Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex

Previous studies have shown that multiple serum response factor (SRF)-bindi ng CArG elements were required for smooth muscle cell (SMC)-specific regula tion of smooth muscle (SM) alpha-actin expression. However, a critical ques tion remains as to the mechanisms whereby a ubiquitously expressed transcri ption factor such as SRF might contribute to SMC-specific expression. The g oal of the present study was to investigate the hypothesis that SMC-selecti ve expression of SM a-actin is due at least in part to (1) unique CArG flan king sequences that distinguish the SM alpha-actin CArGs from other ubiquit ously expressed CArG-dependent genes such as c-fos, (2) cooperative interac tions between CArG elements, and (3) SRF-dependent binding of SMC-selective proteins to the CArG-containing regions of the promoter. Results demonstra ted that specific sequences nanking CArG B were important for promoter acti vity in SMCs but not in bovine aortic endothelial cells. We also provided e vidence indicating that the structural orientation between CArGs A and B wa s an important determinant of promoter function. Electrophoretic mobility s hift assays and methylation interference footprinting demonstrated that a u nique SRF-containing complex formed that was selective for SMCs and, furthe rmore, that this complex was probably stabilized by protein-protein interac tions and not by specific interactions with CArG flanking sequences. Taken together, the results of these studies provide evidence that SM alpha-actin expression in SMCs is complex and may involve the formation of a unique mu ltiprotein initiation complex that is coordinated by SRF complexes bound to multiple CArG elements.