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• W2034234282 abstract "Glucocorticoids play pivotal roles in the maintenance of homeostasis but, when dysregulated, may also have deleterious effects. Smad6, one of the transforming growth factor β (TGFβ) family downstream transcription factors, interacts with the N-terminal domain of the glucocorticoid receptor (GR) through its Mad homology 2 domain and suppresses GR-mediated transcriptional activity in vitro. Adenovirus-mediated Smad6 overexpression inhibits glucocorticoid action in rat liver in vivo, preventing dexamethasone-induced elevation of blood glucose levels and hepatic mRNA expression of phosphoenolpyruvate carboxykinase, a well known rate-limiting enzyme of liver gluconeogenesis. Smad6 suppresses GR-induced transactivation by attracting histone deacetylase 3 to DNA-bound GR and by antagonizing acetylation of histone H3 and H4 induced by p160 histone acetyltransferase. These results indicate that Smad6 regulates glucocorticoid actions as a corepressor of the GR. From our results and known cross-talks between glucocorticoids and TGFβ family molecules, it appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anticatabolic and pro-wound healing properties of the TGFβ family of proteins. Glucocorticoids play pivotal roles in the maintenance of homeostasis but, when dysregulated, may also have deleterious effects. Smad6, one of the transforming growth factor β (TGFβ) family downstream transcription factors, interacts with the N-terminal domain of the glucocorticoid receptor (GR) through its Mad homology 2 domain and suppresses GR-mediated transcriptional activity in vitro. Adenovirus-mediated Smad6 overexpression inhibits glucocorticoid action in rat liver in vivo, preventing dexamethasone-induced elevation of blood glucose levels and hepatic mRNA expression of phosphoenolpyruvate carboxykinase, a well known rate-limiting enzyme of liver gluconeogenesis. Smad6 suppresses GR-induced transactivation by attracting histone deacetylase 3 to DNA-bound GR and by antagonizing acetylation of histone H3 and H4 induced by p160 histone acetyltransferase. These results indicate that Smad6 regulates glucocorticoid actions as a corepressor of the GR. From our results and known cross-talks between glucocorticoids and TGFβ family molecules, it appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anticatabolic and pro-wound healing properties of the TGFβ family of proteins. Glucocorticoids play crucial roles in the regulation of basal and stress-related homeostasis (1Chrousos G.P. N. Engl. J. Med. 1995; 332: 1351-1362Crossref PubMed Scopus (2184) Google Scholar). They are necessary for maintenance of many important biologic activities, such as homeostasis of intermediary metabolism, central nervous and cardiovascular system functions, and the immune/inflammatory reaction (1Chrousos G.P. N. Engl. J. Med. 1995; 332: 1351-1362Crossref PubMed Scopus (2184) Google Scholar). Glucocorticoids also act as potent immunosuppressive and anti-inflammatory agents at “pharmacologic” doses, properties that make them irreplaceable therapeutic means for many inflammatory, autoimmune, allergic, and lymphoproliferative diseases (2Chrousos G.P. Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill Inc., New York2001: 609-632Google Scholar). At high levels and over a long duration, glucocorticoids have neurotoxic, catabolic, and anti-wound healing properties as well as promoting gluconeogenesis, adipogenesis, and a shift of the T helper 1 to T helper 2 balance toward T helper 2 predominance (3Franchimont D. Kino T. Galon J. Meduri G.U. Chrousos G. Neuroimmunomodulation. 2002; 10: 247-260Crossref PubMed Scopus (104) Google Scholar, 4Elenkov I.J. Chrousos G.P. Trends Endocrinol. Metab. 1999; 10: 359-368Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). Many extracellular and intracellular factors influence the actions of glucocorticoids at the level of their target tissues (5Kino T. Chrousos G.P. Steckler T. Kalin N.H. Reul J.M.H.M. Handbook on Stress and the Brain, Part 1. Elsevier BV, Amsterdam2004: 295-312Google Scholar). Some of these are physiologically important, whereas others may also be associated with pathologic processes (5Kino T. Chrousos G.P. Steckler T. Kalin N.H. Reul J.M.H.M. Handbook on Stress and the Brain, Part 1. Elsevier BV, Amsterdam2004: 295-312Google Scholar, 6Kino T. De Martino M.U. Charmandari E. Mirani M. Chrousos G.P. J. Steroid Biochem. Mol. Biol. 2003; 85: 457-467Crossref PubMed Scopus (138) Google Scholar). The actions of glucocorticoids are mediated by the ubiquitous intracellular glucocorticoid receptor (GR), 2The abbreviations used are: GRglucocorticoid receptorDBDDNA-binding domainBMPbone morphogenetic proteinMH1 and MH2Mad homology domain 1 and 2, respectivelyMRmineralocorticoid receptorPRprogesterone receptorARandrogen receptorERαestrogen receptor αGREglucocorticoid response elementMMTVmouse mammary tumor virusCREBcAMP-response element-binding proteinTSAtricostatin AsiRNAsmall interfering RNATATtyrosine aminotransferasePEPCKphosphoenolpyruvate carboxykinaseRPLP0ribosomal phosphoprotein P0ChIPchromatin immunoprecipitationADactivation domainGβguanine nucleotide-binding protein βHAhemagglutininHDAChistone deacetylase.2The abbreviations used are: GRglucocorticoid receptorDBDDNA-binding domainBMPbone morphogenetic proteinMH1 and MH2Mad homology domain 1 and 2, respectivelyMRmineralocorticoid receptorPRprogesterone receptorARandrogen receptorERαestrogen receptor αGREglucocorticoid response elementMMTVmouse mammary tumor virusCREBcAMP-response element-binding proteinTSAtricostatin AsiRNAsmall interfering RNATATtyrosine aminotransferasePEPCKphosphoenolpyruvate carboxykinaseRPLP0ribosomal phosphoprotein P0ChIPchromatin immunoprecipitationADactivation domainGβguanine nucleotide-binding protein βHAhemagglutininHDAChistone deacetylase. which functions as a hormone-activated transcription factor of glucocorticoid target genes (3Franchimont D. Kino T. Galon J. Meduri G.U. Chrousos G. Neuroimmunomodulation. 2002; 10: 247-260Crossref PubMed Scopus (104) Google Scholar, 7Kino T. Chrousos G.P. Essays Biochem. 2004; 40: 137-155Crossref PubMed Scopus (58) Google Scholar). The GR consists of three domains, the N-terminal or “immunogenic” domain, the central, DNA-binding domain (DBD), and the C-terminal, ligand-binding domain. The functions of the latter two domains have been studied extensively, whereas those of the immunogenic domain are less well known (7Kino T. Chrousos G.P. Essays Biochem. 2004; 40: 137-155Crossref PubMed Scopus (58) Google Scholar). In the unliganded state, the GR is located primarily in the cytoplasm (7Kino T. Chrousos G.P. Essays Biochem. 2004; 40: 137-155Crossref PubMed Scopus (58) Google Scholar). After binding to its agonist ligand, the GR undergoes conformational changes and translocates into the nucleus. Ligand-activated GR then binds to the glucocorticoid response elements (GREs) as a dimer and attracts several so-called coactivators and chromatin-remodeling factors to the promoter region through its two transactivation domains, activation function (AF)-1 and AF-2 (7Kino T. Chrousos G.P. Essays Biochem. 2004; 40: 137-155Crossref PubMed Scopus (58) Google Scholar, 8McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1629) Google Scholar). Among them, the p160 type histone acetyltransferase coactivators play an essential role in GR-induced transcriptional activity, being attracted to the promoter region in an early phase of transcriptional activation and facilitating access of other transcription-related molecules on the chromatin through acetylation of lysine residues located in several histone tails, such as those of histone H3 and H4 (8McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1629) Google Scholar, 9Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1358) Google Scholar, 10Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar, 11Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar, 12Peterson C.L. Laniel M.A. Curr. Biol. 2004; 14: R546-R551Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar). In contrast, corepressors, such as the nuclear receptor corepressors and the silencing mediator for retinoid and thyroid hormone receptor, and associated histone deacetylases cause deacetylation of histones, silencing gene transcription by preventing access of cis-acting molecules to the promoter region (8McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1629) Google Scholar). glucocorticoid receptor DNA-binding domain bone morphogenetic protein Mad homology domain 1 and 2, respectively mineralocorticoid receptor progesterone receptor androgen receptor estrogen receptor α glucocorticoid response element mouse mammary tumor virus cAMP-response element-binding protein tricostatin A small interfering RNA tyrosine aminotransferase phosphoenolpyruvate carboxykinase ribosomal phosphoprotein P0 chromatin immunoprecipitation activation domain guanine nucleotide-binding protein β hemagglutinin histone deacetylase. glucocorticoid receptor DNA-binding domain bone morphogenetic protein Mad homology domain 1 and 2, respectively mineralocorticoid receptor progesterone receptor androgen receptor estrogen receptor α glucocorticoid response element mouse mammary tumor virus cAMP-response element-binding protein tricostatin A small interfering RNA tyrosine aminotransferase phosphoenolpyruvate carboxykinase ribosomal phosphoprotein P0 chromatin immunoprecipitation activation domain guanine nucleotide-binding protein β hemagglutinin histone deacetylase. Members of the Smad family of proteins transduce signals of transforming growth factor β (TGFβ) superfamily members, such as TGFβ, activin, and bone morphogenetic proteins (BMPs), by associating with the cytoplasmic side of the type I cell surface receptors of these hormones (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 14Miyazono K. J. Cell Sci. 2000; 113: 1101-1109Crossref PubMed Google Scholar). Nine distinct vertebrate Smad family members have been identified, and they have been classified into three groups: receptor-regulated Smads (R-Smads), such as Smad1, -2, -3, -5, and -8; a common partner Smad (Co-Smad), Smad4; and inhibitory Smads (I-Smads) like Smad6 and Smad7 (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). All Smads have two characteristic domains, the Mad homology domains 1 and 2 (MH1 and -2), in their N-terminal and C-terminal portions, respectively, separated by a proline-rich linker region (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The MH1 domain of R- and Co-Smads is important for complex formation with other Smads, transcriptional activation and repression, and interaction with other transcription factors and target DNA sequences (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The MH2 domain of R-Smads mediates their interaction with cell surface receptors (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Lo R.S. Chen Y.G. Shi Y. Pavletich N.P. Massague J. EMBO J. 1998; 17: 996-1005Crossref PubMed Scopus (207) Google Scholar), whereas the highly conserved MH2 domain of I-Smads interacts with type I receptors and is sufficient for their inhibitory activity (14Miyazono K. J. Cell Sci. 2000; 113: 1101-1109Crossref PubMed Google Scholar). Upon binding of TGFβ, activin, or BMP to their receptors, cytoplasmic R-Smads are phosphorylated by the receptor kinases, translocate into the nucleus, and stimulate the transcriptional activity of TGFβ-, activin-, or BMP-responsive genes by binding to their response elements located in their promoter regions as a heterotrimer with Co-Smad (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). I-Smads, such as Smad6 and Smad7, act as inhibitory molecules in the TGFβ family signaling by forming stable associations with activated type I receptors, which prevent the phosphorylation of R-Smads (13ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Smad6 also competes with Smad4 in the heteromeric complex formation induced by activated Smad1 (16Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (575) Google Scholar). In addition, I-Smads directly suppress the transcriptional activity of TGFβ family signaling by binding to promoter DNA and attracting histone deacetylases and/or the C-terminal binding protein (17Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 18Bai S. Cao X. J. Biol. Chem. 2002; 277: 4176-4182Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Lin X. Liang Y.Y. Sun B. Liang M. Shi Y. Brunicardi F.C. Feng X.H. Mol. Cell. Biol. 2003; 23: 9081-9093Crossref PubMed Scopus (82) Google Scholar). Since I-Smads are produced in response to activation of TGFβ family signaling (20Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.H. Heldin N.E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (295) Google Scholar), they literally function in the negative feedback regulation of the Smad signaling pathways. Smad6 preferably inhibits BMP signaling, whereas Smad7 is a more general inhibitor, repressing TGFβ and activin signaling in addition to that of BMP (14Miyazono K. J. Cell Sci. 2000; 113: 1101-1109Crossref PubMed Google Scholar). In this study, we found that Smad6 interacts with the GR and suppresses the latter's transcriptional activity by attracting histone deacetylase HDAC3. HDAC3 antagonized histone acetylation induced by p160 type histone acetyltransferase coactivators. This inhibitory effect of Smad6 was present in vivo, suppressing glucocorticoid-stimulated gluconeogenesis in the rat liver. It is likely that Smad6 functions as a target tissue regulator of glucocorticoid action. Plasmids—pLexA-GR-(263-419) was described previously (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). pLexA-GR-(263-319), pLexA-GR-(263-367), pLexA-GR-(319-367), and pLexA-GR-(367-427) were constructed by subcloning the coding sequences of the corresponding human GR fragments into pLexA (Clontech, Palo Alto, CA). pB42AD-GR-(2-419), pB42AD-MR-(2-603), pB42AD-PR-A-(2-567), pB42AD-AR-(2-559), and pB42AD-ERα-(2-180) were constructed by inserting the coding sequences of the indicated portions of the human GRα, mineralocorticoid receptor (MR), progesterone receptor-A (PR-A), androgen receptor (AR), and estrogen receptor α (ERα) into pB42AD (Clontech). pLexA-Smad6-(1-496), -(1-330), and -(331-496) were constructed by subcloning the indicated portions of Smad6 coding sequences into pLexA. pB42AD-Smad6-(318-496) is a clone obtained in the original yeast two-hybrid screening using GR-(263-419) as bait (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). pB42AD-Gβ2-(55-226) was described previously (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). pcDEFFlag(N)-mSmad6WT, -mSmad7WT, -mSmad6N, -mSmad6C, -mSmad7/6, and -mSmad6/7 are all kind gifts from Dr. K. Miyazono (University of Tokyo, Tokyo, Japan) (22Hanyu A. Ishidou Y. Ebisawa T. Shimanuki T. Imamura T. Miyazono K. J. Cell Biol. 2001; 155: 1017-1027Crossref PubMed Scopus (178) Google Scholar). pRShGRα and pRShGRα-(Δ262-404), which express the full-length human GRα and its fragment that lacks amino acids 262-404, respectively, were generous gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA). pMMTV-Luc, which expresses the luciferase under the control of the full-length mouse mammary tumor virus (MMTV) promoter that contains four functional GREs (23Bresnick E.H. John S. Berard D.S. LeFebvre P. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3977-3981Crossref PubMed Scopus (114) Google Scholar), was kindly provided by Dr. G. L. Hager (National Institutes of Health, Bethesda, MD). pSG5-GRIP1 was a generous gift from Dr. M. R. Stallcup (University of Southern California, Los Angels, CA). pCDNAI/Amp-MR, p5HBhAR-A, and pRc/CMV-hp53, which express the human MR, AR, and p53, respectively, are kind gifts from Drs. N. Warriar (Centre Recherche Hôtel-Dieu Québec and Laval University, Québec, Canada), E. R. Barrack (Henry Ford Health Sciences Center, Detroit, MI), and P. Chumakov (Princeton University, Princeton, NJ), respectively. pSVPRA, NE0, pRSV-RelA, pRc/RSV-CREB341, and RSV-PKA, which express the human PR-A, ERα, the p65 component of NF-κB, the full-length CRE-binding protein (CREB), and a constitutive active form of the protein kinase A, respectively, were described previously (24Kino T. Gragerov A. Kopp J.B. Stauber R.H. Pavlakis G.N. Chrousos G.P. J. Exp. Med. 1999; 189: 51-62Crossref PubMed Scopus (186) Google Scholar). pERE-E1B-Luc was also described previously (24Kino T. Gragerov A. Kopp J.B. Stauber R.H. Pavlakis G.N. Chrousos G.P. J. Exp. Med. 1999; 189: 51-62Crossref PubMed Scopus (186) Google Scholar). pG13-Py-Luc, which expresses the luciferase under the control of the p53-responsive elements, was a kind gift from Dr. B. Vogelstein (The Johns Hopkins University, Baltimore, MD). (κB)3-Luc, which contains three κB-responsive elements upstream of the luciferase gene, was described previously (25Mirani M. Elenkov I. Volpi S. Hiroi N. Chrousos G.P. Kino T. J. Immunol. 2002; 169: 6361-6368Crossref PubMed Scopus (76) Google Scholar). pCRE-Luc, which has a CREB-response element in front of the luciferase gene, was purchased from Clontech. HA-HDAC3-expressing plasmid was kindly provided by Dr. M. S. Featherstone (McGill University, Montréal, Canada). FLAG-tagged HDAC1, -4, -5, and -6 were generous gifts from Dr. S. L. Schreiber (Harvard University, Cambridge, MA). pSG5, pCDNA3, pMAM-neo-Luc, and pSV40-β-Gal are purchased from Stratagene (La Jolla, CA), Invitrogen, Clontech, and Promega (Madison, WI), respectively. Yeast Two-hybrid Screening and Assay—The yeast two-hybrid screening was performed using GR-(263-419) as bait in the human Jurkat cell cDNA library with the LexA system (Clontech). For a yeast two-hybrid assay, yeast strain EGY48 (Clontech) was transformed with pOP8-LacZ and the indicated pLexA- and pB42AD-based plasmids. β-Galactosidase activity was then measured in the cell suspension as previously described (26Kino T. Gragerov A. Valentin A. Tsopanomihalou M. Ilyina-Gragerova G. Erwin-Cohen R. Chrousos G.P. Pavlakis G.N. J. Virol. 2005; 79: 2780-2787Crossref PubMed Scopus (59) Google Scholar). The β-galactosidase activity was normalized for A600 nm. -Fold induction was calculated by the ratio of adjusted β-galactosidase values of transformed cells cultured in the presence of galactose/raffinose versus those in the medium containing glucose. Cell Cultures and Transfection—Human colon carcinoma HCT116 and uterine cervical carcinoma HeLa cells were purchased from the American Type Culture Collection (Manassas, VA) and were maintained in McCoy's 5A or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units of penicillin, and 50 μg/ml streptomycin. HCT116/MMTV cells, which were stably transformed with pMAM-neo-Luc that has the full-length MMTV promoter upstream of the luciferase gene, were maintained in the McCoy's medium containing 0.2 mg/ml neomycin and the same supplements. Rat hepatoma HTC cells were described previously (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). HCT116 and HCT116/MMTV cells do not contain functional GR, whereas HeLa and HTC cells express fully active GR. HCT116 and HCT116/MMTV cells were transfected as previously described (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). For the experiments using pMMTV-Luc or other reporter genes for indicated nuclear receptors and transcription factors, different amounts of Smad6- or Smad7-related plasmids were cotransfected with 0.5 μg/well of the indicated nuclear receptor- or transcription factor-expressing plasmid, 1.5 μg/well of luciferase-expressing reporter plasmid, and 0.5 μg/well of pSV40-β-Gal. For stimulation of p53, CREB, or NF-κB transcriptional activity, 0.5 μg/well of pRc/CMV-hp53, RSV-PKA, or pRSV-RelA was cotransfected as well. Empty vectors were used to maintain the same amounts of transfected DNA. 10-6 m dexamethasone or progesterone, or 10-8 m aldosterone, dehydrotestosterone, or estradiol was added to the medium after 24 h of transfection. The indicated concentrations of tricostatin A (TSA) (Sigma), the histone deacetylase inhibitor, were also added to the medium at the same time. The cells were harvested after an additional 24 h, and luciferase and β-galactosidase assays were performed as previously described (24Kino T. Gragerov A. Kopp J.B. Stauber R.H. Pavlakis G.N. Chrousos G.P. J. Exp. Med. 1999; 189: 51-62Crossref PubMed Scopus (186) Google Scholar). Introduction of Smad6 Small Interfering RNAs (siRNAs) into HTC Cells, the Tyrosine Aminotransferase (TAT) Assay, and the Real Time PCR—The rat Smad6 siRNA (5′-GCCACUGGAUCUGUCCGAUd-TdT-3′), which targets nucleotides 945-964 of the coding region (Gen-Bank™ accession number XM_345947), was produced by Qiagen (Valencia, CA). This sequence portion has the complete match to corresponding parts of the mouse and the human Smad6 sequences. The negative control siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′) was also purchased from Qiagen. HTC cells were transfected with siRNAs by using the Nucleofector system (Amaxa GmbH, Cologne, Germany) with nearly 80% transfection efficiency, as previously described (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). Twenty-four hours after plating the cells in the 24-well plates, they were stimulated with 10-6 m dexamethasone. After an additional 24 h of incubation, cell lysates for the tyrosine aminotransferase (TAT) assay and total RNA for the real time PCR were harvested. TAT assays were performed as previously reported (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). The reverse transcription reaction was carried out as previously described (27De Martino M.U. Bhattachryya N. Alesci S. Ichijo T. Chrousos G.P. Kino T. Mol. Endocrinol. 2004; 18: 820-833Crossref PubMed Scopus (39) Google Scholar). To detect mRNA levels of rat Smad6 and control rat acidic ribosomal phosphoproteinP0(RPLP0),primerpairs(Smad6,forward(5′-GAAGTCGTGTGGTCCCTGATC-3′) and reverse (5′-CTCGCAGTCACTCTCAG-3′); RPLP0, forward (5′-GACATGCTGCTGGCCAATAAG-3′) and reverse (5′-CAACATGTTCAGCAGTGTG-3′)) were used (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar). The real time PCR was performed in triplicate using the SYBR Green PCR Master Mix (Applied Biosystems) in an ABI PRIZM 7700 SDS light cycler (Applied Biosystems), as previously described (27De Martino M.U. Bhattachryya N. Alesci S. Ichijo T. Chrousos G.P. Kino T. Mol. Endocrinol. 2004; 18: 820-833Crossref PubMed Scopus (39) Google Scholar). Obtained CT (threshold cycle) values of Smad6 were normalized for those of RPLP0, and their relative mRNA expression was demonstrated as -fold induction to the base line. The dissociation curves of the used primer pairs showed a single peak, and samples after PCRs had a single expected DNA band in an agarose gel analysis (data not shown). FLAG-Smad6-expressing Adenovirus, Injection into Rats, and Detection of the Phosphoenolpyruvate Carboxykinase (PEPCK) and TAT Gene mRNAs—The following animal study was approved by the NICHD Animal Care and Use Committee (protocol number ASP04-008). FLAG-tagged Smad6-expressing and control LacZ-expressing adenoviruses were described previously (28Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (366) Google Scholar). 1 × 1010 colony-forming units of these adenoviruses were injected into the peritoneal cavity of 175-200-g Sprague-Dawley rats, and 1 mg/kg dexamethasone was injected intramuscularly after 24 h. After an additional 24 h, blood glucose levels were examined in total blood obtained by tail cut using a OneTouch monitor (LifeScan, Milpitas, CA). The rats were then sacrificed with CO2, and the livers were removed and stored at -70 °C until further use. Total RNA and whole homogenate of the liver were subsequently purified using the RNAeasy Midi kit (Quiagen) and homogenation/centrifugation at 200 × g for 10 min in the buffer containing 50 mm Tris-HCl (pH 7.4), 50 mm NaCl, 0.2% Nonidet P-40, and Complete™ tablets (1 tablet/50 ml), respectively. Levels of the PEPCK, TAT, and RPLP0 mRNA were then determined with the real time PCR reaction in quadruplicate using the SYBR Green PCR Master Mix, as described above. Primer pairs for detecting mRNAs of the hepatic PEPCK and TAT were as follows: PEPCK, forward (5′-CAGGCTGGCTAAGGAGGAAG-3′) and reverse (5′-CATCACTTGTCTCAGCCAC-3′); TAT, forward (5′-GTCGCTTCTTACTACCAC-3′) and reverse (5′-CAGGCAGGAGATTGTAGAG-3′) (21Kino T. Tiulpakov A. Ichijo T. Chheng L. Kozasa T. Chrousos G.P. J. Cell Biol. 2005; 169: 885-896Crossref PubMed Scopus (93) Google Scholar, 27De Martino M.U. Bhattachryya N. Alesci S. Ichijo T. Chrousos G.P. Kino T. Mol. Endocrinol. 2004; 18: 820-833Crossref PubMed Scopus (39) Google Scholar). Whole homogenate of the liver was run on 8% SDS-polyacrylamide gels, and levels of GR and FLAG-Smad6 were examined in Western blots using anti-GR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-FLAG (M2) (Sigma) antibodies, respectively. Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was performed in HCT116/MMTV and HTC cells, which have the genomically integrated MMTV-luciferase gene (HCT116/MMTV) and endogenous glucocorticoid-responsive TAT gene (HTC), respectively, using a chromatin immunoprecipitation kit (Upstate, Charlottesville, VA) with minor modifications as previously described (27De Martino M.U. Bhattachryya N. Alesci S. Ichijo T. Chrousos G.P. Kino T. Mol. Endocrinol. 2004; 18: 820-833Crossref PubMed Scopus (39) Google Scholar, 29Charmandari E. Raji A. Kino T. Ichijo T. Tiulpakov A. Zachman K. Chrousos G.P. J. Clin. Endocrinol. Metab. 2005; 90: 3696-3705Crossref PubMed Scopus (83) Google Scholar). HCT116/MMTV cells were transfected with FLAG-Smad6- and/or HA-HDAC3-expressing plasmids together with pRShGRα using Lipofectin®. HTC cells were transfected with FLAG-Smad6- or GRIP1 (GR-interacting protein 1)-expressing plasmid using the Nucleofector system. Both cells were exposed to either 10-6 m dexamethasone or vehicle overnight. They were then fixed, DNA and bound proteins were cross-linked, and ChIP assays were performed by co-precipitating the DNA-protein complexes with anti-GRα (Affinity Bioreagents, Golden, CO), anti-FLAG (M2), anti-HA (Santa Cruz Biotechnology), anti-GRIP1 antibodies (Santa Cruz Biotechnology), or rabbit control IgG (Santa Cruz Biotechnology). Antibodies reacting to the acetylated histone H3 (Lys14) and H4 were purchased from Upstate. The promoter region -219 to -47 of the MMTV long terminal repeat (fragment size 173 bp), which contains two functional GREs, was amplified from the prepared DNA samples using a primer pair: 5′-AACCTTGCGGTTCCCAG-3′ and 5′-GCATTTACATAAGATTTGG-3′ (29Charmandari E. Raji A. Kino T. Ichijo T. Tiulpakov A. Zachman K. Chrousos G.P. J. Clin. Endocrinol. Metab. 2005; 90: 3696-3705Crossref PubMed Scopus (83) Google Scholar). Tandem endogenous GREs of the rat TAT promoter, which are located ∼2,500 bp upstream of its transcription initiation site, were amplified by a primer pair: 5′-TCTTCTCAGTGTTCTCTATCAC-3′ and 5′-CAGAAACCGACAGGCGACTACG-3′ (fragment size 220 bp), as described previously (27De Martino M.U. Bhattachryya N. Alesci S. Ichijo T. Chrousos G.P. Kino T. Mol. Endocrinol. 2004; 18: 820-833Crossref PubMed Scopus (39) Google Scholar). Amplified products were then run on a 3% agarose gel, and visualized DNA bands were photographed. The real time PCR was directly performed using the primer pair that detects TAT GREs for some of the ChIP samples using the SYBR Green PCR Master Mix (Applied Biosystems) as described above. Obtained CT values of ChIP samples were normalized for those of corresponding inputs, and their relative precipitations were demonstrated as -fold precipitation of the base line. Statistical Analyses—Statistical analysis was carried out by analysis of variance, followed by Student" @default.
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• W2034234282 title "The Smad6-Histone Deacetylase 3 Complex Silences the Transcriptional Activity of the Glucocorticoid Receptor" @default.
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