Interaction and Functional Cooperation of NF-κB with Smads

The transforming growth factor−β (TGF−β) family of cytokines regulates diverse cellular processes through control of the expression of target genes. Smad proteins are a recently identified family of signal transducers for members of the TGF−β family. Smads act as transcriptional regulators through binding to DNA and interacting with a variety of transcription factors. Here, we identified a κB site as a TGF-β-responsive region in the 3′-downstream junB promoter region. We also demonstrate that κB sites alone are sufficient to mediate immediate transcriptional activation by TGF-β. Transactivation of κB sites by TGF-β requires an intact NF-κB pathway, cooperates with known activators of this pathway, and is mediated by Smad family members. Furthermore, we show that Smad3 interacts with p52 in vivo. These data expand the model in which Smad proteins undergo multiple interactions with several transcription factors that could induce either activation or repression of gene expression. The transforming growth factor−β (TGF−β) family of cytokines regulates diverse cellular processes through control of the expression of target genes. Smad proteins are a recently identified family of signal transducers for members of the TGF−β family. Smads act as transcriptional regulators through binding to DNA and interacting with a variety of transcription factors. Here, we identified a κB site as a TGF-β-responsive region in the 3′-downstream junB promoter region. We also demonstrate that κB sites alone are sufficient to mediate immediate transcriptional activation by TGF-β. Transactivation of κB sites by TGF-β requires an intact NF-κB pathway, cooperates with known activators of this pathway, and is mediated by Smad family members. Furthermore, we show that Smad3 interacts with p52 in vivo. These data expand the model in which Smad proteins undergo multiple interactions with several transcription factors that could induce either activation or repression of gene expression. transforming growth factor-β bone morphogenetic proteins Smad DNA-binding element CREB-binding protein N-acetyl-Leu-Leu-norleucinal phorbol dibutyrate mitogen-activated protein/ extracellular signal-regulated kinase kinase-1 constitutively active TGF-β type I receptor (T204D) TGF-β activated kinase-1 Dulbecco's modified Eagle's medium nitrilotriacetic acid Members of the transforming growth factor-β (TGF-β)1 superfamily play an essential role in the control of proliferation, differentiation, and apoptosis and are therefore important for the development and maintenance of most tissues. Many members of this family have been isolated, and three major subfamilies have been described as follows: TGF-βs, activins/inhibins, and bone morphogenetic proteins (BMPs) (reviewed in Refs. 1Derynck R. Feng X.-H. 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Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar, 4Kretzschmar M. Massagué J. Curr. Opin. Genet. & Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar, 5Kawabata M. Miyazono K. J. Biochem. (Tokyo). 1999; 125: 9-16Crossref PubMed Scopus (58) Google Scholar, 6Zhang Y. Derynck R. Trends Cell Biol. 1999; 9: 274-279Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The receptor-regulated or pathway-restricted Smads (R-Smads) are directly phosphorylated by type I receptors. Smad2 and Smad3 propagate TGF-β and activin signals, whereas Smad1, Smad5, and possibly Smad8 are specific for BMP (2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar, 3Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar, 4Kretzschmar M. Massagué J. Curr. Opin. Genet. & Dev. 1998; 8: 103-111Crossref PubMed Scopus (433) Google Scholar, 5Kawabata M. Miyazono K. J. 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Chem. 1998; 273: 13053-13057Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Some of them, such as plasminogen activator inhibitor-1, include Smad-binding elements, and multimerization of these elements confers TGF-β inducibility (13Dennler S. Itoh S. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar). Oligonucleotide screening and the resolution of the crystal structure of Smad3 MH1 domain defined the tetranucleotide CAGA as their minimal Smad DNA-binding element (SBE) (14Shi Y. Wang Y.F. Jayaraman L. Yang H. Massagué J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 15Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar, 16Li J.-M. Nichols M.A. Chandrasekharan S. Xiong Y. Wang X.-F. J. Biol. Chem. 1995; 270: 26750-26753Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 17Datto M.B., Yu, Y. 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A. 1999; PubMed Scopus Google Scholar). interaction has also been between Smads and a of the family of transcriptional K. H. P. L. G. R. D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A with between and or Smad3 and However, in these interactions the of such as or receptor K. M. H. T. Kawabata M. Miyazono K. T. 1999; PubMed Scopus Google Scholar, J. Y. Y. M. M. K. T. Kawabata M. Miyazono K. S. 1999; PubMed Scopus Google Scholar). The family regulate transcription of several in or and cell growth A of and this transcription Jr., Annu. Rev. 1996; PubMed Scopus Google Scholar). members of the family have been identified in and NF-κB is in the of most cell as or NF-κB is an complex through association with the Jr., Annu. Rev. 1996; PubMed Scopus Google Scholar, S. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). are to of is is a signal for and of by the T. 1997; PubMed Scopus Google Scholar). NF-κB is to translocate to the and transcription of target genes. in indicate that different NF-κB have different transcriptional activation evidence that interactions between NF-κB and transcription factors the of NF-κB to regulate gene expression in a with S. Y. T. S. A. 1997; 94: PubMed Scopus Google Scholar) have that and CREB-binding protein to and cell In receptor interacts with with and NF-κB J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). sequences have been in the transcriptional of a of the family of transcription in the and the region of this gene G. D. S. A. PubMed Scopus Google Scholar, J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Itoh S. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) identified Smad-binding sequences in the junB promoter region mediate by several members of the TGF-β In this we have the transcriptional of the immediate by TGF-β. We the region of the gene and identified an NF-κB site as a region. We demonstrate that NF-κB sites are sufficient to mediate transcriptional activation by TGF-β. activation requires an intact NF-κB and is mediated by Smad family members. we show that Smad proteins with NF-κB These data a role for Smads as transcriptional in to their role as DNA-binding transcription factors. and J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), by and and as follows: for for for and their phosphorylated and with the minimal junB gene or with the minimal which NF-κB sites in of the minimal and by P. Smad1, and Smad4 by J. type and and their and constitutively active by R. whereas J. for NF-κB and by J. and cell in with The by J. in Eagle's medium with and in a with Cell by the as described L. Kon A. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. J. Biol. 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T. J.L. R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar and data which to the described by Itoh S. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). We by the 3′-downstream region of this which has been to mediate of expression by growth phorbol or G. D. S. A. PubMed Scopus Google Scholar, J. Biol. 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However, the TGF-β growth to the that TGF-β could induce the of a to the extracellular which for NF-κB We with TGF-β and which is a of of proteins to P. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). either or for either the κB or the We then the of κB in in with TGF-β or the of A also with which Smads and confers immediate to TGF-β (13Dennler S. Itoh S. ten Dijke P. Huet S. Gauthier J.M. 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We that the by TGF-β also activation of by the of and of phosphorylated that TGF-β induce activation of the with the between control and that has TGF-β signaling or cell these indicate that gene expression κB binding sites requires NF-κB Many such as phorbol and and the of TGF-β to NF-κB we effects in with with a of the with TGF-β of a gene the with a of TGF-β and of of transcription the several have revealed the of the family of in signal that NF-κB through R. S. L. R. Genes Dev. 1997; PubMed Scopus Google Scholar). the role of these proteins in the TGF-β κB we different members of the family and their and constitutively active We also the which is in as that of the TGF-β In expression of constitutively active and only activation also transcriptional by TGF-β. The of TGF-β to with of the NF-κB which NF-κB in such we of with with NF-κB and with an NF-κB in of with and revealed a whereas with or These data that TGF-β cooperates with complexes and the functional role of these in of the by TGF-β, we also members of the family alone and in different of NF-κB TGF-β to induce the NF-κB in with of NF-κB In TGF-β the of p52 and studies have in to the of NF-κB by factors which control the specific interaction of NF-κB with transcription factors is to an important that could gene activation or repression Jr., Annu. Rev. 1996; PubMed Scopus Google Scholar). The that the main transducing molecules of TGF-β could in such this we different Smads alone or in with NF-κB and their κB a and Smad4 induced transcription and to TGF-β, whereas Smad1, specific for with control of p52 or with Smads the which by growth These to indicate Smad4, and p52 in this specific TGF-β transcriptional the of these we the of interaction between the NF-κB and the Smads in vivo. in by The interaction between Smad3 and p52 in the of TGF−β by of constitutively active type I receptor However, in we to direct interactions between Smad4 and p52 These show association of Smad3 and p52 and indicate that of Smad3 and p52 the in the of their of the region of the revealed an in activation by several TGF-β family members Itoh S. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). 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These kinase could activation of NF-κB through However, we activation of by TGF-β in and of of either or an activator of N-terminal inducibility that interactions between Smads and NF-κB are for these Here, we evidence a role for Smads as transcriptional through interaction with NF-κB of Smad3 and Smad4 of κB sites in of Smads with NF-κB p52 and We also demonstrate interaction of p52 and Smad3 in the of which is by of an active TGF-β These are with data by J.M. Wang X.-F. Mol. Cell. Biol. 1998; 18: PubMed Google Scholar) that could activation of a promoter by DNA-binding is to interaction upon Smad3 and of Smad3 or of C-terminal also to bind These NF-κB to the of transcription factors that with interactions of Smads with transcription factors have been to through their binding to adjacent sites as described for or or through of binding and/or through a as described for receptor, receptor, or E. Wrana J.L. L. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. L. 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Biol. 1998; 18: PubMed Google Scholar), most of the effects of TGF−β and cytokines that NF-κB are to in of and (2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). In the identified an NF-κB site as a TGF-β-responsive region in the junB We demonstrate that NF-κB sites alone are sufficient to induce immediate transcriptional activation by TGF-β. activation requires an intact NF-κB and ligand-induced of Smads where they with NF-κB as transcriptional These data expand the that Smads undergo multiple interactions with different transcription factors to induce either activation or repression of gene expression. We and for and J. J. J. K. P. P. ten and for of and We also for