Regulation of plasminogen activator inhibitor-1 expression by transforminggrowth factor-beta-induced physical and functional interactions between Smads and Sp1

Members of the transforming growth factor-beta (TGF-beta) superfamily media te a broad range of biological activities by regulating the expression of t arget genes. Smad proteins play a critical role in this process by binding directly to the promoter elements and/or associating with other transcripti on factors. TGF-beta1 up-regulates several genes transcriptionally through Sp1 binding sites; however, the mechanism of TGF-beta induction of gene exp ression through Sp1 sites is largely unknown. Here we report the identifica tion of a novel 38-base pair TGF-beta -responsive element in the human plas minogen activator inhibitor-1 (PAI-1) promoter, which contains two Sp1 bind ing sites, and is required for TGF-beta -induced Smad-dependent transcripti onal activation. Three canonical Sp1 binding sites also support strong tran scriptional activation by TGF-beta and Smads from a minimal heterologous pr omoter. TGF-beta induction of PAI-1 and p21 is blocked by the Sp1 inhibitor mithramycin, implicating Sp1 in the in vivo regulation of these genes by T GF-beta, We show that the association between endogenous Sp1 and Smad3 is i nduced by TGF-beta in several cell lines; however, Smad4 shows constitutive interaction with Sp1, These data provide novel insights into the mechanism by which TGF-beta up-regulates several gene expression by activating Sp1-d ependent transcription through the induction of Smad/Sp1 complex formation.