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Endocrinology Vol. 142, No. 4 1546-1553
Copyright © 2001 by The Endocrine Society

ARTICLES

Requirement of Sp1 and Estrogen Receptor {alpha} Interaction in 17ß-Estradiol-Mediated Transcriptional Activation of the Low Density Lipoprotein Receptor Gene Expression1

Cong Li, Michael R. Briggs, Thomas E. Ahlborn, Fredric B. Kraemer and Jingwen Liu

Veterans Affairs Palo Alto Health Care System (C.L., T.E.A., F.B.K., J.L.), Palo Alto, California 94304; Pharmacia Corp. (M.R.B.), St. Louis, Missouri 63017

Address all correspondence and requests for reprints to: Jingwen Liu, Ph.D. (154P), Vetarans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen is one of the most important physiological regulators of low density lipoprotein receptor (LDLR) expression. Despite many studies conducted in animals and humans showing increased expressions of LDLR messenger RNA by hormone treatment, the molecular basis of the effect of estrogen on LDLR transcription has not been clearly elucidated. By using HepG2 cells that transiently express functional estrogen receptor {alpha} (ER{alpha}) and LDLR promoter constructs, we show that the specific interaction of ER{alpha} with the transcription factor Sp1 bound to the LDLR promoter is responsible for the activation of LDLR transcription by estrogen. We demonstrate that 1) mutations to abrogate the binding of Sp1 to its recognition sequences present in repeat 1 and repeat 3 elements of the LDLR promoter completely abolish the ER{alpha}-mediated activation of the LDLR promoter activity; 2) mutations that abolish the selective DNA-binding activity or inactivate the C-terminal transcription activation function (AF2) of ER{alpha} had no effect on the ability of ER{alpha} to activate LDLR transcription; however, transcriptional activation was completely lost by deletion of the N-terminal transcription activation region (AF1); 3) a subregion of AF1 (amino acids 67–139) was further identified to be important for ER{alpha} to activate the LDLR promoter; and 4) ER{alpha} enhanced the formation of Sp1-repeat 3 DNA complexes. We also show that mutation at the sterol-responsive element-1 site diminishes the activity of ER{alpha} on LDLR transcription, thereby suggesting that the sterol-responsive element-1-binding protein may interact with the Sp1-ER{alpha} complex to trans-activate LDLR gene transcription. This study for the first time provides a molecular basis for an understanding of the regulation of LDLR transcription by estrogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CLINICAL STUDIES among postmenopausal women have found that the use of estrogen is associated with a reduction in the risk of cardiovascular disease (1, 2, 3). Estrogens administered to postmenopausal women increase low density lipoprotein (LDL) clearance (4) and lower plasma LDL cholesterol levels (5, 6). As the majority of plasma LDL cholesterol is removed from the circulation by liver through a process of LDL receptor-mediated endocytosis (7), it is highly likely that the decreased plasma LDL cholesterol level in women taking estrogens is caused at least in part by increased hepatic LDL receptor (LDLR) expression. This postulation is supported by animal studies showing that treatment of rats or rabbits with pharmacological doses of 17{alpha}-ethinyl estradiol increased hepatic LDLR messenger RNA and protein levels (8, 9, 10, 11).

LDLR expression is mainly controlled at the transcriptional level under the influence of the intracellular cholesterol content (12, 13). When cellular cholesterol levels rise, LDLR transcription is reduced, and the amount of LDLR on the cell surface decreases. When cellular cholesterol storage is depleted, LDLR transcription is activated. This leads to increased expression of LDLR on the cell surface of hepatocytes, thereby reducing plasma LDL cholesterol levels through increased binding of LDL particles to the receptor and subsequent internalization of the ligand-receptor complex.

The promoter region and the cis-acting elements that control basal and cholesterol-mediated transcription of LDLR have been localized to three GC-rich imperfect 16 bp direct repeats (14, 15, 16, 17). These three repeats lie within 100 bp upstream of the transcriptional start site. Repeat 1 (R1) and repeat 3 (R3) contain Sp1-binding sites that support the basal transcriptional activity of the LDLR. Interactions of Sp1 to the proximal binding site in R3 and to the distal binding site in R1 are required for the normal maximal transcriptional activity of the LDLR gene. Interruption of Sp1 binding to either repeat severely decreases basal transcription. For example, a 3-bp deletion in the R1 region of the LDLR promoter was identified in a patient with a clinical diagnosis of familial hypercholesterolemia (18). Sp1 binding to the mutated R1 sequence was abolished, and LDLR gene expression in fibroblasts from the patient was severely decreased.

Cholesterol regulation of LDLR transcription is mediated through a 10-bp sequence (5'-ATCACCCCAC-3') within repeat 2 (R2), designated sterol-responsive element-1 (SRE-1) (19, 20). Under low intracellular cholesterol conditions, SRE-1-binding proteins, SREBP1 and SREBP2, can bind to the SRE-1 sequence and activate transcription (21, 22). Recent studies showed that the interaction of SREBPs with SRE-1 increases the binding of Sp1 to R3, thereby resulting in a synergistic activation (23, 24).

To understand the beneficial effect of estrogen on the plasma LDL cholesterol level, a number of studies, using a liver hepatoma cell line HepG2 as an in vitro system, were carried out to directly examine the effects of estrogen on LDLR expression. Semenkovich et al. reported that treatment of HepG2 cells with 10 µg/ml (38 µM) 17ß-estradiol (E2) increased the number of LDLR on the cell surface by measuring [125I]LDL binding and ligand blotting (25), whereas a subsequent study reported by Wade et al. showed that E2 at concentrations up to 500 ng/ml (1.9 µM) did not increase LDLR expression in HepG2 cells (26). However, another study using 10 µg/ml E2 showed that LDLR promoter activity was increased in HepG2 cells (27). Apparently, very high and unphysiological concentrations of E2 (38 µM) were required to produce an effect on LDLR expression in HepG2 cells, which was probably due to a lack of adequate expression of estrogen receptors (ERs). The actions of estrogen are mediated primarily by ER{alpha} (28) and ERß (29, 30). Liver cells express only the ER{alpha} (29, 30). Although HepG2 cells retain many liver-specific functions, the expression of ER{alpha} is below the level of detection (31). With transfection of an ER{alpha} expression vector into HepG2 cells, LDLR promoter activity was increased by E2 at a much lower physiological concentration (1 nM) (32). Collectively, these studies suggest that E2 can increase LDLR expression through activation of gene transcription; however, the cis-acting regulatory elements and the trans-acting factors that mediate the effect of estrogen on LDLR transcription have not been clearly characterized.

The current report shows that ER{alpha}- and E2-mediated activation of LDLR promoter activity in HepG2 cells requires functional Sp1-binding sites. Mutations disrupting Sp1 binding to R1 and R3 completely abolished E2 activity on the LDLR promoter. By using vectors containing mutations or deletions of different functional domains of ER{alpha}, we identified a region within activation function domain 1 (AF1) of ER{alpha} that was important for activation of LDLR transcription by ER{alpha}. This functional region overlaps a domain of AF1 that was recently shown to be required for ER{alpha} to activate a synthetic promoter that contains an Sp1-binding site (33). Electrophoresis mobility shift assays (EMSA) demonstrated that the binding of Sp1 to R3 was enhanced by ER{alpha}. Taken together, these studies for the first time document that the effect of estrogen on LDLR transcription is mediated through specific interactions between ER{alpha} and the transcription factor Sp1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents
The human hepatoma cell line HepG2 was obtained from American Type Culture Collection (Manassas, VA) and was cultured in phenol red-free Eagle’s MEM supplemented with 10% charcoal-absorbed FBS (HyClone Laboratories, Inc., Logan UT). Antibodies specific to human Sp1 and ER{alpha} (SC-543) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), for use in EMSA. Human recombinant Sp1 was purchased from Promega Corp. (Madison, WI). Human recombinant ER{alpha} was obtained from PanVera (Madison, WI). E2 and anti-ß-actin monoclonal antibody were purchased from Sigma (St. Louis, MO).

Plasmid vectors
LDLR promoter luciferase reporters pLDLR234, pLDLR234-R1, pLDLR234-R2, pLDLR234-R3, and pLDLR234-R1/R3 have been previously described (34). Briefly, vector pLDLR234-R2 contains a two-base mutation (underlined) within the core SRE-1 region (AAAATCACCCCACTGC to AAAATCACggCACTGC). The promoter activity of this vector was decreased to 12% of the wild-type promoter and was no longer regulated by cholesterol. The vector pLDLR234-R3 contains a two-base substitution (underlined) within the core Sp1 site (AAACTCCTCCCCCTGC to AAACTCtTtCCCCTGC). This mutation abolished Sp1 binding to R3 and decreased promoter activity to 7% that in the wild-type vector. The vector pLDLR234-R1 contains the same two-base substitution (underlined) within the core Sp1 site as the R3 mutant (AAACTCCTCCTCTTGC to AAACTCtTtCTCTTGC). The mutation of R1 eliminated Sp1 binding and decreased LDLR promoter activity to 10% of the wild-type activity. The vector pLDLR234-R1/R3 contains mutations within both R1 and R3.

The wild-type human ER{alpha} expression vector (pRST7hER) and a luciferase reporter vector (pMTVERE4-LUC) containing four copies of the estrogen response element (ERE) were provided by Dr. Kathy Fosnaugh (Ligand Pharmaceuticals, Inc., San Diego, CA). The ER{alpha} mutant expression vectors AF1-DBD-X, X-DBD-X, AF1-X-AF2, and X-DBD-AF2 have been previously described (31) and were provided by Dr. Douglas C. Harnish (Wyeth-Ayerst Laboratories, Inc., Radnor, PA). The ER{alpha} mutant expression vectors HE1, HE2, HE3, HE10, HE18, and HE19 have been previously described (35) and were provided by Dr. Pierre Chambon (IGBMC, Strasbourg, France).

Preparation of nuclear extracts and EMSAs
HepG2 cells were seeded at 4.5 x 106 cells/100-mm dish 3 days before harvesting. Nuclear extracts were prepared by the method of Dignam et al. (36), except buffer A was supplemented with 1 mM Na3VO4 and 1 µg/ml each of pepstatin and leupeptin. Nuclear extracts were quick-frozen by liquid nitrogen and stored in aliquots. Protein concentrations were determined using a modified Bradford assay using BSA as a standard (Pierce Chemical Co., Rockford, IL). Protein concentrations of nuclear extracts from different preparations were typically 2–3 mg/ml. Oligonucleotide probes were annealed and end-labeled with T4 polynucleotide kinase in the presence of [{gamma}-32P]ATP.

Each binding reaction was composed of 10 mM HEPES (pH 7.8), 2 mM MgCl2, 2 mM dithiothreitol, 80 mM NaCl, 10% glycerol, 1 µg poly(dI-dC), 1 µg BSA, and 0.1–5 µg nuclear extract in a final volume of 20 µl. Nuclear extracts were incubated with 0.4–0.5 ng 32P-labeled, double stranded, synthetic oligonucleotide probe (40–80 x 103 cpm) for 10 min at room temperature. For EMSA with recombinant human Sp1, the reaction mixture contained 10 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 6% glycerol, 1 µg poly(dI-dC), 2 µg BSA, and 1–10 ng affinity-purified Sp1 in the presence or absence of recombinant human ER{alpha} (0.5–2 pmol) in a final volume of 20 µl. The reaction mixtures were loaded onto a 6% polyacrylamide gel and run in TBE buffer (90 mM Tris, 90 mM borate, and 2 mM EDTA, pH 8.3) at 20 mA for 2 h at 4 C. The gels were dried and visualized on a PhosphorImager. For supershift assays, antibody was incubated with recombinant Sp1 for 30 min at room temperature before addition of the probe.

The sequences of EMSA probes are as follows, and the consensus binding sites of Sp1 are underlined: R23, 5'-TTTGAAAATCACCCCACTGCAAACTCCTCCCCCTGCT-3'; R23D, 5'-TTTGAAAATCACCCCACTGCAAACTCtTtCCCCTGCT-3'; and R1, 5'-TTCGAAACTCCTCCTCTTGCAGTGAGGTGAAGACATTTG-3'. The oligonucleotide R23D contains the same mutation as the vector pLDLR234-R3. The mutated nucleotides in R23D are in lowercase italic.

Transient transfection assays
HepG2 cells cultured in a 24-well plate at a density of 0.12 x 106 cells/well were transiently transfected with plasmid DNA by the method of calcium phosphate coprecipitation with 20 µg total DNA in each precipitation in the ratio of 10 µg LDLR-luciferase reporter, 5 µg ER{alpha} expression vector, and 5 µg pRSV-ß-galactosidase. The ratio of pLDLR reporter and ER{alpha} (10:5) was shown to give a maximal response to E2 at both 1-µM and 100-nM concentrations in pilot experiments that examined the ER{alpha} dose-dependent effect on pLDLR promoter activity. After 40-min precipitation, 50 µl precipitate containing 880 ng DNA were added to each well. The cells were incubated with the DNA precipitate for 4 h, washed with PBS, and refed with fresh medium containing 10% charcoal-absorbed FBS without or with various concentrations of E2. After 40-h treatment, cells were lysed, and luciferase and ß-galactosidase activities were assayed. Absolute luciferase activity was normalized against ß-galactosidase activity to correct for transfection efficiency.

Statistical analysis
Comparisons of experimental data were analyzed by a two-tailed Student’s t test. P < 0.05 was considered to indicate a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time- and dose-dependent activation of LDLR promoter activity by E2
To examine the endogenous expression of ER{alpha} in HepG2 cells and the exogenous expression of ER{alpha} after transient transfection, total cell lysates were prepared from HepG2 cells, mock-transfected or transfected with pRST7hER. Cell lysate from MCF-7 cells that express normal ER{alpha} was also prepared to provide a positive control. Fifty micrograms of soluble protein from each sample were loaded on a 10% SDS gel and separated by electrophoresis, transferred to polyvinylidene membrane, and blotted with anti-ER{alpha} polyclonal antibody. Figure 1Go shows that HepG2 cells transfected with pRST7hER express ER{alpha} at a level similar to MCF-7 cells, whereas no ER{alpha} was detected from mock-transfected HepG2 cells. Immunoblotting with anti-ß-actin antibody showed comparable amounts of soluble protein in each sample. To determine the effect of E2-activated ER{alpha} on LDLR transcription, HepG2 cells were transfected with LDLR promoter luciferase reporter construct pLDLR234 with control DNA or with pRST7hER and then treated with different concentrations of E2 for 40 h. Analysis of luciferase activities in transfected cells shows that E2 produced a dose-dependent activation of LDLR promoter activity in ER{alpha}-transfected cells. The promoter activity was increased 3-fold by E2 at 1 nM and was increased 5-fold at 1 µM. In contrast, the LDLR promoter activity in HepG2 cells transfected with a negative control vector was not changed by E2 in the same concentration range. To determine the kinetics of the E2 action, E2 (100 nM) was added at different times after transfection, and all cells were harvested after 48 h posttransfection. The results show that the LDLR promoter activity was increased 3-fold at 24 h and reached a plateau (3.7- to 4-fold) after 32-h treatment with E2. These experiments established that E2 activates LDLR transcription of HepG2 cells in a time- and a dose-dependent manner and is mediated through ER{alpha}.



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  		Figure 1. Western blot analysis of ER{alpha}. Total cell lysates were isolated from MCF-7, HepG2 cells mock-transfected, and HepG2 cells transfected with plasmid pRST7hER. Soluble proteins (50 µg/lane) were applied to SDS-PAGE. Detections of ER{alpha} and ß-actin were performed by immunoblotting as described in Materials and Methods.

 
Sp1 binding sites are the primary responsive elements that mediate the effect of E2-ER{alpha} in activation of LDLR transcription
Vector pLDLR234 contains the LDLR promoter sequence -142 to +35 relative to the transcription start site. This promoter region includes the SRE-1 in R2 and the two Sp1-binding sites in R1 and R3. To determine which regulatory element is involved in the E2-ER{alpha}-mediated activation of LDLR promoter activity, we compared the activity of E2 on the wild-type vector pLDLR234 with the activities of E2 on mutant vectors that either abolish the SREBP binding or the Sp1 binding. Figure 2Go shows that the stimulatory effect of E2 on LDLR promoter activity was partially reduced from 4.7- to 1.9-fold by mutation in the SRE-1 site (pLDLR234-R2). The E2 activity was also significantly diminished in the construct carrying mutations in either R1 (pLDLR234-R1, 2.4-fold) or R3 (pLDLR234-R3, 2.0-fold). In contrast, E2 activity in cells transfected with pLDLR-R1/R3 was completely lost (1.3 ± 0.2-fold), thereby demonstrating that interference of Sp1 binding to both R1 and R3 eliminates the activity of E2 on LDLR transcription. These results suggest that although a functional SRE-1 is required for maximal E2 activation, interaction of Sp1 with the LDLR promoter may be critically involved in the ER{alpha}-mediated activation of LDLR transcription.



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  		Figure 2. Effects of E2 on LDLR promoter constructs containing mutations in repeats 1, 2, 3, and 1+3. Cells were transfected with pRST7hER and pLDLR234, pLDLR234-R1, pLDLR234-R2, pLDLR234-R3, or pLDLR234-R1/R3, respectively. After transfection, cells were stimulated with E2 (100 nM) for 40 h before lysis. The normalized luciferase activity of transfected cells that were untreated is expressed as 1. The data (mean ± SD) shown were derived from three independent transfection experiments in which triplicate wells were assayed. Significant differences in luciferase activities (indicated by an asterisk) were observed between E2-treated cells and control cells in pLDLR234 (P = 0.0003), pLDLR234-R1 (P = 0.002), pLDLR234-R2 (P = 0.004), and pLDLR234-R3 (P = 0.016) transfected cells.

 
ER{alpha} transcription activation function 1 (AF1) is required for activation of LDLR transcription
To further investigate the mechanism by which ER{alpha} and E2 activate LDLR transcription, vectors expressing wild-type ER{alpha} or ER{alpha} with a deletion in the N-terminal transcription activation function (AF1), point mutations in the DNA binding domain, or point mutations in the C-terminal transcription activation function (AF2), were individually cotransfected with pLDLR234 into HepG2 cells. Figure 3AGo shows that inactivation of the AF2 functional domain (AF1-DBD-X) did not affect the ability of E2 to stimulate LDLR promoter activity. Similarly, E2 activity was retained in the vector that contains mutations within the DNA-binding domain (AF1-X-AF2) that convert ER{alpha} binding selectivity from an ERE to a glucocorticoid response element (37). In contrast, deletion of the AF1 functional domain (X-DBD-AF2) completely abolished E2 activity. Likewise, mutant vector (X-DBD-X) containing the AF1 deletion and AF2 mutations did not respond to E2. The lack of E2-ER{alpha} response on LDLR promoter activity by deletion of AF1 was not due to the absence or poor expression of the mutant receptor, as Western blot analysis showed comparable levels of expressions for wild-type ER{alpha} and mutant ER{alpha} in cells transfected with the various vectors (Fig. 3BGo). To demonstrate the promoter-specific effect of the AF1 deletion, vectors pRST7hER and X-DBD-AF2 were cotransfected into HepG2 cells with pMTVERE4-LUC, which contains four copies of ERE. Stimulation of cells with E2 produced 7.5- and 2.8-fold increases in luciferase activities in cells transfected with the wild-type ER{alpha} and the X-DBD-AF2, respectively. This demonstrates that the AF1 deletion impaired the trans-activating activity of ER{alpha}, but did not completely eliminate the ability of ER{alpha} to bind to ERE and to trans-activate. These results suggest that the N-terminal region of ER{alpha} (AF1) is critically involved in the interaction of ER{alpha} and LDLR promoter, possibly through the Sp1-binding sites.



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  		Figure 3. Requirement of the AF1 domain in ER{alpha}-mediated activation of LDLR promoter activity. A, HepG2 cells were cotransfected with pLDLR234 and wild-type ER{alpha}, mutant ER{alpha} expression vectors, or mock DNA (pUC18), respectively. After transfection, cells were stimulated with E2 for 40 h before lysis. The normalized luciferase activity of transfected cells that were untreated is expressed as 1. The data (mean ± SD) shown were derived from three to five independent transfection experiments in which triplicate wells were assayed. Significant differences in luciferase activities (indicated by an asterisk) were observed between E2-treated cells and control cells in wild-type ER{alpha} (P = 9 x 10-5), AF1-X-AF2 (P = 0.001), and AF1-DBD-X (P = 0.003) transfected cells. (B) HepG2 cells were transfected with vectors expressing wild-type or mutant ER{alpha} protein. Two days after transfection, total cell lysates were harvested, and 50 µg soluble protein/sample were used to detect ER{alpha} protein by immunoblotting. The upper arrow indicates the full-length ER{alpha} (lanes 2 and 4). It should be noted that a nonspecific band present in both untransfected and transfected cells was also detected by the anti-ER{alpha} antibody, and this band migrated slightly faster than the truncated ER{alpha} (X-DBD-AF2, lane 3), which is indicated by the lower arrow.

 
Identification of a subdomain within AF-1 of ER{alpha} responsible for activation of LDLR transcription
To further localize the functional region in the AF1 domain that is responsible for induction of LDLR transcription, vectors containing different AF-1 deletions of ER{alpha} were cotransfected into HepG2 cells with pLDLR234, and the transfected cells were treated with E2 for 40 h. Figure 4Go shows that deletion of amino acids 9–62 (HE1) lowered the E2 activation of pLDLR234 from 4.4- to 3-fold, retaining 60% activity of the wild-type ER{alpha}. Deletion of amino acids 67–131 (HE2) decreased the E2 activity to 1.7-fold, and deletion of amino acids 132–198 (HE3) nearly eliminated the ER{alpha} activity. Similar to HE3, constructs HE18 and HE19 that contain deletions of amino acids 1–139 and 1–178 did not activate LDLR promoter activity. In contrast, deletion of amino acids 141–170 (HE10) had no effect on the induction of LDLR promoter activity by ER{alpha} and E2. These data suggest that the region encompassing amino acids 67–139 is important for ER{alpha} to activate LDLR transcription through Sp1-binding sites. A recent study has localized the ER{alpha} and Sp1 interaction to a similar region of AF1 (amino acids 79–117) (33).



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  		Figure 4. Localization of a functional region within AF1 that is important for ER{alpha} to activate LDLR transcription. Mutant vectors expressing ER{alpha} with different AF1 deletions were cotransfected with pLDLR234. After transfection, cells were stimulated with E2 (100 nM) for 40 h before lysis. The normalized luciferase activity of transfected cells that were untreated is expressed as 1. Experiments were repeated and results were confirmed three times.

 
ER{alpha} enhances Sp1 binding to repeat 3 element of the LDLR promoter
To determine whether the interaction of Sp1 with the LDLR promoter is affected by ER{alpha}, EMSA using a labeled oligonucleotide probe R23 containing the R2 and R3 elements was performed with purified recombinant Sp1. Figure 5AGo shows a dose-dependent binding of Sp1 to the probe (lanes 2, 4, and 6). Figure 5BGo shows the specific interaction of Sp1 with the R23 probe, as binding of Sp1 to the probe was inhibited by competition with a 100-fold molar excess of the unlabeled oligonucleotide R23 (lane 2), but was not inhibited by oligonucleotide R23d that contains a CTCCTCCCCC to CTCtTtCCCC double nucleotide substitution (underlined) within the core Sp1 binding site (lane 3). Furthermore, the complex was completely supershifted by anti-Sp1 antibody (lane 4). Addition of purified recombinant ER{alpha} (1 pmol) to the reaction mixture increased the intensity of the Sp1-DNA complex approximately 2-fold within the concentration range of Sp1 tested (Fig. 5AGo, lanes 3, 5, and 7), but formation of additional complexes of different mobility were not detected. The enhancement of Sp1 binding was also ER{alpha} dose dependent (Fig. 5CGo, lanes 1–4). The enhancement by ER{alpha} of Sp1-binding activity within the context of HepG2 cells was also detected by EMSA using HepG2 nuclear extracts (Fig. 5DGo). The increased DNA-binding activity of Sp1 in the presence of ER{alpha} was also observed using oligonucleotide probe containing the R1 sequence of the LDLR promoter (data not shown). These results are consistent with other studies using different GC-rich regulatory elements showing that ER{alpha} enhanced the DNA-binding activity of Sp1 without formation of the Sp1/ER{alpha}-DNA ternary complex (38, 39, 40, 41).



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  		Figure 5. EMSA analysis of Sp1 interacting with the R3 sequence in the presence or absence of ER{alpha} protein. A, The double stranded oligonucleotide R23 was radiolabeled and incubated with different amounts of purified recombinant Sp1 in the absence (lanes 2, 4, and 6) or presence (lanes 3, 5, and 7) of recombinant ER{alpha} (1 pmol) for 10 min at 22 C. The reaction mixtures were loaded onto a 6% polyacrylamide gel and run in 0.25 x TBE buffer at 20 mA for 2 h at 4 C. B, Gel shift assays with Sp1 (5 ng) and the 32P-labeled R23 probe were conducted in the absence or presence of unlabeled wild-type probe or oligonucleotide R23D containing a two-base substitution within the Sp1 site. For supershift assay shown in lane 4, the anti-Sp1 monoclonal antibody was added to the reaction mixture and incubated for 30 min at 22 C before the addition of the labeled probe. C, EMSA was conducted with Sp1 (2.5 ng) and the R23 probe in the presence of different amounts of ER{alpha} (0–2 pmol). D, EMSA was performed with HepG2 nuclear extract and R23 probe in the presence or absence of ER{alpha} (1 pmol). The arrows indicate the Sp1- and Sp3-DNA complexes that were previously identified by using anti-Sp1 or anti-Sp3 antibodies (30 ). All experiments were repeated and confirmed at least two or three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ERs are normally present in the nucleus as inactive forms bound by heat shock proteins. Upon ligand binding, the E2-ER{alpha} complex uncouples from the heat shock proteins and binds, as homodimers or heterodimers, to a specific ERE present in the promoter region of target genes, thereby activating gene transcription. It is also well known that ER can regulate the transcription of genes whose promoters do not contain consensus ERE sites. In such cases, direct interactions of ER{alpha} with other transcription factors bound to their cognate sites in the promoters have been demonstrated (42, 43, 44, 45). The promoter region of the LDLR does not contain a functional ERE site; thus, the effect of E2-ER{alpha} on LDLR transcription is most likely mediated through specific interaction of ER{alpha} with other transcription factors that regulate LDLR transcription through their recognition sequences present in the promoter region. In this study we demonstrate that the ER/Sp1 interaction through Sp1-binding sites present in the R3 and R1 sequences are responsible for activation of LDLR promoter activity in HepG2 cells when functional ER{alpha} is present.

We show that the activity of E2 on the LDLR promoter was significantly impaired by mutations that interrupt Sp1 binding to R1 (pLDLR234-R1) or R3 (pLDLR234-R3) sequences, whereas inhibition of Sp1 binding to both repeats completely abolished the effect of ER{alpha} on LDLR transcription (vector R1/R3). These data suggest that Sp1-binding sites in the promoter are the critical regulatory cis-acting elements that mediate the activation of LDLR transcription by ER{alpha}. We also showed that mutation at the SRE-1 site (pLDLR234-R2), which abolished SREBP binding, partially inhibited the activity of ER{alpha} on LDLR promoter activity. Although E2 activity was decreased, the promoter activity in cells stimulated with E2 was still statistically significantly higher than that in controls (P = 0.004). Previously, using a synthetic promoter reporter construct that contains multiple copies of R2 and R3 in tandem, Croston et al. showed that mutation within the SRE-1 site drastically decreased basal and E2-stimulated promoter activities in HepG2 cells cotransfected with ER{alpha}. Based on those results, it was concluded that the effect of E2 was mediated through the SRE-1 site despite the fact that the activity of the promoter with SRE-1 mutation in E2-stimulated cells was still higher than the control value (~2-fold) (32). The synthetic promoters used by Croston et al. were created to be much more sensitive to signaling through the SRE-1 site, and therefore, differences are likely to be magnified in their system. As the native LDLR promoter system is used in the current study, it is highly likely that results from the present study more accurately reflect the physiological regulation of the LDLR. It has been shown that SREBP increases the binding of Sp1 to R3 in an in vitro study (23). This observation was further confirmed in intact cells, showing that activation of SREBP by sterol depletion results in an increased binding of Sp1 to a site adjacent to SRE-1 in the LDLR promoter (24). Therefore, it is possible that mutation of SRE-1 indirectly affected the action of ER{alpha} on the LDLR promoter by affecting the binding of a Sp1-ER{alpha} complex to the R3 element. Alternatively, ER{alpha} may form a trans-activation complex with Sp1 and SREBP. Requirement of another transcription factor to interact with the ER/Sp1 complex to trans-activate has been described in the regulation of the E2F1 gene (39). Due to the extremely low amounts of SREBP present in the HepG2 nuclear extracts, we were not able to detect the binding of SREBP to the R23 probe in the presence or the absence of ER{alpha}. Therefore, the effect of SREBP on formation of the Sp1/ER{alpha}-DNA complex could not be directly examined in this study.

The critical involvement of Sp1 binding sites of the LDLR promoter in ER{alpha}-mediated LDLR transcription suggests a direct interaction between ER{alpha} and Sp1. It has been shown that different functional domains of ER{alpha} are involved in interactions of ER{alpha} with different transcription factors or cofactors in a cell type- and promoter-specific manner (46). The ER{alpha} mutant vector, AF1-X-AF2, expresses ER{alpha} with point mutations in the DNA-binding domain, which had been shown to convert the selectivity of DNA binding of ER{alpha} from the ERE to the glucocorticoid response element (37). Cotransfection of AF1-X-AF2 with pLDLR234 produced an E2 response similar to that of wild-type ER{alpha}. In addition, our gel shift assays could not detect direct binding of ER{alpha} to the labeled R23 probe, but showed strong binding of ER{alpha} to the ERE probe (data not shown). Moreover, it has been previously shown that the ER{alpha}/Sp1 interaction does not require the DNA-binding domain of ER{alpha} (38). Together, these results demonstrate that regulation of LDLR transcription by ER does not occur through direct binding to the promoter element. Similar to the DBD mutation, inactivation of AF2 (mutant AF1-DBD-X) had no effect on ER{alpha}-mediated activation of LDLR transcription. In contrast, the activity of ER{alpha} on the LDLR promoter was completely lost when the AF1 domain was deleted (X-DBD-AF2). Interestingly, a recent report using the same mutant vectors showed that the ER{alpha}-mediated suppression of apoA1 promoter activity in HepG2 cells was not affected by AF1 deletion, but was affected by mutations that inactivate DBD or AF2 (31). Thus, the loss of ER{alpha} activity by AF1 deletion is LDLR promoter specific. The different effects of AF1 deletion on LDLR vs. apoA1 further illustrate the promoter-specific regulation by ER{alpha}.

Interactions of ER{alpha} with Sp1 have been demonstrated in several promoters that contain GC-rich elements, such as E2F (39) and Bcl-2 (41). A recent study has localized a region of AF1 (amino acids 79–117) that is important for activation at an Sp1 element (33). By using deletion constructs, we showed that deletions of amino acids 67–139 drastically reduced the activity of E2-activated ER{alpha} on the LDLR promoter, whereas deletion of amino acids 141–170 had no effect. Thus, it appears that the functional region of ER{alpha} (aa 67–139) for activating the LDLR promoter overlaps with a functional region (aa 79–117) that is required for activation of a synthetic promoter containing an Sp1 site. These data strongly support our conclusion that the Sp1 binding sites of the LDLR promoter constitute the primary ER{alpha}-responsive element.

Finally, gel shift assays were performed to demonstrate an enhancement of Sp1 binding to the LDLR promoter by ER{alpha}. The binding of Sp1 to the R23 probe was increased by ER{alpha} in a dose-dependent manner. The intensities of the Sp1-DNA complex varied between different experiments. Typically, a 2- to 3-fold increase in Sp1 binding in the presence of ER{alpha} was detected. This is presumably caused by enhancing the on rate of Sp1-[32P]R3 formation by ER{alpha} as described in other studies (38, 39, 40, 41). Our findings that ER{alpha} enhances Sp1 DNA-binding activity without formation of a Sp1/ER{alpha}-R23 ternary structure are consistent with a number of studies in which Sp1/ER{alpha}-DNA ternary complex was not detected by EMSA (33, 38, 39, 40, 41).

In this study we provide strong evidence demonstrating that regulation of LDLR transcription by ligand-activated ER{alpha} is primarily mediated through the Sp1-binding sites present in the R1 and R3 sequences of the LDLR promoter. This regulation requires the AF1 functional domain of ER{alpha}. We hypothesize that upon E2 binding ER{alpha} undergoes a conformational change that exposes the AF1 domain to Sp1 and perhaps also to SREBP, thereby resulting in trans-activation of the LDLR gene transcription. However, our studies could not exclude the possibility that other cofactors might also be recruited in the Sp1/ER{alpha} complex during the activation process. Previously, it has been shown that in MCF-7 cells, E2 and the estrogen antagonists 4'-hydroxytamoxifen and ICI 182,780 induced a synthetic promoter (pSp1) activity that contains a consensus Sp1-binding site presumably through Sp1/ER{alpha} interaction. However, we found that 4'-hydroxytamoxifen and two other ER-selective modulators, namely idoxifen and raloxifen, inhibited E2-stimulated LDLR promoter activity in HepG2 cells cotransfected with the wild-type ER{alpha}. The different activities of ER-selective modulators on pSp1 in MCF-7 cells and on pLDLR in HepG2 cells suggest the possible involvement of different cofactors in these two assay systems.

Previously, our laboratory has identified a sterol-independent regulatory element, located downstream of the SRE-1- and Sp1-binding sites, that is responsible for cytokine oncostatin M- and cAMP-mediated activation of LDLR transcription (47). In addition, several studies have linked mitogen-activated protein kinase activation with LDLR transcription (31, 48, 49). The current studies demonstrate another route of activation of LDLR transcription by ER{alpha}. Together, these studies illustrate that LDLR transcription is regulated by multiple mechanisms, including sterol-dependent and sterol-independent pathways.


   Acknowledgments
 
We thank Dr. Douglas C. Harnish for providing ER{alpha} mutant vectors and giving technical advise concerning our transfection experiments.


   Footnotes
 
1 This work was supported by a Merit Review from the Department of Veterans Affairs and a grant (98–218) from the American Heart Association Western States Affiliate. Back

Received October 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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