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Endocrinology Vol. 143, No. 6 2155-2168
Copyright © 2002 by The Endocrine Society

RECEPTORS

Estrogen Activates the High-Density Lipoprotein Receptor Gene via Binding to Estrogen Response Elements and Interaction with Sterol Regulatory Element Binding Protein-1A

Dayami Lopez1, Mark D. Sanchez1, Wendy Shea-Eaton and Mark P. McLean

Departments of Obstetrics & Gynecology (D.L., M.D.S., W.S.-E., M.P.M.) and Biochemistry & Molecular Biology (M.P.M.), College of Medicine, University of South Florida, Tampa, Florida 33606

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of E2 on the high-density lipoprotein receptor (HDL-R) scavenger receptor class B type I (SR-BI) gene were examined. Four putative estrogen response element half-site motifs (ERE1/2) (-2176, -1726, -1622, and -1211, designated ERE1/2-1, 2, 3, and 4, respectively) were identified in the HDL-R SR-BI promoter. Transfection studies and mutation analysis demonstrated that E2 significantly increased HDL-R SR-BI promoter activity and that mutating ERE1/2-1, 2, and 4 resulted in a loss of E2 responsiveness. Both ER{alpha} and ERß formed specific complexes with ERE1/2-1, 2, and 4 but did not bind ERE1/2-3 in vitro. Interestingly, ERE1/2-3 was the motif shown not to be important for E2-activation of the HDL-R SR-BI promoter in the mutational analysis studies. The influence of SREBP-1a (sterol regulatory element binding protein-1a) on E2 regulation of the HDL-R SR-BI gene was also examined. SREBP-1a was able to bind directly to the ERE1/2 motifs and enhanced ER binding when both ER subtypes were present. ER{alpha} and ß also bound to a sterol response element motif, but they did not enhance SREBP-1a binding. Cotransfection studies demonstrated that the presence of the three factors, ER{alpha}, ERß, and SREBP-1a, enhanced the overall luciferase activity produced from the HDL-R SR-BI promoter construct in the presence of only one of the factors. Interaction of SREBP-1a with both ERs was demonstrated using a mammalian two-hybrid assay. The data confirmed that E2 through the ERs can positively regulate the HDL-R SR-BI through binding and activation of three ERE1/2 motifs and identified SREBP-1a as a potential coactivator of the E2-ER-dependent effects on the HDL-R SR-BI gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASES ARE the leading cause of death and disability in the United States for both sexes (1, 2). Although women show less cardiovascular diseases than age-matched men in the premenopausal period (3), the risk of death from cardiovascular diseases increases with a woman’s age, with the sharpest increases in the postmenopausal years (4). These changes appear to correlate with decreases in estrogen levels after menopause (3, 5). In addition, epidemiological studies consistently report that endogenous and exogenous estrogens reduced cardiovascular morbidity and mortality in women (6, 7) and oophorectomized primates (8, 9). One putative mechanism by which estrogen may reduce the risk of atheroma formation is by increasing high-density lipoprotein (HDL) cholesterol levels (10, 11) and reverse cholesterol transport, and through this protective mechanism, decrease the risk of developing cardiovascular diseases (12).

The HDL receptor (HDL-R) scavenger receptor class B type I (SR-BI) has been shown to mediate cholesterol efflux to lipoproteins from atheromatous arteries (13) and HDL cholesterol uptake by the liver (reverse cholesterol transport) (14, 15) and steroidogenic tissues (15, 16, 17). Several studies have identified this receptor as the major determinant of plasma HDL concentrations (18, 19) and hence, a key element in cholesterol homeostasis. In addition to its role in lipid metabolism, the HDL-R SR-BI has also been shown to be critical in reproductive physiology as demonstrated by infertility of female HDL-R SR-BI knockout mice (17). Thus, elucidation of the regulatory mechanisms underlying HDL-R SR-BI gene expression, especially by factors known to alter HDL levels such as estrogen (10, 11), is extremely important. Landschulz et al. (15) reported that estrogen treatment increased HDL-R SR-BI expression in steroidogenic tissues but dramatically decreased hepatic HDL-R SR-BI gene expression demonstrating tissue specific regulation. The mechanisms by which tissue specific factors influence estrogen regulation of the HDL-R SR-BI gene have not been clearly defined.

Estrogen influences gene transcription through activation of its nuclear receptors (20). In the classical pathway, once activated by estrogen, the ERs can bind as a homo- or heterodimer to the EREs found in the promoter of estrogen-sensitive genes, to activate or repress transcription (21, 22, 23, 24, 25). Estrogen has also been shown to alter transcription of an estrogen-sensitive gene through nonclassical pathways (26, 27, 28, 29, 30, 31). In these cases, activated ER does not bind to EREs in the promoter of the target gene (26, 27, 28, 29, 30, 31). Instead, the ER interacts with other transcription factors bound to their response elements resulting in enhancement or repression of transcription (26, 27, 28, 29, 30, 31). Transcriptional factors that have been shown to interact with the ER include steroidogenic factor-1 (SF-1; 26), specific factor-1 (Sp1; 27, 28, 29), nuclear factor-Y (29), and activator protein-1 (30, 31).

Currently, two ER isoforms have been identified, {alpha} and ß (32, 33). Both isoforms exhibit a high degree of homology within the DNA-binding domain and appear to bind to the same ERE motifs (25) but differ in their transactivation domains suggesting distinct roles in gene activation. In addition, their transcriptional activity has been shown to be influenced not only by ligands, but also by coregulatory proteins with which they associate (34, 35). It has been suggested that the ERß is the form responsible for the cardiovascular benefits of estrogen (36). However, one cannot exclude the possible mechanisms of HDL-R SR-BI gene regulation by ER{alpha}.

In the current investigation, transfection studies followed by luciferase assays and EMSAs were used to examine the effects of E2 on the regulation of the HDL-R SR-BI gene. The influence of a known regulator of HDL-R SR-BI gene transcription, SREBP-1a (37), on estrogen-dependent regulation of this gene was also examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Integrated DNA Technologies, Inc. (Coralville, IA) synthesized all oligonucleotides and primers. The pGL3-basic luciferase vector, the renilla luciferase vector, and the Dual Luciferase Reporter Assay System were obtained from Promega Corp. (Madison, WI). The full-length rat ERß cDNA, which encodes a protein of 549 amino acids, cloned in the p-cytomegalovirus (CMV)-5 vector (ERß-pCMV5) was obtained from Dr. Jan-Åke Gustafsson (Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden). The human breast carcinoma MCF-7 cells, the human bladder carcinoma HTB-9 cells, and the human ER{alpha} cDNA in pBluescript were obtained from American Type Culture Collection (Manassas, VA). For the experiments, the full-length ER{alpha} cDNA was cloned into the pCMV5 expression vector. The rat luteal GG-CL cell line was kindly provided by Dr. Geula Gibori (Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL). ER subtype ({alpha} and ß) specific antibodies and recombinant proteins were purchased from PanVera (Madison, WI). The NH2-terminal segment (active fragment) of SREBP-1a under the control of the CMV promoter (SREBP-1a-pCMV5) and SREBP-1a-polyhistidine-tagged in the pRSET B vector were kindly provided by Dr. Tim Osborne (Department of Molecular Biology and Biochemistry, University of California, Irvine, CA). The rabbit polyclonal SREBP-1 specific antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [{alpha}32P]-deoxy-CTP (3000 Ci/mmol) and the polydeoxyinosinic-deoxycytidylic acid were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). The QuikChange Site-directed Mutagenesis Kit was purchased from Stratagene (La Jolla, CA). All restriction enzymes and the Fugene 6 Transfection reagent were obtained from Roche Biochemicals Inc. (Indianapolis, IN). DMEM: nutrient mixture F-12 (DMEM: F-12) and the Roswell Park Memorial Institute medium were obtained from Life Technologies, Inc./BRL (Grand Island, NY). FBS was purchased from Summit Biotechnology (Ft. Collins, CO). BioMax-MR films were obtained from Fisher Scientific (Norcross, GA). All other chemicals were purchased from Fisher Scientific or Sigma (St. Louis, MO).

Preparation of MCF-7 and GG-CL nuclear extracts
Cells (5 x 107) were treated with 0.25% trypsin in 1 mM EDTA and then centrifuged. Nuclear extracts were prepared from the cell pellets as previously described (37). Protein samples were concentrated 4- to 6-fold using Centricon-10 concentrators (Millipore Corp., Bedford, MA), and concentrations were determined using the Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay. Nuclear proteins were then used in Western blot analysis.

Western blot analysis
Nuclear proteins (50 µg) were denatured at 100 C in loading buffer for 10 min and subjected to electrophoresis on a 12% SDS-PAGE as previously described (37). After electrophoresis, samples were electroblotted onto nitrocellulose membranes (0.2-µm pore) in buffer containing 0.25 M Tris hydrochloride (Tris-HCl; pH 8.3) and 1.92 M glycine for 16 h at 4 C. To verify equal protein loading, nitrocellulose membranes were stained with 0.1% Ponceau S (in 5% acetic acid) and destained in water. Western blot analysis was carried out with a 1:1000 dilution of the rabbit polyclonal specific antiserum to ER{alpha} or ERß in 3% nonfat dry milk. Immunoreactive proteins were then visualized using a 1:6000 dilution of horseradish peroxidase-conjugated goat antirabbit antisera (Santa Cruz Biotechnology, Inc.) in 3% nonfat dry milk and the SuperSignal ULTRA Chemiluminescent Substrate method (Pierce Chemical Co., Rockford, IL).

Cell transfections and luciferase assays
The p-2267 HDL-R SR-BI promoter-luciferase gene construct used in these experiments was prepared as previously described (37). MCF-7 cells were cultured in 24-well (2.5 x 105 cells per well) plates DMEM: F-12 medium without phenol red + 5% charcoal-stripped FBS. GG-CL cells were cultured in 12-well (5 x 105 cells per well) tissue culture plates in Roswell Park Memorial Institute medium without phenol red + 5% charcoal-stripped FBS at 33 C for 24 h and then transferred to 40 C for 24 h before transfection. Cells were transfected with 0.5 (for 24-well) or 1.0 (for 12-well) µg each of appropriate plasmid using Fugene 6 (ratio 1:2) and incubated for 48 h. Twenty-four hours before the end of the incubation period, cells were incubated with 17ß-E2 (0.001–100 nM; E2; Sigma), 4-hydroxytamoxifen (4-OHT; 100 nM), or an equal volume of ethanol (vehicle). Cells were washed twice with PBS and treated with a passive lysis buffer for 20 min. Lysates were transferred to a microcentrifuge tube and stored at -80 C until the determination of luciferase activity. Luciferase assays were performed as previously described (37). Cotransfection of a plasmid containing the renilla luciferase gene under control of the simian virus 40 early enhancer/promoter region (0.25 µg for 24-well or 0.5 µg for 12-well plates) was used as a control to correct for differences in transfection efficiencies. Luciferase data were expressed as mean fold induction ± SEM, where the value of luciferase activity for the promoter construct in the absence of E2 or any exogenous transcriptional factor was set to 1.0. Each luciferase assay experiment was performed in triplicate and repeated for the indicated number of times in the figure legends. Data from the individual parameters were compared by ANOVA followed by Student’s-Newman-Keuls multiple comparison test when applicable (38). A value of P < 0.05 was considered significant for all tests.

Site-directed mutagenesis
Site-directed mutants were obtained using the QuikChange Site- Directed Mutagenesis Kit (Stratagene) as previously described (39). Oligonucleotides used for site-directed mutagenesis to mutate the four ERE1/2 motifs were 5'-GACTCCTCCTGAATCACATAGAATG-3' (for ERE1/2-1), 5'-GGTCTCACTAATCAGAGAAGTCTGG-3' (for ERE1/2-2), 5'-GTGCACCATTTCTGAGGAGG-3' (for ERE1/2-3), and 5'-CTGACTCTGAATCATGTTGCTGAAG-3' (for ERE1/2-4), and their complements. The nucleotides that are underlined correspond to the mutated bases. All mutations were confirmed by sequencing using the T7 Sequenase DNA sequencing kit and [35S]-deoxy-ATP.

Fusion protein production
Histidine-tagged SREBP-1a protein was overexpressed in Escherichia coli by induction of mid-logarithmic-phase cultures with 1 mM isopropyl-ß-D-thiogalactopyranoside. After incubating for 6 h at 27 C, cells were sedimented by centrifuging at 7700 x g for 10 min at 4 C. The cell pellet after the initial centrifugation was resuspended in guanidium lysis buffer provided in the XPRESS System (Invitrogen, Carlsbad, CA). Purification was performed using an immobilized metal affinity column according to the manufacturer’s recommendations. Recombinant protein samples were concentrated 4- to 6-fold using Centricon-10 concentrators (Millipore Corp.). Protein concentrations were determined using the Bio-Rad Laboratories, Inc. protein assay. The purified SREBP-1a protein was then used in mobility shift assays.

EMSA
Oligonucleotides corresponding to each putative ERE1/2 motif and to the distal SRE (dSRE) found in the promoter of the HDL-R SR-BI gene 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 oligonucleotides used were ERE1/2-1 (5'-GACTCCTCCTGGGTCACATAGAATGGC-3'), ERE1/2-2 (5'-CGGGTCTCACTGGTCAGAGAAGTCTGG-3'), ERE1/2-3 (5'-TCTGTGCACCAGGTCAGAGGAGGGCAT-3'), ERE1/2-4 (5'-TCCTGACTCTGGGTCATGTTGCTGAAG-3'), and dSRE (5'-CTGCCCCCCTCACACCCTCCTCTGTAG-3'). An oligonucleotide (5'-CCAGGTCAGAGTGACCTGAGCTAAAAT-3') that contains a consensus ERE motif was also synthesized and used as control in some experiments. Nucleotides in boldface underlined letters correspond to the putative ERE1/2 sites. The oligonucleotide probes were labeled using the Klenow fragment of DNA polymerase and [{alpha}32P] deoxy-CTP (3000 Ci/mmol). Unlabeled oligonucleotides were used as competitors in some experiments. Recombinant proteins (100–200 ng) were incubated either in the presence or absence of competitor for 15 min at 15 C 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 of polydeoxyinosinic-deoxycytidylic acid, and 1 µg of nonfat dry milk proteins. After incubation, 50,000–100,000 cpm of the radiolabeled probe was added and the mixture incubated for 15 min at 15 C. Where indicated, ER{alpha}, ERß (PanVera), or SREBP-1 (Santa Cruz Biotechnology, Inc.) specific antibody was also added to the reaction for supershift analysis. The protein/DNA complexes were resolved on a 4% nondenaturing acrylamide gel in 0.5x TGE (0.25 M Tris, 1.9 M glycine, 10 mM EDTA final). Gels were vacuum-dried and developed using a Cyclone Storage Phosphor Screen (Packard Bioscience, Meriden, CT) or Kodak BioMax MR films (Fisher Scientific). Densitometric quantitation of the autoradiograms was performed using the Ultra-Violet Products imaging system and Labworks software (Ultra-Violet Products, Upland, CA).

Mammalian two-hybrid assay
The plasmids pBIND, pACT and pG5-luciferase used in these assays were provided in the Mammalian Two-hybrid System (Promega Corp.). The pBIND refers to a vector containing the yeast GAL4 DNA binding domain upstream of a multiple cloning region and the pACT refers to a vector containing the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. The pG5-luciferase refers to a vector containing five GAL4 binding sites upstream of a minimal TATA box in front of the firefly luciferase gene. The plasmid SREBP-1a-pBIND was constructed using full-length human SREBP-1a cDNA inserted into the EcoRI and KpnI sites. The ER{alpha}-pACT and ERß-pACT plasmids containing full-length ER{alpha} and ERß, respectively, were kindly provided by Dr. Katarina Pettersson (Department of Medical Nutrition, Karolinska Institute, Sweden). For this, human bladder carcinoma HTB-9 cells plated in six-well plates (at a density of 3 x 106 cells per well) were cotransfected with the pG5-luciferase vector either in the presence or absence of SREBP-1a-pBIND ± ER{alpha}-pACT (or ERß-pACT). For these experiments, transfections were performed with 2 µg of each plasmid using the Fugene 6 transfection reagent according to the manufacturer’s instructions. After transfection, cells were incubated for 48 h at 37 C before harvesting. Luciferase assays and statistical analysis were performed as described in Cell transfections and luciferase assays. Transfection efficiencies were corrected using the renilla activity expressed by the pBIND plasmid.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because estrogen is known to influence gene transcription through activation of the ERs (20), the differential expression of these receptors within distinct cell lines was examined. For this, nuclear extracts were prepared from human breast carcinoma MCF-7 and rat luteal GG-CL cells, and Western blot analysis using receptor subtype ({alpha} and ß) specific antibodies was performed as described in Materials and Methods. MCF-7 cells, the first candidate for these studies, were selected because these cells have been extensively used for investigating the effects of estrogen/ER{alpha} on gene expression (27, 29, 40, 41). Although they have not been as widely used as the MCF-7 cells, the GG-CL cells were also chosen for these studies because they have been previously shown to express functional ERß (42, 43). Figure 1AGo demonstrates that ER{alpha} (66.4 kDa) was mainly expressed in MCF-7 cells, whereas ERß (53.4 kDa) was mainly expressed in GG-CL cells. These results correlate with the previous reports showing the presence of ER{alpha} and ERß in MCF-7 (40, 41) and GG-CL (42, 43) cells, respectively.



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  		Figure 1. Effects of E2 on the HDL-R SR-BI promoter activity in human breast carcinoma MCF-7 (B) and rat luteal GG-CL (C) cells. A, Expression of ER{alpha} and ß proteins in MCF-7 and GG-CL cells. Nuclear extracts were prepared from each cell type and Western blot analysis was performed as described in Materials and Methods using ER subtype ({alpha} and ß) specific antibodies. Typical Western blots are shown. B and C, Effects of adding increasing amounts of E2 on the HDL-R SR-BI promoter activity. MCF-7 (B) and GG-CL (C) cells were transfected with the p-2264 HDL-R SR-BI promoter construct as described in Materials and Methods. E2 (0.001–100.0 nM) was added 24 h before lysing the cells. 4-OHT (100.0 nM) was used as a negative control in these experiments and was also added 24 h before lysing the cells. The data are represented as mean fold-induction ± SEM, where the value of luciferase activity for the promoter construct without treatment was set to 1.0. Data are from a typical experiment performed in triplicate. These experiments were repeated two times. *, P < 0.01; **, P < 0.002; and ***, P < 0.001, were obtained by comparing to the HDL-R SR-BI promoter activity in the absence of any treatment.

 
To begin examining the possible mechanisms through which estrogen and its receptors regulate the HDL-R SR-BI gene, the HDL-R SR-BI promoter sequence (37) was analyzed for ERE whole and half-site motifs using the MacVector Program (Oxford Molecular Group). The consensus ERE motif consists of a palindromic repeat of the sequence GGTCA separated by three nucleotides (reviewed in Ref. 44). Four putative ERE half-sites (ERE1/2s) at positions -2176 (ERE1/2-1), -1726 (ERE1/2-2), -1622 (ERE1/2-3) and -1211 (ERE1/2-4) relative to the translation start site were identified. All four ERE1/2s had 100% identity to the consensus ERE1/2 sequence.

To determine whether estrogen regulates the HDL-R SR-BI gene, transfection studies followed by luciferase assays were performed. For this, the p-2267 HDL-R SR-BI promoter construct, which contained all four putative ERE1/2 motifs, was transfected into MCF-7 (Fig. 1BGo) and GG-CL (Fig. 1CGo) cells. Some plates received increasing amounts of E2 (0.001 to 100 nM) 24 h before lysing the cells. The data were represented as fold-induction where the value of luciferase activity for the construct with no treatment was set to 1.0 (Fig. 1Go, B and C). Using these conditions, it was shown that E2 activated, in a dose-dependent manner, the p-2267 HDL-R SR-BI promoter in both MCF-7 and GG-CL cells (Fig. 1Go, B and C). Higher concentrations of E2 (0.1–2.5 µM), however, did not further induce the HDL-R SR-BI promoter-driven luciferase activity (data not shown). Maximum activation levels (7.5-fold in MCF-7 and 6.8-fold in GG-CL) of the HDL-R SR-BI promoter were obtained at the E2 dose of 100 nM (Fig. 1Go, B and C). Even though the GG-CL cells appeared to express more ERß than MCF-7 cells express ER{alpha} (see Fig. 1AGo), the levels of luciferase activity produced from the HDL-R SR-BI promoter construct were comparable in both cell lines (Fig. 1Go, B and C). These effects appear to be specific for E2 because the addition of 100 nM of the antiestrogen 4-OHT was unable to increase the activity of the HDL-R SR-BI promoter (Fig. 1Go, B and C). To determine whether 4-OHT was able to block E2’s effects on the HDL-R SR-BI promoter, 1 nM of E2 was added to the cells in the presence or absence of increasing amounts of 4-OHT (1–100 nM). As shown, 1 nM of 4-OHT was able to prevent E2’s enhancement of the HDL-R SR-BI in both cell lines (Fig. 2Go, A and B).



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  		Figure 2. Effects of 4-OHT on E2-dependent activation of the HDL-R SR-BI promoter in MCF-7 (A) and GG-CL (B) cells. Cells were transfected with the p-2264 HDL-R SR-BI promoter construct as described in Materials and Methods. E2 (1 nM) and 4-OHT (1–100.0 nM) were added 24 h before lysing the cells. The data are represented as mean fold-induction ± SEM, where the value of luciferase activity for the promoter construct without treatment was set to 1.0. Data are from a typical experiment performed in triplicate. *, P < 0.001, was obtained by comparing to the HDL-R SR-BI promoter activity in the presence of E2 but not 4-OHT.

 
To examine whether any of the ER subtypes ({alpha} or ß) could bind to the putative ERE1/2 motifs found in the promoter of the HDL-R SR-BI gene, EMSAs and supershift analysis were performed. For this, 32P-labeled double-stranded oligonucleotide probes containing each ERE1/2 motif were incubated with recombinant ER{alpha} or ß proteins in the presence or absence of unlabeled oligonucleotide (Cold) or ER subtype ({alpha} or ß) specific antibodies (Ab). As shown in Fig. 3Go, both ER subtypes ({alpha} and ß) bound to all four ERE1/2 motifs. Both subtypes bound with high intensity to ERE1/2-1, 2, and 4 (lanes 6, 10, and 18 in both Fig. 3Go, A and B) and with low intensity to ERE1/2-3 (lane 14 in both Fig. 3Go, A and B). ER ({alpha} or ß)-DNA complexes were specifically competed by addition of cold oligonucleotide (Cold; lanes 7, 11, and 19 in both Fig. 3Go, A and B) and supershifted with subtype ({alpha} or ß) specific Ab (lanes 8, 12, and 20 in both Fig. 3Go, A and B). Control refers to an oligonucleotide containing a consensus ERE1/2 motif (see Materials and Methods). As shown, both ER subtypes ({alpha} and ß) bound to the control oligonucleotide (Fig. 3Go, A and B).



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  		Figure 3. EMSA depicting ER subtype ({alpha} or ß) binding to the ERE1/2 motifs found in the HDL-R SR-BI promoter. 32P- labeled double-stranded oligonucleotide probes (100,000 cpm per lane) containing each ERE1/2 motif found in the HDL-R SR-BI promoter were incubated with recombinant ER{alpha} (A) or ß (B) proteins (100 ng) in the presence or absence of unlabeled oligonucleotide (Cold; 125x) or ER{alpha} (A) or ERß (B) specific Ab (5 µg of IgG). Control refers to an oligonucleotide containing a consensus ERE (see Materials and Methods). The DNA-protein complexes (indicated by arrows) were resolved on a 4% nondenaturing acrylamide gel at 4 C in 0.5x Tris-glycine-EDTA buffer. Typical mobility shift assay autoradiograms are shown. This experiment was repeated two times.

 
To further confirm the specificity of ER ({alpha} or ß) binding to the putative ERE1/2 motifs found in the HDL-R SR-BI promoter, competition studies using unlabeled ERE1/2’s were performed (Fig. 4Go). As shown, the addition of increasing levels (12.5–125x) of unlabeled ERE1/2-4 (Fig. 4AGo) or ERE1/2-1 (Fig. 4BGo) to the binding reactions gradually diminished the ER-ERE1/2 complexes (ER{alpha}-ERE1/2-4 in Fig. 4AGo or ERß-ERE1/2-1 in Fig. 4BGo). Adding 125x-fold molar excess of unlabeled ERE1/2-4 and ERE1/2-1 oligonucleotides to their binding reactions reduced ER-ERE1/2 complex formation by 87% (ER{alpha}; Fig. 4AGo) and 88% (ERß; Fig. 4BGo), respectively. Plotting the percent of ER-ERE1/2 complex remaining against the picomoles of competitor used on that reaction demonstrated that the picomoles of competitor required to decrease the ER-DNA complex by 50% were 74.9 pmol in case of the ER{alpha}-ERE1/2-4 and 137.8 pmol in case of the ERß-ERE1/2-1 (Fig. 4Go, B and C). Similar results were obtained for the other ERE1/2 motifs (data not shown).



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  		Figure 4. Competition analysis of ER subtype ({alpha} or ß) binding to ERE1/2s found in the HDL-R SR-BI promoter. 32P-Labeled double-stranded oligonucleotide probes (100,000 cpm per lane) containing ERE1/2-4 (A) or ERE1/2-1 (B) were incubated with recombinant ER{alpha} (A) or ERß (B) (100 ng) protein in the presence or absence of increasing competing amounts (12.5–125x) of unlabeled ERE1/2-4 (A) or the ERE1/2-1 (B). The DNA-protein complexes (indicated by arrows) were resolved as described in Fig. 3Go. Representative gel mobility shift assay autoradiographs are shown. C, Calculating the amount of competitor required to achieve 50% competition. Densitometric quantitation of the autoradiograms shown in A and B was performed using the Ultra-Violet Products imaging system and Labworks software (Ultra-Violet Products Laboratory Products). The percent remaining was calculated by comparing the density of the major protein/DNA band seen with ER subtype ({alpha} or ß) alone with the band formed in the presence of ER ({alpha} or ß) and competitor. The percent remaining was then plotted against the picomoles of competitor used in each lane. The amount of competitor required to achieve 50% competition was then obtained from the graph and the results shown in D. These experiments were repeated twice for each ERE1/2 motif.

 
To identify which ERE1/2 motif was important in estrogen-dependent activation of the HDL-R SR-BI gene, site-directed mutagenesis was used to introduce a mutation in each ERE1/2. All the mutations used in these studies were tested in mobility shift assays and failed to bind ER{alpha} and ERß. The mutated promoter constructs were then transfected into MCF-7 and GG-CL cells, and E2 (1 nM) was added 24 h before lysing the cells. Variations in basal promoter activity due to the introduction of these mutations were not statistically significant (data not shown). However, significant changes on the HDL-R SR-BI promoter activity in the presence of E2 were observed (Table 1Go). Representing the data as percentage of the wild-type promoter activity in the presence of E2, demonstrated that mutating two or more of the ERE1/2 motifs resulted in a significant (P < 0.005) reduction in HDL-R SR-BI promoter activity (Table 1Go). Figure 5Go summarizes the most significant mutations. As shown, each half-site motif (ERE1/2’s 1, 2, and 4), with the exception of ERE1/2-3, contributed to the total activity produced from the HDL-R SR-BI promoter construct in the presence of E2. These data correlated with the mobility shift assay results showing that ERE1/2-3 bound ER with low intensity (see Fig. 3Go).


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  		Table 1. Mutation analysis of the HDL-R SR-BI ERE1/2 motifs

 


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  		Figure 5. Mutation analysis of the ERE1/2 motifs in the HDL-R SR-BI promoter. Site-directed mutagenesis and transfections of the mutated constructs into the MCF-7 (A) and GG-CL (B) cells were carried out as described in Materials and Methods. E2 (1 nM) was added 24 h before lysing the cells. The data are represented as mean fold-induction ± SEM, where the value of luciferase activity for the wild-type (WT) HDL-R SR-BI promoter construct without treatment was set to 1.0. Numbers after the "M" (mutation) refer to the specific ERE1/2 motif (1, 2, 3, or 4) that has been mutated in that promoter construct. Data are from a typical experiment performed in triplicate. *, P < 0.001, was obtained by comparing to the activity of the WT HDL-R SR-BI promoter treated with E2.

 
It has been previously shown that estrogen treatment differentially affects the HDL-R SR-BI gene expression in a tissue- or cell-specific manner (15, 45). Both ER subtypes are found in most cell types within the adrenal gland and the ovary although their ratio may vary from cell to cell (46, 47, 48, 49). In the liver, however, ER{alpha} is the only subtype expressed within the hepatocytes (46, 47). Thus, one explanation for this differential regulation of the HDL-R SR-BI gene by E2 could be that the ratio between the two ER subtypes ({alpha} and ß) varies from tissue to tissue. To examine this possibility, EMSAs were performed in the presence of both ERs ({alpha} and ß). Figure 6AGo illustrates ER{alpha} and ß binding to HDL-R SR-BI ERE1/2-1. As shown, incubation of both ERs ({alpha} and ß) and the ERE1/2-1 probe resulted in the formation of at least two major complexes, which migrated at a similar position as the major complex observed in the presence of each receptor subtype ({alpha} or ß) alone (compare lanes 2 and 3 with lane 4; Fig. 6AGo). The addition of cold oligonucleotide (Cold) diminished all the DNA-protein complexes (lane 5), whereas the addition of ER subtype ({alpha} or ß) specific Ab ({alpha}Ab or ßAb) formed at least two supershift complexes and diminished the two major complexes seen in the presence of both ER subtypes ({alpha} or ß; Fig. 6AGo, lane 6 and 7). These data suggest that both ER{alpha} and ß were involved in the formation of the major complexes observed when both receptor subtypes were added to the binding reaction. This suggests that the ERs could bind as homo- or heterodimers to the HDL-R SR-BI promoter. It is important to note that a preferential formation of homodimers over heterodimers by the ERs was observed. Similar results were obtained for ERE1/2’s 2 and 4 (data not shown). To determine whether the binding of the two ER subtypes to the DNA influences the response of the HDL-R SR-BI promoter to E2, cotransfection studies were performed in MCF-7 cells. For this, cells were transfected with the p-2267 HDL-R SR-BI promoter construct in the presence or absence of ERß-pCMV5 ± ER{alpha}-pCMV5 expression plasmids, and E2 (1 nM) was added 24 h before lysing the cells. Figure 6BGo demonstrates that adding ERß exogenously to MCF-7 cells, which only express ER{alpha} (see Fig. 1AGo), enhanced both basal and E2-dependent activation of the HDL-R SR-BI (15-fold, in both cases). Interestingly, the addition of ER{alpha} did not further enhance the levels obtained by the endogenous ER{alpha} (Fig. 6Go). Similar results were obtained in the case of the GG-CL cells (data not shown). These results suggest that the presence of both receptors (ER{alpha} and ß) could enhance the overall luciferase activity produced from the HDL-R SR-BI promoter construct.



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  		Figure 6. Effects of both ERs ({alpha} and ß) on the HDL-R SR-BI promoter. A, Mobility shift assay depicting ER{alpha} and ß binding the ERE1/2-1 motif in the HDL-R SR-BI promoter. Binding reactions were performed using recombinant ER{alpha} and ß proteins (100 ng each) in the presence or absence of unlabeled oligonucleotide (Cold; 125x) or ER subtype ({alpha} or ß) specific Ab ({alpha}Ab and ßAb, respectively; 5 µg of IgG). A representative mobility shift assay autoradiograph is shown. This experiment was repeated three times. The DNA-protein and supershifted complexes are indicated. B, Effects of ER{alpha} and ERß on the HDL-R SR-BI promoter activity in MCF-7 cells. Cells were transfected with the p-2267 HDL-R SR-BI promoter construct in the presence or absence of ER{alpha}-pCMV5 ± ERß-pCMV5 expression plasmids as described in Materials and Methods. E2 (1 nM) was added 24 h before lysing the cells. The data are represented as mean fold-induction ± SEM, where the value of luciferase activity for the promoter construct without treatment was set to 1.0. Data are from a typical experiment performed in triplicate. These experiments were repeated four times. *, P < 0.001, was obtained by comparing to the HDL-R SR-BI promoter activity in the absence of any exogenous factor or treatment.

 
Another explanation for the differential regulation of the HDL-R SR-BI gene in the liver and steroidogenic tissues by estrogen (15) could be that tissue specific factors or factors whose expression is also differently affected by estrogen in these tissues, influence this regulatory process. In an effort to examine which factors could be involved in estrogen regulation of the HDL-R SR-BI gene, SREBP-1a, a known regulator of the HDL-R SR-BI promoter (37), was added to the transfection studies. As shown, SREBP-1a significantly enhanced basal and E2-dependent activation of the HDL-R SR-BI promoter in both MCF-7 (Fig. 7AGo) and GG-CL cells (Fig. 7BGo). In MCF-7 cells, SREBP-1a increased basal and E2-dependent activation of the HDL-R SR-BI promoter by 73- and 246-fold, respectively (Fig. 7AGo); whereas, in the case of the GG-CL cells, SREBP-1a increased basal and E2-dependent activation of the HDL-R SR-BI promoter by 5.5- and 15.8-fold, respectively (Fig. 7BGo). It is important to note that the extent of the fold-activation of the HDL-R SR-BI promoter activity in response to SREBP-1a in the presence or absence of E2, occurs in a cell-specific manner, perhaps, due to the presence of ER{alpha} in the MCF-7 and ERß in the GG-CL cells. Another explanation could be that cell-specific factors may mask or enhance SREBP-1a’s effects on the E2-dependent activation of the HDL-R SR-BI gene. Once again, 4-OHT was able to significantly (P < 0.001) reduce E2-dependent activation of the HDL-R SR-BI promoter even in the presence or absence of SREBP-1a (95% in MCF-7 cells and 97% in GG-CL cells; data not shown).



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  		Figure 7. Effects of SREBP-1a on E2-dependent HDL-R SR-BI promoter activity in MCF-7 (A) and GG-CL (B) cells. Cells were transfected with the p-2267 HDL-R SR-BI promoter construct in the presence or absence of SREBP-1a-pCMV5 expression plasmid. E2 (1 nM) was added 24 h before lysing the cells. The data are represented as mean fold-induction ± SEM, where the value of luciferase activity for the HDL-R SR-BI promoter construct without treatment was set to 1.0. The data are from a typical experiment performed in triplicate. These experiments were repeated four times in the MCF-7 cells and two times in the GG-CL cells. *, P < 0.001, and **, P < 0.01, were obtained by comparing to the HDL-R SR-BI promoter activity in the absence of any factor.

 
Mobility shift assays performed in the presence of SREBP-1a, ER{alpha} (or ERß), and the ERE1/2-1 demonstrated that although SREBP-1a was able to form a slow migrating complex with the ERE1/2-1 (see below), SREBP-1a did not enhance the binding of each ER subtype to the DNA independently (data not shown). Thus, the effects of SREBP-1a on the binding of the two ERs to the HDL-R SR-BI ERE1/2-1 simultaneously were examined. As shown above (Fig. 8AGo), ER{alpha} and ß formed two major complexes with the ERE1/2-1 (referred to here as ER{alpha} and ß-ERE1/2 complexes in this figure; Fig. 8AGo, lane 2). Although the amounts of ER{alpha} and ERß proteins used in Fig. 8AGo and Fig. 6AGo (lane 2) were the same, the relative intensities of the ER{alpha}/{alpha} and ERß/ß complexes appeared different in these experiments. This could be due to the use of two different ER protein preparations in these studies. It has been previously reported (50) that a significant amount of the recombinant PanVera ER protein is unable to bind to an ERE motif. Thus, it is possible that the binding capacity of the ER{alpha} protein used in the binding reactions shown in Fig. 8AGo was much lower than the binding capacity of the ER{alpha} protein used in the binding reactions shown in Fig. 6AGo. Interestingly, SREBP-1a enhanced the formation of the top ER{alpha} and ß-ERE1/2 complex in addition to an intermediate complex which was observed between the two major ER{alpha} and ß-ERE1/2 complexes (Fig. 8AGo, lane 3). A fourth complex, referred to as the SREBP-1a-DNA complex, located near the top of the gel, was also observed when SREBP-1a was added to the binding reaction (Fig. 8AGo, lane 3). Adding ER{alpha} specific antibody to the binding reactions diminished the top and intermediate ER{alpha} and ß-DNA complexes as the SREBP-1a-DNA complex increased (Fig. 8AGo, lane 5). Adding ERß specific antibody, however, diminished all protein-DNA complexes including the one for SREBP-1a (Fig. 8AGo, lane 6), whereas the addition of SREBP-1 specific antibody completely supershifted the SREBP-1a-DNA complex but not the ER-DNA complexes (Fig. 8AGo, lane 7). Similar results were obtained with the other ERE1/2 motifs (2–4; data not shown). To examine whether SREBP-1a could bind to the ERE1/2-1 in the absence of the ERs, binding reactions were performed in the presence of SREBP-1a alone and the results are presented in Fig. 8BGo. As shown, SREBP-1a bound to the ERE1/2-1 motif in the absence of ER (Fig. 8BGo, lane 2) with similar intensity as it would bind to the HDL-R SR-BI dSRE (Fig. 8BGo, lane 5). Competition with cold oligonucleotide (lane 3) and supershift analysis (lane 4) demonstrated that the binding of SREBP-1a to the ERE1/2-1 was specific (Fig. 8Go).



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  		Figure 8. Binding of ER{alpha}, ERß, and SREBP-1a to the ERE1/2-1 in the HDL-R SR-BI promoter. A, Binding of SREBP-1a to ERE1/2-1 in the presence of the ERs ({alpha} and ß). A 32P-labeled double-stranded oligonucleotide probe (100,000 cpm per lane) corresponding to ERE1/2-1 was incubated with recombinant ER{alpha} and ERß (100 ng each) in the presence or absence of SREBP-1a protein (200 ng). Unlabeled ERE1/2-1 was added to one lane as a competitor (Cold; 125x). ER subtype ({alpha} or ß) ({alpha}Ab and ßAb, respectively) and SREBP-1 (1Ab) specific antibodies (5 µg of IgG each) were also added to some of the binding reactions. B, Binding of SREBP-1a to the ERE1/2-1 in the absence of the ERs ({alpha} and ß). Binding reactions were performed with SREBP-1a protein in the presence or absence of unlabeled ERE1/2-1 motif (Cold) or SREBP-1 (1Ab) specific antibody. Binding to a probe containing the dSRE of the HDL-R SR-BI was used as a positive control. Representative gel mobility shift assay autoradiographs are shown in both A and B. These experiments were repeated four times. The DNA protein and supershifted complexes are indicated.

 
To examine whether the ERs could also bind to a SRE motif in the absence of SREBP-1a, mobility shift assays were performed with both ERs and the dSRE found in the HDL-R SR-BI promoter. As found for SREBP-1a and the ERE1/2-1 (Fig. 9BGo), the ERs bound to the HDL-R SR-BI dSRE but with lower intensity than to the ERE1/2-1 used here as a positive control (Fig. 9AGo). Competition with cold oligonucleotide (lanes 3 and 7) demonstrated that the binding of the ERs to the SRE was specific (Fig. 9Go). Incubation with the ER subtype specific antibodies caused an abrogation (Fig. 9AGo, lanes 4 and 8) of the ER-SRE binding complex instead of supershifting as in the case of the ER-ERE complexes (Fig. 3Go). To determine whether the ERs ({alpha} and/or ß) have any effect on SREBP-1a binding to the dSRE, mobility shift assays were performed and the results presented in Fig. 9BGo. SREBP-1a bound to the HDL-R SR-BI dSRE as a single complex (referred as SREBP-1a-DNA, Fig. 9BGo, lane 2). The presence of the three proteins (SREBP-1a, ER{alpha}, and ERß) formed several minor complexes in addition to the major complex formed in the presence of SREBP-1a alone (Fig. 9BGo, lane 3). Adding SREBP-1 specific antibody completely supershifted the SREBP-1a-DNA complex but not the minor complexes designated ERs-DNA complexes (Fig. 9BGo, lane 5). Both ER subtype ({alpha} and ß) specific antibody diminished some of the minor complexes but had no effect on the SREBP-1a containing protein-DNA complex (Fig. 9BGo, lanes 6 and 7). Interestingly, the addition of ER{alpha} and ß to the binding reaction partially diminished the SREBP-1a-DNA complex (Fig. 9BGo, lane 3). This appears to occur mainly in the presence of all three proteins (ER{alpha}, ß and SREBP-1a; 49% reduction) because the addition of each ER subtype individually had a small effect (12% reduction in case of the ER{alpha} and 28% reduction in the case of ERß) on the SREBP-1a-DNA complex formation (Fig. 9BGo, lanes 8 and 9). These results demonstrated that the presence of the three factors, SREBP-1a, ER{alpha} and ß, altered the binding of each individual factor to its corresponding DNA binding motif. To determine whether the interactions of these proteins, ER{alpha}, ERß, and SREBP-1a, and the DNA have any effect on the HDL-R SR-BI promoter activity, cotransfection studies were performed in the presence of all three factors. As shown in Table 2Go, maximal activation levels of both basal and E2-dependent HDL-R SR-BI promoter activity were obtained in the presence of all three factors, ER{alpha}, ERß, and SREBP-1a. Exogenous addition of the same factor that is present in the specific cell line (ER{alpha} in the MCF-7 and ERß in the GG-CL) did not enhance the activation of the HDL-R SR-BI promoter. This suggests that the endogenous ER-isoform expression levels were sufficient for maximal activation of the HDL-R SR-BI promoter by that factor. Another interesting fact is the finding that activation of the HDL-R SR-BI promoter was higher in the MCF-7 cells than in the GG-CL cells (Table 2Go). This could be due to the presence of cell-specific factors that modulate the extent of the promoter activation by ER{alpha}, ERß, and/or SREBP-1a.



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  		Figure 9. Binding of SREBP-1a, ER{alpha} and ERß to the dSRE in the rat HDL-R SR-BI promoter. A, Binding of ER{alpha} and ERß to the dSRE in the absence of SREBP-1a. A 32P-labeled double-stranded oligonucleotide probe (100,000 cpm per lane) corresponding to the dSRE was incubated with recombinant ER{alpha} or ERß (100 ng) in the presence or absence of unlabeled dSRE (Cold; 125x) or ER subtype ({alpha} or ß) ({alpha}Ab and ßAb, respectively; 5 µg of IgG) specific antibodies. Binding to ERE1/2-1 was used as positive control. B, Binding of ER{alpha} and ERß to dSRE in the presence of SREBP-1a. Binding reactions were performed with SREBP-1a protein (200 ng) in the presence or absence of recombinant ER{alpha} ± ERß (100 ng each). Unlabeled dSRE was added to some of the lanes as a competitor (Cold; 125x). ER subtype ({alpha} or ß) ({alpha}Ab and ßAb, respectively) and SREBP-1 (1Ab) specific antibodies (5 µg of IgG) were also added to some of the binding reactions. Representative gel mobility shift assay autoradiographs are shown in both (A) and (B). These experiments were repeated two times. The DNA-protein and supershifted complexes are indicated.

 

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  		Table 2. Effects of SREBP-1a on ER activation of the HDL-R SR-BI promoter

 
To examine whether SREBP-1a, ER{alpha}, and ERß directly interact with each other, mammalian two-hybrid assay was performed as described in Materials and Methods. As shown in Fig. 10Go, cotransfecting ER{alpha}- (or ERß-pACT) and SREBP-1a-pBIND with the pG5-basic vector caused a 7.3- (or 9.6-) fold increase (P < 0.001) in luciferase activity compared with cells cotransfected with SREBP-1a-pBIND and pACT empty vector. These results demonstrated that SREBP-1a was able to interact with both ERs ({alpha} and ß).



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  		Figure 10. Interaction between the ERs ({alpha} and ß) and SREBP-1a using the mammalian two-hybrid assay. Human bladder carcinoma HTB-9 cells (plated in six-well plates) were transfected with the pG5-luciferase vector in the presence of the indicated vectors (2 µg of each plasmid DNA per well). ER{alpha} and ß were cloned into the pACT vector, whereas SREBP-1a was cloned into the pBIND vector. The data are represented as mean fold induction ± SEM, where the value of luciferase activity for the pG5-basic plasmid transfected in the presence of the pACT/pBIND negative controls was set to 1.0. The data are from a typical experiment performed in triplicate. These experiments were repeated two times. *, P < 0.001, was obtained by comparing to the pACT/pBIND negative control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this investigation provide evidence that E2 via the ERs activates the HDL-R SR-BI promoter. E2/ER-dependent regulation of the HDL-R SR-BI gene demonstrated herein appears to occur through the classical pathway that involves binding and activation of EREs. Four putative ERE1/2 motifs were identified in the promoter of this gene, but only three (ERE1/2s 1, 2, and 4) bound both ER subtypes ({alpha} and ß) with high intensity and were shown to be important for E2-activation of this gene in both MCF-7 and GG-CL cells. It is commonly proposed that ER binding requires a minimal consensus ERE motif containing two half-sites in inverted position: 5'-GGTCAnnnTGACC-3' [where "n" is any nucleotide] (44). Aligning the sequence of the consensus ERE motif with the sequence of three ERE1/2 identified in the HDL-R SR-BI promoter (see Table 3Go) demonstrated that the promoter of this gene contains imperfect EREs consistent with what has been reported for most estrogen-sensitive genes (50, 51). While the first half-site of the HDL-R SR-BI EREs was 100% identical to the consensus ERE1/2 sequence, the second half-site differs from the consensus sequence by up to four nucleotides (Table 3Go; nucleotides in underlined letters). Independent of these differences, the HDL-R SR-BI EREs bound both ER{alpha} and ERß, and in the case of the ER{alpha}, binding exceeded the binding intensity displayed by the perfect palindromic control ERE motif. It has been previously demonstrated that ER binding to nonconsensus EREs depends on the nucleotides flanking the core sequence (50). The background sequence included in the mobility shift assay oligonucleotides appears to be sufficient for ER binding to the HDL-R SR-BI EREs. Because this flanking sequence came from the promoter itself, it is very likely that this sequence plays an important role in ER binding to the HDL-R SR-BI motifs in vivo as well. The second half-site of the response element located at position -1622, originally designated ERE1/2-3, does not match the consensus ERE at a single nucleotide suggesting that this flanking region may not stabilize ER binding to this motif rendering it unresponsive to E2. Both the mutation analysis studies and the mobility shift assays demonstrated that the ERE1/2-3 is not important for estrogen regulation of the HDL-R SR-BI gene. Sequence analysis and preliminary reporter-construct assays have shown that the promoter element located at -1622 resembles a peroxisome proliferator-responsive element half-site motif through which the PPAR{alpha} and the RXR bind and activate the HDL-R SR-BI gene (Lopez, D., and M. P. McLean, unpublished observations).


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  		Table 3. Comparison of the EREs found in the promoter of the HDL-R SR-BI gene with the consensus ERE motif

 
It is proposed, albeit less frequently, that ERs could also bind and activate half-site motifs (52). These half-site motifs, although widely spaced, may cooperate to generate an efficient ERE (52). Examples of genes in which ERE1/2 motifs play an important role include the rat PRL (53), the human cathepsin D (54), and the ovalbumin gene (55). The ERE1/2 motifs found in the HDL-R SR-BI are widely separated as well suggesting that this gene may be regulated by a similar mechanism. However, according to Anderson and Gorski (53), the ER{alpha} binding complex formed with an ERE1/2 has faster mobility on a 4% polyacrylamide gel than the ER{alpha} binding complex formed with an consensus ERE motif. Because the mobility of the complexes formed between the HDL-R SR-BI ERE1/2 motifs and the ER{alpha} and/or ß was similar to the mobility of the consensus ERE-ER complexes, it could be concluded that the ERs had to be bound to two half-sites similarly to the consensus ERE.

Another important finding of the current report is that similar to other genes regulated by estrogen (24, 25), the ERs were able to bind as homo- or heterodimers to the HDL-R SR-BI EREs as shown by the supershift analysis although the formation of homodimers was preferred over heterodimers. However, the possibility that the preferential formation of homodimers over heterodimers resulted from the use of recombinant protein in the experiment cannot be eliminated. In addition, it was demonstrated that the presence of both ERs in the transfection studies enhanced the overall luciferase activity produced in the presence of only one ER subtype. It is well known that in humans and rodents, both ER{alpha} and ERß are found in most cell types within steroidogenic tissues (46, 47, 48, 49). Reports have shown that ER{alpha} and ß are both expressed in ovarian follicles (56) and the corpus luteum of pregnancy (43). Thus, the presence of the ERs in these tissues simultaneously may contribute to maximize the response of HDL-R SR-BI gene to estrogen treatment. In the liver, however, although both ERs could be found, only ER{alpha} is expressed in hepatocytes (46, 47), the main cell type within this tissue that mediates selective uptake of HDL-cholesterol esters (45, 57). Herein, it was shown that ER{alpha} could also mediate activation of the HDL-R SR-BI gene by estrogen. These data would appear to conflict with previous findings using a rat model or human liver cells in culture that demonstrated that estrogen treatment dramatically decreased hepatic (15) and HepG2 cell (58) HDL-R SR-BI expression and HDL-cholesterol uptake (59). The human cell culture experiments (58) demonstrated that treating HepG2 cells with 10 pM of E2 for 12 h eliminated the expression of the HDL-R SR-BI gene and reduced selective-cholesterol ester uptake (58), confirming the rat liver studies (15). Interestingly, that same study (58) showed that the scavenger receptor class B type II (SR-BII), an alternatively spliced product of the HDL-R SR-BI, significantly increased after E2 treatment in both HepG2 cells and rat liver. Normally, SR-BII represents approximately 12% of the total immunodetectable SR-BI/SR-BII present in the liver (60). SR-BI and SR-BII bind HDL with similar affinity but SR-BII is 4-fold less efficient at selective HDL-uptake compared with SR-BI (60), which explains the decrease in overall cholesterol ester uptake (58) following E2 administration. It appears that E2 reduces hepatic HDL-R SR-BI gene expression by altering the posttranscriptional processing of the HDL-R SR-BI RNA. However, the finding that E2-treatment was able to increase SR-BII protein levels 3.2-fold more than the starting level of SR-BI (58) suggests that transcription of the HDL-R SR-BI gene may also increase in response to estrogen to compensate for the increase in the alternative form. Because the liver expresses only ER{alpha} (46, 47), the increase in transcription may not be as high as in steroidogenic tissues that express both ERs but is adequate to enhance the increase in SR-BII. This would correlate with the findings from the current report, but additional study will be required to confirm transcriptional control of the shift in receptor subtype expression.

SREBP-1a was shown in a previous report to activate the HDL-R SR-BI gene (37). The experiments reported here now identify an additional function for SREBP-1a as a coactivator of the ERs for the HDL-R SR-BI gene. These studies demonstrate that SREBP-1a enhances both binding and activation of the HDL-R SR-BI promoter by E2/ERs. The mechanism for this regulatory process involves direct protein-protein interaction between SREBP-1a and the ERs as demonstrated using a mammalian two-hybrid assay. Interaction of the ERs with other factors have been previously demonstrated by GST-pull-down and two-hybrid assays (26, 29, 31, 61, 62, 63). These factors include SF-1 (26), c-Jun (31, 62), Sp1 (29, 62), and dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome gene-1 (63). However, this is the first study to demonstrate interaction between ERs and SREBP-1a. It was also demonstrated that SREBP-1a was able to bind directly to the ERE1/2 containing oligonucleotides in the absence of ERs with the same intensity, as it would bind to an SRE. Binding of SREBP-1a to non-SRE sequences has been previously demonstrated (64). SREBP-1 has been shown to bind E-box motifs (CANNTG) (64). None of the ERE1/2 oligonucleotides used in these studies contain E-box or SRE motifs. Thus, the possibility that SREBP-1a could be binding directly to the ERE1/2 core sequence cannot be discarded. The ERs also bound to the SRE but with lower intensity than it would bind to an ERE. Recently, Li et al. (28) demonstrated that mutation at the SRE-1 motif found in the low density lipoprotein receptor (LDL-R) promoter diminished ER{alpha} activation of this gene. This suggests that SREBP-1a may be involved in the ER-dependent regulation of the LDL-R (28), or that ER{alpha} may bind to the SRE-1 and that the mutation prevents the binding. In contrast to the HDL-R SR-BI, the LDL-R promoter does not contain a functional ERE site and consequently, estrogen regulation of this gene occurs through nonclassical pathways via the Sp1 binding sites that surround the SRE-1 (28). Thus, interaction of the ER{alpha} with nonconsensus motifs such as an SRE could be crucial in this case. Whether the binding of the ERs to the SRE and/or the binding of SREBP-1a to an ERE occurs in vivo will required additional analysis.

Because SREBP-1a is not a tissue-specific factor (65), the question remains whether this transcription factor could, under certain conditions, enhance estrogen’s effects on the HDL-R SR-BI gene. Steroidogenic tissues use HDL-cholesterol mainly as substrate for steroid hormone production (16, 66, 67). E2 has been shown to enhance steroid hormone production in tissues such as the adrenal and the ovary (15, 68, 69). Thus, to satisfy the high demand for substrate after E2 treatment, cholesterol synthesis and transport as well as lipoprotein receptor content of these tissues, including the HDL-R SR-BI, are dramatically enhanced (15, 68, 69). Due to the increase in steroidogenesis, intracellular cholesterol levels may decrease rapidly resulting in the activation of SREBP-1a. In fact, depletion in ovarian cholesteryl ester content and increased cell-surface HDL binding in response to estrogen stimulation has been previously reported (69) suggesting that synergistic ER-SREBP-1a activation of the HDL-R SR-BI may occur in the ovary. While SREBP-1a is also expressed in the liver (65), it is well known that estrogen treatment enhances the expression of hepatic LDL-R (28, 70, 71, 72) and 3- hydroxy-3-methylglutaryl-coenzyme A reductase (71, 72) genes. Thus, in this tissue cholesterol accumulation may reduce SREBP-1a and consequently, the interaction between ER{alpha} and SREBP-1a is prevented.

In summary, E2 through its nuclear receptors (ER{alpha} and ERß) positively activated the HDL-R SR-BI promoter through the classical pathway by the binding and activation of three EREs. This activation of the HDL-R SR-BI by E2 also involved the formation of heterodimers between the two ER subtypes and interaction with the coactivator SREBP-1a. Although the current data suggest that SREBP modulation of the ER-dependent transactivation could occur via DNA- dependent and/or -independent (coactivation) pathways, further analysis will be required to determine the exact mechanism for this modulation. The results presented here are the first to report on a possible mechanism through which estrogen via its nuclear receptors activates the HDL-R SR-BI promoter in steroidogenic tissues.


   Acknowledgments
 
We thank Drs. Geula Gibori, Jan-Åke Gustafsson, Katarina Pettersson, and Tim Osborne for gifts of materials.


   Footnotes
 
This work was supported by grants from the American Heart Association Florida Affiliate Grant-in-Aid 0150973B and the National Institute of Health R01-HD-35163 (to M.P.M.). D.L. was supported by a National AHA Scientist Development Grant 0030172N.

1 These individuals contributed equally to the research presented in this article. Back

Abbreviations: Ab, Antibody; CMV, cytomegalovirus; dSRE, distal SRE; ERE, estrogen response element; ERE1/2, ERE half-site motifs; HDL, high-density lipoprotein; HDL-R, HDL receptor; 4-OHT, 4-hydroxytamoxifen; SF-1, steroidogenic factor-1; Sp1, specific factor-1; SRE, sterol response element; SR-BI, scavenger receptor class B type I; SREBP-1a, sterol regulatory element binding protein-1a.

Received November 2, 2001.

Accepted for publication February 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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