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 biological processes, such as proliferation, differentiation, extracellular matrix production, and apoptosis. In cultured vascular endothelial cells, TGF-beta induces the expression of platelet-derived growth factor (PDGF) B-chain, a mitogen and chemoattractant, at the level of transcription. The molecular mechanism(s) underlying this process are not presently understood. In this study, we performed serial 5' deletion and transient transfection analysis to define a region in the PDGF-B promoter mediating inducible responsiveness 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 physical association of Smad4 with the promoter. Overexpression of Smad4 activated the PDGF-B promoter and superinduced PDGF-B promoter-dependent expression in cells exposed to TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the PDGF-B promoter. This effect was attenuated when 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 transactivated the PDGF-B promoter in a synergistic manner. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the PDGF-B promoter mediating responsiveness to TGF-beta.
Taylor
L M
LM
Centre for Thrombosis and Vascular Research, The University of New South Wales and Prince of Wales Hospital, Sydney, New South Wales 2052, Australia.
Khachigian
L M
LM
D000818:Animals; D001483:Base Sequence; D000077214:Becaplermin; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020574:Proto-Oncogene Proteins c-sis; D015534:Trans-Activators / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*
D017931:DNA Primers; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D000077214:Becaplermin