A TRANSFORMING GROWTH-FACTOR-BETA (TGF-BETA) CONTROL ELEMENT DRIVES TGF-BETA-INDUCED STIMULATION OF SMOOTH-MUSCLE ALPHA-ACTIN GENE-EXPRESSION IN CONCERT WITH 2 CARG ELEMENTS

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta (TGF beta) increases smooth m uscle (SM) alpha-actin expression. Confluent, growth-arrested rat aort ic smooth muscle cells (SMC) were transiently transfected with various SM alpha-actin promoter/chloramphenicol acetyltransferase deletion mu tants and stimulated with TGF beta (2.5 ng/ml). Results demonstrated t hat the first 125 base pairs of the SM alpha-actin promoter were suffi cient to confer TGF beta responsiveness. Three cis elements were shown to be required for TGF beta inducibility: two highly conserved CArG b oxes, designated A (-62) and B (-112) and a novel TGF beta control ele ment (TCE) (-42). Mutation of any one of these elements completely abo lished TGF beta-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGF beta -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 TGF beta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h(1) calponin, whose promoters also contain ed a TCE-like element. In summary, we identified a TGF beta response e lement in the SM alpha-actin promoter that may contribute to coordinat e regulation of expression of multiple cell-type specific proteins dur ing SMC differentiation.