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INTRODUCTION |
Nuclear hormone receptors are transcription factors that are
involved in numerous processes, including reproduction, development, and general metabolism (1). Most of these receptors are comprised of a
ligand-independent transcriptional activation function (AF-1) at the
amino terminus, a DNA binding domain, a hinge region and a ligand
binding domain, a dimerization interface, and a
ligand-dependent activation function (AF-2) at the carboxyl
terminus (2, 3). In many cases entry of a specific ligand into the
pocket formed by the ligand binding domain results in a conformational
change of the receptor, recruitment of co-activators, and
transcriptional activation (4-13).
Nuclear hormone receptors have been classified into sub-groups
depending on whether they bind DNA as homodimers, heterodimers, or
monomers (14). A few family members have been identified that do not
bind DNA directly but instead function by interacting with other
transcription factors and altering their activity (15, 16).
Nonetheless, the major sub-group contains members that bind to DNA as
heterodimers with the common partner, retinoid X receptor
(RXR)1 (14). The farnesoid X
receptor (FXR, NR1H4) falls into this category.
FXR was isolated by screening a rat cDNA library using PCR and
degenerate primers corresponding to the highly conserved DNA binding
domain of nuclear receptors (17). Independently, two mouse homologues
of rat FXR, termed RIP14-1 and RIP14-2, were isolated using the yeast
two-hybrid assay and the human RXR ligand binding domain as bait (18).
Northern blot assays and in situ hybridization indicate that
FXR expression was limited to the liver, small intestine, kidney, and
adrenal gland (17, 19).
In the initial studies, supraphysiological levels of farnesol were
shown to activate the rat (17) but not the murine FXR (20). In 1999, several groups independently identified bile acids as endogenous
ligands that activated FXR at physiological concentrations (21-23).
The finding that bile acids not only bound to FXR but that this
interaction resulted in recruitment of co-activators (21, 22) provides
compelling evidence that bile acids are physiologically important
hormones that function to activate the FXR/RXR heterodimer.
The recent characterization of FXR null mice (24), the synthesis and
utilization of a high affinity ligand for FXR (25), and the
identification of a number of FXR target genes provide important
insights into the role of FXR in controlling lipid metabolism. FXR
target genes include ileal bile acid-binding protein (I-BABP) (21, 26),
phospholipid transfer protein (27, 28), apolipoprotein C-II (29),
multidrug resistance-associated protein 2 (ABCC2) (30), the bile salt
export pump (BSEP) (31), and the small heterodimer partner receptor
(SHP) (25, 32) (for review, see Ref. 33). These genes are involved in
various aspects of bile acid, lipoprotein, and lipid metabolism (33).
The demonstration that FXR null mice are unable to respond
appropriately to diets enriched in fat or bile acids (24) further
emphasized the critical role of FXR in controlling lipid homeostasis.
Two forms of murine FXR (RIP14-1 and RIP14-2) that differ at their
amino terminus were originally isolated (18). RIP14-2, in contrast to
RIP14-1, contained an additional 12 bp that results in the insertion of
four amino acids in the hinge region, adjacent to the DNA binding
domain. Analysis of the cDNA encoding rat FXR indicates that it
does not contain the 12-bp insert but otherwise corresponds to murine
RIP14-1. It is not known whether these different isoforms have
different functions.
Taken together, these results suggested that there might be at least
four FXR isoforms that differ either at their amino terminus and/or at
the site of the four-amino acid insertion in the hinge region. Because
the hinge region is thought to have a role in the DNA binding
properties of nuclear receptors (34-36), we hypothesized that these
different isoforms might differentially bind to DNA and/or
differentially activate target genes. The current report provides
evidence to support these proposals.
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EXPERIMENTAL PROCEDURES |
Animals--
C57BL/6J female mice were fed a standard rodent
chow diet in a temperature-controlled room (23 °C) on a 12-h
light/dark cycle. Eight to 12-week-old wild type mice were sacrificed,
and tissues were snap-frozen in liquid nitrogen and stored at
80 °C until use.
Plasmids and Reagents--
Four different mouse FXR cDNAs
were isolated from the liver tissue using gene-specific primers (GSP1
and GSP2) and adapter primers (ADP1 and ADP2) in the Sure-RACE panels
according to the manufacture's protocol (OriGene Technologies, Inc.,
Rockville, MD). The first round of PCR utilized ADP1 and GSP1 (Fig.
1A). The generated cDNA was further amplified in a
second round of PCR utilizing ADP2 and GSP2. The full-length coding
regions of four murine FXR isoforms were amplified by PCR using
gene-specific primers and cloned into BamHI/XhoI
sites of CMX-PL1 vector to produce expression constructs CMX-FXR-
1,
CMX-FXR
2, CMX-FXR
1, and CMX-FXR
2. To make retroviral
expression constructs, the full-length coding regions of four different
isoforms were separately excised from the CMX expression constructs
using BamHI/XhoI restriction enzymes and
subcloned into BglII/XhoI sites of the
MSCV-IRES-neo vector to make constructs MSCV-FXR
1, -FXR
2,
-FXR
1, and -FXR
2. The mouse BSEP promoter (
1050 to +25) was
amplified using gene-specific primers and cloned into
SacI/XhoI-digested-pGL3-LUC vector (Promega) to
create pGL3-BSEP-Luc. All the plasmids have been confirmed by
sequencing. Plasmids pIBABP1031-Luc and
pIBABP142mut-Luc were kind gifts from Dr. David Mangelsdorf
(University of Texas Southwestern Medical Center) (21). pGL3-hSHP-Luc
was kindly provided by Dr. Bryan Goodwin (GlaxoSmithKline) (25). The
retroviral vector MSCV-IRES-neo plasmid was a gift from Dr. Owen Witte
(University of California, Los Angeles). The sources of other plasmids
and synthetic ligands have been described elsewhere (30).
5' Rapid Amplification of cDNA Ends (5' RACE) and Southern
and Northern Blot Analysis--
Sure-RACE mouse panels (OriGene
Technologies) contain double-stranded cDNAs synthesized from 24 tissues. A 5' adapter, containing sequences corresponding to ADP1 and
ADP2, was ligated at the 5' ends. The cDNAs were amplified using
gene-specific primers (GSP1 and GSP2) and adapter-specific primers
(ADP1 and ADP2). The PCR products were then isolated on a 1.2% agarose
gel and transferred to a nylon membrane, and the membranes were probed
with a mouse FXR cDNA probe. The bands corresponding to FXR
or
FXR
were recovered from the gel and cloned into pCR2.1-TOPO vector
(Invitrogen). After transformation, the white colonies were patched
onto duplicate ampicillin-positive LB plates. The colonies from the
plate were first screened to identify whether they represent FXR
or
FXR
using P3 and P4 (see Fig. 1) followed by using P1 (for screening the isoform with 12-bp insert) and then P2 (for screening the isoform
without 12-bp insert) (Fig. 1). The sequences for P1 and P2 are
5'-TGGCTGAATGTATGTATACAGGTTTGTTAA-3' and
5'-ATGTTGGCTGAATGTTTGTTAACTGA-3', respectively. All positive colonies
were further confirmed either by sequencing or PCR. For Northern blot
analysis, total RNA was isolated using Trizol reagent (Invitrogen), and
10 µg of RNA was denatured, electrophoresed, transferred to a nylon
membrane, and probed with the indicated cDNA probe.
Quantitative PCR--
Real time PCR was performed essentially as
described (37). Briefly, 1 µg of DNase I-treated total RNA was
reverse-transcribed with random hexamers using the Taqman reverse
transcription kit (Applied Biosystems) according to the manufacturer's
protocol. Each amplification mixture (50 µl) contained 50 ng of
cDNA, 900 nM forward primer, 900 nM reverse
primer, 250 nM fluorogenic probe, and 25 µl of Universal
PCR Master Mix (Applied Biosystems). PCR thermocycling parameters were
50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for
15 s, and 60 °C for 1 min. Real time PCR was carried out
using Applied Biosystems 7700 sequence detector. Samples were analyzed
simultaneously for cyclophilin expression. Quantitative expression
values were extrapolated from separate standard curves. Each sample was
assayed in duplicate and normalized to cyclophilin. The
sequences for primers and probes are as follows: FXR
,
5'-TGGGCTCCGAATCCTCTTAGA-3' (forward primer, F),
5'-TGGTCCTCAAATAAGATCCTTGG-3' (reverse primer, R),
5'-CCTTGGACATCTCTGGCCCAAAGCA-3' (Probe, P); FXR
,
5'-GGGCTTAGAAAATCCAATTCAGATTA-3' (F), 5'-CGTCCGGCACAAATCCTG-3' (R),
5'-TCTTCACCACAGCCACCGGCTG-3' (P); I-BABP, 5'-CAAGGCTACCGTGAAGATGGA-3' (F), 5'-ACCTCCGAAGTCTGGTGATAGTTG-3'(R), 5'-GGAACTCTGCCACCACCTTGCCA-3' (P); BSEP, 5'-ACAGAAGCAAAGGGTAGCCATC-3' (F), GGTAGCCATGTCCAGAAGCAG-3' (R), CCGCGCCCTCATACGGAAACC-3' (P); cyclophilin, 5'-GGCCGATGACGAGCCC-3' (F), 5'-TGTCTTTGGAACTTTGTCTGCAA-3' (R), 5'-TGGGCCGCGTCTCCTTCGA-3' (P).
All probes were dually labeled at the 5' end with
6-carboxyfluorescein and at the 3' end with 6- carboxytetramethylrhodamine.
Electrophoretic Mobility Shift Assay (EMSA)--
EMSAs were
performed essentially as described (27). Mouse FXR isoforms or human
RXR
was synthesized in vitro using the TNT T7-coupled
reticulocyte system (Promega). To compare transcription/translation efficiency of the expression constructs expressing different mouse FXR
isoforms, equal volumes of 35S-labeled lysates were loaded
and separated on an 8% SDS-polyacrylamide gel. The gel was dried and
autoradiographed. The bands were quantitated using a PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA). Binding reactions were
carried out in a buffer containing 10 mM HEPES, pH 7.8, 100 mM KCl, 0.2% Nonidet P-40, 6% glycerol, 0.3 mg/ml bovine
serum albumin, 1 mM dithiothreitol, 2 µg of poly(dI-dC), 1-3 µl each of in vitro translated receptors and
32P end-labeled oligonucleotide. DNA-protein complexes were
resolved on a 5% polyacrylamide gel in 0.5× TBE (45 mM
Tris borate, 1 mM EDTA) at 4 °C. Gels were dried and
autoradiographed. The sequences for mouse I-BABP (mI-BABP) probe
and hSHP probe were 5'-GTTTTCCTTAAGGTGAATAACCTTGGGGCTC-3' and
5'-GTACAGCCTGGGTTAATGACCCTGTTTATGC-3', respectively.
Cell Culture, Transfection, and Stable Cell Lines--
CV-1 and
HepG2 cells were maintained in modified Eagle's medium, 10% fetal
bovine serum. Transient transfections were performed in triplicate in
48-well plates as described (27). Cells were treated with ligands or
vehicle (Me2SO) in super-stripped fetal bovine serum
(HyClone, Logan, UT) as indicated in the legends. Luciferase activity
was measured and normalized to
-galactosidase activity. To produce
stable cell lines, 293T cells were transfected with MSCV-FXR
1,
-FXR
2, -FXR
1, -FXR
2, or MSCV-neo along with
E helper virus.
The supernatants of the culture media were then used to infect NIH3T3
cells followed by selection with 800 µg/ml G418 sulfate
(Geneticin®, Invitrogen) for 3-4 weeks. The selected stable cells
were subsequently treated with the indicated ligands, as described in
the legend to Fig. 6.
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RESULTS |
Isolation of Four Murine FXR Isoforms--
Based on the previous
reports on rat (17) and murine (18) FXR, we hypothesized that there
might be four murine FXR isoforms. For clarity, the four isoforms that
have been characterized in the present report have been termed FXR
1,
FXR
2, FXR
1, and FXR
2 (Fig.
1A). FXR
2 and FXR
1
correspond to RIP14-1 and RIP14-2, previously identified by Seol
et al. (18).

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Fig. 1.
Isolation and characterization of four murine
FXR isoforms. A, schematic diagram of four mouse FXR
mRNAs. Four mouse FXR isoforms termed FXR 1, FXR 2, FXR 1,
and FXR 2 were isolated as described in "Experimental
Procedures." The 5' ends of FXR 1 and FXR 2 are different
from those of FXR 1 and FXR 2. FXR 1 and FXR 2 correspond to
RIP14-2 and RIP14-1, respectively. An additional 10 base pairs
(GTTGCCGTGA) were identified at the 5' end of FXR 1 and FXR 2
mRNAs compared with RIP14-1. The first ATG (asterisk)
and stop codon ( ) are denoted. The 12-bp insert is
denoted by a vertical black bar. The probes (P1, P2, P3, P4)
and gene-specific primers (GSP1, GSP2) are indicated. B,
schematic representation of the organization of murine FXR gene. The
mouse FXR gene consists of 11 exons and 10 introns. FXR and FXR
are transcribed from exon 1 and exon 3, respectively. The 12-bp insert
is located at the 3' end of exon 5. Alternative splicing between exon 5 and exon 6 produces two forms of FXR that contain or do not contain the
12-bp insert. *, initiation and/or in-frame methionines are indicated.
C, schematic diagram of mouse FXR receptors. FXR and
FXR have the same amino acid sequence, except FXR has an
additional 37 amino acids at its amino terminus. FXR 1 and FXR 1
have a four-amino acid insert (MYTG) that is located within the hinge
region (D domain). The DNA binding domain (DBD) and
ligand binding domain (LBD) are indicated.
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To identify all possible FXR isoforms we employed 5'-RACE and cDNAs
generated from 24 murine tissues that had been ligated to a 5' adaptor
(OriGene Technologies). Gene-specific primers (GSP1 or GSP2 in Fig.
1A) together with adapter-specific primers (ADP1 or ADP2)
were used in a series of PCR reactions to amplify FXR-specific
cDNAs (see "Experimental Procedures"). Southern blot analysis
identified the PCR products that corresponded to FXR cDNAs, and
these were subsequently cloned into pCR2.1-TOPO vector. Radiolabeled
oligonucleotides P3 or P4 (Fig. 1A) were then used to
differentiate colonies corresponding to FXR
from FXR
(data not
shown). Filters containing either FXR
- or FXR
-positive colonies were probed with radiolabeled oligonucleotides P1 or P2 (Fig. 1A) to distinguish whether these colonies did or did not
contain the 12-bp insert (Fig.
2A). This approach coupled
with DNA sequencing of selected inserts identified four murine FXR
isoforms (Fig. 1A). Analysis of the 5' RACE data and various
databases suggests that (i) murine FXR consists of 11 exons and 10 introns, (ii) FXR
transcription is initiated from exon 1, (iii)
FXR
transcription is initiated from exon 3, (iv) FXR
and FXR
share exons 4~11, and (v) the 12-bp insert is located at the 3'
terminus of exon 5 (Fig 1; Table I).
Thus, alternative splicing between exon 5 (that contains the variable
12 bp) and exon 6 produces FXR isoforms that include (
1,
1) or
exclude (
2,
2) the four-amino acid (12 bp) insert (Fig.
1B). Table I provides details of the genomic organization
and intron-exon junctions of the murine FXR gene; the gene encompasses
76,997 bp and contains 11 exons that range in size from 100 to 572 bp
and introns that vary from 328 to 16,388 bp. Almost all of the
exon/intron boundaries display the canonical GT/AG sequence
(Table I). The data were obtained by comparison of the sequence of the
FXR cDNA with the publicly available genomic sequence of murine
chromosome 10 (www.genome.ucsc.edu). Fig. 1C illustrates
the domain structures of the FXR isoforms and shows that the FXR
isoforms contain an additional 37 amino acids at the amino terminus
that are absent from FXR
. The four-amino acid (MYTG) insert is
located in the hinge region, adjacent to the DNA binding domain (Fig.
1C).

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Fig. 2.
Relative expression of murine FXR isoforms in
different tissues. A, Southern blot analysis of the
relative expression of FXR 1 to FXR 2 or FXR 1 to FXR 2. Mouse
cDNAs were amplified using adapter primers and gene-specific
primers and then separated on a 1.2% agarose gel. The DNA bands
corresponding to FXR or FXR were recovered from the gel and
cloned into pCR2.1-TOPO vector. After transformation, the white
colonies were transferred to a nylon membrane, and the membranes were
probed with 32P-labeled oligonucleotides P3 or P4 followed
by P1 (containing the12-bp insert) and then P2 (lacking the 12-bp
insert). The results obtained with PCR products derived from the liver
and corresponding to FXR 1 (left panel) and FXR 2
(right panel) are shown. B, quantitative analysis
of the relative expression of FXR and FXR in different tissues.
Total RNA was isolated from different tissues in mice
(n = 5) fed a normal chow diet, and real-time
quantitative PCR assays were performed in duplicate as described under
"Experimental Procedures" and normalized to cyclophilin.
SI-D, SI-J, SI-I represent duodenum,
jejunum, and ileum portions of small intestine, respectively.
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FXR Isoforms Are Differentially Expressed in Tissues--
To
determine the relative expression of FXR
1:FXR
2 and
FXR
1:FXR
2 in different tissues, we transferred
33 bacterial
colonies that contained DNA corresponding to either FXR
or FXR
to
filters. These filters were probed sequentially with probes P1 and P2
(Fig. 1A and Fig. 2A). As illustrated in Fig.
2A, this approach distinguished between FXR
colonies that
contained (Fig. 2A, left panel) or did not
contain (Fig. 2A, right panel) the 12-bp insert.
Thus, the ratio of FXR
1:FXR
2 in the liver was 1:1.9 (Fig.
2A; Table II). Table II
summarizes the results obtained from similar assays utilizing cDNAs
generated from six tissues. The data indicate that the ratio of
FXR
1:FXR
2 and FXR
1:FXR
2 in different tissues varies
significantly. For example, the ratio of FXR
1:FXR
2 (+12 bp/
12
bp) is 1:51 in the heart and 1:0.75 in the adrenal gland (Table II).
Analysis of the PCR products also indicated that some tissues,
including the heart, kidney, stomach, and adrenal gland expressed
predominantly one FXR isoform, either FXR
or FXR
(Table II).
Because the DNA was analyzed after a PCR amplification step, the ratios
of the four FXR isoforms are considered to be semi-quantitative.
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Table II
Relative expression of FXR 1:FXR 2 and FXR 1:FXR 2 in mouse
tissues
Colonies ( 33 colonies) were screened for analysis of the relative
expression of FXR 1 and FXR 2 or FXR 1 and FXR 2 using the
method as described in Fig. 2A. The ratio of FXR 1 to
FXR 2 or FXR 1 to FXR 2 in different tissues is shown. ND, not
detected.
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To determine the relative expression of FXR
versus
FXR
, total RNA from 13 tissues in C57BL/6J mice (n = 5) fed a normal chow diet was isolated, and real time quantitative PCR
was performed. As shown in Fig. 2B, FXR
and FXR
were
most abundantly expressed in the liver. The liver was the only organ
that expressed similar levels of both isoforms. FXR
was abundantly
expressed in ileum, moderately in kidney, and at low levels in stomach,
duodenum, and jejunum (Fig. 2B). FXR
was moderately
expressed in ileum and adrenal gland. In addition, heart, lung, and fat
contained low but measurable levels of both FXR
and FXR
(Fig.
2B). In contrast, FXR was undetectable in brain, spleen, or
muscle (Fig. 2B). Interestingly, the ratio of FXR
:FXR
in different tissues varies significantly; the ratio was 7:1, 1:1, and
1:3 in the adrenals, liver, and ileum, respectively (Fig.
2B). The differences in expression of the FXR isoforms in
different tissues suggest that the physiological functions of the four
isoforms may vary.
FXR Target Gene Promoters Are Differentially Transactivated by FXR
Isoforms--
To investigate if the four FXR isoforms have different
binding affinities to DNA, the full-length coding regions of the four isoforms were cloned into an expression vector. In vitro
transcription/translation experiments show that the expression
constructs for FXR
1 and FXR
2 produce proteins of the expected
molecular weight (Fig. 3A).
Based on the incorporation of radioactive methionine into the proteins,
more FXR
1 and FXR
2 isoforms were synthesized as compared with
FXR
1 and FXR
2 (Fig. 3A). Consequently, different volumes of lysate, containing equivalent amounts of each individual FXR
isoform, were used in EMSAs. The results of multiple EMSAs consistently
show that FXR
2 and FXR
2 bind to the mI-BABP FXR response element
(FXRE) or the hSHP FXRE with a higher affinity as compared with FXR
1
or FXR
1 (Fig. 3B). The interaction of FXR/RXR with the
radiolabeled DNA probe was attenuated in the presence of excess wild
type competitor DNA but not by the competitor DNA containing a mutated
FXRE (data not shown). Similar results to those shown in Fig.
3B were obtained when the radiolabeled DNA contained FXREs
from mouse BSEP, mouse SHP, human phospholipid transfer protein, rat
multidrug resistance-associated protein 2, or human apoC-II genes (data
not shown).

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Fig. 3.
Murine FXR isoforms bind to FXR response
elements with different affinity. A, CMX-FXR 1,
-FXR 2, -FXR 1, and -FXR 2 were synthesized in vitro
in the presence of [35S]methionine. Three µl of
in vitro translated lysates were analyzed on an 8%
SDS-polyacrylamide gel. The gel was dried and subjected to
autoradiography. B, equivalent amounts of in
vitro synthesized RXR and either FXR 1, FXR 2, FXR 1, or
FXR 2 protein were incubated with 32P-labeled DNA probes
containing the mI-BABP or hSHP FXRE. The DNA-protein complex was
resolved on a 5% polyacrylamide gel and analyzed by autoradiography.
Free probes are not shown. NS, nonspecific.
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Results from EMSAs indicate that inclusion of the four-amino acid
insert in the hinge region decreases the ability of FXR/RXR to bind to
a number of FXREs (Fig. 3). To determine whether transactivation is
dependent upon the FXR isoform, CV-1 cells, derived from the kidney of
an African green monkey, were transiently transfected with various
reporter genes, RXR, and specific FXR isoforms. The cells were then
treated with Me2SO (vehicle), CDCA (physiological FXR
ligand) or GW4064 (synthetic FXR ligand) in the presence or absence of
LG100153 (RXR ligand). The hSHP promoter (Fig.
4A) and mouse BSEP promoter
(Fig. 4B) were transactivated to similar levels by each of
the four FXR isoforms. In contrast, the mI-BABP promoter-reporter gene
was differentially induced by the four ligand-activated FXR isoforms
(Fig. 4C). The rank order of activation of the mI-BABP
promoter-reporter gene was FXR
2 > FXR
2
FXR
1 = FXR
1. When the FXRE in the mI-BABP promoter was mutated, the reporter gene was no longer activated by any of the FXR isoforms (Fig.
4D). These data suggest that activation of the mI-BABP
promoter by the four FXR isoforms is mediated through the FXRE in the
promoter.

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Fig. 4.
Four murine FXR isoforms differentially
transactivate reporter constructs. Promoter-reporter plasmids
pGL3-hSHP-Luc (A), pGL3-mBSEP-Luc (B and
F), pIBABP1031-Luc (C and
E), or pIBABP142mut-Luc (D) were
transiently co-transfected into CV-1 cells (A-D) or HepG2
cells (E and F) together with the indicated FXR
isoform or no receptor (NR). Cells were then treated with
Me2SO (DMSO, vehicle), CDCA (100 µM) or GW4064 (1 µM) in the presence or
absence of LG100153 (LG; 100 nM) for 42 h.
Luciferase activities were assayed and normalized to -galactosidase
activity. The data (mean ± S.E.) are derived from three
experiments, each performed in triplicate. Numbers on the
top of the bars refer to the fold increase of
luciferase when the activity was compared with that obtained from
Me2SO-treated cells that were not transfected with nuclear
receptors. RLU, relative light units.
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To ensure that the results obtained with CV-1 cells were not cell- or
species-specific, we also transiently transfected HepG2 cells, a human
hepatoma cell line that has been used extensively in the study of FXR
target genes, with the mI-BABP and mBSEP promoter-reporter genes. As
shown in Fig. 4E, the mI-BABP reporter gene was potently activated in the presence of FXR
2 and FXR
2 and ligands for FXR and RXR. The same reporter gene was refractory to activation by FXR
1
and FXR
1, the isoforms containing the additional four-amino acids in
the hinge region (Fig. 4E). In contrast, the mBSEP reporter gene was activated to similar levels by all four FXR isoforms, demonstrating that transactivation is target gene-specific. Thus, the
transfection experiments, which utilize either CV-1 or HepG2 cells and
mI-BABP and mBSEP promoter-reporter genes, give essentially identical results.
Transcriptional Activation of Specific FXR Target Genes Is Affected
by the Ratio of FXR Isoforms--
The results illustrated above
indicate that activation of certain murine genes such as I-BABP might
depend on the relative nuclear ratio of FXR isoforms that do or do not
contain the four-amino acid insert in the hinge region. To test this
hypothesis, we transiently transfected HepG2 cells with the mI-BABP
reporter gene and different ratios of FXR
1 (which contains the
four-amino acid insert) and FXR
2 (which lacks the four amino acids).
The data of Fig. 5 show that relatively
high levels of FXR
1 (25 ng of plasmid) induce the reporter gene less
than 3-fold in the presence of ligands for FXR and RXR. In contrast,
the reporter gene was potently activated (28-fold) in the presence of
low levels of FXR
2 (5 ng of plasmid) (Fig. 5). Most importantly,
this high level of activation in response to ligand-activated FXR
2
was greatly attenuated in cells after co-transfection of FXR
1 (Fig.
5). The data demonstrate that the fold activation of the mI-BABP
reporter gene decreased from 28-fold (in the presence of FXR
2 and no
FXR
1) to 15-, 11-, and 8-fold as the ratio of FXR
1:FXR
2 was
increased from 1:1 to 2:1 to 5:1 (Fig. 5).

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Fig. 5.
The relative ratio of
FXR 1:FXR 2 affects the
transcriptional activation of the murine I-BABP reporter gene.
HepG2 cells were transiently transfected in triplicate with the mI-BABP
reporter gene and plasmids encoding RXR, FXR 1, FXR 2, and
-galactosidase. The amount (ng) of each plasmid used in the
transfection is shown. Cells were treated with vehicle or the indicated
ligand(s) for 24 h, and the results (mean ± S.E.; two
experiments) were determined as described in the legend to Fig. 4.
RLU, relative light units.
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Mouse I-BABP Gene Is Differentially Induced by FXR
Isoforms--
Based on the transient transfection experiments, we
hypothesized that the mI-BABP gene may be differentially regulated by the four FXR isoforms in vivo. To test this hypothesis, we
utilized NIH3T3 cells, a murine fibroblast that lacks endogenous FXR
(Fig. 6A). NIH3T3 cells were
infected with a retrovirus that expressed one specific murine FXR
isoform, and stable cell lines were then selected by growth in media
containing G418. Results from Northern blot assays indicate that the
stable NIH3T3 cell lines express murine FXR
1, FXR
2, FXR
1, and
FXR
2 at similar high levels (Fig. 6A). No FXR was
detected in NIH3T3 cells infected with the empty retroviral vector
(Fig. 6A). These 5 different cell lines were each treated
for 24 h with vehicle or ligands for FXR (GW4064) or FXR and RXR
(LG100153) before quantitation of specific mRNAs. Fig.
6B shows that mouse BSEP mRNA levels were induced to
similar levels by each of the four FXR isoforms. In contrast, the
endogenous mI-BABP mRNA levels were induced from 2.6- to 141-fold
in an FXR isoform-specific manner. The rank order of potency is the
same as that observed in transient transfection experiments
(FXR
2 > FXR
2
FXR
1 = FXR
2) (Fig.
6C). Taken together, these results strongly suggest these
four FXR isoforms function differentially in vivo.

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Fig. 6.
Mouse I-BABP mRNA levels are
differentially induced in NIH3T3 cells stably expressing individual FXR
isoforms. A, NIH3T3 cells were infected with
retroviruses expressing neomycin alone or neomycin and either FXR 1,
FXR 2, FXR 1, or FXR 2. After 3-4 weeks selection with G418 (800 ng/ml), total RNA was isolated from the cells, and Northern blot
analysis was performed. B, stably infected NIH3T3 cells were
treated with Me2SO (DMSO), GW4064 (1 µM), or GW4064 plus LG100153 (LG, 100 nM) for 24 h. Total RNA was isolated, and real-time
quantitative PCR was performed as described under "Experimental
Procedures" to test the relative expression of mBSEP (B)
or mI-BABP (C) in response to ligands. Values were
normalized to cyclophilin.
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DISCUSSION |
Herein, we report the cloning and functional properties of four
murine FXR isoforms, termed FXR
1, FXR
2, FXR
1, and FXR
2. Before this report, two murine FXR isoforms corresponding to FXR
1 and FXR
2 had been identified but not characterized (18). The FXR
isoforms are derived from the use of an internal promoter that
generates transcripts which are 187 bp shorter than the FXR
transcripts but encode proteins that contain an additional 37 amino
acids at the amino terminus. However, our data suggest that it is the
presence or absence of the four-amino acid residues in the hinge region
(Fig. 1) that plays a critical role in modulating the function of FXR.
Similarly, a recent report, published after the completion of the
current studies, identified four hamster as well as four human FXR
isoforms (38). The fact that these isoforms appear to be conserved
across a number of species provides additional significance to these
collective findings.
A limited number of studies have investigated the importance of the
hinge region (D domain) of other nuclear receptors. For example,
mutagenesis of the D domain reduces the transcriptional activation
properties of the glucocorticoid receptor (34) but has no effect on the
function of the estrogen receptor (35, 36). The availability of FXR
isoforms with natural variations in the amino acid sequence in the
hinge region provided a unique opportunity that allowed us to
investigate the importance of these changes on receptor function. EMSAs
demonstrated that the isoforms containing the additional four amino
acids (FXR
1, FXR
1) bind to several FXREs with a lower affinity
than FXR
2 and FXR
2. Because the additional four amino acids are
separated from the DNA binding domain by only four amino acids, it is
possible that the extra amino acids result in minor alterations in the
structure of the receptor that affect either DNA binding and/or the
ability of FXR to dimerize with RXR on the FXRE and/or the interaction
of FXR with co-repressors or co-activators.
Studies that involved FXR-dependent activation of various
promoter-luciferase reporter genes and endogenous genes demonstrated that induction of mI-BABP by FXR ligands is particularly sensitive to
the FXR isoforms, with the rank order being FXR
2 > FXR
2
FXR
1 = FXR
1 (Figs. 4 and 6). I-BABP is thought to be
primarily expressed in intestinal cells. The finding that murine
duodenum, jejunum, and ileal cells express at least 3-fold more FXR
than FXR
mRNA (Fig. 2B) and more FXR
2 than FXR
1
(Table II) is consistent with the high expression of I-BABP in this
organ. In contrast to I-BABP, two other genes (BSEP, SHP) are induced
to similar levels by each FXR isoform. In preliminary studies we
replaced the FXRE in the mI-BABP promoter-reporter with the FXRE from
the mSHP gene; induction of this novel reporter gene by GW4064 was much
less sensitive to the FXR isoforms than the original wild type reporter
gene (data not shown). These data suggest that the FXR isoforms
differentially regulate target genes in various tissues and that these
differences are due at least in part to the specific sequences that
comprise the FXREs.
Murine FXR expression has been reported to be limited to the liver,
small intestine, kidney, and adrenal gland (17, 19). In the current
study we utilized real time PCR to demonstrate that FXR is also
expressed in stomach, heart, lung, and fat, albeit at lower levels
(Fig. 2B). The relative expression of the four FXR isoforms
differs significantly in these eight tissues (Fig. 2B; Table
I). Because the FXR isoforms differentially activate specific genes
such as I-BABP, we hypothesize that a change in the relative ratio of
the FXR isoforms will significantly affect gene expression. Studies are
currently under way to determine whether the two promoters that control
the expression of FXR
and FXR
are differentially regulated. A
number of FXR target genes have been identified in liver, intestine,
and kidney, tissues that are known to be involved in bile acid
synthesis and metabolism. The current demonstration that there are
multiple FXR isoforms in lung, adipose tissue, heart, and stomach,
tissues that are not currently known to be involved in bile acid
metabolism, raises the possibility that additional FXR ligands remain
to be identified that function in these tissues. Identification of
these target genes and putative ligands may provide important clues as
to the function of FXR in multiple murine tissues.