Abstract 1011: Cbfa1 is Involved in Arteriosclerosis Formation by Promoting Phenotypic Conversion of Vascular Smooth Muscle Cells
[Objective] Cbfa1, a key transcription factor in osteoblast differentiation, is expressed in calcified atherosclerotic plaques and has been implicated in the pathogenesis of vascular calcification. Previous studies using cultured vascular smooth muscle cells (SMC) suggest that SMC transform to osteoblast-like cells via up-regulation of Cbfa1. However, precise molecular mechanism by which Cbfa1 induces dedifferentiation of SMC remains unanswered. In this study, we tested the hypothesis that Cbfa1 represses SMC differentiation by inhibiting SRF-dependent transcription of the SMC genes.
[Methods and Results] RT-PCR analyses showed that overexpression of Cbfa1 repressed the expression of myocardin-induced SMC marker genes (SM22α and SM myosin heavy chain) in 10T1/2 pluripotent mesenchymal stem cells. Luciferase assays showed that Cbfa1 remarkably reduced the transcriptional activation of these promoters by myocardin. Consistent with these data, we found that myocardin-induced expression of the SMC marker genes and their promoter activity were more pronounced in embryonic fibroblasts derived from Cbfa1 deficient mice compared with those from wild type mice. We further noted that SMC marker gene expression was decreased in human aortic SMC (HASMC) transduced with adenovirus-expressing Cbfa1, whereas osteogenic-marker genes expression and the alkaline phosphatase activity were up-regulated in these cells. Co-immunoprecipitation assay (Co-IP) demonstrated that Cbfa1 physically bound to serum response factor (SRF), an essential transcription factor collaborating with myocardin to induce SMC marker genes expression. Co-IP further revealed that overexpression of Cbfa1 disrupted SRF-myocardin complex. We excluded the possibilities that the recruitment of repressor proteins such as histone deacetylase, TLE and HERP1 and inhibition of SRF-DNA binding play a major role in the repression of SMC marker gene expression by Cbfa1.
[Conclusion] Results of the present study indicates that Cbfa1 represses SMC differentiation by disrupting SRF-myocardin complex through the preferential formation of Cbfa1-SRF complex. Our data suggest that Cbfa1 regulates SMC phenotypic change that is inevitably coupled with vascular calcification.