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Sterol Regulatory Element-Binding Protein-1a Binds to cis Elements in the Promoter of the Rat High Density Lipoprotein Receptor SR-BI Gene¹

Departments of Obstetrics and Gynecology and Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, Florida 33606

Address all correspondence and requests for reprints to: Dr. Mark P. McLean, Departments of Obstetrics and Gynecology, 4 Columbia Drive, Room 529, Tampa, Florida 33606. E-mail: mmclean{at}com1.med.usf.edu

	Abstract

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The high density lipoprotein (HDL) receptor, or scavenger receptor class B type I (SR-BI), is critical for cholesterol transport and a potential target for hypercholesterolemic drugs. Thus, elucidation of the mechanism underlying regulation of the HDL receptor SR-BI gene is essential. It has been previously shown that there is a correlation between depletion in ovarian cholesteryl ester content and increased HDL receptor SR-BI expression in response to hormonal stimulation. We wanted to determine whether the levels of mature sterol response element-binding protein-1a (SREBP-1a), a key protein in the transcriptional regulation of several genes by sterols, are affected under these conditions. Thus, Western blot analysis was carried out. Consistent with the possibility that SREBP-1a may be involved in the regulation of the HDL receptor SR-BI gene, we found that mature SREBP-1a levels increased up to 11-fold in the ovary after treatment with 50 U hCG. This increase in mature SREBP-1a protein levels correlated with a 30% decrease in ovarian cholesterol levels. These changes in both SREBP-1a and cholesterol levels preceded a 2-fold induction of HDL receptor SR-BI protein levels. To determine whether SREBP-1a could directly regulate the expression of the rat HDL receptor SR-BI gene, approximately 2.2 kb of the receptor SR-BI promoter were cloned and sequenced, and deletion analysis and mobility shift assays were performed. The results of these studies demonstrate that the rat HDL receptor SR-BI promoter contains two sterol response elements (pSRE and dSRE) through which SREBP-1a can bind and activate transcription of this gene. These motifs are similar to known SRE motifs reported for sterol-sensitive genes, and the pSRE is located between two Sp1 sites, similar to the SRE-1 motif in the low density lipoprotein receptor. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, which inhibits SREBP degradation, enhanced the effect of SREBP-1a on the regulation of the rat HDL receptor SR-BI gene. It has previously been shown that tropic hormones such as hCG can also influence gene expression by increasing cAMP levels. Consistent with this fact, we have recently shown that steroidogenic factor-1 (SF-1) mediates cAMP activation of the HDL receptor SR-BI gene. Thus, we decided to examine whether SREBP-1a could cooperate with SF-1 to enhance transcription this gene. The results confirm that indeed both SF-1 and SREBP-1a synergize to induce HDL receptor SR-BI gene expression.

	Introduction

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IT IS WELL known that plasma concentrations of high density lipoprotein (HDL) cholesterol exhibit an inverse relationship with the incidence of atherosclerosis and coronary heart disease (1). This protective mechanism is thought to be due to the ability of a HDL particle to transport excess cholesterol from cells in the arterial wall to the liver for its disposal as bile acid (reverse cholesterol transport) (2, 3). This lipoprotein is also involved in providing cholesterol to nonplacental steroidogenic tissues, such as the adrenal, ovary, and testis, for steroid hormone synthesis (4, 5). HDL cholesterol is delivered to these tissues by a mechanism called selective lipid uptake, a process that differs from the low density lipoprotein (LDL) uptake pathway in that the HDL uptake pathway does not involve endocytosis and lysosomal degradation of the lipoprotein particle (6, 7). Instead, cholesterol (primarily in the form of cholesteryl esters) is selectively transported into the cell from a bound HDL, which is then released into the extracellular fluid after its lipid content has been depleted (6, 7).

The selective cholesteryl ester uptake from a HDL particle by the liver and steroidogenic tissues has been shown to be mediated by the recently identified HDL receptor, or scavenger receptor class B type I (SR-BI) (8, 9, 10, 11, 12). This receptor has been shown to be the major determinant of plasma HDL concentrations (8, 9, 10, 11, 12), and hence, it is a key element in cholesterol homeostasis. It has also been proposed that the HDL receptor SR-BI could be considered a potential target for hypercholesterolemic drugs (13, 14). Based on this, elucidating the molecular mechanism involved in the regulation of the HDL receptor SR-BI gene expression is extremely important. One possible regulator of this gene appears to be cholesterol. Several reports have shown that increased adrenal HDL receptor SR-BI gene expression was associated with depleted cholesterol stores resulting from a deficiency in apolipoprotein A-I (15), hepatic lipase (15), or lecithin cholesterol acyl transferase (16) genes. These effects were not restricted to the adrenal gland, because reductions in ovarian cholesterol levels due to LH-induced desensitization also caused up-regulation of the HDL receptor SR-BI gene (17). In fact, depletion of ovarian cholesteryl ester content and increased cell surface HDL binding in response to hormonal stimulation were reported even before the HDL receptor SR-BI was identified (18, 19). On the contrary, feeding rats a high cholesterol diet for 2 weeks lowered HDL receptor SR-BI expression in liver parenchymal cells (20). These changes in HDL receptor SR-BI expression appear to be accounted for by changes in receptor messenger RNA (mRNA) levels (16) suggesting possible transcriptional regulation of this gene by cholesterol.

Transcriptional regulation by cholesterol has been reported for several genes (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). Each of these sterol-sensitive genes contains at least one sterol regulatory element (SRE) in their promoter, through which a pair of proteins, known as sterol regulatory element-binding protein-1a and -2 (SREBP-1a and -2), activate transcription (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). SREBPs are synthesized as 125-kDa precursors that are attached to the endoplasmic reticulum membrane and the nuclear envelope (34, 35, 36). In sterol-depleted cells, a two-step proteolytic process releases the 65-kDa NH₂-terminal segment of the SREBPs, which then enters the nucleus, binds to the SRE, and activates transcription of the target gene (34, 35, 36). The importance of these proteins in activating transcription of several SRE-containing genes has been clearly demonstrated in experiments using transgenic mice overexpressing the NH₂-terminal segment of SREBP-1a (37). In these mice, the levels of sterol-sensitive genes were increased severalfold over control levels (37).

Sequence analysis of the human HDL receptor SR-BI promoter has revealed the presence of a 9-bp sequence (38) containing an E box previously shown to bind SREBP-1a (39). In addition, the mRNA for both SREBPs (-1 and -2) have been found in the adrenal and liver (35, 36). These results suggest that SREBPs may be involved in the regulation of the HDL receptor SR-BI gene.

In view of the current data, we cloned the rat HDL receptor SR-BI promoter and examined whether this gene is directly controlled by SREBP-1a. We found that SREBP-1a induces the expression of the HDL receptor SR-BI gene. This investigation indicates that intracellular cholesterol levels may control HDL receptor SR-BI gene transcription, like other sterol-sensitive genes. In addition, we investigated whether SREBP-1a can cooperate with steroidogenic factor-1 (SF-1) in controlling HDL receptor SR-BI transcription.

	Materials and Methods

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Materials
The Advantage Tth polymerase mix and the PromoterFinder DNA Walking Kit were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). All oligonucleotides and primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The pGL3 basic luciferase vector, Renilla luciferase vector, and the Dual Luciferase Reporter Assay System were obtained from Promega Corp. (Madison, WI). The African green monkey kidney COS-7, human liver HepG2, rat liver Clone 9 (L9), Chinese hamster ovary CHO-K1, mouse adrenal Y1, and human bladder HTB-9 cell lines were obtained from American Type Culture Collection (Manassas, VA). The QuickChange Site-directed Mutagenesis Kit was purchased from Stratagene (La Jolla, CA). The NH₂-terminal segment (active fragment) of SREBP-1a under the control of the cytomegalovirus (CMV) promoter (SREBP-1a-pCMV5) was provided by Dr. Tim Osborne (Department of Molecular Biology and Biochemistry, University of California-Irvine). The murine steroidogenic factor-1 (SF-1) complementary DNA (cDNA) under control of CMV promoter (SF-1-pCMV) was provided by Dr. Kieth L. Parker (University of Texas Southwestern Medical Center, Dallas, TX). [-³²P]Deoxy (d)-CTP (3000 Ci/mmol) and the T7 Sequenase DNA Sequencing Kit were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). [-³²P]ATP (3000 Ci/mmol) and [³⁵S]dATP (1000–1500 Ci/mmol) were obtained from DuPont/New England Nuclear (Wilmington, DE). pCR 2.1 TA cloning vector was purchased from Invitrogen (San Diego, CA). All restriction enzymes and the calpain inhibitor I [N-acetyl-leucyl-leucyl-norleucinal (ALLN)]) were obtained from Roche Diagnostic Corp. (Indianapolis, IN). DMEM-nutrient mixture F-12 (DMEM/F12) and the 5'-Rapid Amplification of cDNA Ends Kit were obtained from Life Technologies, Inc./BRL (Grand Island, NY). FBS was purchased from Summit Biotechnology (Ft. Collins, CO). Poly(dI-dC) was obtained from Pharmacia Biotech (Piscataway, NJ). BioMax-MR films were obtained from Fisher Scientific (Norcross, GA). The rabbit polyclonal anti-SREBP-1 and the horseradish peroxidase-conjugated goat antirabbit antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The SuperSignal ULTRA Chemiluminescent Substrate was obtained from Pierce Chemical Co. (Rockford, IL). Nitrocellulose membrane was obtained from Schleicher & Schuell, Inc. (Keene, NH). Rivastatin from Merck & Co. (Rahway, NJ) was provided by Dr. Gene C. Ness (Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, FL). All other chemicals were purchased from Fisher Scientific or Sigma (St. Louis, MO).

Animals
Twenty-eight-day-old Sprague Dawley rats were purchased from Harlan Industries (Madison, WI). All protocols to treat animals were approved by the University of South Florida animal care committee. Throughout the experiment, animals had free access to food and water and were housed under a 12-h dark, 12-h light cycle. Follicular development and ovulation were induced in rats by injection of 8 IU PMSG. By this method, rats ovulate approximately 72 h after treatment with PMSG (40). Ten days postovulation, rats were injected with 50 U hCG. Ovaries, which consist mainly of luteal tissue using this protocol (40), were removed before hCG injection (controls) and at 3, 6, 12, and 24 h post-hCG treatment.

Western blot analysis
Ovarian tissue (100–150 mg) was homogenized in 1.5 ml ice-cold homogenization buffer, as previously described (41). Ovarian homogenates were assayed for protein concentration by the method developed by Bio-Rad Laboratories, Inc. (Hercules, CA), using BSA as the standard. Ovarian proteins (50 µg) were denatured at 100 C in loading buffer (42) for 10 min and subjected to electrophoresis on a 7.5–18% gradient SDS-PAGE according to the method of Laemmli (43). After electrophoresis, samples were electroblotted onto nitro-cellulose membranes (0.2 µm pore) in buffer containing 0.25 M Tris-HCl (pH 8.3) and 1.92 M glycine for 16 h at 4 C. To verify equal protein loading, nitro-cellulose membranes were stained with 0.1% Ponceau S (in 5% acetic acid) and destained in water. Western blot analysis of SREBP-1a protein was carried out with a 1:1000 dilution of the rabbit polyclonal antiserum to SREBP-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in 3% milk. Western blot analysis of HDL receptor SR-BI protein was performed using a rabbit polyclonal antiserum (Research Genetics, Inc., Huntsville, AL) raised against amino acids 489–509 (AYSESLMSPAAKGTVLQEAKL) of the rat protein (44). HDL receptor SR-BI protein immunoreactivity was identified at its molecular size of 82 kDa. Immunoreactive proteins were then visualized using a 1:10,000 dilution of horseradish peroxidase-conjugated goat antirabbit antisera (Santa Cruz Biotechnology, Inc.) in 3% milk and the SuperSignal ULTRA Chemiluminescent Substrate method (Pierce Chemical Co.). Immunoreactive actin protein detected using a rabbit polyclonal antibody (Sigma; 1:1000 dilution in 3% milk) was used as an internal control.

Determination of cholesterol levels
Ovarian tissue (10–70 mg) was homogenized in 500 µl chloroform-methanol (2:1). After homogenization, 125 µl 0.15 M NaCl were added, and samples were centrifuged at 2000 x g for 15 min. The lower phase was used in cholesterol determination. Total cholesterol levels were determined using the Cholesterol 20 Kit (Sigma) according to the manufacturer’s instructions. Lipid Lin-Trol (Sigma) was used as the assay standard.

Oil Red O lipid staining
Oil Red O lipid staining of ovarian cryosections was carried out as previously described (45). Briefly, unfixed sections were placed in Oil Red O staining solution for 5 min. Sections were then washed five times in distilled water and counterstained in hematoxylin for 1 min. Cryosections were washed twice with acid water (2 ml concentrated HCl in 250 ml water), twice with saturated aqueous sodium bicarbonate solution, and eight times with distilled water. Oil Red O lipid staining was determined using an Olympus Corp. BX60 microscope (Melville, NY).

Cloning of the HDL receptor SR-BI promoter
Cloning of the HDL receptor SR-BI promoter was performed using the Advantage Tth Polymerase Mix and the PromoterFinder DNA Walking Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). For this, two nested primers, 1GSP1 (¹³⁵CTTGATGAGCGAGGGCACCATGAG¹⁵⁹) and 1GSP2 (¹⁰⁵GATAACGCCGAGTGCAGCACACAG¹²⁹), were designed to the rat HDL receptor SR-BI cDNA sequence (44, 46). These primers were then used with adaptor-specific primers in screening five separate adaptor-ligated rat genomic libraries by PCR. The conditions for PCR were 7 cycles of denaturation at 94 C for 2 sec and elongation at 72 C for 3 min, followed by 40 cycles of denaturation at 94 C for 2 sec and elongation at 67 C for 3 min. Nested PCR products were analyzed by 1.2% agarose/ethidium bromide gel electrophoresis. A second round of nested PCR reactions was carried out using primers 2GSP1 (5'-CCTTGATGCACTGTCCCCTCGGAAT-3') and 2GSP2 (5'- CTCCCAGGACCTTCGCACACC-CTT-3'), designed to one of the PCR products obtained from the first nested PCR reaction. All DNA fragments were cloned into the pCR 2.1 TA cloning vector and sequenced in both directions using the T7 Sequenase DNA sequencing kit (Amersham Pharmacia Biotech, Arlington Heights, IL) and [³⁵S]deoxy-ATP (1000–1500 Ci/mmol; DuPont/New England Nuclear). HDL receptor SR-BI promoter fragments obtained from both sets of PCR reactions (designated clones I and II, respectively) were ligated together using a shared XhoI site.

5'-Rapid amplification of cDNA ends
The transcription start site of the rat HDL receptor SR-BI gene was mapped using the 5'-Rapid Amplification of cDNA End Kit from Life Technologies, Inc./BRL. For this, 1 µg rat ovarian total RNA was reverse transcribed using the primer 3GSP (5'-GACCAGGATGTTAGGCAGTA-3'), designed to the HDL receptor SR-BI cDNA, and reverse transcriptase. After degrading the RNA with ribonuclease H, the single stranded cDNA was tailed with terminal deoxynucleotide transferase and dCTP. The tailed cDNA was then used as a template for PCR using 1GSP1 primer (see above) and the abridged anchor primer (5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'). The parameters for PCR were denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 1 min, annealing at 55 C for 2 min, and extension at 72 C for 3 min. The PCR product was then cloned into the pCR 2.1 TA cloning vector and sequenced to determine the transcription start site.

Plasmids for luciferase assays
All HDL receptor SR-BI promoter-luciferase gene constructs were derivatives of the pGL3-basic luciferase vector. Standard molecular biology techniques were used in all cloning procedures (47). To make the p-2267 plasmid, the entire HDL receptor SR-BI promoter obtained from the PromoterFinder DNA walking PCR reactions was used. This promoter fragment was subcloned into pGL3-basic luciferase vector using MluI and HindIII sites. Nested deletions of the HDL receptor promoter were carried out using restriction enzymes. The restriction enzymes used were BglII and HindIII for the p-1421 fragment, XhoI and HindIII for the p-719 fragment, and SmaI and HindIII for the p-170 fragment. All promoter fragments were then subcloned into the pGL3-Basic luciferase vector using those restriction enzyme sites.

Cell transfections
Cells were transfected with the specified HDL receptor SR-BI promoter-luciferase reporter gene construct in either the presence or absence of SREBP-1a-pCMV5 using the calcium phosphate method (48). SF-1-pCMV was used in some experiments. Cells were first plated out in six-well tissue culture plates at a density of 1.5–3.3 x 10⁶ cells/well (depending on the cell type) and incubated for 24 h at 37 C (5% CO₂). Fresh DMEM/F12 medium and 10% FBS were added 2 h before transfections. The DNA to be transfected was purified by ethanol precipitation, resuspended in 0.25 M calcium chloride (CaCl₂), and reprecipitated in an equal volume of 2 x HEPES buffer for 20 min at room temperature. Precipitated DNA was then added to the culture plates. After incubating for 4 h, the medium was replaced, and the cells were allowed to incubate for 48 h at 37 C (5% CO₂). Either 50 µM rivastatin, 50 µg/ml ALLN, or 1 mM 8-bromo-cyclic 3',5'-AMP (8-Br-cAMP) was added to some plates 24 h before the end of the incubation time. Cells were washed twice with PBS, treated with a passive lysis buffer for 20 min, and then scraped with a rubber policeman. Lysates were transferred to a microcentrifuge tube and stored at -80 C until determination of luciferase activity. Cotransfection of a plasmid containing the Renilla luciferase gene under control of the simian virus 40 early enhancer/promoter region was used as a control to correct for differences in transfection efficiencies.

Luciferase assays
Luciferase assays were performed using the Dual Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega Corp., Madison, WI). Briefly, 100 µl luciferase substrate were added to 20 µl lysate, and luciferase activity was measured using a Turner Designs-20/20 luminometer (Sunnyvale, CA).

Site-directed mutagenesis
Site-directed mutants were obtained using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Briefly, 10 ng plasmid were incubated with 125 ng of the appropriate complementary oligonucleotides (see below) and 1 µl dNTPs in 50 µl reaction buffer [100 mM KCl, 100 mM (NH₄)₂SO₄, 200 mM Tris-HCl (pH 8.8), 20 mM MgSO₄, 1% Triton X-100, and 1 mg/ml nuclease-free BSA]. One microliter of Pfu DNA polymerase (2.5 U/µl) was added to the reaction, and each reaction was heated to 95 C for 30 sec followed by 35 cycles of denaturation at 95 C for 30 sec, annealing at 55 C for 1 min, and extension at 68 C for 12 min. After the cycling reaction, samples were subjected to digestion with DpnI for 1 h at 37 C to remove the parental DNA template. One microliter of the mutant samples was used to transform XL-1 Blue bacterial cells. The mutations were confirmed by sequencing using the T7 Sequenase DNA sequencing kit and [³⁵S]dATP.

Complementary oligonucleotides used for site-directed mutagenesis to mutate E box-1 were M1 (5'-GGCGCATAAAACCTCTGGCTTTTTTCAGGGCTACTGCTGC-3') and M2 (5'-CATAAAACCTCTGGCTCCCAACAGGGCTAC-3'). M1 was prepared in both the p-719 and the p-170 constructs, whereas M2 was only prepared in the p-170 construct. The oligonucleotide E2-M (5'-CTGGCGACTGTAATTTATGCAGGGG-3') and its complement were used to mutate the second E box found in the p-719 construct. Complementary oligonucleotides used to mutate dSRE and pSRE were MD (5'-CTGCCCCCCTCCCCCCCCCCTCTG-3') and MP (5'-CCATCAGAGCCCCGCCCCCTCCCC-3'), respectively. MD was prepared in the p-2267 construct, whereas MP was prepared in the constructs p-2267, p-719, and p-719 with E boxes mutated. The nucleotides that are underlined correspond to the mutated bases.

Nuclear extracts
Nuclear proteins were prepared either from COS-7 cells overexpressing the NH₂-terminal segment (mature form) of SREBP-1a or from ovaries isolated from rats treated with hCG for 6 h. COS 7 cells at a density of 10⁸ cells/flask were transiently transfected with SREBP-1a-pCMV5 plasmid (50 µg/flask) using the calcium phosphate method (48) as described above. After transfections, fresh DMEM/F12 medium and 10% FBS were added, and cells were incubated for 48 h at 37 C (5% CO₂). Cells were then washed twice with PBS and treated with 10 ml 0.25% trypsin and 1 mM EDTA for 10 min. The cell pellet was recovered by centrifugation at 500 x g for 5 min. Nuclei were prepared as previously described (49). The nuclear pellet was resuspended in 2.5 vol extract buffer [15 mM Tris-HCl (pH 7.5), 15 mM NaCl, 60 mM KCl, 0.34 M sucrose, 15 mM ß-mercaptoethanol, 0.15 mM spermine, 0.5 mM spermidine, 5% glycerol, and 0.2 mM phenylmethylsulfonylfluoride]. The suspension was gently agitated for 30 min at 4 C. KCl was then added to a final concentration of 300 mM. After incubation for another 30 min at 4 C, the samples were centrifuged in a microcentrifuge at top speed for 30 min. Nuclear extracts were stored at -80 C until used. Protein concentrations were determined using the Bio-Rad Laboratories, Inc., protein assay.

Gel mobility shift assay
Complementary oligonucleotides corresponding to the HDL receptor SR-BI promoter regions from -1966 to -1940 (dSRE, 5'-CTGCCCCCCTCACACCCTCCTCTGTAG-3') and from -246 to -220 (pSRE, 5'-CCATCAGAGCACCGCCCACTCCCCGCC-3'), with GGG overhangs at the 5'-ends, were synthesized and annealed in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 25 mM NaCl, 10 mM MgCl₂, and 1 mM dithiothreitol. The oligonucleotide probe was then labeled using the Klenow fragment of DNA polymerase and [-³²P]dCTP (3000 Ci/mmol). In some experiments, an oligonucleotide containing the human LDL receptor SRE-1 (5'-ATCACCCCACTGCAAACTCCTCCCCCTGC-3') (21) was also used either ³²P-labeled as a positive control or unlabeled as a competitor. Mutated-dSRE (5'-CTGCCCCCCTCCCCCCCCC-CTCTGTAG-3') and mutated-pSRE (5'-CCATCAGAGCCCCGCCCCCTCCCCGCC-3') oligonucleotides were also used in some experiments. The underlined letters correspond to the mutated bases. In other experiments, unlabeled distal or proximal SRE were also used as competitors. Twenty micrograms of nuclear proteins were incubated in either the presence or absence of competitor for 30 min at room temperature in binding buffer [12 mM HEPES (pH 7.9), 12% glycerol, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 4 mM Tris-HCl (pH 8.0)], 2 µg poly(dI-dC), and 0.4 µg BSA. After incubation, 100,000 cpm of the radiolabeled probe were added, and the mixture was incubated for 15 min at 30 C. The DNA-protein complexes were resolved on a 4% nondenaturing acrylamide gel at 4 C in 1 x Tris-borate buffer (0.05 M Tris, 0.05 M boric acid, and 0.001 M EDTA). Gels were then vacuum-dried and exposed to BioMax-MR films at -80 C for 12–24 h.

Data analysis
Luciferase, cholesterol, and Western blot data were expressed as the mean ± SEM. Each luciferase assay experiment was performed in triplicate and repeated for the indicated number of times as listed in the figure legends. Quantitation of Western blot and DNA-protein complex signals in the mobility shift assay autoradiographs was performed using a Hoefer scanning densitometer (San Francisco, CA). Data from the individual parameters were compared by ANOVA, followed by Student-Newman-Keuls multiple comparison test when applicable (50).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
As hormonal stimulation has been shown to be associated with depletion of ovarian cholesteryl ester content (17, 18, 19), we examined whether these conditions affect mature SREBP-1a protein levels. To accomplish this, Western blot analysis was performed. As shown in Fig. 1, the 65-kDa mature SREBP-1a protein was increased 3-fold at 3 h (P < 0.002) and 11-fold (P < 0.001) at 6 h after hCG injection. These results correlate with our previous report showing that HDL receptor SR-BI mRNA levels increased 2-fold at 3 h and 4-fold at 6 h post-hCG treatment (44). However, significant increases in HDL receptor SR-BI protein levels (2-fold; P < 0.05) were not observed until 24 h after hCG injection (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Figure 1. Regulation of SREBP-1 protein levels in rat ovaries by hCG. Twenty-eight-day-old-Sprague Dawley rats were treated as described in Materials and Methods. Ovaries were obtained before hCG (50 U) and 3 and 6 h post-hCG injection. A, A representative Western blot is presented. B, Western blot results were analyzed using a Hoefer scanning densitometer. The data are presented as the mean ± SEM from two experiments (n = 5/treatment).

View larger version (28K): [in this window] [in a new window]   Figure 2. Regulation of HDL receptor protein levels in rat ovaries by hCG. Twenty-eight-day-old Sprague Dawley rats were treated as described in Materials and Methods. Ovaries were obtained before hCG (50 U) and 6, 12, and 24 h post-hCG injection. A, A representative Western blot for controls and 24 h post-hCG injection is presented. B, Western blot results were analyzed using a Hoefer scanning densitometer. The data are presented as the mean ± SEM (n = 6/treatment).

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of hCG treatment on ovarian cholesterol levels. Total cholesterol levels were determined as described in Materials and Methods. Cholesterol values were corrected by sample weight, and the data are presented as the percentage of ovarian cholesterol remaining where the value for controls was set at 100%. The SEMs for the different time points were 0.04 for 6 h, 0.05 for 12 h, and 0.08 for 24 h post-hCG injection. *, P < 0.01; **, P < 0.05 (n = 6/treatment).

The nucleotide sequence of the rat HDL receptor SR-BI promoter is shown in Fig. 4. This DNA segment contained a TATA box (CATAAAA), which is 40 bp upstream of the transcription start site mapped by 5'-rapid amplification of cDNA ends as described in Materials and Methods (Fig. 4). The sequence of this TATA box is identical to that reported for the human HDL receptor SR-BI promoter (38). The transcription start site identified in the rat promoter is about 10 bp closer to the ATG initiation site than that identified for the human gene (38) (Fig. 4). Similar to the human gene, no CAT box motif was identified in the rat promoter (38) (Fig. 4). After searching the entire sequence for other putative consensus sites, we found that this DNA fragment also contains 11 E boxes (CANNTG) (39), 7 Sp1 sites, 4 estrogen receptor half-sites, 2 activator protein-1 (AP-1) sites, and 1 SF-1 site (Fig. 4). Many of these motifs are also found in the human HDL receptor SR-BI gene at similar positions relative to the translation start site (38) (Fig. 4). These include 2 E boxes (at -1140 and -142) and 2 Sp1 sites (at -282 and -260). The SF-1 site (5'-TCAAGGCC-3') identified in the rat HDL receptor SR-BI promoter is 78% identical to the site found in the human gene (38) (Fig. 4). However, their positions within the promoter are different (38) (Fig. 4). The SF-1 site in the rat gene is located further upstream (at -480) (38) (Fig. 4).

View larger version (53K): [in this window] [in a new window]   Figure 4. Nucleotide sequence of the rat HDL receptor SR-BI promoter. Nucleotide position +1 is assigned to the A of the ATG start codon (boldface), and negative numbers refer to promoter sequences. Putative SRE, AP-1, SF-1, Sp1, estrogen receptor half-site (E1/2), E box, and TATA box motifs are underlined. The transcription start site mapped by 5'-rapid amplification of cDNA ends is indicated by an arrow.

View larger version (14K): [in this window] [in a new window]   Figure 5. Effect of SREBPs on the expression of luciferase activity under the control of the HDL receptor SR-BI promoter in HepG2 cells. The p-2267 construct was prepared by cloning the entire HDL receptor SR-BI promoter obtained as described in Materials and Methods in front of the luciferase gene. Cells (1.5 x 106/well) were transfected with this construct in either the presence or absence of SREBP-1a-pCMV5 plasmid. Rivastatin (50 µM) was added to some of the plates 24 h before lysing the cells. Luciferase activity was measured in cell lysates 48 h after transfections. The data are presented as relative luciferase units ± SEM. The data are from a typical experiment, performed in triplicate. This experiment was repeated twice.

View larger version (21K): [in this window] [in a new window]   Figure 6. SREBP-1a induces the expression of the luciferase gene through two SREs found in the HDL receptor SR-BI gene. Constructs used in these experiments were prepared as described in Materials and Methods. HTB9 cells (3.3 x 106/well) were transfected with the indicated construct in either the presence or absence of SREBP-1a-pCMV5 plasmid. Luciferase activity was measured in cell lysates 48 h after transfections. The data are presented as relative luciferase units ± SEM and are from two experiments, each performed in triplicate. Numbers next to the bars indicate fold induction by SREBP-1a.

View larger version (20K): [in this window] [in a new window]   Figure 7. SREBP-1a activates an E box found in the HDL receptor SR-BI promoter. A, Schematic representation of the wild-type (WT) and mutated (M1 and M2) HDL receptor SR-BI promoter-reporter constructs. The sequences of the wild-type and mutated E box motifs are shown. Mutations were prepared by site-directed mutagenesis as described in Materials and Methods. M1 was prepared in both p-170 (B) and p-719 (C) constructs, whereas M2 was only prepared in the p-170 construct (B). HTB9 cells (3.3 x 106/well) were then transfected with the indicated construct in either the presence or absence of SREBP-1a-pCMV5 plasmid. Luciferase activity was measured in cell lysates 48 h after transfection. The data are presented as the fold induction (mean ± SEM), where the value of luciferase activity for the construct transfected in the absence of SREBP-1a was set at 1.0. The data are from three experiments, each performed in triplicate.

View larger version (11K): [in this window] [in a new window]   Figure 8. Effect of calpain inhibitor I (ALLN) on the expression of the luciferase gene under the expression of the p-719 HDL receptor SR-BI promoter. HTB9 cells (3.3 x 106/well) were transfected with the p-719 construct in either the presence or absence of SREBP-1a-pCMV5 plasmid as described in Materials and Methods. ALLN (50 µg/ml) was added to some of the plates 24 h before lysing the cells. The data are presented as relative luciferase units ± SEM and are from a typical experiment, performed in triplicate. This experiment was repeated twice.

View larger version (55K): [in this window] [in a new window]   Figure 9. Binding of SREBP-1a to the distal and proximal SRE sites in the HDL receptor SR-BI promoter. 32P-Labeled, double stranded, oligonucleotide probes (100,000 cpm/lane) containing either dSRE (A) or pSRE (B) SREBP-binding sites were incubated with nuclear extracts (20 µg) prepared from COS-7 cells overexpressing SREBP-1a in the presence or absence of a 250-fold molar excess of competitor as described in Materials and Methods. Representative gel mobility shift assay autoradiographs are presented. LDL receptor SRE-1, which refers to the SRE motif found in the promoter of the human LDL receptor gene, was used as both positive control (A) and competitor. X and Y refer to the two complexes formed by SREBP-1a and probe. This experiment was repeated four times.

View larger version (52K): [in this window] [in a new window]   Figure 10. Binding of ovarian SREBP-1a to wild-type and mutated SRE motifs. 32P-Labeled, double-stranded, oligonucleotide probes (100,000 cpm/lane) containing either the wild-type or mutated dSRE (A) or the wild-type or mutated pSRE (B) SREBP-binding sites were incubated with ovarian nuclear extracts (20 µg) prepared from animals treated with hCG for 6 h. Representative gel mobility shift assay autoradiographs are presented. This experiment was repeated twice.

View larger version (28K): [in this window] [in a new window]   Figure 11. Mutational analysis of the distal and proximal SRE sites found in the HDL receptor SR-BI promoter. Mutations were prepared by site-directed mutagenesis as described in Materials and Methods. The mutations analyzed were MD (mutated dSRE), MP (mutated pSRE), and MB (both SRE motifs mutated) in the p-2267 construct (A) and MP in constructs p-719 and p-719 with both E boxes mutated (M-E-boxes; B). The cell type used is indicated. Transfections were carried out as described in Materials and Methods. Rivastatin (50 µM) was added to some of the plates 24 h before lysing the cells. Luciferase activity was measured in cell lysates 48 h after transfection. A, The data are presented as the fold induction (mean ± SEM), where the value of luciferase activity for the construct not incubated with rivastatin was set at 1.0. B, The data are presented as relative luciferase units ± SEM. In both cases, the data are from two experiments, each performed in triplicate.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present report, we have shown that the increase in mature SREBP-1a protein levels correlates with a 30% decrease in ovarian cholesterol levels. These changes in both SREBP-1a protein and cholesterol levels correlated with an increase in HDL receptor SR-BI mRNA levels (44) and preceded an increase in HDL receptor SR-BI protein levels. Ovarian cholesterol levels returned to control levels by 24 h post-hCG treatment, when HDL receptor protein levels were maximal. These results suggest that the increase in HDL receptor SR-BI protein assisted in replenishing ovarian cholesterol levels. Most importantly, these results suggested that the HDL receptor SR-BI gene may be regulated by the cellular cholesterol status and that SREBPs may mediate this regulation. Interestingly, the increased expression of the HDL receptor SR-BI gene in response to depleted cholesterol stores in the LH-induced desensitized ovary paralleled increases in both HMGCoA reductase and LDL receptor mRNA levels (17), two genes known to be regulated by the cellular cholesterol status. The finding that SREBPs are expressed in the adrenal gland (35, 36) suggests that SREBPs may also regulate the HDL receptor SR-BI gene in that tissue.

In the case of the liver, inconsistent results have been obtained (20). Feeding rats a high cholesterol diet for 2 weeks decreased HDL receptor SR-BI expression in parenchymal cells in agreement with the findings reported here (20). However, in Kupffer cells, the same treatment caused an increase in receptor SR-BI expression (20). It has been shown that in rats, parenchymal cells are the main cells responsible for both HDL receptor SR-BI expression and selective uptake of HDL cholesteryl esters within the liver (57, 58). Thus, it appears that in cell types responsible for selective lipid uptake from an HDL particle, HDL receptor SR-BI expression is up-regulated in response to depleted cellular cholesterol levels. In the case of the liver Kupffer cells and macrophages, where the HDL receptor SR-BI may be involved mainly in cholesterol efflux (59, 60), receptor SR-BI expression would be up-regulated as a result of increased cellular cholesterol levels. This is critical considering the function of this receptor in reverse cholesterol transport (2, 3). Whether sterol regulation of the HDL receptor SR-BI in macrophages is mediated by SREBPs will require further analysis.

Although SREBPs are the key regulatory factors involved in sterol balance, it has been shown that other factors acting as coactivators are also required. For example, in the human LDL receptor (21) and fatty acid synthase (25) promoters, the coactivator is Sp1, whereas in the HMGCoA synthase (28) and farnesyl diphosphate synthase (26) promoters, the coactivator is nuclear factor-Y. To date, we have identified seven putative binding sites for Sp1, but none for nuclear factor-Y. The pSRE is located between two Sp1 sites, similar to the SRE motif in the human LDL receptor promoter (21). In the case of the dSRE, the closest putative Sp1 is 130 bp downstream. Whether any of the Sp1 sites found in the HDL receptor SR-BI promoter are important for sterol regulation of this gene will require further analysis.

The finding that SREBP regulation of a gene in response to the cellular cholesterol status is mediated exclusively through the SRE motifs is common to most sterol-sensitive genes (52). However, the fact that the HDL receptor SR-BI gene contains 11 E boxes and that some of these elements are conserved in other species (38), one must consider the possibility that the E box motifs may still play some role in the regulation of this gene. As all basic helix-loop-helix proteins, including SREBP-1a, have been shown to bind E boxes (61), further analysis is necessary to determine whether the other helix-loop-helix proteins might regulate HDL receptor SR-BI gene expression through the these motifs. One candidate for this type of regulation is the upstream stimulatory factor, which has been shown to bind and activate E boxes found in the promoter of the SF-1 gene (62).

Another important consideration is whether SREBP regulation of the HDL receptor SR-BI is critical in steroidogenic tissues. It has been reported that both human (38) and rat (56) HDL receptor SR-BI promoters contain an SF-1-binding site through which SF-1 protein binds and activates their transcription. The gonadotropic hormone hCG is known to induce cAMP levels (54, 55) in the ovary. In contrast to the human gene, no cAMP response element was identified in the rat HDL receptor SR-BI promoter (56). In the rat gene, SF-1 appears to be the major mediator of cAMP regulation of the HDL receptor SR-BI gene (56). However, as has been shown for a number of genes (63, 64), the simple binding and activation of transcription by SF-1 are insufficient. For example, the regulation of the fish gonadotropin II ß-subunit gene requires SF-1 and estrogen receptor (63), whereas the regulation of the bovine cholesterol side-chain cleavage cytochrome P450 gene (CYP11A) requires SF-1 and Sp1 (64). Thus, in the present study, we decided to investigate whether SREBP-1a may cooperate with SF-1 to mediate HDL receptor SR-BI induction in response to tropic hormones. In the present study we demonstrated that SREBP-1a and SF-1 act synergistically to activate HDL receptor SR-BI gene transcription. By combining these two mechanisms, steroidogenic tissues may ensure that cholesterol is available when required.

In summary, the rat HDL receptor SR-BI promoter contains two SRE motifs through which SREBP-1a binds and activates transcription of the luciferase reporter gene. These motifs have significant identity to SREs found in the promoters of several other sterol-regulated genes (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). This investigation indicates that intracellular cholesterol levels may control HDL receptor SR-BI gene transcription, like other sterol-sensitive genes. Furthermore, in steroidogenic tissues, SREBP-1a may synergize with SF-1 to induce transcription of the HDL receptor SR-BI gene in response to hormones such as hCG.

	Acknowledgments

 
We thank K. Warden, F. R. Rodriguez, and T. W. Sandhoff for their technical assistance, and Drs. T. Osborne, K. L. Parker, and G. C. Ness for gifts of materials.

	Footnotes

 
¹ This work was supported by NIH Grants R29-HD-31644 and R01-HD-35163 (to M.P.M.) and American Heart Association Florida Affiliate Post-Doctoral Fellowship 9703004 (to D.L.).

Received April 9, 1999.

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