Estrogen Response Elements Can Mediate Agonist Activity of Anti-estrogens in Human Endometrial Ishikawa Cells

Abstract

Anti-estrogens like hydroxytamoxifen (OHT) have mixed agonist/antagonist activities, leading to tissue-specific stimulation of cellular proliferation. Partial agonist activity of OHT can be observed in vitro in endometrial carcinoma cells like Ishikawa. Here, we have compared several anti-estrogens (including extensively characterized OHT and pure anti-estrogens such as ICI164,384 and RU58,668, which are devoid of uterotrophic activity) for their capacity to stimulate promoters containing estrogen response elements (EREs) or AP1-binding sites (12-O-tetradecanoylphorbol-13-acetate response elements, TREs), the two types of DNA motifs known to mediate transcriptional stimulation by estrogen receptors. Assays were performed in Ishikawa cells either by transient transfection or by using cell lines with stably propagated reporter vectors. In transient transfection experiments, none of the anti-estrogens displayed agonist activity on the promoters tested. In contrast, significant transcriptional stimulation was observed with low concentrations of OHT and RU39,411 in Ishikawa cells stably propagating reporter constructs containing a minimal ERE3-TATA promoter. In addition, micromolar concentrations of OHT, but not of RU39,411, stimulated stably propagated AP1-responsive reporter constructs. No transcriptional stimulation of ERE- or TRE-containing promoters was observed with the pure anti-estrogens ICI164,384 and RU58,668. These results indicate that the presence of estrogen response elements in promoters is sufficient to mediate cell-specific agonism of anti-estrogens at the transcriptional level, and that stimulation of AP1 activity may be restricted to a subset of anti-estrogens possessing agonist activity on EREs. In addition, our results suggest that transient transfections do not fully recapitulatein vivo conditions required to observe agonist activity of anti-estrogens. Anti-estrogens like hydroxytamoxifen (OHT) have mixed agonist/antagonist activities, leading to tissue-specific stimulation of cellular proliferation. Partial agonist activity of OHT can be observed in vitro in endometrial carcinoma cells like Ishikawa. Here, we have compared several anti-estrogens (including extensively characterized OHT and pure anti-estrogens such as ICI164,384 and RU58,668, which are devoid of uterotrophic activity) for their capacity to stimulate promoters containing estrogen response elements (EREs) or AP1-binding sites (12-O-tetradecanoylphorbol-13-acetate response elements, TREs), the two types of DNA motifs known to mediate transcriptional stimulation by estrogen receptors. Assays were performed in Ishikawa cells either by transient transfection or by using cell lines with stably propagated reporter vectors. In transient transfection experiments, none of the anti-estrogens displayed agonist activity on the promoters tested. In contrast, significant transcriptional stimulation was observed with low concentrations of OHT and RU39,411 in Ishikawa cells stably propagating reporter constructs containing a minimal ERE3-TATA promoter. In addition, micromolar concentrations of OHT, but not of RU39,411, stimulated stably propagated AP1-responsive reporter constructs. No transcriptional stimulation of ERE- or TRE-containing promoters was observed with the pure anti-estrogens ICI164,384 and RU58,668. These results indicate that the presence of estrogen response elements in promoters is sufficient to mediate cell-specific agonism of anti-estrogens at the transcriptional level, and that stimulation of AP1 activity may be restricted to a subset of anti-estrogens possessing agonist activity on EREs. In addition, our results suggest that transient transfections do not fully recapitulatein vivo conditions required to observe agonist activity of anti-estrogens. The estrogen 17β-estradiol (E2) 1The abbreviations used are: E2, estradiol; EBV, Epstein-Barr virus; ER, estrogen receptor; ERE, estrogen response element; PR, progesterone receptor; FBS, fetal bovine serum; TAM, tamoxifen; OHT, 4-hydroxytamoxifen; ICI, ICI164,384; RUp, RU39,411; RUf, RU58,668; CAT, chloramphenicol acetyltransferase; tk, thymidine kinase; STR, rat stromelysin promoter; Vit, Xenopusvitellogenin A2 promoter; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response element; RT, reverse transcriptase; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; GRE, glucocorticoid response element(s). regulates gene transcription by binding to the estrogen receptor (ER), which interacts with specific target DNA sequences known as estrogen response elements (EREs). When bound to DNA the ER stimulates transcription via two transcription activation domains, AF-1 and AF-2 (1Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (890) Google Scholar, 2Metzger D. Ali S. Bornert J.-M. Chambon P. J. Biol. Chem. 1995; 270: 9535-9542Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 3Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (664) Google Scholar). AF-1 is located in the poorly conserved N-terminal A/B domain of the ER (2Metzger D. Ali S. Bornert J.-M. Chambon P. J. Biol. Chem. 1995; 270: 9535-9542Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 4McInerney E.M. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 24172-24178Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), whereas AF-2 is found in the C-terminus of region E, the hormone-binding region (5Hollenberg S.M. Evans R.M. Cell. 1988; 55: 899-906Abstract Full Text PDF PubMed Scopus (549) Google Scholar, 6Webster N.J. Green S. Jin J.R. Chambon P. Cell. 1988; 54: 199-207Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 7Henttu P.M. Kalkhoven E. Parker M.G. Mol. Cell. Biol. 1997; 17: 1832-1839Crossref PubMed Scopus (193) Google Scholar, 8Danielian P.S. White R. Lees J.A. Parker M.G. EMBO J. 1992; 11: 1025-1033Crossref PubMed Scopus (722) Google Scholar). Binding of E2 to the ER is thought to induce a conformational change in the hormone binding domain, stimulating its transactivation properties. Different types of synthetic compounds have been developed that are capable of antagonizing ER action in reproductive tissues and, in particular, of blocking estradiol stimulation of cellular growth in breast and uterine tissues. These anti-estrogens act by competing with E2 for binding to the ER and block ER-mediated activation of transcription when co-administered with hormone (9Katzenellenbogen B.S. Montano M.M. Le Goff P. Schodin D.J. Kraus W.L. Bhardwaj B. Fujimoto N. J. Steroid Biochem. Mol. Biol. 1995; 53: 387-393Crossref PubMed Scopus (107) Google Scholar). However, tamoxifen, one of the most widely used anti-estrogens in breast cancer treatment (10Jordan V.C. Breast Cancer Res. Treat. 1990; 15: 125-136Crossref PubMed Scopus (115) Google Scholar), can induce uterine cell growth in vivo in animal models (11Gottardis M.M. Robinson S.P. Satyaswaroop P.G. Jordan V.C. Cancer Res. 1988; 48: 812-815PubMed Google Scholar) and in humans (12Van Leeuwen F.E. Benraadt J. Coebergh J.W. Kiemeney L.A. Gimbrere C.H. Otter R. Schouten L.J. Damhuis R.A. Bontenbal M. Diepehhorst F.W. van den Belt-Dusebout A.W. van Tinteren H. Lancet. 1994; 343: 448-452Abstract PubMed Scopus (576) Google Scholar, 13Fisher B. Costantino J.P. Redmond C.K. Fisher E.R. Wickerham D.L. Cronin W.M. J. Natl. Cancer Inst. 1994; 86: 527-537Crossref PubMed Scopus (1255) Google Scholar). Hydroxytamoxifen also induces cellular proliferation (14Anzai Y. Holinka C.F. Kuramoto H. Gurpide E. Cancer Res. 1989; 49: 2362-2365PubMed Google Scholar, 15Jamil A. Croxtall J.D. White J.O. J. Mol. Endocrinol. 1991; 6: 215-221Crossref PubMed Scopus (53) Google Scholar) and transcription of endogenous estrogen target genes such as the progesterone receptor (PR) gene in human endometria and cultured human endometrial carcinoma cells (15Jamil A. Croxtall J.D. White J.O. J. Mol. Endocrinol. 1991; 6: 215-221Crossref PubMed Scopus (53) Google Scholar,16Schwartz L.B. Krey L. Demopoulos R. Goldstein S.R. Nachtigall L.E. Mittal K. Am. J. Obstet. Gynecol. 1997; 176: 129-137Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Other anti-estrogens, like ICI164,384 (17Wakeling A.E. J. Steroid Biochem. Mol. Biol. 1990; 37: 771-775Crossref PubMed Scopus (39) Google Scholar) or the more recently developed RU58,668 (18Van de Velde P. Nique F. Bouchoux F. Brémaud J. Hameau M.-C. Lucas D. Moratille C. Viet S. Philibert D. Teutsch G. J. Steroid Biochem. Mol. Biol. 1994; 48: 187-196Crossref PubMed Scopus (119) Google Scholar), were reported not to stimulate uterine cell growth and may therefore prove more appropriate for breast cancer therapy (17Wakeling A.E. J. Steroid Biochem. Mol. Biol. 1990; 37: 771-775Crossref PubMed Scopus (39) Google Scholar). To better understand the mechanisms of tissue-specific estrogenic activity of anti-estrogens, we compared transcriptional activation of ERE-containing reporter constructs by full or partial anti-estrogens in the estrogen-dependent breast carcinoma cell line MCF7 and in the endometrial carcinoma cell line Ishikawa. We also assessed whether anti-estrogens may regulate transcription of target genes through AP1-binding sites (TPA response elements, TREs) rather than, or as well as, through EREs. Indeed, estrogenic stimulation of promoters containing TRE sites has been documented (19Gaub M.P. Bellard M. Scheuer I. Chambon P. Sassone-Corsi P. Cell. 1990; 63: 1267-1276Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 20Philips A. Chalbos D. Rochefort H. J. Biol. Chem. 1993; 268: 14103-14108Abstract Full Text PDF PubMed Google Scholar, 21Umayahara Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Y. T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, P. R.M. Mol. Endocrinol. 1995; 9: PubMed Google Scholar), a ER and AP1 P. R.M. Mol. Endocrinol. 1995; 9: PubMed Google Scholar). Assays used for the of or in transcriptional stimulation by anti-estrogens with partial agonist activity in to transient transfection in MCF7 or Ishikawa stimulation of reporter stably propagated as in cell The was of for transcriptional stimulation and of of the two types of and for the of cell-specific agonism by are (E2) was RU58,668 and RU39,411 were D. ICI164,384 was by T. OHT and were fetal bovine and were and reporter L. M. Cell. Full Text PDF PubMed Scopus Google Scholar) and to in S. S. M. C. Chambon P. EMBO J. 1990; 9: PubMed Scopus Google Scholar) were by P. Chambon was by of of containing the A2 L. E. Res. 1988; PubMed Scopus Google Scholar) and sites and sites of was in several S. White Natl. S. A. 1993; PubMed Scopus Google Scholar). of the gene in was by with containing the ERE3-TATA was the M. White Chambon P. EMBO J. 1990; 9: PubMed Scopus Google Scholar) by with with and with was which been with with and with to the promoter. of the and sites and the motifs were and by containing a TPA response and and were by of containing the minimal gene transcriptional the and also by S. 1994; PubMed Scopus Google Scholar). MCF7 and Ishikawa cells were in Eagle's with and fetal bovine and to Y. Katzenellenbogen J.A. Katzenellenbogen B.S. Natl. S. A. PubMed Scopus Google Scholar) with for transient were and using the J. S. Cell. Full Text PDF PubMed Scopus Google Scholar) with of DNA of of reporter of and to was to and were for a in were by in of by at for were by of in and for activity was by of for with of and by with and B. 1988; PubMed Scopus Google Scholar). To for of progesterone receptor cells in with were for in the or presence of estradiol or of anti-estrogens. was and were cells were cells in and in with of S. White Natl. S. A. 1993; PubMed Scopus Google Scholar), of and as DNA was of by two were and activity was as were a of cells were in with were at a of and with of or vectors. were and were in with by of on were with of in for on by in the presence of hormone or and of for on and for at were by for Ishikawa or MCF7 cells were with of or cells were using containing and in for of cells in S. 1994; PubMed Scopus Google Scholar). cells in were and for estrogen or TPA of Different of cells the reporter were found to of cell lines containing reporter Ishikawa cells were with of and of gene cells were and Eagle's containing and were and for stimulation of activity by with TPA for cell lines were in containing the of used for cells were for in with with estrogen or anti-estrogens was for when cells in DMEM, were with E2 OHT or for and of by of were in of containing of for at and for at and on were using reverse and in a of with at for by for of were by DNA polymerase in a of with and and reverse or as and and of for for and for were performed for of and were performed for for and for for of by a for The were on a Partial anti-estrogens such as hydroxytamoxifen are thought to induce a specific of the estrogen which of and a ER and estrogen or OHT of in Full anti-estrogens like ICI164,384 induce in a that is to that of ER D. M. Ali S. Chambon P. Mol. Endocrinol. 1995; 9: PubMed Google Scholar). We used a to ER conformational by two anti-estrogens, RU39,411 and RU58,668 which are of estradiol and were reported to as partial and full anti-estrogens, (18Van de Velde P. Nique F. Bouchoux F. Brémaud J. Hameau M.-C. Lucas D. Moratille C. Viet S. Philibert D. Teutsch G. J. Steroid Biochem. Mol. Biol. 1994; 48: 187-196Crossref PubMed Scopus (119) Google Scholar, L. M. M. N. G. 1995; PubMed Scopus Google Scholar). cells with for estrogen receptor were with anti-estrogens, or with a estrogen response were by specific be in the of Indeed, was when using cells with the or when were with a of not treatment of the cellular the of the E2, OHT on the in a and treatment a at a to that of OHT RUp, ER and the at a of and RUf, and that of the ICI, results were using the ER that were observed in the presence of estrogen or of the anti-estrogens and not in the of not with the of the on ER L. A. Metzger D. M. I. Chambon P. EMBO J. 1989; PubMed Scopus Google Scholar, J.P. R. White J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of ER with or are with their as partial and full anti-estrogens, Hydroxytamoxifen was reported to induce of the estrogen target gene human progesterone receptor in Ishikawa cells (15Jamil A. Croxtall J.D. White J.O. J. Mol. Endocrinol. 1991; 6: 215-221Crossref PubMed Scopus (53) Google Scholar, L.B. Krey L. Demopoulos R. Goldstein S.R. Nachtigall L.E. Mittal K. Am. J. Obstet. Gynecol. 1997; 176: 129-137Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We to whether RUp, which ER in can also induce of in Ishikawa and MCF7 were with estrogen or anti-estrogens to transient transfection with by of progesterone for No stimulation of activity by progesterone be observed in of cells in the of treatment with estrogen or anti-estrogens In cells with estrogen or with the ER agonist the of estradiol J. D. C. J.P. J. Steroid Biochem. PubMed Scopus Google Scholar, J.P. P.M. M.M. T. Cancer Res. Google Scholar), activity was in the presence of progesterone and In cells with OHT, were of with estrogen The to whereas stimulation be with to was observed with Ishikawa none of the anti-estrogens stimulated transcriptional activity in MCF7 cells using and These that partial agonist activity of anti-estrogens on of endogenous estrogen target genes can be observed in Ishikawa cells (15Jamil A. Croxtall J.D. White J.O. J. Mol. Endocrinol. 1991; 6: 215-221Crossref PubMed Scopus (53) Google Scholar,16Schwartz L.B. Krey L. Demopoulos R. Goldstein S.R. Nachtigall L.E. Mittal K. Am. J. Obstet. Gynecol. 1997; 176: 129-137Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In to the mechanisms of transcriptional by anti-estrogens with partial agonist activity in Ishikawa we whether synthetic promoters can be stimulated by anti-estrogens in transient transfection reporter or were Ishikawa cells in the of estrogen receptor Anti-estrogens not stimulate or conditions estradiol stimulated reporter constructs and and results were when were with more in to that none of the anti-estrogens stimulated activity not of for ER with or in in the of treatment and with anti-estrogens not compared with in the presence of OHT or were with anti-estrogens. in the of treatment binding of in which be by with anti-estrogens. ER may be a transcriptional the conditions of to observed in Ishikawa cells were by transient transfection of MCF7 cells not In cell the concentrations of anti-estrogens used were sufficient to stimulation by that estrogen are fully by anti-estrogens the conditions used to for agonist activity not In agonist activity of anti-estrogens not be in transient transfection of ERE-containing reporter either in Ishikawa or in MCF7 The to significant transcriptional activation by OHT or anti-estrogens in transient transfections the that in vivo conditions required for agonist activity of OHT are not fully in we Ishikawa and MCF7 cell lines stably propagating the of cells by of in cell that for cell lines stably propagating be by the with estrogen of cells to a stimulation of No stimulation was observed with the pure anti-estrogens or In contrast, OHT and stimulated activity to and of the with and These results were with of cells by two of the the partial anti-estrogens OHT and the full anti-estrogens or stimulated more in that the stimulation by estrogen was in stably MCF7 cells in cell lines activity of OHT was and stimulation of activity be with concentrations as low as whereas or not induce of activity at of the concentrations that none of the anti-estrogens were capable of in a cell line Ishikawa cells by transfection of containing glucocorticoid response elements of S. 1994; PubMed Scopus Google Scholar), that the presence of is required for transcriptional activation by anti-estrogens not The of stimulation by estrogen and OHT for were by in to that the OHT stimulation were of with whereas not the These results are in with by of The agonist of OHT and when were performed for for with OHT or compared with treatment with full anti-estrogens or In to the that in the used for transient transfection and with cell lines be the of the in the results observed with partial anti-estrogens stimulation of activity in transient transfection stimulation in cell we in using Ishikawa and with or transfection of cells transfection with DNA stimulation of activity was in the transient transfection with the cell line and whereas of activity were and and the of stimulation with E2, observed with transient transfections performed in the presence of OHT or were with and observed with OHT or in cells also transfection of cells stimulation with estrogen and the stimulation by anti-estrogens but not in the results observed using the two to be at in to the of reporter of cell results suggest that anti-estrogens with partial agonist but not full anti-estrogens, can stimulate transcription by endogenous of estrogen bound to minimal ERE-containing promoters in Ishikawa activity is in transient transfection using the reporter vectors. have of AP1 activity by estrogen treatment in several cell lines (19Gaub M.P. Bellard M. Scheuer I. Chambon P. Sassone-Corsi P. Cell. 1990; 63: 1267-1276Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 20Philips A. Chalbos D. Rochefort H. J. Biol. Chem. 1993; 268: 14103-14108Abstract Full Text PDF PubMed Google Scholar, 21Umayahara Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Y. T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, P. R.M. Mol. Endocrinol. 1995; 9: PubMed Google Scholar) and by anti-estrogens in transient transfection of Ishikawa cells P. R.M. Mol. Endocrinol. 1995; 9: PubMed Google Scholar). To whether TRE motifs are sufficient to mediate stimulation by estrogen and anti-estrogens in Ishikawa we reporter containing a minimal of TRE motifs of a or a reporter containing the AP1-responsive rat stromelysin also S. S. M. C. Chambon P. EMBO J. 1990; 9: PubMed Scopus Google Scholar). of the reporter be observed in the presence of estrogen to in but not in the presence of anti-estrogens. and was in the presence of estrogen or anti-estrogens No stimulation of the minimal reporter constructs be observed of the cells with either estrogen or anti-estrogens When the was a activity was observed not and a stimulation by estrogen be in the but not in the presence of TPA to and Anti-estrogens on reporter and and experiments, we that stimulation of promoters containing TRE motifs by estrogen is on the we not observe transcriptional activation by anti-estrogens using we the of To the of anti-estrogens in stimulation of the AP1 cell lines were Ishikawa cells by transfection of by the cellular or of the or by for of the reporter to TPA stimulation by in to for was used to whether estrogen anti-estrogens can stimulate activity the conditions as used for the cell lines with with estradiol or anti-estrogens not in either in the presence or in the of TPA and results were with the not that estrogen stimulate reporter in that of of the AP1 in cell lines was not to of ER not of cells were for of the the cell line activity was stimulated by TPA in not of cells as well as of cells with estrogen or not to stimulation of activity whereas in the agonist activity be observed using the cells results were in the presence of TPA, that of activity were in the presence of TPA in the cells containing AP1-responsive promoters but not in the cell lines not the conditions used for in the containing may stimulation of AP1 activity by estrogen or anti-estrogens, we performed in the of activity was by estrogen and anti-estrogens in the cells but not in cells or in cells which containing two TPA response However, when concentrations of anti-estrogens were used of but of activity were observed with OHT or with and to but not with estrogen or with anti-estrogens results were observed using or cells not These results indicate that TRE elements with a can mediate transcriptional stimulation by concentrations of OHT in Ishikawa whereas estrogen response elements can be at concentrations by OHT and the the partial agonist activity of anti-estrogens are poorly (9Katzenellenbogen B.S. 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In addition, are also capable of transcriptional stimulation by anti-estrogens, in a that is restricted by the and the of the transient transfection has to be a for our that the conditions required for of transcription in We are to D. Philibert and to T. for the of and to P. Chambon for reporter and ER vectors. We also J. White for of the