The Relative Contribution Exerted by AF-1 and AF-2 Transactivation Functions in Estrogen Receptor α Transcriptional Activity Depends upon the Differentiation Stage of the Cell
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m402148200
ISSN1083-351X
AutoresYohann Mérot, Raphaël Métivier, Graziella Penot, Dominique Manu, Christian Saligaut, Frank Gannon, Farzad Pakdel, Olivier Kah, Gilles Flouriot,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoThe activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor α (ERα) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ERα AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ERα activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ERα transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ERα signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ERα in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ERα-positive cell lines tested strongly suggest that the transcriptional activity of ERα relies on its AF-1 in most estrogen target cell types. The activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor α (ERα) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ERα AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ERα activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ERα transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ERα signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ERα in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ERα-positive cell lines tested strongly suggest that the transcriptional activity of ERα relies on its AF-1 in most estrogen target cell types. A wide range of physiological processes, including female reproductive function, are controlled by 17β-estradiol (1Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1573) Google Scholar). Most of the biological actions exerted by this steroid hormone are mediated by a specific receptor, the estrogen receptor α (ERα) 1The abbreviations used are: ERα, estrogen receptor α; ERE, estrogen responsive element; TK, thymidine kinase; NR, nuclear receptor; AF, activation function; rtER, rainbow trout estrogen receptor; cVg, chicken vitellogenin; CMV, cytomegalovirus; E2, estradiol; 4-OHT, 4-hydroxytamoxifen; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase.1The abbreviations used are: ERα, estrogen receptor α; ERE, estrogen responsive element; TK, thymidine kinase; NR, nuclear receptor; AF, activation function; rtER, rainbow trout estrogen receptor; cVg, chicken vitellogenin; CMV, cytomegalovirus; E2, estradiol; 4-OHT, 4-hydroxytamoxifen; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase. (2Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar). ERα has a critical role in the control of the balance between cell proliferation and differentiation and is therefore intimately associated with the biology of endometrium and breast cancers (3Jordan V.C. Breast Cancer Res. Treat. 1995; 36: 267-285Crossref PubMed Scopus (136) Google Scholar). The biological effects of ERα often result from modifications in the pattern of expression of specific target genes. These transcriptional regulations are achieved through recruitment of ERα to the promoter region of the target gene, either directly through interaction with cognate DNA sequences (ERE or estrogen responsive elements), or through protein/protein interaction with other transcriptional factors (4Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 5Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2841) Google Scholar, 6Paech K. Webb P. Kuiper G.G. Nilsson S. Gustafsson J. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2061) Google Scholar).ERα is a ligand-inducible transcription factor that belongs to the nuclear receptor (NR) subfamily whose members include steroid, thyroid hormone, retinoic acid, and orphan receptors. Based on structural and functional similarities, the sequences of NRs were divided into six functional domains designated A to F (4Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 5Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2841) Google Scholar). The central well conserved cysteine-rich C domain constitutes the signature of the NR superfamily, and mediates DNA binding. Hormone lodges into a hydrophobic pocket located within the C-terminal E/F domains that constitute the ligand binding domain. Ligand-induced transcription involves the action of distinct transactivation functions (AFs), located in the N-terminal A/B (AF-1) and the C-terminal E/F (AF-2) domains (7Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (887) Google Scholar).The respective contribution that these AFs make toward the activity of the full-length ERα is both promoter- and cell-specific (7Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 8Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (660) Google Scholar, 9Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (608) Google Scholar, 10Metzger D. Ali S. Bornert J.M. Chambon P. J. Biol. Chem. 1995; 270: 9535-9542Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). For instance, a maximal transcriptional activity of ERα can require both AFs in some cells, but only a specific one in others. This suggests that ERα does not interact with the transcriptional machinery in an identical manner in all cells. Functional and physical links between ERα and the transcriptional machinery involves the sequential recruitment by ERα of a group of proteins, called coactivators, on the target promoter, on which they build large protein complexes (11McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1235) Google Scholar, 12Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Structurally, these recruitments occur after ligand binding has induced specific conformational changes within the protein. So far, two classes of NR coactivator complexes, directly interacting with AF-2 in a ligand-dependent manner, have been identified. The first is formed by CBP/p300, the p160 nuclear receptor coactivator family (SRC-1/TIF2/AIB1), an RNA coactivator (SRA), and probably other unknown components (11McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1235) Google Scholar, 12Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). This complex allows a strong decondensation of the chromatin, mainly because of the histone acetyltransferase activity of several of its components (13Bannister J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar, 14Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1054) Google Scholar). The second coactivator complex includes proteins of the SMCC/TRAP/DRIP/ARC/Mediator class, and allows the physical link between ERα and the general transcription apparatus, facilitating the activation of polymerase II (12Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar).A cell-type specific activity of both AFs was suggested to result from a specific expression of distinct coactivators. However, the majority of the coactivators are widely expressed in a similar amount in most cells (15McDonnell D.P. Norris J.D. Science. 2002; 296: 1642-1644Crossref PubMed Scopus (487) Google Scholar, 16Smith C.L. O'Malley B.W. Endocr. Rev. 2004; 25: 45-7115Crossref PubMed Scopus (796) Google Scholar). Furthermore, several of the coactivators primarily identified as AF-2 specific have now been shown to also interact with the N-terminal region of ERα and to mediate AF-1 activity (17Webb P. Nguyen P. Shinsako J. Anderson C. Feng W. Nguyen M.P. Chen D. Huang S.M. Subramanian S. McKinerney E. Katzenellenbogen B.S. Stallcup M.R. Kushner P.J. Mol. Endocrinol. 1998; 12: 1605-1618Crossref PubMed Scopus (0) Google Scholar, 18Onate S.A. Boonyaratanakornkit V. Spencer T.E. Tsai S.Y. Tsai M.J. Edwards D.P. O'Malley B.W. J. Biol. Chem. 1998; 273: 12101-12108Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 19Benecke A. Chambon P. Gronemeyer H. EMBO Rep. 2000; 1: 151-157Crossref PubMed Scopus (126) Google Scholar, 20Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Therefore, although considerable advances have been made in understanding the mechanisms allowing the receptor to modulate the transcription of a target gene, no clear scheme is emerging with regard to the differential sensitivity of cell types to AF-1 and AF-2.In the present study, we demonstrate that the relative contribution exerted by AF-1 and AF-2 on the transcriptional activity of ERα varies in a cell differentiation stage-dependent manner. Precisely, we show that the more a cell is differentiated, the more this cell mediates ERα signaling through its AF-1. In contrast, AF-2 becomes the dominant AF involved in the transcriptional activity of ERα in undifferentiated or de-differentiated cells. These results, together with data obtained in different ERα-positive cell lines strongly suggest that the transcriptional activity of ERα relies on its AF-1 in most estrogen target cell types.EXPERIMENTAL PROCEDURESPlasmids—The expression vectors used in this study were pSG5, pSG human (h)ERα (HEO), pSG hERα CF (21Green S. Kumar V. Theulaz I. Wahli W. Chambon P. EMBO J. 1988; 7: 3037-3044Crossref PubMed Scopus (173) Google Scholar, 22Flouriot G. Brand H. Denger S. Métivier R. Kos M. Reid G. Sonntag-Buck V. Gannon F. EMBO J. 2000; 19: 4688-4700Crossref PubMed Google Scholar), and pCMV-β-galactosidase (Promega). Four luciferase reporter plasmids with different estrogen-sensitive promoters were employed in the transfection experiments: an artificial promoter containing one ERE upstream of the TK promoter (ERE-TK-LUC) (22Flouriot G. Brand H. Denger S. Métivier R. Kos M. Reid G. Sonntag-Buck V. Gannon F. EMBO J. 2000; 19: 4688-4700Crossref PubMed Google Scholar), the promoter of the human complement C3 (C3-LUC) (23Métivier R. Penot G. Flouriot G. Pakdel F. Mol. Endocrinol. 2001; 15: 1953-1970PubMed Google Scholar), the rainbow trout estrogen receptor promoter (rtER-LUC) (24Lazennec G. Kern L. Valotaire Y. Salbert G. Mol. Cell. Biol. 1997; 17: 5053-5066Crossref PubMed Scopus (38) Google Scholar), and the chicken vitellogenin promoter (cVg-LUC) (25Griffin C. Flouriot G. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1999; 13: 1571-1587Crossref PubMed Google Scholar).Cell Culture—All cell lines (HeLa, HepG2, MCF7, T47D, BT20, MDA-MB 231, LNCaP, PC3, DU 145, TSU PR1, P19, Ishikawa, and αT3) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (Sigma), penicillin (100 units/ml), streptomycin (100 μg/ml), and amphotericin (25 μg/ml) (Sigma) at 37 °C in a 5% CO2 incubator. The MDA-MB 231 cell line stably transfected with a hERα expression vector (26Reid G. Hubner M.R. Métivier R. Brand H. Denger S. Manu D. Beaudouin J. Ellenberg J. Gannon F. Mol. Cell. 2003; 11: 695-707Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar) were grown in Dulbecco's modified Eagle's medium containing 5% charcoal-stripped serum, antibiotics (see above), and hygromycin (0.4 mg/ml). The differentiation of the pluripotent stem cell line P19 into neurons and glial cells was achieved by treating P19 aggregates with retinoic acid (1 μm) during 48 h (27Jones-Villeneuve E.M. Rudnicki M.A. Harris J.F. McBurney M.W. Mol. Cell. Biol. 1983; 3: 2271-2279Crossref PubMed Scopus (267) Google Scholar).Transient Transfection Experiments—Cell lines were transfected with FuGENE™ 6 as recommended by the manufacturer (Roche Diagnostics). One day before transfection, cells were plated in 24- or 6-well plates at a density previously determined as giving the best transfection efficiency. One hour prior to transfection, the medium was replaced with phenol red-free Dulbecco's modified Eagle's medium/F-12 containing 2.5% charcoal-stripped calf serum. Transfection was carried out with 250 ng of total DNA per well for the 24-well plates or with 1 μg of total DNA for the 6-well plates. Total DNA was composed by the expression vector (50 ng for the 24-wells plate or 200 ng for the 6-well plates), the reporter gene (100 or 400 ng, respectively), and the CMV-β-galactosidase internal control (100 or 400 ng, respectively). Following an overnight incubation with the transfection mixture, the cells were treated with 10 nm estradiol (E2), 2 μm 4-hydroxytamoxifen (4-OHT), or ethanol (control). After 36 h of transient transfection, cells were harvested and luciferase and β-galactosidase assays were performed as previously described (23Métivier R. Penot G. Flouriot G. Pakdel F. Mol. Endocrinol. 2001; 15: 1953-1970PubMed Google Scholar). The reporter gene activity was obtained after normalization of the luciferase activity with the β-galactosidase activity.Western Blot Analysis—The expression of estrogen receptor, E-cadherin, vimentin, Tau, as well as actin, was examined by Western blot analysis. Subconfluent cells from 10-cm diameter dishes were washed with phosphate-buffered saline and lysed in RIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0,1% SDS) containing a mixture of protease inhibitors (Roche Diagnostics). Following sonication and quantification, 20 μg of these whole cell extracts were denatured in Laemmli buffer at 95 °C for 5 min, resolved on a 10% SDS-PAGE, and electrotransferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were blocked in phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat milk powder, during 1.5 h at room temperature. The blots were then incubated with either the polyclonal anti-hERα HC20 (1:1000, TEBU), the monoclonal anti-E-cadherin SHE78-7 (1:1000, Zymed Laboratories), the polyclonal anti-vimentin C-20 (1:500, TEBU), the polyclonal anti-Tau C-17 (1:500, TEBU), or the monoclonal anti-β-actin AC-15 (1:5000, Sigma) in phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat milk powder for 1.5 h at room temperature. After three washings with phosphate-buffered saline, 0.1% Tween, the blots were incubated with peroxidase-conjugated goat anti-rabbit (1:5000, Pierce), peroxidase-conjugated horse anti-goat (1:5000, TEBU), or peroxidase-conjugated goat anti-mouse (1:5000, Pierce) for 1 h. Membrane-bound secondary antibodies were detected using the SuperSignal West Dura kit from Pierce according to the manufacturer's instructions.RESULTSDistinct Sensitivities of HepG2 and HeLa Cell Lines to ERα AF-1 and AF-2: A Result from Their Divergent Phenotype?—Both ERα AF-1 and AF-2 have been shown to exert their transcriptional activity in a cell-specific manner. Accordingly, cell contexts can be defined as AF-1 or AF-2 permissive, depending upon which AF is principally involved in ERα activity: cell lines can be either equally or exclusively sensitive to either AFs (7Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 8Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (660) Google Scholar, 9Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (608) Google Scholar, 22Flouriot G. Brand H. Denger S. Métivier R. Kos M. Reid G. Sonntag-Buck V. Gannon F. EMBO J. 2000; 19: 4688-4700Crossref PubMed Google Scholar, 23Métivier R. Penot G. Flouriot G. Pakdel F. Mol. Endocrinol. 2001; 15: 1953-1970PubMed Google Scholar, 28Norris J.D. Fan D. Kerner S.A. McDonnell D.P. Mol. Endocrinol. 1997; 11: 747-754Crossref PubMed Scopus (92) Google Scholar). Selection of an adequate cell line is therefore of primary importance when studying a specific AF of ERs. For instance, the hepatocarcinoma cell line HepG2 is frequently used to measure the AF-1 activity, whereas AF-2 function is mainly studied in HeLa cells that derive from a cervix carcinoma.We first hypothesized that a distinct cell phenotype may explain the so far misunderstood cell preference for one given AF. Indeed, HeLa and HepG2 cells exhibit very distinct growth rates and phenotypes, despite their common epithelial origin. The cell proliferation rate of HeLa cells is in fact ∼15-fold higher than for HepG2 cells, as demonstrated by a kinetic cell counting (Fig. 1A). Furthermore, contrasting to HeLa cells that exhibit a poorly differentiated phenotype associated with an elongated morphology and little cell-cell interactions, HepG2 cells appear more differentiated, establishing many cell contacts (data not shown). These observations are corroborated by the fact that HeLa cells do not express E-cadherin, a calcium-dependent cell-cell adhesion molecule considered as a marker for epithelial differentiation (29Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2913) Google Scholar), as assessed by Western blot using HeLa whole cell protein extracts (Fig. 1B). Furthermore, HeLa cells express vimentin, an intermediate filament protein whose expression is associated with increased invasive and metastatic potency (30Thompson E.W. Paik S. Brunner N. Sommers C.L. Zugmaier G. Clarke R. Shima T.B. Torri J. Donahue S. Lippman M.E. J. Cell. Physiol. 1992; 150: 534-544Crossref PubMed Scopus (484) Google Scholar) (Fig. 1B). This is in sharp contrast to HepG2 cells that produce a high level of E-cadherin but no vimentin (Fig. 1B).These results suggest a correlation between the distinct permissiveness of HepG2 and HeLa cells to either ERα AF-1 or AF-2 and the divergent phenotype of these cells. However, the different tissue origins of these cell lines may also account for the observed differences. We thus decided to verify whether the cell-dependent involvement of AF-1 or AF-2 in ERα activity is associated with the differentiation stage of cells from a similar tissue origin. However, this study was subordinated to the development of adequate test systems to define the relative cell sensitivity to either ERα transactivation functions.Design of Accurate Test Systems to Probe Distinct Cell Sensitivity to ERα AF-1 and AF-2—Two different approaches were selected to define the relative sensitivity of a cell line to AF-1 and AF-2. In the first system, cell contexts were evaluated in transient transfection experiments by comparing the transcriptional activity of full-length ERα with that of an ERα deleted from the A/B domain (ERα CF) and thus devoid of AF-1 activity. A similar activity of both receptors would define a strict AF-2 permissive cell context, whereas an AF-1 permissive cell context would be inferred from the observation of a transcriptional activity of full-length ERα but not ERα CF. The respective involvement of the AFs of ERα is also promoter-specific (7Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 8Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (660) Google Scholar, 9Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (608) Google Scholar, 10Metzger D. Ali S. Bornert J.M. Chambon P. J. Biol. Chem. 1995; 270: 9535-9542Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Therefore, the assessment of the permissiveness of different cell lines for AF-1 or AF-2 required the design of a reporter system having no intrinsic preference for a specific AF and whose response would therefore only reflect cellular variations in the relative contribution that both AF-1 and AF-2 make toward ERα transcriptional activity. From the different estrogen-sensitive promoters tested, a synthetic ERE-TK promoter and the promoters from the complement 3 (C3), rtERα, and cVg genes, the ERE-TK promoter only gave the expected responses (Fig. 2A). Indeed, on this promoter, whereas full-length and truncated ERα exhibit the same activity in the strictly AF-2 permissive HeLa cells, the truncated form is inactive in strictly AF-1 permissive HepG2 cells. Although to a lesser extent, the C3 promoter was also permissive to both AFs. Finally, as the ERα CF was transcriptionally inactive on the rtER and cVg promoters in both HeLa and HepG2 cell lines, these two promoters did not fulfill the criteria of selection for a promoter adequate to our aims. Western blots controlled similar expression of both full-length ERα and ERα CF proteins in the tested cells (Fig. 2B), confirming the relevance of the results. The ERE-TK promoter was therefore selected as a reporter system to probe the distinct cell sensitivity of the ERα AF-1 and AF-2.Fig. 2Design of accurate experimental approaches to define cell sensitivity to ERα AF-1 and AF-2.A, to select estrogen responsive systems assessing cell permissiveness to either ERα AF, we transiently transfected into AF-1 (HepG2) and AF-2 permissive (HeLa) cells several promoter constructs (100 ng of ERE-TK-LUC, 100 ng of C3-LUC, 400 ng of rtER-LUC, or 400 ng of cVg-LUC) together with 50 ng of the expression vectors pSG5, pSG hERα, or pSG hERα CF. 100 ng of CMV-β-galactosidase was used as internal control. Cells were treated for 36 h with 10 nm E2 or with ethanol (EtOH) (control). Quantified luciferase and β-galactosidase activities were normalized, and the values standardized to the reporter activity measured in the presence of pSG hERα without E2. B, Western blot analysis controlling the correct and similar expression of ERα and ERα CF in HeLa and HepG2 cells transiently transfected with the corresponding expression vectors. C, identification of cell sensitivity to either AFs through the ability of ERα CF to repress the AF-1 activity of the full-length ERα. HeLa and HepG2 cells were cotransfected with 100 ng of ERE-TK-LUC together with 50 ng (+) of pSG5, 200 ng (++) of pSG hERα CF, or 50 ng (+) of pSG hERα alone or with increasing amounts of pSG hERα CF (50 ng (+) and 200 ng (++)). 100 ng of CMV-β-galactosidase was used as internal control. Results are expressed as the percentage of the reporter gene activity measured in the presence of pSG hERα and E2. D, use of the agonistic activity of the 4-OHT to characterize cell permissiveness to AF-1. HeLa and HepG2 cells were cotransfected with 200 ng of C3-LUC, 100 ng of CMV-β-galactosidase, and 50 ng of pSG hERα. Cells were treated 36 h with EtOH (control), 10 nm E2, or 2 μm 4-OHT. Results are expressed as in panel A. All experiments were performed at least 3 times, and the values correspond to the average ± S.E. of the results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although this approach is particularly pertinent in defining the AF permissiveness of ERα negative cell lines, this test is not appropriate in cells endogenously expressing ERα, whose transcriptional activity is likely to mask that of the transfected receptor. Alternatively, the relative sensitivity of ERα positive cell lines to either AFs can be characterized through the ability of the ERα CF to repress the transcriptional activity of full-length ERα in cell contexts sensitive to AF-1 only (22Flouriot G. Brand H. Denger S. Métivier R. Kos M. Reid G. Sonntag-Buck V. Gannon F. EMBO J. 2000; 19: 4688-4700Crossref PubMed Google Scholar). Indeed, as shown in Fig. 2C, whereas an increased expression of ERα CF does not impact ERα transcriptional activity in HeLa cells, ERα CF becomes a potent inhibitor of the full-length form in HepG2 cells. This variant of the first approach was therefore used to estimate the sensitivity of ERα positive cell lines for either AFs.The second system, designed to confirm the relative sensitivity of a cell line to ERα AFs, relies on the partial estrogen agonistic activity of 4-OHT. Actually, the estrogenic activity of 4-OHT exclusively depends upon the AF-1 of ERα, and is therefore observed only in cells sensitive to this AF (8Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (660) Google Scholar). Transfection experiments carried out with the human complement C3 promoter, a well characterized 4-OHT-responsive promoter (31Norris J.D. Fan D. Wagner B.L. McDonnell D. Mol. Endocrinol. 1996; 10: 1605-1616Crossref PubMed Scopus (123) Google Scholar), confirmed that this molecule induces the reporter gene activity via the AF-1 of ERα only. Indeed, an OHT-induced response of the C3 promoter was observed in AF-1 permissive HepG2 cells but not in AF-2 permissive HeLa cells (Fig. 2D). We therefore used this test to further substantiate the AF-1 sensitivity of cells assayed by the first approach.Correlation between the Relative Activity of ERα AFs and the Differentiation Status of Cell Lines Deriving from a Common Tissue—To verify whether the differentiation status of a given cell line influences the respective activity of both ERα transactivation functions, four breast cancer cell lines exhibiting distinct differentiation phenotypes were tested for their ability to mediate ERα transactivation through AF-1 or AF-2. According to the literature, the selected cell lines can be classified from a more to a less differentiated phenotype as follows: MCF7, T47D, BT20, and MDA-MB 231 (30Thompson E.W. Paik S. Brunner N. Sommers C.L. Zugmaier G. Clarke R. Shima T.B. Torri J. Donahue S. Lippman M.E. J. Cell. Physiol. 1992; 150: 534-544Crossref PubMed Scopus (484) Google Scholar, 32Coradini D. Biffi A. Pellizzaro C. Pirronello E. Di Fronzo G. Tumor Biol. 1997; 18: 22-29Crossref PubMed Scopus (26) Google Scholar). We first wished to confirm these data by probing through Western blots the expression levels of two differentiation markers (ERα and E-cadherin), and one invasive and metastatic marker (vimentin) in these cells. Results of these experiments (Fig. 3A) confirm that MDA-MB 231 cells (ERα and E-cadherin negative; vimentin positive) and to a lesser extend BT20 cells (E-cadherin positive; ERα and vimentin negative) are less differentiated than MCF7 or T47D cells (ERα and E-cadherin positive; vimentin negative). Importantly, MCF7 and T47D cells are ERα positive, in contrast to BT20 and MDA-MB 231 cells.Fig. 3Correlation between the relative activity of ERα AFs and the differentiation status of MCF7, T47D, BT20, and MDA-MB 231 breast cancer cell lines.A, Western blot analysis probing the expression of endogenous ERα, E-cadherin, vimentin, and β-actin in the four indicated cell lines. B, the permissiveness of MCF7 and T47D ERα positive cell lines to either ERα AFs was assessed using the dominant negative property of ERα CF in AF-1-sensitive cells. Cells were transfected with 100 ng of ERE-TK-LUC together with 50 ng of pSG5 or pSG hERα in the absence or presence of 200 ng of hERα CF. In the ERα negative BT20 and MDA-MB 231 cell lines, the direct comparison of ERα and ERα CF transcriptional activities evaluated the sensitivity of these cells to either AFs. Cells were transfected using 100 ng of ERE-TK-LUC with 50 ng of pSG5, pSG hERα, or pSG hERα CF. In both ERα positive and negative cell lines, 100 ng of CMV-β-galactosidase was used as internal control. Cells were treated for 36 h with 10 nm E2 or with EtOH vehicle (control). Luciferase activities were normalized by β-galactosidase values, and the results are figured as the percentage of the reporter gene activity measured in the presence of pSG hERα and E2. C, agonistic act
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