The Farnesoid X Receptor (FXR) Controls Gene Expression in a Ligand-

and Promoter- Selective Fashion

Jane-L. Lew, Annie Zhao, Jinghua Yu, Li Huang, Nuria de Pedro§, Fernando Peláez§, Samuel

D. Wright and Jisong Cui*

Department of Atherosclerosis and Endocrinology Merck Research Laboratories Rahway, New Jersey 07065

§ Merck Sharp & Dohme de España, S. A. Josefa Valcarcel 38, 28027 Madrid, Spain *Correspondence: Jisong Cui Department of Atherosclerosis and Endocrinology Merck Research Laboratories 126 E. Lincoln Avenue P. O. Box 2000, RY80W-107 Rahway, New Jersey 07065 tel: 732-594-6369 fax: 732-594-7926 email: jisong_cui@merck.com

Abbreviations: FXR, farnesoid X receptor; BSEP, bile salt export pump; Cyp7a, cholesterol 7α-hydroxylase; CDCA, chenodeoxycholate; DCA, deoxycholate; CA, cholate; UDCA, ursodeoxycholate; LCA, lithocholate; RXRα, retinoid X receptor α; SRC-1, steroid receptor coactivator protein-1; FBS, fetal bovine serum; CS-FBS, charcoal striped FBS; DMEM, Dulbecco’s modified Eagle’s medium. GST, glutathione-S-transferase; SPA, scintillation proximity binding assay.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Running Title: Bile acid regulation on FXR target expression

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Abstract

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, while CA partially activated FXR and DCA and UDCA had negligible

activities. Similar results were also obtained from a GST 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, while 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.

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Introduction

Bile acids are the end products of cholesterol catabolism. The five major bile acids in

humans are chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA), ursodeoxycholate

(UDCA) and lithocholate (LCA) (1). 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 (2-4). Agonist

bound-FXR activates expression of the bile salt export pump (BSEP) (5,6), intestinal bile acid

binding protein (7), phospholipid transfer protein (8), dehydroepiandrosterone sulfotransferase

(9), apolipoprotein C-II (10), apolipoprotein E (11) and kininogen (12). FXR represses

expression of cholesterol 7α-hydroxylase (Cyp7a) (11,13-15), sterol 12 α-hydroxylase (16), the

Na+/taurocholate co-transporting polypeptide (17) and apolipoprotein A-I (18).

A few functional distinct classes of FXR ligands have been identified. We have

previously shown that LCA is an FXR antagonist and guggulsterones are selective bile acid

receptor modulators (SBARMs) (6,19). GW4064 is a synthetic FXR agonist (20). Bile acids,

such as CDCA, DCA and CA, have been previously shown to be FXR agonists that regulate

expression of FXR targets (2-4). 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, 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, while 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

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acids have unique properties as FXR agonists and reveals an auto-regulatory loop in FXR

signaling pathways.

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Materials and Methods

Reagents. The following reagents were obtained from Invitrogen (CarIsbad, CA): DMEM and

Optimem I; regular fetal bovine serum (FBS) and charcoal striped-FBS (CS-FBS); TRIZOL

reagents; L- 35S-methionine (1000 Ci/mmol) was obtained from Amersham Pharmacia Biotech

(Arlington Heights, IL). The TNT T7 Quick Coupled transcription/translation kit and reagents

for β-Galactosidase and luciferase assays were from Promega (Madison, WI). FuGENE6

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 18S RNA were purchased from Applied Biosystems (Foster City, CA).

FXR coactivator association assays. Human GST-FXR-LBD fusion protein was prepared from

E. coli strain BL21. A homogeneous time-resolved fluorescence (HTRF) based FXR and

coactivator SRC-1 interaction assay was used to examine the interaction of FXR-LBD with

various ligands according to previously described for other nuclear receptors (21) with minor

modifications: briefly, 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-LBD, 2 nM anti-GST-(Eu)K, 10 nM biotin-

SRC-1 fragment (human SRC-1, animo acids NSPSRLNIQP to VKVKVEKKEQ) and 20 nM

SA/XL665 (streptavidin-labeled allophycocyanin)] was added to each well, followed by addition

of 2 µl DMSO 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.

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FXR scintillation proximity binding assay (SPA). The assay was performed in a 96-well

microtiter plate in a total volume of 100 µl. The assay mixture includes the GST-hFXR fusion

protein at 100 ng/well, goat anti-GST antibody (Amersham Pharmacia Biotech, at 1:400 fold

dilution), Protein A-Yttrium Silicate SPA beads (Amersham Pharmacia Biotech, at 250 µg /well)

and a 3H-labeled FXR radioligand (Alan Adams et al., 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 PMSF 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 previously described (21). 35S-

labeled human FXR was synthesized using the TNT T7 Quick Coupled

Transcription/Translation kit (Promega) according to the manufacturer’s instructions.

Approximately 2 µg GST-SRC-1 protein on beads was incubated with 2 µl 35S-labeled full-

length human FXR (19) in the presence of various FXR ligands. The mixture was incubated

overnight at 4oC with gentle agitation in a total volume of 116 µl (8 mM Tris-HCl, pH 7.4, 0.12

M KCl, 8% glycerol, 0.5% w/v CHAPS, 4 mM DTT, 1mg/ml BSA). 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, 2 mM DTT) prior to electrophoresis on a 4-20% SDS-PAGE and

visualization by autoradiography.

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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

previously described (19). Twenty µg of total nuclear proteins was separated by electrophoresis.

Western blotting was carried out following the manufacturer’s instructions (Amersham) using

polyclonal rabbit anti-human FXR antibody (Cat # 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.

FXR transactivation. HepG2 cells were transfected in 96-well plates using the FuGENE6

transfection reagent as described previously (22). FXR transactivation assay using pGL3-

enhancer-hBSEP-Promoter-Luc construct was performed as described previously (19).

Treatment of transfected cells with various FXR ligands, assays for luciferase and β-

galactosidase activities were also performed as previously described (22). This assay was carried

out at Merck Sharp & 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.

Twenty-four hours 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

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Biosystems). Sequence-specific amplification was detected with an increased fluorescent signal

of FAM (reporter dye) during the amplification cycles. Amplification of human 18S RNA was

used in the same reaction of all samples as an internal control. Gene specific mRNA was

subsequently normalized to 18S RNA. Levels of human BSEP and Cyp7a mRNA were

expressed as fold difference of compound-treated cells against DMSO-treated cells.

Oligonucleotide primers and probes for human BSEP and Cyp7a were previously described (6).

Primers and probe for human 18S RNA were purchased from Applied Biosystems.

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Results

Bile acids in FXR coactivator association assays. A homogeneous time-resolved fluorescence

(HTRF) 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

while LCA is an antagonist in this assay (6). Here we determined activities of other bile acids

including CA, DCA and UDCA on FXR. Consistent with our previous results, CDCA robustly

activated FXR with a half-maximum activation (EC50) at 2-5 µM. However, CA showed partial

activation with a maximum 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 assay (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 SPA

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, while DCA and UDCA had an IC50 of

131 µM 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 un-

conjugated 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 since CA, a

much weaker binder, was able to activate FXR.

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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 approximately 1-2 µM and a saturation concentration of between 8-16 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 to 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 FXRE in the BSEP promoter (5).

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- to 30- fold (Fig. 3A). In the same experiment, DCA,

CA and UDCA maximally increased luciferase activity by 20-, 15- and 8-fold respectively,

approximately 80%, 60% and 30% respectively of the CDCA activity (Fig. 3B-D). DCA, CA

and UDCA also had a higher EC50 than that of CDCA (Fig. 3A-D). Thus, DCA, CA and UDCA

act as partial agonists of FXR when compared to CDCA.

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Bile acids in expression of endogenous BSEP. It was previously reported that FXR agonists

strongly induced BSEP expression in HepG2 cells (6). 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 (6), CDCA strongly induced BSEP expression up

to 750-fold with an EC50 of 25 to 50 µM (Fig. 4A), while 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 CPF (14,15). We thus 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 around 10 µM (Fig. 5A). Interestingly, DCA, CA and UDCA

also effectively repressed Cyp7a expression although with higher EC50 values (Fig. 5B-D). In

particular, UDCA, which was a very weak FXR agonist in other assays, decreased Cyp7a mRNA

by 80% at 400 µM (Fig. 5D).

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Bile acids significantly increase FXR protein expression. Bile acids increased FXR expression in

rabbits (23). 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

hours (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.

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Discussion

We previously demonstrated that LCA was an FXR antagonist that decreased the FXR

agonist-induced BSEP transcription (6). 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.

Bile salt export pump (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 (24). BSEP is a direct FXR target and FXR

agonists strongly induce BSEP mRNA in both primary human hepatocytes and HepG2 cells

(5,6). The efficacy and potency of bile acids in FXR transactivation assays correlate well with

that in BSEP expression (Figs. 3 and 4).

Cholesterol 7α-hydroxylase (Cyp7a) catalyzes the first and rate-limiting step in the

conversion of cholesterol to bile acids. It is well established that bile acids feedback inhibit

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 CPF, a critical positive regulator of Cyp7a (13-

15,25,26). The importance of this pathway is indicated by the fact that Cyp7a expression was

elevated in SHP KO mice (27,28). However, the bile acid-mediated Cyp7a repression was not

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completely abolished in SHP KO mice (27,28), suggesting the existence of other redundant

pathways. The other two most plausible pathways are 1) the xenobiotic receptor PXR as the

second bile acid receptor that can repress Cyp7a expression (29) and 2) C-Jun N-terminal kinase

JNK-mediated Cyp7a repression (30). 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 PPARγ (31),

RARα and RARγ (32), LXRα (33) and others. In this study we demonstrated that all 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 reported that DCA, CA or UCA increased FXR mRNA in rabbits (23), indicating

that this auto-regulatory loop is at least in part transcriptional, and is conserved across species.

The existence of an FXR auto-regulatory 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.

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Acknowledgments

We thank Drs. Jilly Evans and Gerard M. Waters for critically reading the manuscript.

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Figure Legends

Figure 1. Effect of bile acids on SRC-1 recruitment in an FXR coactivator association

assay. Four nM of purified GST-FXR were incubated with 2 nM anti-GST-(Eu)K, 10 nM biotin-

SRC-1-(568-780), 20 nM SA/XL665 and various concentrations of bile acids (A) or CDCA and

CA conjugates (B). The mixture was incubated overnight at 4°C. The fluorescent signal was

measured, and results were calculated as described in Material and Methods. Each value

represents the mean ± SD of three determinations.

Figure 2. Effects of bile acids on interaction of FXR with SRC-1 in the GST pull-down

assay. Two µg GST-SRC-1 was incubated with 2 µl of in vitro translated 35S-labeled FXR in the

presence of various concentrations of CDCA (A) or 32 µM of the indicated bile acids or 50 nM

GW4064 (B). Bound proteins were separated by SDS-PAGE and visualized by autoradiography

as described in Material and Methods.

Figure 3. Effects of bile acids in FXR transactivation. HepG2 cells at a density of 3.2 x 104

cells/well in 96-well plates were transfected with 0.405 µl of FuGENE6, 10.4 ng of pcDNA3.1-

hFXR, 10.4 ng of pcDNA3.1-hRXRα, 10.4 ng of pGL3-enhancer-hBSEP-Promoter-Luc and

103.8 ng of pCMV-lacZ in serum free Opti-MEM I medium using the FuGENE6 transfection

reagent according to the manufacturer’s instructions. The transfected cells were treated with

various concentrations of CDCA (A), DCA (B), CA (C) or UDCA (D). After 24 h treatment,

cells were harvested and the cell lysate was used for determination of luciferase and β-

galactosidase activities as described in Material and Methods. Luciferase activities were

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normalized to β-galactosidase activities individually for each well. Each value represents the

mean ± SD of three determinations. Note the different scales of the Y-axes.

Figure 4. Induction of BSEP mRNA by various bile acids. HepG2 cells at a density of 1

million cells/well in 6-well plates were treated with various concentrations of CDCA (A), DCA

(B), CA (C) or UDCA (D) for 24 h in DMEM containing 0.5% CS-FBS. Total RNA was

prepared and BSEP mRNA was analyzed by TaqMan-PCR as described in Materials and

Methods. Results are normalized as fold of control (treated cells vs. vehicle), and data are the

mean ± SD of three determinations.

Figure 5. Repression of Cyp7a mRNA by various bile acids. HepG2 cells at a density of 1

million cells/well in 6-well plates were treated with various concentrations of CDCA (A), DCA

(B), CA (C) or UDCA (D) for 24 h in DMEM containing 0.5% CS-FBS. Total RNA was

prepared and Cyp7a mRNA was analyzed by TaqMan-PCR as described in Materials and

Methods. Results are normalized as fold of control (treated cells vs. vehicle), and data are the

mean ± SD of three determinations.

Figure 6. Induction of FXR protein expression by various FXR agonists. A, HepG2 cells

were treated with 50 µM CDCA at various time points, and the FXR protein was determined by

Western blot analysis followed by quantitation with densitometry. Results are normalized as fold

of control (treated cells vs. vehicle), and data are the mean of two determinations. B and C,

HepG2 cells were treated with indicated concentration (µM) of bile acids (B) or GW4064 (C) for

24 h in DMEM containing 0.5% CS-FBS. At the end of the incubation, nuclear extracts were

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prepared using a Nuclear and Cytoplasmic Extraction kit according to the manufacturer’s

instructions. Twenty µg of total nuclear proteins was separated by 4-20% SDS-PAGE. Western

blotting was carried out following the manufacturer’s instructions using the polyclonal rabbit

anti-human FXR antibody and donkey anti-rabbit IgG conjugated to horseradish peroxidase and

the ECL chemiluminescence kit.

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Table I Binding affinity of bile acids on human FXR

Bile Acid IC50 µM) CDCA 17 ± 3 DCA 131 ± 8 CA 586 ± 64

UDCA 185 ± 26 LCA 3 ± 0.5

Glyco CDCA 32 ± 4 Tauro CDCA 19 ± 0

Glyco CA 800 ± 0 Tauro CA 733 ± 0

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Fig. 1

10 -1 10 0 10 1 10 2 10 30

1000

20003000

40005000

60007000

8000

CADCAUDCA

CDCA

[Bile acid] µM

Rel

ativ

e flu

ores

cenc

e

10 -1 10 0 10 1 10 2 10 30

20

40

60

80

100

120CDCA

Glyco CDCATauro CDCA

CAGlyco CATauro CA

[Bile acid] µM

Rel

ativ

e flu

ores

cenc

e

A

B

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Fig. 2

CDCADCA

CA UDCALCA

GW4064

DMSO

A

B

0 2 4 8 1610.5 (µM)

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Fig. 3

5 10 20 40 60 800

5

10

15

20

25

30

[CDCA] µM

Luci

fera

se a

ctiv

ity(f

old)

10 20 50 100 150 200 3000

2

4

6

8

10

[UDCA] µM

Luci

fera

se a

ctiv

ity(f

old)

A B

C D

10 20 40 50 100 1500

5

10

15

20

25

[DCA] µM

Luci

fera

se A

ctiv

ity(f

old)

20 50 100 200 300 400 6000

5

10

15

20

[CA] µM

Luci

fera

se a

ctiv

ity(f

old)

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Fig. 4

0 5 10 25 50 750

250

500

750

1000

[CDCA] µM

BSE

P m

RN

A(f

old)

0 10 25 50 75 1000

50

100

150

200

250

300

[DCA] µMB

SEP

mR

NA

(fol

d)

0 50 100 200 400 6000

25

50

75

100

[CA] µM

BSE

P m

RN

A(f

old)

0 20 50 100 200 4000

5

10

15

20

[UDCA] µM

BSE

P m

RN

A(f

old)

A B

C D

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Fig. 5

0 5 10 25 50 750.0

0.5

1.0

1.5

[CDCA] µM

Cyp

7a

mR

NA

(fol

d)

0 10 25 50 75 1000.0

0.5

1.0

1.5

[DCA] µM

Cyp

7a

mR

NA

(fol

d)

0 50 100 200 400 6000.0

0.5

1.0

1.5

[CA] µM

Cyp

7a

mR

NA

(fol

d)

0 20 50 100 200 4000.0

0.5

1.0

1.5

[UDCA] µM

Cyp

7a

mR

NA

(fol

d)

A B

C D

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0 3 16 24 480

1

2

3

4

Time (hour)

FXR

pro

tein

(fol

d of

con

trol)

Fig. 6

10

FXR10 50 50 600 50 200 DMSO

50

0.05 1 DMSO

FXR

CDCA CADCA UDCA

GW4064

B

C

10

FXRFXR10 50 50 600 50 200 DMSO

50

0.05 1 DMSO

FXRFXR

CDCA CADCA UDCA

GW4064

B

C

A

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D. Wright and Jisong CuiJane-L. Lew, Annie Zhao, Jinghua Yu, Li Huang, Nuria de Pedro, Fernando Peláez, Samuel

selective fashionThe farnesoid X receptor (FXR) controls gene expression in a ligand- and promoter-

published online December 18, 2003J. Biol. Chem. 

  10.1074/jbc.M306422200Access the most updated version of this article at doi:

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