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• W2007163552 abstract "Insulin-like growth factor (IGF-1) is a potent mitogen for vascular smooth muscle cells. Both IGF-1 and its receptor have been shown to be highly expressed in atherosclerotic lesions. Here we investigated whether part of the vasculoprotective properties of E2 may be mediated by its negative regulation of the IGF-1 system. HeLa cells, which do not contain endogenous estrogen receptors (ER), were transiently transfected with IGF-1R promoter constructs with or without a plasmid encoding human ERα or ERβ and treated with 100 nm 17β-estradiol (E2) for 24 h. E2 treatment decreased basal luciferase activity by 51%, and this effect was dependent on co-expression of ERα, whereas no repression was observed with ERβ. A mutation within the DNA binding domain of the ERα abolished the repressor function of the ER receptor. Similarly, E2 decreased IGF-1R transcription by 21% in rat aortic smooth muscle cells (RASMC), which express endogenous ER. This effect was specific for E2, because it was inhibited by an antiestrogen and because progesterone did not have any effect on IGF-1R expression in HeLa or RASMC transfected with progesterone receptor. Accordingly, E2 decreased IGF-1R and IGF-1 mRNA in RASMC by 47% and 33%. Western blot analysis and radioligand binding studies showed that E2 also dose-dependently decreased IGF-1R protein expression in RASMC by 40% and 30%, respectively, and that IGF-1 protein was reduced by 43%. Repression of IGF-1R promoter activity by a combination of ERα and E2 did not appear to be mediated via direct binding of ER to the IGF-1R promoter but rather by inhibition of SP1 binding to the IGF-1R promoter. Thus, E2down-regulates IGF-1R and IGF-1 expression in vascular smooth muscle cells. This may have important implications for the understanding of the beneficial effects of estrogen in the cardiovascular system. Insulin-like growth factor (IGF-1) is a potent mitogen for vascular smooth muscle cells. Both IGF-1 and its receptor have been shown to be highly expressed in atherosclerotic lesions. Here we investigated whether part of the vasculoprotective properties of E2 may be mediated by its negative regulation of the IGF-1 system. HeLa cells, which do not contain endogenous estrogen receptors (ER), were transiently transfected with IGF-1R promoter constructs with or without a plasmid encoding human ERα or ERβ and treated with 100 nm 17β-estradiol (E2) for 24 h. E2 treatment decreased basal luciferase activity by 51%, and this effect was dependent on co-expression of ERα, whereas no repression was observed with ERβ. A mutation within the DNA binding domain of the ERα abolished the repressor function of the ER receptor. Similarly, E2 decreased IGF-1R transcription by 21% in rat aortic smooth muscle cells (RASMC), which express endogenous ER. This effect was specific for E2, because it was inhibited by an antiestrogen and because progesterone did not have any effect on IGF-1R expression in HeLa or RASMC transfected with progesterone receptor. Accordingly, E2 decreased IGF-1R and IGF-1 mRNA in RASMC by 47% and 33%. Western blot analysis and radioligand binding studies showed that E2 also dose-dependently decreased IGF-1R protein expression in RASMC by 40% and 30%, respectively, and that IGF-1 protein was reduced by 43%. Repression of IGF-1R promoter activity by a combination of ERα and E2 did not appear to be mediated via direct binding of ER to the IGF-1R promoter but rather by inhibition of SP1 binding to the IGF-1R promoter. Thus, E2down-regulates IGF-1R and IGF-1 expression in vascular smooth muscle cells. This may have important implications for the understanding of the beneficial effects of estrogen in the cardiovascular system. insulin-like growth factor-1 insulin-like growth factor-1 receptor rat aortic smooth muscle cells estrogen receptor 17β-estradiol progesterone receptor B dextran-coated charcoal-treated 4-hydroxytamoxifen ICI 164,384 insulin-like growth factor binding protein estrogen response element electrophoretic mobility shift assay Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis base pair(s) interleukin 1 Several studies, but not all, have suggested that estrogens are cardioprotective in postmenopausal women (1Hulley S. Grady D. Bush T. Furberg C. Herrington D. Riggs B. Vittinghoff E. Jama. 1998; 280: 605-613Crossref PubMed Scopus (5701) Google Scholar, 2Mendelsohn M.E. Karas R.H. N. Engl. J. Med. 1999; 340: 1801-1811Crossref PubMed Scopus (2511) Google Scholar). The mechanisms of estradiol-induced reduction in the risk of coronary artery disease remain unclear. Although these atheroprotective effects of estrogen were principally attributed to the hormone's effects on serum lipid concentrations (3Hong M.K. Romm P.A. Reagan K. Green C.E. Rackley C.E. Am J Cardiol. 1992; 69: 176-178Abstract Full Text PDF PubMed Scopus (162) Google Scholar, 4Nabulsi A.A. Folsom A.R. White A. Patsch W. Geiss G. Wu K.K. Szklo M. N. Engl. J. Med. 1993; 328: 1069-1075Crossref PubMed Scopus (922) Google Scholar, 5Cauley J.A. LaPorte R.E. Kuller L.H. Bates M. Sandler R.B. Atherosclerosis. 1983; 49: 31-40Abstract Full Text PDF PubMed Scopus (50) Google Scholar), recent findings suggest that the majority of the vasculoprotective effects of estrogen are due to direct effects on the vasculature (6Mendelsohn M.E. Karas R.H. Curr. Opin. Cardiol. 1994; 9: 619-626Crossref PubMed Scopus (276) Google Scholar). Direct effects of estrogens have been demonstratedin vitro and in vivo both in animal and human models. These include effects on gene expression (7Koike H. Karas R.H. Baur W. O'Donnell T.F.J. Mendelsohn M.E. J. Vasc. Surg. 1996; 23: 477-482Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 8Orimo A. Inoue S. Ikegami A. Hosoi T. Akishita M. Ouchi Y. Muramatsu M. Orimo H. Biochem. Biophys. Res. Commun. 1993; 195: 730-736Crossref PubMed Scopus (126) Google Scholar), ion channel function (9Zhang F. Ram J.L. Standley P.R. Sowers J.R. Am. J. Physiol. 1994; 266: C975-C980Crossref PubMed Google Scholar, 10White R.E. Darkow D.J. Lang J.L.F. Circ. Res. 1995; 77: 936-942Crossref PubMed Scopus (337) Google Scholar), response to vasoactive substances (11Jiang C. Sarrel P.M. Poole-Wilson P.A. Collins P. Am. J. Physiol. 1992; 263: H271-H275PubMed Google Scholar, 12Collins P. Shay P.J. Jiang P. Moss J. Circulation. 1994; 90: 1964-1968Crossref PubMed Scopus (182) Google Scholar, 13Redmond E.M. Cherian M.N. Wetzel R.C. Circulation. 1994; 90: 2519-2524Crossref PubMed Scopus (26) Google Scholar, 14Bhalla R.C. Toth K.F. Bhatty R.A. Thompson L.P. Sharma R.V. Am. J. Physiol. 1997; 272: H1996-H2003PubMed Google Scholar), as well as vascular smooth muscle cell proliferation and migration (14Bhalla R.C. Toth K.F. Bhatty R.A. Thompson L.P. Sharma R.V. Am. J. Physiol. 1997; 272: H1996-H2003PubMed Google Scholar, 15Kolodgie F.D. Jacob A. Wilson P.S. Carlson G.C. Farb A. Verma A. Virmani R. Am. J. Pathol. 1996; 148: 969-976PubMed Google Scholar). The possible involvement of insulin-like growth factor-1 (IGF-1)1 and IGF-1 receptor (IGF-1R) in cardiovascular pathology has recently raised interest.In vitro data have shown that IGF-1 is a potent vascular smooth muscle cell (VSMC) mitogen (16Pfeifle B. Ditschuneit H.H. Ditschuneit H. Horm. Metab. Res. 1982; 4: 409-414Crossref Scopus (40) Google Scholar, 17Clemmons D.R. Van Wyk J.J. J. Clin. Invest. 1985; 75: 1914-1918Crossref PubMed Scopus (206) Google Scholar), and several reports have documented that VSMCs express IGF-1 and its receptor (18Delafontaine P. Bernstein K.E. Alexander R.W. Hypertension. 1991; 17: 693-699Crossref PubMed Scopus (78) Google Scholar, 19Delafontaine P. Ku L. Ververis J.J. Cohen C. Runge M.S. Alexander R.W. J. Mol. Cell Cardiol. 1994; 26: 1659-1673Abstract Full Text PDF PubMed Scopus (4) Google Scholar, 20Sidawy A.N. Termanini B. Nardi R.V. Harmon J.W. Korman L.Y. Surgery. 1990; 108: 165-171PubMed Google Scholar). We and others have shown that several growth factors up-regulate IGF-1R on VSMC and this ability of growth factors to increase the number of IGF-1R is likely critical for their mitogenic effects (17Clemmons D.R. Van Wyk J.J. J. Clin. Invest. 1985; 75: 1914-1918Crossref PubMed Scopus (206) Google Scholar, 21Coppola D. Ferber A. Miura M. Sell C. D'Ambrosio C. Rubin R. Baserga R. Mol. Cell. Biol. 1994; 14: 4588-4595Crossref PubMed Scopus (267) Google Scholar, 22Delafontaine P. Anwar A. Lou H. Ku L. J. Clin. Invest. 1996; 97: 139-145Crossref PubMed Scopus (74) Google Scholar, 23Clemmons D.R. Van Wyk J.J. Pledger W.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6644-6648Crossref PubMed Scopus (69) Google Scholar, 24Delafontaine P. Meng X.P. Ku L. Du J. J. Biol. Chem. 1995; 270: 14383-14388Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Furthermore, regulation of IGF-1 binding proteins (IGFBPs) by growth factors may be physiologically important (25Anwar A. Zahid A.A. Phillips L. Delafontaine P. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 370-376Crossref PubMed Scopus (15) Google Scholar). Steroid receptors, including the estrogen receptors (ER) α and β, mediate the specific response of cells to their respective ligands by virtue of their ability to bind cis-acting regulatory sequences termed steroid response elements (for review see Ref. 26Evans R. Science. 1988; 240: 889-895Crossref PubMed Scopus (6326) Google Scholar). Although much is known about mechanisms of gene activation by ER, less information exists about repression of gene expression by ER. Although activation of genes by estrogens is typically mediated by binding of the activated receptor to the respective response element(s) present upstream of or within target genes, negative regulation by these hormones cannot always be explained by receptor-DNA interaction (27Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2851) Google Scholar, 28Lucas P.C. Granner D.K. Annu. Rev. Biochem. 1992; 61: 1131-1173Crossref PubMed Scopus (163) Google Scholar). To our knowledge, the inhibition of IL-6 in HeLa cells, lipoprotein lipase in 3T3-L1 cells, tumor necrosis factor α in U937 cells, IGF-1 gene expression in primary rat osteoblasts, and the mannose-6 IGF-II receptor gene in breast cancer cells by estrogens are the only documented examples of repression by estrogens (29Adler S. Waterman M.L. He X. Rosenfeld M.G. Cell. 1988; 52: 685-695Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 30Homma H. Kurachi H. Nishio Y. Takeda T. Yamamoto T. Adachi K. Morishige K. Ohmichi M. Matsuzawa Y. Murata Y. J. Biol. Chem. 2000; 275: 11404-11411Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 31An J. Ribeiro R.C. Webb P. Gustafsson J.A. Kushner P.J. Baxter J.D. Leitman D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15161-15166Crossref PubMed Scopus (180) Google Scholar, 32McCarthy T.L. Ji C. Shu H. Casinghino S. Crothers K. Rotwein P. Centrella M. J. Biol. Chem. 1997; 272: 18132-18139Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 33Mathieu M. Vignon F. Capony F. Rochefort H. Mol. Endocrinol. 1991; 5: 815-822Crossref PubMed Scopus (88) Google Scholar). It was therefore of interest to us to explore the effects of estradiol on IGF-1R and IGF-1 expression in vascular cells such as RASMC and to determine the molecular mechanisms of ER-mediated action on IGF-1R gene expression. We show that E2 dose-dependently decreases IGF-1R and IGF-1 expression and that the antiproliferative activity of E2 involves a down-regulation of IGF-1R and IGF-1. However, we found no direct binding of ER to sequences in the IGF-1R promoter that were sufficient to confer repression by ER in functional experiments. Nevertheless, results obtained from bandshift experiments indicated that there was an interaction between SP1 and ER, because ER decreased SP1 binding to the IGF-1R promoter. These data indicate that ER can modulate transcription from promoters that lack classical estrogen response elements (ERE) and have important implications for understanding cardiovascular effects of estrogens. Cell culture media and LipofectAMINE were purchased from Life Technologies (Basel, Switzerland). 4-Hydroxytamoxifen (OHT), 17β-estradiol, and progesterone were from Sigma (St. Louis, MO). The antiestrogen ICI 164,384 was a kind gift from Dr. A. Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK). The Dual-Luciferase reporter assay, the TNT-T7 Quick kit, and human recombinant SP1 were from Promega (Wallisellen, Switzerland). Recombinant ERα was from Panvera (Madison, WI), and anti-ERα (314) was from NeoMarkers (Fremont, CA). Antibodies against the β-chain of the IGF-1 receptor and SP1 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Peroxidase-conjugated anti-mouse IgG was from Transduction Laboratories (Lexington, KY), and horseradish peroxidase-conjugated anti-rabbit immunoglobulin was from Amersham Pharmacia Biotech (Dübendorf, Switzerland). Iodinated IGF-1 was purchased from PerkinElmer Life Sciences (Boston, MA). RASMC (kindly provided by Dr. K. Griendling, Emory University, Atlanta, GA) were grown in DMEM supplemented with 10% heat-inactivated calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin and incubated at 37 °C in a humidified 5% CO2 atmosphere. HeLa cells were cultured under similar conditions with 5% fetal bovine serum. The ER-expressing, human breast tumor cells, MCF-7 (34Horwitz K.B. Zara D.T. Thilagar A.K. Jensen E.M. McGuire W.L. Cancer Res. 1978; 38: 2434-2437PubMed Google Scholar), were maintained in DMEM and 10% fetal bovine serum. Prior to experiments, cell media were changed to DMEM without Phenol Red, containing dextran-coated, charcoal-treated, heat-inactivated (DCT) fetal bovine serum. The full-length promoter of the IGF-1R p(−2350/+640-Luc) and the shorter construct p(−476/+640-Luc) were a generous gift from Dr. H. Werner (National Institutes of Health, Bethesda, MD). Deletion fragments were made from the full-length promoter construct and subcloned upstream of the firefly luciferase cDNA (35Scheidegger K.J. Du J. Delafontaine P. J. Biol. Chem. 1999; 274: 3522-3530Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The plasmids encoding human ERα (HEG0), HE82, human ERβ (pCMV-hERβ), and pCMV-SP1 were kind gifts from Dr. P. Chambon (Strasbourg, France), Dr. S. Mader (Montreal, Canada), Dr. J.-Å. Gustafsson (Huddinge, Sweden), and Dr. R. Tjian (University of California at Berkeley, CA), respectively. The following constructs have previously been described: the empty vector pSG5 (36Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (547) Google Scholar), and XETL (37Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (849) Google Scholar). In brief, HEG0 carries human wild-type ERα cDNA, HE82 contains human ERα cDNA with a mutation in the DNA binding domain resulting in the recognition of a glucocorticoid response element instead of an ERE (38Mader S. Kumar V. Verneuil de H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (361) Google Scholar), and the reporter plasmid XETL expresses firefly luciferase under control of one vitellogenin A2 ERE upstream of the herpes simplex virus thymidine kinase promoter. The coding sequence of the human progesterone receptor B (PRB) was subcloned into the unique EcoRI site of pSG5 resulting in pSG5/hPR. HeLa cells were plated in 24-well and RASMC in 12-well plates and transfected with 1 μg of reporter plasmid and 5 ng of pRL-TK per well with or without 200 ng of HEG0, pSG5/hPR, HE82, pCMV-hERβ, or pCMV-SP1 with LipofectAMINE reagent. 20 h after transfection, the DNA-containing medium was changed and the cells were treated with or without E2 (100 nm) or progesterone (100 nm) for 12–24 h. In some experiments transfected cells were incubated with OHT (1 μm) or ICI (0.1 μm) for 1 h prior to the addition of E2. Luciferase activity was measured with the Dual-Luciferase kit according to the manufacturer's recommendations. Firefly luciferase activity was normalized to the internal control Renillaluciferase (Luc/Ren). RNase protection assays were performed as described previously (18Delafontaine P. Bernstein K.E. Alexander R.W. Hypertension. 1991; 17: 693-699Crossref PubMed Scopus (78) Google Scholar). In brief, 20 μg of total RNA was hybridized with [32P]UTP-labeled antisense IGF-1R and IGF-1 riboprobe and cohybridized with an 18 S probe (Ambion, Austin, TX). After overnight hybridization at 42 °C and RNase digestion, samples were proteinase K-treated, phenol-extracted, and analyzed by 6% polyacrylamide/8 m urea denaturing gel electrophoresis. Densitometric analyses were performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Radioligand binding assays were performed as described previously (39Ververis J.J. Ku L. Delafontaine P. Circ. Res. 1993; 72: 1285-1292Crossref PubMed Scopus (60) Google Scholar). Briefly, RASMC cultured under DCT serum conditions treated with or without E2 in 24-well plates were incubated with 0.1 nm125I-IGF-1 and 0–0.1 μm unlabeled IGF-1 for 90 min at room temperature. Cells were washed in ice-cold binding buffer and solubilized in 0.2 n NaOH before counting. All assays were performed in duplicate for each experimental point. Data were analyzed using the LIGAND program. Prior to the experiments, cultured RASMC were switched to Phenol Red-free DMEM containing DCT FBS for 48 h before adding E2 at various concentrations for 24 h. Cells were washed in ice-cold phosphate-buffered saline and lysed as previously published (35Scheidegger K.J. Du J. Delafontaine P. J. Biol. Chem. 1999; 274: 3522-3530Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Lysates were subjected to SDS-PAGE on 7.5% gels, and separated proteins were transferred to polyvinylidene difluoride membranes. Blots were blocked with 5% dry milk and incubated with anti-IGF-1Rβ antibody and secondary peroxidase-conjugated donkey anti-rabbit antibody. Immunopositive bands were visualized by enhanced chemiluminescence. Specific IGF-1 immunoreactivity of cell-conditioned medium was determined as described previously (18Delafontaine P. Bernstein K.E. Alexander R.W. Hypertension. 1991; 17: 693-699Crossref PubMed Scopus (78) Google Scholar). Briefly, cell medium was dialyzed, lyophilized, and chromatographed using Bio-Gel P-30 polyacrylamide columns (Bio-Rad Laboratories AG, Glattbrugg, Switzerland). IGF-1 fractions were assayed using a polyclonal anti-IGF-1 rabbit antiserum (kindly provided by Dr. A. F. Parlow, UCLA). Standard curves were generated using human recombinant IGF-1. RASMC were plated in 24-well plates in DMEM without Phenol Red alone or containing DCT FBS. After 48 h cells were treated with or without E2, for 24 h in complete medium. 1 μCi/ml of [3H]thymidine was added during the last 2 h of the incubation period. Cells were washed three times with ice-cold phosphate-buffered saline, incubated for 30 min in 10% trichloroacetic acid on ice, washed two times in ice-cold 95% ethanol, and lysed in 0.2 n NaOH. Samples were measured by liquid scintillation spectrophotometry. All experiments were performed in quadruplicates. The human ER, PRB, and HE82 proteins were synthesized in vitro in TNT-T7-coupled rabbit reticulocyte lysates. Nuclear extracts from untransfected MCF-7 and RASMC, or HeLa cells transfected with HEG0, were incubated with recombinant human SP1, NFκB, or ERα proteins in binding buffer containing 10 mm HEPES, pH 7.9, 10% glycerol, 100 mm KCl, 2 mm dithiothreitol, 0.1 mm EDTA, 5 mm MgCl2, 2 μg of poly(dI-dC), 0.3 μg/μl bovine serum albumin, and32P-labeled DNA in a final volume of 20 μl at room temperature. Preincubations containing ligand, antibody, and/or cold competitor (200-fold excess) as indicated were performed at room temperature for 15 min. After the incubation step the probe was added and binding conducted for additional 20 min. Reaction mixtures were loaded onto a 6% PAGE gel in 0.5 × Tris borate-EDTA (TBE). The following oligonucleotide and its complement were used as labeled probes and cold/unlabeled competitors: ERE, 5′-GATCTCTTTGATCAGGTCACTGTGACCTGACTTTG-3′. The probe for the IGF-1R promoter extended from nucleotides −476/+21. All experiments were performed at least three times. Statistical significance was measured by Student's ttest. A value of p < 0.05 was considered statistically significant. To measure the effect of E2 on IGF-1R gene expression, HeLa cells, which do not contain endogenous ER, were transiently transfected with the full-length IGF-1R promoter reporter construct with or without cotransfecting HEG0, a plasmid encoding human ERα. In HeLa cells estradiol treatment (100 nm for 24 h) decreased basal luciferase activity to 49 ± 5% (Fig. 1 A). This effect was ER-dependent, because E2 did not reduce basal IGF-1R expression in HeLa cells when human ER was not cotransfected. Similarly, E2 decreased by 21% IGF-1R transcription in RASMC-expressing endogenous ER (p = 0.005) (Fig. 1 B). Using specific primers for rat ERα and rat ERβ, we found both transcripts in RASMC (data not shown) as has been previously published by others (40Kuiper G.G.J.M. Carlsson B. Grandien K. Enmark E. Haeggblad J. Nilsson S. Gustafsson J.-A. Endocrinology. 1997; 138: 863-870Crossref PubMed Scopus (3678) Google Scholar, 41Bayard F. Clamens S. Meggetto F. Blaes N. Delsol G. Faye J.-C. Endocrinology. 1995; 136: 1523-1529Crossref PubMed Scopus (108) Google Scholar, 42Li G. Chen Y.-F. Greene G.L. Oparil S. Thompson J.A. Circulation. 1999; 100: 1639-1645Crossref PubMed Scopus (56) Google Scholar, 43Makela S. Savolainen H. Aavik E. Myllarniemi M. Strauss L. Taskinen E. Gustafsson J.A. Hayry P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7077-7082Crossref PubMed Scopus (234) Google Scholar). The fact that E2 stimulated transactivation of a minimal ERE promoter reporter construct in RASMC without transfecting HEG0 (data not shown) supports the notion that the endogenous ERs were functional as has been previously shown by others (41Bayard F. Clamens S. Meggetto F. Blaes N. Delsol G. Faye J.-C. Endocrinology. 1995; 136: 1523-1529Crossref PubMed Scopus (108) Google Scholar). Accordingly, these effects of E2 appeared to be specific, because progesterone did not have any effect on IGF-1R expression in HeLa, or RASMC transfected with progesterone receptor (data not shown). In addition, the E2 antagonist ICI 164,384 reversed the reduction in IGF-1R promoter activity induced by E2, demonstrating a specific ER-mediated effect, whereas the partial antagonist OHT acted in a synergistic way by further decreasing luciferase activity (Fig. 1 C). Interestingly, the repression of IGF-1R promoter activity by E2 was abrogated when smaller IGF-1R promoter deletion mutants were used. Indeed, the reduction in luciferase activity was maintained with the construct p(−476/+21) and p(−416/+21), however, the reduction disappeared with p(−330/+21), suggesting that the E2-responsive region was located 5′ of base pair −330 in the IGF-1R promoter (data not shown). Importantly, HE82, an ER mutant carrying a mutation within the DNA binding domain and thus recognizing a glucocorticoid response element instead of an ERE, was unable to repress expression from the IGF-1R promoter, suggesting that an ER with an intact DNA binding domain is required (Fig. 1 D). Since the identification of a second ER subtype, termed the ERβ (44Kuiper G.G.J.M. Enmark E. Pelto-Huikko M. Nilsson S. Gustafsson J.-A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5925-5930Crossref PubMed Scopus (4224) Google Scholar, 45Mosselman S. Polman J. Dijkema R. FEBS Lett. 1996; 392: 49-53Crossref PubMed Scopus (2054) Google Scholar), much research has been focused on the potentially distinct role of ERα and ERβ in vasculoprotection. It was therefore of interest to determine whether the reduction in IGF-1R transcription by the combination of E2 and ERα was subtype-specific or whether it could also be observed using ERβ. Most interestingly, E2 did not repress IGF-1R transcription when HeLa cells were transfected with human ERβ, which suggests a ERα subtype-specific effect (Fig. 1 D). To confirm the results obtained in transfection studies, endogenous IGF-1 and IGF-1R mRNA levels were measured by RNase protection assay in RASMC treated with or without E2. In agreement with the transfection studies, E2 significantly and dose-dependently reduced basal levels of IGF-1 and IGF-1R by 47% and 33%, respectively (Fig. 2 A), whereas OHT had similar effects as E2 (Fig. 2 B). Similarly to the transcriptional assays, the antiestrogen ICI reversed the decreasing effect of E2 on IGF-1 and IGF-1R (Fig. 2 B). A dose-response with ICI on IGF-1R mRNA showed that 10−7m and 10−6m ICI, but not 10−8m ICI, blocked the effect of E2: control, 100 ± 0%; E2, 65 ± 11%; ICI 10−8m/E2, 63 ± 12%; ICI 10−7m/E2, 100 ± 17%; ICI 10−6m/E2, 109 ± 37%; ICI 10−8m, 88 ± 1%; ICI 10−7m, 114 ± 19%; ICI 10−6m, 104 ± 19%; n = 3. To assess whether E2 decreased IGF-1R protein levels, cell lysates of RASMC treated with or without increasing doses of E2 were assayed for IGF-1R protein level by Western immunoblot and radioligand binding. E2dose-dependently decreased IGF-1R protein expression after 24 h, starting with doses of 1 nmE2 and resulting in a 40% reduction with 100 nm E2 (Fig. 3 A). Similarly, E2(100 nm) reduced basal IGF-1 binding sites by approximately 30% as measured by radioligand binding studies, further confirming the results seen in Western blots (percentage change in IGF-1R number: control = 100 ± 0% and E2 = 71 ± 7%, respectively, n = 4). In addition, IGF-1 protein levels in RASMC were also significantly reduced by E2 (43% reduction with 100 nm E2) as measured by RIA of cell-conditioned medium (Fig. 3 B). IGF-1 is a potent mitogen, and a functional IGF-1R is required for the mitogenic effects of various growth factors (22Delafontaine P. Anwar A. Lou H. Ku L. J. Clin. Invest. 1996; 97: 139-145Crossref PubMed Scopus (74) Google Scholar, 24Delafontaine P. Meng X.P. Ku L. Du J. J. Biol. Chem. 1995; 270: 14383-14388Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To determine whether the reduced levels of IGF-1 and IGF-1R expression induced by E2could explain the reduced DNA synthesis observed after E2treatment (14Bhalla R.C. Toth K.F. Bhatty R.A. Thompson L.P. Sharma R.V. Am. J. Physiol. 1997; 272: H1996-H2003PubMed Google Scholar, 46Somjen D. Kohen F. Jaffe A. Amir-Zaltsman Y. Knoll E. Stern N. Hypertension. 1998; 32: 39-45Crossref PubMed Scopus (114) Google Scholar), we measured [3H]thymidine incorporation in confluent RASMC. As shown in TableI, E2dose-dependently reduced DNA synthesis under serum conditions by approximately 50%. Exogenous addition of IGF-1 (50–100 ng/ml), however, was not able to reverse the E2-induced decrease in thymidine incorporation (data not shown).Table IE2 decreases serum-induced DNA synthesis in RASMCE2dosecpm/wellnm% of control0100.0 ± 00.0177.5 ± 4.90.169.6 ± 4.61.056.9 ± 3.710.056.2 ± 6.5100.049.8 ± 7.9DNA synthesis of RASMC treated with increasing doses of E2 was determined by measuring [3H]thymidine incorporation. Shown is the percentage change from control. Data are mean ± S.E. from four separate experiments performed in quadruplicates. Open table in a new tab DNA synthesis of RASMC treated with increasing doses of E2 was determined by measuring [3H]thymidine incorporation. Shown is the percentage change from control. Data are mean ± S.E. from four separate experiments performed in quadruplicates. The IGF-1R promoter contains multiple SP1 sites in both the 5′-flanking and 5′-untranslated regions (47Werner H. Bach M.A. Stannard B. Roberts Jr., C.T. LeRoith D. Mol. Endocrinol. 1992; 6: 1545-1558Crossref PubMed Scopus (66) Google Scholar). Consensus EREs consist of an inverted repeat of the palindrome GGTCA separated by a 3-base pair spacer (27Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2851) Google Scholar, 48Tora L. Gaub M.P. Mader S. Dierich A. Bellard M. Chambon P. EMBO J. 1988; 7: 3771-3778Crossref PubMed Scopus (129) Google Scholar, 49Naar A.M. Boutin J.-M. Lipkin S.M., Yu, V.C. Holloway J.M. Glass C.K. Rosenfeld M.G. Cell. 1991; 65: 1267-1279Abstract Full Text PDF PubMed Scopus (463) Google Scholar). However, no evident ERE is present within the IGF-1R promoter. In preliminary experiments, we tested our EMSA conditions by determining classical ER binding to its consensus response element and concurrent supershift with anti-ER antibodies (Fig. 4 A). To investigate the binding of ER to the IGF-1R promoter sequences, the ER-regulated promoter construct p(−476/+21) was used. Nuclear extracts from MCF-7 cells formed retarded bands with the 32P-labeled IGF-1R probe, which could be supershifted by anti-ERα antibody (data not shown). However, neither in vitro synthesized or purified ER bound the IGF-1R promoter probe, whereas SP1 and NFκB proteins formed a retarded band complex (data not shown). Interestingly, the intensity of the two main SP1-dependent bands was significantly reduced by co-incubation with in vitro synthesized ER or purified ER (Fig. 4 B), whereas both ER preparations had no effect on the NFκB-dependent band (data not shown). Although ER protein diminished the SP1/probe band, in vitrotranslated PRB or HE82, human ERα carrying a mutation in the DNA binding domain, had no effect, indicating not only an ER-specific effect but also an effect dependent on a conserved ER DNA binding domain (Fig. 4 B). Because recent studies have demonstrated that physical and functional interactions exist between ERα and the transcription factor SP1 (50Batistuzzo de Medeiros S.R. Krey G. Hihi A.K. Wahli W. J. Biol. Chem. 1997; 272: 18250-18260Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 51Porter W. Saville B. Hoivik D. Safe S. Mo" @default.
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• W2007163552 title "Estradiol Decreases IGF-1 and IGF-1 Receptor Expression in Rat Aortic Smooth Muscle Cells" @default.
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