Transforming Growth Factor-β Signaling Through the Smad Pathway: Role in Extracellular Matrix Gene Expression and Regulation
2002; Elsevier BV; Volume: 118; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2002.01641.x
ISSN1523-1747
AutoresFranck Verrecchia, Alain Mauviel,
Tópico(s)Kruppel-like factors research
ResumoTransforming growth factor (TGF)-β represents a prototype of multifunctional cytokine. Its broad activities include, among others, context-specific inhibition or stimulation of cell proliferation, control of extracellular matrix (ECM) synthesis and degradation, control of mesenchymal–epithelial interactions during embryogenesis, mediation of cell and tissue responses to injury, control of carcinogenesis, and modulation of immune functions. Regulation of production and turnover of ECM components is essential for tissue homeostasis and function. TGF-β exerts its effects on cell proliferation, differentiation, and migration in part through its capacity to modulate the deposition of ECM components. Specifically, TGF-β isoforms have the ability to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Deregulation of these functions is associated with abnormal connective tissue deposition, as observed, for example, during scarring or fibrotic processes. In this review we discuss the current understanding of the signaling mechanisms used by TGF-β to elicit its effects on target genes, focusing primarily on Smad proteins and their role in the transcriptional regulation of ECM gene expression. Other signaling mechanisms, such as the MAP/SAP kinase or Ras pathways, although potentially important for transmission of some of the TGF-β signals, will not be described. Transforming growth factor-β (TGF-β) plays a critical role in the regulation of extracellular matrix gene expression. Its overexpression is believed to contribute to the development of tissue fibrosis. The recent identification of Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, has increased our understanding of the molecular mechanisms underlying TGF-β action. This review focuses primarily on the mechanisms underlying Smad modulation of gene expression and how they relate to wound healing. Potential implications for the development of therapeutic approaches against tissue fibrosis are discussed. Transforming growth factor (TGF)-β represents a prototype of multifunctional cytokine. Its broad activities include, among others, context-specific inhibition or stimulation of cell proliferation, control of extracellular matrix (ECM) synthesis and degradation, control of mesenchymal–epithelial interactions during embryogenesis, mediation of cell and tissue responses to injury, control of carcinogenesis, and modulation of immune functions. Regulation of production and turnover of ECM components is essential for tissue homeostasis and function. TGF-β exerts its effects on cell proliferation, differentiation, and migration in part through its capacity to modulate the deposition of ECM components. Specifically, TGF-β isoforms have the ability to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Deregulation of these functions is associated with abnormal connective tissue deposition, as observed, for example, during scarring or fibrotic processes. In this review we discuss the current understanding of the signaling mechanisms used by TGF-β to elicit its effects on target genes, focusing primarily on Smad proteins and their role in the transcriptional regulation of ECM gene expression. Other signaling mechanisms, such as the MAP/SAP kinase or Ras pathways, although potentially important for transmission of some of the TGF-β signals, will not be described. Transforming growth factor-β (TGF-β) plays a critical role in the regulation of extracellular matrix gene expression. Its overexpression is believed to contribute to the development of tissue fibrosis. The recent identification of Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, has increased our understanding of the molecular mechanisms underlying TGF-β action. This review focuses primarily on the mechanisms underlying Smad modulation of gene expression and how they relate to wound healing. Potential implications for the development of therapeutic approaches against tissue fibrosis are discussed. LTGF-β-binding protein latent precursor molecules The TGF-β super-family of growth factors includes the various forms of TGF-β, bone morphogenic proteins (BMP), nodals, activins, the anti-Mullerian hormone, and many other structurally related factors found in vertebrates, insects, and nematodes (Massagué, 1998Massagué J. TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3848) Google Scholar). There are three mammalian isoforms of TGF-β (TGF-β1–3), structurally nearly identical, with a knot motif composed of six cysteine residues joined together by three intrachain disulfide bonds that stabilize β-sheet bands. One free cysteine forms an interchain disulfide bond with an identical monomeric chain to generate the mature TGF-β dimer. TGF-β are secreted as latent precursor molecules (LTGF-β) requiring activation into a mature form for receptor binding and subsequent activation of signal transduction pathways. The LTGF-β molecules consist of 390–414 amino acids. They contain an amino-terminal hydrophobic signal peptide region, the latency-associated peptide (LAP) region, of 249 residues, and the C-terminal, potentially bioactive region that contains 112 amino acids per monomer. LTGF-β is usually secreted as a large latent complex covalently bound via the LAP region to LTGF-β-binding protein (LTBP;Roberts, 1998Roberts A.B. Molecular and cell biology of TGF-beta.Miner Electrolyte Metab. 1998; 24: 111-119Crossref PubMed Scopus (336) Google Scholar) or as a small latent complex without LTBP. The LAP confers latency to the complex, whereas LTBP serves to bind TGF-β to the ECM and to enable its proteolytic activation (Nunes et al., 1997Nunes I. Gleizes P.E. Metz C.N. Rifkin D.B. Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta.J Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (329) Google Scholar). Activation of TGF-β is a complex process involving conformational changes of LTGF-β, induced by either cleavage of the LAP by various proteases such as plasmin, thrombin, plasma transglutaminase, or endoglycosylases, or by physical interactions of the LAP with other proteins, such as thrombospondin-1, leading to the release of bioactive, mature, TGF-β (Roberts, 1998Roberts A.B. Molecular and cell biology of TGF-beta.Miner Electrolyte Metab. 1998; 24: 111-119Crossref PubMed Scopus (336) Google Scholar). Upon activation, TGF-β superfamily members initiate their cellular action by binding to serine/threonine kinase receptors. The TGF-β receptor family consists of two structurally similar subfamilies, type I and type II receptors, with small cysteine-rich extracellular regions and intracellular portions consisting mainly of their kinase domains. Type I receptors have a region rich in glycine and serine residues (GS domain) preceding the receptor kinase domain (Huse et al., 1999Huse M. Chen Y.G. Massagué J. Kuriyan J. Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12.Cell. 1999; 96: 425-436Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). To exert their signal, type II and type I receptors act in sequence: TGF-β first binds to the type II receptor (TβRII), which occurs in the cell membrane in an oligomeric form with intrinsic kinase activity; TGF-β type I receptor (TβRI) is then recruited and phosphorylated in its GS domain by TβRII, leading to activation of its kinase activity and subsequent intracellular signaling (Massagué, 1998Massagué J. TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3848) Google Scholar;Piek et al., 1999Piek E. Heldin C.H. Ten Dijke P. Specificity, diversity, and regulation in TGF-beta superfamily signaling.FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (727) Google Scholar). Betaglycan, a transmembrane proteoglycan also known as TβRIII (Lopez-Casillas et al., 1991Lopez-Casillas F. Cheifetz S. Doody J. Andres J.L. Lane W.S. Massagué J. Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system.Cell. 1991; 67: 785-795Abstract Full Text PDF PubMed Scopus (528) Google Scholar), allows high-affinity binding of TGF-β to TβRII but does not itself transduce signal (Rodriguez et al., 1995Rodriguez C. Chen F. Weinberg R.A. Lodish H.F. Cooperative binding of transforming growth factor (TGF)-beta 2 to the types I and II TGF-beta receptors.J Biol Chem. 1995; 270: 15919-15922Crossref PubMed Scopus (74) Google Scholar;Brown et al., 1999Brown C.B. Boyer A.S. Runyan R.B. Barnett J.V. Requirement of type III TGF-beta receptor for endocardial cell transformation in the heart.Science. 1999; 283: 2080-2082Crossref PubMed Scopus (317) Google Scholar). Following ligand activation, signaling from TβRI to the nucleus occurs predominantly by phosphorylation of cytoplasmic mediators belonging to the Smad family (Piek et al., 1999Piek E. Heldin C.H. Ten Dijke P. Specificity, diversity, and regulation in TGF-beta superfamily signaling.FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (727) Google Scholar;Massagué and Chen, 2000Massagué J. Chen Y.G. Controlling TGF-beta signaling.Genes Dev. 2000; 14: 627-644PubMed Google Scholar;Massagué and Wotton, 2000Massagué J. Wotton D. Transcriptional control by the TGF-beta/Smad signaling system.EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). Type I receptors specifically recognize and phosphorylate the ligand-specific receptor-activated Smad (R-Smad, Figure 1). The latter are recruited to activated TβRI by a membrane bound cytoplasmic protein called SARA (Smad Anchor for Receptor Activation;Tsukazaki et al., 1998Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor.Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). R-Smad include Smad1, Smad5, and Smad8 downstream of the BMP, and Smad2 and Smad3 downstream of TGF-β and activin. They all consist of two conserved Mad-homology (MH) domains that form globular structures separated by a linker region (Shi et al., 1997Shi Y. Hata A. Lo R.S. Massagué J. Pavletich N.P. A structural basis for mutational inactivation of the tumour suppressor Smad4.Nature. 1997; 388: 87-93Crossref PubMed Scopus (357) Google Scholar). The N-terminal MH1 domain has DNA-binding activity, whereas the C-terminal MH2 domain has protein-binding properties. Phosphorylation of R-Smad by type I receptors occurs principally on two serine residues within a conserved –SS(M/V)S–motif at their C-terminus (Massagué and Chen, 2000Massagué J. Chen Y.G. Controlling TGF-beta signaling.Genes Dev. 2000; 14: 627-644PubMed Google Scholar;Massagué and Wotton, 2000Massagué J. Wotton D. Transcriptional control by the TGF-beta/Smad signaling system.EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). Upon phosphorylation by TβRI, R-Smad form heteromeric complexes with co-Smad, such as Smad4. Co-Smad act as a convergent node in the Smad pathways downstream of the TGF-β superfamily receptors, complexing R-Smad, regardless of the TGF-β ligand specificity of the latter. R-Smad/Smad4 complexes are then translocated into the nucleus by a mechanism involving the cytoplasmic protein importin (Xiao et al., 2000Xiao Z. Liu X. Lodish H.F. Importin beta mediates nuclear translocation of Smad 3.J Biol Chem. 2000; 275: 23425-23428Crossref PubMed Scopus (139) Google Scholar;Kurisaki et al., 2001Kurisaki A. Kose S. Yoneda Y. Heldin C.H. Moustakas A. Transforming growth factor-beta induces nuclear import of Smad3 in an Importin-beta1 and Ran-dependent manner.Mol Biol Cell. 2001; 12: 1079-1091Crossref PubMed Scopus (148) Google Scholar). They may then function as transcription factors, binding DNA either directly or in association with other DNA binding proteins (Piek et al., 1999Piek E. Heldin C.H. Ten Dijke P. Specificity, diversity, and regulation in TGF-beta superfamily signaling.FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (727) Google Scholar;Massagué and Chen, 2000Massagué J. Chen Y.G. Controlling TGF-beta signaling.Genes Dev. 2000; 14: 627-644PubMed Google Scholar;Massagué and Wotton, 2000Massagué J. Wotton D. Transcriptional control by the TGF-beta/Smad signaling system.EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). Maximal affinity of recombinant Smad3 and Smad4 to DNA is observed with the 5 bp sequence, CAGAC (Shi et al., 1998Shi Y. Wang Y.F. Jayaraman L. Yang H. Massagué J. Pavletich N.P. Crystal structure of a Smad MH1 domain bound to DNA. insights on DNA binding in TGF-beta signaling.Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar;Zawel et al., 1998Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Human Smad3 and Smad4 are sequence-specific transcription activators.Mol Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar). Smad2, on the other hand, does not bind DNA directly, requiring a nuclear DNA-binding protein of the Fast family to bind DNA, in association with Smad4, and activate transcription in response to TGF-β and activin (Chen et al., 1997Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Smad4 and FAST-1 in the assembly of activin-responsive factor.Nature. 1997; 389: 85-89Crossref PubMed Scopus (483) Google Scholar;Labbé et al., 1998Labbé E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Smad2 and Smad3 positively and negatively regulate TGF beta-dependent transcription through the forkhead DNA-binding protein FAST2.Mol Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar;Liu et al., 1999Liu B. Dou C.L. Prabhu L. Lai E. FAST-2 is a mammalian winged-helix protein which mediates transforming growth factor beta signals.Mol Cell Biol. 1999; 19: 424-430Crossref PubMed Scopus (81) Google Scholar). A third group of Smad proteins, the inhibitory Smad (I-Smad), such as Smad6 or Smad7, prevent R-Smad phosphorylation and subsequent nuclear translocation of R-Smad/Smad4 heterocomplexes (Imamura et al., 1997Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Smad6 inhibits signalling by the TGF-beta superfamily.Nature. 1997; 389: 622-626Crossref PubMed Scopus (841) Google Scholar;Nakao et al., 1997Nakao A. Afrakhte M. Moren A. et al.Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.Nature. 1997; 389: 631-635Crossref PubMed Scopus (1489) Google Scholar). Following target gene transcription, Smad complexes are released from the chromatin and undergo ubiquitination, followed by proteasomal degradation (Zhu et al., 1999Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation.Nature. 1999; 400: 687-693Crossref PubMed Scopus (654) Google Scholar). A summary of the various steps of TGF-β signaling through the Smad cascade is provided in Figure 2. Several cross-signaling mechanisms have been described that implicate Smad proteins. For example, Smad3 interacts with the vitamin D receptor to mediate the effect of TGF-β on vitamin D3-induced transcription (Yanagisawa et al., 1999Yanagisawa J. Yanagi Y. Masuhiro Y. et al.Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators.Science. 1999; 283: 1317-1321https://doi.org/10.1126/science.283.5406.1317Crossref PubMed Scopus (405) Google Scholar). Smad3 also interacts with TFE3 and Sp1 to activate transcription from the PAI-1 and p21cip promoters, respectively (Hua et al., 1998Hua X. Liu X. Ansari D.O. Lodish H.F. Synergistic cooperation of TFE3 and smad proteins in TGF-beta-induced transcription of the plasminogen activator inhibitor-1 gene.Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (251) Google Scholar;Moustakas and Kardassis, 1998Moustakas A. Kardassis D. Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members.Proc Natl Acad Sci USA. 1998; 95: 6733-6738Crossref PubMed Scopus (316) Google Scholar). Smad/AP-1 interactions have also been reported, which may lead to either additive (Zhang et al., 1998Zhang Y. Feng X.H. Derynck R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription.Nature. 1998; 394: 909-913Crossref PubMed Scopus (656) Google Scholar;Liberati et al., 1999Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.F. Smad bind directly to the Jun family of AP-1 transcription factors.Proc Natl Acad Sci USA. 1999; 96: 4844-4849Crossref PubMed Scopus (267) Google Scholar;Verrecchia et al., 2001cVerrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (153) Google Scholar) or antagonistic (Verrecchia et al., 2000Verrecchia F. Pessah M. Atfi A. Mauviel A. Tumor necrosis factor-alpha inhibits transforming growth factor-beta/Smad signaling in human dermal fibroblasts via AP-1 activation.J Biol Chem. 2000; 275: 30226-30231Crossref PubMed Scopus (149) Google Scholar;Verrecchia et al., 2001cVerrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (153) Google Scholar) activities on gene transactivation. The outcome is dependent on the structure of target promoters, as not only the presence of AP-1- and/or Smad-specific cis-elements, but also their respective positions, may influence the transcriptional response (Liberati et al., 1999Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.F. Smad bind directly to the Jun family of AP-1 transcription factors.Proc Natl Acad Sci USA. 1999; 96: 4844-4849Crossref PubMed Scopus (267) Google Scholar;Verrecchia et al., 2001cVerrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (153) Google Scholar). For example, in a promoter context exhibiting Smad-specific boxes distant from functional AP-1 sites, such as in the PAI-1 and c-jun promoters, transcriptional cooperation between Smad and Jun has been observed, without formation of Smad/Jun heterocomplexes on DNA (Liberati et al., 1999Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.F. Smad bind directly to the Jun family of AP-1 transcription factors.Proc Natl Acad Sci USA. 1999; 96: 4844-4849Crossref PubMed Scopus (267) Google Scholar;Verrecchia et al., 2001cVerrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (153) Google Scholar). In the context of the COL7A1 promoter that possesses an AP-1 site located within a bipartite Smad-specific TGF-β-response element, inhibition of Smad-driven transactivation by Jun members was observed, again without formation of Smad/Jun heterocomplexes on DNA. In the latter case, Jun was shown to act as a direct repressor of Smad function, binding to and blocking transcriptional activation domains intrinsic to Smad3, and preventing its binding to DNA (Verrecchia et al., 2001cVerrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (153) Google Scholar). Another possibility for Smad to interfere with other transcription factors occurs through direct interactions with transcriptional coactivators such as the Ski-interacting protein (SKIP) or CBP/p300. The latter proteins modify transcription either by altering chromatin structure so that the underlying DNA sequences are exposed to the transcriptional apparatus (Workman and Kingston, 1998Workman J.L. Kingston R.E. Alteration of nucleosome structure as a mechanism of transcriptional regulation.Annu Rev Biochem. 1998; 67: 545-579Crossref PubMed Scopus (935) Google Scholar), or by directly recruiting the RNA polymerase II holoenzyme to the promoter (Snowden and Perkins, 1998Snowden A.W. Perkins N.D. Cell cycle regulation of the transcriptional coactivators p300 and CREB binding protein.Biochem Pharmacol. 1998; 55: 1947-1954Crossref PubMed Scopus (53) Google Scholar). Smad–CBP/p300 interaction is required for transcriptional activation of several TGF-β-dependent promoters (Feng et al., 1998Feng X.H. Zhang Y. Wu R.Y. Derynck R. The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for smad3 in TGF-beta-induced transcriptional activation.Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (442) Google Scholar;Janknecht et al., 1998Janknecht R. Wells N.J. Hunter T. TGF-beta-stimulated cooperation of smad proteins with the coactivators CBP/p300.Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (424) Google Scholar;Nishihara et al., 1998Nishihara A. Hanai J.I. Okamoto N. Yanagisawa J. Kato S. Miyazono K. Kawabata M. Role of p300, a transcriptional coactivator, in signalling of TGF-beta.Genes Cells. 1998; 3: 613-623Crossref PubMed Scopus (130) Google Scholar;Pouponnot et al., 1998Pouponnot C. Jayaraman L. Massagué J. Physical and functional interaction of SMADs and p300/CBP.J Biol Chem. 1998; 273: 22865-22868Crossref PubMed Scopus (283) Google Scholar;Shen et al., 1998Shen X. Hu P.P. Liberati N.T. Datto M.B. Frederick J.P. Wang X.F. TGF-beta-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein.Mol Biol Cell. 1998; 9: 3309-3319Crossref PubMed Scopus (182) Google Scholar). SKIP, a nuclear hormone receptor coactivator, enhances TGF-β-dependent transactivation by interacting with Smad2 and Smad3 proteins (Leong et al., 2001Leong G.M. Subramaniam N. Figueroa J. Flanagan J.L. Hayman M.J. Eisman J.A. Kouzmenko A.P. Ski-interacting protein interacts with smad proteins to augment transforming growth factor-beta-dependent transcription.J Biol Chem. 2001; 276: 18243-18248Crossref PubMed Scopus (74) Google Scholar). In addition, several nuclear proteins, including the viral oncoprotein E1A (Nishihara et al., 1999Nishihara A. Hanai J. Imamura T. Miyazono K. Kawabata M. E1A inhibits transforming growth factor-beta signaling through binding to Smad proteins.J Biol Chem. 1999; 274: 28716-28723Crossref PubMed Scopus (90) Google Scholar), the proto-oncogenes c-Ski and SnoN (Akiyoshi et al., 1999Akiyoshi S. Inoue H. Hanai J. Kusanagi K. Nemoto N. Miyazono K. Kawabata M. c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads.J Biol Chem. 1999; 274: 35269-35277Crossref PubMed Scopus (329) Google Scholar;Stroschein et al., 1999Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.Science. 1999; 286: 771-774https://doi.org/10.1126/science.286.5440.771Crossref PubMed Scopus (427) Google Scholar), TGIF (Wotton et al., 1999Wotton D. Lo R.S. Lee S. Massagué J. A Smad transcriptional corepressor.Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), Snip-1 (Kim et al., 2000Kim R.H. Wang D. Tsang M. et al.A novel smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction.Genes Dev. 2000; 14: 1605-1616PubMed Google Scholar), SIP1 (Verschueren et al., 1999Verschueren K. Remacle J.E. Collart C. et al.SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5′-CACCT sequences in candidate target genes.J Biol Chem. 1999; 274: 20489-20498Crossref PubMed Scopus (390) Google Scholar), and c-Jun (Verrecchia et al., 2000Verrecchia F. Pessah M. Atfi A. Mauviel A. Tumor necrosis factor-alpha inhibits transforming growth factor-beta/Smad signaling in human dermal fibroblasts via AP-1 activation.J Biol Chem. 2000; 275: 30226-30231Crossref PubMed Scopus (149) Google Scholar) compete for R-Smad/Smad4 binding to CBP or p300, thereby interfering with Smad-dependent gene expression. Despite extensive research directed toward the elucidation of the role played by Smad proteins downstream of TGF-β, very few direct Smad target genes have been characterized. Indeed, it was shown that transactivation of the fibronectin gene downstream of TGF-β is a JNK-specific, Smad-independent, mechanism (Hocevar et al., 1999Hocevar B.A. Brown T.L. Howe P.H. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway.EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar), suggesting that pathways that alternate to the Smad cascade also play an important part in the modulation of ECM gene expression by TGF-β. Recently, using a combined cDNA microarray/promoter transactivation approach, we have identified several new Smad gene targets among which are COL1A1, COL3A1, COL5A2, COL6A1, COL6A3, and TIMP-1. Most notably, these data indicate that the Smad signaling pathway is crucial for simultaneous activation of several fibrillar collagen genes by TGF-β. About 60 other ECM-related genes were also identified as immediate-early genes targets downstream of TGF-β (Verrecchia et al., 2001aVerrecchia F. Chu M.L. Mauviel A. Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.J Biol Chem. 2001; 276: 17058-17062Crossref PubMed Scopus (508) Google Scholar). Our group identified the first described genuine Smad binding sequence (SBS) within the human COL7A1 promoter (Vindevoghel et al., 1998aVindevoghel L. Lechleider R.J. Kon A. de Caestecker M.P. Uitto J. Roberts A.B. Mauviel A. SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.Proc Natl Acad Sci USA. 1998; 95: 14769-14774Crossref PubMed Scopus (155) Google Scholar, Vindevoghel et al., 1998bVindevoghel L. Kon A. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-beta.J Biol Chem. 1998; 273: 13053-13057Crossref PubMed Scopus (97) Google Scholar). Specifically, TGF-β upregulation of COL7A1 gene expression is mediated by rapid and transient binding of a Smad-containing complex to the region -496/-444 of the COL7A1 promoter, a bipartite element consisting of a CAGA tandem repeat in its 5′ end, and a Medea-like (Drosophila Smad4 homolog) binding site in 3′, both framing a potential AP-1 binding site whose integrity is not necessary for either Smad-driven transactivation or Smad/DNA complex formation (Verrecchia et al., 2001cVerrecchia F. Vindevoghel L. Lechleider R.J. Uitto J. Roberts A.B. Mauviel A. Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (153) Google Scholar). A few other ECM-related genes whose expression is modulated by TGF-β have also been identified as potential Smad targets, and CAGA-like elements have been identified within their promoter regions, that bind Smad complexes. These genes include PAI-1, COL1A2, and the β5 integrin gene (INTB5) (Chen et al., 1998Chen S.J. Artlett C.M. Jimenez S.A. Varga J. Modulation of human alpha1(I) procollagen gene activity by interaction with Sp1 and Sp3 transcription factors in vitro.Gene. 1998; 215: 101-110Crossref PubMed Scopus (69) Google Scholar;Dennler et al., 1998Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1518) Google Scholar;Lai et al., 2000Lai C.F. Feng X. Nishimura R. Teitelbaum S.L. Avioli L.V. Ross F.P. Cheng S.L. Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression via Sp1 and Smad signaling.J Biol Chem. 2000; 275: 36400-36406Crossref PubMed Scopus (95) Google Scholar). In these three cases, TGF-β-induced promoter activation may also involve functional interactions between Smad and Sp1, a mechanism that has also been shown to be critical for activation of p21/WAF1/CIP1 (Pardali et al., 2000Pardali K. Kurisaki A. Moren A. ten Dijke P. Kardassis D. Moustakas A. Role of Smad proteins and transcription factor Sp1 in p21 (Waf1/Cip1) regulation by transforming growth factor-beta.J Biol Chem. 2000; 275: 29244-29256Crossref PubMed Scopus (334) Google Scholar) and p15/Ink4B (Feng et al., 2000Feng X.H. Lin X. Derynck R. Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15 (Ink4B) transcription in response to TGF-beta.EMBO J. 2000; 19: 5178-5193Crossref PubMed Scopus (331) Google Scholar) by TGF-β in the control of cell proliferation. Regarding COL1A2, initial observations demonstrated that a 135 bp region of the COL1A2 promoter within 330 bp of the transcription start site confers responsiveness to TGF-β (Inagaki et al., 1994Inagaki Y. Truter S. Ramirez F. Transforming growth factor
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