Farnesoid X Receptor Regulates Bile Acid-Amino Acid Conjugation*
Parinaz C. Pircher,
Jennifer L. Kitto,
Mary L. Petrowski,
Rajendra K. Tangirala,
Eric D. Bischoff,
Ira G. Schulman and
Stefan K. Westin
From the
Department of Biology, X-Ceptor Therapeutics Inc., San Diego, California
92121
Received for publication, February 28, 2003
, and in revised form, May 7, 2003.
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ABSTRACT
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The farnesoid X receptor (FXR; NR1H4) regulates bile acid and lipid
homeostasis by acting as an intracellular bile acid-sensing transcription
factor. Several identified FXR target genes serve critical roles in the
synthesis and transport of bile acids as well as in lipid metabolism. Here we
used Affymetrix micro-array and Northern analysis to demonstrate that two
enzymes involved in conjugation of bile acids to taurine and glycine, namely
bile acid-CoA synthetase (BACS) and bile acid-CoA: amino acid
N-acetyltransferase (BAT) are induced by FXR in rat liver. Analysis
of the human BACS and BAT genes revealed the presence of functional response
elements in the proximal promoter of BACS and in the intronic region between
exons 1 and 2 of the BAT gene. The response elements resemble the consensus
FXR binding site consisting of two nuclear receptor half-sites organized as an
inverted repeat and separated by a single nucleotide (IR-1). These response
elements directly bind FXR/retinoid X receptor (RXR) heterodimers and confer
the activity of FXR ligands in transient transfection experiments. Further
mutational analysis confirms that the IR-1 sequence of the BACS and BAT genes
mediate transactivation by FXR/RXR heterodimers. Finally, Fisher rats treated
with the synthetic FXR ligand GW4064 clearly show increased transcript levels
of both the BACS and BAT mRNA. These studies demonstrate a mechanism by which
FXR regulates bile acid amidation, a critical component of the enterohepatic
circulation of bile acids.
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INTRODUCTION
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Bile acids act as signaling molecules that regulate their own synthesis and
transport, at least in part, by serving as physiological ligands for the
farnesoid X receptor
(FXR1; NR1H4), a
member of the nuclear receptor superfamily
(14).
For example, bile acid-activated FXR exerts inhibitory effects on the gene
encoding cholesterol-7
hydroxylase (CYP7A1), the rate-limiting enzyme
in bile acid synthesis, indirectly by inducing the gene encoding the short
heterodimer partner (SHP). SHP then inactivates CYP7A1 by binding to liver
receptor homolog 1, a competence factor for CYP7A1 expression
(5,
6). A similar mechanism has
also been shown for bile acid feedback repression of the sodium/taurocholate
cotransporter peptide (NTCP), the major bile acid uptake protein in the liver
(7). In contrast, bile
acid-activated FXR directly induces the transcription of the gene for the bile
salt export pump (BSEP), the principal bile salt efflux pump in the liver
(8). Thus, regulation of
hepatic gene expression by FXR promotes a net efflux of bile acids from the
liver.
FXR induces transcription of SHP and BSEP by binding DNA sequences composed
of two inverted repeats separated by one nucleotide (IR-1) as a heterodimer
with the retinoid X receptor (RXR)
(5,
6,
8). FXR also activates
transcription of the ileal bile acid-binding protein (IBABP) and phospholipid
transfer protein (PLTP) genes via IR-1 elements in the promoters of these
genes (9,
10). In contrast to this IR-1
arrangement, FXR has also been shown to bind and activate an inverted repeat
without a spacing nucleotide (IR-0). This IR-0 arrangement was found to be the
cognate FXR element in the dehydroepiandrosterone sulfotransferase gene, which
encodes an enzyme with bile acid sulfo-conjugating activity
(11).
Bile acids are synthesized in the liver from cholesterol and conjugated to
glycine or taurine before they are secreted into bile canaliculi
(12,
13). In humans conjugated bile
acids are the major solutes in bile, whereas unconjugated bile acids are
almost nondetectable. Importantly, conjugated bile acids are less toxic and
are more efficient promoters of intestinal absorption of dietary lipid than
unconjugated bile acids (14).
The primary bile acids in humans, cholic acid and chenodeoxycholic acid, are
synthesized via the concerted action of enzymes located in the endoplasmic
reticulum, cytosol, mitochondria, and peroxisomes
(13). The immediate precursors
of the C24 bile acids cholate and chenodeoxycholate are the
C27 compounds
3
,7
,12
-trihydroxy-5-
-cholestanoic acid (THCA) and
3
, 7
,12
-dihydroxy-5-
-cholestanoic acid (DHCA),
respectively. Before chain shortening of THCA and DHCA via peroxisomal
-oxidation can take place, the C27 compounds have to be
activated to their CoA thioesters. Current knowledge of the synthesis of bile
acid-amino acid conjugates in human liver involves two independent enzyme
reactions (15). An
ATP-dependent microsomal enzyme, bile acid-CoA synthetase (BACS), catalyzes
the formation of the thioester intermediate and is considered the
rate-limiting step in bile acid amidation
(16). Interestingly, it was
recently demonstrated that human very long chain acyl CoA synthetase homolog 2
(VLCS-H2) is a bile acid-CoA synthetase
(17). VLCS-H2 belongs to a
family that includes very long chain synthetase (VLCS) and fatty acid
transport proteins. In the second reaction, the thioester bond is cleaved and
an amide bond is formed between the bile acid and the amino acids glycine or
taurine. The bile acid-CoA:amino acid N-acetyltransferase (BAT)
catalyzes this reaction
(18).
Here we show that FXR regulates the expression of genes required for bile
acid conjugation to amino acids. We have used Northern analysis and
micro-array technology to identify BACS and BAT as FXR target genes. Induction
of BACS and BAT gene expression occurs through the direct interaction of
FXR/RXR heterodimers with conserved IR-1 elements in the promoter of BACS and
in the first intron of the human BAT gene. Finally we show that these genes
are up-regulated in rats treated with a synthetic FXR ligand. These findings
provide further evidence for the critical role of FXR in regulating bile acid
homeostasis.
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EXPERIMENTAL PROCEDURES
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Affymetrix GeneChip Expression AnalysisOligonucleotide
microarray experiments were performed on 10 µg of total RNA and analyzed
according to protocols developed by Affymetrix (Santa Clarita, CA). Briefly,
after quality determination on test arrays, samples were hybridized for 16 h
at 45 °C to Affymetrix Rat Genome arrays (U34A). Arrays were washed and
then stained with streptavidin-phycoerythrin (genome arrays were amplified
with an anti-streptavidin Ab). Stained arrays were scanned with the GeneArray
scanner (Agilent Technologies, Palo Alto, CA). Raw data were collected and
analyzed by using Affymetrix Microarray Suite and Data Mining Tools software.
Experiments were done in two replicates from two rat primary hepatocyte pools.
Each of the two compound-treated samples was compared with vehicle-treated
samples resulting in four pairwise comparisons for each treatment. Genes with
concordance exceeding 75% were considered statistically significant
(p < 0.05).
RNA Isolation and Northern Blot AnalysisTotal RNA was
isolated from rat primary hepatocytes and rat liver RNA using TRIzol reagent
(Invitrogen) and further purified by using an RNeasy kit (Qiagen, Valencia,
CA). Poly(A)+ RNA was purified from rat liver total RNA using Oligotex
(Qiagen) according to the manufacturer's instructions. Five micrograms of
poly(A)+ RNA was resolved on a 1% agarose/formaldehyde gel, transferred to
nylon membrane, and cross-linked to the membrane with UV light. cDNA probes
were radiolabeled with [
-32P]dCTP using the Rediprime II
labeling kit (Amersham Biosciences). Membranes were hybridized using the Rapid
hybridization buffer (Amersham Biosciences), washed according to the
manufacturer's instructions, and quantified using a Molecular Imager
(Bio-Rad).
Taqman Primers and ProbesOligonucleotide primers and probes
for rat SHP and BSEP were designed using the Primer Express program and were
synthesized by Integrated DNA Technologies Inc. The sequences
(5'
3') were as follows: Rat SHP, forward primer
(ttggatttcctcggtttgc), probe (6FAM-cagtgtttgactaadtgtccagcaggcc-TAMRA), and
reverse primer (acccaggtaagggaaggcata); rat BSEP, forward primer
(tgcatgtcaggagacggc), probe (6FAM-tcacattgtggaactcaatttcacccttg-TAMRA), and
reverse primer (tcacgtccggtctagaagga).
Primary Hepatocyte Cultures and Cell LinesPrimary rat
hepatocytes were obtained from In Vitro Technologies plated on collagen-coated
plates. Upon arrival (2 days after collagenase isolation), shipping media was
replaced by fresh hepatocyte growth media (In Vitro Technologies). After a 2-h
recovery period the cells were treated with vehicle (Me2SO) or the
following FXR ligands: 100 µM
3
,7
-dihydroxy-5
-cholanic acid (CDCA), 1 µM
3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole
(GW4064) (19). After 48 h,
total RNA was isolated from the cells. CV-1 cells were obtained from and
maintained according to ATCC depository.
cDNA Probes and Reporter GenesHuman cDNAs for BAT and BACS
were PCR-amplified from reverse-transcribed HepG2 mRNA using primers
5'-atgatccagttgacagctacccctgtg-3' and
5'-tcaagcatgttcctgtgcagctgcgtg-3' for BAT, and
5'-gtaccatgggtgtcaggcaacag-3' and
5'-cagagctcagcacagagtgcgc-3' for BACS. The PCR amplicons, 1230 and
667 bp for BAT and BACS, respectively, were radiolabeled and used as probes
for Northern analysis. Human BAT and BACS gene sequences were obtained from
GenBankTM. A fragment spanning
1.5 kb of 5'-flanking sequence
of the BACS (VLCS-H2) gene was PCR-amplified from human genomic DNA using
primers 5'-gctgtgagcacctggatcagtgc-3' and
5'-gtgacgactgtcaccgaccaggag-3' and cloned into
XhoI-HindIII sites of pGL3-basic vector (Promega). A
fragment spanning nucleotide 383 to + 2751 of the BAT gene was
PCR-amplified from human genomic DNA using primers
5'-ccctggtctcctgcggtaccctcaggc-3' and
5'-gcacaagcagggcacgcatgtggg-3' and cloned into
SacI-XhoI sites of pGL3-basic vector (Promega). The
three-copy BACS IR-1 construct was generated by annealing the oligonucleotides
5'-agcttcccaaggggcagagacctgcggggcagagacctgcggggcagagacctgggag-3'
and
5'-gatcctcccaggtctctgccccgcaggtctctgccccgcaggtctctgccccttggga-3'
before ligation into HindIII/BamHI-digested TK-Luc. The
three-copy BAT IR-1 was done using the oligonucleotides
5'-agcttcttggaggtcaagtgcctcgaggtcaagtgcctcgaggtcaagtgcctcgttg-3'
and
5'-gatccaacgaggcacttgacctcgaggcacttgacctcgaggcacttgacctccaaga-3'.
Point mutations of FXRE (IR-1) sequences were done using the QuikChange
mutagenesis kit from Stratagene. For mutation of the BACS IR-1, an antisense
primer (5'-gtggtgcccaaggaagcagagatttgggaacccaga-3')
and a sense primer
(5'-tctgggttcccaaatctctgcttccttgggcaccac-3')
(mutated bases indicated in boldface type) were used in a PCR according to the
manufacturer's directions. For mutation of the IR-1 in the BAT gene, the
antisense primer
(5'-aggcatcttggaaatcaagtgtttcgttcatccttg-3') and the
sense primer (5'-caaggatgaacgaaacacttgatt-
tccaagatgcct-3') were used. All oligonucleotides primers were ordered
from Integrated DNA Technologies Inc. The sequences of all constructs were
verified by automated DNA sequencing.
Transient Transfections and Reporter Gene AssaysCV-1 cells
were transfected using the FuGENE 6 reagent (Roche Applied Science). Briefly,
30 ng of reporter plasmid, 10 ng of pCMX-hFXR, 10 ng of pCMX-hRXR
, and
10 ng of pCMV-
-galactosidase and 0.28 µl of FuGENE 6 were used in a
final volume of 10 µl. After a 15-min incubation at room temperature, the
mixture was added to cells in 96-well plates and incubated for5hat37 °C.
The cells were then treated with medium containing 10%
charcoal/dextran-stripped fetal bovine serum and vehicle (Me2SO) or
one of the following ligands: 100 µM CDCA, 1 µM
GW4064, or 0.1 µM LG1305. After 24 h, cells were lysed and
assayed for luciferase and
-galactosidase activity. The normalized
luciferase units (RLUs) were determined by dividing the luciferase activity by
the
-galactosidase activity.
Electrophoretic Mobility Shift AssaysCore sequences of the
BAT, BACS, and IBABP IR-1s are shown below in
Fig. 2A. Annealed
double-stranded oligonucleotides were radiolabeled with
[
-32P]dCTP using the Klenow fragment of DNA polymerase II.
hFXR and hRXR
were synthesized from pCMX-FXR and pCMX-RXR
expression vectors using the TNT T7 Coupled Reticulocyte System
(Promega, Madison, WI). Binding reactions contained 20 mM Hepes, pH
7.2, 75 mM KCl, 2 mM dithiothreitol, 0.2% Nonidet P-40,
15% glycerol, 1 µg of poly(dI-dC)-poly(dI-dC), 3 µl each of synthesized
receptor proteins, and 0.05 pmol of 32P-labeled double-stranded
oligonucleotide. Competitor oligonucleotides were added at 50- and 150-fold
molar excesses. The oligonucleotides used for BAT and BACS IR-1s were
5'-agcttcttggaggtcaagtgcctcgttg-3' and
5'-agcttcccaaggggcagagacctgggag-3', respectively (only one
strand is shown, and the IR-1 is indicated in boldface). IBABP IR-1 was
5'-agcttttccttaaggtgaataaccttggggct-3'.

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FIG. 2. The BACS and BAT genes contain potential FXREs. A,
comparison of BACS and BAT FXREs to an IR-1 found in the mouse IBABP gene and
an idealized IR-1 consensus sequence. B, location of FXREs (IR-1s) in
the BACS and BAT genes and comparison to the IBABP consensus IR-1. Exon
1 indicates the first exon, and the star marks the translation
start site of each gene.
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Animal StudiesMale F344/NHsd rats were fed ad
libitum and were kept under standard light/dark cycle. The GW4064
compound was suspended in 0.5% carboxymethyl cellulose vehicle for a dosing
volume of 1 ml. Animals were dosed bis in die for 7 days with a
16-gauge 5.08-cm feeding needle to give a final concentration of 100
mg/kg.
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RESULTS
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Identification of BAT as a FXR Target GeneTo identify FXR
target genes expressed in the liver, primary rat hepatocytes were used for
gene expression profiling experiments. Cells were treated for 48 h with CDCA
(100 µM) or the synthetic FXR ligand GW4064 (1
µM), whereas control cells were treated with Me2SO
(vehicle) before isolation of total RNA. To confirm that FXR target genes were
regulated in this experiment, quantitative reverse transcription-PCR analysis
of total RNA was performed using specific primers for BSEP and SHP. SHP mRNA
levels were significantly increased with the synthetic FXR agonist GW4064,
whereas an increase of
2-fold was seen with CDCA compared with
vehicle-treated cells (Fig.
1A) consistent with studies indicating that CDCA is a
weak activator of SHP in vivo
(20). BSEP mRNA levels were
also induced by GW4064, and a 2-fold induction was seen with CDCA
(Fig. 1B). Having
verified regulation of FXR target genes in this experiment, total RNA from
each sample was processed and hybridized to Affymetrix rat genomic U34A
GeneChips according to Affymetrix protocols. Differentially expressed genes
were identified by comparing hybridization signals for FXR ligand treated
samples to signals for vehicle treated samples using the Affymetrix Microarray
Suite and Data Mining Tools software. Expression profiling by a natural
versus a synthetic FXR agonist of primary hepatocytes indicated some
degree of overlap but also separation of activities by these ligands (Tables
I,
II,
III). Two known FXR target
genes, BSEP and SHP, and one novel target gene, namely bile acid-CoA:amino
acid N-acetyltransferase (BAT), were clearly differentially expressed
in this experiment (Tables I
and II). We decided to focus on
the BAT gene for further characterization, because FXR has previously been
shown to play a critical role in bile acid homeostasis. The BAT transcript was
induced
2-fold by GW4064 as well as with CDCA in the micro-array
experiment.

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FIG. 1. Induction of FXR target genes in rat primary hepatocytes. Cells were
cultured as described under "Experimental Procedures" and treated
with FXR ligands GW4064 (1 µM) and CDCA (100 µM)
for 48 h. Total RNA was prepared, and mRNA levels of FXR targets were
determined by quantitative RT-PCR. A, SHP mRNA levels; B,
BSEP mRNA levels.
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TABLE I Genes regulated by GW4064 and CDCA in rat primary hepatocytes
Listed are genes that showed 1.8-fold regulation in at least one of the
treatments and above 1.5-fold in the other treatment. Induction/repression is
expressed as -fold over vehicle-treated cells.
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TABLE II Genes regulated by GW4064 but not CDCA in rat primary
hepatocytes
Listed are genes that showed above a 2.0-fold regulation.
Induction/repression is expressed as -fold over vehicle-treated cells.
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TABLE III Genes regulated by CDCA but not GW4064 in rat primary
hepatocytes
Listed are genes that showed above a 2.0-fold regulation.
Induction/repression is expressed as -fold over vehicle-treated cells.
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The Human BACS and BAT Genes Contain Potential FXREsThe
finding that BAT is transcriptionally activated by FXR ligands suggested that
the rate-limiting enzyme in bile acid-amino acid conjugation, bile acid-CoA
synthetase (BACS) may also be an FXR target gene. To date, most functional
binding sites (FXRE) identified in FXR target genes correspond to two inverted
repeats spaced by one nucleotide as exemplified by the IR-1 from the mouse
IBABP promoter (Fig.
2A). Analysis of the proximal promoter of the human BACS
gene identified a potential IR-1 (5'-GGGGCAaAGACCT-3') in the
flanking region located
230 bp upstream of the transcription start site.
Similar analysis of the proximal promoter and intron 1 of the human BAT gene
identified an IR-1 (5'-AGGTCAaGTGCCT-3') located in intron 1 at
+2615 to +2628 relative to the transcription start site
(Fig. 2B).
Electrophoresis mobility gel shift assays (EMSA) and transient transfection
experiments were next performed to investigate direct binding and
functionality of the FXR/RXR binding to these sequences.
FXR/RXR Heterodimers Bind to FXREs in the BACS and BAT
GenesEMSA experiments with a radiolabeled oligonucleotide spanning
the BACS IR-1 showed that when incubated with in vitro translated FXR
and RXR protein this element produced a significant band shift that was
competed with 150-fold molar excess of unlabeled BACS or IBABP IR-1. No
shifted DNA-protein complex was observed when either FXR or RXR was omitted
from the EMSAs (Fig.
3A). An oligonucleotide containing the BAT IR-1 sequence
was next radiolabeled and used in a gel-shift experiment
(Fig. 3B). The BAT
IR-1 produced a significant band shift when incubated with FXR and RXR
proteins but not when incubated by RXR alone. Interestingly, a weak band shift
was detected when the BAT IR-1 was incubated with FXR alone, indicating that
FXR may bind as a monomer or homodimer to this DNA element. The binding of
FXR/RXR was blocked by 50- and 150-fold excesses of either unlabeled BAT IR-1
or IBABP IR-1. Conversely, the binding of FXR/RXR heterodimers to the
radiolabeled IBABP IR-1 was efficiently competed by 50- and 150-fold molar
excesses of either unlabeled IBABP IR-1, BAT IR-1
(Fig. 3C), or BACS
IR-1 (data not shown).

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FIG. 3. FXR/RXR heterodimers bind to IR-1 elements in the proximal BACS gene
promoter and an intronic element in the BAT gene.
AC, FXR/RXR proteins bind to radiolabeled IR-1
sequences from the BACS and BAT genes with similar affinities as binding to an
IR-1 from the mouse IBABP gene. Electrophoretic mobility shift assays were
performed using in vitro translated FXR/RXR proteins and radiolabeled
IR-1s as outlined under "Experimental Procedures." Competitions
were done using unlabeled oligonucleotides at 50- and 150-fold molar excesses
as indicated.
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The BACS and BAT IR-1s Are Functional FXREs in the Context of a
Heterologous PromoterTo test the ability of the BACS and BAT IR-1
elements to function as FXREs, three copies of each of the IR-1s were cloned
into the thymidine kinase luciferase (TK-Luc) reporter plasmid. The resultant
3xBACS-IR-1-TK-Luc and 3xBAT-IR-1-TK-Luc reporters were cotransfected with
expression plasmids for FXR and RXR into CV-1 cells. After treatment with
vehicle (Me2SO), GW4064, and CDCA the cells were lysed and assayed
for luciferase activity. The BACS and BAT IR-1 elements were able to confer
FXR-dependent transactivation as demonstrated by activation of the
3xBACS-IR-1-TK-Luc (Fig.
4A) and 3xBAT-IR-1-TK-Luc
(Fig. 4B) reporters by
GW4064 and CDCA, whereas no significant induction was seen with the empty
TK-Luc vector (Fig. 4,
AB). Similar activities were achieved with a
three-copy IR-1 from the mouse IBABP promoter (data not shown). These data
together with the band-shift results in
Fig. 3 suggest that the two
IR-1s from the BACS and BAT genes are functional FXR binding sites.

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FIG. 4. FXR and FXR ligands activate reporter genes under control of BACS and
BAT IR-1 sequences. A and B, the IR-1 sequences from the
BACS and BAT genes are functional FXREs in the context of a heterologous
promoter. CV-1 cells were transfected with either 3xBACS-IR-1-TK-Luc,
3xBAT-IR-1-TK-Luc, or TK-Luc reporters, together with pCMX-RXR ,
pCMX-FXR, and pCMV- -galactosidase. Cells were then treated with vehicle
control (Me2SO), 1 µM GW4064, or 100 µM
CDCA, and lysed cells were assayed for luciferase activity 24 h later. The
results were normalized to -galactosidase activity, expressed as
relative light units (RLUs), and represent the mean ± S.D. of
triplicate determinations. Transfections were performed at least three times,
and one representative experiment is shown.
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The BACS and BAT IR-1s Are Functional FXREs in the Context of Their
Native GeneTo demonstrate that the BACS IR-1 is a functional FXRE
in the context of its natural promoter,
1.5 kb of the 5' flanking
region of the human BACS gene was cloned in pGL3b, and the resultant reporter
gene construct was tested in transient transfection experiments using CV-1
cells (Fig. 5A). This
region of the BACS gene conferred significant FXR activity mediated by GW4064
as well as CDCA. The BACS promoter was also significantly activated by the
synthetic RXR ligand LG1305, and an additive effect was seen with the
combination of LG1305 with GW4064 or CDCA. Mutation of the putative IR-1 in
the BACS gene reporter construct significantly reduced the FXR/RXR-mediated
activity (Fig. 5A). It
is concluded from these experiments that the BACS gene confers FXR regulation
via a functional FXRE located in the proximal promoter of the gene. To
demonstrate that the BAT IR-1 element identified in the above experiments is a
functional FXRE in the context of the BAT gene promoter, a fragment spanning
the human BAT gene from 383 to + 2751 was cloned into the luciferase
vector pGL3 and the resultant reporter gene (BAT-wt-Luc) was tested for
FXR-dependent transactivation. No activation of the BAT-wt-Luc by FXR or RXR
ligands was observed in CV-1 cells when cotransfected with FXR and RXR
expression plasmids (data not shown). FXR ligand-dependent activation of the
BAT promoter/intron 1 construct was observed, however, when a VP16-FXR fusion
protein was introduced (Fig.
5B). Importantly, the ligand-dependent activation
detected with VP16-FXR requires the IR-1 sequence in the first intron, because
mutation of this site eliminates the response
(Fig. 5B). The failure
to see activation of the BAT promoter/intron by wild-type FXR most likely
arises from the relatively low transcriptional activity of this reporter in
CV-1 cells. A similar VP16-FXR construct was used to define the FXR-dependent
regulation of apoCII (21). We
conclude from these experiments that the BAT gene contains a functional FXRE
located in the first intron of the gene.

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FIG. 5. The IR-1 sequences in the BACS and BAT genes mediate transactivation by
FXR/RXR heterodimers. A, the BACS IR-1 is a functional FXRE in
the context of the native BACS gene. CV-1 cells were transfected with either
wild-type or mutated BACS reporter genes, together with pCMX-RXR ,
pCMX-FXR, and pCMV- -galactosidase. B, the IR-1 in intron 1 of
the BAT gene is a functional FXRE in the context of the native gene. CV-1
cells were transfected with either wild-type or mutated BAT
promoter/intron-Luc reporters and pCMV- -galactosidase with or without
plasmids for pCMX-VP16FXR and pCMX-RXR . Transfected cells were treated
with vehicle control (Me2SO), 1 µM GW4064, 100
µM CDCA, 0.1 µM LG1305, or a combination of FXR
and RXR ligands, and lysed cells were assayed for luciferase activity 24 h
later. The results were normalized to -galactosidase activity, expressed
as relative light units (RLUs), and represent the mean ± S.D. of
triplicate determinations. Transfections were performed at least three times,
and one representative experiment is shown.
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Hepatic BACS and BAT mRNAs Are Induced in VivoTo confirm
that the FXR-dependent regulation of BAT and BACS observed in vitro
also occurs in vivo, Fisher rats (six vehicle- and six
GW4064-treated) were treated for 7 days with the synthetic FXR agonist GW4064.
Northern blot analysis demonstrated that treatment of rats with GW4064
(lanes 16) results in the induction of BAT mRNA compared with
vehicle (lanes 712)-treated rats
(Fig. 6A). Similarly,
the BACS transcript was also significantly induced by the GW4064 ligand in rat
liver. After normalization to the glyceraldehyde-3-phosphate dehydrogenase
control transcript, the BAT and the BACS transcripts showed an average
induction of
2- to 3-fold by GW4064 compared with vehicle-treated rats
(Fig. 6B). Taken
together these results support the conclusion that both the BAT and the BACS
genes are direct targets of FXR.

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FIG. 6. Northern blot analysis of BAT and BACS mRNAs from vehicle- or
GW4064-treated rats. A, rats were administered vehicle
(Me2SO) (lanes 16) or GW4064 (lanes
712) for 7 days as described under "Experimental
Procedures." Total RNA was prepared, and 5 µg of
poly(A)+-enriched RNA was loaded in each lane. After blotting the
filter was hybridized with the indicated radiolabeled cDNAs. B,
relative mRNA levels for BAT and BACS were determined for rats treated as
described in panel A. Values (±S.D.) are the average obtained
for each group of rats (n = 6) after normalization to
glyceraldehyde-3-phosphate dehydrogenase mRNA. Significant differences
compared with vehicle treated rats; ***, p < 0.0005.
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DISCUSSION
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Bile acids are not simply by-products of cholesterol metabolism but play an
essential role in solubilizing cholesterol and facilitating the uptake of
dietary lipids. In addition, recent discoveries have established bile acids,
including CDCA and CA, and their respective conjugated metabolites, as
signaling molecules that bind and activate the nuclear receptor FXR
(22). FXR is highly expressed
in the enterohepatic system where it acts as a bile acid sensor that protects
the body from elevated bile acid concentrations. The role of FXR in
coordinating the expression of genes involved in bile acid homeostasis has
been firmly established by the use of natural and synthetic FXR agonists and
FXR knockout mice (19,
23,
24). The bile acid-sensing
function of FXR is largely achieved through the regulation of genes involved
in bile acid transport and biosynthesis
(25). These include the
transport proteins BSEP, NTCP, and IBABP, as well as proteins affecting bile
acid synthesis like CYP7A1, CYP8B1, and SHP. In addition,
dehydroepiandrosterone-sulfotransferase, an enzyme with bile acid
sulfo-conjugating activity, is directly regulated by FXR suggesting
involvement also in bile acid detoxification and clearance
(11).
In the present study we show that the genes encoding two key enzymes in
bile acid-amino acid conjugation, BAT and BACS, are direct targets of the bile
acid receptor FXR further expanding and confirming the critical role of this
receptor in bile acid homeostasis. We used micro-array technology and
identified BAT as a novel FXR target gene and then hypothesized that BACS,
which catalyzes the rate-limiting reaction in conjugation of bile acid to
taurine or glycine, may also be a target gene for FXR. A search for FXR
binding sites in the human BACS gene identified one potential IR-1 in the
proximal promoter of this gene. We presented evidence that the FXR-mediated
activation of BACS gene transcription is mediated through this IR-1 and based
upon gel-shift and competition experiments that RXR/FXR heterodimers directly
bind to this element. Further mutational studies established that induction of
a reporter gene was dependent on an intact IR-1 element. The observation that
the proximal promoter of the BAT gene was not induced by FXR ligands (data not
shown) coupled with the fact that an intronic sequence did confer FXR
responsiveness led us to conclude that bile acid activation of the BAT gene is
likely mediated by the FXRE in the first intron. Conceivably, other FXREs
located further upstream to the BAT gene sequences used in the present study
could contribute to the FXR-dependent induction of this gene. However,
analysis of the rat genomic sequence for BAT showed a similar location (intron
1) of an IR-1 as for the human gene (data not shown), further supporting the
conclusion that FXR regulates the BAT gene via an intronic FXRE. Functional
intronic nuclear receptor binding sites have recently been found in the
lipoprotein lipase gene and the gene encoding acyl-CoA-binding protein
(26,
27). The current study
demonstrates that BACS and BAT mRNAs are induced in rat liver in response to
natural and synthetic FXR ligands. Interestingly, the FXR-dependent induction
of BACS and BAT mRNA may be specific to rats, because similar studies in mice
failed to demonstrate regulation of these mRNAs. On the other hand, human gene
promoter sequences were used in the present study, suggesting FXR-dependent
regulation in humans.
The two-step process of conjugation of bile acids with the amino acids
taurine and glycine is a result of the successive action of BACS and BAT. Our
results establish a role of FXR in regulating these processes. One question
raised by these findings is what is the physiological basis for the regulation
of bile acid-amidation by FXR. High concentrations of bile acids in the
hepatocyte repress further synthesis mediated by CYP7A1, the rate-limiting
enzyme of bile acid biosynthesis
(13). Current understanding is
that bile acids, by acting as FXR ligands, inhibit bile acid synthesis partly
via activation of SHP, which in turn represses CYP7A1
(5,
6). FXR activators also
decrease the intracellular bile acid concentration by directly inducing BSEP,
a bile acid export pump, and by indirectly repressing NTCP, which extracts
bile acids from the blood. In the enterocytes of the ileum, bile acids are
efficiently reclaimed for return to the liver. In these ileal enterocytes,
bile acids induce the expression of the cytosolic-binding protein IBABP,
another FXR target gene that has been proposed to buffer intracellular bile
acids and promote their translocation into the portal circulation. Based on
the current study we propose that FXR, by controlling the level of bile acid
amidation, regulates intracellular levels of unconjugated bile acids, which
can be cytotoxic unless they are conjugated. Importantly, BSEP does not
translocate unconjugated bile acids into bile
(28). Individuals with
mutations in the BAT gene have no conjugated bile acids. Consequently, small
quantities of bile acids enter into bile. In these patients most of the
unconjugated bile acids diffuse out of hepatocytes and into plasma, leading to
high serum bile acid concentrations and low intestinal concentrations
(29).
In conclusion, our results show that two genes encoding bile acid-amino
acid conjugation enzymes are induced by FXR ligands in the liver via IR-1
elements cognate to the FXR/RXR heterodimer. The identification of FXR target
genes involved in bile acid amidation is consistent with an important role of
this nuclear receptor in regulating cholesterol and bile acid metabolism in
the liver, excretion of bile acids into bile, and the re-uptake of bile acids
from the intestinal lumen. These observations thus couple the process of bile
acid conjugation to bile acid synthesis and transport via transcriptional
regulation by FXR.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Biology, X-Ceptor
Therapeutics Inc., 4757 Nexus Centre Dr., San Diego, CA 92121. Tel.:
858-458-4522; Fax: 858-458-4501; E-mail:
swestin{at}x-ceptor.com.
1 The abbreviations used are: FXR, farnesoid X receptor; CYP7A1,
cholesterol-7
hydroxylase; SHP, short heterodimer partner; NTCP,
sodium/taurocholate cotransporter peptide; BSEP, bile salt export pump; IR,
inverted repeat; RXR, retinoid X receptor; IBABP, ileal bile acid receptor;
THCA, trihydroxycholestanoic acid; DHCA, dihydroxycholestanoic acid; BACS,
bile acid-CoA synthetase; VLCS-H2, very long chain acyl CoA synthetase homolog
2; BAT, bile acid-CoA:amino acid N-acetyltransferase; 6-FAM,
6-carboxyfluorescein; 6-TAMRA, 6-carboxytetramethylrhodamine; CA, cholic acid;
CDCA, chenodeoxycholic acid; FXRE, farnesoid X receptor element; RLUs,
relative light units; EMSA, electrophoretic mobility shift assay. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Richard Martin and Jeff Kahl for the chemical synthesis of
GW4064; Chris Daige and Calvin Vu for help with animal studies; and Bosun Kim
for technical assistance.
 |
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