A Transforming Growth Factor b (TGFb) Control Element Drives TGFb-induced Stimulation of Smooth Muscle a-Actin Gene Expression in Concert with Two CArG Elements*

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor b (TGFb) increases smooth muscle (SM) a-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM a-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFb (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM a-actin promoter were sufficient to confer TGFb responsiveness. Three cis elements were shown to be required for TGFb inducibility: two highly conserved CArG boxes, designated A (262) and B (2112) and a novel TGFb control element (TCE) (242). Mutation of any one of these elements completely abolished TGFbinduced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFb-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFb also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFb response element in the SM a-actin promoter that may contribute to coordinate regulation of expression of multiple celltype specific proteins during SMC differentiation.