Convergence of Progesterone with Growth Factor and Cytokine Signaling in Breast Cancer
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.31317
ISSN1083-351X
AutoresJennifer K. Richer, Carol A. Lange, Nicole Manning, Gareth I. Owen, Roger Powell, Kathryn B. Horwitz,
Tópico(s)Medicinal Plant Pharmacodynamics Research
ResumoSTATS (signal transducers and activators of transcription) are latent transcription factors activated in the cytoplasm by diverse cell surface signaling molecules. Like progesterone receptors (PR), Stat5a and 5b are required for normal mammary gland growth and differentiation. These two proteins are up-regulated during pregnancy, a period dominated by high levels of progesterone. We now show that progestin treatment of breast cancer cells regulates Stat5a and 5b, Stat3, and Stat1 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the association of PR and Stat5. Last, progesterone pretreatment enhances the phosphorylation of Stat5 on tyrosine 694 induced by epidermal growth factor. Functional data show that progestin pretreatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a β-casein promoter. Progesterone and epidermal growth factor synergize to control transcription from p21WAF1 and c-fospromoters. These data demonstrate the convergence of progesterone and growth factor/cytokine signaling pathways at multiple levels, and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and differentiative events in the mammary gland. STATS (signal transducers and activators of transcription) are latent transcription factors activated in the cytoplasm by diverse cell surface signaling molecules. Like progesterone receptors (PR), Stat5a and 5b are required for normal mammary gland growth and differentiation. These two proteins are up-regulated during pregnancy, a period dominated by high levels of progesterone. We now show that progestin treatment of breast cancer cells regulates Stat5a and 5b, Stat3, and Stat1 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the association of PR and Stat5. Last, progesterone pretreatment enhances the phosphorylation of Stat5 on tyrosine 694 induced by epidermal growth factor. Functional data show that progestin pretreatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a β-casein promoter. Progesterone and epidermal growth factor synergize to control transcription from p21WAF1 and c-fospromoters. These data demonstrate the convergence of progesterone and growth factor/cytokine signaling pathways at multiple levels, and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and differentiative events in the mammary gland. progesterone receptors signal transducers and activators of transcription Janus kinase prolactin epidermal growth factor epidermal growth factor receptor glucocorticoid receptor minimal essential medium phenylmethylsulfonyl fluoride progesterone response element glucocorticoid response element polyacrylamide gel electrophoresis chloramphenicol acetyltransferase 1,4-piperazinediethanesulfonic acid. Progesterone, acting through progesterone receptors (PR),1 is important in the control of breast cell proliferation and differentiation (1Clarke C.L. Sutherland R.L. Endocr. Rev. 1990; 11: 266-301Crossref PubMed Scopus (590) Google Scholar). Mice lacking PR exhibit incomplete mammary gland ductal branching and failure of lobulo-alveolar development (2Lydon J.P. DeMayo F.J. Funk C.R. Mani S.K. Hughes A.R. Montgomery Jr., C.A. Shyamala G. Conneely O.M. O'Malley B.W. Genes Dev. 1995; 9: 2266-2278Crossref PubMed Scopus (1504) Google Scholar). Interestingly, similar disruptions of mammary gland development and lactation are observed in mice upon deletion of several other genes including cyclin D1 (3Sicinski P. Donaher J.L. Parker S.G. 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 (890) Google Scholar), prolactin receptors (4Ormandy C.J. Camus A. Barra J. Damotte D.L., B. Buteau H. Edery M. Brousse N. Babinet C. Binart N. Kelly P.A. Genes Dev. 1997; 11: 167-178Crossref PubMed Scopus (632) Google Scholar), the activin/inhibin βB gene (5Vassalli A. Matzuk M.M. Gardner H.A.R. Lee K.F. Jaenisch R. Genes Dev. 1994; 8: 414-427Crossref PubMed Scopus (339) Google Scholar) and most recently, two members of the STAT (signal transducers and activators of transcription) family, Stat5a (6Liu X. Robinson G.W. Wagner K. Garrett L. Wunshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (912) Google Scholar) and Stat5b (7Udy G.B. Towers R.P. Snell R.G. Wilkens R.J. Park S.H.P. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7243Crossref PubMed Scopus (824) Google Scholar). It is evident that the products of these genes are involved in pathways that influence the mammary gland; however, it remains to be determined whether they function in distinct or convergent pathways. STAT family members are latent cytoplasmic transcription factors activated via diverse cell surface signaling molecules (8Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar). Although Stat5 is functional in many cell types, Stat5a-deficient mice are normal except for lack of proliferative mammary lobulo-alveolar outgrowth, as well as inability of females to lactate, thus demonstrating a mandatory and specific role for this STAT in both processes (6Liu X. Robinson G.W. Wagner K. Garrett L. Wunshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (912) Google Scholar). The role of Stat5a (originally identified as mammary gland factor in extracts from lactating mice) as a mediator of prolactin-induced transcription of milk protein genes during lactation is well documented (9Wakao H.F. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (713) Google Scholar). Stat5b-deficient mice show a phenotype mainly in sexual dimorphism of growth rates and liver gene expression; however, females also have impaired mammary gland development. Stat5b−/− females consistently abort their pups, a problem that can be overcome by progesterone administration during pregnancy (7Udy G.B. Towers R.P. Snell R.G. Wilkens R.J. Park S.H.P. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7243Crossref PubMed Scopus (824) Google Scholar). In relation to progesterone action, both Stat5a and 5b are particularly interesting since mRNA and protein levels rise during pregnancy (10Philp J.A.S. Burdon T.G. Watson C.J. FEBS Lett. 1996; 396: 77-80Crossref PubMed Scopus (96) Google Scholar), a period dominated by high levels of progesterone. STATs are known to be activated by the ligand-induced intrinsic tyrosine kinase activity of members of the growth factor receptor family or by cytokine receptors that lack intrinsic tyrosine kinase activity, but associate with soluble tyrosine kinases known as Janus kinases (JAKs), as reviewed in Refs. 11Leaman D.W. Leung S. Li X. Stark G.R. FASEB J. 1996; 10: 1578-1588Crossref PubMed Scopus (271) Google Scholar and 12Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Thierfelder W.E. Kreider B. Silvennoinen O. Trends Biochem. Sci. 1994; 19: 222-227Abstract Full Text PDF PubMed Scopus (596) Google Scholar. Phosphorylation of a tyrosine residue (tyrosine 694 in Stat5) conserved in all STAT family members induces their dimerization, which is followed by translocation into the nucleus, DNA binding, and regulation of numerous genes involved in growth and differentiation (8Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3356) Google Scholar). In the lactating mammary gland, the cytokine prolactin, acting via prolactin receptors and JAK2, induces phosphorylation of Stat5a and 5b, which then bind sites on the promoters of mammary-specific genes such as β-casein. In addition, growth factors such as epidermal growth factor (EGF) can also stimulate Stat5, as well as Stat1 and Stat3, which then bind to STAT sites on the promoters of growth regulatory genes such as p21WAF1(13Xie W. Su K. Wang D. Paterson A.J. Kudlow J. Anticancer Res. 1997; 17: 2627-2634PubMed Google Scholar, 14Ruff-Jamison S. Chen K. Cohen S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4215-4218Crossref PubMed Scopus (134) Google Scholar, 15Chin Y.E. Kitagawa M. Su W.C. You Z. Iwamoto Y. Fu X. Science. 1996; 272: 719-722Crossref PubMed Scopus (727) Google Scholar) and c-fos (16Sadowski H.B. Shuai K. Darnell J.E. Gilman M.Z. Science. 1993; 261: 1739-1743Crossref PubMed Scopus (640) Google Scholar). Despite conflicting in vivo and in vitro data regarding the proliferative versus growth inhibitory role of progesterone in the breast, the lack of mammary gland ductal branching and failure of lobulo-alveolar development observed in the PR-knockout mouse (2Lydon J.P. DeMayo F.J. Funk C.R. Mani S.K. Hughes A.R. Montgomery Jr., C.A. Shyamala G. Conneely O.M. O'Malley B.W. Genes Dev. 1995; 9: 2266-2278Crossref PubMed Scopus (1504) Google Scholar) demonstrates that PR must play a proliferative role during development. Interestingly, in vitro, progestins stimulate breast cancer cells to progress through one round of cell division accompanied by the induction of cyclin D1, p21WAF1, EGF, EGFR, c-myc, and c-fos. This is followed by growth arrest at the G1/S phase of the second cycle (17Musgrove E.A. Lee C.S. Sutherland R.L. Mol. Cell. Biol. 1991; 11: 5032-5043Crossref PubMed Google Scholar, 18Groshong S. Owen G.I. Grimison B. Schauer I.E. Todd M.C. Langan T.A. Sclafani R.A. Lange C.A. Horwitz K.B. Mol. Endocrinol. 1997; 11: 1593-1607Crossref PubMed Scopus (229) Google Scholar, 19Musgrove E.A. Lee C.S.L. Cornish A.L. Swarbrick A. Sutherland R.L. Mol. Endocrinol. 1997; 11: 54-66Crossref PubMed Scopus (61) Google Scholar). We have proposed that progesterone-arrested cells are poised to respond to secondary proliferative or differentiative signals ((18) and the accompanying paper (20Lange C.A. Richer J.K. Shen T. Horwitz K.B. J. Biol. Chem. 1998; 273: 31308-31316Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar)). There is considerable evidence that progesterone and EGF have complementary effects on the mammary gland. Like progestins and PR, EGF and EGFR are required in the proliferative phase of mammary gland development (21Coleman S. Silberstein G.B. Daniel C.W. Dev. Biol. 1988; 127: 304-315Crossref PubMed Scopus (182) Google Scholar, 22Edery M. Pang K. Larson L. Colosi T. Nandi S. Endocrinology. 1985; 117: 405-411Crossref PubMed Scopus (73) Google Scholar). Mice carrying a spontaneous mutation resulting in a critical amino acid substitution in the kinase domain of the EGFR have underdeveloped ductal trees and impaired lactation (23Fowler K.J. Walker F. Alexander W. Hibbs M.L. Nice E.C. Bohmer R.M. Mann G.B. Thumwood C. Maglitto R. Danks J.A. Chetty R. Burgess A.W. Dunn A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1465-1469Crossref PubMed Scopus (186) Google Scholar). Furthermore, progestin treatment: 1) up-regulates EGFR (17Musgrove E.A. Lee C.S. Sutherland R.L. Mol. Cell. Biol. 1991; 11: 5032-5043Crossref PubMed Google Scholar, 24Murphy L.C. Murphy L.J. Shiu R.P.C. Biochem. Biophys. Res. Commun. 1988; 150: 192-196Crossref PubMed Scopus (45) Google Scholar, 25Murphy L.J. Sutherland R.L. Stead B. Murphy L.C. Lazarus L. Cancer Res. 1986; 46: 728-734PubMed Google Scholar) and other type I growth factor receptors (20Lange C.A. Richer J.K. Shen T. Horwitz K.B. J. Biol. Chem. 1998; 273: 31308-31316Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) in breast cancer cell lines; 2) enhances the ability of EGF to induce proliferation of breast cancer cells (18Groshong S. Owen G.I. Grimison B. Schauer I.E. Todd M.C. Langan T.A. Sclafani R.A. Lange C.A. Horwitz K.B. Mol. Endocrinol. 1997; 11: 1593-1607Crossref PubMed Scopus (229) Google Scholar); and 3) potentiates EGF mediated signaling pathways (20Lange C.A. Richer J.K. Shen T. Horwitz K.B. J. Biol. Chem. 1998; 273: 31308-31316Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Since STAT proteins are downstream effectors of EGFR (13Xie W. Su K. Wang D. Paterson A.J. Kudlow J. Anticancer Res. 1997; 17: 2627-2634PubMed Google Scholar, 14Ruff-Jamison S. Chen K. Cohen S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4215-4218Crossref PubMed Scopus (134) Google Scholar, 15Chin Y.E. Kitagawa M. Su W.C. You Z. Iwamoto Y. Fu X. Science. 1996; 272: 719-722Crossref PubMed Scopus (727) Google Scholar, 16Sadowski H.B. Shuai K. Darnell J.E. Gilman M.Z. Science. 1993; 261: 1739-1743Crossref PubMed Scopus (640) Google Scholar) we postulated that cross-talk between progesterone and EGF occurs at the level of STATs. That steroid hormone and growth factor signaling pathways converge at STAT proteins is suggested by the recent report of a functional interaction between glucocorticoid receptors (GR) and Stat5 in which GR act as coactivators for Stat5-mediated induction of the β-casein promoter by glucocorticoids and prolactin (26Stocklin E. Wissler M. Gouilleux F. Groner B. Nature. 1996; 383: 726-728Crossref PubMed Scopus (571) Google Scholar). Conversely, Stat3 can act as a coactivator of GR-mediated transcription on a mouse mammary tumor virus promoter in the presence of interleukin-6 and dexamethasone (27Zhang Z. Jones S. Hagood J.S. Fuentes N.L. Fuller G.M. J. Biol. Chem. 1997; 272: 30607-30610Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). We now report that progestin treatment of breast cancer cells up-regulates Stat5 and Stat3 protein levels in a PR-dependent manner. In addition, progestin treatment induces translocation of Stat5 into the nucleus, possibly mediated by the physical association of PR and Stat5 proteins. Functional data show that progestin treatment of breast cancer cells enhances the ability of prolactin to stimulate the transcriptional activity of Stat5 on a β-casein promoter, and synergizes with EGF to control transcription of the p21WAF1 and c-fos promoters. These data demonstrate the regulation of a key growth factor signaling molecule by progesterone and suggest a mechanism for coordination of PR and Stat5-mediated proliferative and/or differentiative events in the mammary gland. The wild type PR-positive T47Dco breast cancer cell line and its clonal derivatives T47D-Y, T47D-YA, and T47D-YB, have been described (28Sartorius C.A. Groshong S.D. Miller L.A. Powell R.L. Tung L. Takimoto G.S. Horwitz K.B. Cancer Res. 1994; 54: 3668-3877PubMed Google Scholar). Cells are routinely cultured in 75-cm2 plastic flasks and incubated in 5% CO2at 37 °C in a humidified environment. The stock medium consists of Eagle's minimum essential medium with Earle's salts (MEM), containingl-glutamine (292 μg/liter) buffered with sodium bicarbonate (2.2 g/liter), insulin (6 ng/ml), and 5% fetal bovine serum (Hyclone, Logan, UT) without antibiotics. For routine subculturing, cells are diluted 1:20 into new flasks once per week, and medium replaced every 2–3 days. Cells are harvested by incubation in Hank's-EDTA for 10 min at 37 °C. For time course experiments, cells are plated at 1 million cells per plate in MEM with supplements described above and were treated with 10 nm progesterone (Sigma) or R5020 (NEN Life Science Products Inc., Boston, MA). Cells were harvested at specified time points in RIPA buffer (10 mm sodium phosphate, pH 7.0, 150 mm NaCl, 2 mm EDTA, 1% deoxycholic acid, 1% Nonidet P-40, 0.1% SDS, 0.1% β-mercaptoethanol, 1 mm PMSF, 50 mmsodium fluoride, 200 μm Na3VO4, and one Complete Protease Inhibitor Mixture tablet (Boehringer Mannheim, GmbH Germany) per 50 ml). EGF (Collaborative Biomedical Products, Bedford, MA) was used at 10 nm and prolactin (Sigma) at 5 μg/ml. Protein extracts were equalized to 100 μg by the Bradford assay (Bio-Rad), resolved by SDS-PAGE, and transferred to nitrocellulose. Equivalent protein loading was confirmed by Ponceau S staining. Following incubation with the appropriate antibodies, protein bands were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and quantitated using a Molecular Dynamics Series 300 Computing Densitometer and Molecular Dynamics ImageQuant Program. The following antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA: Stat5 C-17 (recognizes both Stat5a and 5b isoforms); Stat3 (C-20); JAK2 (HR-758); and p21 (C-18). Anti-phosphotyrosine (monoclonal 4G10), anti-human Stat5A, and anti-human Stat5B (specific for the Stat5a or 5b isoform), anti-phospho-Stat5 (specific for Tyr-694) and anti-human cdc2 kinase (PSTAIR) were purchased from Upstate Biotechnology, Lake Placid, NY. The anti-PR monoclonal antibody AB-52 was produced in our laboratories. Breast cancer cells stably expressing either the PR-A (T47D-YA) or PR-B (T47D-YB) isoform were treated for 12 or 24 h with 10 nm progesterone or ethanol vehicle. Total RNA was isolated by ultracentrifugation of guanidinium isothiocyanate lysates through a CsCl cushion (29Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1976; 18: 5294-5299Crossref Scopus (16648) Google Scholar). RNA (30 μg) from each treatment group was transferred to a Hybond nylon membrane (Amersham) and hybridized sequentially with the cDNA inserts described below labeled by random priming with [32P]dCTP using the Mega-Prime DNA Labeling Kit (Amersham). The membrane was stripped in boiling 0.1 × SSC, 0.1% SDS between hybridizations. A 2-kilobase insert was removed from a cDNA clone encoding fatty acid synthetase by restriction digest with EcoRI and HindIII followed by gel isolation and purification. The cyclin D1 cDNA was a 1.1-kilobase fragment removed from the vector by restriction digest with XbaI and HindIII. The Stat5a cDNA probe consisted of a 2.3-kilobase fragment cut from the vector by EcoRI digest. Last, a glyceraldehyde-3-phosphate dehydrogenase cDNA probe representing a non-regulated gene served as a control for RNA loading. Fatty acid synthetase clone pG8 (30Chalbos D. Westley B. May F. Alibert C. Rochefort H. Nucleic Acids Res. 1986; 14: 965-982Crossref PubMed Scopus (34) Google Scholar) was obtained from D. Chalbos, INSERM, Montpellier, France; the cyclin D1 cDNA in a modified pUC19 vector was obtained from A. Arnold, Massachusetts General Hospital, Boston, MA, via R. Sclafani, University of Colorado Health Sciences Center, Denver, CO; Stat5a cDNA in pcDNA3 vector was obtained from A. D'Andrea, Harvard Medical School, Boston, MA, via A. Kraft, University of Colorado Health Sciences Center, Denver, CO. Cells in 10-cm dishes were washed twice with ice-cold phosphate-buffered saline and lysed by scraping in extraction buffer (EB: 1% Triton X-100, 10 mm Tris-HCl (pH 7.4), 5 mm EDTA, 50 mm NaCl, 50 mm sodium fluoride, 2 mm Na3VO4, and 1 mmPMSF). Lysates were clarified by centrifugation for 10 min at maximum speed in a Savant (μSpeedfuge SFR13K) bench-top centrifuge and equal amounts of protein (1 mg/ml) were immunoprecipitated with the anti-phosphotyrosine monoclonal antibody 4G10 (4 μg/mg) by rotation at 4 °C for 2 h to overnight. Immuno complexes were captured by adding 30 μl of washed protein A ((insoluble formalin-fixed StaphA-derived Sorbin (Sigma)) that had been preincubated with rabbit anti-mouse antibody, incubated by constant rotation at 4 °C for an additional 2 h, then collected by centrifugation at 10,000 rpm for 3 min in a Savant bench-top centrifuge. Immunoprecipitates were washed twice in EB (1 ml), twice in PAN (10 mm PIPES (pH 7.0), 100 mm NaCl) containing 0.25% Nonidet P-40, and twice in PAN without Nonidet P-40. Washed pellets were resuspended in Laemmli sample buffer, boiled for 3 min, and analyzed by SDS-PAGE and immunoblotting. To facilitate immunoprecipitation studies, hPR1, the full-length PR-B cDNA cloned into the mammalian expression vector pSG5 from P. Chambon (Strasbourg, France) (31Kastner P. Krust A. Turcotte B. Stropp U. Tora L. Gronemeyer H. Chambon P. EMBO J. 1990; 9: 1603-1614Crossref PubMed Scopus (1323) Google Scholar), was modified to disrupt the stop codon and add a carboxyl-terminal flag epitope consisting of amino acids DYKDDDDK. To verify that the FLAG epitope-tagged PRB (PRB:f) was expressed and functional in vivo, HeLa cells were co-transfected with PRB:f and the PRE2-TATAtk-CAT reporter using calcium phosphate precipitation as described previously (32Sartorius C.A. Tung L. Takimoto G.S. Horwitz K.B. J. Biol. Chem. 1993; 268: 9262-9266Abstract Full Text PDF PubMed Google Scholar), and treated with the synthetic progestin R5020. The chloramphenicol acetyltransferase activity produced by the PRB:f was equal to that of wild-type hPR1 (data not shown). A stable cell line expressing PRB:f was established by co-transfecting HeLa cells with 4.5 μg of PRB:f plasmid and 0.5 μg of a plasmid encoding the neomycin resistance gene, pSV2neo. DNA precipitate was removed 18 h later, and cells were grown in MEM + 5% fetal bovine serum containing 700 μg/ml of the neomycin analog G418 (Life Technologies, Inc., Gaithersburg, MD) to kill non-transfected cells. Surviving neomycin-resistant colonies were expanded and cells were analyzed by immunoblotting and chloramphenicol acetyltransferase assay for clones expressing high levels of functional PRB:f. For co-immunoprecipitations, wild type HeLa and HeLa PRB:f were cultured in MEM supplemented with 5% twice charcoal-stripped heat-inactivated fetal bovine serum. Cells were treated with progesterone or R5020 (10 nm) for 1 h. Nuclear extracts were prepared according to the methods described in Ref. 33Dignam J.D. Lebovitz R.M. Roeder R. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar. After harvesting in Hanks'; EDTA, cells were washed in phosphate-buffered saline then resuspended in 5 packed cell volumes of buffer A (10 mm (pH 7.9) at 4 °C, 1.5 mmMgCl2, 10 mm KCl, and 0.5 mmdithiothreitol, 0.5 mm PMSF, one complete protease inhibitor mixture tablet (Boehringer Mannheim, GmbH Germany) per 50 ml of buffer) and allowed to stand at 4 °C for 10 min. The cells were collected by centrifugation at 2,000 rpm for 10 min, then resuspended in two packed cell volumes of buffer A and lysed by 10 strokes of a Kontes all glass Dounce homogenizer (B type pestle). The homogenate was centrifuged for 10 min at 2,000 rpm to pellet the nuclei. The supernatant was removed and the pellet subjected to a second centrifugation at 25,000 × g for 20 min (Beckman Optima Le-80K Ultracentrifuge, 70.1 Ti rotor) to remove residual cytoplasmic material. The pellet was resuspended in 3 ml of buffer C per 5 × 108 cells and re-homogenized as above. Buffer C consisted of 20 mm Hepes, 25% (v/v) glycerol, 0.42m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm PMSF, 0.5 mmdithiothreitol, and protease inhibitors. The homogenate was mixed by rotation for 30 min at 4 °C then centrifuged for 30 min at 25,000 × g. The resulting clear supernatant was dialyzed at 4 °C for 5 h to overnight in 20 mmHepes (pH 7.9), 20% glycerol, 0.1 m KCl, 0.2 mEDTA, 0.5 mm dithiothreitol, 0.5 mm PMSF, and protease inhibitors. One mg of nuclear extract was incubated with 100 μl of a 50% slurry of Anti-Flag M2 Affinity Gel (Eastman Kodak, New Haven, CT) at 4 °C for 4 h. The anti-Flag resin was then washed twice with TEDG containing 0.1 m NaCl, twice with TEDG containing 0.3 m KCl, and twice with TEDG containing 0.1m NaCl and 0.1% Nonidet P-40, using 1 ml of buffer for each wash. The PRB:f protein was then eluted by competition with 0.2 mg/ml Flag peptide (N-DYKDDDDK-C) (Eastman Kodak) in 200 μl of TEDG containing 0.3 m KCl and 0.1% Nonidet P-40, for 30 min at 4 °C. Laemmli sample buffer was added to the eluate, and proteins were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose, and identified by immunoblotting. PR positive breast cancer cells plated at 1 million cells per 10-cm dish in MEM supplemented with 5% fetal bovine serum were treated with 10 nm R5020 or ethanol vehicle for 48 h prior to transfection. Cells were then transiently transfected with 3 μg of β-casein −2300/+490 promoter in the luciferase reporter plasmid pGL2-E (provided by Paul A. Kelly, Molecular Endocrinology, INSERM, Paris, France), 3 μg of the β-galactosidase expression plasmid pCH110 (Amersham Pharmacia Biotech) to check transfection efficiency, and Bluescript carrier plasmid (Stratagene, La Jolla, CA) for a total of 20 μg of DNA using calcium phosphate precipitation as described previously (32Sartorius C.A. Tung L. Takimoto G.S. Horwitz K.B. J. Biol. Chem. 1993; 268: 9262-9266Abstract Full Text PDF PubMed Google Scholar). Three hours after transfection, the medium was aspirated and the cells were shocked with 2 ml of phosphate-buffered saline containing 20% glycerol. Cells were then washed twice with serum-free MEM to remove the glycerol and 10 ml of MEM containing 5% charcoal-stripped fetal bovine serum was added for 18 h. Cells were then treated with either 10 nm R5020 or 5 μg/ml prolactin in triplicate dishes, and harvested 24 h after hormone addition in 300 μl of lysis solution (Analytical Luminescence Laboratories, Ann Arbor, MI), and 100 μl of lysate was analyzed for luciferase activity using the Enhanced Luciferase Assay Kit and a Monolight 2010 Luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI) as described by the manufacturer. In experiments using the T47D-YB breast cancer cells to study the effects of progesterone plus EGF in combination on STAT site containing promoters p21WAF1 and c-fos, the transfection protocol was as described above except cells were transfected 24 h after plating and treated with ethanol vehicle, progesterone, EGF, or both hormones together 18 h after glycerol shocking. Cells were harvested 24 h after hormone treatment. The −2320-base pair p21 promoter construct, a gift of A. Kraft and J. Biggs, Division of Oncology, University of Colorado Health Sciences Center, Denver, CO, was cloned into a pA3-LUC vector as described previously (34Owen G.I. Richer J.K. Tung L. Takimoto G. Horwitz K.B. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). The c-fos promoter, c-fos-81TK-luc, containing two copies of the −357/−276 promoter linked to a minimal (81 base pairs) thymidine kinase promoter was obtained from A. Gutierrez-Hartmann, Division of Endocrinology, University of Colorado Health Sciences Center, Denver, CO (35Guthridge C.J. Eidlen D. Arend W.P. Gutierrez-Hartmann A. Smith M.F. Mol. Cell. Biol. 1997; 17: 1118-1128Crossref PubMed Scopus (15) Google Scholar), and was modified from c-fos-TK-luc (36Chen W.S. Lazar C.S. Poenie M. Tsien R.Y. Gill G.N. Rosenfeld M.G. Nature. 1987; 328: 820-823Crossref PubMed Scopus (411) Google Scholar). To examine the effects of progestins on STAT protein levels, PR-positive T47Dco breast cancer cells were treated with progesterone (not shown), 10 nm R5020, or ethanol vehicle (Fig. 1 A) and cells were harvested 8–72 h later. Total Stat5 protein present in whole cell lysates was detected using a polyclonal antibody that cross-reacts with both Stat5a and 5b. Stat5 protein levels were elevated by 8 h and remained elevated until 72 h after R5020 treatment with increases of 4–7-fold (Fig. 1 A). To test the PR dependence of this effect, the experiment was repeated using PR-negative T47D-Y cells (28Sartorius C.A. Groshong S.D. Miller L.A. Powell R.L. Tung L. Takimoto G.S. Horwitz K.B. Cancer Res. 1994; 54: 3668-3877PubMed Google Scholar) (Fig. 1 B). In the absence of PR, R5020 did not up-regulate Stat5. The two isoforms of Stat5, Stat5a and Stat5b, share 96% similarity at the protein level (Fig. 2). Aside from the non-conserved 5′- and 3′-untranslated regions, the main difference between the two isoforms is in the COOH terminus. The last 8 amino acids of the two isoforms are completely divergent, and Stat5a is 7 amino acids longer than Stat5b (37Liu X. Robinson G.W. Gouilleux F. Groner B. Henninghausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (456) Google Scholar). To determine which of the two isoforms are regulated by PR, T47Dco cells were treated with or without R5020 for 8–60 h. Protein blots of whole cell lysates were probed with antibodies that recognize epitopes unique to Stat5a or Stat5b, as well as with an antibody that recognizes both isoforms (Fig. 2 A). This study shows that both the longer (95 kDa) Stat5a and the shorter (92 kDa) Stat5b isoform are up-regulated by progestin treatment. Stat5a was below detectable levels in the absence of R5020 (Fig. 2 A). Note that a nonspecific (NS) protein migrating just above the 95-kDa Stat5a is recognized by Stat5a antibody, but is not regulated by R5020 and serves as a loading control. Similar studies using anti-Stat3 (Fig. 2 B) and anti-Stat1 (Fig. 2 C) antibodies show that these STAT family members are also progestin regulated. While Stat3 is clearly up-regulated by progestin treatment, Stat1 is slightly down-regulated. The same blot, after probing with anti-Stat1 antibody, was stripped and reprobed with antibody recognizing total Stat5, which showed that Stat5 was again strongly up-regulated as in Figs. 1 and 2 A (data not shown). To determine whether progesterone, via PR, up-regulates Stat5a mRNA levels, total RNA was isolated from two T47Dco breast cancer cell lines stably expressing either the PR-A or PR-B isoform (28Sartorius C.A. Groshong S.D. Miller L.A. Powell R.L. Tung L. Takimoto G.S. Horwitz K.B. Cancer Res. 1994; 54: 3668-3877PubMed Google Scholar) that had been treated with progesterone or vehicle for 12 or 24 h (Fig. 3). Northern blot analysis demonstrates hormone-dependent induction of Stat5a message at 12 and 24 h following progestin treatment (Fig. 3). Interestingly, Stat5a appears to be more strongly induced by the PR-B than the PR-A isoform (compare lanes 2 and 4; 6 and 8). In contrast, the same Northern blot hybridized
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