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• W2076234060 abstract "A major unanswered question of glucocorticoid and progesterone action is how different whole cell responses arise when both of the cognate receptors can bind to, and activate, the same hormone response elements. We have documented previously that the EC50 of agonist complexes, and the partial agonist activity of antagonist complexes, of both glucocorticoid receptors (GRs) and progesterone receptors (PRs) are modulated by increased amounts of homologous receptor and of coregulators. We now ask whether these components can differentially alter GR and PR transcriptional properties. To remove possible cell-specific differences, we have examined both receptors in the common environment of a line of mouse mammary adenocarcinoma (1470.2) cells. In order to segregate the responses that might be due to unequal nucleosome reorganization by the two receptors from those reflecting interactions with other components, we chose a transiently transfected reporter containing a simple glucocorticoid response element (i.e. GREtkLUC). No significant differences are found with elevated levels of either receptor. However, major, qualitative differences are seen with the corepressors SMRT and NCoR, which afford opposite responses with GR and PR. Studies with chimeric GR/PR receptors indicate that no one segment of PR or GR is responsible for these properties and that the composite response likely involves interactions with both the amino and carboxyl termini of receptors. Collectively, the data suggest that GR and PR induction of responsive genes in a given cell can be differentially controlled, in part, by unequal interactions of multiple receptor domains with assorted nuclear cofactors. A major unanswered question of glucocorticoid and progesterone action is how different whole cell responses arise when both of the cognate receptors can bind to, and activate, the same hormone response elements. We have documented previously that the EC50 of agonist complexes, and the partial agonist activity of antagonist complexes, of both glucocorticoid receptors (GRs) and progesterone receptors (PRs) are modulated by increased amounts of homologous receptor and of coregulators. We now ask whether these components can differentially alter GR and PR transcriptional properties. To remove possible cell-specific differences, we have examined both receptors in the common environment of a line of mouse mammary adenocarcinoma (1470.2) cells. In order to segregate the responses that might be due to unequal nucleosome reorganization by the two receptors from those reflecting interactions with other components, we chose a transiently transfected reporter containing a simple glucocorticoid response element (i.e. GREtkLUC). No significant differences are found with elevated levels of either receptor. However, major, qualitative differences are seen with the corepressors SMRT and NCoR, which afford opposite responses with GR and PR. Studies with chimeric GR/PR receptors indicate that no one segment of PR or GR is responsible for these properties and that the composite response likely involves interactions with both the amino and carboxyl termini of receptors. Collectively, the data suggest that GR and PR induction of responsive genes in a given cell can be differentially controlled, in part, by unequal interactions of multiple receptor domains with assorted nuclear cofactors. hormone response elements dexamethasone glucocorticoid receptors progesterone receptors androgen receptor estrogen receptor ligand binding domain DNA binding domain dexamethasone mesylate glucocorticoid response element dexamethasone oxetanone mouse mammary tumor virus Among the longstanding conundrums of steroid hormone action is why different whole cell responses are observed for androgen, glucocorticoid, mineralocorticoid, and progestin steroid hormones (1Truss M. Beato M. Endocr. Rev. 1993; 14: 459-479Crossref PubMed Scopus (588) Google Scholar) even though each receptor-steroid complex can bind to the same DNA sequences to induce gene transcription (2Roche P.J. Hoare S.A. Parker M.G. Mol. Endocrinol. 1992; 6: 2229-2235Crossref PubMed Scopus (184) Google Scholar, 3Lieberman B.A. Bona B.J. Edwards D.P. Nordeen S.K. Mol. Endocrinol. 1993; 7: 515-527Crossref PubMed Scopus (84) Google Scholar). Steroid hormone-regulated gene transactivation requires ligand binding to the cognate intracellular receptor. After binding to biologically active DNA sequences, called hormone response elements (HREs),1 the activated complexes are thought to recruit transcriptional coregulators and components of the transcriptional complex prior to modifying the transcription rates of target genes. Different ligands bind to the various steroid receptors with different affinities. However, this specificity for the different steroid hormones would seem to soon disappear as the activated form of each complex of the above four steroid receptors can bind to, and activate transcription from, the same HREs. The actions of glucocorticoid receptors (GRs) and progesterone receptors (PRs) have been extensively studied in an effort to understand how biological diversity can be maintained when the activated receptor complexes act on a common HRE. Several explanations have been proposed, including different levels of the receptor within a given cell (4Strahle U. Boshart M. Klock G. Stewart F. Schutz G. Nature. 1989; 339: 629-632Crossref PubMed Scopus (100) Google Scholar). HRE mutations have also been reported to affect differentially GR versus PR transactivation (5Ricousse S.L. Gouilleux F. Fortin D. Joulin V. Richard-Foy H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5072-5077Crossref PubMed Scopus (22) Google Scholar), although the differences seem to be less pronounced when receptor concentrations are about equal (6Thackray V.G. Lieberman B.A. Nordeen S.K. J. Steroid Biochem. Mol. Biol. 1998; 66: 171-178Crossref PubMed Scopus (16) Google Scholar). Nelson et al. (7Nelson C.C. Hendy S.C. Shukin R.J. Cheng H. Bruchovsky N. Koop B.F. Rennie P.S. Mol. Endocrinol. 1999; 13: 2090-2107Crossref PubMed Google Scholar) found that the flanking and spacer DNA of the palindromic HRE can contribute to the affinity and specificity of receptor binding. Similarly, DNA binding specificity has been implicated in the androgen receptor (AR)versus GR-specific induction of the probasin gene (8Schoenmakers E. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. J. Biol. Chem. 2000; 275: 12290-12297Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and in GR versus AR activation of the aspartate aminotransferase gene (9Massaad C. Garlatti M. Wilson E.M. Cadepond F. Barouki R. Biochem. J. 2000; 350: 123-129Crossref PubMed Google Scholar). Thus, subtle differences in HRE sequence may regulate the relative activities of GR and PR. Chromatin architecture has recently emerged as another promising modifier for receptor-regulated expression of some, but not all (10Archer T.K. Lee H.-L. Cordingley M.G. Mymryk J.S. Fragoso G. Berard D. Hager G.L. Mol. Endocrinol. 1994; 8: 568-576Crossref PubMed Scopus (85) Google Scholar), genes. Chromatin structure can repress gene expression (11Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 12Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6623) Google Scholar), thereby increasing the fold induction by receptor-steroid complexes, especially in cell-free systems (13Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar, 14Liu Z. Wong J. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9485-9490Crossref PubMed Scopus (66) Google Scholar). Chromatin environment can control gene inducibility by PR (15Smith C.L. Archer T.K. Hamlin-Green G. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11202-11206Crossref PubMed Scopus (38) Google Scholar, 16Archer T.K. Zaniewski E. Moyer M.L. Nordeen S.K. Mol. Endocrinol. 1994; 8: 1154-1162Crossref PubMed Scopus (67) Google Scholar, 17Lambert J.R. Nordeen S.K. J. Biol. Chem. 1998; 273: 32708-32714Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and chromatin structure has been proposed to be a determinant for PR induction (10Archer T.K. Lee H.-L. Cordingley M.G. Mymryk J.S. Fragoso G. Berard D. Hager G.L. Mol. Endocrinol. 1994; 8: 568-576Crossref PubMed Scopus (85) Google Scholar, 15Smith C.L. Archer T.K. Hamlin-Green G. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11202-11206Crossref PubMed Scopus (38) Google Scholar). However, alterations in chromatin structure do not appear to be a prerequisite for all steroid receptor-induced gene transactivation. In several cases, chromatin reorganization appears to precede the actions of receptor-steroid complexes in inducing gene expression (14Liu Z. Wong J. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9485-9490Crossref PubMed Scopus (66) Google Scholar, 18Fryer C.J. Archer T.K. Nature. 1998; 393: 88-91Crossref PubMed Scopus (411) Google Scholar, 19Wong J. Patterton D. Imhof A. Guschin D. Shi Y.-B. Wolffe A.P. EMBO J. 1998; 17: 520-534Crossref PubMed Scopus (140) Google Scholar, 20Kinyamu H.K. Fryer C.J. Horwitz K.B. Archer T.K. J. Biol. Chem. 2000; 275: 20061-20068Abstract Full Text Full Text PDF PubMed Google Scholar), whereas in other cases chromatin disruption or remodeling is not sufficient for transactivation, which requires a second step (21Mymryk J.S. Archer T.K. Genes Dev. 1995; 9: 1366-1376Crossref PubMed Scopus (43) Google Scholar). In T47D cells lacking both PR and GR, or expressing only GR, the responsive B nucleosome of the MMTV enhancer is in a constitutively “open” state, indicating that GR transcriptional activation is independent of chromatin remodeling (20Kinyamu H.K. Fryer C.J. Horwitz K.B. Archer T.K. J. Biol. Chem. 2000; 275: 20061-20068Abstract Full Text Full Text PDF PubMed Google Scholar). The low homology of the amino-terminal halves of GR and PR (<15%) has been advanced as an additional possible cause for selective gene expression (22Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6326) Google Scholar). One mechanism by which this could be achieved is through differential interactions with the recently discovered transcriptional coregulators such as coactivators and corepressors. Although the initially determined interactions of these coregulators were with the ligand binding domain (LBD) of receptors (23McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1655) Google Scholar, 24Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 25Robyr D. Wolffe A.P. Wahli W. Mol. Endocrinol. 2000; 14: 329-347Crossref PubMed Scopus (325) Google Scholar), several recent reports (26Lavinsky R.M. Jepsen K. Heinzel T. Torchia J. Mullen T.-M. Schiff R. Del-Rio A.L. Ricote M. Ngo S. Gemsch J. Hilsenbeck S.G. Osborne C.K. Glass C.K. Rosenfeld M.G. Rose D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2920-2925Crossref PubMed Scopus (583) Google Scholar, 27Webb 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, 28Alen P. Claessens F. Verhoeven G. Rombauts W. Peeters B. Mol. Cell. Biol. 1999; 19: 6085-6097Crossref PubMed Scopus (217) Google Scholar, 29Hittelman A.B. Burakov D. Iniguez-Lluhi J.A. Freedman L.P. Garabedian M.J. EMBO J. 1999; 18: 5380-5388Crossref PubMed Scopus (243) Google Scholar, 30Lanz R.B. McKenna N.J. Onate S.A. Albrecht U. Wong J. Tsai S.Y. Tsai M.-J. O'Malley B.W. Cell. 1999; 97: 17-27Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 31Yang Z. Hong S.-H. Privalsky M.L. J. Biol. Chem. 1999; 274: 37131-37138Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) describe interactions with the amino-terminal domain of receptors. Unfortunately, receptor-specific interactions with coregulators appear limited. ARA70 (compare Ref. 32Yeh S. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5517-5521Crossref PubMed Scopus (530) Google Scholar with Refs. 33Alen P. Claessens F. Schoenmakers E. Swinnen J.V. Verhoeven G. Rombauts W. Peeters B. Mol. Endocrinol. 1999; 13: 117-128Crossref PubMed Scopus (116) Google Scholar and 34Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar) and FHL2 (35Muller J.M. Isele U. Metzger E. Rempel A. Moser M. Pscherer A. Breyer T. Holubarsch C. Buettner R. Schule R. EMBO J. 2000; 19: 359-369Crossref PubMed Scopus (288) Google Scholar) have been described to be specific coactivators for ARs, whereas a 68-kDa protein (p68) appears to be a coactivator for estrogen receptor (ER) α but not for ERβ, AR, mineralocorticoid receptors, or retinoic acid receptor α (36Endoh H. Maruyama K. Masuhiro Y. Kobayashi Y. Goto M. Tai H. Yanagisawa J. Metzger D. Hashimoto S. Kato S. Mol. Cell. Biol. 1999; 19: 5363-5372Crossref PubMed Google Scholar). Repressor of estrogen receptor activity selectively interacts with ERs to decrease both the activity of agonists and the concentration of antiestrogens required for half-maximal inhibition of estrogens (37Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (246) Google Scholar). NRIF3 is a ligand-dependent specific coactivator that binds (and augments transactivation of) thyroid and retinoid receptors but not retinoic acid, vitamin D, or steroid receptors (38Li D. Desai-Yajnik V. Lo E. Schapira M. Abagyan R. Samuels H.H. Mol. Cell. Biol. 1999; 19: 7191-7202Crossref PubMed Scopus (54) Google Scholar). More evidence exists for differential effects of coactivators and corepressors on GR versus PR activities. The nuclear orphan receptor estrogen receptor-related receptor 2 is described to be a repressor of GR transcriptional activity but does not affect PR activity (39Trapp T. Holsboer F. J. Biol. Chem. 1996; 271: 9879-9882Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Conversely, PIAS1 enhances GR transactivation but represses PR transactivation in the same system (40Tan J.-a. Hall S.H. Hamil K.G. Grossman G. Petrusz P. Liao J. Shuai K. French F.S. Mol. Endocrinol. 2000; 14: 14-26Crossref PubMed Scopus (86) Google Scholar). A 130-kDa auxiliary protein increases the DNA binding of full-length GR but not truncated PRs (41Kupfer S.R. Marschke K.B. Wilson E.M. French F.S. J. Biol. Chem. 1993; 268: 17519-17527Abstract Full Text PDF PubMed Google Scholar). Similar quantitative differences have been noted for other steroid receptors. SRC-1 has a greater effect on ER than AR action in the brain of developing rats (42Auger A.P. Tetel M.J. McCarthy M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7551-7555Crossref PubMed Scopus (163) Google Scholar), which may result from SRC-1 interacting with the ER LBD via the LXXLL motifs of SRC-1 but with the amino-terminal region of AR in a manner that does not depend on the SRC-1 LXXLL motifs (34Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar). SRC-1e interacts with the fragments containing the DNA binding domain and LBD of ER but not of AR (28Alen P. Claessens F. Verhoeven G. Rombauts W. Peeters B. Mol. Cell. Biol. 1999; 19: 6085-6097Crossref PubMed Scopus (217) Google Scholar). RIP140 represses GR (43Subramaniam N. Treuter E. Okret S. J. Biol. Chem. 1999; 274: 18121-18127Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) but increases AR (44Ikonen T. Palvimo J.J. Janne O.A. J. Biol. Chem. 1997; 272: 29821-29828Crossref PubMed Scopus (311) Google Scholar) transactivation. Whether Zac1 augments or represses the activity of GRIP1 with AR versus ER depends on the target gene and cell (45Huang S.-M. Stallcup M.R. Mol. Cell. Biol. 2000; 20: 1855-1867Crossref PubMed Scopus (130) Google Scholar). Recently we have found that varying concentrations of the homologous receptor, coactivators, and corepressors can alter the EC50of agonist complexes, and the partial agonist activity of antagonist complexes, for both GRs (46Szapary D. Xu M. Simons Jr., S.S. J. Biol. Chem. 1996; 271: 30576-30582Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 47Szapary D. Huang Y. Simons Jr., S.S. Mol. Endocrinol. 1999; 13: 2108-2121Crossref PubMed Scopus (116) Google Scholar, 48Chen S. Sarlis N.J. Simons Jr., S.S. J. Biol. Chem. 2000; 275: 30106-30117Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and PRs (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). Preliminary evidence suggested that the responses of GR and PR to these factors might be different (47Szapary D. Huang Y. Simons Jr., S.S. Mol. Endocrinol. 1999; 13: 2108-2121Crossref PubMed Scopus (116) Google Scholar, 48Chen S. Sarlis N.J. Simons Jr., S.S. J. Biol. Chem. 2000; 275: 30106-30117Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). Therefore, the purpose of this study was to examine whether the quantitative activities of those factors known to alter PR and GR transactivation properties are the same or different for PR and GR. To answer this question, we have performed multiple dose-response curves to determine the EC50 of agonists and the partial agonist activity of antagonists. It was important to conduct these assays in the same cells so that cell-specific contributions to transcriptional activities could be eliminated. Similarly, we needed to use the same reporter so that effects of chromatin organization would be minimized. In the context of such an assay system, we found two instances in which the induction properties of PR and GR were qualitatively different, with almost opposite effects being produced by the same added component. Further studies with PR/GR chimeras indicated that no one segment of PR or GR was responsible for these differences. Collectively, the data suggest that the differences between GR and PR induction in a selected cell can be controlled, in part, by unequal responses from the combination of amino- and carboxyl-terminal domains of each receptor to assorted nuclear components. Unless otherwise indicated, all operations were performed at 0 °C. [3H]Dexamethasone (Dex, 91 Ci/mmol) was obtained from PerkinElmer Life Sciences and non-radioactive Dex from Sigma. Dex-Ox (50Pons M. Simons Jr., S.S. J. Org. Chem. 1981; 46: 3262-3264Crossref Scopus (33) Google Scholar) and Dex-Mes (51Simons Jr., S.S. Pons M. Johnson D.F. J. Org. Chem. 1980; 45: 3084-3088Crossref Scopus (95) Google Scholar) were prepared as described. Restriction enzymes and digestions were performed according to the manufacturer's specifications (New England Biolabs, Beverly, MA). The Renilla null luciferase reporter was purchased fromPromega (Madison, WI). GREtkLUC (52Sarlis N.J. Bayly S.F. Szapary D. Simons Jr., S.S. J. Steroid Biochem. Mol. Biol. 1999; 68: 89-102Crossref PubMed Scopus (28) Google Scholar) has been described previously. The cDNA plasmids of GR (pSVLGR from Keith Yamamoto, University of California, San Francisco), MMTVLUC (pLTRLUC from Gordon Hager, National Institutes of Health, Bethesda), TIF2, and the B form of human progesterone receptor (hPR-B) (Hinrich Gronemeyer, IGBMC, Strasbourg, France), NCoR (Michael Rosenfeld, University of California, San Diego), and s-SMRT (53Ordentlich P. Downes M. Xie W. Genin A. Spinner N.B. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2639-2644Crossref PubMed Scopus (140) Google Scholar) (Ron Evans, Salk Institute, La Jolla, CA) were each received as gifts. The cDNA encoding GR or PR was recombined through a compatible site (NspI/SphI) that coincides with the position of cysteine 495 of the rat GR. No amino acids were added or subtracted or changed at this junction. Both expression vectors for the chimeric receptors start with the unrelated sequence ASGSWP, which is due to the thymidine kinase promoter AUG followed by a BamHI linker. The PR/GR chimera bears a PR, which is lacking the first 24 amino acids of PR. This deletion has no observable influence on the transactivation capacity of the PR. 2B. Huse, unpublished data. The GR/PR chimera starts with a rat GR that misses 3 amino acids of the amino terminus. Various experiments since this construct was first employed (54Severne Y. Wieland S. Schaffner W. Rusconi S. EMBO J. 1988; 7: 2503-2508Crossref PubMed Scopus (134) Google Scholar) have confirmed that there is no substantial difference between this GR and the wild type GR. 3S. Rusconi, unpublished data. Monolayer cultures of COS-7 and 1470.2 cells were grown at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc., and Dulbecco's modified Eagle's medium with 4.5 g glucose/liter, Quality Biologicals, Inc., respectively) supplemented with 5 and 10% of fetal bovine serum, respectively. We had previously used charcoal-stripped serum with 1470.2 cells to prevent any PR-mediated induction by endogenous progestins (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). However, we have confirmed the observations of others (55Smith C.L. Wolford R.G. O'Neill T.B. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar) that this is not necessary (data not shown). Therefore, charcoal-stripped serum was no longer used with 1470.2 cells. CV-1 cells were grown as described (46Szapary D. Xu M. Simons Jr., S.S. J. Biol. Chem. 1996; 271: 30576-30582Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Coregulator plasmids were transiently cotransfected into 1470.2 cells using LipofectAMINE (LIfe Technologies, Inc.) with hPR-B receptor-containing plasmid, 1 μg of GREtkLUC, and 50 ng Renilla null luciferase, with the total transfected DNA brought up to 3 μg/60-mm dish with pBSK+ DNA (56Kaul S. Blackford Jr., J.A. Chen J. Ogryzko V.V. Simons Jr., S.S. Mol. Endocrinol. 2000; 14: 1010-1027Crossref PubMed Scopus (45) Google Scholar). In experiments with varying amounts of receptor or coregulator cDNA plasmids, equimolar amounts of the same plasmid vector were cotransfected to control for artifacts of the vector DNA. The cells were treated for 24 h with 1% ethanol ± steroids in media containing 10% fetal bovine serum and harvested in 1× Passive Lysis Buffer (0.5 ml/dish, Promega). Fifty μl of the cell lysates were used to assay for luciferase activity using the Dual-luciferase Assay System from Promega (Madison, WI) according to the supplier. The data were then normalized for the cotransfected Renilla activity. Transient transfection of COS-7 cells with 1 μg/10-cm plate of GR or PR/GR plasmid DNA and 19 μg of single-stranded DNA was performed as described (57Szapary D. Oshima H. Simons Jr., S.S. Mol. Endocrinol. 1993; 7: 941-952PubMed Google Scholar). Cytosols of transfected cells containing the steroid-free receptors were obtained by the lysis of cells on dry ice and centrifugation at 15,000 ×g (58Simons Jr., S.S. Miller P.A. Biochemistry. 1984; 23: 6876-6882Crossref PubMed Scopus (26) Google Scholar). Thirty percent cytosol with 20 mm sodium molybdate was added to varying concentrations of [3H]Dex ±100-fold excess of non-radioactive Dex and incubated at 0 °C for 18 h. Unbound [3H]Dex was removed by dextran-coated charcoal. Whole cell steroid binding was performed by incubating suspensions of cells (1.5–2 × 106) with increasing concentrations of [3H]Dex (1.5 to 50 nm) in 200 μl of serum-free medium in the presence or absence of a 100-fold molar excess of unlabeled Dex (each with 1.2% ethanol) for 30–45 min at 37 °C. The binding was terminated by the addition of 2 ml of phosphate-buffered saline, followed by centrifugation for 15 s, all at room temperature. Cells were washed three more times with phosphate-buffered saline at room temperature. In both cases, the total binding was determined by liquid scintillation counting. The specific binding was calculated by subtracting the background disintegrations/min (100-fold Dex) from the total [3H]Dex binding. The binding capacity and affinity were determined by Scatchard plot analysis by plotting the ratio of bound steroid/free steroidversus bound steroid. The activity for subsaturating concentrations of agonist, or saturating concentrations of antagonist, was expressed as percent of maximal activity with saturating concentrations of agonist (30 nm R5020 or 1 μm Dex unless otherwise noted). The fold induction was calculated as the luciferase activity (relative firefly light units/relative Renilla light units) with 30 nmR5020, or 1 μm Dex, divided by the basal activity obtained with ethanol. Individual values were generally within ±20% of the average, which was plotted. The dose-response curves were constructed from the theoretical sigmoidal curve for the binding isotherm, which is described by the equation of y = x/(x + k), wherey is the fractional response; x is the concentration of free steroid, and k is an arbitrary value for the binding affinity of steroid to receptor. This theoretical curve was then aligned with the experimental data so as to give the best visual fit. Unless otherwise noted, all statistical analyses were by two-tailed Student's t test using the program “InStat 2.03” for Macintosh (GraphPad Software, San Diego, CA). A bioassay with transiently transfected receptors and reporters to analyze possible differences in the biological properties of PR and GR was chosen for several reasons. First, bioassays measure the cumulative effect of the proceeding steps in the induction of protein synthesis. Bioassays are also often more sensitive than other assays, like DNA binding (8Schoenmakers E. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. J. Biol. Chem. 2000; 275: 12290-12297Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Furthermore, not allin vitro interactions are sufficiently strong to elicit an effect in whole cell bioassays (59Chen D. Huang S.M. Stallcup M.R. J. Biol. Chem. 2000; 275: 40810-40816Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). A transiently transfected template, in which nucleosome reorganization does not occur (60Archer T.K. Lefebvre P. Wolford R.G. Hager G.L. Science. 1992; 255: 1573-1576Crossref PubMed Scopus (350) Google Scholar), was used in order to minimize the possible complications of differential chromosomal reorganization by receptors. The ideal cells for this study would lack both GR and PR but would display dynamic, induction responses over a range of transiently transfected receptors. CV-1 cells lack both GR and PR and respond well to transfected GR (46Szapary D. Xu M. Simons Jr., S.S. J. Biol. Chem. 1996; 271: 30576-30582Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, in our hands, CV-1 cells give a low fold induction with transfected PR over a very narrow range of transfected receptors (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). 4D. Szapary and S. S. Simons, Jr., unpublished results. The 1470.2 mouse mammary adenocarcinoma cells do contain some GR but possess excellent properties regarding gene induction by transfected human PR-B (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). As both transiently and stably transfected PR induce transactivation with transiently transfected MMTVLUC reporters (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar, 55Smith C.L. Wolford R.G. O'Neill T.B. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar,61Smith C.L. Htun H. Wolford R.G. Hager G.L. J. Biol. Chem. 1997; 272: 14227-14235Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the use of transiently transfected PR should not pose a problem. PR is limiting for transactivation and capable of displaying increasing levels of gene induction over a range of transiently transfected receptors. PR also responds to a variety of coactivators and corepressors in these cells (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). Increasing concentrations of transiently transfected PR produce a progressive left shift in the dose-response curve to lower EC50 values for R5020 induction of transiently transfected MMTVLUC reporters in 1470.2 cells (49Giannoukos G. Szapary D. Smith C.L. Meeker J.E.W. Simons Jr., S.S. Mol. Endocrinol. 2001; 15: 255-270Crossref PubMed Scopus (54) Google Scholar). Steroid hormone induction of MMTV is complicated, due to the binding of NF1 and Oct1 to the MMTV promoter (62Lee H.-L. Archer T.K. Mol. Cell. Biol. 1994; 14: 32-41Crossref PubMed Google Scholar). In order to avoid these additional complications, we elected to use the simpler GREtkLUC reporter, which does not contain cis-acting binding sequences for other transcription factors. We first determined that higher concentrations of hPR-B plasmid afford increased total reporter activity, indicating that PR is limiting in this range (Fig.1 A). Under these conditions, the dose-response curve (or EC50) for R5020 induction of the transi" @default.
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• W2076234060 title "Transactivation Specificity of Glucocorticoid VersusProgesterone Receptors" @default.
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