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Originally published In Press as doi:10.1074/jbc.M002188200 on May 17, 2000

J. Biol. Chem., Vol. 275, Issue 30, 22769-22779, July 28, 2000
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Inhibition of Vascular Endothelial Growth Factor Expression in HEC1A Endometrial Cancer Cells through Interactions of Estrogen Receptor alpha  and Sp3 Proteins*

Matthew StonerDagger , Fan WangDagger , Mark WormkeDagger , Thu NguyenDagger , Ismael SamudioDagger , Carrie VyhlidalDagger , Dieter MarmeDagger , Gunter Finkenzeller§, and Stephen SafeDagger

From the Dagger  Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466 and the § Institute of Molecular Medicine, Tumor Biology Center, D-79106 Freiburg, Germany

Received for publication, March 15, 2000, and in revised form, May 15, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment of HEC1A endometrial cancer cells with 10 nM 17beta -estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from -2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor alpha  (ERalpha ) and reversed by the anti-estrogen ICI 182,780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between -66 to -47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERalpha proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERalpha proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERalpha -Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiogenesis is an important physiological process associated with neovascularization and growth and metastasis of many different tumors (1-7). The complex process of angiogenesis involves tissue-specific interplay between multiple growth factors, cytokines, kinases, and other growth regulatory proteins including vascular endothelial growth factor (VEGF)1 or vascular permeability factors. VEGF is thought to play a key role in blood vessel formation; disruption of one VEGF allele in transgenic mice leads to embryonic death associated with impaired blood vessel formation, suggesting an important role for VEGF in vascularization (8, 9). Angiogenesis is an important process for the growth of solid tumors, and anti-angiogenic drugs are rapidly being developed for treatment of this disease (10-13). Not surprisingly, VEGF expression is a prognostic factor for many tumor types, and this protein plays a key role in tumor angiogenesis (14-19).

VEGF and related angiogenic factors are expressed in mammary tumors, and increased levels of VEGF are a negative prognostic factor for survival of women with breast cancer (20-27). Hormonal regulation of VEGF expression has been reported in the rodent uterus and mammary and in human breast cancer cells (28-34). Hyder et al. (28, 29) show that 17beta -estradiol (E2), tamoxifen, and structurally related triphenylethylene-derived anti-estrogens induced VEGF in the rat uterus, and E2 also induced VEGF mRNA and protein levels in carcinogen-induced rat mammary tumors (30). Regulation of VEGF in breast/endometrial cancer cell lines by steroid hormones has given variable results. One study showed that progestins, but not estrogens, androgens, or glucocorticoids, induced secretion of VEGF protein in T47D human breast cancer cells, whereas the progesterone-induced response was not observed in other breast (MCF-7, ZR-75, and MDA-MB-231) and endometrial (Ishikawa) cancer cell lines (32). Another study reported that E2 and tamoxifen induced VEGF mRNA levels, and VEGF protein was also increased by the hormone in breast cancer cells (33). HEC1A endometrial cancer cells express ERalpha protein, and both E2 and tamoxifen induce cell proliferation and reporter gene activity in cells transfected with E2-responsive constructs containing promoter inserts from the complement C3, creatine kinase B, and cathepsin D genes (35). Therefore, we have used HEC1A cells for investigating the molecular mechanisms of estrogen-dependent regulation of VEGF gene expression. The results showed that E2 decreased VEGF mRNA levels in HEC1A cells, and deletion analysis of the VEGF gene promoter identified a minimal sequence (-66 to -47) that was required for hormone-dependent decreased expression. Subsequent mutational analysis and results of gel mobility shift and DNA-footprinting assays indicated that hormonal effects were associated with ERalpha /Sp3 interactions at G/GC-rich sites. Transfection studies in Drosophila SL2 Schneider cells in culture also showed that ERalpha /Sp3 mediated decreased transactivation of a VEGF gene promoter-derived construct, and both ERalpha and Sp3 proteins physically interact, as determined in coimmunoprecipitation and pull-down assays. Thus, ligand-activated ERalpha /Sp3 interaction with G/GC-rich elements represents a novel pathway for down-regulation of VEGF, and these interactions may be important for hormone-dependent regulation of other genes given the appropriate cellular context.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Lines, Chemicals, and Biochemicals-- The human endometrial carcinoma cell line HEC1A was obtained from American Type Culture Collection (ATCC, Manassas, VA), and cells were cultured in Dulbecco's modified Eagle's medium/F12 (Sigma) supplemented with 5% fetal bovine serum (Intergen, Des Plains, IA or JRH Biosciences, Lenexa, KS). Medium was further supplemented with sodium bicarbonate, antibiotic/antimycotic solution, bovine serum albumin (Fraction V), with or without dimethyl sulfoxide (Me2SO) or 10 nM E2 (Sigma) treatment; cells were maintained at 37 °C with a humidified CO2:air (5:95) mixture. Schneider's SL-2 Drosophila cells (ATCC) were cultured at room temperature in Schneider's Drosophila medium (Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal bovine serum. ICI 182,780 was kindly provided by Dr. Alan Wakeling (AstraZeneca, Macclesfield, UK). Tamoxifen and progesterone were obtained from Sigma. Antibodies for the following proteins were also obtained: Sp1 (Sp1 PEP-2), Sp3 (Sp3 D-20), Egr-1 (Egr-1 588) (Santa Cruz Biotechnology, Santa Cruz, CA) and human estrogen receptor (hERalpha ) (H222) (Abbott Laboratories, North Chicago, IL). Pure hERalpha protein was obtained from PanVera (Madison, WI), and pure Sp1 protein was purchased from Promega (Madison, WI). All other chemicals were obtained from commercial sources at the highest quality available.

Oligonucleotides and Plasmids-- VEGF promoter-derived oligonucleotides, PCR primers, and primers employed in plasmid construction were synthesized by Genosys/Sigma (The Woodlands, TX), Gene Technologies Laboratory (Texas A&M University (TAMU), College Station, TX), or by the laboratory of Dr. James Derr (Department of Veterinary Pathobiology, TAMU). Consensus and mutant Sp1 wild type and mutant and Egr-1 wild type (5'-GGA TCC AGC GGG GGC GAG CGG GGG CGA-3') and mutant (5'-GGA TCC AGC TAG GGC GAG CTA GGG CGA-3') oligonucleotides were designed according to published sequences; Sp1 oligonucleotides were sometimes synthesized with HindIII and BamHI restriction sites at either end, represented by lowercase letters (consensus Sp1, 5'-agc ttA TTC GAT CGG GGC GGG GCG AGC g-3'; and mutant Sp1, 5'-agc ttA TTC GAT CGA AGC GGG GCG AGC g-3'). Occasionally, consensus Sp1 and mutant Sp1 oligonucleotides were synthesized with KpnI and NheI overhangs. The consensus estrogen-responsive element probe used in electrophoretic mobility shift assays (EMSA) competition experiments was 5'-GTC CAA AGT CAG GTC ACA GTG ACC TGA TCA AAG TT-3'.

VEGF promoter luciferase constructs pVEGF1, pVEGF2, and pVEGF3 were previously named pLuc 2068, pLuc 840, and pLuc 318, as described (36). pVEGF4 was kindly provided by Dr. Gregg Semenza (The Johns Hopkins University, Baltimore, MD). pVEGF5 (-131/+54) was made by digesting pVEGF4 with NheI to excise +54/+379; the vector was then religated. pVEGF6 and pVEGF7 were produced by PCR amplification using pVEGF4 as the template and primers and methodologies previously described (37). pVEGF8 and pVEGF8m were constructed by insertion of oligonucleotides -66/-47 (5'-cTC CCG GCG GGG CGG AGC CAT G-3') and -66/-47m (5'-cTC CCG GCT TTT CGG AGC CAT G-3') into TATA-pGL2 digested with KpnI and NheI. pS1 and pSp1m contained consensus and mutant Sp1 promoter elements, respectively, and were made by insertion of oligonucleotides Sp1(5'-cAT TCG ATC GGG GCG GGG CGA GCG-3') and Sp1m (5'-cAT TCG ATC GGT TCG GGG CGA GCG-3') in TATA-pGL2. Restriction enzyme sequences are in lowercase, with only the sense strand shown; complementary bottom strands had 5' and 3' overhangs to complete KpnI and NheI when oligonucleotides were annealed. For EMSA analysis, oligonucleotides -66/-47 (5'-GGT CCC GGC GGG GCG GAG CCA TG-3') and -66/-47m (5'-GGT CCC GGC TTT TCG GAG CCA TG-3') were used without restriction sites.

hERalpha expression plasmid was kindly provided by Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). hERalpha deletion constructs HE11C, HE15C, and HE19C were originally obtained from Dr. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France) and inserted into vectors pCDNA3 and pCDNA3.1/His C (InVitrogen, Carlsbad, CA) in this laboratory for in vitro translation and expression in mammalian cells. Eukaryotic expression plasmids for human Sp1 and Sp3 proteins were made by excising the Sp1 or Sp3 cDNAs from pPacSp1 (generously supplied by Dr. Robert Tjian, University of California, Berkeley, CA) or pPacUSp3 (kindly provided by Dr. Guntram Suske, Institute fur Molekularbiologie und Turmorforschung, Marburg, Germany) using XhoI and NotI restriction enzymes, respectively. Sp1 and Sp3 cDNAs were then ligated into appropriately digested, calf intestinal phosphatase-treated pCDNA3.1 expression plasmid with oligonucleotide modifications to produce Kozak sequences with in-frame start codons for each protein. pPac-hERalpha was produced by removal of hERalpha cDNA from pCDNA3 or pCDNA3.1 by EcoRI digest and ligation into a modified Drosophila expression plasmid pPacUbx multiple cloning site. Before insertion into pPacUbx, oligonucleotide linkers were added to hERalpha cDNA to ensure proper frame and expression as described (38). Human expression plasmid for progesterone receptor-B form was also a generous gift of Dr. Pierre Chambon. Rat c-Fos expression plasmid was kindly provided by Dr. Tom Curran (Roche Institute of Molecular Biology, Nutley, NJ) and was digested with HindIII and BamHI to release c-Fos cDNA; the insert was then cloned into pCDNA3.1 expression vector. Egr-1 expression plasmid was made by digesting pJDM1196 (a kind gift of Dr. Jeffrey Milbrandt, Washington University School of Medicine, St. Louis, MO) with EcoRI and XbaI to release a 1.8-kilobase pair rat Egr-1 cDNA, which was then ligated into pCDNA3 empty vector digested with the same restriction enzymes. pCDNA3-Egr-1 was used as the template to produce Egr-1 protein in a rabbit reticulocyte-coupled transcription/translation reaction (Promega). Plasmid pCMV-DNSp3 contains the DNA binding domain and dominant negative form of Sp3 in pCDNA3.1/His C and was generously provided by Drs. Yoshihiro Sowa and Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan).

Transient Transfection Assays-- Appropriate VEGF-luciferase reporter plasmid, hERalpha expression plasmid, and pCDNA3.1-His-LacZ (InVitrogen) expression plasmid (for normalization of transfection efficiency) were transiently cotransfected into HEC1-A cells using the calcium phosphate-DNA coprecipitation method or LipofectAMINE (Life Technologies, Inc.). pCDNA3 or pCDNA3.1 empty vectors were also transfected to maintain DNA mass balance among different transfection groups. An estrogen-responsive pC3-Luc construct, containing the mouse complement-3 (C3) gene promoter insert, was kindly provided by Dr. Donald P. McDonnell (Duke University Medical School, Durham, NC) and used as a positive control in most experiments to confirm hormone responsiveness of the transfected cells. After 12-24 h, the transfection mixture was aspirated from cells, and the cells, which were then treated for 24-72 h with fresh serum-free medium containing 10 nM E2 dissolved in Me2SO, served as a solvent control. One µM concentrations of ICI 182,780 or tamoxifen were used in experiments with anti-estrogens. Drosophila Schneider SL-2 cells were cultured as described above, seeded in 6-well culture dishes, and transfected with pVEGF6, pPacSp1, or pPacUSp3 and hERalpha expression plasmid; SL-2 cells were then treated with Me2SO or 10 nM E2. After treatment for 24-72 h, all cells were harvested by manual scraping in 1× lysis buffer (Promega). An aliquot of soluble protein was obtained by freezing cells in liquid nitrogen (30 s), vortexing (30 s), and centrifuging at 12,000 × g for (1 min). Lysates were assayed for luciferase activity using luciferase assay reagent (Promega); beta -galactosidase activity was measured using Tropix Galacto-Light Plus assay system (Tropix, Bedford, MA) according to the manufacturer's conditions in a Lumicount micro-well plate reader (Packard Instrument Co.). Relative luciferase activity was normalized to relative beta -galactosidase units for each transfection experiment.

Reverse Transcriptase (RT)-PCR Analysis-- VEGF PCR primers were: VEGF(F), 5'-CCG CCT CGG CTT GTC ACA TCT CCT TAA TGT CAC GCA GC-3'; and VEGF(R), 5'-CAC ATA GGA GAG ATG AGC TTC GTG GGG CGC CCC AGG CAC CA-3'. beta -Actin PCR primers were: beta -actin (F), 5'-GTG GGG CGC CCC AGG CAC CA-3': and beta -actin (R), 5'-CTC CTT ATT GTC ACG CAC GAT TTC-3'.

Cells were cultured in serum-free medium for 3 days before treatment with 10 nM E2 and also in serum-free media supplemented with bovine serum albumin. Total RNA was extracted using RNAzol B (Tel-Test, Friendswood, TX), following manufacturer's protocol. RNA was quantitated by measuring the 260/280-nm absorption ratio, and concentrations of each sample were adjusted to 100-200 ng/µl RNA for use in RT-PCR. RNA were reverse-transcribed at 42 °C for 25 min using oligo-d(T) primer, followed by PCR amplification of RT product using 2 mM MgCl2, 1 µM each gene-specific primer, 1 mM dNTPs, and 2.5 units AmpliTaq DNA polymerase (Perkin-Elmer). Primer sets for both VEGF and beta -actin were added to each mixture, and the gene products were coamplified (22 cycles). Ater amplification in a PTC-200 thermal cycler (MJ Research, Watertown, MA), 25 µl of each sample was loaded on a 5% polyacrylamide gel. Electrophoresis was performed at 110 V in 1× TBE (0.09 M Tris-base, 0.09 M boric acid, 2 mM EDTA, pH 8.3) for 3-4 h, the gel was dried, and individual VEGF bands were quantitated and normalized relative to beta -actin using an Instant Imager (Packard) and Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Band intensity values were obtained by autoradiography on X-Omat AR film (Eastman Kodak Co.) and scanning densitometry with a Sharp JX-330 scanner (Sharp Electronics, Mahwah, NJ), followed by analysis with a software package, Zero-D, (Scanalytics, Sunnyvale, CA).

Preparation of HEC1A Nuclear Extracts-- Cells were cultured in medium without phenol red, supplemented with 2.5% fetal bovine serum, and pretreated with dextran-coated charcoal to remove growth factors and endogenous estrogens. Treatments were added to cells for 1-24 h prior to harvesting by trypsinization or manual scraping. Resuspension of cells in HED (25 mM HEPES, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.6) to allow swelling was followed by cellular homogenization in HEGD (HED with 10% glycerol), using a drill/pestle apparatus or Type-B Dounce homogenizer (Kontes Glass Co., Vineland, NJ) on ice. Cell viability was checked by trypan-blue staining, and homogenization was stopped when approximately 80-90% of cells were broken. The cellular homogenate was centrifuged at 800 × g/10 min/4 °C to give a nuclear pellet that was incubated in high salt HEGDK+ buffer (HEGD containing 0.5 M KCl) on ice for 1 h. Nuclei in high salt buffer were centrifuged at 12,000 × g for 30 min at 4 °C, and protein concentration of the nuclear extract (supernatant) proteins was determined (39); the nuclear extracts were then stored in small aliquots at -80 °C for use in gel shift assays.

EMSA-- VEGF promoter-derived oligonucleotides were synthesized and annealed, and 5 pmol were 5'-end-labeled using T4 kinase (Roche Molecular Biochemicals) and [gamma -32P]ATP (NEN Life Science Products). A 20-30-µl EMSA reaction mixture contained 75-150 mM buffer (a mixture of HEGD and HEGDK+ to achieve the proper salt concentration), 1-10 µg of nuclear protein, 500-1000 ng of poly(dI-dC) or poly(dA-dT) (Roche Molecular Biochemicals) with or without unlabeled competitor oligonucleotide, and 10 fmol of labeled probe; the mixture was incubated for 15 min at room temperature or on ice. Commercially available antibodies to Sp1, Sp3, and hERalpha proteins were sometimes added to the incubation mixtures immediately after the addition of the oligonucleotide probe and incubated for 15-60 min further. Protein-DNA complexes were resolved by 5% PAGE at 110-200 V at room temperature or 4 °C for 1.5-4 h. Specific antibody-protein interactions were observed as supershifted or immuno-depleted complexes.

The assay for determining Egr-1 DNA binding used a similar buffering system supplemented with 40 µM ZnCl2. Specific nuclear extract-VEGF promoter complexes formed were separated from free probe by electrophoresis at room temperature or at 4 °C on a 3.5-5% polyacrylamide gel in 1× TBE, 100-180 V, for 2-4 h. For EMSA of pure proteins or in vitro transcribed/translated proteins, 3-5 µl of rabbit reticulocyte lysate containing the protein of interest was incubated in similar conditions as used for nuclear extract. All reactions were balanced for equal amounts of rabbit reticulocyte lysate that "mock"-transcribed/translated empty pCDNA3 or pCDNA3.1 vector. To verify specific components of DNA-protein complexes formed, a commercially available antibody against Egr-1 was added to some reaction mixtures immediately after labeled probe, incubated on ice for 15-60 min, and analyzed as described above.

SssI Methylase Footprinting-- The protocol for SssI methylase footprinting was used as described (40, 41), with some modifications. Briefly, a plasmid containing the human VEGF 5'-untranslated region was restricted at a single site to linearize the vector. The VEGF gene promoter, at a concentration of 10 ng/µl, was incubated with varying concentrations of nuclear extracts from Me2SO- or E2-treated cells or in vitro translated Sp1 and Sp3 proteins in a reaction buffer containing 5% glycerol, 17.65 mM MgCl2, 0.18 mM S-adenosylmethionine, and 5 mM dithiothreitol. The reaction was incubated on ice for 5 min, then at room temperature for 20 min to allow protein-DNA interactions. Diluted SssI methylase (New England Biolabs Inc., Beverly, MA) was added to each reaction, and all components were incubated at 30 °C for 15 min to allow for the enzyme to methylate CpG islands. Samples were then heat-inactivated at 75 °C for 15 min, and a deamination buffer was added to each tube. Fresh deamination buffer consisted of 0.9 M NaOH, 25 mM EDTA, and 200 µg/ml sonicated fish sperm DNA. After the addition of saturated aqueous sodium metabisulfite, the samples were boiled for 5 min, and the deaminated VEGF promoter product was subjected to PCR using modified gene-specific primers. PCR products were then separated by agarose gel electrophoresis, extracted from the gel using a commercially available kit (Qiagen Inc., Santa Clarita, CA), and sequenced using either one of the VEGF gene-specific primers mentioned above and Sequitherm Thermostable DNA polymerase (Epicentre Technologies, Madison, WI). Dideoxy-GTP was used for chain termination, and the products were resolved on a denaturing 6% polyacrylamide-urea gel. After gel drying and exposure to a phosphor storage screen, sequence data were visualized on a Storm scanner (Molecular Dynamics) for determination of specific footprint patterns.

hERalpha /Sp3 Immunoprecipitation-- For immunoprecipitation of hERalpha by polyclonal antibodies against Sp3, in vitro transcribed/translated proteins were obtained using the rabbit reticulocyte lysate system (Promega), and hERalpha was labeled with [35S]methionine (Amersham Pharmacia Biotech). Equal volumes (10 µl) of lysate containing [35S]Met-hERalpha and cold Sp3 protein were incubated for 1 h at room temperature. To this mixture, Sp3 polyclonal antibodies were added, and the samples were subsequently incubated for 2 h on ice. Protein G-agarose beads (Santa Cruz) were added then added, and the samples were incubated at 4 °C for 3-5 h. Immunoprecipitation of protein G-bound proteins was conducted according to the manufacturer's recommendations. Proteins were separated by SDS-PAGE (10% gel) and visualized by Storm PhosphorImager (Molecular Dynamics).

GST-Sp3/hERalpha Pull-down Assay-- Plasmid constructs containing the glutathione S-transferase (GST) cDNA linked to human Sp3 cDNA were also generously provided by Dr. Guntram Suske and included pGEX-Sp3 II (or Sp3-full), pGEX-Sp3-B, and pGEX-Sp3-BC. Empty vector pGEX-2TK was produced from pGEX-Sp3-BC by removing the Sp3 cDNA fragment with an XbaI and XhoI digest and religating the vector. Subsequently, pGEX-Sp3-ZD was generated by PCR amplification of the DNA binding domain of Sp3 using primers previously described (42). The Sp3-ZD product was digested with BamHI and EcoRI and ligated into the correspondingly cut pGEX-2TK vector. BL21 Escherichia coli competent cells (ATCC) were transformed with the appropriate pGEX plasmid and induced by 1 mM isopropyl-1-thio-beta -D-galactopyranoside (Promega) treatment, and bacterial extracts of GST-Sp3 fusion proteins were isolated according to the manufacturer's protocol (Pierce). Cell lysates were resolved by 10% SDS-PAGE and visualized by Coomassie Blue staining to verify protein expression ratios for each Sp3 deletion. GST-Sp3 fusion proteins were purified from bacterial cell lysates by incubation with Sepharose 4B beads conjugated to glutathione (Amersham Pharmacia Biotech). Subsequently, beads and attached Sp3 protein deletions were incubated at 4 °C for 2-12 h with in vitro translated [35S]methionine-labeled hERalpha proteins (WT and variants described above) in hER binding buffer (250 mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES, pH 7.5, 5.0 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/µl aprotinin), as described (43). Beads were then washed with the same buffer 4×, and specific protein interactions were eluted by boiling in 1× Laemmli's solution. Eluted proteins were resolved on a 10% SDS-PAGE gel, and the gel was dried and visualized by exposure to a phospho-storage screen scanned on a Storm PhosphorImager or by autoradiography.

Western Immunoblot-- HEC1A cells in serum-free medium were treated with Me2SO or 10 nM E2 at various times and harvested in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml aprotinin, 50 mM phenylmethylsulfonyl fluoride, 50 mM sodium orthovanadate), placed on a rocker at 4 °C to extract soluble protein, and centrifuged at 14,000 × g for 5 min at 4 °C. Protein was quantitated (37), and a 50-µg aliquot of protein diluted with loading buffer was boiled and loaded on a 7.5% SDS-polyacrylamide gel. Samples were electrophoresed at 150-180 V for 3-4 h, and the separated proteins were transferred (in a buffer containing 48 mM Tris-HCl, 29 mM glycine, and 0.025% SDS) to PVDF membrane (Bio-Rad). Specific proteins were detected by incubation with polyclonal primary antibodies Sp1-PEP2 and Sp3-D20 (both 1:1000 dilution) against Sp1 and Sp3 proteins, respectively, followed by blotting with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000 dilution). Blots were then exposed to chemiluminescent substrate (ECL) (NEN Life Science Products) and placed on Kodak X-Omat AR autoradiography film. Band intensities were evaluated by scanning laser densitometry (Sharp Electronics Corp.) and background subtraction using Zero-D Scanalytics software (Scanalytics Corp.).

Statistical Analysis-- Experiments were repeated two or more times, and representative results are expressed as the mean ± S.E. for at least three replicates for each treatment group. Data were subjected to analysis of variance, and statistical differences between treatment groups were demonstrated by Scheffe's test. Treatments were considered statistically significantly different from controls if p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

E2-dependent Down-regulation of VEGF mRNA and Reporter Gene Activity in HEC1A Endometrial Cancer Cells-- Semiquantitative RT-PCR analysis of VEGF mRNA levels showed that treatment of HEC1A endometrial cells with 10 nM E2 caused a decrease 4 h after treatment, and there was a significant (>50%) decrease in mRNA levels after 24 h (Fig. 1A). E2 also decreased luciferase activity in HEC1A cells transiently transfected with pVEGF1, pVEGF2, and pVEGF3 constructs containing the -2018 to +50, -789 to +50, and -267 to +50 regions of the VEGF gene promoter linked to a bacterial luciferase reporter gene (Fig. 1B). Further deletion analysis with pVEGF4 (-131 to +379), pVEGF5 (-131 to +54), and pVEGF6 (-66 to +54) showed that E2 decreased luciferase activity 40-60% in HEC1A cells transfected with all three constructs; however, a significant decrease in basal activity was also observed with pVEGF6 (data not shown), indicating that the -131 to -66 region of the VEGF gene promoter was important for basal activity in HEC1A cells.


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  		Fig. 1.   Transcriptional repression of VEGF mRNA by E2. A, RT-PCR analysis of VEGF mRNA levels in HEC1A cells. Cells were seed in 2.5% charcoal-stripped serum medium for 1 day and cultured under serum-free conditions for 3 days and treated with Me2SO (DMSO) (vehicle) or 10 nM E2 for the indicated times, and mRNA levels were determined as described under "Materials and Methods." E2 significantly (*p < 0.05) decreased steady state VEGF mRNA levels after 24 h. Results are expressed as mean ± S.E. B, effects of E2 treatment on VEGF gene promoter-luciferase reporter activity in HEC1A cells. Cells were transiently transfected with 500 ng of VEGF gene promoter-luciferase reporter construct (pVEGF1-6), 500 ng of hERalpha expression plasmid, and 500 ng of beta -galactosidase expression vector and then treated for 24-72 h with Me2SO or 10 nM E2. Luciferase activity was determined as described under "Materials and Methods" and normalized to beta -galactosidase activity; results are presented as percent Me2SO for each transfection group. In most experiments, cells were transfected in parallel with a highly E2-inducible promoter-reporter construct pC3-Luc to verify E2 responsiveness. Results are expressed as mean ± S.E., and significant (p < 0.05) inhibition (*) was observed with all constructs.

The specificity of the ERalpha -mediated down-regulation response was further investigated using pVEGF4 and pVEGF5, which contained VEGF 5'-regulatory sequences up to -131 and pVEGF6 containing only the -66 to +54 region of the promoter. pVEGF4, pVEGF5, and pVEGF6 gave similar responses in transient transfection studies showing that ERalpha , but not progesterone receptor (PR), mediates the hormone-dependent decrease in luciferase activity (Fig. 2A). Moreover, the decreased response in HEC1A cells transfected with pVEGF4 or pVEGF5 caused by E2 is reversed after cotreatment with 1 µM concentration of the anti-estrogen ICI 182,780 (Fig. 2B). In parallel studies with pVEGF6, the results were similar to those obtained for the longer constructs (pVEGF4 and pVEGF5); wild-type ERalpha , but not PR, mediated a hormone-dependent decrease in activity, and the anti-estrogen ICI 182,780 reversed the estrogenic response (Fig. 2C). The requirement for activation function 1 (AF1) or AF2 domains of ERalpha were also investigated in HEC1A cells cotransfected with pVEGF6 and variants expressing AF2 (HE19), AF1 (HE15), or both AF1 and AF2 but not the DNA binding domain (HE11). The results showed that only wild-type ERalpha mediated hormone-dependent down-regulation of luciferase activity in HEC1A cells transfected with pVEGF6. The downstream -66 to +54 region of the VEGF gene promoter contains two overlapping G/GC-rich regions (-66/-47) and a third downstream GC-rich site; results of deletion (pVEGF7) and mutational (pVEGF6m) analysis indicate the former site was required for the hormone-induced response (Fig. 2D). Moreover, the -66/-47 site alone (pVEGF8) was sufficient for significant E2-dependent decrease in luciferase activity, but the magnitude of this response was lower than observed for constructs containing the endogenous proximal promoter region. Mutation of the overlapping G/GC-rich site (pVEGF8m) resulted in loss of hormone responsiveness, and similar results were obtained using constructs containing a wild-type consensus GC-rich Sp1 binding site (pSp1) and a mutant Sp1 binding site (pSp1m). In all of these experiments, E2 responsiveness of the cells was monitored (data not shown) by showing that 10 nM E2 induced a 5-20-fold increase of luciferase activity in HEC1A cells transfected with pC3-luc, a construct containing the mouse complement C3 gene promoter insert (35).


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  		Fig. 2.   Deletional analysis and hormone or anti-estrogen responsiveness of pVEGF constructs in HEC1A cells. A, comparative effects of E2 and progesterone. Cells were transiently transfected with 500 ng of reporter (pVEGF4-6), 500 ng of hERalpha or human PR-B form, and 500 ng of beta -galactosidase expression plasmid, treated with Me2SO (DMSO), 10 nM E2, or 10 nM progesterone (P) for 24-72 h, and luciferase activity was determined as described under "Materials and Methods." E2 significantly (*p < 0.05) decreased pVEGF4-6 luciferase activity, whereas progesterone had no effect on activity. B, effect of the anti-estrogen ICI 182,780. Cells were transiently transfected as described above and treated with Me2SO, 10 nM E2, 10 nM E2 + 1 µM ICI 182,780 (I), or 1 µM ICI 182,780 alone for 24-72 h. The pure anti-estrogen (ICI 182,780 (I)) significantly (*p < 0.05) reversed the effects of E2 on luciferase activity but did not affect control activity. C, comparative effect of wild-type and variant hERalpha . Cells were transiently transfected with 500 ng of pVEGF6, 500 ng of WT, or variant (HE11C, HE15C, and HE19C) hERalpha as described above and treated with Me2SO (vehicle), 10 nM E2, 1 µM ICI 182,780 (I), or E2 + ICI 182,780 for 24-72 h. Decreased luciferase activity was only observed with WT hERalpha , and this activity was reversed by cotreatment with ICI 182,780 (*p < 0.05). D, mutational analysis of pVEGF6. HEC1A cells were transiently transfected with pVEGF6, pVEGF6m (mutation in both GC-rich sites), pVEGF7 (GC-rich sequence deleted), pVEGF8 (GC-element alone inserted upstream of the TATA box), or pVEGF8 m (GC-rich site mutated and placed upstream of the TATA box), as described above, and treated with Me2SO or 10 nM E2 for 24-72 h. The GC-rich -66/-47 VEGF promoter sequence was necessary and sufficient for mediating E2-decreased (*p < 0.05) luciferase activity. As a control, consensus Sp1 and mutant Sp1 oligonucleotides were synthesized, inserted in TATA-pGL2, and assayed for response to E2 treatment. Down-regulation of luciferase activity through a consensus Sp1 site was significant (*p < 0.05) and similar to results obtained with pVEGF8. Oligonucleotide sequences are included, with lowercase letters representing 5'-KpnI and 3'-NheI restriction site overhangs; bold-faced letters denote mutated G right-arrow T sequences.

Identification of Proteins Interacting with the -66/-47 Region of the VEGF Gene Promoter-- The G-rich -66 to -47 region of the VEGF gene promoter is sufficient for down-regulation by ERalpha and incubation of nuclear extracts from HEC1A cells with 32P-(-66/-47), and analysis by gel electrophoretic mobility shift assays gave a high molecular weight broad band (Fig. 3A, lane 2) and a less intense lower band. Intensities of the higher molecular weight band were decreased after competition with wild-type (but not mutant -66/-47) and consensus GC-rich oligonucleotides (lanes 3-6). Moreover, after coincubation with antibodies to Sp3 or Sp1 proteins or nonspecific IgG (lanes 8-10), Sp1 antibody supershifted the major band (Sp1-SS), whereas a smaller component of the major band and the lower molecular weight complex were immunodepleted by Sp3 antibody. Nonspecific IgG had no effect. In a separate experiment using a different Sp3 antibody, a supershifted complex was observed (data not shown). This same region of the VEGF promoter did not bind recombinant human ERalpha (data not shown), and competition with unlabeled estrogen-responsive element did not decrease intensities of the retarded bands formed with 32P-(-66/-47) (lane 7). Thus, both Sp1 and Sp3 protein in nuclear extracts from HEC1A cells bind to the -66 to -47 region of the VEGF gene promoter.


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  		Fig. 3.   Binding of nuclear extracts and Egr-1 to 32P-(-66/-47) (EMSA analysis). A, nuclear extracts from E2-treated HEC1A cells bind 32P-(-66/-47) and consensus [32P]Sp1 oligonucleotides. Binding of nuclear extracts to oligonucleotides and antibody supershifts were determined by EMSA, as described under "Materials and Methods." Nuclear protein extracts formed specific Sp1 and Sp3 complexes with 32P-(-66/-47) (lanes 1-8) and [32P]Sp1 (lanes 9-15), and these bands were supershifted with antibodies to Sp1 (lanes 5 and 12) and Sp3 (lanes 7 and 14) proteins. Normal rabbit and goat IgG did not supershift any bands (lanes 6 and 8; lanes 13 and 15). B, binding of nuclear extracts from E2-treated HEC1A cells with 32P-(-66/-47) or consensus [32P]Egr. This assay was carried out as described in A; Egr-1 protein does not bind 32P-(-66/-47) (lanes 1-6) but forms a specifically bound complex with [32P]Egr (lanes 7-12) that is supershifted (S.S.) with Egr-1 antibody (lane 11). C, binding of in vitro translated rat Egr-1 protein and 32P-(-66/-47) or [32P]Egr. In vitro Egr-1 does not bind 32P-(-66/-47) (lanes 1-7) but binds [32P]Egr (lanes 8-14) and forms an immuno-reactive complex with consensus [32P]Egr (lane 13).

Previous studies show that Egr-1 is induced by E2 in some uterine-derived cells and MCF-7 cells (44, 45), and Egr-1 can exert anti-proliferative signals in some tumor cells (46, 47) and inhibit transactivation by competing for Sp1 binding sites (48, 49). Therefore, we investigated the role of Egr-1 in mediating the down-regulation of VEGF since a putative Egr-1 binding site was identified at -60 to -51 in the VEGF gene promoter, although direct binding by Egr-1 protein was not demonstrated (36). Results summarized in Fig. 3B compare binding of nuclear extracts from E2-treated HEC1A cells to 32P-(-66/-47) and [32P]Egr-1 oligonucleotides. Protein binding to the -66 to -47 region gave bands (lanes 1-6) that were not competitively decreased with consensus Egr-1 oligonucleotide or supershifted with Egr-1 antibodies. In contrast, the same nuclear extracts from HEC1A cells formed a more mobile specifically bound band [32P]Egr-1 (lanes 7-12) that was competitively decreased by wild-type (but not mutant) Egr-1 oligonucleotide and supershifted by Egr-1 antibodies. These results indicate that, whereas Egr-1 is expressed in HEC1A cells, this protein does not bind 32P-(-66/-47). We further investigated potential binding of Egr-1 to the VEGF gene promoter by comparing interactions of in vitro translated protein with a consensus [32P]Egr-1 oligonucleotide and with 32P-(-66/-47) (derived from the VEGF gene promoter) (Fig. 3C). In vitro translated Egr-1 bound consensus [32P]Egr-1 to give a retarded band (lanes 9-14), and intensity of this band was decreased after competition with unlabeled consensus wild-type Egr-1 but not mutant Egr-1 oligonucleotides (lanes 11 and 12). Moreover, Egr-1 antibodies supershifted the Egr-1-DNA complex (lane 13). In contrast, in vitro translated Egr-1 did not bind 32P-(-66/-47) to form a retarded band (lanes 2-7), suggesting that Egr-1 did not play a significant role in down-regulating VEGF through direct interactions with the -66/-47 region of the promoter.

Fig. 4A confirms that recombinant human Sp1 protein binds 32P-(-66/-47) (lanes 2-5) and competition with excess unlabeled wild-type -66/-47, but not mutant -66/-47 oligonucleotide (lanes 6 and 7), decreased intensity of the Sp1-DNA retarded band. In addition, Sp1 protein bound a consensus wild-type oligonucleotide to give a retarded band complex (lane 8) with mobility similar to the Sp1-32P-(-66/-47) band. Incubation of reticulocyte lysate alone with 32P-(-66/-47) gave two nonspecific bands (Fig. 4B, lane 1), and incubation with in vitro translated Sp3 protein primarily gave a single higher molecular weight retarded band (lanes 2-5) that was decreased in intensity after incubation with 100-fold excess of unlabeled -66/-47 oligonucleotide (lane 6) but not mutant -66/-47m oligonucleotide (lane 7). Thus, the G/GC-rich -66/-47 region of the VEGF gene promoter binds Sp1 and Sp3 proteins but not Egr-1. In some studies, variability of Sp1 and Sp3 ratios can account for treatment-dependent effects on transactivation (50-52), and therefore, the effects of 10 nM E2 on immunoreactive Sp1 and Sp3 proteins were determined in HEC1A cells (Fig. 4C). The results show that levels of Sp1 protein do not significantly change over a period of 24 h, whereas a 20-30% increase of immunoreactive Sp3 protein was observed within 3 h after treatment with E2. This shows that there is an overall significant increase in Sp3 protein (at 3 and 24 h); however, the increased levels are modest.


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  		Fig. 4.   Role of Sp1 and Sp3 proteins in E2-responsiveness of VEGF. A, binding of recombinant human Sp1 protein with 32P-(-66/-47) or consensus [32P]Sp1. EMSAs were carried out as described under "Materials and Methods." Pure Sp1 protein specifically binds 32P-(-66/-47) (lanes 1-7) and consensus [32P]Sp1 (lane 8) with similar affinities. B, binding of in vitro translated Sp3 protein with 32P-(-66/-47) or consensus [32P]Sp1. Binding studies were carried out as described above in A. In vitro translated Sp3 protein specifically binds 32P-(-66/-47) (lanes 1-7) and consensus [32P]Sp1 (lane 8) with similar affinities. C, Western immunoblot analysis of Sp1 and Sp3 protein levels in HEC1A cells. Cells were seeded in 2.5% charcoal-stripped serum medium and treated with Me2SO (DMSO) or 10 nM E2 for the indicated times. Cells were harvested, and Western analysis was performed as described under "Materials and Methods." E2 treatment resulted in a time-dependent increase in Sp3, but not Sp1, protein levels. Results expressed as mean ± S.E. and significance (p < 0.05) are indicated (*).

Transactivation of pVEGF6 by ERalpha /Sp1 and ERalpha /Sp3 in Schneider SL2 Cells and Effect of Dominant Negative Sp3 in HEC1A Cells-- Previous studies in this laboratory have demonstrated functional interactions of Sp1 and ERalpha at GC-rich sites in several gene promoters where ERalpha /Sp1 is associated with hormone-dependent up-regulation of gene expression in human breast cancer cell lines (38, 53-61). Therefore, we investigated ERalpha (+E2) interactions with both Sp1 and Sp3 proteins in Drosophila Schneider SL-2 cells that do not constitutively express ERalpha , Sp1, or Sp3 proteins. In SL-2 cells transiently transfected with pVEGF6 and expression plasmids for Sp1 and ERalpha , the results show that ERalpha enhances Sp1-dependent activation of pVEGF6, and a >3-fold increase in luciferase activity was observed using 500 ng of ERalpha expression plasmid (Fig. 5A). Similar results were observed using a G/GC-rich construct from the bcl-2 gene promoter (38). Enhanced activity was lower using 1000 ng of ERalpha , whereas ERalpha expression plasmid alone (2000 ng) did not enhance transactivation compared with controls (no plasmids added). Using a similar approach in SL-2 cells cotransfected with Sp3 expression plasmid (100 ng) and pVEGF6 (1 µg), cotransfection with increasing amounts of ERalpha expression plasmid (200-1000 ng) resulted in a >50% decrease in luciferase activity. These data are consistent with the observed hormone-dependent ERalpha /Sp3-mediated down-regulation of VEGF gene expression in HEC1A cells, and in this same cell line, overexpression of dominant negative Sp3 expression plasmid partially reversed the hormone-dependent response (Fig. 5C).


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  		Fig. 5.   Activation of pVEGF6 by Sp1, Sp3, and hERalpha expression in Drosophila Schneider SL-2 cells and effects of dominant negative (DN)-Sp3 on pVEGF6 in HEC1A cells. A, effects of hERalpha cotransfection on Sp1-mediated transactivation of pVEGF6. In SL-2 cells transfected with Sp1 expression plasmid (100 ng), cotransfection with increasing amounts of hERalpha (20-500 µg) along with 10 nM E2 treatment significantly (*p < 0.05) increased luciferase activity, whereas hERalpha alone was inactive. B, effects of hERalpha cotransfection on Sp3 mediated transactivation of pVEGF6. Cells were transfected with Sp3 expression plasmid (100 ng) and treated with 10 nM E2; hERalpha (500 and 1000 ng) significantly (*p < 0.05) decreased Sp3-dependent activation of pVEGF6 (a*, plotted from a separate experiment in which a new batch of cells, culture medium, and fetal bovine serum were used). Luciferase activities were corrected for total protein instead of beta -galactosidase activity. C, effects of dominant negative (DN)-Sp3 on E2-decreased pVEGF6 activity in HEC1A cells. Cotransfection of increasing amounts (0.2-3.0 µg) of pCMV-DNSp3 (expressing a DNA binding form of Sp3 that competes with endogenous Sp3 proteins but does not transactivate) resulted in a dose-dependent reversal of E2-mediated down-regulation of luciferase reporter activity in cells transiently transfected with pVEGF6 (0.5 µg) and hERalpha (0.5 µg). Significant (p < 0.05) reversal of the hormone-decreased response was observed using 0.5, 2.0, and 3.0 µg of pCMV-DNSp3.

Physical Interactions of ERalpha and Sp3 Proteins-- Previous studies have shown the ERalpha does not supershift an Sp1-DNA complex in gel mobility shift assays but enhances retarded band intensity. This type of interaction has been observed for other proteins in gel mobility shift assays; for example, human T cell leukemia virus type-1 Tax, sterol regulatory element-binding protein, and cyclin D1 enhanced bZIP (and GATA-4), Sp1, and ER binding to their respective cognate DNA enhancer sequences but did not form a supershifted band even though the binary mixture of proteins physically interacts (43, 62-65). The results illustrated in Fig. 6A show that recombinant human ERalpha protein enhances the intensity of in vitro translated Sp1 protein-32P-(-66/-47) interaction (lanes 2-4), and the enhanced band is supershifted by Sp1 antibody (lane 5). Similar result were also observed for the in vitro translated Sp3-32P-(-66/-47) retarded band (lanes 6-8), and at the highest concentration of ERalpha , a 160% increase in retarded band intensity was observed (lane 8). The Sp1-DNA retarded band was both supershifted and immuno-depleted using Sp3 antibodies (lane 9), whereas only immuno-depletion was observed with a different Sp3 antibody preparation (see Fig. 3A). Physical interactions between ERalpha and Sp3 were determined in coimmunoprecipitation assays using in vitro translated [35S]Sp3 and ERalpha (Fig. 6B, lanes 2 and 3). Sp3 antibody immunoprecipitates [35S]Sp3 protein alone (lane 5) and [35S]ERalpha plus unlabeled Sp3 protein (lane 6). In control experiments, Sp3 antibody does not immunoprecipitate a radiolabeled band after incubation of Sp3 protein plus [35S]lysate (lane 6) or [35S]ERalpha alone (data not shown) or rat c-Fos protein (negative control). ERalpha and Sp3 interactions were also investigated using pull-down assays with GST-Sp3 (wild-type and truncated) fusion proteins. The results (Fig. 6C) show that [35S]ERalpha interacts with GST-Sp3 (wild-type) (lane 5) and variants expressing the N-terminal ZD domain (lane 8) as well as fusion proteins expressing B and BC domains (lanes 6 and 7). In contrast, ERalpha primarily interacted with the ZD domain of Sp1, and only minimal interaction with other regions of the protein were observed (65). Interactions of wild-type radiolabeled ERalpha (Fig. 6D, lanes 1-3), HE11 (lanes 4-6), HE15 (lanes 7-9), and HE19 (lanes 10-12) with GST-Sp3 demonstrated that Sp3 specifically bound with wild-type ERalpha , HE11, and HE15, whereas only nonspecific interactions were observed for HE19, suggesting specific interactions of Sp3 with the C-terminal (AF-1 and DNA binding domain) region of ERalpha . In parallel studies, GST-Sp1 interacted with wild-type ERalpha , HE11, HE15, and H19 (data not shown).


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  		Fig. 6.   Interactions of hERalpha and Sp3 proteins. A, effects of hERalpha on Sp1 and Sp3 protein binding 32P-(-66/-47). EMSA assays were carried out as described under "Materials and Methods." In vitro translated Sp1 and Sp3 proteins were incubated with 32P-(-66/-47), and binding of both Sp1 (lanes 2-5) and Sp3 (lanes 6-9) proteins was enhanced (2-3-fold) after coincubation with increasing amounts of recombinant hERalpha protein in the presence of 3.3 nM E2 treatment. However, hERalpha protein alone did not bind 32P-(-66/-47). B, coimmunoprecipitation assay. This assay was performed as described under "Materials and Methods," and E2 was not added. In vitro translated [35S]hERalpha protein is specifically precipitated after incubation with in vitro translated Sp3 protein and antibody to Sp3 (alpha Sp3) (lane 6). As a negative control, incubation of [35S]hERalpha with in vitro translated rat c-Fos protein and c-Fos antibody did not precipitate [35S]hERalpha (data not shown). C, GST pull-down assay. GST-Sp3 fusion proteins were expressed in BL21 E. coli and isolated; fusion protein concentrations and sizes were verified by SDS-PAGE and Coomassie Blue staining, and pull-down assays were carried out as described under "Materials and Methods" without the addition of E2. [35S]Met-hERalpha interacts specifically with full-length (lane 5), B (activation domain B alone) (lane 6), BC (activation domains B and C) (lane 7), and ZD (zinc finger and D domains) (lane 8) regions of Sp3 protein fused to GST. D, GST pull-down assay with full-length GST-Sp3 fusion protein and hERalpha variant proteins. In vitro translated [35S]Met-hERalpha WT, [35S]Met-HE11C (DNA binding domain deleted), [35S]Met-HE15C (AF-2, C-terminal deletion), and [35S]Met-HE19C (AF-1, N-terminal deletion) were resolved by SDS-PAGE; relative protein input amounts were normalized by autoradiography and densitometric analysis. Full-length GST-Sp3 fusion protein specifically interacted with ERalpha WT (lane 3), HE11C (lane 6), and HE15C (lane 9). Interaction of Sp3 protein with HE19C was nonspecific, and the radiolabeled band was not higher than background binding observed for GST-Sp3, GSH-conjugated beads, or GST alone (lanes 10-12). Negative control, rat c-Fos protein (lane 13), does not bind GST-Sp3 fusion protein (lanes 14-16). S.S., supershift.

We have also used a highly sensitive viral SssI CpG methylase (40, 41) to footprint the G/GC-rich region of the VEGF gene promoter (Fig. 7). Incubation with increased amounts of nuclear extracts from HEC1A cells treated with 10 nM E2 increase footprinting in the GC-box area, and there was a >50% increase in the cytosine footprint at -56 and -64 (data not shown). In vitro footprinting using expressed recombinant proteins (Fig. 7) showed that Sp1 (lanes 5 and 6) and Sp3 (lanes 7 and 8) proteins alone or in combination with ERalpha (lanes 10 and 11) protected several GC sites within the proximal region of the VEGF promoter, and this was particularly evident at cytosines -56 and -61. Surprisingly, coincubation with Sp1 plus Sp3 proteins (lane 9) significantly reversed the footprint observed for the proteins alone, and this may be due to competitive protein-protein interactions resulting in decreased protein-DNA binding.


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  		Fig. 7.   SssI methylase footprint analysis. Binding of in vitro translated Sp1 and Sp3 proteins to VEGF gene promoter elements. In vitro translated Sp1 and Sp3 proteins were incubated with the VEGF promoter and SssI methylase footprint analysis was carried out as described under "Materials and Methods." Sp1 and Sp3 proteins dose-dependently enhanced the footprint of GC-rich elements in the VEGF gene promoter (lanes 5-8); Sp1 and Sp3 footprints were enhanced by pure hERalpha protein (lanes10-11). hERalpha enhanced footprints of Sp1 and Sp3 proteins at the GC-site critical for E2-mediated down-regulation of VEGF and also at adjacent 5' and 3' GC-rich sequences.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VEGF plays a pivotal role in angiogenesis and endothelial cell growth as well as the growth and metastasis of various primary tumors (1-9). Not surprisingly, cell type-specific regulation of VEGF expression has been extensively investigated, and multiple mechanisms and factors can affect VEGF mRNA and/or protein levels. VEGF and related family members are essential components for endothelial cell growth and, in turn, a variety of growth factors, cytokines, and kinases also up-regulate VEGF mRNA and/or protein levels in different cell types (36, 37, 66-75). Hypoxia also induces VEGF (76-84), and there are also several cellular conditions or factors that down-regulate expression of this gene product, including the tumor suppressor gene p53 and the von Hippel-Lindau tumor suppressor gene product (85-89).

Previous studies on hormone-dependent regulation of VEGF expression indicate that the responses are cell/tissue-specific (28-34). Recent studies in this laboratory have demonstrated that the HEC1A endometrial carcinoma cell line expresses ERalpha and is E2-responsive (35); however, analysis of VEGF mRNA levels and activities of constructs containing VEGF gene promoter inserts (Fig. 1) show that E2 down-regulates VEGF. This hormone-mediated response is dependent on ERalpha and can be reversed by the pure anti-estrogen ICI 182,780 (but not tamoxifen; data not shown); however, in cells cotransfected with pVEGF4 or pVEGF5 and PR expression plasmid, treatment with progesterone did not alter reporter gene activity. Thus down-regulation of VEGF by E2 parallels results of previous studies showing that p53 and von Hippel-Lindau factor also decreased VEGF activity and/or VEGF gene promoter-dependent constructs (85-89). Analysis of the VEGF gene promoter has shown that high basal activity of constructs is dependent on a GC/G-rich sequence from -88 to -50 that contains putative binding sites for Sp1-like proteins, Egr-1 and AP-2 (36, 37, 74, 75), and a similar pattern of basal activity was also observed in HEC1A cells (data not shown). Interestingly transforming growth factor-alpha and p42/p44 mitogen-activated protein kinase activate VEGF gene promoter constructs at GC-rich regions between -88 to -66 that bind AP-2 and Sp1 proteins, and both transcription factors play a role in this response in hamster CCL39 fibroblast and A431 human epidermoid carcinoma cells (37, 74). In contrast, mutation of the AP-2 site did not affect platelet-derived growth factor-induced responses that appeared to be dependent on Sp1 and/or Sp3 interactions with three GC-rich sites between -85 to -50 (36). Results of deletion analysis of the VEGF gene promoter in HEC1A cells showed (Figs. 1B and 2D) that comparable ERalpha -mediated down-regulation was observed for both pVEGF6 and pVEGF8, but not pVEGF7, suggesting that the -66 to -52 region of the promoter was required for the hormone-dependent inhibitory response.

This region of the promoter contains two GC-rich binding sites for Sp-like proteins and an overlapping Egr-1 site at -60 to -51. Results of gel mobility shift assays indicated that ERalpha did not bind directly to this region of the promoter and nuclear extracts formed retarded bands that primarily contained Sp1 and Sp3 proteins. Egr-1 competition with Sp1 binding to GC-rich sites can result in repressed gene expression (48, 49), and previous studies indicate the Egr-1 can be induced by E2 in some hormone-dependent cell lines (44). Our studies show the in vitro expressed Egr-1 did not bind the -66 to -47 oligonucleotide, suggesting that Sp1 and/or Sp3 are the major proteins directly interacting with this region of the gene promoter.

Previous studies in this laboratory show that ERalpha interacts with Sp1 protein (65), and ligand-activated ERalpha /Sp1 interactions with GC-rich sites are associated with induction of several E2-responsive genes in breast cancer cells (53-61, 65). Although both Sp1 and Sp3 activate some GC-rich promoters, Sp3 overexpression has also been associated with repression of Sp1-mediated transactivation (50-52) or decreased gene expression (90, 91). Sp1 or Sp3 alone or in combination with ERalpha footprinted G/GC-rich sites in the proximal region of the VEGF gene promoter (Figs. 7), and the pattern of Sp3-DNA interactions has previously been observed for ERalpha -Sp1 binding to other GC-rich gene promoters (53-61, 65). Coimmunoprecipitation and pull-down assays (Figs. 6, B-D) demonstrate that like Sp1 (65), Sp3 also physically interacts with ERalpha ; however, there are some differences in their interacting domains since ERalpha preferentially binds to the C-terminal (ZD) domain of Sp1 protein (65), whereas ERalpha interacts with multiple (ZD, BC, and B) domains of Sp3. In contrast, GST-Sp3 fusion protein preferentially interacted with the AF1-DBD (HE15) of ERalpha , whereas GST-Sp1 interacted with ER variant proteins expressing AF1 (HE15) or AF2 (HE19) alone (data not shown). Although Sp1 and Sp3 proteins alone activated pVEGF6 in SL-2 cells (Fig. 5) as described previously for other GC-rich promoters in these cells (90-92), cotransfection with ERalpha expression plasmid enhanced ERalpha /Sp1 but decreased ERalpha /Sp3 action. This novel ERalpha /Sp3 pathway was further confirmed in HEC1A cells using a dominant negative form of Sp3 that partially reversed ERalpha /Sp3 down-regulation of pVEGF6 (Fig. 5C). Since treatment-dependent variations in Sp3/Sp1 levels can affect promoter activity (51), we also investigated immunoreactive Sp1 and Sp3 protein levels after treatment with E2 (Fig. 4C). Sp1 protein was not affected, whereas a small but significant increase (<30%) in Sp3 levels was observed, and this may also contribute to the overall decreased transcriptional activation of VEGF by E2 in HEC1A cells.

Sp1 physically and functionally interacts with several nuclear receptor superfamily proteins including ERalpha (53-61, 65), retinoic acid receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF), and steroidgenic factor-1, and the progesterone receptor interacts with Sp1 and basal transcription element binding protein (an Sp family member) to modulate gene expression through GC-rich promoter elements (93-99). This paper describes a novel ERalpha /Sp3 pathway for hormone-mediated decreased gene expression through a G/GC-rich element in the VEGF gene promoter, and this mechanism may be important for hormone-mediated down-regulation of other genes. Results of preliminary studies with pVEGF constructs in other breast and endometrial cancer cell lines suggest that changes in Sp3/Sp1 protein ratios do not necessarily dictate differential ERalpha /Sp3 or ERalpha /Sp1 action at GC-rich sites. Therefore, current research is focused on identifying other cellular factors that differentially regulate ERalpha interactions with Sp1 or Sp3 proteins.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA76636 and ES09106 and a grant from the Texas Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Sid Kyle Professor of Toxicology. To whom correspondence should be addressed. Tel.: 409-845-5988; Fax: 409-862-4929; E-mail: ssafe@cvm.tamu.edu.

Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M002188200

    ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; E2, 17beta -estradiol; ERalpha , estrogen receptor alpha ; hERalpha , human ERalpha ; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; RT, reverse transcription; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; WT, wild type; AF1 and AF2, activation functions 1 and 2, respectively; PR, progesterone receptor.

    REFERENCES