DIRECT BINDING OF SMAD3 AND SMAD4 TO CRITICAL TGF-BETA-INDUCIBLE ELEMENTS IN THE PROMOTER OF HUMAN PLASMINOGEN-ACTIVATOR INHIBITOR-TYPE-1 GENE

Smad proteins play a key role in the intracellular signalling of trans forming growth factor beta (TGF beta), which elicits a large variety o f cellular responses. Upon TGF beta receptor activation, Smad2 and Sma d3 become phosphorylated and form heteromeric complexes with Smad4. Th ese complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transc riptional regulation is largely unknown. Human plasminogen activator i nhibitor-1 (PAI-1) is a gene that is potently induced by TGF beta. Her e we report the identification of Smad3/Smad4 binding sequences, terme d CAGA boxes, within the promoter of the human PAI-I gene. The CAGA bo xes confer TGF beta and activin, but not bone morphogenetic protein (B MP) stimulation to a heterologous promoter reporter construct. Importa ntly, mutation of the three CAGA boxes present in the PAI-I promoter w as found to abolish TGF beta responsiveness. Thus, CAGA elements are e ssential and sufficient for the induction by TGF beta. In addition, TG F beta induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 pro teins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGF beta-responsive regions of several other genes suggests that this may be a widely used moth in TG F beta-regulated transcription.