Abstract 18326: p38MAPK is a Novel Negative Regulator for Smooth Muscle Differentiation

p38MAPK belongs to members of the MAPK family which can be activated by various environmental stressors and inflammatory cytokines. While more and more studies have defined the critical roles of p38MAPK in different biological processes involving cell growth, differentiation and apoptosis, its actual contribution to smooth muscle cell (SMC) phenotypic modulation is largely unexplored. Using the mouse complete carotid artery ligation model, we found both total p38 and phosphorylated p38 to be predominantly located in the neointima of the vessel wall where SMCs undergo hyper-proliferation and SMC differentiation is compromised, suggesting a negative regulatory role of p38MAPK in SMC differentiation. To test this hypothesis, we applied siRNA studies in primary human coronary artery SMC (HCASM) cultures and examined SMC contractile gene expression. Depletion of p38MAPK evoked a dramatic increase in SMC contractile gene expression. The stimulatory effect of SMC contractile gene expression upon knockdown of p38MAPK was reproduced in a variety of SMC culture models including human aortic SMCs, rat aortic SMCs, PAC1 cells and primary human myofibroblasts. Consistent with the siRNA studies, cotransfecting p38 with its upstream kinase MKK3 or MMK6 abolished promoter activity of several SMC-restricted genes, while little effect was found with the dominant negative mutants of p38. Furthermore, adenovirus carrying the constitutively active MKK6 completely abrogated TGF-beta1 induced SMC differentiation as assessed by Western blotting and immunfluorescence mirocsopy, indicating that phosphorylated p38 is required for the repressive effect of p38MAPK in SMC differentiation. Interestingly, in HCASMs, we found myocardin-related transcription factor A (MRTFA) was mainly located in the nucleus when the endogenous p38MAPK was knocked down; conversely, over-expression of constitutively active MKK6 was found to shift the majority of MRTFA to the cytoplasm of HCASMs, suggesting that phosphorylated p38MAPK is able to block nuclear import of MRTFA in SMCs. Taken together, our studies revealed an unexpected role for p38MAPK in the negative regulation of SMC differentiation.