at Merck (Rahway, NJ). TaqMan reagents for cDNA synthesis and real-time PCR and TaqMan oligonucleotide primers and probes for human 18 S RNA were purchased from Applied Biosystems (Foster City, CA). FXR Coactivator Association Assays—Human GST-FXR-ligand binding domain fusion protein was prepared from Escherichia coli strain BL21. A homogeneous time-resolved fluorescence based FXR and coactivator SRC-1 interaction assay was used to examine the interaction of FXR-ligand binding domain with various ligands according to those described previously for other nuclear receptors (21Zhou G. Cummings R. Li Y. Mitra S. Wilkinson H.A. Elbrecht A. Hermes J.D. Schaeffer J.M. Smith R.G. Moller D.E. Mol. Endocrinol. 1998; 12: 1594-1604Crossref PubMed Scopus (160) Google Scholar) with minor modifications. 198 μl of reaction mixture (100 mm HEPES, 125 mm KF, 0.125% (w/v) CHAPS, 0.05% dry milk, 4 nm human GST-FXR-ligand binding domain, 2 nm anti-GST-Eu3+-cryptate 10 nm biotin-SRC-1 fragment (human SRC-1, amino acids NSPSRLNIQP to VKVKVEKKEQ), and 20 nm SA/XL665 (streptavidin-labeled allophycocyanin)) was added to each well followed by the addition of 2 μl of Me2SO or various concentration of bile acids into appropriate wells. Plates were incubated overnight at 4 °C followed by measurement of fluorescent signals on a Packard Discovery instrument. Data were expressed as the ratio of the emission intensity at 665 nm to that at 620 nm multiplied by a factor of 104. FXR Scintillation Proximity Binding Assay—The assay was performed in a 96-well microtiter plate in a total volume of 100 μl. The assay mixture includes the GST-human FXR fusion protein at 100 ng/well, goat anti-GST antibody (at 1:400-fold dilution, Amersham Biosciences), protein A-Yttrium Silicate scintillation proximity binding assay beads (at 250 μg/well, Amersham Biosciences), and a 3H-labeled FXR radioligand 2A. Adams, V. Rusiecki-Lombardo, and Y. S. Tang, unpublished data. at 2 nm in an assay buffer consisting of 10 mm Tris-HCl, pH 7.2, 1 mm EDTA, 10% glycerol, 10 mm sodium molybdate, 1 mm DTT, 2 μg/ml benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, and 0.05% dry milk. Plates were incubated at 4 °C for 16 h with shaking. Radioactivity was quantified in a Packard Topcount scintillation counter. GST Pull-down Assay—The GST-SRC-1-(568–780) fusion protein was expressed in E. coli strain BL21 and purified using glutathione-Sepharose 4B beads as described previously (21Zhou G. Cummings R. Li Y. Mitra S. Wilkinson H.A. Elbrecht A. Hermes J.D. Schaeffer J.M. Smith R.G. Moller D.E. Mol. Endocrinol. 1998; 12: 1594-1604Crossref PubMed Scopus (160) Google Scholar). 35S-Labeled human FXR was synthesized using the TnT T7 QuickCoupled transcription/translation kit (Promega) according to the manufacturer’s instructions. Approximately, 2 μg of GST-SRC-1 protein on beads was incubated with 2 μl of 35S-labeled full-length human FXR (19Cui J. Huang L. Zhao A. Lew J.L. Yu J. Sahoo S. Meinke P.T. Royo I. Pelaez F. Wright S.D. J. Biol. Chem. 2003; 278: 10214-10220Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) in the presence of various FXR ligands. The mixture was incubated overnight at 4 °C with gentle agitation in a total volume of 116 μl(8mm Tris-HCl, pH 7.4, 0.12 m KCl, 8% glycerol, 0.5% w/v CHAPS, 4 mm DTT, and 1 mg/ml bovine serum albumin). At the end of the incubation, the beads were washed four times with wash buffer (20 mm Tris-HCl, pH 8.0, 100 mm KCl, 0.5% Tween 20, and 2 mm DTT) prior to electrophoresis on a 4–20% SDS-PAGE and visualization by autoradiography. Western Blot Analysis for Expression of FXR Protein—Treatment of HepG2 cells with various FXR agonists and extraction of total nuclear proteins from treated cells were performed as described previously (19Cui J. Huang L. Zhao A. Lew J.L. Yu J. Sahoo S. Meinke P.T. Royo I. Pelaez F. Wright S.D. J. Biol. Chem. 2003; 278: 10214-10220Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). 20 μg of total nuclear proteins was separated by electrophoresis. Western blotting was carried out following the manufacturer’s instructions (Amersham Biosciences) using polyclonal rabbit anti-human FXR antibody (catalog number H-130, Santa Cruz Biotechnology). Donkey anti-rabbit IgG conjugated to horseradish peroxidase and the ECL chemiluminescence kit used for detection were purchased from Amersham Biosciences. FXR Transactivation—HepG2 cells were transfected in 96-well plates using the FuGENE 6 transfection reagent as described previously (22Cui J. Heard T.S. Yu J. Lo J.L. Huang L. Li Y. Schaeffer J.M. Wright S.D. J. Biol. Chem. 2002; 277: 25963-25969Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). FXR transactivation assay using pGL3-enhancer-human BSEP-promoter-Luc construct was performed as described previously (19Cui J. Huang L. Zhao A. Lew J.L. Yu J. Sahoo S. Meinke P.T. Royo I. Pelaez F. Wright S.D. J. Biol. Chem. 2003; 278: 10214-10220Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Treatment of transfected cells with various FXR ligands, assays for luciferase, and β-galactosidase activities were also performed as described previously (22Cui J. Heard T.S. Yu J. Lo J.L. Huang L. Li Y. Schaeffer J.M. Wright S.D. J. Biol. Chem. 2002; 277: 25963-25969Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This assay was carried out at Merck Sharp and Dohme de España in Spain. Treatment of HepG2 Cells for Gene Expression—HepG2 cells were seeded in 6-well plates at a density of 1 million cells/well in DMEM containing 10% FBS, 1% Pen/Strep, and 5 mm HEPES. 24 h after seeding, cells were treated with various concentrations of compounds for 24 h in DMEM containing 0.5% CS-FBS, 1% Pen/Strep, and 5 mm HEPES. RNA Isolation and Real-time Quantitative PCR—Total RNA was extracted from HepG2 cells using the TRIzol reagent according to the manufacturer’s instructions. Reverse transcription and TaqMan-PCR reactions were performed according to the manufacturer’s instructions (Applied Biosystems). Sequence-specific amplification was detected with an increased fluorescent signal of carboxyfluorescein (reporter dye) during the amplification cycles. Amplification of human 18 S RNA was used in the same reaction of all of the samples as an internal control. Gene-specific mRNA was subsequently normalized to 18 S RNA. Levels of human BSEP and Cyp7a mRNA were expressed as fold difference of compound-treated cells against Me2SO-treated cells. Oligonucleotide primers and probes for human BSEP and Cyp7a were described previously (6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Primers and probe for human 18 S RNA were purchased from Applied Biosystems. Bile Acids in FXR Coactivator Association Assays—An homogeneous time-resolved fluorescence-based FXR coactivator association assay was used to assess agonist/antagonist activities of bile acids on FXR in a cell-free system. This assay measures ligand-dependent association of FXR with the coactivator SRC-1. We have previously shown that CDCA is an FXR agonist, whereas LCA is an antagonist in this assay (6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Here we determined the activities of other bile acids including CA, DCA, and UDCA on FXR. Consistent with our previous results, CDCA robustly activated FXR with a half-maximal activation (EC50) at 2–5 μm. However, CA showed partial activation with a maximal stimulation that was 40% of that induced by CDCA, and DCA and UDCA were virtually inactive up to 200 μm in this assay (Fig. 1A). Tauro- or glyco-conjugated CDCA or CA was similar to their unconjugated homologues but with slightly higher EC50 values (Fig. 1B). Similar to the unconjugated molecule, tauro- or glyco-conjugated DCA or UDCA had no activity in these assays (data not shown). These results indicate that each bile acid results in a different maximal recruitment of SRC-1 by FXR. The binding affinity of the same panel of bile acids was determined by a FXR scintillation proximity binding assay. As shown in Table I, LCA had the highest binding affinity on FXR with an IC50 of 3 μm. CDCA bound to FXR with an IC50 of 17 μm, whereas DCA and UDCA had an IC50 of 131 and 185 μm, respectively. CA was the weakest binder among the five bile acids with an IC50 of 586 μm. The conjugated CA and CDCA had a similar IC50 to the cognate unconjugated bile acid. These data suggest that lack of the agonist activity for DCA and UDCA in the FXR coactivator association assay was not due to lack of the binding of FXR because CA, a much weaker binder, was able to activate FXR.Table IBinding affinity of bile acids on human FXRBile acidIC50μmCDCA17 ± 3DCA131 ± 8CA586 ± 64UDCA185 ± 26LCA3 ± 0.5Glyco CDCA32 ± 4Tauro CDCA19 ± 0Glyco CA800 ± 0Tauro CA733 ± 0 Open table in a new tab CDCA, but Not Other Bile Acids, Promotes the Interaction of FXR and SRC-1 in a GST Pull-down Assay—To confirm the results that unique bile acids can result in different levels of recruitment of SRC-1 by FXR, these bile acids were also evaluated in a GST pull-down assay. Similar to the FXR coactivator association assay, the GST pull-down assay measures interaction of SRC-1 with the ligand-bound FXR in a cell-free environment. Consistent with the results in FXR coactivator association assay, CDCA promoted the interaction of SRC-1 with FXR in a dose-dependent manner with an EC50 of ∼1–2 μm and a saturation concentration between 8 and 16 μm determined by densitometry (Fig. 2A). However, DCA, CA, UDCA, or LCA at 32 μm were inactive in this assay (Fig. 2B). GW4064, a synthetic FXR agonist, also promoted the interaction of SRC-1 with FXR. At 50 nm, GW4064 showed a comparable level of activity with that of 32 μm CDCA (Fig. 2B). These results demonstrate again that distinct FXR ligands can differentially promote FXR-coactivator interaction. Bile Acids in FXR Transactivation with the BSEP Promoter—BSEP, the major bile acid transporter in the liver, is transcriptionally activated by FXR through an FXR-responsive element in the BSEP promoter (5Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. J. Biol. Chem. 2001; 276: 28857-28865Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). The activity of each bile acid was also compared in FXR transactivation assays using a BSEP promoter-driven luciferase construct. CDCA increased luciferase activity in a dose-dependent fashion with a maximum induction of 25–30-fold (Fig. 3A). In the same experiment, DCA, CA, and UDCA maximally increased luciferase activity by 20-, 15-, and 8-fold, respectively, ∼80, 60, and 30%, respectively, of the CDCA activity (Fig. 3, B–D). DCA, CA, and UDCA also had a higher EC50 than that of CDCA (Fig. 3, A–D). Thus, DCA, CA, and UDCA act as partial agonists of FXR when compared with CDCA. Bile Acids in Expression of Endogenous BSEP—It was previously reported that FXR agonists strongly induced BSEP expression in HepG2 cells (6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In this study, these bile acids were also evaluated for their ability to regulate BSEP expression to quantify their agonist activities on FXR relative to CDCA, which we operationally define as a full agonist. HepG2 cells were treated with each of CDCA, CA, DCA, or UDCA, and BSEP mRNA was quantified by real-time PCR (TaqMan). Consistent with our previous report (6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), CDCA strongly induced BSEP expression up to 750-fold with an EC50 of 25–50 μm (Fig. 4A), whereas DCA showed a partial induction up to 250-fold with an EC50 of 50–75 μm (Fig. 4B). CA treatment resulted in a much lower induction up to 75-fold at 600 μm (Fig. 4C), and UDCA had the weakest induction of only 15-fold (Fig. 4D). These data indicate that various bile acids display different abilities in induction of BSEP expression. Bile Acids in Repression of Cyp7a Expression—It has been well established that FXR agonists repress Cyp7a mRNA. This repression is believed to be mediated primarily by the orphan nuclear receptors SHP and Cyp7a promoter binding factor (14Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar, 15Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar). Thus, we evaluated the same panel of bile acids for repression of Cyp7a mRNA to further assess relative activities on another FXR target. Consistent with the result in BSEP expression, again, CDCA was the most potent bile acid in decreasing Cyp7a expression with an EC50 of around 10 μm (Fig. 5A). Interestingly, DCA, CA, and UDCA also effectively repressed Cyp7a expression but with higher EC50 values (Fig. 5, B–D). In particular, UDCA, which was a very weak FXR agonist in other assays, decreased Cyp7a mRNA by 80% at 400 μm (Fig. 5D). Bile Acids Significantly Increase FXR Protein Expression— Bile acids increased FXR expression in rabbits (23Xu G. Pan L.X. Li H. Forman B.M. Erickson S.K. Shefer S. Bollineni J. Batta A.K. Christie J. Wang T.H. Michel J. Yang S. Tsai R. Lai L. Shimada K. Tint G.S. Salen G. J. Biol. Chem. 2002; 277: 50491-50496Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We investigated whether these bile acids and the FXR synthetic agonist GW4064 could increase FXR protein expression in HepG2 cells. Our results indicate that CDCA induced FXR protein expression in a time-dependent manner, and this induction reached maximum at 24 h (Fig. 6A). In addition to CDCA, the other three bile acids, DCA, CA, and UDCA, also significantly increased FXR protein levels (Fig. 6B). Again, CDCA was the most potent inducer of FXR protein expression (Fig. 6B). Similar to the results in down-regulation of Cyp7a mRNA, both DCA and UDCA showed a significant induction of FXR protein expression (Fig. 6B). The synthetic FXR agonist GW4064 also effectively increased FXR protein (Fig. 6C). These data indicate that both endogenous and synthetic FXR agonists can effectively increase FXR protein expression and that the extent of induction was similar for full, high partial, and low partial agonists of FXR. The efficient induction of FXR by various FXR agonists suggests that this auto-regulatory loop may play an important role in FXR-mediated gene regulation. We previously demonstrated that LCA was an FXR antagonist that decreased the FXR agonist-induced BSEP transcription (6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In this study, we systematically characterized the other four bile acids in FXR function using a series of cell-free and cell-based assays. We showed here that CDCA was the most potent FXR agonist in cell-free assays and effectively regulated expression of BSEP and Cyp7a and also strongly increased FXR protein expression in HepG2 cells. Interestingly, we observed that DCA, CA, and UDCA were partial FXR agonists in FXR transactivation assays yet regulated FXR targets in a gene-selective fashion. The three bile acids partially increased BSEP expression but they repressed Cyp7a mRNA and increased FXR protein expression with nearly equal effects as CDCA. BSEP is a major hepatic bile acid transporter whose deficiency in humans results in progressive familial intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage (24Strautnieks S.S. Bull L.N. Knisely A.S. Kocoshis S.A. Dahl N. Arnell H. Sokal E. Dahan K. Childs S. Ling V. Tanner M.S. Kagalwalla A.F. Nemeth A. Pawlowska J. Baker A. Mieli-Vergani G. Freimer N.B. Gardiner R.M. Thompson R.J. Nat. Genet. 1998; 20: 233-238Crossref PubMed Scopus (854) Google Scholar). BSEP is a direct FXR target, and FXR agonists strongly induce BSEP mRNA in both primary human hepatocytes and HepG2 cells (5Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. J. Biol. Chem. 2001; 276: 28857-28865Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 6Yu J. Lo J.L. Huang L. Zhao A. Metzger E. Adams A. Meinke P.T. Wright S.D. Cui J. J. Biol. Chem. 2002; 277: 31441-31447Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The efficacy and potency of bile acids in FXR transactivation assays correlate well with that in BSEP expression (Figs. 3 and 4). Cyp7a catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. It is well established that bile acid feedback inhibits Cyp7a production. Three pathways have been postulated to be responsible for this feedback repression. The first pathway, probably the predominant one, involves FXR up-regulation of SHP that in turn suppresses the activity of Cyp7a promoter binding factor, a critical positive regulator of Cyp7a (13Chiang J.Y. Kimmel R. Weinberger C. Stroup D. J. Biol. Chem. 2000; 275: 10918-10924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 14Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. 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A. 2001; 98: 3369-3374Crossref PubMed Scopus (1138) Google Scholar) and 2) c-Jun N-terminal kinase-mediated Cyp7a repression (30Gupta S. Stravitz R.T. Dent P. Hylemon P.B. J. Biol. Chem. 2001; 276: 15816-15822Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Thus, although it is likely that the discordance between bile acid activities in FXR transactivation and in Cyp7a expression is the result of promoter selectivity by ligand-bound FXR, it is possible that the bile acid-mediated Cyp7a repression involves FXR-independent mechanisms. Auto-regulatory loops have been identified for nuclear receptors such as peroxisome proliferator-activated receptor γ (31Camp H.S. Whitton A.L. Tafuri S.R. FEBS Lett. 1999; 447: 186-190Crossref PubMed Scopus (51) Google Scholar), retinoic acid receptor α and retinoic acid receptor γ (32Takeyama K. Kojima R. Ohashi R. Sato T. Mano H. Masushige S. Kato S. Biochem. Biophys. Res. Commun. 1996; 222: 395-400Crossref PubMed Scopus (25) Google Scholar), liver X receptor (33Li Y. Bolten C. Bhat B.G. Woodring-Dietz J. Li S. Prayaga S.K. Xia C. Lala D.S. Mol. Endocrinol. 2002; 16: 506-514Crossref PubMed Scopus (86) Google Scholar), and others. In this study, we demonstrated that all of the bile acids tested as well as the synthetic FXR agonist GW4064 strongly increased FXR protein, despite the fact that DCA, CA, and UDCA are partial agonists of FXR. Consistent with our observations here, Xu et al. (23Xu G. Pan L.X. Li H. Forman B.M. Erickson S.K. Shefer S. Bollineni J. Batta A.K. Christie J. Wang T.H. Michel J. Yang S. Tsai R. Lai L. Shimada K. Tint G.S. Salen G. J. Biol. Chem. 2002; 277: 50491-50496Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) reported that DCA, CA, or UCA increased FXR mRNA in rabbits (23Xu G. Pan L.X. Li H. Forman B.M. Erickson S.K. Shefer S. Bollineni J. Batta A.K. Christie J. Wang T.H. Michel J. Yang S. Tsai R. Lai L. Shimada K. Tint G.S. Salen G. J. Biol. Chem. 2002; 277: 50491-50496Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), indicating that this auto-regulatory loop is at least in part transcriptional and is conserved across species. The existence of an FXR autoregulatory loop would lead to more efficient propagation of bile acid signals. In conclusion, we have shown that different bile acids have unique properties as FXR ligands, ranging from antagonist to partial agonist to full agonist. Our data here suggest that each of the endogenous bile acids interacts with FXR in a unique fashion that leads to a ligand and promoter selectivity for FXR-mediated gene transcription. These selective properties of bile acid activation of FXR may greatly facilitate FXR gene regulation in appropriate tissues and cell types. We thank Drs. Jilly Evans and Gerard M. Waters for critically reading the paper.