Regulation of Plasminogen Activator Inhibitor-1 Expression by Transforming Growth Factor-β-induced Physical and Functional Interactions between Smads and Sp1

Members of the transforming growth factor-β (TGF-β) superfamily mediate a broad range of biological activities by regulating the expression of target genes. Smad proteins play a critical role in this process by binding directly to the promoter elements and/or associating with other transcription factors. TGF-β1 up-regulates several genes transcriptionally through Sp1 binding sites; however, the mechanism of TGF-β induction of gene expression through Sp1 sites is largely unknown. Here we report the identification of a novel 38-base pair TGF-β-responsive element in the human plasminogen activator inhibitor-1 (PAI-1) promoter, which contains two Sp1 binding sites, and is required for TGF-β-induced Smad-dependent transcriptional activation. Three canonical Sp1 binding sites also support strong transcriptional activation by TGF-β and Smads from a minimal heterologous promoter. TGF-β induction of PAI-1 and p21 is blocked by the Sp1 inhibitor mithramycin, implicating Sp1 in the in vivo regulation of these genes by TGF-β. We show that the association between endogenous Sp1 and Smad3 is induced by TGF-β in several cell lines; however, Smad4 shows constitutive interaction with Sp1. These data provide novel insights into the mechanism by which TGF-β up-regulates several gene expression by activating Sp1-dependent transcription through the induction of Smad/Sp1 complex formation. Members of the transforming growth factor-β (TGF-β) superfamily mediate a broad range of biological activities by regulating the expression of target genes. Smad proteins play a critical role in this process by binding directly to the promoter elements and/or associating with other transcription factors. TGF-β1 up-regulates several genes transcriptionally through Sp1 binding sites; however, the mechanism of TGF-β induction of gene expression through Sp1 sites is largely unknown. Here we report the identification of a novel 38-base pair TGF-β-responsive element in the human plasminogen activator inhibitor-1 (PAI-1) promoter, which contains two Sp1 binding sites, and is required for TGF-β-induced Smad-dependent transcriptional activation. Three canonical Sp1 binding sites also support strong transcriptional activation by TGF-β and Smads from a minimal heterologous promoter. TGF-β induction of PAI-1 and p21 is blocked by the Sp1 inhibitor mithramycin, implicating Sp1 in the in vivo regulation of these genes by TGF-β. We show that the association between endogenous Sp1 and Smad3 is induced by TGF-β in several cell lines; however, Smad4 shows constitutive interaction with Sp1. These data provide novel insights into the mechanism by which TGF-β up-regulates several gene expression by activating Sp1-dependent transcription through the induction of Smad/Sp1 complex formation. extracellular matrix plasminogen activator inhibitor-1 transforming growth factor-β rat intestinal epithelial hemagglutinin dominant negative type II receptor histone deacetylase 1 Extracellular matrix (ECM)1 remodeling plays an important role in several biological processes ranging from cell proliferation and differentiation to cell adhesion, migration, and tissue morphogenesis (1Matrisian L.M. Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1555) Google Scholar). ECM is a complex and dynamic meshwork of several proteoglycans and proteins, and its degradation is inhibited by TGF-β. TGF-β stabilizes ECM by down-regulating the expression of the ECM proteases and by stimulating the expression of some ECM protease inhibitors, including the plasminogen activator inhibitor (PAI)-1 (2Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3070) Google Scholar). PAI-1 is the primary inhibitor of both tissue-type and urokinase-type plasminogen activator. The induction of PAI-1 mRNA and protein by TGF-β has been observed in several types of cultured cells (3Westerhausen Jr., D.R. Hopkins W.E. Billadello J.J. J. Biol. Chem. 1991; 266: 1092-1100Abstract Full Text PDF PubMed Google Scholar). Recent work has identified potential consensus Smad3-Smad4 DNA binding motifs in the distal region of the PAI-1 promoter, AG(C/A)CAGACA (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar) and AGACAAGGTTGT (5Song C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), which contain the 4-base pair AGAC element in common and have been shown to mediate TGF-β-transcriptional induction. Information about the mechanism of TGF-β-mediated activation of transcription of target genes is increasing rapidly (6Massague J. Wotton D. EMBO J. 2000; 19: 745-754Crossref Google Scholar). Although several studies demonstrate that Smad3-Smad4 binding sites are required for TGF-β-mediated promoter regulation, they do not completely explain the broad spectrum of TGF-β responsiveness in activating target genes. Biochemical studies, under conditions of overexpression, have suggested that a Smad3-Smad4 complex and an AP-1 complex synergize in the transcriptional activation from the c-Jun promoter (7Wong C. Rougier-Chapman E.M. Frederick J.P. Datto M.B. Liberati N.T. Li J.-M. Wang X.-F. Mol. Cell. Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (231) Google Scholar). Cooperation between Smad2- or Smad3-Smad4 complexes and other sequence-specific DNA-binding proteins such as FAST1 and FAST2 has been demonstrated to regulate TGF-β-responsive promoters (8- 10). Although no physical association between the basic helix-loop-helix protein TFE3 and Smad3/Smad4 has been shown, all three proteins interact with a segment of PAI-1 promoter to induce transcription in response to TGF-β (11Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (261) Google Scholar). AML-Smad complexes target the IgA promoter in response to TGF-β (12Pardali E. Xie X.Q. Tsapogas P. Itoh S. Arvanitidis K. Heldin C.H. ten Dijke P. Grundstorm T. Sideras P. J. Biol. Chem. 2000; 275: 3552-3560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Several reports demonstrate that Smads can also activate transcription from specific promoters in response to TGF-β by interacting with non-DNA-binding proteins (6Massague J. Wotton D. EMBO J. 2000; 19: 745-754Crossref Google Scholar). Functional analysis of the promoters of some TGF-β target genes has led to the identification of putative cis-acting TGF-β response elements and transacting factors associated with those elements. TGF-β1 activates the transcription of several cell cycle regulatory genes, including the cyclin-dependent kinase inhibitors p21 and p15 genes (13Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 14Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (324) Google Scholar, 15Li J.-M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.-F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar) or genes involved in extracellular matrix formation including the α2(I) collagen, fibronectin, and PAI-1 genes (2Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3070) Google Scholar, 3Westerhausen Jr., D.R. Hopkins W.E. Billadello J.J. J. Biol. Chem. 1991; 266: 1092-1100Abstract Full Text PDF PubMed Google Scholar, 16Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 17Ignotz R.A. Massague J. J. Biol. Chem. 1986; 261: 4337-4345Abstract Full Text PDF PubMed Google Scholar, 18Keeton M.R. Curriden S.A. Zonneveld A.V. Loskutoff D.J. J. Biol. Chem. 1991; 266: 23048-23052Abstract Full Text PDF PubMed Google Scholar). Some of these studies have provided convincing evidence that Sp1 consensus sites in several promoters are required to support TGF-β-mediated activation. However, it is not known how Sp1 binding sites in those promoters mediate the TGF-β transactivating signal or how TGF-β signaling and the transcription factor Sp1 is linked to the TGF-β-regulated expression of various genes. In the present study, we elucidate a novel mechanism by which TGF-β up-regulates gene expression through Sp1 binding sites. We have identified a 38-base pair element containing two Sp1 binding sites in the proximal region of PAI-1 promoter, which is required, in part, for TGF-β-mediated induction of this promoter. Mithramycin, an inhibitor of Sp1-DNA binding, blocks TGF-β stimulation of PAI-1 and p21 proteins. Here, we show that Sp1 interacts with Smad3 and Smad4 in vivo and that the association between endogenous Smad3 and Sp1 proteins is induced by TGF-β in several cell lines. Our results indicate that TGF-β-induced physical and functional interactions between Sp1 and Smads may explain how TGF-β induces gene expression through Sp1 binding sites. Mv1Lu, HepG2, COS-1, 293, NIH 3T3, and rat intestinal epithelial (RIE) cells were obtained from the American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Mv1Lu and HepG2 cells were supplemented with nonessential amino acids. Cells were transfected as described previously (19Datta P.K. Moses H.L. Mol. Cell. Biol. 2000; 20: 3157-3167Crossref PubMed Scopus (149) Google Scholar). COS-1 cells were transfected with expression constructs as indicated. After 40 h cells were washed, scraped, and solubilized in lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10 mm EDTA, 0.5% Nonidet P-40, 0.5 mmdithiothreitol, 5 mm sodium fluoride, 0.5 mmsodium orthovanadate, 1.0 mm phenylmethylsulfonyl fluoride, 2 μg (each) of leupeptin, pepstatin, and aprotinin per ml). Cleared cell lysates were incubated with anti-FLAG or for 2 h by with protein for 1 were by as described previously (19Datta P.K. Moses H.L. Mol. Cell. Biol. 2000; 20: 3157-3167Crossref PubMed Scopus (149) Google Scholar). in and and were for and and were for endogenous proteins the cells with TGF-β1 for h and for h lysis in serum. PAI-1 promoter and have been described (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar, Li Y. Mol. Cell. Biol. PubMed Scopus Google Scholar, M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). HepG2 and Mv1Lu cells were transfected with various constructs as and the and In of DNA were cells were incubated in medium containing fetal bovine with and as indicated. and were as described previously (19Datta P.K. Moses H.L. Mol. Cell. Biol. 2000; 20: 3157-3167Crossref PubMed Scopus (149) Google Scholar). to to for in studies have shown that the Sp1 binding sites present in several promoters are required for TGF-β-induced gene expression (13Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 14Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (324) Google Scholar, 15Li J.-M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.-F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar, 16Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). how TGF-β activates target genes through Sp1 binding sites, we the of TGF-β and Smads a promoter containing three Sp1 consensus sites of a gene 1 of this into HepG2 cells in a of which in response to TGF-β. of Smad3 induced the promoter both in the and of TGF-β 1 of or Smad4 the promoter in the or of TGF-β. of or Smad4 with Smad3 not transcriptional activation. TGF-β induces of the of which is for signaling X. Y. E. R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). a the type in promoter 1 We observed of TGF-β and Smads other heterologous promoters containing or two Sp1 sites not These data that Smad3 is required for TGF-β-mediated activation of Sp1 sites of Smad DNA binding Sp1-dependent is involved in the TGF-β stimulation of PAI-1 gene we of the PAI-1 promoter. of the PAI-1 promoter region and a of regulatory elements in the proximal promoter is shown in 1 A. have shown that the between and contains three Smad binding elements involved in the induction of PAI-1 promoter in response to TGF-β (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar, C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (261) Google Scholar). are regulatory elements in the proximal region and including a response AP-1 and two Sp1 sites M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). in or binding of Sp1 not shown and M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) to those sites. HepG2 cells were transfected with these promoter constructs and Smad3 expression and for to TGF-β and promoter constructs and which contain three strong induction by TGF-β and/or Smad3 as 1 of the promoter from to in a in promoter and this promoter by TGF-β and Smad3 the of TGF-β-responsive elements this proximal The which contains a in the Sp1 in promoter in the of TGF-β and the to induced by TGF-β and in the Sp1 in of promoter 1 However, this by TGF-β and Smad3 in both Sp1 sites in a of promoter both in the and of TGF-β and these data that both Sp1 sites in the proximal region to are required, in part, for induction of the PAI-1 promoter in response to TGF-β. The type human PAI-1 promoter contains three Smad binding elements in the distal region (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar). the of the Sp1 consensus sites in the proximal region in transcription of the PAI-1 promoter by we type PAI-1 promoter and a promoter all three Smad binding sites are The type promoter, containing distal and proximal induced by TGF-β both in and of Smad3 1 all three Smad sites were the promoter of induced by TGF-β in the and of Smad3 with the promoter these data demonstrate that in to the Smad binding sites, Sp1 consensus sites in the proximal region are also important for the of TGF-β induction of the PAI-1 the of Smad proteins in the TGF-β-induced regulation of PAI-1 promoter through Sp1 sites in the proximal HepG2 cells were with the with expression for Smad proteins and the dominant negative type II receptor TGF-β induced the promoter by of Smad3 in a in the both in the and of TGF-β 2 In a induction of the promoter by or Smad4 of and/or Smad4 with Smad3 not have promoter of Smad3 through TGF-β-induced required in the induction of the minimal PAI-1 promoter, in the blocked the of Smad3 to the promoter in or of TGF-β. or which TGF-β TGF-β-mediated induction of transcription from the minimal promoter. These in with the results indicate that Smad3 is in the of PAI-1 promoter by TGF-β through Sp1 binding sites. results support the that this TGF-β-mediated transcriptional activation of the not the DNA binding of the of Smads to through Sp1 sites as transcriptional of the proximal PAI-1 promoter by Smads and TGF-β Sp1 binding to the promoter. into HepG2 cells with the expression for Smads and dominant negative type II receptor and TGF-β were as described in the to 1 or with or Smad3 as indicated. to TGF-β and were as described under Sp1 binding to its DNA element required for TGF-β and Smad-dependent activation of PAI-1 promoter, or into HepG2 cells with Cells were with TGF-β and mithramycin, which Sp1-DNA binding by sites P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). blocked the activation of both by TGF-β and Smad3 2 Sp1 is known to transcription through its binding by A. M. E. C. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). is to that TGF-β induce Sp1-dependent transcription by the complex between Sp1 and this not to the TGF-β and induction of the promoter activities were not by a inhibitor the association between Sp1 and in vivo not by TGF-β in HepG2, Mv1Lu, and cells Our demonstrate that Smad proteins the PAI-1 promoter by interacting with Sp1 the proximal region that contains two Sp1 binding sites. studies that Sp1 binding sites are required for TGF-β activation of the p21 promoter (13Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 14Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (324) Google Scholar). the of Sp1-dependent transcription through binding to its consensus sites in endogenous PAI-1 and p21 expression by we with lysates from cells with TGF-β and as indicated. TGF-β induced the expression of PAI-1 and p21 proteins as previously (3Westerhausen Jr., D.R. Hopkins W.E. Billadello J.J. J. Biol. Chem. 1991; 266: 1092-1100Abstract Full Text PDF PubMed Google Scholar, M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). The induction of PAI-1 protein inhibited by and The of is PAI-1 promoter is induced by TGF-β not through Sp1 sites in the proximal region also through the distal region Smads can to the elements directly (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar, C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (261) Google Scholar). In induction of p21 by TGF-β completely blocked by We not in Sp1 under the conditions Sp1 can mediate TGF-β-induced transcription through binding to sites in the of Sp1 inhibitor TGF-β-induced expression of the endogenous PAI-1 and p21 proteins. cells were with or or 150 and/or TGF-β cell lysates were to of Sp1 were by cells were as that of and cell lysates were for Mv1Lu cells were transfected with and increasing of Sp1 as indicated. Cells were with TGF-β. is as the of Sp1 is known to interact and with several proteins and transcription factors to regulate the transcription of genes J. Cell Biol. 1997; PubMed Scopus Google Scholar, M. Rev. Google Scholar). Sp1 has an transcription through Smad binding sites in response to Mv1Lu cells were with a TGF-β-responsive (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar), which contains of a and element of a minimal promoter, with expression for and Sp1. of Smad3 and/or Smad4 induced the promoter both in the and of TGF-β of Sp1 or in with Smad3 and/or not in Smad-dependent both in the or of TGF-β. We observed results in HepG2 cells TGF-β-responsive Massague J. EMBO J. 1995; PubMed Scopus Google Scholar) not These results that Sp1 not regulate transcription through Smad binding elements in response to TGF-β. Our data demonstrated a functional interaction between Smads and Sp1 to induce TGF-β-mediated transcription through Sp1 sites. Sp1 and Smads interact in or of Smad proteins were in COS-1 cells with Sp1. Cell lysates were to anti-FLAG or by with Sp1 in an complex with Smad3 or Smad4 and In a we observed that Smad3 or Smad4 with the association of Sp1 with Smad3 or Smad4 We were to physical association between Sp1 and other Smads for which a which of the Sp1 protein involved in the interaction with we or with or in COS-1 In these Smad3 or Smad4 with type Sp1 and not with and the of the of Sp1 in binding with Smad3 and We also for interaction between endogenous Sp1 and Smad3 or Smad3 and Smad4 were with from and NIH cell lysates Sp1 by and In Smad4 in the complex from cell an no Smad4 the with the In an to the association between endogenous Sp1 and Smads is by Smad3 or Smad4 from Mv1Lu, 293, or HepG2 cells with or TGF-β. Sp1 in the Smad3 complexes from cells the of Sp1 and Smad3 were not in response to TGF-β from In of Sp1 with Smad4 not TGF-β TGF-β of the cells the association of Smad3 with Smad4 as described previously (6Massague J. Wotton D. EMBO J. 2000; 19: 745-754Crossref Google Scholar). Sp1 has been shown to as a transcriptional by A. M. E. C. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar), we TGF-β Sp1-dependent transcription by the complex between Sp1 and We not in the of with Sp1 under these conditions the association between Sp1 and not between Sp1 and is induced by TGF-β. These results a mechanism for TGF-β-induced transcription of several genes through the Sp1 binding sites in The of Smads to induce specific transcriptional in response to TGF-β results from a functional with other transcription factors in complexes in the (6Massague J. Wotton D. EMBO J. 2000; 19: 745-754Crossref Google Scholar). TGF-β extracellular which may linked to the regulation of matrix formation by the regulation of plasminogen and inhibitors, including induction of the PAI-1 promoter by several promoter elements in the distal region have been which Smad3 and Smad4 binding (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar, C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (261) Google Scholar). Here, we have identified a promoter element in the proximal region to which contains two Sp1 binding sites that are required for TGF-β-induced and Smad-dependent transcriptional activation of the PAI-1 promoter. The of Sp1-dependent transcription in TGF-β-induced gene expression is by the that the expression of endogenous PAI-1 and p21 is by mithramycin, an inhibitor of Sp1-DNA We demonstrate that Smads as transcriptional for TGF-β-mediated induction of PAI-1 promoter through Sp1 sites. Sp1 consensus sites are required in the regulation of genes, including genes, genes, and (13Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 14Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (324) Google Scholar, 15Li J.-M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.-F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar, 16Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, J. Cell Biol. 1997; PubMed Scopus Google Scholar, M. Rev. Google Scholar). The of the gene regulation by Sp1 is by or that interact with Sp1 or by the of Sp1 sites and other regulatory elements the promoter. Although Sp1 is to for the transcription of genes, this work the that Sp1 can also play a role in the regulation of genes in response to specific several by which TGF-β activates gene expression through the transcription factor Sp1 activate transcription through Smad binding sites in the TGF-β-responsive promoters by interacting with Smads and by as the DNA binding activities of Sp1 by the of Sp1 through the of by or the transactivating of Sp1 through its binding to the elements in the promoter by TGF-β signal through specific interaction between Sp1 and The of TGF-β-mediated activation through Sp1 not by We not regulation of transcription through Smad binding to the promoter by Sp1 in response to as by the not and Our data indicate that both Sp1 sites in the proximal PAI-1 promoter are required, in part, for and transcriptional activation. of both Sp1 sites in the proximal promoter TGF-β Our work not and work M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) that Sp1 not to these sites. Although both Sp1 and are and to the the of involved in this TGF-β-mediated induction is by reports J.-M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.-F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar, J. Cell Biol. 1997; PubMed Scopus Google Scholar), that is a transcriptional and that not the responsiveness to TGF-β. We have shown that blocks and Sp1 containing promoter including PAI-1 promoter 2 and TGF-β stimulation of endogenous PAI-1 In TGF-β induction of p21 is completely inhibited by by is the PAI-1 promoter is induced by TGF-β through Smad binding sites in the distal region (4Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar, C.-Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (261) Google Scholar), and through proximal region containing Sp1 sites data and p21 promoter is induced by TGF-β through the Sp1 sites (13Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 14Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (324) Google Scholar). Sp1-DNA binding by sites, it that Sp1 binding to its consensus in several promoters is required for TGF-β-mediated transcriptional activation through Sp1 binding sites. studies demonstrated that TGF-β of cells not the DNA binding activities of Sp1 or of Sp1 J.-M. Datto M.B. Shen X. Hu P.P., Yu, Y. Wang X.-F. Nucleic Acids Res. 1998; 26: 2449-2456Crossref PubMed Scopus (92) Google Scholar). Our that the association between Sp1 and is not by and that the inhibitor has no Sp1-dependent transcription in response to that is not involved in TGF-β-induced Sp1-dependent We demonstrate that Smad3 and Smad4 Sp1 in vivo and that the association between endogenous Smad3 and Sp1 is induced by TGF-β in several cell lines. Smad3 induces the minimal PAI-1 promoter, and the heterologous promoter containing three Sp1 sites in the or of the of to induce the PAI-1 promoter is induced by TGF-β by two this promoter is by TGF-β-induced binding of a Smad3/Smad4 containing complex to in the distal the proximal promoter, containing two Sp1 sites, induction through TGF-β-induced interaction between Smad3 and of these are required for induction of the PAI-1 promoter. In results a for how Smads mediate TGF-β-induced transcription Sp1 binding sites in promoters through physical and functional interaction with and this a novel mechanism of gene regulation by TGF-β. We D. E. P. J. J. J. X.-F. J. C. and J.-M. for of We are also to K. and for critical of the