The regulation of myocardin factor dependent transcription in vascular smooth muscle

This work explores the role of the myocardin family of serum response factor (SRF) co-factors in smooth muscle cell (SMC) differentiation, and identifies novel modes of regulating their activities. Extensive evidence indicates that SRF regulates muscle-specific gene expression by binding to conserved promoter regions known as CArG boxes. Myocardin and the myocardin-related transcription factors MRTF-A and MRTF-B are all capable of potently transactivating SRF-dependent gene expression. The sub-cellular localization of MRTFs is regulated by RhoA signaling, an established determinant of SMC marker gene activity. Studies included here demonstrate that the MRTFs are expressed in primary and cultured SMC and in multiple organs with a large SMC component, and that they are capable of upregulating SMC-specific gene activity in multipotential 10T1/2 cells. We demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists is likely mediated by increased nuclear translocation of the MRTFs. Gel shift assays were used to show that myocardin factor activity correlates well with SRF/CArG ternary complex formation, and that MRTF-SRF interactions are partially dependent upon CArG sequence. In a yeast-two-hybrid screen for novel SRF binding partners in aortic SMC, we identified four and a half LIM domain protein 2 (FHL2). We showed that FHL2 also interacted with all three myocardin factors and enhanced myocardin and MRTF-A-dependent transactivation of the SM α-actin, SM22, and cardiac ANF promoters by increasing the half-lives of these proteins. Treatment of cells with the proteasome inhibitors MG132 and lactacystin strongly upregulated myocardin factor protein levels and resulted in a substantial increase in ubiquitin immunoreactivity in MRTF-A immunoprecipitants. Importantly, these data are the first to indicate that the myocardin factors are regulated by proteasome-mediated degradation, and that SRF-dependent gene activity can be upregulated via inhibition of their degradation. We have further shown that the muscle-specific ring finger protein MuRF3 physically interacts with the myocardin factors and inhibits transactivation of the SM α-actin and SM22 promoters. MuRF3 also inhibited myocardin factor protein expression, and may act as an E3 ubiquitin ligase for this very important family of SRF co-factors