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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go).



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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. 1AGo. The results shown in panels A–E are representative of two to three experiments.

 
As demonstrated in Fig. 1AGo, 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. 1BGo) and elevated basal levels of apoC-II mRNA (Fig. 1AGo, 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. 1AGo, 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. 1BGo). Induction of apoC-II mRNA was maximal at 100 µM CDCA (Fig. 1CGo). 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. 1CGo), 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. 1DGo). 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. 1EGo; 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. 2AGo) (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{alpha} 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.

 
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. 2AGo). We then used recombinant human (h) RXR{alpha} and FXR proteins in EMSAs to determine whether the FXR/RXR heterodimer can bind to these potential FXREs (Fig. 2Go). 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. 2BGo). 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. 2DGo). 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. 2DGo).

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. 2Go 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. 3Go). 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. 3AGo 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. 3AGo).



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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{alpha} 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{alpha}. 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{alpha} 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{alpha} plasmids for 24 h. C, FXR and RXR{alpha} 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{alpha}. 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.

 
Figure 3BGo 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. 3BGo, 2xGoMutIR-1a). The data of Fig. 3CGo 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. 3CGo). The thymidine kinase (TK)-luciferase empty vector was not induced under these conditions (Fig. 3CGo). These data, taken together with those of Fig. 2Go, demonstrate that the FXRE (IR-1A), identified in both the HCR.1 and HCR.2 enhancer elements (Fig. 2AGo), 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. 4AGo 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 4BGo 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).

 
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. 5Go). 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. 5AGo, 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. 5AGo, 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. 5AGo 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. 1Go using the indicated probes. Normalization and quantitation (shown as fold induction) were as described in Fig. 1Go. 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.

 
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 5BGo 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. 5BGo, 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. 5BGo, 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 2Go) (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. 2AGo), we constructed reporter genes containing two copies of either the wild-type or mutant IR-1A. Figure 3Go 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. 3AGo), 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. 2Go), 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. 2AGo), 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. 1–3GoGoGo, 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. 5BGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, 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 manufacturer’s 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 [{alpha}-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 manufacturer’s 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{Delta}110 and hRXR{alpha} (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{alpha}, 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{alpha},7{alpha}-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. 4Go 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. 5Go 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.


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