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Molecular Endocrinology, doi:10.1210/me.2007-0089
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Molecular Endocrinology 21 (6): 1359-1369
Copyright © 2007 by The Endocrine Society

Regulation of Hepatic Insig-2 by the Farnesoid X Receptor

Melissa L. Hubbert, Yanqiao Zhang, Florence Y. Lee and Peter A. Edwards

Departments of Biological Chemistry (M.L.H., F.Y.L., P.A.E.) and Medicine (M.L.H., Y.Z., F.Y.L., P.A.E.), David Geffen School of Medicine, and the Molecular Biology Institute at UCLA (P.A.E.), Los Angeles, California 90095

Address all correspondence and requests for reprints to: Peter A. Edwards, Department of Biological Chemistry, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, 33-257 CHS, Los Angeles, California 90095. E-mail: pedwards{at}mednet.ucla.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of the farnesoid X receptor (FXR{alpha}) affects genes controlling many pathways, including those involved in bile acid and glucose homeostasis. Here we report that a critical gene involved in cholesterol homeostasis, Insig-2, was induced when mice or cultured cells were treated with FXR{alpha} agonists or infected with constitutively active FXR{alpha}. No such induction was observed in agonist-treated FXR{alpha}–/– mice. Further analysis, which included EMSAs, reporter gene activation, and chromatin immunoprecipitation, identified two functional FXR{alpha} response elements within intron 2 of the mouse Insig-2 gene. In addition to increasing hepatic Insig-2 protein levels in wild-type mice, FXR{alpha} activation also reduced lanosterol 14{alpha}-demethylase mRNA levels and 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein levels. Together, these changes likely account for the decrease in cholesterol synthesis observed after activation of FXR in primary hepatocytes. In conclusion, the current study links hepatic FXR{alpha} activation to regulation of genes involved in cholesterol synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HEPATIC CONVERSION of cholesterol to bile acids and their subsequent excretion into the intestine represents the major route of cholesterol catabolism and excretion in mammals. The farnesoid X receptor (FXR{alpha}; NR1H4), a member of the nuclear receptor superfamily, plays a central role in this process by regulating key genes in bile acid synthesis and transport (reviewed in Ref. 1). Expression of the mammalian FXR{alpha} gene is largely limited to the liver, intestine, kidney, and adrenals, although low levels of FXR{alpha} mRNA are detectable in adipose tissue (2, 3, 4, 5). Four FXR{alpha} transcripts (FXR{alpha}1, FXR{alpha}2, FXR{alpha}3, and FXR{alpha}4) are produced from a single mammalian gene as a result of the use of two alternative promoters and/or alternative splicing between exons 5 and 6 (3, 5). These transcripts encode four FXR{alpha} isoforms that differ in their amino terminus and/or by the inclusion or exclusion of four amino acids (MYTG) in the hinge region, adjacent to the DNA binding domain (3, 5). Specific bile acids, that include chenodeoxycholic acid (CDCA) and cholic acid, were identified as the natural FXR{alpha} agonists in 1999 (6, 7, 8) and subsequently shown to activate all four FXR{alpha} isoforms (3, 5). Interestingly, the four FXR{alpha} isoforms are not functionally equivalent as they have been shown to differentially activate specific target genes (5, 9, 10).

Studies using FXR{alpha}–/– mice have illustrated the importance of this nuclear receptor in regulating bile acid metabolism. FXR{alpha}–/– mice exhibit increased levels of bile acid and cholesterol in serum, elevated hepatic levels of cholesterol and triglyceride (11), decreased bile acid pool size, and decreased fecal bile acid excretion (11). Activation of FXR{alpha} mediates a negative feedback pathway on bile acid synthesis, mainly through repressing the transcription of CYP8B1 and CYP7A1, the rate-limiting enzymes in bile acid synthesis, by processes that involve induction of small heterodimer partner (SHP) (12, 13, 14) and FGF15/19 (15, 16). FXR{alpha} also controls bile acid homeostasis by regulating the expression of numerous genes involved in bile acid conjugation, detoxification, and transport (reviewed in Ref. 1).

In addition to its role in bile acid homeostasis, FXR{alpha} has been shown to play an important role in carbohydrate and lipid metabolism. FXR{alpha}–/– mice have reduced glucose tolerance and impaired response to insulin in the liver, white adipose tissue, and muscle (17, 18, 19). In agreement with these observations, activation of FXR{alpha} in wild-type or diabetic mice results in hypoglycemia and increased insulin sensitivity (17, 18, 19). Administration of FXR{alpha} agonists also reduced plasma triglyceride levels in murine models (20, 21) presumably due in part to the increased hepatic expression of the very low-density lipoprotein receptor (22) and to the inhibitory effects on sterol regulatory element-binding protein (SREBP)-1c expression (9, 23), a key regulator of fatty acid and triglyceride synthesis.

One recent study reported that activated FXR{alpha} repressed 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase mRNA levels by a mechanism that involved SHP and liver receptor homolog 1 (LRH-1), thus suggesting that FXR{alpha} might also control genes involved in cholesterol synthesis (24). Studies over the last three decades have demonstrated that cholesterogenic genes are regulated by SREBPs, transcription factors that are modulated, at least in part, by the formation of Insig/SREBP cleavage-activating protein (SCAP)/SREBP complexes (reviewed in Ref. 25); when cellular sterol levels are depleted a SREBP/SCAP complex translocates from the endoplasmic reticulum to the Golgi where SREBP is sequentially cleaved by two proteases to release a functional, mature SREBP (25). This mature transcription factor enters the nucleus, binds to sterol regulatory elements and induces several genes involved in sterol and lipid biosynthesis (reviewed in Ref. 26). In contrast, when cellular sterol levels are elevated SCAP/SREBP complexes with Insig proteins that are resident in the endoplasmic reticulum, thus preventing the SCAP/SREBP complex from translocating to the Golgi (25). Elevated cellular sterol levels also lead to the enhanced degradation of HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway (27, 28). Additionally, increasing cellular lanosterol levels by treating cells with ketoconazole, an inhibitor of lanosterol 14{alpha}-demethylase (Cyp51), also reduces HMG-CoA reductase levels (29). Recent studies have shown that interaction of HMG-CoA reductase with lanosterol is the initial event that results in its association with Insig in the endoplasmic reticulum and the subsequent ubiquitination and degradation of HMG-CoA reductase by the proteosome (30, 31).

The two Insig genes, Insig-1 and Insig-2, are differentially expressed and differentially regulated in various mouse tissues (25). In addition, the Insig-2 gene contains two functional promoters that are also differentially regulated (32). Nonetheless, the two mRNAs derived from the Insig-2 gene, Insig-2a and Insig-2b, encode an identical protein (32). The control of Insig-1 and Insig-2 expression is even more complicated because the two Insig proteins are also differentially regulated at the level of protein stability (33). It is now clear that the Insig proteins play key roles in both regulating the maturation of SREBPs and in controlling HMG-CoA reductase stability (25). However, the relative importance of each of the complex regulatory steps that control Insig protein expression under different physiological conditions remains to be determined.

In the current report, we demonstrate that activation of FXR{alpha} both in cultured liver-derived cells and in the livers of wild-type mice results in induction of the Insig-2 gene and to increased expression of Insig-2a mRNA and Insig-2 protein. In addition, we use EMSAs, reporter gene assays, and chromatin immunoprecipitation to confirm that the mouse Insig-2 gene contains two functional FXREs. Finally, we demonstrate that activation of FXR{alpha} results in reduced hepatic levels of both lanosterol 14{alpha}-demethylase mRNA and HMG-CoA reductase protein. Together, these data suggest that activation of FXR{alpha} results in unexpected changes in key genes involved in sterol homeostasis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Insig-2 as an FXR{alpha} Target Gene
Previous studies have identified FXR{alpha} target genes that are involved in bile acid synthesis and transport, glucose metabolism, and resistance to intestinal bacterial infection (reviewed in Ref. 1). To identify FXR{alpha} target genes that are involved in additional metabolic pathways, murine primary hepatocytes were infected with adenovirus expressing constitutively active individual FXR{alpha}-VP16 isoforms. After 48 h, RNA was isolated to generate cDNAs for probing Codelink microarrays. This approach identified a number of putative FXR{alpha} target genes, including Insig-2, and the known FXR{alpha} target genes bile salt export pump (BSEP) and SHP. Analysis of the microarray data suggested that other genes of the cholesterol biosynthetic pathway including Insig-1, HMG-CoA synthase, HMG-CoA reductase, SREBP-1, and SREBP-2 were not induced in the infected cells (data not shown).

Because the Codelink microarray analysis did not distinguish between the two Insig-2 transcripts, Insig-2a and Insig-2b, RT-qPCR (real-time quantitative PCR) was performed using the RNA isolated from adenovirus-infected primary hepatocytes. As expected, each FXR{alpha} isoform induced the expression of SHP, a well-characterized FXR{alpha} target gene (Fig. 1AGo). The data of Fig. 1AGo also show that Insig-2a mRNA levels were increased significantly after infection with adenovirus expressing each of the FXR{alpha}-VP16 isoforms as compared with uninfected cells or cells infected with adenovirus that expresses VP16 alone. In contrast, Insig-2b and Insig-1 mRNA levels did not change after infection with any of the FXR{alpha} isoforms (Fig. 1AGo). To assess whether activation of endogenous FXR{alpha} also induced Insig-2a mRNA levels, primary mouse hepatocytes were treated for 24 h with either dimethyl sulfoxide (DMSO) or GW4064 (compound no. 4064, synthetic FXR{alpha} ligand; GlaxoSmithKline, Research Triangle Park, NC), a potent FXR{alpha} agonist. RT-qPCR analysis demonstrated that activation of endogenous FXR{alpha} also increased Insig-2a mRNA levels (Fig. 1BGo). This effect was specific because neither Insig-2b nor Insig-1 mRNA levels were affected (Fig. 1BGo).


Figure 1
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  		Fig. 1. Induction of Insig-2a in Primary Hepatocytes in Response to Activated FXR{alpha}

RNA was isolated from adenovirus-infected primary hepatocytes. Specific mRNAs were quantified using RT-qPCR performed in triplicate. Untreated cells are indicated (–) (A). Triplicate dishes of mouse primary hepatocytes were treated for 24 h with DMSO or 1 µM GW4064. Specific RNA expression was quantified using RT-qPCR. Data are reported as mean ± SEM (n = 3); *, P < 0.05 (B).

 
Reduced Expression of Insig-2 Protein in FXR{alpha}–/– Mice
To determine whether loss of FXR{alpha} affects Insig-2 expression, wild-type and FXR{alpha}–/– mice were fed a chow diet ad libitum before being euthanized at the beginning of the light cycle. Under these nonfasted conditions, the mRNA levels of hepatic Insig-1 and Insig-2a, but not Insig-2b, were reduced in the FXR{alpha}–/– mice (Fig. 2AGo). Western blot analysis indicated that Insig-2 protein levels were reduced in the FXR{alpha}–/– mice, consistent with the decrease in mRNA levels (Fig. 2BGo). Surprisingly, Insig-1 protein levels were not reduced in the FXR{alpha}–/– mice (Fig. 2BGo).


Figure 2
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  		Fig. 2. Hepatic Insig Expression Is Altered in FXR{alpha}–/– Mice

Wild-type (WT) and FXR{alpha}–/– mice were fed normal chow ad libitum (n = 5 mice/group). Livers were isolated at the start of the light cycle and mRNA levels determined using RT-qPCR. The data are reported as mean ± SEM. *, P < 0.05 (A). Western blot analysis was performed in duplicate using 50 µg of pooled cell lysates and antibodies raised against Insig-1 (~28 kDa), Insig-2 (~26 kDa), or ß-actin (B). The bands for Insig-1 and Insig-2 on the Western blot correspond to proteins of ~28 kDa and 26 kDa, respectively.

 
Hepatic Insig-2a mRNA Levels Are Induced in Vivo after Activation of FXR{alpha}
To determine whether hepatic Insig-2a mRNA levels could be induced in vivo, wild-type mice were infected with adenovirus expressing either constitutively active FXR{alpha}-VP16 isoforms or VP16. The data show that each FXR{alpha} isoform significantly induced hepatic Insig-2a mRNA levels (Fig. 3AGo). In contrast, mRNA levels of Insig-1 and Insig-2b were unaffected (Fig. 3AGo).


Figure 3
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  		Fig. 3. Activation of FXR{alpha} in Vivo Induces Hepatic Insig-2a mRNA and Insig-2 Protein

Wild-type (WT) mice (three mice per group) were infected with adenovirus encoding VP16 or the indicated FXR{alpha}-VP16 fusion proteins. After 7 d, hepatic RNA was isolated and mRNA quantified as described in Fig. 2Go (A). Wild-type and FXR{alpha}–/– mice (six mice per group) were administered either vehicle or GW4064 by oral gavage for 10 d and fasted for 5 h before isolation of the liver. Hepatic mRNA levels were determined using RT-qPCR. The data are reported as mean ± SEM. *, P < 0.05; **, P < 0.01 (B–D). Pooled liver lysates were analyzed in duplicate by Western blot analysis using the indicated antibodies (E).

 
To overcome any possible artifact resulting from infection with adenovirus and/or overexpression of the constitutively active FXR{alpha} isoforms, we treated wild-type and FXR{alpha}–/– mice for 10 d with vehicle or the specific FXR{alpha} agonist, GW4064. Mice were then fasted for 5 h before isolation of RNA and protein. Such treatment (fasting) is known to result in induction of Insig-2a and repression of Insig-1 mRNA (32). Indeed, the data in supplemental Fig. 1 (which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) show that Insig-2a mRNA levels were induced to similar levels when either wild-type or FXR{alpha}–/– mice were fasted for 12 h. Thus, induction of Insig-2a by fasting is independent of FXR. Consistent with the observed mRNA levels, hepatic Insig-2a protein levels were similar in fasted wild-type and FXR{alpha}–/– mice (Fig. 3EGo, compare lanes 1 and 2 with 5 and 6).

Importantly, the data of Fig. 3Go, B and C, demonstrate that Insig-2a, but not Insig-1 or Insig-2b, mRNA levels were significantly increased after treatment of wild-type mice with GW4064. This induction was FXR{alpha}-dependent because Insig-2a mRNA levels were unchanged after treatment of FXR{alpha}–/– mice with GW4064 (Fig. 3CGo). As expected, Insig-2b mRNA levels were unaffected after treatment of FXR{alpha}–/– mice with GW4064 (Fig. 3CGo). Induction of hepatic BSEP and SHP mRNA and repression of SREBP-1c mRNA in wild-type, but not FXR{alpha}–/– mice (Fig. 3DGo) served as controls consistent with FXR activation by GW4064 (19).

Western blot analysis of liver lysates from these same mice indicate that Insig-2 protein levels were increased after GW4064 treatment of wild-type, but not FXR{alpha}–/– mice (Fig. 3EGo, lanes 3 and 4 vs. 1 and 2). Thus, activation of FXR{alpha} in vivo resulted in induction of hepatic Insig-2 mRNA and protein (Fig. 3Go). Surprisingly, GW4064 treatment reduced Insig-1 protein levels in wild-type mice (Fig. 3EGo). This decrease was dependent upon FXR{alpha} because no such decrease was observed in FXR{alpha}–/– mice (Fig. 3EGo).

Identification of FXREs in the Insig-2 Gene
The FXR{alpha}/retinoid X receptor (RXR) heterodimer has been shown to bind DNA containing IR-1, DR-1 (direct repeat-1 separated by one nucleotide), or ER-8 (everted repeat separated by eight nucleotides) sequences (10, 34, 35). Using the Nubiscan program, four potential IR-1 elements were identified within the first and second introns of the mouse Insig-2 gene (Fig. 4AGo). No such elements were identified within 7 kb of the proximal promoter (data not shown). EMSAs, using oligonucleotides corresponding to the four putative FXREs (IR-1A, IR-1B, IR-1C, and IR-1D), demonstrated that FXR{alpha}/RXR heterodimers were able to bind to IR-1C or IR-1D but not to IR-1A or IR-1B (Fig. 4BGo). A radiolabeled oligonucleotide containing the FXRE from the promoter of the phospholipid transfer protein (PLTP) gene (36) served as a positive control (Fig. 4BGo). To determine whether the Insig-2 IR-1 elements bound to FXR{alpha} with a high affinity, competitive EMSAs were performed. The binding of the 32P-labeled PLTP FXRE probe to FXR{alpha} and RXR proteins was competed with increasing concentrations of oligonucleotides corresponding to the PLTP FXRE, or wild-type or mutant Insig-2 IR-1C and Insig-2 IR-1D (Fig. 4CGo). The data of Fig. 4CGo demonstrate that unlabeled oligonucleotides corresponding to the PLTP FXRE or wild-type Insig-2 IR-1C and IR-1D efficiently competed for the formation of the radiolabeled DNA/FXR{alpha}/RXR complex. Indeed, the data show that IR-1C and IR-1D were more potent competitors than the oligonucleotide containing the PLTP FXRE (Fig. 4CGo). The competition was specific because oligonucleotides containing mutant IR-1C or IR-1D sequences failed to affect the radioactive DNA/protein complex (Fig. 4CGo).


Figure 4
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  		Fig. 4. FXR{alpha}/RXR Heterodimers Bind to Two Putative FXREs in the Mouse Insig-2 Gene

Analysis of the murine Insig-2 gene ± 7 kb with the Nubiscan program identified four potential FXREs (IR-1 elements) (shown as A, B, C, or D) within intron 1 and intron 2 (A). EMSAs were performed with FXR{alpha}/RXR heterodimers and 32P-labeled oligonucleotides corresponding to the indicated putative FXRE (B). Competitive EMSAs were performed with FXR{alpha}/RXR heterodimers and a 32P-labeled PLTP probe with increasing amounts of PLTP, wild-type (WT) or mutant Insig-2 FXRE as cold competitor (C).

 
IR-1C and IR-1D Are Functionally Active in a Reporter Assay
To better assess the functionality of the Insig-2 IR-1C and IR-1D sequences, HepG2 cells were cotransfected with plasmids that express specific mouse FXR{alpha} isoforms, RXR, and luciferase reporter constructs under the control of either two copies of the wild-type or mutant Insig-2 IR-1C or IR-1D elements. The cells were then treated with DMSO or GW4064 (1 µM) for 48 h and luciferase activities determined and corrected for transfection efficiency. The data demonstrate that activation of FXR{alpha}1 or FXR{alpha}2 by GW4064 increased luciferase activity for both reporter constructs controlled by either IR-1C or IR-1D (Fig. 5Go). Mutated IR-1C or IR-1D elements prevented the binding of FXR{alpha}/RXR heterodimers (Fig. 4CGo) and abrogated the reporter construct’s response to GW4064 (Fig. 5Go).


Figure 5
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  		Fig. 5. FXR{alpha} Activation of Insig-2 IR-1 Elements in a Heterologous Promoter

Triplicate dishes of HepG2 cells were transiently transfected with pTK-luciferase reporter constructs under the control of two copies of either wild-type (WT) or mutant Insig-2 FXREs, ß-galactosidase, hRXR{alpha}, and either mFXR{alpha}1 or mFXR{alpha}2. The mutant FXREs contained 2 mutations in each half-site (supplemental Table 1). After treatment for 48 h with either DMSO or 1 µM GW4064, cells were lysed and luciferase activity was determined and the values corrected for variations in transfection efficiency (n = 3). RLU, Relative light units. The results are representative of two studies.

 
In Vivo Association of FXR{alpha} with the Insig-2 Gene
The data of Figs. 4Go and 5Go demonstrate that FXR{alpha}/RXR heterodimers bind with high affinity to IR-1C and IR-1D elements, and that these elements are sufficient to mediate FXR{alpha}-dependent activation of reporter constructs. To investigate whether the two FXREs bind FXR{alpha} in vivo, we performed chromatin immunoprecipitation using AML12 cells, an established murine hepatocyte cell line (37). The data of Fig. 6AGo show that total FXR{alpha} mRNA levels in AML12 cells approximated 33% of the levels expressed in mouse liver. These data suggested that there was sufficient endogenous FXR{alpha} in AML12 cells such that addition of FXR agonists would alter gene expression.


Figure 6
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  		Fig. 6. Chromatin Immunoprecipitation Demonstrates that FXR{alpha} Binds to Two FXREs in Intron 2 of the Insig-2 Gene

Triplicate dishes of AML12 cells were treated with DMSO, 1 µM GW4064, or 100 µM CDCA for 48 h. FXR{alpha} mRNAs were quantified by RT-qPCR and compared with values obtained from mouse liver (A). mRNA levels of BSEP, HMG-CoA reductase and SREBP-1c mRNA (B) and Insig (C) in the same AML12 cells are shown. Data are reported as mean ± SEM. *, P < 0.05; **, P < 0.01. Chromatin immunoprecipitation was performed as described in Materials and Methods (D).

 
Consistent with this proposal, GW4064 treatment of AML12 cells resulted in increased expression of BSEP mRNA and a decrease in SREBP-1c mRNA expression (Fig. 6BGo). The remarkable induction of BSEP mRNA in response to GW4064 is likely a result of the very low basal levels noted in the vehicle-treated cells (data not shown). In contrast, HMG-CoA reductase mRNA levels were unaffected after activation of FXR in AML 12 cells (Fig. 6BGo) or primary hepatocytes (see below). In other studies we failed to observe any significant changes in hepatic HMG-CoA reductase mRNA levels 7 d after tail vein injection of C57BL/6 mice with adenovirus expressing either VP16 or constitutively active FXR{alpha}-VP16 despite evidence that expression of known FXR target genes was affected in mice expressing FXR{alpha}-VP16 (supplemental Fig. 2). GW4064 treatment of AML12 cells resulted in a significant increase in Insig-2a mRNA levels, a small yet statistically significant increase in Insig-2b mRNA levels, but no change in Insig-1 mRNA levels (Fig. 6CGo). As expected, treatment of the AML12 cells with CDCA, an endogenous bile acid that functions as a physiological FXR{alpha} agonist (6, 7, 8), also induced Insig-2a (Fig. 6CGo). This latter induction was specific because neither Insig-1 nor Insig-2b mRNA levels were affected by CDCA treatment (Fig. 6CGo).

Taken together, these studies with AML12 cells suggested that they would be a suitable cell line for chromatin immunoprecipitation experiments because they contained sufficient FXR to alter the expression of numerous genes after activation of the nuclear receptor with either natural or synthetic agonists.

After treatment of AML12 cells with GW4064, cells were treated with formaldehyde/PBS to cross-link-bound proteins to DNA. Subsequent sonication of the nuclei and immunoprecipitation with mouse IgG or anti-FXR{alpha} antibody were performed as described in Materials and Methods. As shown in Fig. 6DGo, FXR{alpha} bound to regions of the Insig-2 gene that contained the FXREs IR-1C and IR-1D. In contrast, FXR{alpha} did not bind to the region of the Insig-2 gene containing IR-1B (Fig. 6DGo). These data are consistent with EMSAs that demonstrate that FXR{alpha}/RXR binds to IR-1C and IR-1D, but not IR-1B (Fig. 4Go). As a positive control we also analyzed the pregnane X receptor (PXR) gene (38). Consistent with a recent report (38), we show that FXR{alpha} binds to an FXRE in the second intron of the PXR gene (Fig. 6DGo). We performed similar immunoprecipitation studies that used the livers of wild-type mice that had been gavaged twice daily with GW4064 for 5 d; the data demonstrate that FXR was bound to the intronic FXREs in the Insig-2 gene that correspond to IR-1C and IR-1D, but not to IR-1B (supplemental Fig. 3). Taken together, these data demonstrate that FXR{alpha} binds to two intronic FXREs in the Insig-2 gene in vivo, consistent with transcriptional activation by FXR{alpha} agonists.

Activation of FXR Inhibits Cholesterogenesis
Because Insig proteins are known to affect the expression of enzymes involved in cholesterol biosynthesis, we next determined whether treatment of primary mouse hepatocytes with GW4064 affected the incorporation of radiolabeled acetate into cholesterol. The data show that activation of FXR inhibited cholesterol synthesis by 46% (Fig. 7Go). mRNA levels of HMG-CoA reductase were unchanged in cells treated in an identical manner (data not shown).


Figure 7
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  		Fig. 7. FXR Activation Inhibits Cholesterol Synthesis in Primary Hepatocytes

Hepatocytes were isolated from wild-type mice, allowed to adhere to plastic dishes and incubated in triplicate for 24 h in the presence or absence of GW4064 (1 µM). Fresh media containing radiolabeled [1-14C]acetate were then added and after 2 h the 14C-radiolabeled cholesterol was isolated. The values shown were determined after correction for percent recovery and cell protein as described in Materials and Methods. Values are given as mean ± SEM (n = 3). *, P < 0.05.

 
Activation of FXR{alpha} in Vivo Affects Specific Cholesterogenic Genes
To better understand how activated FXR might result in repression of cholesterol synthesis, we treated wild-type mice with GW4064 for 5 d and determined the microsomal protein level of HMG-CoA reductase and the mRNA levels of selected cholesterogenic genes. The data show that under these conditions neither HMG-CoA reductase, squalene synthase nor SREBP-2 mRNA levels were affected in the mice receiving the FXR agonist (Fig. 8AGo). In contrast, hepatic Insig-2a mRNA levels increased and lanosterol 14{alpha}-demethylase mRNA levels were decreased in the GW4064-treated mice (Fig. 8AGo). Similar results were obtained when mice had been treated for 10 d with GW4064 (data not shown). The repression of Cyp7a1 mRNA levels is consistent with activation of hepatic FXR by GW4064 in these same mice (Fig. 8AGo). Western blot assays demonstrate that the GW4064-treated mice had decreased levels of hepatic microsomal HMG-CoA reductase protein (Fig. 8BGo).


Figure 8
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  		Fig. 8. Activation of FXR{alpha} Reduces HMG-CoA Reductase Protein and Lanosterol 14{alpha}-Demethylase mRNA Levels

Wild-type mice were treated with either vehicle or 50 mg/kg of GW4064 once a day at the beginning of the light cycle for 5 d (n =4/group). On d 5, mice were fasted for 4 h and hepatic microsomes and RNA isolated. RT-qPCR was performed using individual cDNA samples (n = 4/group) (A). HMG-CoA reductase protein levels were determined by Western blot analysis on pooled microsome samples (B). Mouse primary hepatocytes were treated with DMSO or GW4064 (1 µM) for 24 h and mRNA levels were determined by RT-qPCR (n =3) (C). Data are reported as mean ± SEM; *, P < 0.05.

 
In other studies, we show that the mRNA levels of lanosterol 14{alpha}-demethylase, but not HMG-CoA reductase or squalene synthase, also decreased significantly when primary mouse hepatocytes were treated with GW4064 (Fig. 8CGo).

Taken together, these data suggest that FXR activation results in repression of lanosterol 14{alpha}-demethylase gene expression, and in decreased levels of microsomal HMG-CoA reductase protein.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The current study identifies the mouse Insig-2 gene as an FXR{alpha} target gene. This conclusion is based on the induction of both Insig-2a mRNA and protein levels, in either cultured hepatocytes and in mice after treatment with bile acids or the synthetic FXR{alpha} agonist GW4064, or after infection with constitutively active FXR{alpha}-VP16 (Figs. 1Go and 3Go). FXR{alpha} agonists failed to activate Insig-2a in FXR{alpha}–/– mice, consistent with the essential role for FXR{alpha} (Fig. 3Go). In addition, based on EMSAs, chromatin immunoprecipitation and reporter gene assays, we identified two functional FXREs in intron 2 of the mouse Insig-2 gene (Figs. 4–6GoGoGo). One previous study that used reporter gene assays demonstrated that the Insig-2 promoter contained a vitamin D response element, but lacked an LXRE or an FXRE (39). The current study is consistent with this conclusion because we also failed to identify an FXRE in the proximal promoter (Fig. 4Go).

Insig proteins play a critical role in controlling both SREBP processing and HMG-CoA reductase degradation (25). Recent reports have shown that the binding of lanosterol to HMG-CoA reductase promotes the formation of an Insig/HMG-CoA reductase/lanosterol complex. This, in turn, leads to enhanced ubiquitination and proteosomal degradation of HMG-CoA reductase (30, 31). The finding that addition of lanosterol to cells or inhibition of lanosterol 14{alpha}-demethylase by ketoconazole resulted in a reduction in HMG-CoA reductase protein levels is consistent with this model (29). These latter studies provide a mechanism to explain the decrease in HMG-CoA reductase protein levels that follows activation of FXR{alpha} (Fig. 8Go). We propose that the FXR{alpha}-dependent increase in Insig-2 protein coupled with a decrease in lanosterol 14{alpha}-demethylase expression results in enhanced degradation of HMG-CoA reductase.

At the present time, the mechanism involved in the repression of lanosterol 14{alpha}-demethylase by FXR{alpha} is unknown. However, this inhibitory effect appears to be relatively specific because the mRNAs of other cholesterogenic genes, including HMG-CoA reductase, HMG-CoA synthase, and squalene synthase, are unchanged after activation of FXR{alpha} in AML12 cells, primary mouse hepatocytes or the livers of wild-type mice (Figs. 6BGo and 8Go and supplemental Fig. 2). One possibility is that lanosterol 14{alpha}-demethylase mRNA levels are reduced in the FXR agonist-treated mouse liver or primary hepatocytes as a result of increased expression of SHP. SHP, an atypical nuclear receptor, is known to function as a transcriptional repressor of a number of genes including Cyp7a1. The findings that HMG-CoA reductase mRNA levels are refractory to FXR activation contrast with a recent study wherein it was reported that hepatic HMG-CoA reductase mRNA levels declined after treatment of mice with 0.5% cholic acid for 12 wk or 1 d after administration of GW4064 in the food (24). Additional experiments will be required to determine whether these differences result from the use of alternative experimental procedures, from the use of mice of different genetic strains, or from the possible hepato-toxic effects that can occur after long-term administration of high levels of cholic acid.

Quantitative trait loci analysis has linked the Insig-2 gene with the control of plasma cholesterol levels (40) and obesity (41, 42). Additionally hepatic overexpression of Insig-2 in Zucker diabetic fatty (fa/fa) rats reduced plasma and hepatic triglyceride concentrations (43). We have noted that hepatic Insig-2a mRNA levels are increased in diabetic (db/db) mice in response to FXR{alpha} activation (supplemental Fig. 4). Additional studies will be required to determine whether such changes in Insig-2a contribute to the decreased hepatic and plasma triglyceride and cholesterol concentrations noted in db/db mice after treatment with GW4064 (19).

The current study suggests that FXR{alpha} plays a role in regulating hepatic cholesterol levels in part by inducing Insig-2 expression, decreasing lanosterol demethylation and decreasing HMG-CoA reductase protein levels. Figure 9Go shows a model consistent with the data presented in this report.


Figure 9
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  		Fig. 9. Model of FXR{alpha}-Mediated Negative Feedback Inhibition of Genes Involved in Bile Acid and Cholesterol Synthesis

Activation of FXR{alpha} induces the expression of SHP (12 13 14 ) and FGF15 (16 ) in the liver and enterocytes, respectively. SHP and FGF15 repress the expression of hepatic Cyp7a1 and Cyp8a1, key enzymes in bile acid synthesis thus inhibiting the conversion of cholesterol to bile acids (49 ). FXR{alpha} also induces the expression of Insig-2, represses lanosterol 14{alpha}-demethylase, and reduces HMG-CoA reductase protein levels. Details are discussed in the text.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Codelink microarrays were purchased from GE Healthcare Life Sciences (formerly Amersham Biosciences, Piscataway, NJ). GW4064 was kindly provided by Drs. T. Wilson and P. Maloney (GlaxoSmithKline, Research Triangle Park, NC) (20). DMSO, CDCA, (2-hydroxypropyl)-ß-cyclodextrin, and 2-nitrophenyl D-galactopyranoside were purchased from Sigma-Aldrich (St. Louis, MO). Antibody to the transferrin receptor and ß-actin were purchased from Zymed Laboratories (San Francisco, CA) and Abcam Inc. (Cambridge, MA), respectively. The anti-FXR{alpha} antibody (C-20) was purchased from Santa Cruz Biotechnology, Inc. (Santa, Cruz, CA). Collagenase I was from Worthington Biochemical Corporation (Lakewood, NJ). Insig-1 and Insig-2 antibodies were kindly provided by Dr. M. Brown (University of Texas Southwestern Medical Center, Dallas, TX). AML12 and HepG2 cells were purchased from ATCC (Manassas, VA).

Animal Treatments
Animals were fed a standard chow diet and housed in a pathogen-free barrier facility with a 12-h light, 12-h dark cycle. In experiments in which wild-type and FXR{alpha}–/– (11) mice were fed a chow diet ad libitum, the animals were euthanized at the start of the light cycle. Adenovirus treatment of animals was performed as previously described (19). Two separate FXR{alpha} agonist treatments were performed; in one series of experiments wild-type and FXR{alpha}–/– mice (n = 6/group) were gavaged with either vehicle (2-hydroxypropyl-ß-cyclodextrin) or GW4064 in vehicle at a dose of 30 mg/kg twice a day for 10 d (19). On the last day, mice were dosed and then fasted 5 h before euthanization (19). In studies that involve the isolation of liver microsomes, wild-type male mice (n = 4/group) were administered either vehicle (2-hydroxypropyl-ß-cyclodextrin) or GW4064 in vehicle at a dose of 50 mg/kg via oral gavage once a day for 4 d at the beginning of the light cycle. On the last day mice were gavaged, fasted for 4 h, euthanized, and livers excised for microsome preparation and mRNA analysis. Microsomes were prepared from fresh liver samples as previously described (44). In all experiments, mice were matched for age and sex. All animal studies were performed in accordance with accepted standards of humane animal care and were conducted in accordance with the National Institutes of Health animal care guidelines.

Isolation and Culture of Primary Hepatocytes
Primary hepatocytes were isolated and cultured as previously described (45). After 16 h, the media were replaced with fresh media containing 10% charcoal-stripped FBS supplemented with either DMSO (0.01%) or GW4064 (1 µM, dissolved in DMSO). RNA was isolated after 24 h. For adenovirus infection, primary hepatocytes were cultured for 3 d before infection with 10 multiplicity of infection of adenovirus containing VP16 alone or the individual FXR{alpha} isoforms fused to the transactivation domain of VP16 (FXR{alpha}-VP16) (19). At the time of infection, the cells were also treated with either 0.01% DMSO or 2 µM GW4064. After 48 h, RNA was isolated using Trizol. Microarray analysis, using this RNA, was performed by the BioMedical Genomics Microarray Facility (BIOGEM) at the University of California, San Diego. The same RNA samples were also used to generated cDNAs to confirm potential gene targets by RT-qPCR.

Cholesterol Synthesis
Four hours after plating primary mouse hepatocytes on collagen-coated dishes, the cells were washed and then incubated for an additional 16 h in media supplemented with super-stripped FBS and either DMSO or GW4064 (1 µM). Cells were then incubated in fresh media (2 ml) containing [1-14C]acetate (0.5 µCi/ml) ± GW4064. After 2 h, the media were removed, the cells washed in PBS and lipids extracted into 2 ml isopropanol containing [3H]cholesterol (25,000 dpm). Samples were saponified and aliquots then applied to thin-layer chromatography plates. Cholesterol was recovered from the plates after chromatography using a hexane:ether:acetic acid (73:25:2 vol/vol) solvent system. The 14C present in the band corresponding to cholesterol was determined and corrected for recovery using the [3H]cholesterol internal control and normalized to cell protein.

RT-qPCR Analysis
RNA was isolated from mouse livers and cultured cells were isolated using Trizol. DNA-free RNA (Turbo DNA-free kit; Ambion, Austin, TX) was used to generate cDNAs using a TaqMan reverse transcription kit (Applied Biosystems-Roche, Branchburg, NJ). The Bio-Rad iQ SYBR green supermix (Hercules, CA) was used for RT-qPCR and cycle threshold values were detected using Bio-Rad’s MyiQ Single-Color Real-Time PCR Detection System. Cycle threshold values were normalized to cyclophilin A and/or 36B4. A list of the RT-qPCR primers used is provided in supplemental Table 1.

Analysis of FXR{alpha} Response Element
Four putative FXR{alpha} response elements were identified in the mouse Insig-2 gene using the NUBIScan computer algorithm (www.nubiscan.unibas.ch/). Oligonucleotides containing these putative FXREs (supplemental data Table 1) were used for EMSAs and competition studies as previously described (36).

Cell Culture and Reporter Gene Assays
HepG2 and AML12 cells, maintained as described (9, 37), were incubated for 48 h in media containing 10% charcoal-stripped FBS supplemented with DMSO (0.01%), GW4064 (1 µM, dissolved in DMSO), or CDCA (100 µM). HepG2 cells were transiently transfected (36) with plasmids encoding human (h) or mouse (m) proteins (hRXR{alpha} and either mFXR{alpha}1 or mFXR{alpha}2), ß-galactosidase, and pTK-luciferase reporter constructs containing two copies of the specified FXR{alpha} response element. pTK-reporter plasmids were constructed as previously described (34) using the oligonucleotides shown in supplemental Table 1. After treatment, cells were lysed and luciferase activity was determined using the Promega Luciferase Assay System (Madison, WI) and detected using the MicroLumat Luminometer from EG&G Berthold (Aliquippa, PA). Transfection efficiency was normalized after assays for ß-galactosidase activity.

Chromatin Immunoprecipitation
AML12 cells were treated for 48 h with DMSO (0.01%) or GW4064 (1 µM). Chromatin immunoprecipitation was carried out using the Active Motif ChIP-IT kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, the cells were fixed, the isolated nuclear fraction sonicated for 15 sec followed by a 40-sec incubation on ice (repeated five times), and the sheared DNA visualized on an agarose gel to ensure shearing efficiency resulted in fragments approximating 500 bp. The sheared chromatin was precleared with Protein G beads and an aliquot used as a positive control (input). Aliquots of the precleared sheared chromatin were then immunoprecipitated using mouse IgG or anti-FXR{alpha} antibody (C-20; Santa Cruz Biotechnology). The resulting DNA was used for PCR analysis and the amplified DNA fragments visualized on an agarose gel. PCR primers are listed in supplemental Table 1. Chromatin immunoprecipitation from mouse livers used a similar approach that has been described in detail (46).

Western Blot Analysis
Protein concentrations were determined using the Bio-Rad protein reagent assay (Hercules, CA). Western blot analysis of Insig-1 and Insig-2 was performed using 75 µg of liver lysates from wild-type and FXR{alpha}–/– mice treated with either vehicle or GW4064 for 10 d. ß-Actin was used as a load control. Western blot analysis of HMG-CoA reductase was performed as described (47) using an anti-HMG-CoA reductase antibody (48) and 45 µg of pooled microsomes from wild-type mice treated with either vehicle or GW4064 for 4 d (four mice per group). The protein levels of Insig-1, Insig-2, and transferrin receptor were analyzed as previously described (44).

Statistical Analysis
Data are expressed as mean ± SEM as determined by the analysis of multiple independent samples, as indicated in the figure legends. A two-tailed Student’s t test was used to calculate P values. Differences with P values <0.05 were considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Drs. T. Wilson and P. Maloney (GlaxoSmithKline, Research Triangle Park, NC) for providing the GW4064 compound and Dr. M. Brown (University of Texas Southwestern Medical Center, Dallas, TX) for providing antibody to Insig-1 and -2.


   FOOTNOTES
 
This work is supported by National Institutes of Health Grants HL30568, HL68445 (to P.A.E.), Beginning Grant-in-Aid 0565173Y from AHA (to Y.Z.), and Public Health Service National Research Service Award GM07185 (to F.Y.L.).

The authors have nothing to disclose.

First Published Online April 17, 2007

Abbreviations: BSEP, Bile salt export pump; CDCA, chenodeoxycholic acid; CHO, Chinese hamster ovary; CoA, coenzyme A; DMSO, dimethyl sulfoxide; FXR, farnesoid X receptor; GFP, green fluorescent protein; GW4064, GlaxoSmithKline compound no. 4064, synthetic FXR{alpha} ligand; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A; IR-1, inverted repeat-1 separated by one nucleotide; PLTP, phospholipid transfer protein; PXR, pregnane X receptor; RT-qPCR, real-time quantitative PCR; RXR, retinoid X receptor; SCAP, SREBP cleavage-activating protein; SHP, small heterodimer partner; SREBP, sterol regulatory element-binding protein; VP16, herpes simplex virus transactivation domain VP16.

Received for publication February 16, 2007. Accepted for publication April 9, 2007.


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Nuclear Receptors:   SHP  |  FXRα
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