Abstract 435: Myosin Phosphatase-Rho Interacting Protein Modulation of Vascular Smooth Muscle Phenotype
Vascular smooth muscle cells (VSMCs), unlike skeletal or cardiac muscle, retain a degree of plasticity and can transition between contractile and synthetic phenotypes. Transition of VSMCs to a synthetic phenotype is a well recognized event in the pathogenesis of atherosclerosis and the response to vascular injury. Recent studies in our laboratory suggest a potential role for myosin phosphatase-Rho interacting protein (M-RIP) in the phenotypic modulation of human aortic smooth muscle cells (Ao184). M-RIP was initially identified as a protein that binds both the myosin binding subunit of myosin phosphatase (MP) and RhoA and targets MP to stress fibers to regulate the phosphorylation state of myosin light chains. Intriguingly, it was also found that silencing M-RIP expression by RNAi resulted in morphological changes in Ao184 cells including increased stress fiber number and cell area. Since M-RIP is known to be a cytoskeletal protein, we have investigated its intracellular localization by subcellular fractionation, and found that M-RIP is also present in the nucleus. In an effort to understand how M-RIP functions to regulate VSMC morphology we performed a yeast 2-hybrid screen using M-RIP as bait. We found an interaction with a notch pathway regulatory gene, CBF-1 interacting corepressor (CIR) and confirmed the interaction in vitro using protein interaction assays. We next examined the effect of M-RIP on the expression of both smooth-muscle specific as well as notch-responsive genes, as it is well known that the notch signaling pathway can modulate VSMC phenotype. We determined in Ao184 cells that silencing M-RIP increased both message and protein for smooth muscle α-actin and calponin. Additionally, M-RIP silencing caused decreased expression of two notch-responsive genes, Hes1 and Hes7. Using reporter analyses in C3H10T1/2 cells we determined that M-RIP expression enhanced Hes1 luciferase promoter activation by the constitutively active notch 1 intracellular domain. Our studies have thus established a new role for M-RIP in the modulation of smooth muscle cell phenotype. Our experiments support that nuclear M-RIP regulates smooth muscle-specific gene expression and that one mechanism may be through regulation of notch pathway transcription factors.