Induction of platelet-derived growth factor B-chain expression by transforming growth factor-beta involves transactivation by Smads

Transforming growth factor-beta (TGF-beta) regulates a diverse array of bio logical processes, such as proliferation, differentiation, extracellular ma trix production, and apoptosis. In cultured vascular endothelial cells, TGF -beta induces the expression of platelet-derived growth factor (PDGF) B-cha in, a mitogen and chemoattractant, at the level of transcription. The molec ular mechanism(s) underlying this process are not presently understood. In this study, we performed serial 5' deletion and transient transfection anal ysis to define a region in the PDGF-B promoter mediating inducible responsi veness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. Electrophoretic mobility shift analysis revealed that nuclear proteins bound to this site in a transient and specific manner. Supershift studies demonstrated the phy sical association of Smad4 with the promoter. Overexpression of Smad4 activ ated the PDGF-B promoter and superinduced PDGF-B promoter-dependent express ion in cells exposed to TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the PDGF-B promoter. This effect was attenuated w hen Smad4 was substituted with its dominant negative counterpart. Mutation of the (-81)CAGA(-78) motif in the PDGF-B promoter abrogated Smad-inducible promoter-dependent expression. Overexpression of Smad2 and Smad3 transacti vated the PDGF-B promoter in a synergistic manner. These findings demonstra te the existence of a novel, functional binding element in the proximal reg ion of the PDGF-B promoter mediating responsiveness to TGF-beta.