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• W2090897053 abstract "Induction of cyclin D1 gene transcription by estrogen receptor α (ERα) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERα has been mapped to an alternative response element, a cyclic AMP-response element at −57, with possible participation of an activating protein-1 site at −954. The action of ERβ at the cyclin D1 promoter is unknown, although evidence suggests that ERβ may inhibit the proliferative action of ERα. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERα, ERαK206A (a derivative that is superactive at alternative response elements), or ERβ. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERβ occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERα, inhibit expression with ERβ. Strikingly, the presence of ERβ completely inhibits cyclin D1 gene activation by estrogen and ERα or even by estrogen and the superactive ERαK206A. The observation of the opposing action and dominance of ERβ over ERα in activation of cyclin D1 gene expression has implications for the postulated role of ERβ as a modulator of the proliferative effects of estrogen. Induction of cyclin D1 gene transcription by estrogen receptor α (ERα) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERα has been mapped to an alternative response element, a cyclic AMP-response element at −57, with possible participation of an activating protein-1 site at −954. The action of ERβ at the cyclin D1 promoter is unknown, although evidence suggests that ERβ may inhibit the proliferative action of ERα. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERα, ERαK206A (a derivative that is superactive at alternative response elements), or ERβ. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERβ occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERα, inhibit expression with ERβ. Strikingly, the presence of ERβ completely inhibits cyclin D1 gene activation by estrogen and ERα or even by estrogen and the superactive ERαK206A. The observation of the opposing action and dominance of ERβ over ERα in activation of cyclin D1 gene expression has implications for the postulated role of ERβ as a modulator of the proliferative effects of estrogen. estrogen receptor α and β, respectively ERα and ERβ knock-out, respectively estrogen receptor estrogen response element activating protein-1 cyclic AMP-response element activation function cyclin D1 luciferase 17-β-estradiol diethylstilbesterol raloxifene tamoxifen Estrogen stimulates proliferation of epithelial cells in the female reproductive tract and mammary gland and in the prostate. In the female tissues and most likely in the prostate as well, it also plays a role in the development of cancer (for recent reviews, see Refs. 1Signoretti S. Loda M. Am. J. Pathol. 2001; 159: 13-16Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 2Katzenellenbogen B.S. Katzenellenbogen J.A. Breast Cancer Res. 2000; 2: 335-344Crossref PubMed Scopus (253) Google Scholar, 3Dickson R.B. Stancel G.M. J. Natl. Cancer Inst. Monogr. 2000; 27: 135-145Crossref PubMed Scopus (159) Google Scholar, 4Tonetti D.A. Jordan V.C. J. Mammary Gland Biol. Neoplasia. 1999; 4: 401-413Crossref PubMed Scopus (10) Google Scholar). Two related proteins, estrogen receptor α (ERα)1 and β (ERβ), which function as transcription factors to regulate expression of target genes, carry out and modulate the effects of estrogen (2Katzenellenbogen B.S. Katzenellenbogen J.A. Breast Cancer Res. 2000; 2: 335-344Crossref PubMed Scopus (253) Google Scholar, 5Pettersson K. Gustafsson J.A. Annu. Rev. Physiol. 2001; 63: 165-192Crossref PubMed Scopus (435) Google Scholar). Studies with mice carrying disrupted estrogen receptors indicate that ERα mediates the major proliferative effects of estrogen (6Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar). Thus, female mice in which ERα has been knocked out (αERKOs) lack estrogen-provoked proliferation of the uterus, cervix, and vagina and have rudimentary mammary glands (6Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar, 7Lubahn D.B. Moyer J.S. Golding T.S. Couse J.F. Korach K.S. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11162-11166Crossref PubMed Scopus (1611) Google Scholar, 8Bocchinfuso W.P. Lindzey J.K. Hewitt S.C. Clark J.A. Myers P.H. Cooper R. Korach K.S. Endocrinology. 2000; 141: 2982-2994Crossref PubMed Scopus (176) Google Scholar). Male αERKO mice are completely resistant to estrogen-provoked prostate metaplasia and cancer (9Risbridger G. Wang H. Young P. Kurita T. Wang Y.Z. Lubahn D. Gustafsson J.A. Cunha G. Wong Y.Z. Dev. Biol. 2001; 229: 432-442Crossref PubMed Scopus (151) Google Scholar, 10Prins G.S. Birch L. Couse J.F. Choi I. Katzenellenbogen B. Korach K.S. Cancer Res. 2001; 61: 6089-6097PubMed Google Scholar). In contrast, ERβ knockout (βERKO) female mice have full estrogen responses of the reproductive tract, and males have a full response of the prostate. Indeed, there is suggestive evidence that ERβ may modulate the proliferative effects of ERα. Thus, the βERKOs are reported to have exaggerated estrogen responses in the uterus and to have spontaneous hyperplasia of the prostate, although the latter observation is not free of controversy (11Weihua Z. Saji S. Makinen S. Cheng G. Jensen E.V. Warner M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5936-5941Crossref PubMed Scopus (466) Google Scholar, 12Weihua Z. Makela S. Andersson L.C. Salmi S. Saji S. Webster J.I. Jensen E.V. Nilsson S. Warner M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6330-6335Crossref PubMed Scopus (386) Google Scholar, 13Dupont S. Krust A. Gansmuller A. Dierich A. Chambon P. Mark M. Development. 2000; 127: 4277-4291Crossref PubMed Google Scholar). Furthermore, there is a progressive loss of ERβ expression in prostate cancer and in mammary multistage carcinogenesis, consistent with a loss of a potential inhibitor of proliferation (14Horvath L.G. Henshall S.M. Lee C.S. Head D.R. Quinn D.I. Makela S. Delprado W. Golovsky D. Brenner P.C. O'Neill G. Kooner R. Stricker P.D. Grygiel J.J. Gustafsson J.A. Sutherland R.L. Cancer Res. 2001; 61: 5331-5335PubMed Google Scholar, 15Pasquali D. Rossi V. Esposito D. Abbondanza C. Puca G.A. Bellastella A. Sinisi A.A. J. Clin. Endocrinol. Metab. 2001; 86: 2051-2055Crossref PubMed Scopus (87) Google Scholar, 16Roger P. Sahla M.E. Makela S. Gustafsson J.A. Baldet P. Rochefort H. Cancer Res. 2001; 61: 2537-2541PubMed Google Scholar). Estrogen receptors (ERs) regulate gene expression in two ways. In the classical mode of action, ERs bind directly to classical estrogen response elements (EREs) within promoters of estrogen-regulated target genes. Tethered on the DNA, the ERs recruit p160-CBP and possibly DRIP/TRAP coactivator complexes that remodel chromatin and mediate function of the transcriptional machinery (17McKenna N.J., Xu, J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 1999; 69: 3-12Crossref PubMed Scopus (365) Google Scholar). In a second mode of action, ERs regulate transcription at promoter elements that directly bind heterologous transcription factors. These promoter elements include AP-1 sites that bind Jun/Fos (18Kushner P.J. Agard D.A. Greene G.L. Scanlan T.S. Shiau A.K. Uht R.M. Webb P. J. Steroid Biochem. Mol. Biol. 2000; 74: 311-317Crossref PubMed Scopus (742) Google Scholar), variant cyclic AMP-response elements (CREs) that bind c-Jun/ATF-2 proteins (19Webb P. Keneally M.-R. Shinsako J. Uht R. Anderson C. Paech K. Scanlan T.S. Kushner P.J. Gronemeyer H. Fuhrmann U. Parczyk K. Molecular Basis of Sex Hormone Receptor Function. Springer, Berlin1998: 121-140Crossref Google Scholar, 20Sabbah M. Courilleau D. Mester J. Redeuilh G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11217-11222Crossref PubMed Scopus (294) Google Scholar), and Sp1 sites (21Saville B. Wormke M. Wang F. Nguyen T. Enmark E. Kuiper G. Gustafsson J.A. Safe S. J. Biol. Chem. 2000; 275: 5379-5387Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 22Safe S. Vitam. Horm. 2001; 62: 231-252Crossref PubMed Google Scholar). These sites for AP-1, CRE, or Sp1 do not bind directly ERs, and regulation is presumed to occur through protein-protein interactions. The two ERs have a similar domain structure and similar actions at EREs (5Pettersson K. Gustafsson J.A. Annu. Rev. Physiol. 2001; 63: 165-192Crossref PubMed Scopus (435) Google Scholar). The central DNA binding domain is highly conserved between ERα and ERβ, and the C-terminal ligand binding domain, which is responsible for high affinity ligand binding, dimerization, and hormone-dependent activation (AF-2), is moderately conserved. Only the N-terminal region is poorly conserved; ERα, but not ERβ, has a hormone-independent activation function (AF-1) (23Delaunay F. Pettersson K. Tujague M. Gustafsson J.A. Mol. Pharmacol. 2000; 58: 584-590Crossref PubMed Scopus (143) Google Scholar). Given the conservation, it is not surprising that ERα and ERβ interact with the same spectrum of EREs and exhibit similar patterns of gene activation at classical ERE-containing target genes. Thus, both ERα and ERβ activate gene expression from either consensus or divergent EREs. There are nonetheless subtle differences between the two ERs. In particular, ERβ requires higher levels of 17-β-estradiol (E2) for activation at an ERE than does ERα (24Hall J.M. McDonnell D.P. Endocrinology. 1999; 140: 5566-5578Crossref PubMed Google Scholar, 25Pettersson K. Delaunay F. Gustafsson J.A. Oncogene. 2000; 19: 4970-4978Crossref PubMed Scopus (321) Google Scholar). In cells that have both ERα and ERβ, heterodimers are the dominant species. In these heterodimers, ERβ functions as a transdominant inhibitor of ERα with subsaturating hormone levels, although ERβ does not interfere with ERα-activated transcription at saturating levels of hormone (24Hall J.M. McDonnell D.P. Endocrinology. 1999; 140: 5566-5578Crossref PubMed Google Scholar, 25Pettersson K. Delaunay F. Gustafsson J.A. Oncogene. 2000; 19: 4970-4978Crossref PubMed Scopus (321) Google Scholar). Despite their similar action at EREs, ERα and ERβ have completely different effects at AP-1 sites. ERα activates and ERβ inhibits transcription from an AP-1 site when the receptors are complexed to 17-β-estradiol (26Paech K. Webb P. Kuiper G.G. Nilsson S. Gustafsson J. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2074) Google Scholar, 27Webb P. Nguyen P. Valentine C. Lopez G.N. Kwok G.R. McInerney E. Katzenellenbogen B.S. Enmark E. Gustafsson J.A. Nilsson S. Kushner P.J. Mol. Endocrinol. 1999; 13: 1672-1685Crossref PubMed Google Scholar). At Sp1 sites, the differences between the ERs are less dramatic; ERα activates with multiple ligands, but in a cell-specific manner, and ERβ is nearly inactive (21Saville B. Wormke M. Wang F. Nguyen T. Enmark E. Kuiper G. Gustafsson J.A. Safe S. J. Biol. Chem. 2000; 275: 5379-5387Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 22Safe S. Vitam. Horm. 2001; 62: 231-252Crossref PubMed Google Scholar). To explain ER action at Sp1 sites, it has been proposed that ER binding to Sp1 increases the binding of Sp1 to its cognate element, thereby enhancing transcription (21Saville B. Wormke M. Wang F. Nguyen T. Enmark E. Kuiper G. Gustafsson J.A. Safe S. J. Biol. Chem. 2000; 275: 5379-5387Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 22Safe S. Vitam. Horm. 2001; 62: 231-252Crossref PubMed Google Scholar). The model for ER action at AP-1 sites is more complicated. It has been proposed that ERα is present at AP-1 sites through contact with p160-CBP coactivators that have been recruited by Jun/Fos. ERα-estrogen is believed to trigger the ability of coactivator to stimulate transcription (18Kushner P.J. Agard D.A. Greene G.L. Scanlan T.S. Shiau A.K. Uht R.M. Webb P. J. Steroid Biochem. Mol. Biol. 2000; 74: 311-317Crossref PubMed Scopus (742) Google Scholar). It has been proposed that ERβ-raloxifene, in contrast, is not present at the AP-1 site within the promoter and functions instead by serving as a decoy for inhibitors that would otherwise dampen transcription (18Kushner P.J. Agard D.A. Greene G.L. Scanlan T.S. Shiau A.K. Uht R.M. Webb P. J. Steroid Biochem. Mol. Biol. 2000; 74: 311-317Crossref PubMed Scopus (742) Google Scholar). One important target gene through which estrogen-complexed ERα mediates its proliferative action on mammary cancer cells in culture is cyclin D1, a major regulator of entry into the proliferative stage of the cell cycle (28Planas-Silva M.D. Weinberg R.A. Mol. Cell. Biol. 1997; 17: 4059-4069Crossref PubMed Scopus (235) Google Scholar, 29Prall O.W. Rogan E.M. Sutherland R.L. J. Steroid Biochem. Mol. Biol. 1998; 65: 169-174Crossref PubMed Scopus (135) Google Scholar). Thus, there is a strong correlation between increased proliferative response and increased levels of cyclin D1 mRNA with increased levels of ERα overexpression in MCF-7 breast cancer cells (30Wilcken N.R. Prall O.W. Musgrove E.A. Sutherland R.L. Clin. Cancer Res. 1997; 3: 849-854PubMed Google Scholar). The effect of ERα−estrogen on cyclin D1 protein expression appears to be predominantly transcriptional with increased expression of cyclin D1 mRNA preceding changes in cyclin D1 protein (31Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 32Prall O.W. Rogan E.M. Musgrove E.A. Watts C.K. Sutherland R.L. Mol. Cell. Biol. 1998; 18: 4499-4508Crossref PubMed Scopus (216) Google Scholar). The abundance of cyclin D1 rises during estrogen-provoked proliferation and declines following exposure to antiestrogens in ERα-positive MCF-7 cells (33Watts C.K. Brady A. Sarcevic B. deFazio A. Musgrove E.A. Sutherland R.L. Mol. Endocrinol. 1995; 9: 1804-1813Crossref PubMed Scopus (153) Google Scholar, 34Musgrove E.A. Hui R. Sweeney K.J. Watts C.K. Sutherland R.L. J. Mammary Gland Biol. Neoplasia. 1996; 1: 153-162Crossref PubMed Scopus (36) Google Scholar, 35Sutherland R.L. Hamilton J.A. Sweeney K.J. Watts C.K. Musgrove E.A. Ciba Found. Symp. 1995; 191: 218-234PubMed Google Scholar). Strikingly, the estrogen-provoked entry into the cell cycle is blocked by antisense cyclin D1 or by microinjection of anti-cyclin D1 antibodies, whereas the blockade imposed by antiestrogens can be overcome by cyclin D1 gene expression in MCF-7 cells (32Prall O.W. Rogan E.M. Musgrove E.A. Watts C.K. Sutherland R.L. Mol. Cell. Biol. 1998; 18: 4499-4508Crossref PubMed Scopus (216) Google Scholar, 36Lukas J. Bartkova J. Bartek J. Mol. Cell. Biol. 1996; 16: 6917-6925Crossref PubMed Scopus (297) Google Scholar, 37Carroll J.S. Prall O.W. Musgrove E.A. Sutherland R.L. J. Biol. Chem. 2000; 275: 38221-38229Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Although cyclin D1 gene transcription is directly inducible by estrogen, there is no ERE-related sequence in the promoter region (38Herber B. Truss M. Beato M. Mueller R. Oncogene. 1994; 9: 1295-1304PubMed Google Scholar). Instead, the cyclin D1 promoter contains multiple regulatory elements, including binding sites for AP-1, STAT5, NF-κB, E2F, Oct1, Sp1, Myc/Max, Egr, Ets, CRE, and TCF/LEF (see Ref. 39Allan A.L. Albanese C. Pestell R.G. LaMarre J. J. Biol. Chem. 2001; 276: 27272-27280Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar and references therein). Sabbah et al. (20Sabbah M. Courilleau D. Mester J. Redeuilh G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11217-11222Crossref PubMed Scopus (294) Google Scholar) showed that E2induced reporter gene activity in MCF-7 cells transfected with a construct containing the −973 to +139 region of the cyclin D1 promoter. Deletion analysis of this promoter in ER-negative HeLa cells identified a variant CRE at −57 as the E2-responsive region (20Sabbah M. Courilleau D. Mester J. Redeuilh G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11217-11222Crossref PubMed Scopus (294) Google Scholar). This variant CRE appeared to bind Jun/ATF-2. Altucciet al. mapped the estrogen-responsive region to a fragment between −994 and −136 of the cyclin D1 promoter (40Altucci L. Addeo R. Cicatiello L. Dauvois S. Parker M.G. Truss M. Beato M. Sica V. Bresciani F. Weisz A. Oncogene. 1996; 12: 2315-2324PubMed Google Scholar). Several potential binding sites for known transcription factors can be found in this region of the promoter including the AP-1 site at −954, indicating that this AP-1 site has a potential contribution. Recently, Safe and colleagues (41Castro-Rivera E. Samudio I. Safe S. J. Biol. Chem. 2001; 276: 30853-30861Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) reported that the Sp1 site in the cyclin D1 promoter also contributed to the estrogen response with ERα. The cyclin D1 gene and its induction by ERα also appear to play a key role in estrogen-provoked proliferation in vivo (42Steeg P.S. Zhou Q. Breast Cancer Res. Treat. 1998; 52: 17-28Crossref PubMed Scopus (104) Google Scholar). The cyclin D1 gene is amplified in up to 20% of human breast cancers, whereas cyclin D1 protein is overexpressed in over 50% of human mammary carcinomas, including those that are estrogen-responsive (43Dickson C. Fantl V. Gillett C. Brookes S. Bartek J. Smith R. Fisher C. Barnes D. Peters G. Cancer Lett. 1995; 90: 43-50Crossref PubMed Scopus (157) Google Scholar, 44Schuuring E. Gene (Amst.). 1995; 159: 83-96Crossref PubMed Scopus (249) Google Scholar, 45Frierson H.F., Jr. Gaffey M.J. Zukerberg L.R. Arnold A. Williams M.E. Mod. Pathol. 1996; 9: 725-730PubMed Google Scholar, 46Hosokawa Y. Arnold A. Genes Chromosomes Cancer. 1998; 22: 66-71Crossref PubMed Scopus (135) Google Scholar). Targeted overexpression of cyclin D1 protein in mammary epithelial cells leads to ductal hyperproliferation and eventual tumor formation (47Wang T.C. Cardiff R.D. Zukerberg L. Lees E. Arnold A. Schmidt E.V. Nature. 1994; 369: 669-671Crossref PubMed Scopus (896) Google Scholar). Mice nullizygous for cyclin D1 show profound defects in estrogen-driven mammary lobuloalveolar development during pregnancy, indicating that the induction of the cyclin D1 gene plays a critical role in the maturation of this tissue (48Sicinski P. Donaher J.L. Parker S.B., Li, T. Fazeli A. Gardner H. Haslam S.Z. Bronson R.T. Elledge S.J. Weinberg R.A. Cell. 1995; 82: 621-630Abstract Full Text PDF PubMed Scopus (897) Google Scholar, 49Fantl V. Stamp G. Andrews A. Rosewell I. Dickson C. Genes Dev. 1995; 9: 2364-2372Crossref PubMed Scopus (606) Google Scholar, 50Fantl V. Edwards P.A. Steel J.H. Vonderhaar B.K. Dickson C. Dev. Biol. 1999; 212: 1-11Crossref PubMed Scopus (76) Google Scholar). It should be noted, however, that cyclin D1 gene knockout mice still had estrogen-stimulated ductal elongation and branching during puberty and early pregnancy. Nonetheless, a recent study has shown that cyclin D1-deficient mice are resistant to breast cancers induced by theneu and ras oncogenes (51Yu Q. Geng Y. Sicinski P. Nature. 2001; 411: 1017-1021Crossref PubMed Scopus (833) Google Scholar). Whereas the above studies suggest a pathway for ERα action at the cyclin D1 gene and subsequent proliferation, they leave the role of ERβ unclear. The suggestion from the ER knock-out studies is that ERβ may modulate the proliferative effects of ERα, but the matter is controversial. Furthermore, there is some uncertainty as to the exact elements in the cyclin D1 promoter that mediate the effects of ERα. We therefore sought to confirm the cis-elements in the cyclin D1 promoter that are required for the transcriptional activation of the gene targeted by human ERs and determine how ERβ acts at the cyclin D1 promoter. In the present study, the response elements of the cyclin D1 promoter targeted by ERs were mapped to the CRE as well as the AP-1 site, with the CRE predominant. Unlike ERα, ERβ complexed to 17-β-estradiol repressed cyclin D1 gene transcription and blocked ERα-mediated induction when both receptors were present. This suggests that ERβ may indeed modulate the proliferative effects of ERα−estrogen by blocking its action at the cyclin D1 gene or other key pro-proliferative target genes containing CRE or AP-1 sites. HeLa cells were grown in Dulbecco's modified Eagle's/F-12 Coon's modified medium (Sigma) with 15 mmHepes, l-glutamine (0.438 g/liter), NaHCO3(1.338 g/liter), 8% iron-supplemented calf serum (Sigma), and penicillin/streptomycin. E2, tamoxifen (Tam), and diethylstilbestrol (DES) were purchased from Sigma. Imperial Chemical Industries (ICI) 182780 was a gift from Dr. A. Wakeling (Astra/Zeneca, Macclesfield, UK). Raloxifene (Ral) was a gift from Paul Webb and was extracted from Evista. Human cyclin D1 promoter reporter constructions used for luciferase assays in pA3LUC termed −963CD1LUC, −963AP1mtCD1LUC, −163CD1LUC, and −163ΔSp1CD1LUC have been previously described (52Albanese C. Johnson J. Watanabe G. Eklund N., Vu, D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar, 53Albanese C. D'Amico M. Reutens A.T., Fu, M. Watanabe G. Lee R.J. Kitsis R.N. Henglein B. Avantaggiati M. Somasundaram K. Thimmapaya B. Pestell R.G. J. Biol. Chem. 1999; 274: 34186-34195Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 54Watanabe G. Albanese C. Lee R.J. Reutens A. Vairo G. Henglein B.D. Pestell R.G. Mol. Cell. Biol. 1998; 18: 3212-3222Crossref PubMed Scopus (145) Google Scholar, 55Watanabe G. Howe A. Lee R.J. Albanese C. Shu I.W. Karnezis A.N. Zon L. Kyriakis J. Rundell K. Pestell R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12861-12866Crossref PubMed Scopus (197) Google Scholar, 56Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines G.K. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). −963CREmtCD1LUC and −1745AP1/CREmtCD1LUC were generated by primer-based PCR and confirmed by sequencing. ERE-II-LUC and the full-length human ERα cDNA (HEO) in pSG5 expression vector have been previously described (27Webb P. Nguyen P. Valentine C. Lopez G.N. Kwok G.R. McInerney E. Katzenellenbogen B.S. Enmark E. Gustafsson J.A. Nilsson S. Kushner P.J. Mol. Endocrinol. 1999; 13: 1672-1685Crossref PubMed Google Scholar). The full-length human ERβ cDNA in pCMV5 expression vector was moved into pSG5 vector as anEcoRI and HindIII fragment, and the orientation of the insert was confirmed by sequencing. The ERαK206A mutant was introduced into the full-length ERα cDNA in pSG5 vector by PCR-based site-directed mutagenesis (QuikChange kit; Stratagene) and confirmed by sequencing. The ERαK206A creates a point mutation in the codon coding for lysine 206 into alanine in the first zinc finger of the human ERα DNA binding domain. The oligonucleotide containing the desired nucleotide changes for ERαK206A was 5′-CTGTGAGGGCTGCGCTGCCTTCTTCAAGAG-3′, and its complementary strand is 5′-CTCTTGAAGAAGGCA- GCGCAGCCCTCACAG-3′. Cells were grown to a density of not more than 5 × 104cells/cm2. HeLa cells were transfected by electroporation. 2–3 million cells were trypsinized and resuspended in 0.5 ml of phosphate-buffered saline supplemented with 10% glucose and 10 μg/ml BioBrene (Applied Biosystems, Foster City, CA) in a single 0.4-cm gap electroporation cuvette with 2.5 μg of the luciferase reporter plasmid and the expression vector plasmid and 1.0 μg of actin-β-galactosidase plasmid internal control. Cells were electroporated at 0.24 kV, 960 microfarads in a Bio-Rad Gene Pulser II apparatus (Bio-Rad). Following electroporation, the cells were immediately resuspended in growth medium, plated into 12-well dishes at 1 ml/well, and treated with ligands (ICI 182780 (1 μm), raloxifene (1 μm), tamoxifen (5 μm), E2 (0.1 μm), DES (0.1 μm), or ethyl alcohol vehicle control). After 40–48 h of incubation at 37 °C, the cells were lysed by first removing the medium from the wells, washing with phosphate-buffered saline, and then adding 0.1 ml of lysis buffer consisting of 100 mm Tris (pH 7.8) and 0.2% Triton X-100 for 10 min at 4 °C. Luciferase and β-galactosidase activities were then measured using standard luciferase (Promega, Madison, WI) and β-galactosidase detection kits (Applied Biosystems, Bedford, MA). Luciferase activities were normalized for β-galactosidase activities. Individual transfections (each containing data from triplicate wells) were repeated three times. HeLa cells were transfected with human ERα, ERβ, and ERαK206A expression plasmids by electroporation and treated with ligands as above. After 46–48 h of treatment, cells were washed with cold phosphate-buffered saline buffer and harvested in radioimmune precipitation buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors phenylmethylsulfonyl fluoride, sodium orthovanadate, aprotinin (Sigma)). The whole cell extracts were separated by SDS-PAGE in 10% gels and electroblotted to nitrocellulose membranes. The membranes were incubated with a monoclonal antibody against human cyclin D1 (HD11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and an anti-mouse horseradish peroxidase second antibody. The immune complexes were detected with an enhanced chemiluminescence detection kit (Invitrogen). Autoradiograms from Western blots were quantitated using NIH Image 1.62 (National Institutes of Health, Bethesda, MD). A gel quantification macro was used to generate density peaks for each band based on an internal optical density standard. Peaks were user-defined, and the area under the curve for each density peak was measured. Measurements were made on each of three different Western blots. Graphs represent mean values, and error bars represent the S.D. We examined the transactivation properties of human ERα expressed in HeLa cells on a series of cyclin D1 gene promoter constructs driving luciferase. In particular, we compared a reporter plasmid that contained the wild-type cyclin D1 promoter (−963CD1LUC) with a similar reporter carrying mutations in the AP-1 site (−963AP1mtCD1LUC), in CRE (−963CREmtCD1LUC), or in both the AP-1 site and the CRE (−1745AP1/CREmtCD1LUC). The estrogens (17-β-estradiol and diethylstilbesterol) up-regulated the ERα action at the wild-type cyclin D1 gene promoter, and antiestrogens had a modest but valuable effect (Fig.1 A). Induction with E2 and DES was around 3-fold. The estrogen response of the AP-1 mutant was slightly, but consistently, weaker than that of the wild-type cyclin D1 promoter. The response of the CRE mutant, in contrast, was much weaker than wild-type but was not abolished. The response of the AP-1/CRE double mutant was abolished. Thus, ERα up-regulates the cyclin D1 promoter with estrogens through both the CRE and the AP-1 site, which have partly redundant functions. We also examined the expression of the endogenous cyclin D1 gene regulated by ERα in HeLa cells. We used Western blot analysis using a cyclin D1-specific antibody of extracts from ERα transiently transfected HeLa cells. A modest increase of cyclin D1 protein levels was observed in HeLa cells transiently transfected with an expression vector for ERα and treated with estrogens and a smaller increase with antiestrogens (Fig. 1 B). Cyclin D1 protein levels did not increase in HeLa cells transiently transfected with control vector after treatment with antiestrogens and estrogens (data not shown). ERα is unique among the nuclear receptors in its ability to enhance AP-1-mediated transcription with the estrogen. Other nuclear receptors, such as glucocorticoid receptor and thyroid receptor, repress AP-1-dependent transcription in response to their cognate hormone. Interestingly, a point mutation at the base of the first zinc finger of the DNA binding domain converts glucocorticoid receptor and thyroid receptor from inhibitors to AP-1 activators (57Starr D.B. Matsui W. Thomas J.R. Yamamoto K.R. Genes Dev. 1996; 10: 1271-1283Crossref PubMed Scopus (109) Google Scholar). When we introduced the homologous mutation (K206A) into the first zinc finger of the DNA binding domain of human ERα, we found that the mutant receptor (ERαK206A) was superactive at target genes regulated by AP-1 but underactive at target genes with classical EREs. This pattern occurred in cell culture and in transgenic mice. 2B. Anderegg and R. M. Uht, unpublished results. In accord with these results, ERαK206A enhanced transcriptional activity at the cyclin D1 promoter about 20-fold with estrogens and was consistently 7–10-fold more active than ERα (Fig.2 A). No activation occurred with antiestrogens in contrast to the weak activation observed with ERα. Because the transcriptional effect of ERαK206A on cyclin D1 promoter was so profound, we explored whether expression of the endogenous cyclin D1 gene in HeLa cells might also respond to transient transfection with this superactive receptor. Cyclin D1 protein levels consistently rose in HeLa cells transiently" @default.
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• W2090897053 date "2002-07-01" @default.
• W2090897053 modified "2023-10-17" @default.
• W2090897053 title "Opposing Action of Estrogen Receptors α and β on Cyclin D1 Gene Expression" @default.
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• W2090897053 doi "https://doi.org/10.1074/jbc.m201829200" @default.
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