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Originally published In Press as doi:10.1074/jbc.M302387200 on July 10, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36176-36182, September 19, 2003
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Sterol Regulatory Element-binding Protein-2 Interacts with Hepatocyte Nuclear Factor-4 to Enhance Sterol Isomerase Gene Expression in Hepatocytes*

Koichi Misawa {ddagger}, Taro Horiba {ddagger}, Naoto Arimura {ddagger}, Yuko Hirano {ddagger}, Jun Inoue {ddagger}, Noriaki Emoto §, Hitoshi Shimano ¶, Makoto Shimizu {ddagger} and Ryuichiro Sato {ddagger} ||

From the {ddagger}Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, the §Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University of Graduate School of Medicine, Kobe 650-0017, and the Division of Metabolism and Endocrinology, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan 305-8575

Received for publication, March 7, 2003 , and in revised form, July 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the course of an effort to identify unknown targets genes for sterol regulatory element-binding proteins (SREBPs) by PCR, the gene for ATP citrate-lyase was determined to be one such gene. (Sato, R., Okamoto, A., Inoue, J., Miyamoto, W., Sakai, Y., Emoto, N., Shimano, H., and Maeda, M. (2000) J. Biol. Chem. 275, 12497–12502). We here report that gene expression of sterol {Delta}8-isomerase (SI), which catalyzes the conversion of the 8-ene isomer into the 7-ene isomer in the last steps of the cholesterol biosynthetic pathway, is regulated by SREBPs, mainly by SREBP-2. Luciferase assays using the promoter of the human SI gene revealed that a 200-base pair segment upstream region from the transcription start site contains functional elements required for the activity of the SREBPs, Sp1 and NF-Y. Interestingly, SI gene expression was well regulated by sterols in Caco-2 and HepG2 cells, in contrast with HEK293 and HeLa cells. Overexpression of hepatocyte nuclear factor (HNF)-4 in HEK293 cells augmented expression of SREBP-responsive genes including the SI gene, whereas inactivation of HNF-4 by small interfering RNAs in HepG2 cells reduced the SI gene promoter activity. The in vitro pull-down and in vivo co-immunoprecipitation experiments showed the direct interaction between SREBP-2 and HNF-4. These data provide a novel pathway by which HNF-4 potentiates the SREBP functions and stimulates expression of SREBP-responsive genes in enterohepatic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
SREBPs1 are synthesized as membrane-bound precursor proteins. As long as intracellular sterol concentrations are sufficient, SREBPs remain bound to the endoplasmic reticulum and the nuclear envelope. Upon sterol deprivation, the NH2-terminal portion containing the basic helix-loop-helix leucine zipper domain is cleaved by a two-step process that occurs mainly in the Golgi apparatus (1). The mature SREBPs are then transported into the nucleus in a {beta}-importin-dependent manner (2) and stimulate the transcription of genes involved in cholesterol and fatty acid metabolism. The SREBP family comprises three subtypes: SREBP-1a and SREBP-1c, which are derived from a single gene by alternative promoters and splicing, and SREBP-2, which is derived from a different gene. In addition to the sterol regulatory cleavage system, rapid degradation by the ubiquitin-proteasome pathway and another post-translational modification, sumoylation, also regulate SREBP activity in the nucleus (3, 4). All of the data reported so far support the notion that SREBP-1c and SREBP-2 mainly regulate the transcription of genes involved in fatty acid synthesis and cholesterol metabolism, respectively, whereas SREBP-1a regulates both (5–7). SREBP-1a is the more potent activator of transcription than SREBP-1c because of a more extensive transactivation region but is expressed at a much lower level than SREBP-1c in most organs.

We have identified several SREBP-responsive genes by a PCR subtraction method using a Chinese hamster ovary (CHO) cell line (CHO-487) expressing a nuclear form of human SREBP-1a with a LacSwitch inducible mammalian expression system (8). The benefit of this cell line is that it affords a higher induction of target genes, which is a crucial feature so as not to overlook any candidates, rather than a CHO cell line expressing SREBP-1c, which does not afford such a high induction level. Furthermore, the inducible genes in this cell line likely include those that are SREBP-2-responsive. Through this method we have detected SREBP-responsive genes such as the 3-hydroxy-3-methylglutaryl (HMG) CoA synthase and the stearoyl CoA desaturase-1 genes and further the ATP-citrate lyase gene (8), which catalyzes acetyl CoA synthesis. We also detected insulin-inducing gene-1, which was recently demonstrated to be a novel SCAP-binding protein regulating the proteolytic activation of SREBPs (9, 10). In the course of screening for SREBP-responsive genes, we have cloned and characterized the 5'-flanking region of the human SI gene.

Cholesterol biosynthesis in mammals requires more than 30 enzymes, and most of this enzymatic activity is under sterol-mediated feedback control (11). In the later stage of this pathway, SI catalyzes the conversion of the 8-ene isomer into the 7-ene isomer. In addition to its role in sterol isomerization, SI also functions as a multidrug-binding protein for various drugs, including the Ca2+ antagonist emopamil, the immunosuppressant SR31747A, and the antiestrogen tamoxifen (12–15). Furthermore, recent genetic analyses revealed that mutations in the SI gene cause X-linked dominant Conradi-Hünermann syndrome in humans and Tattered in mice, which is one of the disorders with aberrant punctate calcification in cartilage or chondrodysplasia punctata (16, 17). Clinical and biochemical data on the Conradi-Hünermann syndrome patients show an accumulation of 8(9)-cholesterol and 8-dehydrocholesterol in plasma because of a defect at C8-C7 (8-ene–7-ene) isomerization. Although SI is an important enzyme possessing unique features in the cholesterol biosynthesis pathway, to date little is known about its regulation. A lone previous paper has addressed the sterol-mediated regulation of SI mRNA levels (18). Here we report that the SI gene promoter, like other SREBP-responsive genes, contains a cluster of essential responsive elements for SREBPs, Sp1 and NF-Y. Interestingly, the promoter responds well to sterol depletion in HepG2 cells but not in HEK293 cells, and overexpression of HNF-4 in HEK293 cells improves the responsiveness. Various reporter assays reveal that co-expression of SREBP-2 and HNF-4 synergistically stimulates luciferase gene transcription driven by the promoters of several SREBP-responsive genes including the SI gene. Moreover, it is confirmed that HNF-4 is able to interact with SREBP-2. Our findings provide new insights into the complex network between the SREBPs and nuclear receptors that regulate lipid homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cholesterol, 25-hydroxycholesterol and lipoprotein-deficient serum were purchased from Sigma. Restriction enzymes were obtained from New England Biolabs.

Northern Blot Analysis—The stable CHO cell lines, CHO-Lac expressing the Lac repressor, CHO-487 expressing a nuclear form of human SREBP-1a, and CHO-481 expressing a nuclear form of human SREBP-2 were described previously (3, 8). HepG2, Caco-2, and HEK293 cells were set up on day 0 in medium A (Dulbecco's modified Eagle's medium, 100 units/ml penicillin, and 100 µg/ml streptomycin) supplemented with 2.5% fetal calf serum. On day 1, the medium was removed, and the cells were then washed with phosphate-buffered saline and refed with medium A containing 5% lipoprotein-deficient serum supplemented either with 1 µg/ml 25-hydroxycholesterol plus 10 µg/ml cholesterol (the sterol-loaded conditions) or with a 50 µM concentration of a HMG CoA reductase inhibitor (pravastatin) plus 50 µM sodium mevalonate (the sterol-depleted conditions). After 48 h of culture, total RNA was extracted, and Northern blotting was performed as described previously (3, 19). A 300-bp fragment from human HMG CoA synthase, an 800-bp fragment from human fatty acid synthase, a 530-bp fragment from human SI, a 700-bp fragment from 36B4 (3), and a 330-bp fragment from human squalene synthase were used as templates for 32P-labeled probes.

Transgenic Mice Study—Transgenic mice were as described previously (5–7). All of the mice were housed in a controlled environment with a 12-h light/dark cycle and free access to water and food. The animals of each group (n = 3) were fed a high protein/low carbohydrate diet for 2 weeks and fasted overnight prior to sacrifice. An equal aliquot of liver total RNA was pooled (15 µg) and subjected to Northern blot analysis with cDNA probes for human SI. The resulting bands were quantified by exposure to the filters of a BAS2000 with BAStation software (Fuji Photo Film, Co., Ltd.), and the results were normalized to the signal generated from 36B4 mRNA.

Construction of Plasmids—To generate constructs of human HNF-4{alpha}2 (pHNF4 and pFLAGHNF4), a 1.5-kilobase pair EcoRI-NotI fragment obtained by reverse transcription-PCR using total RNA from HepG2 was ligated into a pME18S vector (20) or p3XFLAG-CMV-7.1 (Sigma). An expression plasmid pGal4-SREBP2 was constructed by inserting the fragment for amino acid 2–481 of SREBP-2 into the expression plasmid pM-His, which was engineered from a pM vector (Clontech) to contain an in-frame NH2-terminal His epitope tag (MRGS(H)6). To generate siRNA expression constructs, pSiHNF4 and pSi, one of the 19-bp fragments (5'-CCCTCGTCGACATGGACAT-3' for human HNF-4{alpha}2 and 5'-AGGGAGGGCCATGTCCCTA-3' as an unrelated control, respectively) was inserted into pSilencer1.0-U6 (Ambion) according to the manufacturer's protocol.

Construction of the Reporter Genes for Luciferase Assays—The luciferase reporter plasmids were constructed by cloning the BglII-HindIII PCR fragments coding the 5'-flanking region of human SI gene into the same restriction sites of a pGL3 basic vector (Promega). To generate pSI-2000, pSI-300, pSI-203, pSI-169, and pSI-93, PCR primers were designed to hybridize at the corresponding position (see Fig. 2) and were coupled with the common downstream primer from nucleotide +68. All of the mutant reporter constructs were synthesized by a PCR-assisted method using a site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. All of the mutant SRE sites were replaced by a NotI sequence (GCGGCCGC), and both a GC site and the CCAAT boxes were replaced by an EcoRI sequence (GAATTC). To generate the pG5Luc reporter plasmid, five copies of Gal4-binding sites in the pG5CAT (Clontech) were transferred to pGL3-Basic (Promega).



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  		FIG. 2.
Nucleotide sequence of the 5'-flanking region of the human SI gene. The transcription start site is +1. The putative regulatory cis-elements (six SRE sites, three CCAAT (CAT1, CAT2, and CAT3) boxes, and one GC box) are underlined. The sites used for preparation of truncated reporter gene constructs are indicated by the arrows.

 

Reporter Assays—Reporter assays were performed as described previously (19–21). HEK293 cells were transfected with 0.2 µg of the indicated reporter construct and 0.01 µg of pRL-CMV, an expression plasmid encoding Renilla luciferase (Promega). HepG2 cells were transfected with 2 µg of the indicated reporter construct and 0.1 µg of pRL-CMV and then cultured under either the sterol-depleted or sterol-loaded conditions. After incubation for 48 h, the Dual-LuciferaseTM reporter system (Promega) was used to determine luciferase activity.

Gel Mobility Shift Assay—A 32P-labeled probe (5'-AAAATCACCCCACTGC-3') corresponding to the SRE of the human low density lipoprotein (LDL) receptor gene was incubated with 1.7 µg of recombinant SREBP-2 (1–481) for 20 min at room temperature in 20 µl of binding buffer (12.5 mM Hepes-KOH, pH 7.5, 6 mM MgCl2, 5.5 mM EDTA, 50 mM KCl, 0.5 mM dithiothreitol, 0.25 mg/ml nonfat dry milk, 50 µg/ml sodium poly(dI-dC)-poly(dI-dC), 5% (v/v) glycerol). In competition assays, an excess amount of an unlabeled 16-bp fragment for wild type or mutant SREs in the human SI promoter was added prior to addition of the labeled probe. Recombinant SREBP-2 (1–481) containing six consecutive histidines were expressed using a pET28 vector (Novagen) in Escherichia coli and purified by nickel-nitrilotriacetic acid-agarose affinity chromatography (Qiagen).

Protein-Protein Interaction Assay—To generate a glutathione S-transferase (GST) expression construct (pGEX6PSBP2), the fragment for amino acid 1–481 of SREBP-2 was ligated into pGEX-6P (Amersham Biosciences). The fusion protein, GST-SREBP2 (1–481), and GST were expressed in E. coli and bound to glutathione-Sepharose 4B (Amersham Biosciences) beads following the manufacturer's instructions. Using the TNT-coupled transcription/translation kit (Promega), 35S-labeled HNF-4 was synthesized according to the manufacturer's protocol. 25 µl of either GST- or the GST fusion protein-coupled resin, 10 µl of the in vitro translation reaction, and 300 µl of binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1 µl/ml protease inhibitor mixture (Sigma)) were incubated at 4 °C for 30 min. The reactions were washed five times with 500 µl of buffer, and the specifically bound proteins were pelleted, resuspended in sample buffer, and analyzed by SDS-PAGE followed by autoradiography. To confirm the in vivo interaction between SREBP-2 and HNF-4, HEK293 cells were transfected with pFLAGHNF4, an expression construct for GST-SREBP2 (2–481), and pGSTSREBP2 (2–481; Ref. 4), and proteins bound to glutathione-Sepharose beads were subjected to Western blot analysis (4, 19) using anti-FLAG antibodies (M2; Sigma). Alternatively, the immunoprecipitates with anti-HNF-4 antibodies (H-171, Santa Cruz) from the nuclear extracts of HepG2 cells cultured under the sterol-depleted conditions were subjected to Western blot analysis using anti-SREBP-2 antibodies RS004 (21).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SREBP-2 Preferentially Regulates the SI Gene Expression— Through a PCR subtraction method we obtained several PCR fragments induced by SREBP-1a including fatty acid synthase, HMG CoA synthase, and ATP citrate lyase cDNA (8). One of these fragments was found to have a 90% homology with human phenylalkylamine Ca2+ antagonist-binding protein (emopamil-binding protein), which recently turned out to be identical to SI, an essential enzyme of the sterol biosynthesis pathway (12, 13). Northern blot analyses revealed that expression of SI mRNA was up-regulated 4-fold in response to sterol depletion in human hepatoma cells, HepG2 cells (data not shown). To assess which subtype of SREBPs, SREBP-1a or SREBP-2, preferentially regulates SI gene expression, Northern blot analysis was performed using total RNA from stable CHO cell lines, CHO-487 and CHO-481, expressing a mature form of either human SREBP-1a or SREBP-2 with a LacSwitch inducible mammalian expression system, respectively. These stable cells were cultured with sterols to suppress the activation of endogenous SREBPs and with isopropyl-{beta}-D-thiogalactopyranoside to induce expression of exogenous SREBPs. Fig. 1A shows that the induction of SREBP-1-responsive gene, fatty acid synthase, was significantly higher in CHO-487 cells, whereas a remarkable increase in squalene synthase, an SREBP-2 responsive gene (22, 23), was observed in CHO-481. Expression of SI mRNA was induced more in CHO-487 cells, suggesting that SREBP-2 preferentially regulates SI gene expression. Further evidence was provided by Northern blot analyses using total RNA in the livers of SREBP-1a (TgBP-1a), SREBP-1c (TgBP-1c), and SREBP-2 transgenic (TgBP-2) mice. The highest expression of mRNA for SI was observed in TgBP-2 mice, which expressed a mature form of human SREBP-2 (Fig. 1B). Taken together, these data suggest it is likely that SREBP-2 is a major regulator of the expression of the SI gene, although it was originally found to be one of SREBP-1-responsive genes in the current experiment.



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  		FIG. 1.
Northern blot analysis for the SI gene in CHO stable cells and in the livers from wild type (WT) and TgBP-1a, -1c, and -2 mice. A, CHO-Lac (Control), CHO-487 (SREBP-1a), and CHO-481 (SREBP-2) cells were cultured with 1 µg/ml of 25-hydroxycholesterol, 10 µg/ml of cholesterol, and 1 mM isopropyl-{beta}-D-thiogalactopyranoside for 19 h. Total RNA (20 µg/lane) was subjected to electrophoresis and blot hybridization with the indicated 32P-labeled probe. FAS, fatty acid synthase; SQS, squalene synthase. An 800-bp fragment from human fatty acid synthase, a 530-bp fragment from human SI, a 700-bp fragment from 36B4, and a 330-bp fragment from human squalene synthase were used as templates for 32P-labeled probes. The resulting bands were quantified by exposure of the filters to a FluorImage Analyzer with Image Gauge (Fuji Film), and the results were normalized to the signal generated from 36B4 mRNA. In three separate experiments the same relative mRNA levels were obtained. B, animals of each group (n = 3) were fed a high protein/low carbohydrate diet for 2 weeks and fasted overnight prior to sacrifice. An equal aliquot of liver total RNA was pooled (15 µg) and subjected to Northern blot analysis.

 

Human SI Promoter Sequence—To study the direct effect of SREBP-2 on transcription, we cloned the 5'-flanking region of the human SI gene. After we cloned a 2-kb fragment, the nucleotide sequence around the SI gene (AF196972 [GenBank] ) was submitted to the GenBankTM/EBI Data Bank. The sequence in Fig. 2 (accession number AB103460 [GenBank] ) has a 99.7% homology with AF196972 [GenBank] , and no mismatch was observed in the region -300 bp upstream of the transcription start site. The nucleotide start site was determined by 5' rapid amplification of cDNA ends analysis of the SI mRNA. The 5' end of the longest clone (three of nine clones) is numbered +1. The -300-bp region contains six potential SRE-like motifs, three CCAAT boxes (CAT1, CAT2, and CAT3), and a GC box, which are present as a cluster in the promoter region of most SREBP-responsive genes for coordinate regulation of the genes by SREBPs and one of the ubiquitous transcription factors, NF-Y and Sp1 (8, 20, 24).

Sterol-mediated Transcriptional Regulation of the SI Gene—To determine whether SREBP-2 directly regulates the transcription of the SI gene, we carried out luciferase assays using reporter genes including various versions of the human SI 5'-flanking region. HepG2 cells were transfected with each reporter gene and cultured under either sterol-loaded or -depleted conditions for 2 days. As long as the reporter gene contained the segment from -203 to +68 bp, sterol depletion resulted in an increase in luciferase activities more than 6-fold (Fig. 3). Otherwise, in the absence of this segment, the reporter gene pSI93 no longer responded to sterol depletion, suggesting that the segment from 300 to 94, at least, contains functionally essential elements required for the SREBP-dependent regulation of the SI gene expression.



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  		FIG. 3.
Regulation of SI promoter-luciferase reporter genes by sterols. HepG2 cells in a 35-mm dish were transfected with one of the human SI promoter reporter genes (2 µg) and pRL-CMV (0.1 µg). The cells were cultured under either sterol-loaded or sterol-depleted conditions for 48 h and then lysed, and enzyme activity was determined. The ratio of firefly luciferase to Renilla luciferase activity is given as a normalized luciferase activity. The values given are the averages of data from more than three experiments performed in triplicate.

 

The Elements Responsible for the Sterol-mediated Regulation—The promoters analyzed thus far in sterol-regulated genes contain NF-Y or Sp1 sites adjacent to SREs, and these co-factors have been shown to be indispensable for SREBP-dependent regulation (8, 20, 24, 25). Fig. 4A shows that mutation of either the GC box (GCKO) or the CAT2 sequence (CAT2KO) caused a significant reduction of the response to sterol depletion, and a modest reduction was also observed by mutation in the CAT3 sequence (CAT3KO). This suggests that both the Sp1 site (GC) and the NF-Y site (CAT2) are crucially involved in sterol-mediated induction of the SI promoter. When a mutation was introduced into the SREa or SREc site, a substantial decrease in the fold activation by sterol depletion was observed (Fig. 4B). Mutation of either the SREb or SREd site caused a slight decrease but retained a high induction level (more than 4-fold). Together, these data demonstrate that sterol-mediated induction of the SI promoter is attributable to the NF-Y (CAT2) and Sp1 sites as well as the SREa and SREc sites.



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  		FIG. 4.
Effect of the mutation of the GC box, the CCAAT box and the SRE on sterol-mediated regulation of reporter genes. A and B, HepG2 cells were transfected and cultured as described in the legend to Fig. 3. Reporter gene constructs are shown on the left. Mutated regulatory elements are indicated in an open box (GC), open circles (CAT), and closed circles (sterol regulatory element, SRE). The fold activation (luciferase activity under the sterol-depleted versus the sterol-loaded conditions) is shown. The values given are the averages of data from more than three experiments performed in triplicate.

 

Next, gel mobility shift assays were performed to demonstrate the direct binding of SREBP-2 to the SRE sites. Fig. 5A shows that the SRE probe corresponding to the SRE in the human LDL receptor promoter was shifted after the addition of the SREBP-2 protein (first lane). In competition assays, the shifted band completely disappeared upon the addition of an excess amount of the unlabeled SREc probe (seventh lane) but was only slightly diminished by the SREa and SREd probes. Mutated probes did not compete with the labeled probe, but wild type probes did (Fig. 5B), suggesting that the competition observed in Fig. 5A is attributable to the specific nucleotide sequence in the SREc site. These data indicate that SREBP-2 activates the SI promoter mainly through binding to the SREc but partly also through the SREa, which is consistent with the results of the luciferase assays (Fig. 4B).



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  		FIG. 5.
SREBP-2 binds to the SRE sequence in the SI promoter. A 32P-labeled probe corresponding to the SRE of the human LDL receptor gene was incubated with recombinant SREBP-2 (1–481). A, in competition assays, an increasingly excessive amount (x100, x300, and x600) of an unlabeled 16-bp fragment for the SREs (SREa, SREc, and SREd in Fig. 2) in the human SI promoter was added prior to addition of the labeled probe. B, i5n competition assays, a 600-fold molar excess of an unlabeled 16-bp fragment was added. M, mutant; W, wild type.

 

Regulation of SI Gene Expression in Several Different Cell Lines—When HEK293 cells were transfected with various reporter genes including the SI gene promoter, we found that the luciferase activity was only slightly increased in response to sterol depletion in contrast to a marked rise (more than 8-fold in Fig. 4A) in HepG2 cells. Indeed, the mRNA levels of the SI gene in HEK293 cells were not significantly induced by sterol depletion (1.2-fold) as compared with a 4-fold induction in HepG2 and Caco-2 cells (Fig. 6). The increase in the HMG CoA synthase mRNA levels in HEK293 cells under the sterol-depleted conditions was also smaller than those in other cells but was still substantial (4.8-fold). The same tendency was observed in HeLa cells (data not shown).



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  		FIG. 6.
Northern blot analysis for the SI and HMG CoA synthase (HMG syn) gene in HepG2, Caco-2, and HEK293 cells. Cells were cultured under either sterol-loaded (+) or sterol-depleted conditions (-) for 48 h. Northern blot analysis was carried out as described in the legend to Fig. 1.

 

HNF-4 Stimulates SREBP-responsive Promoter Activity— The above observation allowed us to hypothesize that certain factor(s) such as HNF-4 or HNF-1 in these cells might ameliorate the low level of SREBP activity. Therefore we next determined whether the SI promoter reporter gene, pSI2000, responded to sterol depletion in HEK293 cells when transfected with an expression plasmid for HNF-4. Fig. 7A shows that the fold activation of the SI promoter activity in response to sterol depletion increased by expression of HNF-4 in a dose-dependent manner. It is possible that this HNF-4-mediated induction might be attributed to a direct transcriptional regulation of the SI gene through an as yet unknown HNF-4-responsive element in the SI promoter. However, overexpression of HNF-4 was not able to induce the SI promoter directly when the nuclear SREBPs levels were low under the sterol-loaded conditions, whereas both HNF-4 and SRBP-2 synergistically stimulated the luciferase activities (Fig. 7B). Surprisingly, the same results were obtained with both the HMG CoA synthase and LDL receptor promoter reporter genes. These data indicate that HNF-4 can activate the promoter of the SREBP-responsive genes coordinately with SREBP-2.



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  		FIG. 7.
Effect of HNF-4 on the sterol-mediated or SREBP-dependent expression of reporter genes. A, HEK293 cells were transfected with 0.2 µg of pSI2000, 0.01 µg of pRL-CMV, and the indicated amount of pHNF4, an expression plasmid for human HNF-4{alpha}2. The cells were cultured under either sterol-loaded or sterol-depleted conditions for 48 h and then lysed, and enzyme activities were determined. The fold activation (luciferase activity under the sterol-depleted versus the sterol-loaded conditions) is shown. The values given are the averages of data from more than three experiments performed in triplicate. B, HEK293 cells were transfected with 0.2 µg of the indicated reporter gene and 0.01 µg of pRL-CMV together with either 0.1 µg of pSREBP2 (1–481) or 0.5 µg of pHNF4, or both. pHMG syn, human HMG CoA synthase; pLDLR, human LDL receptor. The cells were cultured under sterol-loaded conditions for 48 h. The promoter activities in the absence of SREBP-2 (1–481) and HNF-4 are represented as 1. The values given are the averages of data from more than three experiments performed in triplicate. C, HEK293 cells were transfected with 0.2 µg of pG5Luc, 0.1 or 0.3 µg of pGAl4-SREBP2 (an expression plasmid for a fusion protein, the Gal4 DNA-binding domain, and SREBP-2 (1–481)) or pGal (an empty vector, pM), and 0.01 µg of pRL-CMV together with the indicated amount of pHNF4. Promoter activities (0.3 µg of pGAl4-SREBP2) in the absence of HNF-4 are represented as 1. The values given are the averages of data from more than three experiments performed in triplicate. D, HepG2 cells were transfected with 0.2 µg of pSI2000, 0.01 µg of pRL-CMV, and 0.8 µg of siRNA expression constructs, pSiHNF4 and pSi. The cells were cultured with medium A containing 2.5% fetal calf serum for 24 h and further cultured under the sterol-depleted conditions for 48 h. Promoter activities when transfected with 0.8 µg of pSi are presented as 100%. The values given are the averages of data from more than three experiments performed in triplicate.

 

To determine the direct effect of HNF-4 on the transcriptional activity of SREBP-2, we employed a heterologous Gal4 system. In this assay luciferase gene transcription is driven by a promoter that contains five consensus Gal4-binding sites, without any co-regulatory transcription factors such as Sp1 and/or NF-Y. HEK293 cells were transfected with a reporter plasmid, pG5Luc, and an expression plasmid encoding SREBP-2 coupled to the DNA-binding domain of yeast Gal4. Although an expression plasmid encoding only the Gal4 DNA-binding domain, pGal4, did not promote luciferase activities, Gal4-SREBP2 significantly enhanced transcription of the reporter gene with an increasing expression of HNF-4 (Fig. 7C). These results suggest that HNF-4 accelerates transcription of SREBP target genes by stimulating the transcriptional activity of SREBP-2, not because of a strengthening of the DNA affinity of SREBP-2 or the interaction between SREBP-2 and the coregulatory transcription factors, Sp1 and NF-Y. Furthermore, the induced SI promoter activity in response to sterol depletion in HepG2 cells significantly decreased by reduction in endogenous HNF-4 gene expression using siRNAs against HNF-4 (Fig. 7D). In these experiments all of the cells were transfected with 0.8 µg of siRNA expression plasmids, pSiHNF4, and/or pSi (unrelated siRNAs). These data indicate the importance of HNF-4 in the regulation of SREBP-responsive gene expression.

Protein-Protein Interaction between SREBP-2 and HNF-4 — We speculated that the direct stimulating effect of HNF-4 is due to its direct binding to SREBP-2. To confirm an interaction between HNF-4 and SREBP-2 proteins, we performed a GST pull-down assay. When GST-SREBP-2-bound agarose beads were incubated with the in vitro translated HNF-4, a 35S-labeled band corresponding to HNF-4 was detected (Fig. 8A, first and second lanes). There was no detectable interaction with the GST control resin (second lane). This indicates that SREBP-2 and HNF-4 interact in solution in the absence of DNA. Fig. 8B shows that HNF-4 is co-precipitable with SREBP-2 when both FLAG-HNF4 and GST-SREBP2 are overexpressed in HEK293 cells. We observed the upper band in the second lane (marked by an asterisk in Fig. 8B) that was not detected in the nuclear extracts (first lane). It is likely that the band was nonspecific, but it is possible that acetylated or phosphorylated HNF-4 (26, 27) might preferentially interact with SREBP-2. To see the direct interaction between endogenous SREBP-2 and HNF-4, co-immunoprecipitation experiments were performed using the nuclear extracts from HepG2 and HEK293 cells cultured under the sterol-depleted conditions. Endogenous SREBP-2 in HepG2 cells was co-immunoprecipitable with HNF-4, whereas SREBP-2 in HEK293 cells was not precipitated in the absence of HNF-4 (Fig. 8C). These results clearly show the direct interaction between HNF-4 and SREBP-2 proteins.



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  		FIG. 8.
The interaction between SREBP-2 and HNF-4. A, GST or GST-SREBP2 (1–481) immobilized on glutathione-Sepharose beads was incubated with in vitro translated and 35S-labeled HNF-4. After the matrix was extensively washed, the labeled proteins retained on the beads were eluted and resolved by SDS-PAGE followed by autoradiography together with 10% of the total radiolabeled protein input used in each binding reaction. B, HEK293 cells were transfected with pFLAGHNF4 and pGSTSREBP2 (2–481). After 48 h of culture, GST fusion proteins were purified with glutathione-Sepharose resins from the nuclear extracts prepared as described previously (4), and subjected to Western blotting (WB) with anti-FLAG antibodies. An asterisk marks the nonspecific band that was not detected in the nuclear extracts. C, HepG2 and HEK293 cells were cultured under the sterol-depleted conditions for 48 h, and then their nuclear extracts were prepared. The immunoprecipitates (IP) with anti-HNF-4 antibodies were subjected to Western blotting with anti-SREBP-2 antibodies.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The current study is the first report showing evidence that SI is one of the SREBP-responsive genes and that the SI promoter contains essential functional elements required for SREBP-2, Sp1, and NF-Y activity. The present finding that expression of the SI gene is regulated by SREBPs is not really surprising, because this gene encodes an enzyme in the cholesterol biosynthesis pathway. But surprising or not, it does provide us with a vital clue to a novel interaction between SREBP-2 and a member of the nuclear receptor superfamily, HNF-4, which is most highly expressed at the highest levels in the liver, intestine, pancreas, and kidney in mammals. In the current experiments we noticed that both the SI gene expression and the SI promoter only weakly responded to sterol depletion in HEK293 cells in contrast to their high response in HepG2 and Caco-2 cells. This led us to hypothesize that certain factor(s) lacking in HEK293 cells might potentiate the SREBP functions in the enterohepatic cells. We examined the stimulating effects of some plausible candidates including HNF-4 and HNF-1, which are highly expressed in the liver and intestine and are tightly involved in the regulation of lipid metabolism in these tissues (28–30), and found that HNF-4, but not HNF-1 (data not shown), augments the transcriptional activity of SREBP-2 via a direct interaction, thereby promoting the induction of SREBP-responsive gene expression in enterohepatic cells. Indeed, the HNF-4 expression levels in HEK293 cells were much less than those in HepG2 and Caco-2 cells (data not shown). Moreover, it is reported that the nuclear thyroid hormone receptor interacts with SREBP-1-enhancing acetyl CoA carboxylase transcription in hepatocytes (31). We also confirmed the stimulating effect of HNF-4 on SREBP-1a activity by reporter assays (data not shown). Based on the fact that the structure of the nuclear receptors is highly conserved among the family members, some members of this family other than just thyroid hormone receptor and HNF-4 might interact with the SREBPs and affect their activities.

The mechanism by which HNF-4 augments the transcriptional activity of SREBPs remains unclear. One possibility is that NHF-4 masks a negatively regulatory domain localizing in the COOH-terminal region of nuclear SREBPs, the truncation of which somehow enhances the transcriptional activities of the SREBPs (32). We indeed found that the COOH-terminal domain containing ~90 amino acids of the nuclear SREBPs does exert a transcriptional repression function when fused to the Gal4 DNA-binding domain plus the SREBP-1a activation domain including the NH2-terminal 50 amino acids (data not shown). HNF-4 might compete with an as yet unidentified factor recruited to the negative regulatory domain, which limits SREBPs functions. In contrast, it has been reported that the SREBPs recruit the cAMP response element binding protein (CREB)-binding protein/p300 as a co-activator for regulating transcription of their target genes (33, 34). Peroxisome proliferator-activated receptor {gamma} co-activator-1 interacts and works with HNF-4 to activate the expression of genes in lipid and glucose homeostasis (35, 36). Moreover, peroxisome proliferator-activated receptor {gamma} co-activator-1 physically interacts with CREB-binding protein in vitro (37). Therefore, it is possible that a protein complex containing the SREBPs, HNF-4, CREB-binding protein, and peroxisome proliferator-activated receptor {gamma} co-activator-1 could effectively form and activate the expression of SREBP-responsive genes in hepatic cells. Now it is under investigation which regions in the SREBPs and HNF-4 molecules are required for mutual interaction and whether a tetrameric protein complex can be formed. Furthermore, it will be of interest to examine whether the SREBPs affect the expression of HNF-4-responsive genes.

Data from the present study indicate that the SREBPs must interact with HNF-4 to be effective in regulating the transcription of cholesterogenic enzymes including SI in enterohepatic tissues. This means that upon sterol deprivation the rate of cholesterol biosynthesis in the liver and intestine is accelerated by the SREBPs and HNF-4 activity. The synergistic activity of the SREBPs and HNF-4 in these organs apparently takes place to distribute lipids to other tissues, which do not have the capacity to biosynthesize sufficient lipids on their own. A recent study that carried out liver-specific disruption of the HNF-4{alpha} gene revealed that HNF-4 is essential for the constitutive expression of several key hepatic genes involved in lipid homeostasis (29, 30, 38). This ostensible role for HNF-4 is in close agreement with our current findings. Further studies will be needed to elucidate the complex network between the SREBPs and the nuclear receptors, not only of HNF-4 but also the peroxisome proliferator-activated receptors, the farnesoid X receptor, and the liver X receptor, all of which play critical roles in lipid homeostasis.


   FOOTNOTES
 
* This work was supported by research grants from the Ministry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. E-mail: aroysato{at}mail.ecc.u-tokyo.ac.jp.

1 The abbreviations used are: SREBP, sterol regulatory element-binding protein; SI, sterol {Delta}8-isomerase; HNF, hepatocyte nuclear factor; siRNA, small interfering RNA; CHO, Chinese hamster ovary; HMG, 3-hydroxy-3-methylglutaryl; LDL, low density lipoprotein; GST, glutathione S-transferase; CREB, cAMP response element binding protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Kevin Boru of Advance Clinical Trials for review of the manuscript



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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