Estrogen Inhibits Glucocorticoid Action via Protein Phosphatase 5 (PP5)-mediated Glucocorticoid Receptor Dephosphorylation
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.021469
ISSN1083-351X
AutoresYong Zhang, Donald Y.M. Leung, Steven K. Nordeen, Elena Goleva,
Tópico(s)14-3-3 protein interactions
ResumoAlthough glucocorticoids suppress proliferation of many cell types and are used in the treatment of certain cancers, trials of glucocorticoid therapy in breast cancer have been a disappointment. Another suggestion that estrogens may affect glucocorticoid action is that the course of some inflammatory diseases tends to be more severe and less responsive to corticosteroid treatment in females. To date, the molecular mechanism of cross-talk between estrogens and glucocorticoids is poorly understood. Here we show that, in both MCF-7 and T47D breast cancer cells, estrogen inhibits glucocorticoid induction of the MKP-1 (mitogen-activated protein kinase phosphatase-1) and serum/glucocorticoid-regulated kinase genes. Estrogen did not affect glucocorticoid-induced glucocorticoid receptor (GR) nuclear translocation but reduced ligand-induced GR phosphorylation at Ser-211, which is associated with the active form of GR. We show that estrogen increases expression of protein phosphatase 5 (PP5), which mediates the dephosphorylation of GR at Ser-211. Gene knockdown of PP5 abolished the estrogen-mediated suppression of GR phosphorylation and induction of MKP-1 and serum/glucocorticoid-regulated kinase. More importantly, after PP5 knockdown estrogen-promoted cell proliferation was significantly suppressed by glucocorticoids. This study demonstrates cross-talk between estrogen-induced PP5 and GR action. It also reveals that PP5 inhibition may antagonize estrogen-promoted events in response to corticosteroid therapy. Although glucocorticoids suppress proliferation of many cell types and are used in the treatment of certain cancers, trials of glucocorticoid therapy in breast cancer have been a disappointment. Another suggestion that estrogens may affect glucocorticoid action is that the course of some inflammatory diseases tends to be more severe and less responsive to corticosteroid treatment in females. To date, the molecular mechanism of cross-talk between estrogens and glucocorticoids is poorly understood. Here we show that, in both MCF-7 and T47D breast cancer cells, estrogen inhibits glucocorticoid induction of the MKP-1 (mitogen-activated protein kinase phosphatase-1) and serum/glucocorticoid-regulated kinase genes. Estrogen did not affect glucocorticoid-induced glucocorticoid receptor (GR) nuclear translocation but reduced ligand-induced GR phosphorylation at Ser-211, which is associated with the active form of GR. We show that estrogen increases expression of protein phosphatase 5 (PP5), which mediates the dephosphorylation of GR at Ser-211. Gene knockdown of PP5 abolished the estrogen-mediated suppression of GR phosphorylation and induction of MKP-1 and serum/glucocorticoid-regulated kinase. More importantly, after PP5 knockdown estrogen-promoted cell proliferation was significantly suppressed by glucocorticoids. This study demonstrates cross-talk between estrogen-induced PP5 and GR action. It also reveals that PP5 inhibition may antagonize estrogen-promoted events in response to corticosteroid therapy. Breast cancer is a leading cause of cancer mortality among women. In 2004, 186,772 women were diagnosed with breast cancer and 40,954 women died from breast cancer in United States (1.United States Cancer Statistics Working GroupUnited States Cancer Statistics. Centers for Disease Control and Prevention, Atlanta, GA2007: 1-684Google Scholar). The female hormone, estrogen, promotes breast cancer cell growth via the estrogen receptor (ER), 2The abbreviations used are: ERestrogen receptorMAPKmitogen-activated protein kinaseJNKc-Jun N-terminal kinaseERKextracellular signal-regulated kinaseGRglucocorticoid receptorGREglucocorticoid-responsive elementshRNAshort hairpin RNATBPTATA-binding proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPBSphosphate-buffered salineDEXdexamethasoneE217β-estradiolPPprotein phosphataseSGKserum/glucocorticoid-regulated kinaseCDKcyclin-dependent kinaseMEKmitogen-activated protein kinase kinasesiRNAsmall interfering RNA. which is expressed in ∼60% of breast cancers (2.McGuire W.L. Recent Prog. Horm. Res. 1980; 36: 135-156PubMed Google Scholar). Another consequence of estrogen is suggested by observations that the course of some allergic, autoimmune, and malignant diseases is more severe and less responsive to corticosteroid treatment in females (3.Tantisira K.G. Colvin R. Tonascia J. Strunk R.C. Weiss S.T. Fuhlbrigge A.L. Childhood Asthma Management Program Research Group.Am. J. Respir. Crit. Care Med. 2008; 178: 325-331Crossref PubMed Scopus (116) Google Scholar, 4.Fagan J.K. Scheff P.A. Hryhorczuk D. Ramakrishnan V. Ross M. Persky V. Ann. Allergy Asthma Immunol. 2001; 86: 177-184Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 5.Rohleder N. Schommer N.C. Hellhammer D.H. Engel R. Kirschbaum C. Psychosom. Med. 2001; 63: 966-972Crossref PubMed Scopus (226) Google Scholar), implicating a role for estrogen in glucocorticoid resistance. estrogen receptor mitogen-activated protein kinase c-Jun N-terminal kinase extracellular signal-regulated kinase glucocorticoid receptor glucocorticoid-responsive element short hairpin RNA TATA-binding protein 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol phosphate-buffered saline dexamethasone 17β-estradiol protein phosphatase serum/glucocorticoid-regulated kinase cyclin-dependent kinase mitogen-activated protein kinase kinase small interfering RNA. There are two forms of ER, ERα and ERβ, that reside in the cell membrane, cytoplasm, and nucleus (6.Levin E.R. J. Appl. Physiol. 2001; 91: 1860-1867Crossref PubMed Scopus (253) Google Scholar, 7.Kushner P.J. Agard D. Feng W.J. Lopez G. Schiau A. Uht R. Webb P. Greene G. Novartis Found. Symp. 2000; 230: 20-40Crossref PubMed Google Scholar). Nuclear ER regulates gene transcription by binding to DNA directly at estrogen-response elements or indirectly through interactions with transcriptional factors (7.Kushner P.J. Agard D. Feng W.J. Lopez G. Schiau A. Uht R. Webb P. Greene G. Novartis Found. Symp. 2000; 230: 20-40Crossref PubMed Google Scholar). Membrane-bound ER participates in cell signal transduction by activating G protein subunits and subsequently augments downstream kinase activities, such as p38 and ERK, in endothelial and breast cancer cells (8.Razandi M. Pedram A. Levin E.R. J. Biol. Chem. 2000; 275: 38540-38546Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9.Razandi M. Pedram A. Merchenthaler I. Greene G.L. Levin E.R. Mol. Endocrinol. 2004; 18: 2854-2865Crossref PubMed Scopus (291) Google Scholar). Binding to estrogen causes a conformational change in the ER that promotes the assembly of an active transcription complex at estrogen-induced genes such as c-myc and cyclin D1, which mediate the promotion of cell proliferation (10.Hermeking H. Rago C. Schuhmacher M. Li Q. Barrett J.F. Obaya A.J. O'Connell B.C. Mateyak M.K. Tam W. Kohlhuber F. Dang C.V. Sedivy J.M. Eick D. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 2229-2234Crossref PubMed Scopus (387) Google Scholar, 11.Altucci L. Addeo R. Cicatiello L. Dauvois S. Parker M.G. Truss M. Beato M. Sica V. Bresciani F. Weisz A. Oncogene. 1996; 12: 2315-2324PubMed Google Scholar). Glucocorticoids are well known for their anti-inflammatory, immunosuppressive, and anti-proliferative actions (12.Barnes P.J. Clin. Sci. 1998; 94: 557-572Crossref PubMed Scopus (1230) Google Scholar, 13.Mouhieddine O.B. Cazals V. Kuto E. Le Bouc Y. Clement A. Endocrinology. 1996; 137: 287-295Crossref PubMed Scopus (47) Google Scholar, 14.Ito K. Chung K.F. Adcock I.M. J. Allergy Clin. Immunol. 2006; 117: 522-543Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). They bind to the glucocorticoid receptor (GR) and regulate gene expression by mechanisms similar to ER. However, direct binding to DNA is accomplished through distinct DNA sequence motifs or glucocorticoid-responsive elements (GRE) to regulate the expression of specific genes, such as mitogen-activated protein kinase phosphatase-1 (MKP-1) and serum/glucocorticoid-regulated kinase (SGK) (15.Beato M. Herrlich P. Schütz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar, 16.Jenkins B.D. Pullen C.B. Darimont B.D. Trends Endocrinol. Metab. 2001; 12: 122-126Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17.Kassel O. Sancono A. Krätzschmar J. Kreft B. Stassen M. Cato A.C. EMBO J. 2001; 20: 7108-7116Crossref PubMed Scopus (407) Google Scholar, 18.Itani O.A. Liu K.Z. Cornish K.L. Campbell J.R. Thomas C.P. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E971-E979Crossref PubMed Scopus (9) Google Scholar). Three pathways have been reported to affect GR phosphorylation and activity. First, MAPK family members p38, JNK, and ERK regulate GR activity differentially. Activation of JNK and ERK inhibits GR transcriptional enhancement, and inhibition of JNK and ERK by inhibitors enhances GR function (19.Wang X. Wu H. Lakdawala V.S. Hu F. Hanson N.D. Miller A.H. Neuropsychopharmacology. 2005; 30: 242-249Crossref PubMed Scopus (48) Google Scholar, 20.Li L.B. Goleva E. Hall C.F. Ou L.S. Leung D.Y. J. Allergy Clin. Immunol. 2004; 114: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21.Rogatsky I. Logan S.K. Garabedian M.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 2050-2055Crossref PubMed Scopus (251) Google Scholar). The role of p38 in modulation of GR activity remains controversial (22.Miller A.L. Webb M.S. Copik A.J. Wang Y. Johnson B.H. Kumar R. Thompson E.B. Mol. Endocrinol. 2005; 19: 1569-1583Crossref PubMed Scopus (184) Google Scholar, 23.Wang X. Wu H. Miller A.H. Mol. Psychiatry. 2004; 9: 65-75Crossref PubMed Scopus (121) Google Scholar). Second, cyclin-dependent kinases (CDK) phosphorylate GR and regulate its activity. CDK2 phosphorylate rat GR at Ser-224 and Ser-232 (24.Wang Z. Garabedian M.J. J. Biol. Chem. 2003; 278: 50897-50901Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 25.Krstic M.D. Rogatsky I. Yamamoto K.R. Garabedian M.J. Mol. Cell. Biol. 1997; 17: 3947-3954Crossref PubMed Scopus (229) Google Scholar), and CDK5 suppresses GR transcriptional activity by attenuating binding of transcriptional cofactors to glucocorticoid-responsive promoters (26.Kino T. Ichijo T. Amin N.D. Kesavapany S. Wang Y. Kim N. Rao S. Player A. Zheng Y.L. Garabedian M.J. Kawasaki E. Pant H.C. Chrousos G.P. Mol. Endocrinol. 2007; 21: 1552-1568Crossref PubMed Scopus (116) Google Scholar). Third, serine/threonine protein phosphatases (PP) negatively regulate GR phosphorylation. Inhibition of PP1, PP2A, PP2B, and PP5 by protein phosphatase inhibitors okadaic acid and calyculin A potentiates GR activity and increases GR phosphorylation (27.Wang Z. Chen W. Kono E. Dang T. Garabedian M.J. Mol. Endocrinol. 2007; 21: 625-634Crossref PubMed Scopus (90) Google Scholar, 28.Nordeen S.K. Moyer M.L. Bona B.J. Endocrinology. 1994; 134: 1723-1732Crossref PubMed Scopus (86) Google Scholar). Unlike PP1 and PP2A, PP5 acts predominantly in protein complexes because the N-terminal domain of PP5 folds over the catalytic site blocking access to substrates in the absence of other proteins (29.Swingle M.R. Honkanen R.E. Ciszak E.M. J. Biol. Chem. 2004; 279: 33992-33999Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 30.Yang J. Roe S.M. Cliff M.J. Williams M.A. Ladbury J.E. Cohen P.T. Barford D. EMBO J. 2005; 24: 1-10Crossref PubMed Scopus (164) Google Scholar). PP5 has been identified in complexes containing GR and heat shock protein 90 (hsp90) (31.Silverstein A.M. Galigniana M.D. Chen M.S. Owens-Grillo J.K. Chinkers M. Pratt W.B. J. Biol. Chem. 1997; 272: 16224-16230Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 32.Chen M.S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), suggesting that PP5 may regulate GR activity. Glucocorticoids have been used in breast cancer therapy to antagonize the growth-promoting effect of estrogen. Nonetheless, clinical trials of glucocorticoid monotherapy in breast cancer have shown only a modest response (33.Keith B.D. BMC Cancer. 2008; 8: 84Crossref PubMed Scopus (85) Google Scholar). In advanced breast cancer meta-analyses, the addition of glucocorticoids to either chemotherapy or other endocrine therapy has resulted in increased response rates, but not increased survival (33.Keith B.D. BMC Cancer. 2008; 8: 84Crossref PubMed Scopus (85) Google Scholar, 34.Ludwig Breast Cancer Study GroupCancer Res. 1985; 45: 4454-4459PubMed Google Scholar). To date, the mechanism of glucocorticoid resistance in breast cancer has not been elucidated but would be important to understand if estrogen-driven corticosteroid resistance is to be circumvented. In this study, we investigated the three GR-regulating pathways discussed above, and we identified PP5 to be involved in the inhibition of GR activity by estrogen providing a novel mechanism of cross-talk between estrogen and glucocorticoids. 17β-Estradiol (E2), dexamethasone (DEX), ICI 182,780, nonimmune rabbit serum, and monoclonal anti-β-actin antibody were purchased from Sigma. PD98059 and roscovitine were purchased from Calbiochem. Purified mouse anti-glucocorticoid receptor antibody was purchased from BD Biosciences. Rabbit polyclonal antibody to glucocorticoid receptor, rabbit polyclonal antibody to phospho-glucocorticoid receptor (Ser-226) antibody, PP5 antibody, and mouse monoclonal antibody to TATA-binding protein (TBP) were purchased from Abcam Inc. (Cambridge, MA). Phospho-glucocorticoid receptor (Ser-211) antibody was purchased from Cell Signaling (Danvers, MA). Rabbit IgG was purchased from Southern Biotechnology Association, Inc. (Birmingham, AL). Normal mouse IgG1 and protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). FuGENE 6 transfection reagent was purchased from Roche Applied Science. NE-PER nuclear and cytoplasmic extraction reagents were purchased from Pierce. SureSilencing shRNA plasmids were purchased from SuperArray Bioscience Corp. (Frederick, MD). ON-TARGETplus SMARTpool siRNA against PP5 and ON-TARGETplus nontargeting pool siRNA were purchased from Dharmacon (Lafayette, CO). CellQuanti-MTTTM cell viability assay kit was purchased from BioAssay Systems (Hayward, CA). SuperBlock was purchased from Skytec (Logan, UT). Nonimmune donkey serum was purchased from Jackson ImmunoResearch (West Grove, PA). Anti-mouse or anti-rabbit horseradish peroxidase-labeled IgG was purchased from Amersham Biosciences. Chemiluminescent reagent was purchased from PerkinElmer Life Sciences. MCF-7, T47D, and MDA-MB-231 cell lines were purchased from American Type Culture Collection. For routine proliferation, MCF-7 and MDA-MB-231 cell lines were cultured in minimum Eagle's medium; T47D was cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum, 50 μg/ml streptomycin, and 50 units/ml penicillin. Cells were cultured in hormone-free medium (phenol red-free minimum Eagle's medium containing 2.5% charcoal-stripped serum) at least 2 days before they were treated with 10 nm E2 and 100 nm DEX for the time length as indicated below. An equal volume of ethanol was used as vehicle control. 6 × 103 MCF-7 cells were plated in flat bottom 96-well plates and cultured in hormone-free medium. Two days later, 10 nm E2 was added. The following day, 100 nm DEX was added alone or in combination with E2, and the cells were allowed to grow for an additional 2 days. In experiments that examined the effect of PP5 knockdown on MCF-7 cell line proliferation, the cells were transfected with 0.05 μg of SureSilencing shRNA plasmid per well 24 h prior E2 treatment. The number of viable cells was determined with CellQuanti-MTTTM cell viability assay kit according to the manufacturer's instructions. 105 cells per well were cultured in hormone-free medium in 24-well plates and treated with hormones and inhibitors as indicated. Total RNA was prepared using RNeasy mini kit (Qiagen, Valencia, CA). After reverse transcription, 500 ng of cDNA from each sample were analyzed by real time PCR using the dual-labeled fluorigenic probe method on an ABI Prism 7300 real time PCR system (Applied Biosystems). All primers were purchased from Applied Biosystems (Foster City, CA). The ΔΔCt method was utilized to calculate the relative change in target gene expression as an approximation of transcription based on the change in threshold values for control versus treated cells (the cycle number at which the fluorescent signaling crosses the "threshold" or logarithmic increases in cDNA concentration). This method assumes that both reference gene (internal control, i.e. β-actin used in this study) and target genes have similar amplification efficiencies. GR nuclear translocation and its phosphorylation in response to DEX was analyzed according to Ref. 20.Li L.B. Goleva E. Hall C.F. Ou L.S. Leung D.Y. J. Allergy Clin. Immunol. 2004; 114: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar with modifications. In brief, 105 MCF-7 cells were cultured on 18-mm round coverslips in 12-well plates. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized 15 min in Permeabilization Buffer (PBS containing 0.1% Tween 20, 0.1% bovine serum albumin, 0.01% saponin), and blocked at 37 °C for 1 h in Blocking Buffer (2.25% bovine serum albumin, 45% SuperBlock, 10% nonimmune donkey serum). Total GR and phospho-GR (Ser-211) antibodies were diluted 1:50 in Permeabilization Buffer and incubated with the cells at 4 °C overnight. Corresponding amounts of mouse IgG1 and rabbit IgG were used as negative controls, respectively. The cells were washed in PBS containing 0.1% Tween 20 for 20 min, followed by incubation with Cy3-conjugated secondary antibody (donkey anti-mouse or donkey anti-rabbit, diluted 1:500 in Permeabilization Buffer containing 300 nm 4′,6-diamidino-2-phenylindole) at room temperature for 1 h. Cells were washed again and mounted on slides. All slides were analyzed by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) with the imaging software Slidebook (Intelligent Imaging Innovations, Denver, CO). Mean fluorescence intensity in the cell nuclei defined by 4′,6-diamidino-2-phenylindole staining was assessed. Fifty to 100 cells were analyzed per slide. Protein samples were resolved on 4–12% BisTris gel (Invitrogen) and transferred to polyvinylidene difluoride membranes. The membranes were incubated in PBS containing specific antibodies, 5% dry milk, and 0.05% Tween 20 at 4 °C overnight. Subsequently, membranes were washed in PBS, 0.05% Tween 20 and incubated for 1 h at room temperature with anti-mouse or anti-rabbit horseradish peroxidase-labeled IgG (1:10,000), washed, incubated with chemiluminescent reagent, and processed for autoradiography. 105 MCF-7 cells were plated in each well of a 24-well plate and cultured in hormone-free medium. 24 h later, cells were transfected with 100 nm siRNA (or 0.1 μg of shRNA) in 1 ml of medium containing 1 μl of FuGENE 6 transfection reagent. Corresponding amounts of control siRNA or shRNA plasmids were used. GR binding to GRE was assessed by chromatin immunoprecipitation assay as described previously (35.Zhang Y. Wang J.S. Chen L.L. Zhang Y. Cheng X.K. Heng F.Y. Wu N.H. Shen Y.F. J. Biol. Chem. 2004; 279: 42545-42551Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) with modifications. Briefly, 2.5 × 106 cells were used in each precipitation. After sonication, chromatin solution was pre-cleared with 60 μl of protein A/G PLUS-agarose beads and 20 μl of nonspecific serum, followed by precipitation with 60 μl of protein A/G PLUS-agarose beads and specific antibody. Precipitated chromatin complexes were removed from the beads through incubation at 65 °C for 30 min with 550 μl of Elution Buffer (50 mm Tris, pH 8.0, 1 mm EDTA, 1% SDS). 500 μl of eluates were mixed with 25 μl of 5 m NaCl, 1 μl of RNase A (10 mg/ml, DNase-free) and incubated at 65 °C overnight. Samples were then digested with proteinase K, and DNA was purified with QIAquick columns (Qiagen, Valencia, CA) as indicated by the manufacturer, except that the sample was first mixed with PBI buffer (supplied by the manufacturer) for 30 min with agitation (36.Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar). Precipitated DNA was quantified by quantitative real time PCR using SYBR green (Applied Biosystems). Primers used to amplify GRE in SGK gene promoter were as follows: 5′-CTTGTTACCTCCTCACGTG-3′ (forward); 5′-GTCGTCTCTGCACTAAAGG-3′ (reverse). Results are expressed as the mean ± S.E. Statistical analysis was conducted using GraphPad Prism, version 5 (GraphPad Software, La Jolla, CA). Responses within an experiment were expressed as fold change over the control setting. These data were analyzed by the paired Student's t test, pairing by experiment. Before testing, paired difference distributions were examined for outliers, which can indicate violation to the normality assumption of the t test. No outliers were apparent. Tests were performed only for specific pre-planned treatment comparisons. Differences were considered significant at p < 0.05. A minimum of three independent experiments were conducted to allow for statistical comparisons. In this study, we chose the MCF-7 breast adenocarcinoma cell line as a model for our experiments. This cell line is known to proliferate in response to estrogen stimulation (37.Urban G. Golden T. Aragon I.V. Scammell J.G. Dean N.M. Honkanen R.E. J. Biol. Chem. 2001; 276: 27638-27646Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). To study whether glucocorticoids can inhibit estrogen-mediated cell growth, a proliferation assay was carried out. MCF-7 cells were first cultured in hormone-free medium for 2 days to deplete hormones in the cells and then pretreated with 10 nm E2 (the only form of estrogen used in this study) or an equal volume of vehicle (ethanol) for 24 h followed by 100 nm DEX treatment. The purpose of pretreating the cells with estrogen was to mimic the in vivo state, because the breast cancer cells are under estrogen influence before glucocorticoid treatment. Estrogen promoted cell growth by 2.09 ± 0.06-fold as compared with mock control. No change in cell proliferation was noted when cells were cultured in the presence of both estrogen and DEX (1.97 ± 0.04-fold as compared with mock control), indicating that estrogen-promoted cell growth was not inhibited by glucocorticoid treatment. To further explore how estrogen affects the action of glucocorticoids, we assessed the effect of estrogen on DEX induction of MKP-1 and SGK, two well known glucocorticoid-responsive genes (17.Kassel O. Sancono A. Krätzschmar J. Kreft B. Stassen M. Cato A.C. EMBO J. 2001; 20: 7108-7116Crossref PubMed Scopus (407) Google Scholar, 18.Itani O.A. Liu K.Z. Cornish K.L. Campbell J.R. Thomas C.P. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E971-E979Crossref PubMed Scopus (9) Google Scholar). DEX alone induced MKP-1 and SGK by 3.23 ± 0.16- and 108.30 ± 9.69-fold, respectively, for 3 h in the MCF-7 cell line. Preincubating MCF-7 cells with estrogen for 24 h prior to DEX treatment significantly inhibited MKP-1 and SGK induction by a mean of 85% (n = 3) and 74% (n = 3), respectively (Fig. 1). Similar estrogen effects were observed in another breast cancer cell line, T47D (Fig. 1). Significantly lower GR expression was found in the T47D cell line as compared with MCF-7 cell line (data not shown). 8 h of stimulation with DEX was determined as an optimal time point for MKP-1 and SGK induction in this cell line. To investigate whether estrogen inhibited DEX induction of MKP-1 and SGK through ER, we employed a selective ER inhibitor ICI 182,780 (inhibitory concentration of 50% (IC50) = 0.29 nm) to antagonize ER in MCF-7 cells. The results demonstrated that with the presence of 1 μm ICI 182,780, DEX induced both MKP-1 and SGK by 2.37 ± 0.36- and 182.90 ± 38.98-fold, respectively, and the DEX-mediated induction of these genes was no longer inhibited by preincubating the cells with estrogen (Fig. 2A). These data indicate that estrogen exerts its inhibitory effect on glucocorticoid action through the ER. In all experiments using inhibitors, MCF-7 cells were treated in parallel with estrogen and DEX as mentioned above without inhibitors. The results showed that the cells were responding to hormones the same way as described in Fig. 1 (data not shown). In the ERα-negative MDA-MB-231 cell line, which expresses only ERβ (38.Im J.Y. Park H. Kang K.W. Choi W.S. Kim H.S. Chem. Biol. Interact. 2008; 172: 235-244Crossref PubMed Scopus (38) Google Scholar, 39.Chen B. Gajdos C. Dardes R. Kidwai N. Johnston S.R. Dowsett M. Jordan V.C. Int. J. Oncol. 2005; 27: 327-335PubMed Google Scholar), DEX induction of MKP-1 and SGK was not affected by estrogen (Fig. 2B). These data indicate that estrogen exerts its inhibitory effect on glucocorticoid action through ERα. Because DEX induces gene expression through GR, which accumulates in the nucleus after ligand binding, and Ser-211 phosphorylation is associated with the transcriptionally active form of GR (40.Ismaili N. Garabedian M.J. Ann. N.Y. Acad. Sci. 2004; 1024: 86-101Crossref PubMed Scopus (243) Google Scholar), we tested GR nuclear translocation and GR phosphorylation at Ser-211 by immunofluorescence (Fig. 3) and Western blot (Fig. 4). DEX alone increased GR nuclear localization with concomitant loss of cytoplasmic GR. This was not affected by preincubation of the cells with estrogen (Fig. 3, A and C). DEX-induced GR phosphorylation at Ser-211 was observed only in the nucleus and was significantly inhibited by estrogen by a mean of 39% (n = 3) (Fig. 3, B and D). Consistent with the immunofluorescence assay results, Western blot of cytoplasmic and nuclear fractions showed that estrogen significantly inhibited DEX-mediated Ser-211 GR phosphorylation by a mean of 55% (n = 3) (Fig. 4, A and C) without affecting GR nuclear localization (Fig. 4, A and B). We also tested GR phosphorylation at Ser-226. No phosphorylation at this site was observed in either the absence or presence of glucocorticoid and/or estrogen (data not shown). The other GR phosphorylation sites were not tested because there were no commercially available antibodies.FIGURE 4DEX-induced GR nuclear translocation and its phosphorylation at Ser-211 in MCF-7 cells treated with estrogen as detected by Western blot. A, cells were treated with hormones as described in Fig. 3. Nuclear and cytoplasmic protein samples were prepared using NE-PER nuclear and cytoplasmic extraction reagents and blotted with antibodies against Ser-211-phosphorylated GR. The membranes were stripped and reprobed with antibodies against total GR. Actin and TBP were used as loading controls for cytoplasmic and nuclear proteins, respectively. Images are representative of three independent experiments. Fold changes in the densitometry readings of nuclear total GR normalized to TBP (B) and Ser-211-phosphorylated nuclear GR normalized to total GR (C) in the cells treated with DEX alone (set as 1) versus cells cultured with E2 and DEX are provided. NS, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chromatin immunoprecipitation was then performed to test GR binding to a well characterized GRE in the promoter region of the SGK gene (18.Itani O.A. Liu K.Z. Cornish K.L. Campbell J.R. Thomas C.P. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E971-E979Crossref PubMed Scopus (9) Google Scholar, 41.Boonyaratanakornkit V. Bi Y. Rudd M. Edwards D.P. Steroids. 2008; 73: 922-928Crossref PubMed Scopus (108) Google Scholar). Within 1 h, DEX induced GR binding to the GRE by 5.49 ± 0.15-fold, and this was significantly inhibited by estrogen by a mean of 82% (n = 3) (Fig. 5). This result suggests that estrogen inhibits DEX induction of SGK by reducing GR recruitment to the SGK promoter. Because MAPK, CDK, and protein phosphatase can regulate GR activity, we examined the effect of estrogens on these three pathways (19.Wang X. Wu H. Lakdawala V.S. Hu F. Hanson N.D. Miller A.H. Neuropsychopharmacology. 2005; 30: 242-249Crossref PubMed Scopus (48) Google Scholar, 20.Li L.B. Goleva E. Hall C.F. Ou L.S. Leung D.Y. J. Allergy Clin. Immunol. 2004; 114: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21.Rogatsky I. Logan S.K. Garabedian M.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 2050-2055Crossref PubMed Scopus (251) Google Scholar, 22.Miller A.L. Webb M.S. Copik A.J. Wang Y. Johnson B.H. Kumar R. Thompson E.B. Mol. Endocrinol. 2005; 19: 1569-1583Crossref PubMed Scopus (184) Google Scholar, 23.Wang X. Wu H. Miller A.H. Mol. Psychiatry. 2004; 9: 65-75Crossref PubMed Scopus (121) Google Scholar, 24.Wang Z. Garabedian M.J. J. Biol. Chem. 2003; 278: 50897-50901Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 25.Krstic M.D. Rogatsky I. Yamamoto K.R. Garabedian M.J. Mol. Cell. Biol. 1997; 17: 3947-3954Crossref PubMed Scopus (229) Google Scholar, 26.Kino T. Ichijo T. Amin N.D. Kesavapany S. Wang Y. Kim N. Rao S. Player A. Zheng Y.L. Garabedian M.J. Kawasaki E. Pant H.C. Chrousos G.P. Mol. Endocrinol. 2007; 21: 1552-1568Crossref PubMed Scopus (116) Google Scholar, 27.Wang Z. Chen W. Kono E. Dang T. Garabedian M.J. Mol. Endocrinol. 2007; 21: 625-634Crossref PubMed Scopus (90) Google Scholar, 28.Nordeen S.K. Moyer M.L. Bona B.J. Endocrinology. 1994; 134: 1723-1732Crossref PubMed Scopus (86) Google Schol
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