Thymine DNA glycosylase is a key regulator of CaMKIIγ expression and vascular smooth muscle phenotype

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII gamma is a multigene family with isoform-specific regulation of vascular smooth muscle (VSM) functions. In previous studies, we found that vascular injury resulted in VSM dedifferentiation and reduced expression of the CaMKII gamma isoform in medial wall VSM. Smooth muscle knockout of CaMKII gamma enhanced injury-induced VSM neointimal hyperplasia, whereas CaMKII gamma overexpression inhibited VSM proliferation and neointimal formation. In this study, we evaluated DNA cytosine methylation/demethylation as a mechanism for regulating CaMKII isoform expression in VSM. Inhibition of cytosine methylation with 5-Aza-2'-deoxycytidine significantly upregulated CaMKII gamma expression in cultured VSM cells and inhibited CaMKII gamma downregulation in organ-cultured aorta ex vivo. With the use of methylated cytosine immunoprecipitation, the rat Camk2g promoter was found hypomethylated in differentiated VSM, whereas injury- or cell culture-induced VSM dedifferentiation coincided with Camk2g promoter methylation and decreased expression. We report for the first time that VSM cell phenotype switching is accompanied by marked induction of thymine DNA glycosylase (TDG) protein and mRNA expression in injured arteries in vivo and in cultured VSM synthetic phenotype cells. Silencing Tdg in VSM promoted expression of CaMKII gamma and differentiation markers, including myocardin, and inhibited VSM cell proliferation and injury-induced neointima formation. This study indicates that CaMKII gamma expression in VSM is regulated by cytosine methylation/demethylation and that TDG is an important determinant of this process and, more broadly, VSM phenotype switching and function. NEW & NOTEWORTHY Expression of the calcium calmodulin- dependent protein kinase II-gamma isoform (CaMKII gamma) is associated with differentiated vascular smooth muscle (VSM) and negatively regulates proliferation in VSM synthetic phenotype (VSMSyn) cells. This study demonstrates that thymine DNA glycosylase (TDG) plays a key role in regulating CaMKTh gamma expression in VSM through promoter cytosine methylation/demethylation. TDG expression is strongly induced in VSMSyn cells and plays key roles in negatively regulating CaMKII gamma expression and more broadly VSM phenotype switching.