Regulation of Transforming Growth Factor-β Signaling by Protein Inhibitor of Activated STAT, PIASy through Smad3

Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-β family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF-β signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT (signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-β-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-β in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-β signaling using the negative feedback loop. Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-β family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. In particular, Smad7 acts as an antagonist of TGF-β signaling, which could determine the intensity or duration of its signaling cascade. In this study we identified a protein inhibitor of activated STAT (signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait. The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells. Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-β-mediated activation of Smad3. PIASy also stimulated the sumoylation of Smad3 in vivo. Furthermore, endogenous PIASy expression was induced by TGF-β in Hep3B cells. These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-β signaling using the negative feedback loop. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF, transforming growth factor; STAT, signal transducers and activators of transcription; PIAS, protein inhibitor of activated STAT; LUC, luciferase; TβR-I, TGF-β type I receptor; Smad, Sma- and MAD-related protein; SUMO, small ubiquitin-related modifier; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PAI-1, plasminogen activator inhibitor-1.1The abbreviations used are: TGF, transforming growth factor; STAT, signal transducers and activators of transcription; PIAS, protein inhibitor of activated STAT; LUC, luciferase; TβR-I, TGF-β type I receptor; Smad, Sma- and MAD-related protein; SUMO, small ubiquitin-related modifier; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PAI-1, plasminogen activator inhibitor-1. is a member of the TGF-β superfamily, which also includes the activins/inhibins, bone morphogenetic proteins, and other members, such as müllerian inhibiting substance and Lefty (1Roberts A.B. Sporn M.B. Sporn M.B. Robert A.B. Pepide Growth Factors and Their Receptor, Part I. Springer-Verlag, Heidelberg1990: 419-472Google Scholar). These factors regulate growth, differentiation, apoptosis, migration, and secretion of important molecules, such as components of the extracellular matrix, adhesion molecules, hormones, and cytokines in a variety of cell types, affecting morphogenesis, tissue repair, tumor suppression, and immunoregulation (1Roberts A.B. Sporn M.B. Sporn M.B. Robert A.B. Pepide Growth Factors and Their Receptor, Part I. Springer-Verlag, Heidelberg1990: 419-472Google Scholar). The TGF-β family also plays important roles in various pathological conditions ranging from abnormal tissue repair state to cancer development (1Roberts A.B. Sporn M.B. Sporn M.B. Robert A.B. Pepide Growth Factors and Their Receptor, Part I. Springer-Verlag, Heidelberg1990: 419-472Google Scholar, 2de Caestecker M.P. Piek E. Roberts A.B. J. Natl. Cancer Inst. 2000; 92: 1388-1402Crossref PubMed Google Scholar). TGF-β signaling is mediated through transmembrane receptors located at the cell surface (TβRs) that are serine/threonine kinases, which in turn use the highly conserved members of the Smad (Sma- and MAD-related protein) family of transcription factors to transduce their signals to the nucleus (3Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3984) Google Scholar). Two of the receptor-regulated Smads (R-Smads), Smad2 and Smad3, transduce signals for TGF-β. On the other hand, Smad4 is a common mediator (Co-Smad) and acts as a heterodimeric partner for Smad2 and Smad3 for efficient DNA binding and transcriptional activation (4Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3341) Google Scholar, 5Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). When TβRs are activated by the binding of their cognate ligands, Smad 2 and Smad3 are phosphorylated by the type I receptor (TβR-I) serine-threonine kinase. Phosphorylated Smad2 and Smad3 then form stable hetero-complexes with Smad4 that translocate into the nucleus and activate transcription. Inhibitory Smads, such as Smad6 and Smad7, bind activated TβRI, thereby preventing phosphorylation of R-Smads (6Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1562) Google Scholar, 7Hayashi H. Abdollah S. Qiu Y. Cai J. Xu Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1159) Google Scholar, 8Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). In addition, Smad7 interacts with the E3-ubiquitin ligases Smurf1 or Smurf2 in the nucleus and induces ubiquitination and degradation of the TGF-β receptors (9Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 10Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). Transcriptional activation by Smads partly requires the aid of nuclear coactivators such as CBP and p300 (11Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (450) Google Scholar, 12Janknecht R. Wells N.J. Hunter T. Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (437) Google Scholar). Conversely, transcriptional repression is effected by interaction with nuclear corepressors such as SnoN and c-Ski (13Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (436) Google Scholar, 14Xu W.K. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar). These findings indicate that Smad7 regulates intensity and/or duration of TGF-β signaling by interaction with the several molecules, and it could be possible another signaling regulatory molecule(s) may co-operate with Smad7. In attempt to identify novel Smad7 partners, we screened a mouse embryo cDNA library with a yeast two-hybrid system using the MH2 domain of Smad7 as bait. Here, we identify PIASy, which is one of the protein inhibitor of activated STAT (signal transducers and activators of transcription) family protein, as a protein that interacts specifically with Smad7. They have been originally identified as a cofactor that inhibits the transcriptional activation potential of STAT, and in mammals five PIAS proteins (PIAS1, 3, xα, xβ, and y) have been reported (15Shuai K. Oncogene. 2000; 19: 2638-2644Crossref PubMed Scopus (300) Google Scholar). PIAS1 and PIAS3 were initially cloned as transcriptional repressors of the Jak-STAT-signaling pathway (16Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (631) Google Scholar, 17Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (802) Google Scholar). PIAS3 was originally identified as a specific corepressor of signal transducer and activator of transcription 3 (STAT3) (17Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (802) Google Scholar). PIAS3 binds to STAT3 and inhibits its DNA binding activity and thereby interferes with STAT3-mediated gene activation. PIAS1, another member of PIAS family, was originally identified as a co-repressor of STAT1 (16Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (631) Google Scholar). PIAS family proteins also function as a transcriptional cofactor for nuclear receptors (18Kotaja N. Aittomaki S. Silvennoinen O. Palvimo J.J. Janne O.A. Mol. Endocrinol. 2000; 14: 1986-2000Crossref PubMed Scopus (145) Google Scholar, 19Junicho A Matsuda T. Yamamoto T. Kishi H. Korkmaz K. Saatcioglu F. Fuse H. Muraguchi A. Biochem. Biophys. Res. Commun. 2000; 278: 9-13Crossref PubMed Scopus (73) Google Scholar, 20Gross M. Liu B. Tan J. French F.S. Carey M. Shuai K. Oncogene. 2001; 20: 3880-3887Crossref PubMed Scopus (148) Google Scholar). Recently, PIAS family proteins have been proposed to function as a small ubiquitin-related modifier (SUMO)-E3 ligase (21Jackson P.K. Genes Dev. 2001; 15: 3053-3058Crossref PubMed Scopus (201) Google Scholar). PIAS1 and PIASy were shown to catalyze sumoylation of p53 and LEF-1, respectively (22Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 23Nelson V. Davis G.E. Maxwell S.A. Apoptosis. 2001; 6: 221-234Crossref PubMed Scopus (57) Google Scholar, 24Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (463) Google Scholar). PIAS family proteins have RING finger-like domain, and their SUMO-E3 ligase activities are dependent on this domain (21Jackson P.K. Genes Dev. 2001; 15: 3053-3058Crossref PubMed Scopus (201) Google Scholar). We find that PIASy interacts with other Smads including Smad3 and antagonizes Smad3-dependent transcriptional activation by TβR-I. Coexpression of PIASy with Smad3 results in the modification of Smad3 with SUMO, suggesting that PIASy functions as a SUMO-E3 ligase for Smad3. We also show that expression of PIASy is induced by TGF-β. Our findings provide additional mechanisms of the negative regulation of TGF-β signaling by PIASy, which may due to the sumoylation of Smad3 by PIASy using the negative feedback loop. Reagents and Antibodies—Human recombinant TGF-β1 was purchased from Strathmann Biotech GmbH (Hamburg, Germany). MG132 was purchased from Peptide Institute (Osaka, Japan). Expression vectors, FLAG-tagged Smad2, Smad3, Smad4, Smad6, Smad7, and a series of Smad3 mutants (25Nishihara A. Hanai J.-i. Okamoto N. Yanagisawa J. Kato S. Miyazono K. Kawabata M. Genes Cells. 1998; 3: PubMed Scopus Google were by K. Miyazono Japan). J. A. J. Mol. Cell. Biol. 15: PubMed Google PIASy, PIASy, and K. T. T. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google in were by J. Cancer H. and T. and were purchased from was purchased from was purchased from and Japan). of with Smad7 or Smad6 MH2 and the DNA mouse Smad7 and Smad6 were from K. Miyazono Japan). a with the MH2 domain of Smad7 and Smad6, was used to the of the cDNA for Smad7 and for Smad6 are The was with and and into with and of the activation were to the of the MH2 was by the Smad7 to the DNA binding domain in the as the with MH2 by with a mouse embryo cDNA library in were that and and been with 3 were screened for growth in the of from were from and were into yeast with MH2 to the and cell was in and in by the T. Yamamoto T. Kishi H. A. Muraguchi A. 2000; PubMed Scopus Google Scholar). cell Hep3B was in T. Matsuda T. Muraguchi A. Miyazono K. Kawabata K. 2001; PubMed Scopus Google Scholar). the were for in by with TGF-β G. P.K. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, K. W. D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Hep3B in a were by using was as T. Yamamoto T. Kishi H. A. Muraguchi A. 2000; PubMed Scopus Google Scholar). The were in of and for and activities to the activities were to the or were for and and were as T. Yamamoto T. Kishi H. A. Muraguchi A. 2000; PubMed Scopus Google Scholar). were and in and of and The from cell were on and to The were then with proteins were using an system 293T were with the vectors, and the were with MG132 for to degradation of Smad3. were and in and of and was as the were in with and PIASy with by the were with a and with a mouse or with a The were then with an or and a and of in FCS, Hep3B were with TGF-β for the were by using and used in to the T. Matsuda T. Muraguchi A. Miyazono K. Kawabata K. 2001; PubMed Scopus Google Scholar). A and cDNA were used was using a Japan). PIAS3 and were used as G. P.K. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, K. W. D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of PIASy as an of identify proteins that could be in the negative regulation of TGF-β signaling, we screened a mouse embryo cDNA library using the MH2 domain of mouse Smad7 as bait. Smad7 proteins were identified from a screening of yeast DNA that one of the that specifically with DNA binding Smad7 MH2 was with a member of the protein inhibitor of activated STAT family PIASy that a in the of the was from the two-hybrid and into S. with Smad7 MH2 domain or Smad6 MH2 domain to the DNA binding domain of or DNA binding domain Smad7 MH2 domain Smad6 MH2 domain in activation of the the growth in the of Smad7 MH2 or Smad6 MH2 that PIASy with the MH2 domain of Smad7 and Smad6 in this the association of PIASy with other Smads including Smad7 in 293T were with FLAG-tagged Smad2, Smad3, Smad4, Smad6, or Smad7 with shown in of Smads were in cell PIASy was in of associated proteins with an that PIASy interacts with Smads in 293T the interaction was shown in the MH2 domain of the Smad, Smad6, and Smad7 with We then PIASy interacts with Smad3 the domain using or mutants of Smad3 (25Nishihara A. Hanai J.-i. Okamoto N. Yanagisawa J. Kato S. Miyazono K. Kawabata M. Genes Cells. 1998; 3: PubMed Scopus Google Expression PIASy and/or FLAG-tagged Smad3 or one of its mutants were into 293T cells. were and to with an were then used in with an shown in the Smad3 with PIASy, the mutants the MH2 or were to bind PIASy In the mutants in which or are with These results indicate that efficient the MH2 domain of Smad3. of PIASy and determine the of PIASy and activated Smad3, expression for PIASy and FLAG-tagged Smad3 with a form of TGF-β type I which Smad3, were into cells. of with Smad3 in in a transcription the were with and and were with and a Smad3 is shown to to translocate into nucleus and as a in L. J. Biol. 2000; PubMed Scopus Google Scholar, Y. T. Derynck R. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Smad3 was in the nucleus with a In the study PIASy was shown to to in the nucleus (21Jackson P.K. Genes Dev. 2001; 15: 3053-3058Crossref PubMed Scopus (201) Google Scholar). shown in PIASy was to in the Our results also that PIASy and a of activated Smad3 were located in the and were found to be in a nuclear of the in which the and These results that activated Smad3 and PIASy in the nucleus of TGF-β by the of the interaction in the of TGF-β signaling we the using a cell The TGF-β-mediated transcriptional were by which is one of the for TGF-β activity J. A. J. Mol. Cell. Biol. 15: PubMed Google Scholar). In a form of TGF-β type I J. A. J. Mol. Cell. Biol. 15: PubMed Google was used that stimulated TGF-β in 293T K. W. D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). When 293T were with with an expression for expression was by The additional expression of Smad3 additional expression of Smad7 in a We then the of PIASy expression on TGF-β signaling in this When 293T were with an expression for PIASy, and PIASy suppressed activity in a 3, A and The of activity was stimulated by PIASy which was by the additional Smad3 expression These results indicate that the of PIASy on transcriptional activity is mediated by the interaction PIASy and Smad3. Furthermore, expression of another member of the PIAS family, on activity We then the of Smad3 that is in the of TGF-β signaling by shown in expression of PIASy activation. Expression of the mutants of Smad3, the mutants of Smad3 the MH2 domain, the of These results also that the MH2 domain of Smad3 the of TGF-β signaling by PIASy of of the possible mechanisms that is with the is that is modification or degradation of Smad3. the of of activation was through a specific the inhibitor MG132 was to at of and for the results show that the inhibitor MG132 could of activation In PIASy were shown to catalyze sumoylation of p53 and as ligase and their transcriptional activation (22Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 23Nelson V. Davis G.E. Maxwell S.A. Apoptosis. 2001; 6: 221-234Crossref PubMed Scopus (57) Google Scholar, 24Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (463) Google Scholar). PIASy modification of Smad3, we FLAG-tagged Smad3, and PIASy in 293T cells. The cell was to with and the were by with A with that of Smad3 was by in the of with Smad3, and PIASy expression These results that Smad3 is with the mediated by we could in Smad3 we Smad3 and PIASy in 293T may that other factors in a pathway (9Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 10Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1102) Google are also in of transcription. PIASy Smad3 in Hep3B the of PIASy on the pathway conditions through endogenous proteins, we first a cell Hep3B G. P.K. Biochem. Biophys. Res. Commun. PubMed Scopus Google and the Hep3B were with with or PIASy and with and activities were shown in PIASy expression a of activation in Hep3B cells. We PIASy expression is by TGF-β in Hep3B cells. were or with and PIASy expression was by PIASy expression was induced by of TGF-β in Hep3B The of PIASy expression at and at and When we the by TGF-β induced plasminogen activator the expression of PIAS3 was by TGF-β These results that expression of PIASy PIAS3 is induced by TGF-β and its signaling in Hep3B cells. the expression by TGF-β was at 3 a PIASy expression by TGF-β was at suggesting that PIASy may be in the regulation or in to TGF-β. Furthermore, we the of PIASy on endogenous expression by TGF-β. Hep3B were with or PIASy and with and expression was by shown in were with PIASy, endogenous expression was by with that of These results that PIASy is induced by TGF-β and inhibits gene expression in Hep3B cells. of the TGF-β signaling have been associated with a variety of including and Caestecker M.P. Piek E. Roberts A.B. J. Natl. Cancer Inst. 2000; 92: 1388-1402Crossref PubMed Google Scholar, N. J. 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