Abstract 637: Role of Nuclear Smooth Muscle Alpha-Actin in the Differentiation of Vascular Smooth Muscle Cells

Abstract

Although the function of the cytoplasmic smooth muscle (SM) α-actin in contractile units has been well characterized in vascular SMCs, whether the SM α-actin is present in the nucleus and its nuclear function are yet to be determined. Our previous study discovered the mutations in ACTA2 , which encodes SM α-actin, predispose to both thoracic aortic disease and occlusive vascular diseases (stroke and coronary artery disease). We hypothesize that nuclear SM α-actin is critical for differentiation of SMCs. The cytoplasmic and nuclear fractions of mouse SMCs were isolated and the SM α-actin was detected in both cytosol and nucleus by immunoblotting. Moreover, both cytoplasmic and nuclear SM α-actin were dramatically upregulated during the differentiation of SMCs stimulated with TGF-β. Two-dimensional gel electrophoresis, which can separate actin isoforms based on distinct isoelectric points, was used to show that the ratio of SM α-actin to β-actin was increased in nuclear fraction when compared to cytoplasmic fraction with differentiation of SMCs, driven by serum starvation and TGF-β treatment. Furthermore, differentiation was associated with a significantly higher nuclear to cytoplasmic ratio of SM α-actin, indicating SM α-actin accumulates in the nucleus during differentiation. Co-immunoprecipitation experiments identified that nuclear SM α-actin is a component of the chromatin remodeling complex, Ino80, and the immunofluorescence confirmed co-localization of SM α-actin and Ino80 in the nucleus. To investigate the transcriptional function of nuclear SM α-actin, we performed the chromatin immunoprecipitation quantitative PCR, which revealed that the SM α-actin, but not β-actin, accumulated to the promoter region of SMC differentiation markers, including Myh11 , Cnn1 , and Tagln , during the differentiation of SMC. As expected, the Acta2 mutant SMCs (pArg149Cys) disrupt the differentiation of SMCs based on decreased contractile protein levels and increased proliferation. Furthermore, specific Acta2 missense mutations disrupt the ability for SM α-actin to localize to the nucleus. Taken together, our study found the SM α-actin concentrates into the nucleus of SMCs and may play a role in chromatin remodeling required for differentiation of SMCs.