From Bristol-Myers Squibb Company, Experimental Station, Wilmington, Delaware 19880
Received for publication, August 14, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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The bile salt export pump (BSEP) plays an
integral role in lipid homeostasis by regulating the canalicular
excretion of bile acids. Induction of BSEP gene expression is mediated
by the farnesoid X receptor (FXR), which binds as a heterodimer with
the retinoid X receptor (RXR) to the FXR response element (FXRE)
located upstream of the BSEP gene. RXR ligands mimic several partner
ligands and show additive effects upon coadministration. Using
real-time quantitative PCR and cotransfection reporter assays, we
demonstrate that the RXR agonist LG100268 antagonizes induction
of BSEP expression mediated by endogenous and synthetic FXR ligands,
CDCA and GW4064, respectively. Moreover, this antagonism is a general
feature of RXR agonists and is attributed to a decrease in binding of
FXR/RXR heterodimers to the BSEP-FXRE coupled with the inability of RXR agonists to recruit coactivators to FXR/RXR. Our data suggest that
FXR/RXR is a conditionally permissive heterodimer and is the first
example of RXR ligand-mediated antagonism of FXR activity. Because FXR
agonists lower triglyceride levels, our results suggest a novel role
for RXR-mediated antagonism of FXR activity in the development of
hypertriglyceridemia observed with RXR agonists in rodents and humans.
The retinoid X receptor
(RXR),1 a member of the
intracellular receptor superfamily, is the common heterodimeric partner
for many receptors, including the thyroid hormone receptor (1), retinoic acid receptor (RAR) (2), vitamin D receptor (3), peroxisome
proliferator-activated receptor (PPAR) (4, 5), liver X receptor (6),
and farnesoid X receptor (FXR) (7). RXR is activated by its natural
ligand 9-cis retinoic acid (8), however, additional high
affinity ligands such as LGD1069 and LG100268 have been synthesized
and shown to be RXR agonists (9, 10). Collectively, these ligands also
activate the PPAR/RXR and liver X receptor/RXR heterodimers, (4, 6)
referred to as permissive heterodimers. In contrast, RXR agonists fail
to activate the thyroid hormone receptor/RXR and RAR/RXR heterodimers in transfection assays (11, 12) and are therefore known as nonpermissive heterodimers. Although RXR ligands alone do not activate
RAR/RXR, the combination of RAR and RXR ligands results in activity
greater than that seen with RAR ligand alone (11). Synergistic
activation is also observed with PPAR and RXR ligands (4, 12).
Therefore, the pleiotropic in vivo activity of RXR agonists
should be evaluated in the presence of the partner's ligand.
Bile acids serve as natural ligands for FXR (7, 13, 14),
which control many aspects of bile acid synthesis and transport. The
FXR/RXR heterodimer binds to farnesoid X receptor response elements
(FXREs) or bile acid response elements found in the promoter of
FXR-responsive genes such as the bile-salt export pump (BSEP), which
controls the excretion of bile acids from hepatocytes. The BSEP gene
promoter is stimulated by bile acid-bound FXR, and transactivation by
FXR/RXR is lost when this FXRE is mutated (15). More recently, we and
others have shown that CDCA induces BSEP mRNA in HepG2 cells (16,
17).
Because RXR is the heterodimeric partner of FXR, we tested whether
LG100268, a high affinity, selective RXR agonist (rexinoid) (Refs. 10,
12, and data not shown), would also induce BSEP expression. We
demonstrate that RXR agonists alone do not induce BSEP expression, but
antagonize ligand-bound FXR-induced expression of BSEP. Furthermore,
RXR agonists fail to recruit certain coactivator peptides to FXR/RXR
heterodimers. Hence, we propose that FXR/RXR heterodimers function as
conditionally permissive heterodimers.
Reagents--
Chenodeoxycholic acid (CDCA), dimethyl sulfoxide
(Me2SO) and 9-cis retinoic acid were purchased
from Sigma (St. Louis, MO). GW4064, LG100268, and LG100641 (18) were
synthesized at Bristol-Myers Squibb Company.
Expression Profiling by Real-time Quantitative PCR--
Total
RNA was prepared from cultured HepG2 cells (ATCC) using the RNeasy
purification system according to the manufacturer's instructions
(Qiagen). cDNA synthesis was performed using the Advantage RT-PCR
system according to the manufacturer's instructions (Clontech). Real-time quantitative PCR was
performed essentially as described (16, 19). Briefly, 1 µg of total
RNA was DNaseI-treated and reverse transcribed using random hexamers
and Moloney murine leukemia virus reverse transcriptase. Twenty five ng
of each cDNA was added to the Taqman Universal PCR Master Mix along
with 900 nM each primer and 200 nM probe
according to the manufacturer's instructions (Applied Biosystems).
Real-time fluorescence monitoring was performed with the Applied
Biosystem 7700. Relative expression levels of the various transcripts
were determined essentially as described (19). Values were
normalized to 18S rRNA and expressed relative to Me2SO
(taken as 1) and represent the average of three determinants carried
out in duplicate ± S.D. Probes for BSEP were modified at the 5'
end with 6-carboxyfluorescein (6-FAM) and at the 3' end with
6-carboxytetramethylrhodamine (TAMRA) (Biosearch Technologies,
Novato, CA); the 18S rRNA probe was modified at the 5' end with
VIC and the 3' end with TAMRA. Primer and probe sequences were
designed using Primer Express (Applied Biosystems) and are as
follows:
BSEP primer, 5'-GGTGAGAAAAGAGAGGTTGAAAGG,
5'-CCACACGAATCCAGTAAAGAATCC; BSEP probe, 5'-CCAACGCTGGGCGAACACAAGATTT;
18S rRNA primer, 5'-CGGCTACCACATCCAAGGAA, 5'-GCTGGAATTACCGCGGCT; 18S
rRNA probe, 5'-TGCTGGCACCAGACTTGCCCTC.
Plasmids and Transfection Assays--
Cloning of the 1.5-kb
human BSEP promoter by PCR has been described previously (15).
Briefly, the forward primer (ccaagcttCCCTTTGACTGCTGTCAATAAC) and the reverse primer (ccaagcttGAGGAAGCCAGAGGAAATAATGG)
were used to amplify the 1.5 kb (
The full-length open reading frame of the human FXR was amplified by
PCR using the published sequence (accession number U68233) and cloned
into pCDNA3.1/TOPO (Invitrogen).
HepG2 cells were transfected in 24-well plates with FuGENE reagent
according to the manufacturer's instruction (Roche Molecular Biochemicals). For each well, transfection mixes contained 0.25 µg of
FXRE-pTA-luc report plasmid, 0.05 µg of human FXR, 0.05 µg of human
RXR Fluorescence Resonance Energy Transfer (FRET)
assays--
Human FXR (amino acids 222-472) or PPAR Electromobility-shift Assays--
In vitro-translated
FXR and RXR were made using the TNT Quick Coupled
transcription/translation system (Promega). Electromobility-shift assay
was performed as described (22) with in vitro-translated FXR, RXR, or the combination (0.3 µl of each per reaction) as indicated. Total lysate was kept constant by the addition of
unprogrammed reticulocyte lysate. Ligands were added from 100× stock
solutions in Me2SO and incubated for 10 min on ice.
32P-labeled probe was then added and incubated at room
temperature for 20 min and the complex resolved on 6%
polyacrylamide gels. The sequence of the oligonucleotide used is
5'-GATCCTGCCCTTAGGGACATTGATCCTTAGGCAAAA. The intensities of the
protein-DNA complexes were quantitated using a PhosphorImager
(Molecular Dynamics).
LG100268 Antagonizes BSEP Expression by FXR Ligands--
Because
RXR is the heterodimeric partner of FXR, we investigated whether the
selective RXR agonist, LG100268, (10, 12) would induce BSEP mRNA.
HepG2 cells were treated with CDCA, GW4064, and LG100268 alone or in
combination, and BSEP mRNA levels were analyzed by real-time
quantitative PCR. Robust induction of BSEP mRNA was observed in
HepG2 cells by CDCA, a natural bile acid ligand and GW4064, a
synthetic, high affinity ligand for FXR (30-fold and 80-fold,
respectively) (23) (Fig. 1A).
Treatment with LG100268 induced BSEP mRNA very poorly (2-fold), but
surprisingly, strongly inhibited CDCA- and GW4064-mediated induction by
80% (p < 0.0005) and 83% (p < 0.00005), respectively. Time course analysis of LG100268-mediated antagonism of FXR activity shows a significant effect
(p < 0.05) as soon as 8 h after treatment,
suggesting an immediate transcriptional effect (Fig.
1B).
To determine whether this antagonism is a general property of RXR
ligands we tested 9-cis retinoic acid, a natural ligand for
RXR (8). In addition, to address ligand selectivity of this effect, we
utilized another synthetic ligand, LG100641 (LG641), which although
structurally similar to LG268, binds very poorly to RXR (18). As with
LG268, very poor induction of BSEP mRNA (less than 2-fold) was
observed with 9-cis retinoic acid and LG641 (Fig.
1C). However, both LG268 and 9-cis retinoic acid
antagonized GW4064-mediated induction. (82 and 65%, respectively)
whereas LG641 inhibited weakly (32%) (Fig. 1C). This is the
first example of RXR ligand-mediated antagonism of FXR activity.
RXR-mediated Antagonism Occurs via the BSEP-FXRE--
To determine
whether RXR agonists also antagonized FXR activation of the natural
BSEP promoter, we cloned a 1445-base pair fragment of the BSEP promoter
into a luciferase reporter. HepG2 cells were transfected with this
reporter and treated with different ligands (Fig.
2A). In the presence of
GW4064. we observed 5-fold induction from the BSEP promoter, but no
effect with LG100268, 9-cis retinoic acid, or LG641.
Coadministration of LG100268 or 9-cis retinoic acid with
GW4064 antagonized FXR-mediated activation by 67% and 63%,
respectively (p < 0.05, Fig. 2A) whereas
LG641 antagonized less than 10%. Hence, RXR agonist-mediated
antagonism of FXR-induced BSEP promoter activity is observed with
endogenous levels of FXR/RXR heterodimers corroborating our real-time
PCR data (Fig. 1).
An inverted repeat (IR-1) element has been identified in the BSEP
promoter and shown to be a functional FXRE (15). In a cotransfection
assay with expression plasmids for FXR, RXR Increased Amounts of RXR Fail to Abrogate Antagonism by
LG100268--
We next determined whether LG100268-mediated antagonism
was caused by limiting amounts of RXR in HepG2 cells. Transient
transfections were performed by increasing the ratio of RXR RXR Agonists Do Not Recruit Coactivators to
FXR/RXR Heterodimers--
To explain the lack of
transcriptional activity of FXR/RXR with LG100268, we determined
whether LG100268-liganded FXR/RXR recruits coactivators. In a FRET
assay, GW4064 induced recruitment of a peptide identical to the
receptor-interacting domain of SRC-1 and CBP (Fig.
3A) to the heterodimer but not
LG100268. This implies that RXR agonists do not recruit these
coactivators to the FXR/RXR heterodimer. To rationalize the mechanism
of LG100268-mediated antagonism of FXR activity, we speculated that
LG100268 may function as an FXR ligand, displacing GW4064 from the FXR
ligand-binding pocket, thereby decreasing coactivator recruitment and
activity of FXR. To test this, we coadministered increasing
concentrations of LG100268 with a fixed concentration of GW4064 in the
presence of FXR/RXR. LG100268 did not alter the signal observed with
GW4064 (Fig. 3B). This suggests that LG100268, by binding to
RXR, does not induce an allosteric conformation that reduces
coactivator recruitment to FXR/RXR. A similar result was also observed
with FXR alone (data not shown), indicating that LG100268 is not a ligand for FXR.
These results are in stark contrast to those observed with PPAR LG100268 Modulates Binding of FXR/RXR Heterodimers to
the BSEP-FXRE--
Previous reports have demonstrated that FXR/RXR
heterodimers bind to the BSEP-FXRE in vitro (15). To
determine whether LG100268 modulated FXR/RXR binding to DNA, we
performed an electromobility-shift assay with in
vitro-translated FXR and RXR protein and a radiolabeled oligonucleotide containing the FXRE sequence from the BSEP gene. No
protein-DNA complex was observed with FXR or RXR alone in the presence
or absence of their respective ligands, (Fig.
4A, lanes 1-4). However, a distinct complex was seen with the
FXR and RXR in the same reaction (lane 5). This DNA-protein
complex formation is enhanced in the presence of GW4064 (lane
6) but not with LG100268 (lane 7). When both ligands
are present, LG100268 decreased the ability of GW4064 to enhance
binding to the FXRE by 47% (p < 0.05, Figs.
4A, lane 8 and 4B). Both LG268 and
9-cis retinoic acid decreased GW4064-bound FXR/RXR complexes
to the BSEP-response element (Fig. 5) in
a dose-dependent manner. This effect was much reduced with LG641. Therefore, RXR ligands decrease the binding of FXR ligand-bound FXR/RXR heterodimers to the BSEP-FXRE.
An important means by which the body eliminates excess dietary
cholesterol is through the conversion of cholesterol into bile acids.
Recently, the nuclear hormone receptors, FXR and RXR and their putative
ligands have been implicated as key regulators in cholesterol/bile acid
metabolism. In this report, we have demonstrated RXR ligand-mediated
antagonism in a gene- and response element-specific manner. This
antagonism in not observed with related compounds that do not bind to
RXR. In contrast to our data involving antagonism of BSEP gene
expression via the BSEP-FXRE, LG100268-mediated activation has been
observed with the FXRE from the ultraspiracle
(usp) gene (7, 25) which was additive to an FXR agonist.
Whether this translates to induction of usp mRNA is not
known. These contrasting results may be due in part to
sequence-specific differences between the BSEP and usp FXRE.
Hence, the same ligand and receptor may induce one gene while
repressing another, a phenomenon reported with another RXR partner, the
vitamin D receptor. RXR ligands have been shown to activate the
25-hydroxyvitamin D3-24-hydroxylase promoter and to
synergize with 1,25-dihydroxyvitamin D3 (26), whereas RXR
ligands inhibit 1,25-dihydroxyvitamin
D3-dependent induction of the rat osteocalcin
gene (27). We now demonstrate the complexity underlying
RXR-permissiveness extends to the FXR/RXR heterodimer which, to date,
has been described as a permissive heterodimer (28). Our data provide
evidence that this may not be true in all cases and we therefore
describe FXR/RXR as a conditionally permissive heterodimer.
One possible mechanism of RXR-mediated antagonism is by induction of
the small heterodimerization partner (29-31), which could dimerize
with FXR forming an inactive transcription complex. However, the small
heterodimerization partner was not induced in HepG2 cells with LG100268
(data not shown). Our results could also be reasonably explained by RXR
agonist-induced formation of RXR homodimers (32), which do not bind to
IR-1 FXREs but precludes formation of FXR/RXR heterodimers, as has been
postulated for vitamin D receptor (33). Although possible, this may not
be the predominant explanation because a similar magnitude of
LG100268-mediated antagonism of FXR was observed with 5-fold excess of
transfected RXR (Fig. 2D). Our data are also consistent with
LG100268-induced dimerization of RXRs with other heterodimeric partners
like PPARs in vivo, thus reducing the availability of RXR
for dimerization with FXR. Reduced BSEP expression by antagonizing FXR
activity is consistent with the phenotype of FXR knockout mice, which
have significantly lower BSEP expression in the liver compared with
wild-type controls (34) and therefore indicates a role for RXR and its
ligand in bile acid transport and homeostasis.
The FXR agonist CDCA decreases triglyceride levels in humans
(35), whereas GW4064 decreases triglyceride levels in rats (23).
Conversely, RXR agonists increase triglyceride levels in mice and
humans (36, 37) which is postulated to occur by a mechanism involving
RXR-mediated activation of liver X receptor and induction of SREBP1-c
(38, 39). Our data suggest that LG100268-mediated antagonism of FXR may
also contribute to the hypertriglyceridemia induced by RXR agonists.
This report is the first example of RXR-mediated antagonism of FXR
activity by decreasing DNA binding coupled with the inability of RXR
agonists to recruit coactivators to the FXR/RXR heterodimer. FXR/RXR
thus falls into a distinct category as a conditionally permissive heterodimer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1445 to +86) human BSEP
promoter and cloned into pGL3-Basic Vector (Promega) to give BSEP-LUC. The reporter plasmid BSEP-3xFXRE-pTA-LUC was constructed by subcloning three copies for the FXRE identified in the BSEP promoter (15) into the
pTA-luc vector (Clontech). Complementary
oligonucleotides containing the FXRE sequences were synthesized
and cloned into the BglII site upstream of the TATA box
using flanking BamHI and BglII sequences
engineered in the oligonucleotides.
expression vectors, and 0.25 µg of pSV
-galactodidase control plasmid (Promega). Twenty-four h later, FXR
and RXR ligands or vehicle (Me2SO, 0.1% final
concentration) were added to cells at the concentration indicated in
the figures. After 24 h, cells were lysed and analyzed for
luciferase and
-galactosidase activities as recommended by the
manufacturer (Promega, Madison, WI and Stratagene, La Jolla, CA, respectively).
(amino acids
196-468) with an N-terminal His tag and RXR (amino acids 225-462,
untagged) were expressed in Escherichia coli. The cells were
disrupted by sonication and FXR and RXR or PPAR
and RXR pastes mixed
and the heterodimers formed purified by nickel column chromatography. Amino acid analysis indicated the complexes were a 1:1 heterodimer of
FXR/RXR or PPAR
/RXR. The FRET assay (20) was performed by incubating
7.5 nM FXR/RXR with 0.84 nM
Europium-labeled anti-His antibody, 0.055 µM
streptavidin-conjugated allophycocyanin, and 100 nM
biotinylated-SRC1 or CBP peptide (SynPEP) in 50 mM Tris (pH
8.0), 50 µM KCl, 0.15 mg/ml bovine serum albumin, 10 µM EDTA, and 1 mM dithiothretiol, in the
presence of ligands (as indicated in the figure legends) for 4 h
at room temperature. Data are expressed as the ratio of the emission
intensity at 665 nm to that at 620 nm. The FRET method for PPAR
and
the sequences for the coactivator peptides have been published
(21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RXR ligands antagonize induction
of BSEP mRNA by FXR agonists in HepG2 cells. A,
cells were treated with various ligands (30 µM CDCA, 1 µM GW4064 (GW),1 µM LG100268
(LG), or combinations) as indicated for 24 h, and
real-time quantitative PCR was used to monitor gene-expression levels.
*, p < 0.0005 compared with CDCA treatment and **,
p < 0.00005 compared with GW4064 treatment determined
by Students' t test. B, time course of induction
of BSEP mRNA by FXR ligand and antagonism by RXR ligand. BSEP
mRNA levels after treatment for 4, 8, 12, and 24 h with
different ligands were monitored and normalized to 18S rRNA. *,
p < 0.05; **, p < 0.005; ***,
p < 0.0005 compared with GW4064 treatment. Values are
expressed relative to Me2SO (taken as 1) and represent the
average of three determinants carried out in duplicate ± S.D.
C, HepG2 cells were treated with GW4064 (1 µM)
or LG268, 9-cis retinoic acid, or LG641 (10 µM) each alone or in combination with GW4064 (1 µM) and analyzed as in A.
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Fig. 2.
RXR agonists antagonize FXR-induced BSEP
promoter activity. RXR agonists antagonize induction of the BSEP
promoter (A) and the BSEP-FXRE (B). C,
dose-response of RXR-mediated antagonism. D, the inability
of LG268 to induce BSEP is not due to limiting RXR. HepG2 cells were
cotransfected with the reporter plasmid BSEP-LUC (A) or
BSEP-3xFXRE-pTA-LUC with expression plasmids for hFXR, hRXR ,
(B-D), and a
-galactosidase plasmid for
normalization, ligand treated for 24 h, and assayed.
Me2SO is the vehicle control. The data show the mean ± S.E. (n = 3). The activity in the presence of GW4064
alone was normalized to 100% in C and D. CDCA, chenodeoxycholic acid (30 µM);
LG, LG268 (1 µM); GW, GW4064 (1 µM); 9-cis RA, 9-cis
retinoic acid (1 µM), and LG641 (1 µM).
, and a reporter
containing three tandem copies of the FXRE from the BSEP gene promoter,
CDCA and GW4064 induced FXR/RXR-mediated transcription from the
BSEP-FXRE (9-fold and 28-fold, respectively) (Fig. 2B). In
contrast, LG100268 weakly activated FXR/RXR (2-fold) and antagonized activation by CDCA and GW4064. LG100268 and 9-cis retinoic
acid antagonized FXR-mediated activation in a
dose-dependent manner with an IC50 of 6 nM and 300 nM, respectively (Fig.
2C). FXR-mediated induction was also antagonized by LGD1069
(9), another synthetic high affinity RXR ligand (data not shown). No
antagonism was observed with LG641 (Fig. 2C). These data
indicate that antagonism by RXR ligands is mediated via FXR/RXR through
the FXR response element in the BSEP promoter and is not observed with
ligands that do not bind to RXR.
to FXR
expression plasmid from 1 to 5. This caused a slight increase in the
signal observed with both ligands compared with GW4064 alone (Fig.
2D). However, even a 5-fold increase in RXR plasmid did not
overcome LG100268-mediated antagonism.
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Fig. 3.
RXR agonists do not recruit coactivators to
FXR/RXR. FRET assays were performed with biotinylated peptides
from the coactivators SRC-1 or CBP (A). B,
LG100268 is not a ligand for FXR. Increasing doses of LG100268 were
added to a fixed concentration (1 µM) of GW4064 and the
signal with GW4064 alone was taken as 100%. C, LG100268
recruits SRC-1 to PPAR /RXR. The data represent the mean ± S.E.
(n = 3).
/RXR
(Fig. 3C) where LG100268 induced SRC-1 recruitment similar to that seen with GW9578 (24), a potent and selective PPAR
agonist.
Furthermore, a decrease in the EC50 and increase in the maximum signal was observed when LG100268 was added in the presence of
a fixed concentration of the PPAR
agonist. These results suggest that the RXR ligand-binding and coactivator recruitment properties of
FXR/RXR are distinct from that of PPAR
/RXR, a well characterized permissive heterodimer.
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Fig. 4.
LG100268 inhibits FXR ligand-induced binding
of FXR/RXR to the FXR response element in the BSEP promoter.
A, electromobility-shift assays were performed with in
vitro translated receptors (0.3 µl each) in the presence or
absence of ligands (1 µM) as indicated. B,
quantitation of the electromobility-shift assays. The data are the
mean ± S.D. of 3 independent experiments. The difference in
intensity with ligand treatment compared with Me2SO is
shown, and the change with GW4064 treatment is set at 100%.
GW, GW4064; LG, LG100268.
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Fig. 5.
RXR ligands inhibit binding of FXR/RXR to the
BSEP-FXRE in a dose-dependant manner. Electromobility-shift assays
were performed as in Fig. 4 with GW4064 (1 µM) in all
lanes and increasing amounts of LG268 (A), 9-cis
retinoic acid (9-cis RA; B), or LG641
(C). The concentration of these three compounds are:
lanes 2, 1 nM; lanes 3, 10 nM; lanes 4, 100 nM; lanes
5, 1 µM; and lanes 6; 10 µM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Jodi
Muckelbauer and Haiying Chen for the FXR, RXR, and PPAR proteins,
Bruce Spiegelman for the pSV-SPORT-RXR
expression plasmid, and Drs.
Jim Trzaskos, Leonard Freedman, and Richard Heyman for critically
reading the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to the work.
§ Present address: Dept. of Laboratory Medicine and Pathobiology, University of Toronto, Medical Sciences Bldg., Rm. 6316, 1 King's College Circle, Toronto ON M5S 1A8, Canada.
¶ Present address: Bristol-Myers Squibb Co., 311 Pennington-Rocky Hill Rd., Pennington, NJ 08543.
Present address: SUGEN Inc., 230 East Grand Ave., South San
Francisco, CA 94080-4811.
** To whom correspondence should be addressed: Bristol-Myers Squibb Co., 311 Pennington-Rocky Hill Rd., Pennington, NJ 08543. Tel.: 609-818-5498; Fax: 609-818-7877; E-mail: ranjan.mukherjee@bms.com.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M208312200
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ABBREVIATIONS |
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The abbreviations used are: RXR, retinoid X receptor; FXR, farnesoid X receptor; FXRE, farnesoid X receptor response element; RAR, retinoic acid receptor; PPAR, peroxisome proliferator-activated receptor; BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; FRET, fluorescence resonance energy transfer.
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