Farnesoid X-Activated Receptor Induces Apolipoprotein C-II Transcription: a Molecular Mechanism Linking Plasma Triglyceride Levels to Bile Acids
Heidi Rachelle Kast,
Catherine M. Nguyen,
Christopher J. Sinal,
Stacey A. Jones,
Bryan A. Laffitte,
Karen Reue,
Frank J. Gonzalez,
Timothy M. Willson and
Peter A. Edwards
Departments of Biological Chemistry and Medicine (H.R.K., C.M.N.,
B.A.L., P.A.E.), University of California, Los Angeles, California
90095; Laboratory of Metabolism (C.J.S., F.J.G.,) Division of Basic
Sciences, National Institutes of Health, Bethesda, Maryland 20892;
Molecular Biology Institute (K.R., P.A.E.), University of California,
Los Angeles, California 90095; GlaxoSmithKline (S.A.J., T.M.W.) Nuclear
Receptor Discovery Research, Research Triangle Park, North Carolina
27709
Address all correspondence and requests for reprints to: Peter A. Edwards, Ph.D., Department of Biological Chemistry, University of California, Los Angeles, California 90095. E-mail:
pedwards{at}mednet.ucla.edu
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ABSTRACT
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The farnesoid X-activated receptor (FXR; NR1H4), a member of the
nuclear hormone receptor superfamily, induces gene expression in
response to several bile acids, including chenodeoxycholic acid. Here
we used suppression subtractive hybridization to identify
apolipoprotein C-II (apoC-II) as an FXR target gene. Retroviral
expression of FXR in HepG2 cells results in induction of the mRNA
encoding apoC-II in response to several FXR ligands. EMSAs demonstrate
that recombinant FXR and RXR bind to two FXR response elements
that are contained within two important distal enhancer elements
(hepatic control regions) that lie 11 kb and 22 kb upstream of the
transcription start site of the apoC-II gene. A luciferase reporter
gene containing the hepatic control region or two copies of the
wild-type FXR response element was activated when FXR-containing cells
were treated with FXR ligands. In addition, we report that hepatic
expression of both apoC-II and phospholipid transfer protein mRNAs
increases when mice are fed diets supplemented with cholic acid, an FXR
ligand, and this induction is attenuated in FXR null mice. Finally, we
observed decreased plasma triglyceride levels in mice fed cholic acid-
containing diets. These results identify a mechanism whereby FXR and
its ligands lower plasma triglyceride levels. These findings may have
important implications in the clinical management of
hyperlipidemias.
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INTRODUCTION
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NUCLEAR RECEPTORS COMPRISE a superfamily of
ligand-activated transcription factors that influence development,
differentiation, and physiological homeostasis (1). These
receptors bind to specific cis-acting elements within the
promoters of their target genes and regulate gene expression, usually
in response to the binding of small lipophilic ligands. A number of
nuclear receptors have been shown to undergo a conformational change
upon ligand binding, which promotes the release of corepressor
proteins, the subsequent binding of coactivator proteins, and increased
levels of transcription (2, 3).
The expression of the farnesoid X-activated receptor (FXR; NR1H4), a
member of this superfamily, is restricted to the liver, kidney,
intestine, and adrenal gland (4). The farnesoid
X-activated receptor (FXR) heterodimerizes with RXR and binds to FXR
response elements (FXREs) within the regulatory regions of target genes
(4). The idealized FXRE, comprised of two hexanucleotide
repeats (AGGTCA) arranged as inverted repeats with one nucleotide
spacing between the two half-sites, is referred to as an IR-1 (4, 5).
Forman et al. (4) originally cloned rat
FXR and reported that farnesol or juvenile hormone III weakly activated
the FXR/RXR heterodimer. Independently, Seol et al.
(6) cloned RIP-14, the murine homolog of rat FXR.
Subsequent studies identified a nonphysiological synthetic
retinoid, TTNPB,
4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]
benzoic acid, as a potent activator of both rat and murine FXR
(7). In 1999, three laboratories made the important and
unexpected observation that bile acids function as ligand activators
for both rodent and human FXR (8, 9, 10). Chenodeoxycholate
(CDCA) (8, 9, 10), a primary bile acid that is synthesized in
the liver as a result of oxidation and catabolism of cholesterol, is
the most potent ligand for FXR (11). At the present time,
primary response genes that are known to be activated in an FXR- and
CDCA-dependent manner are limited to the ileal bile acid-binding
protein (8, 12), the phospholipid transfer protein (PLTP)
(5, 13), and the small heterodimer partner (SHP), an
unusual member of the nuclear receptor family which lacks a DNA-binding
domain (14, 15). Recent studies demonstrate that SHP binds
to and inactivates the liver receptor homolog 1, a transcription factor
that is required for active transcription of the CYP7A1 gene (14, 15). Thus, increased expression of SHP, in response to activated
FXR, results in a decrease in CYP7A1 transcription and bile acid
synthesis (14).
In contrast, the physiological effects that result from induction of
ileal bile acid-binding protein and PLTP by FXR remain unknown.
Insights into other possible roles of FXR in lipid metabolism were
recently reported by Maloney et al.; these authors
identified a novel synthetic FXR ligand (GW4064) that, when
administered to rats for 7 d, resulted in an approximate 50%
decrease in plasma triglyceride levels (16). The mechanism
by which GW4064 decreases plasma triglyceride levels is currently
unknown. These findings, together with the recent observation that
plasma triglyceride levels are increased 150% in FXR null mice,
implicate FXR as a key regulator in the control of plasma lipids
(17).
In the present study, suppression subtractive hybridization was used to
identify apolipoprotein C-II (apoC-II) as a target of FXR in
vivo and in vitro. To maximize the number of induced
genes, we used HepG2 cells that stably overexpressed FXR, as a result
of infection with a retrovirus that encodes FXR. Herein, we report that
both apoC-II and PLTP are induced by FXR in isolated cells and in
livers of mice fed diets supplemented with an FXR ligand. Induction of
these genes in vivo correlates with decreased plasma
triglyceride levels. Consequently, activation of FXR/RXR may provide an
alternative approach in the clinical management of certain
hyperlipidemias.
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RESULTS
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ApoC-II Expression Is Regulated by FXR
To generate a cell line that expresses increased levels of
FXR, full-length rat FXR was cloned upstream of the internal ribosome
entry site (IRES) in the retroviral vector MSCV-IRES-Neo. HepG2 cells
were infected with either MSCV-FXR-IRES-Neo or the empty vector
(MSCV-IRES-Neo) and G418-resistant cells were isolated. In this manner,
pooled cell populations that overexpressed either FXR (HepG2-FXR)
or the vector alone (HepG2-vector) were isolated. We have previously
shown that the retrovirally derived FXR protein is expressed in
HepG2-FXR cells and is functionally active (5). HepG2-FXR
cells were treated for 24 h with CDCA (50 µM), while
the HepG2-vector cells were treated with the vehicle
[dimethylsulfoxide (DMSO)] before isolation of total RNA. Genes
that were induced in the CDCA-treated HepG2-FXR cells were identified
using suppression subtractive hybridization (SSH). The corresponding
cDNAs were isolated, radiolabeled, and used to probe membranes
containing RNA isolated from HepG2-FXR and HepG2-vector cells that had
been treated with CDCA or vehicle. One highly induced mRNA that we
identified using SSH encodes apoC-II (Fig. 1
).

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Figure 1. Induction of apoC-II mRNA by FXR and FXR Ligands
A, ApoC-II mRNA levels are induced by CDCA and by overexpression of
FXR. HepG2-vector (lanes 1 and 2) or HepG2-FXR (lanes 3 and 4) cells
were treated for 24 h with DMSO (lanes 1 and 3) or 100
µM CDCA (lanes 2 and 4). Total RNA (10 µg/lane) was
separated on a 1% agarose/formaldehyde gel, transferred to nylon
membrane, and hybridized to radiolabeled cDNA probes. The fold
regulation shown was determined after normalization to a control probe.
B, Induction of apoC-II mRNA by CDCA is maximal at 48 h. Cells
were incubated in the presence of DMSO or 100 µM CDCA.
RNA isolation and Northern analysis were as described above. C, ApoC-II
mRNA is induced by CDCA and TTNPB. HepG2-FXR cells were incubated for
24 h in the presence of the indicated amount of CDCA or the
synthetic retinoid TTNPB. RNA was isolated and used in the Northern
analysis as described above. D, ApoC-II mRNA levels are induced by
ligands for both FXR and RXR. HepG2-vector or HepG2-FXR cells were
incubated for 24 h with either DMSO (-), 100 µM
CDCA, 100 nM LG100153, or both CDCA and LG100153, as
indicated. Northern analysis and fold induction were as described
above. E, Induction of apoC-II mRNA is dependant on specific bile acids
or androsterone. HepG2-FXR cells were incubated for 24 h with the
indicated bile acid or androsterone. Northern analysis was performed as
described above in Fig. 1A . The results shown in panels AE are
representative of two to three experiments.
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As demonstrated in Fig. 1A
, treatment of HepG2-vector cells with 100
µM CDCA results in a 5-fold increase in apoC-II mRNA
(compare lanes 1 and 2), presumably as a result of very low levels of
endogenous FXR in HepG2 cells. In contrast, the HepG2-FXR cells
expressed detectable levels of FXR mRNA (as the FXR-IRES-Neo
transcript) (Fig. 1B
) and elevated basal levels of apoC-II mRNA (Fig. 1A
, lane 3 vs. 1). Addition of 100
µM CDCA to the HepG2-FXR cells resulted in a
further increase in apoC-II mRNA to levels that are 15-fold greater
than in the DMSO-treated HepG2-vector cells (Fig. 1A
, lanes 4
vs. 1).
Incubation of HepG2-FXR cells with 100 µM CDCA resulted
in induction of apoC-II mRNA as early as 12 h, and maximal levels
were attained after 48 h (Fig. 1B
). Induction of apoC-II mRNA was
maximal at 100 µM CDCA (Fig. 1C
). Although higher levels
of CDCA resulted in a decline in apoC-II mRNA levels, this effect
appears not to be due to toxicity as we have identified other mRNAs
from the SSH screen, such as the canalicular multispecific organic
anion transporter, that are maximally induced at 250 µM
CDCA (data not shown). Addition of TTNPB (5 µM) to
HepG2-FXR cells also resulted in increased apoC-II mRNA levels (Fig. 1C
), consistent with the observation that this synthetic retinoid can
activate FXR (7).
FXR binds to FXREs as an FXR/RXR heterodimer (4, 5),
and RXR functions as a permissive heterodimer, as evidenced by
transactivation of reporter genes by ligands for either RXR or FXR
(18). Consistent with this model, apoC-II mRNA levels
increased when HepG2-FXR cells were treated with either a synthetic
RXR-specific ligand (LG100153) or the FXR ligand, CDCA (Fig.
1D). Addition of both LG100153 and CDCA resulted in an additive
induction of apoC-II mRNA (Fig. 1D
). To determine bile acid
specificity, HepG2-FXR cells were incubated for 24 h in the
presence of various bile acids. The rank order of potency of bile acids
that function to induce apoC-II mRNA (Fig. 1E
; CDCA > deoxycholic
acid (DCA) > lithocholic acid (LCA) >cholic acid (CA)]
matches their activity as FXR agonists (9). ApoC-II mRNA
levels also increased in response to androsterone (Fig. 1E) and
GW4064 (data not shown), consistent with reports that these compounds
activate FXR (16, 19). Preliminary results indicate that
the induction of apoC-II mRNA in response to CDCA is unaffected by
cycloheximide treatment (data not shown), suggesting that CDCA
activates transcription of the apoC-II gene by a process that does not
require continued protein synthesis. Taken together, these observations
demonstrate that apoC-II mRNA levels are induced in a human
hepatoma-derived cell by a mechanism that requires FXR and ligands for
either FXR and/or RXR.
Identification of an FXRE Within the Hepatic Control Region
(HCR)
The human hepatic control regions, HCR.1 and HCR.2, were
originally identified in a series of elegant studies
(20, 21, 22, 23). Using transgenic mice that contained 45 kb of
human genomic DNA, Allan et al. (24)
demonstrated that both HCR.1 and HCR.2 were critical for the hepatic
expression of the apoE/C-I/C-IV/C-II gene cluster. Vorgia et
al. (25) then used promoter reporter genes
under the control of the apoC-II proximal promoter and the HCR to
demonstrate that the HCRs contained elements necessary for maximal
hepatic expression of the apoC-II gene. HCR.1 and HCR.2 have 85%
sequence identity and are located approximately 22 kb and 11 kb
upstream of the apoC-II transcriptional start site, respectively (Fig. 2A
) (24).

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Figure 2. FXR/RXR Heterodimers Bind to the Hepatic Control
Region of the Human apoE/C-I/C-IV/C-II Gene Cluster
A, A schematic of the apoE/C-I/C-IV/C-II gene cluster with the
potential FXREs (IR-1A, -IB, -IC) are indicated. The nucleotide
sequences corresponding to the various FXR/RXR binding sites discussed
in the text are shown. Nucleotides are numbered relative to the
transcription start site (20 ). Nucleotides that differ
from the consensus IR-1 are underlined. Nucleotides in
the mutant FXRE (mutIR-1A) that differ from the wild-type IR-1A are
italicized and shown in lowercase. B,
FXR/RXR binds to the hepatic control region IR-1A. Partially purified
FXR and RXR were incubated with the indicated radiolabeled probes of
similar specific activity before gel electrophoresis, as described in
Materials and Methods. The shifted DNA-protein complexes
were detected by autoradiography. The free probe is not shown. No other
complexes were observed. C, Mutation of IR-1A prevents formation of an
FXR-RXR-DNA complex. FXR and/or RXR were incubated with
32P-labeled IR-1A, mutant IR-1A, or the idealized IR-1
before EMSAs were performed as described in Materials and
Methods. The shifted complex is indicated. The specific
activities of the probes (not shown) were similar. D, Competition
assays demonstrate that FXR/RXR binds to IR-1A and EcRE with similar
affinity. Recombinant FXR and RXR were incubated with
32P-labeled IR-1A in the absence or presence of 100, 500,
or 1,000 molar excess of the indicated unlabeled competitor. The
fluorogram representing the shifted protein-DNA complex is shown. The
free probe has been omitted.
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We scanned the apoC-II proximal promoter and the two hepatic control
regions for sequences that correspond to previously identified FXREs
(5). Three potential FXREs (referred to as IR-1A, IR-1B,
and IR-1C) were identified. The nucleotide sequences and relative
positions in the HCRs and proximal apoC-II promoter are shown (Fig. 2A
). We then used recombinant human (h) RXR
and FXR proteins in
EMSAs to determine whether the FXR/RXR heterodimer can bind to
these potential FXREs (Fig. 2
). Recombinant FXR/RXR bound to a
radiolabeled probe containing IR-1A but not to probes containing
sequences corresponding to IR-1B or IR-1C (Fig. 2B
). Probes containing
an Ecdysone response element (EcRE) or idealized IR-1, to which
FXR/RXR is known to bind (4, 5), served as positive
controls. No shifted DNA-protein complex was observed when either FXR
or RXR was omitted from the EMSAs or when three mutations were
introduced into the core sequence of IR-1A (mutIR-1A) (Fig. 2C).
These data are consistent with a requirement for the presence of both
FXR and RXR to form a stable complex with the FXRE identified in the
HCR.1 and HCR.2. Competition assays were also performed to compare the
relative affinity of FXR/RXR for the IR-1A and the previously
characterized EcRE (4, 5); a
32P-labeled probe containing the IR-1A sequence
was incubated in the presence of increasing amounts of unlabeled IR-1A,
mutIR-1A, or EcRE. The results demonstrate that the unlabeled wild-type
IR-1A and EcRE were equally effective competitors (Fig. 2D
). In
contrast, the formation of the shifted complex containing radiolabeled
IR-1A probe was unaffected by the presence of the unlabeled DNA
containing the mutated IR-1A (Fig. 2D
).
FXR Transactivates the Hepatic Control Region FXRE (IR-1A) in HepG2
Cells
HCR.1 and HCR.2 have previously been identified as important
enhancers that control hepatic expression of apoE, C-I, C-IV and C-II
(20, 25, 26). The data of Fig. 2
suggest that the FXRE in
HCR.1 and HCR.2 may be important for regulated transcription of apoC-II
in response to FXR and its ligands. To test this hypothesis,
we constructed luciferase reporter genes under the control of either
the apoC-II proximal promoter, the HCR.1, or two copies of either the
wild-type or mutant IR-1A (Fig. 3
). Each
reporter construct was transiently transfected into HepG2 cells in the
presence or absence of plasmids encoding RXR and FXR or VP16-FXR, and
the cells were treated with the indicated ligands. VP16-FXR is expected
to be constitutively active as a result of the strong activation domain
of VP16. The data of Fig. 3A
show that VP16-FXR induces the HCR.1
reporter construct even in the absence of ligand. In contrast, no
induction was observed when VP16-FXR was coexpressed with the C-II
proximal promoter reporter gene (Fig. 3A
).

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Figure 3. FXR and FXR-Ligands Activate Reporter Genes Under
the Control of the IR-1A
A, VP16-FXR and RXR activate the HCR.1. HepG2 cells were transiently
transfected with ß-galactosidase and the TK-reporter gene under the
control of the apoC-II proximal promoter (C-II prox) or the HCR.1 and,
where indicated, VP16-FXR and RXR . Cells were treated for 24 h
with DMSO or CDCA (100 µM) and LG100153 (100
nM). Fold induction of the reporter gene is shown after
normalization. B, VP16-FXR and RXR activate the IR-1A from the HCR.
HepG2 cells were transiently transfected as described above with the
TK-luciferase plasmid (vector), the 2xIR-1A reporter gene, or the
mutant 2xIR-1A (2x mutIR-1A) and, as indicated, with VP16-FXR and
RXR plasmids for 24 h. C, FXR and RXR activate the
IR-1A. HepG2 cells were transiently transfected as above with
TK-luciferase, 2xIR-1A-luciferase, or mutant 2xIR-1A-luciferase and,
where indicated, plasmids encoding FXR and RXR . Cells were incubated
with DMSO, 100 µM CDCA, 100 nM LG100153, or
10 µM GW4064, as indicated. Luciferase activities were
normalized for transfection efficiency, and the results are shown as
fold regulation relative to the DMSO-treated vector. All transfections
were done in triplicate, and the averages are given. In each assay the
range is less than 10% and the data are representative of two to three
experiments.
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Figure 3B
shows that a reporter gene controlled by 2xIR-1A is activated
>90 fold by VP16-FXR and RXR. In contrast, no induction of the
reporter gene was observed when three mutations were incorporated into
the IR-1A sequence (Fig. 3B
, 2x
MutIR-1a). The data of Fig. 3C
confirm
and extend these later studies and demonstrate that the
2xIR-1A-luciferase reporter is activated 10- to 27-fold by ligands for
FXR and/or RXR. This induction is dependent on coexpression of FXR and
is abolished when three mutations are introduced into the IR-1A
sequence (Fig. 3C
). The thymidine kinase (TK)-luciferase empty vector
was not induced under these conditions (Fig. 3C
). These data, taken
together with those of Fig. 2
, demonstrate that the FXRE (IR-1A),
identified in both the HCR.1 and HCR.2 enhancer elements (Fig. 2A
), is
functional when placed upstream of a heterologous promoter. These data
suggest that these two FXREs, contained within the two 319-bp enhancers
that are necessary for hepatic expression of apoC-II mRNA, are involved
in the transcriptional activation of the apoC-II gene in response to
FXR and an FXR-activating ligand.
Hepatic ApoC-II and PLTP mRNAs Are Induced in Vivo
by Bile Acids by a Process That Requires FXR
The results described above demonstrate that FXR ligands activate
transcription of the apoC-II gene in cultured HepG2 cells. We next used
wild-type or FXR null mice to determine whether cholic acid, an FXR
ligand (8, 10), could induce these genes
in vivo. The data of Fig. 4A
demonstrate that apoC-II and PLTP mRNAs are induced in wild-type mice
upon feeding a 1% cholic acid diet for 5 d, and that the
induction of both mRNAs is attenuated in FXR null mice. Figure 4B
shows
the relative apoC-II and PLTP mRNA levels that have been corrected for
ß-actin levels. These results provide evidence that FXR and its
ligands directly regulate the expression of genes involved in
lipoprotein metabolism in vivo.

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Figure 4. Northern Blot Analysis of ApoC-II and PLTP mRNAs
from Wild-type and FXR Null Mice
A, Wild-type (+/+) and FXR null mice (-/-) were fed the control
(Chow) or cholic acid (CA)-containing diets for 5 d. Total RNA (10
µg/lane) was used in a Northern analysis as described previously
(17 ) using the indicated radiolabeled probes. A Northern
blot is shown that is representative of five separate experiments of
similarly treated mice. B, Relative apoC-II and PLTP mRNA levels were
determined for mice treated as described in panel A. Values (±
SD) are the average obtained for each group of mice (n
= 5).
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Murine Plasma Triglyceride Levels Decrease in Response to Cholic
Acid
To further investigate the long-term physiological effects
resulting from the administration of cholic acid to rodents, we fed
C57BL/6J mice for 3 wk either a control, an atherogenic (standard chow
supplemented with fat, cholesterol and cholic acid) (27),
or a Western (standard chow supplemented with fat and cholesterol) diet
(Fig. 5
). The latter two diets have been
used in numerous studies to induce atherosclerosis in mice. Hepatic
apoC-II and PLTP mRNAs were detectable in mice fed the control diet,
and both apoC-II and PLTP mRNAs were induced significantly when the
diet was supplemented with cholic acid (Fig. 5A
, lane 2 vs.
1). In contrast, apoC-II and PLTP mRNAs were not induced when the mice
were fed a diet containing fat and cholesterol in the absence of cholic
acid (Fig. 5A
, lane 3 vs. 1). We previously observed that
PLTP mRNA levels were induced when cultured liver cells were exposed to
ligands for FXR (5). The data of Fig. 5A
extend these
earlier studies and demonstrate that PLTP mRNAs are induced in
vivo in response to a cholic acid-containing diet.

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Figure 5. Cholic Acid-Containing Diets Induce Hepatic ApoC-II
and PLTP mRNAs and Affect Plasma Triglyceride Levels
A, C57BL/6J mice (five per group) were fed for 3 wk a control chow diet
(lane1), an atherogenic diet (cholesterol, cholate, and fat, lane 2),
or a Western diet (cholesterol and fat, lane 3). Hepatic polyA RNA was
isolated and pooled and 2 µg/lane were used in a Northern analysis as
described in Fig. 1 using the indicated probes. Normalization and
quantitation (shown as fold induction) were as described in Fig. 1 . B,
Plasma triglyceride levels are decreased by the cholic acid-containing
diet. Plasma lipids were quantitated for each mouse (n = 5).
Values are given as the mean ± SD (n = 5).
Significant differences compared with mice fed the chow diet: **,
P < 0.05; **, P < 0.005; ***,
P < 0.0005.
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Since both apoC-II and PLTP are known to be involved in plasma
lipoprotein metabolism (28, 29), we next determined
whether administration of cholic acid to mice affected plasma
triglyceride and cholesterol levels. Figure 5B
shows that plasma
triglyceride levels (solid bars) decline
50% when
mice are fed a diet enriched in cholic acid, cholesterol, and fat,
whereas plasma cholesterol levels increase > 2-fold (compare
panel A vs. B). In contrast, both triglyceride and
cholesterol levels increased 146% and 57%, respectively, when mice
were fed a diet enriched in fat and cholesterol, but no cholic acid
(Fig. 5B
, panel C vs. A). Thus, inclusion of cholic acid to
the high-fat and cholesterol diet results in an approximate 4-fold
decrease in plasma triglyceride levels, but only a minor change in
plasma cholesterol levels (Fig. 5B
, panel B vs. C).
Preliminary results indicate that this effect is independent of dietary
fat; when mice were fed diets supplemented with cholic acid and
cholesterol, in the absence of fat, we observed a 64% decrease in
plasma triglyceride levels and a 110% increase in plasma cholesterol
levels as compared with mice fed a normal chow diet (data not
shown).
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DISCUSSION
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In addition to solubilizing cholesterol and facilitating the
uptake of dietary lipids, bile acids have recently been shown to act as
signaling molecules that bind and activate the nuclear receptor FXR
(8, 9, 10). To elucidate the spectrum of genes regulated by
bile acid-activated FXR, we used a retroviral expression system to
overexpress FXR in HepG2 cells. The HepG2-FXR cells were then used in a
screen to identify target genes that are activated by FXR and FXR
ligands. The findings that apoC-II mRNA levels increase in both cells
and animals in an FXR-dependent and FXR ligand (bile acids, TTNPB,
GW4064, or androsterone) and/or RXR ligand (LG100153)-dependent manner
suggest that the apoC-II gene may be a direct target gene.
Based upon gel shift and competition studies, a potential FXRE (termed
IR-1A) was identified in the HCR of the apoE/C-I/C-IV/C-II gene
cluster. The affinity of FXR/RXR for IR-1A is similar to that of a well
characterized FXRE termed the EcRE (Fig. 2
) (4, 5). There
are two hepatic control regions, HCR.1 and HCR.2, within the human gene
cluster that mediate the liver-specific expression of the
apoE/C-I/C-IV/C-II locus (24, 25). HCR.1 and HCR.2 are
each approximately 350 bp and have >85% nucleotide identity
(24). Dang et al. (30) used
in vivo footprinting to identify seven liver-specific
protein binding sites within HCR.1. The sequence of the FXRE in HCR.1
and HCR.2 is 100% conserved and is contained within a 52-bp footprint
identified by Dang et al. (30).
Based on the presence of identical potential FXREs in both HCR.1
and HCR.2 (Fig. 2A
), we constructed reporter genes containing two
copies of either the wild-type or mutant IR-1A. Figure 3
demonstrates
that multiple distinct FXR ligands and/or an RXR ligand all activate
the wild-type, but not the mutant promoter-reporter gene. The
observation that reporter genes containing the proximal apoC-II
promoter were not induced by FXR ligands (Fig. 3A
), coupled with the
observation that potential FXREs in the apoC-II proximal promoter did
not form a complex in vitro with purified FXR and RXR (Fig. 2
), lead us to conclude that bile acid activation of the apoC-II gene
is likely to be mediated by the FXREs present in HCR.1 and HCR.2. Since
HCR.1 and HCR.2 are reported to modulate hepatic expression of multiple
genes within the apoE/C-I/C-IV/C-II gene cluster (20, 25, 26), it is possible that other apolipoproteins are also
regulated by FXR and FXR ligands. Consistent with this proposal,
preliminary studies indicate that 1) apoE and apoA-IV mRNAs are induced
after the addition of FXR ligands to FXR expressing cells
(Kast, H., S. Jones, and P. Edwards,
unpublished data); and 2) plasma apoE protein levels are
increased in GW4064-treated rats (Winegar, D., and S. Jones,
unpublished data). Additional studies will be required to determine
whether the changes in expression of these and other apolipoproteins
result from a direct effect of FXR.
ApoC-II is an obligate cofactor for lipoprotein lipase, which in turn
is responsible for the hydrolysis of triglycerides in
chylomicrons and very low density lipoprotein. Individuals with a
homozygous deficiency of apoC-II characteristically have massive
hypertriglyceridemia (31). Interestingly, patients who
have undetectable levels of apoC-II and hyperchylomicronemia, as a
result of an A-to-G substitution at -86 of the proximal apoC-II
promoter have been described (32). Although this
substitution lies within the IR-1C element (Fig. 2A
), the current
studies predict that the decrease in transcription of the apoC-II gene
in these patients is independent of FXR/RXR.
Based on a previous report (5) and the results shown
in
Figs. 13

, we reasoned that treatment of mice with an FXR ligand
(such as cholate) should result in elevated hepatic apoC-II and
PLTP mRNA levels, and that this treatment might alter lipoprotein
levels. There is a significant induction of both apoC-II and PLTP
mRNAs, and a concomitant >50% decrease in triglyceride levels when
mice were fed a cholic acid-containing diet (Fig. 5B
). These data are
consistent with two recent reports: Maloney et al.
(16) demonstrated that plasma triglyceride levels
decreased 50% when rats were treated for 7 d with the FXR
synthetic ligand GW4064, and Sinal et al. (17)
demonstrated that triglyceride levels increased in FXR null mice. The
observation that PLTP mRNA levels increased when mice are fed cholic
acid containing diets is consistent with two recent reports that showed
that the PLTP promoter contained an FXRE (5, 13), and that
induction of a reporter gene was dependent on the intact FXRE, and
ligand-activated FXR/RXR (13).
The current data demonstrate that activators of FXR result in
decreased triglyceride levels and provide a mechanism to explain
studies described more than 25 yr ago in which CDCA was administered to
patients with gallstones (33). This treatment solubilized
cholesterol-rich gallstones and also lowered plasma triglycerides
(33). The current studies suggest that the administration
of FXR ligands results in the coordinate induction of genes that
regulate lipoprotein metabolism and lower plasma triglyceride
levels. Such genes include, but are probably not limited to,
apoC-II, PLTP, apoA-IV, and apoE. Based on the observations that FXR
ligands both activate genes involved in lipoprotein metabolism and
decrease plasma triglyceride levels and the finding that FXR null mice
exhibit a "proatherogenic lipoprotein profile"
(17), we hypothesize that FXR agonists may prove useful as
a novel treatment for metabolic mixed dyslipidemias (low HDL, high LDL
and triglycerides).
 |
MATERIALS AND METHODS
|
---|
Materials
The apoC-II-HCR.1 was a kind gift from Dimitris Kardassis
(University of Crete Medical School, Crete, Greece). The
FXR-specific agonist GW4064 was a gift from Patrick Maloney
(GlaxoSmithKline, Research Triangle Park, NC) (16).
LG100153 was kindly provided by Dr. R. Heyman (Ligand Pharmaceuticals, Inc., San Diego, CA) (34). The
retroviral vector MSCV-IRES-neo plasmid was a gift from Dr. Owen Witte
(University of California, Los Angeles, CA). Mammalian expression
vectors for FXR (pCMX-FXR), hRXR
, and VP16-FXR were gifts from Dr.
Ron Evans (Salk Institute, La Jolla, CA). The sources of other reagents
and plasmids have been noted elsewhere (5).
Cell Culture and Stable Cell Lines
The isolation and maintenance of wild-type and stably infected
HepG2s cells have been described (5).
SSH
Total RNA was isolated from HepG2-vector cells treated with DMSO
(driver) and from HepG2-FXR cells treated with 50 µM CDCA
(tester) for 24 h. The driver and tester RNA were used in SSH
using the PCR-Select Subtraction Kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturers
instructions (35).
RNA Isolation and Northern Blot Hybridization
Unless otherwise indicated, HepG2-derived cell lines were
cultured in medium containing super-stripped FBS for 24 h before
the addition of ligand or DMSO (vehicle) for an additional 24 h.
Total RNA was isolated using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) and was resolved (10 µg/per lane) 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 Pharmacia Biotech,
Arlington Heights, IL). Membranes were hybridized using the QuikHyb
Hybridization Solution (Stratagene) according to the
manufacturers protocol. Blots were normalized for variations of RNA
loading by hybridization to a control probe, either
glyceraldehyde-3-phosphate dehydrogenase, rat 18S ribosomal
cDNA, or the ribosomal protein 36B4. The RNA levels were
quantitated using a PhosphorImager (ImageQuant software,
Molecular Dynamics, Inc., Sunnyvale, CA).
EMSAs
Partially purified FXR
110 and hRXR
(5)
were incubated with the binding buffer [10 mM HEPES, pH
7.9, 0.5 mM dithiothreitol, 2.5 mM
MgCl2, 0.05% (vol/vol) glycerol, 50 mg/ml nonfat
milk, and 50 mM NaCl] at room temperature for 15 min.
Labeled probe (30,000 cpm, 1.5 fmol) was added and allowed to complex
at room temperature for 30 min. Complexes were resolved by 4%
nondenaturing PAGE at 4 C for 2.5 h. The gel was dried and
analyzed by autoradiography and PhosphorImaging. The oligos used were
IR-1A 5'-gctggggcagaggtcagagacctctctggg-3',
IR-1B 5'-gaccttgggggacgtcattgccctttctgtcccc-3',
IR-1C
5'-gttctgttggccaggactttggcctagacaaaggatgggg-3'
and mutant IR-1A (mutIR-1A)
5'-gctggggcagagtgcagagatctctctggg-3' (only one
strand is shown). The wild-type or mutant IR-1 is
boldface and underlined.
Reporter Genes
The proximal promoter of apoC-II (-47 to -540 bp)
(25) was amplified from human genomic DNA using primers
5'-gggggatccggatccttccccagtgtggc-3' and
5'-ggagatctgctccacagccacaaccccat-3' and cloned into
BamHI/BglII sites of the TK-Luc vector. The HCR.1
was amplified from an HCR.1-apoCII proximal promoter clone using
primers 5'-ggggatccggcacacaggagtttctgggctca-3' and
5'-aaagatcttctcacactacctaaaccacgccaggaca-3'. The PCR product was
subsequently ligated into the TK-Luc vector at the
BamHI/BglII location. The two copy IR-1A was
generated by annealing the oligonucleotides
5'-gatcgctggggcagaggtcagagacctctctgggcccatgccaaggtcagagacctctct-3'
and
5'-gatcagagaggtctctgaccttggcatgggcccagagaggtctctgacctctgccccagc-3'
before ligation into BamHI/BglII digested TK-Luc.
The two FXR/RXR binding sites are bolded. The two copy
mutIR-1A was created in a similar manner using
5'-gatcgctggggcagagtgcagagatctctctgggcccatgccaagtgcagagatctctct-3'
and
5'-gatcagagagatctctgcacttggcatgggcccagagagatctctgcactctgccccagc-3'.
Mutations are italic boldface and
underlined.
Transient Transfections and Reporter Gene Assays
HepG2 cells were transiently transfected using the MBS Mammalian
Transfection Kit (Stratagene, La Jolla, CA), with minor
modifications. Reporter plasmid (100 ng), 50 ng pCMX-FXR, 5 ng
pCMX-RXR
, and 50 ng pCMV-ß-galactosidase were transfected into
HepG2 cells in a 48-well dish. After 3.5 h the cells were treated
with 10% superstripped FBS and one of the following ligands: TTNPB
(BIOMOL Research Laboratories, Inc., Plymouth Meeting,
PA), 3
,7
-dihydroxy-5ß-cholanic acid (CDCA)
(Sigma, St. Louis, MO), LG100153 (synthetic RXR-agonist)
(Ligand Pharmaceuticals, Inc.), or
3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole
(GW4064) (GlaxoSmithKline, Research Triangle Park, NC). After
24 h, the cells were lysed and assayed for luciferase and
ß-galactosidase activity (5).
FXR Null Mice and Diets
The FXR null mice were generated as previously described
(17). The mice used in Fig. 4
were maintained on a
standard AIN-93G diet supplemented, where indicated, with 1% cholic
acid for 5 d. RNA was isolated and analyzed as described
previously (17).
Mice and Diets
The data of Fig. 5
were generated using C57BL/6J mice
(The Jackson Laboratory; Bar Harbor, ME). Mice were fed
Purina Mouse Chow 5001 (Ralston Purina Co., Battle Creek,
MI) ad libitum. At 3 months of age, mice (five per group)
were housed individually and fed for 3 wk either control chow (Teklad
Research Diets, Madison, WI), an atherogenic diet (21) (TD
90221), which contained 75% Purina Mouse Chow, 7.5% cocoa butter,
1.25% cholesterol, and 0.5% sodium cholate, or a Western diet (TD
94059, 7.5% cocoa butter and 1.25% cholesterol). The mice were fasted
overnight before harvesting blood for lipid determinations and tissues
for RNA isolation. Plasma total cholesterol and triglyceride
concentrations were determined by enzymatic assays (36).
The care of the mice, as well as all procedures used in this study,
were conducted in accordance with the NIH animal care guidelines.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. D. Kardassis, R. Heyman, O. Witte, P. Maloney, and
R. Evans for providing plasmids and reagents. We thank Dr. Tontonoz and
all the members of the Edwards and Tontonoz laboratories for useful
discussions.
 |
FOOTNOTES
|
---|
This work was supported by grants from the National Institutes of
Health (HL-30568 to P.A.E.), the Laubisch fund (to P.A.E.), and
predoctoral training grants (US Department of Education no. P200A80113)
(to H.R.K.).
Abbreviations: apoC-II, Apolipoprotein C-II; CDCA,
chenodeoxycholic acid; DMSO, dimethylsulfoxide; EcRE, Ecdysone response
element; FXR, farnesoid X-activated receptor; FXRE, FXR response
element; HCR, hepatic control region; IR-I, inverted repeat with one
nucleotide spacing between the two half-sites; IRES, internal ribosome
entry site; LCA, lithocholic acid; SHP, small heterodimer partner; SSH,
suppression subtractive hybridization; TK, thymidine kinase; TTNPB,
2-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]
benzoic acid.
Received for publication April 20, 2001.
Accepted for publication June 28, 2001.
 |
REFERENCES
|
---|
-
Mangelsdorf DJ, Thummel C, Beato M, et al. 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Nolte RT, Wisely GB, Westin S, et al. 1998 Ligand
binding and co-activator assembly of the peroxisome
proliferator-activated receptor-
. Nature 395:137143[CrossRef][Medline]
-
Westin S, Kurokawa R, Nolte RT, et al. 1998 Interactions controlling the assembly of nuclear-receptor heterodimers
and co-activators. Nature 395:199202[CrossRef][Medline]
-
Forman BM, Goode E, Chen J, et al. 1995 Identification of a nuclear receptor that is activated by farnesol
metabolites. Cell 81:687693[Medline]
-
Laffitte BA, Kast HR, Nguyen CM, Zavacki AM, Moore
DD, Edwards PA 2000 Identification of the DNA binding specificity and
potential target genes for the farnesoid X-activated receptor. J
Biol Chem 275:1063810647[Abstract/Free Full Text]
-
Seol W, Choi HS, Moore DD 1995 Isolation of
proteins that interact specifically with the retinoid X receptor: two
novel orphan receptors. Mol Endocrinol 9:7285[Abstract]
-
Zavacki AM, Lehmann JM, Seol W, Willson TM,
Kliewer SA, Moore DD 1997 Activation of the orphan receptor RIP14 by
retinoids. Proc Natl Acad Sci USA 94:79097914[Abstract/Free Full Text]
-
Makishima M, Okamoto AY, Repa JJ, et al. 1999 Identification of a nuclear receptor for bile acids. Science 284:13621365[Abstract/Free Full Text]
-
Parks DJ, Blanchard SG, Bledsoe RK, et al. 1999 Bile acids: natural ligands for an orphan nuclear receptor. Science 284:13651368[Abstract/Free Full Text]
-
Wang H, Chen J, Hollister K, Sowers LC, Forman BM 1999 Endogenous bile acids are ligands for the nuclear receptor
FXR/BAR. Mol Cell 3:543553[Medline]
-
Broughton G 1994 Chenodeoxycholate: the bile acid.
The drug. A review. Am J Med Sci 307:5463[Medline]
-
Grober J, Zaghini I, Fujii H, et al. 1999 Identification of a bile acid-responsive element in the human ileal
bile acid-binding protein gene. Involvement of the farnesoid X
receptor/9-cis-retinoic acid receptor heterodimer. J Biol Chem 274:2974919754[Abstract/Free Full Text]
-
Urizar NL, Dowhan DH, Moore DD 2000 The farnesoid
X-activated receptor mediates bile acid activation of phospholipid
transfer protein gene expression. J Biol Chem 275:3931339317[Abstract/Free Full Text]
-
Goodwin B, Jones SA, Price RR, et al. 2000 Regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1
represses bile acid biosynthesis. Mol Cell 6:517526[Medline]
-
Lu TT, Makishima M, Repa JJ, et al. 2000 Molecular
basis for feedback regulation of bile acid synthesis by nuclear
receptors. Mol Cell 6:507515[Medline]
-
Maloney PR, Parks DJ, Haffner CD, et al. 2000 Identification of a chemical tool for the orphan nuclear receptor FXR.
J Med Chem 43:29712974[CrossRef][Medline]
-
Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G,
Gonzales FJ 2000 Targeted disruption of the nuclear receptor FXR/BAR
impairs bile acid and lipid homeostasis. Cell 102:731744[Medline]
-
Willy PJ, Mangelsdorf DJ 1998 Nuclear orphan
receptors: The search for novel ligands and signaling pathways. In:
OMalley BW, ed. Hormones and signaling, vol. 1. San Diego, CA:
Academic Press, Inc; 307358
-
Howard WR, Pospisil JA, Njolito E, Noonan DJ 2000 Catabolites of cholesterol synthesis pathways and forskolin as
activators of the farnesoid X-activated nuclear receptor. Toxicol Appl
Pharmacol 163:195202[CrossRef][Medline]
-
Simonet WS, Bucay N, Lauer SJ, Taylor JM 1993 A far-downstream hepatocyte-specific control region directs expression
of the linked human apolipoprotein E and C-I genes in transgenic mice.
J Biol Chem 268:82218229[Abstract/Free Full Text]
-
Shachter NS, Zhu Y, Walsh A, Breslow JL, Smith
JD 1993 Localization of a liver-specific enhancer in the apolipoprotein
E/C-I/C-II gene locus. J Lipid Res 34:16991707[Abstract]
-
Simonet WS, Bucay N, Pitas RE, Lauer SJ, Taylor JM 1991 Multiple tissue-specific elements control the apolipoprotein E/C-I
gene locus in transgenic mice. J Biol Chem 266:86518564[Abstract/Free Full Text]
-
Allan CM, Walker D, Taylor JM 1995 Evolutionary
duplication of a hepatic control region in the human apolipoprotein E
gene locus. Identification of a second region that confers high level
and liver-specific expression of the human apolipoprotein E gene in
transgenic mice. J Biol Chem 270:2627826281[Abstract/Free Full Text]
-
Allan CM, Taylor S, Taylor JM 1997 Two hepatic
enhancers, HCR.1 and HCR.2, coordinate the liver expression of the
entire human apolipoprotein E/C-I/C-IV/C-II gene cluster. J Biol
Chem 272:2911329119[Abstract/Free Full Text]
-
Vorgia P, Zannis VI, Kardassis D 1998 A short
proximal promoter and the distal hepatic control region-1 (HCR-1)
contribute to the liver specificity of the human apolipoprotein C-II
gene. Hepatic enhancement by HCR-1 requires two proximal hormone
response elements which have different binding specificities for orphan
receptors HNF-4, ARP-1, and EAR-2. J Biol Chem 273:41884196[Abstract/Free Full Text]
-
Allan CM, Walker D, Segrest JP, Taylor JM 1995 Identification and characterization of a new human gene (APOC4) in the
apolipoprotein E, C-I, and C-II gene locus. Genomics 28:291300[CrossRef][Medline]
-
Paigen B, Morrow A, Brandon C, Mitchell D, Holmes PA 1985 Variation in susceptibility to atherosclerosis among inbred
strains of mice. Atherosclerosis 57:6573[Medline]
-
Jong MC, Hofker MH, Havekes LM 1999 Role of ApoCs in
lipoprotein metabolism: functional differences between ApoC1, ApoC2,
and ApoC3. Arterioscler Thromb Vasc Biol 19:472484[Free Full Text]
-
Jiang XC, Bruce C, Mar J, Lin M, Ji Y, Francone OL,
Tall AR 1999 Targeted mutation of plasma phospholipid transfer protein
gene markedly reduces high-density lipoprotein levels. J Clin
Invest 103:907914[Abstract/Free Full Text]
-
Dang Q, Walker D, Taylor S, et al. 1995 Structure of
the hepatic control region of the human apolipoprotein E/C-I gene
locus. J Biol Chem 270:2257722585[Abstract/Free Full Text]
-
Fojo SS 1992 Genetic dyslipoproteinemias: role of
lipoprotein lipase and apolipoprotein C-II. Curr Opin Lipidol 3:186195
-
Streicher R, Geisel J, Weisshaar C, et al. 1996 A
single nucleotide substitution in the promoter region of the
apolipoprotein C-II gene identified in individuals with
chylomicronemia. J Lipid Res 37:25992607[Abstract]
-
Iser JH, Sali A 1981 Chenodeoxycholic acid: a review
of its pharmacological properties and therapeutic use. Drugs 21:90119[Medline]
-
Boehm MF, Zhang L, Badea BA, et al. 1994 Synthesis
and structure-activity relationships of novel retinoid X
receptor-selective retinoids. J Med Chem 37:29302941[Medline]
-
Diatchenko L, Lau YF, Campbell AP, et al. 1996 Suppression subtractive hybridization: a method for generating
differentially regulated or tissue-specific cDNA probes and libraries.
Proc Natl Acad Sci USA 93:60256030[Abstract/Free Full Text]
-
Hedrick CC, Castellani LW, Warden CH, Puppione DL,
Lusis AJ 1993 Influence of mouse apolipoprotein A-II on plasma
lipoproteins in transgenic mice. J Biol Chem 268:2067620682[Abstract/Free Full Text]