To identify potential regulators of smooth muscle cell (SMC) differentiation, we studied the molecular mechanisms that control the tissue-specific transcriptional expression of SM alpha-actin, the most abundant protein in fully differentiated SMCs. A construct containing the region from -1 to -125 of the promoter (p125CAT) had high transcriptional activity in SMCs (57-fold > promoterless) and endothelial cells (ECs) (18-fold) but not in skeletal myoblasts or myotubes. Mutation of either of two highly conserved CC(AT-rich)6GG (CArG) motifs at -62 and -112 abolished the activity of p125CAT in SMCs but had no effect in ECs. In contrast, high transcriptional activity in skeletal myotubes, which also express SM alpha-actin, required at least 271 base pairs of the promoter (-1 to > or = -271). Constructs containing 547 base pairs or more of the promoter were transcriptionally active in SMCs and skeletal myotubes but had no activity in skeletal myoblasts or ECs, cell types that do not express SM alpha-actin. Electrophoretic mobility shift assays provided evidence for binding of a unique serum response factor-containing complex of factors to the CArG box elements in SMCs. Results indicate that: 1) transcriptional expression of SM alpha-actin in SMCs requires the interaction of the CArG boxes with SMC nucleoprotein(s); 2) expression of SM alpha-actin in skeletal myotubes requires different cis-elements and trans-factors than in SMCs; and 3) negative-acting cis-elements are important in restricting transcription in cells that do not express SM alpha-actin.

Previous studies have demonstrated that angiotensin II (ANG II) and arginine vasopressin (AVP) stimulate increased protein synthesis and cellular hypertrophy in cultured rat aortic smooth muscle cells (SMC). The aim of this study was to explore the hypothesis that ANG II- and/or AVP-induced increases in protein synthesis are mediated by autocrine secretion of platelet-derived growth factor (PDGF)-AA. Results demonstrated that ANG II or AVP increased expression of PDGF-A, but not -B, chain mRNA. Additionally, conditioned media from ANG II- and AVP-treated SMC had increased mitogenic activity for Swiss 3T3 cells, which could be inhibited with a neutralizing antibody to PDGF-AA. However, PDGF-AA-neutralizing antibodies did not inhibit ANG II- or AVP-induced increases in protein synthesis, and exogenous PDGF-AA did not stimulate increased protein synthesis. Furthermore, no PDGF-alpha receptors were evident based on 125I-labeled PDGF-AA binding studies. In summary, results indicate that ANG II- or AVP-induced increases in protein synthesis were not dependent on autocrine secretion of PDGF-AA.

Identification of the regulators of smooth muscle cell (SMC) gene expression is critical to understanding SMC differentiation and alterations in SMC phenotype in vascular disease. Previous studies revealed positive transcriptional activity within the chicken smooth muscle (SM) alpha-actin promoter region from -209 to -257. In the present study, transient transfections of wild-type and mutant chicken SM alpha-actin promoter/reporter gene constructs into rat aortic SMC demonstrated that the positive transcriptional activity of this region was abolished with a two base pair mutation in a conserved sequence motif at -225 to -233 (TGTTTATC to TACTTATC). Electrophoretic mobility shift assays revealed that nuclear factors bound promoter fragments containing this sequence and that specific mutations in the TGTTTATC motif abolished nuclear factor binding. Studies thus provide evidence for binding of a nuclear factor to a positive cis-acting element within the SM alpha-actin promoter. Further characterization of this factor may contribute to a better understanding of the molecular mechanisms that regulate differentiation of SMC in vascular disease.