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

Abstract

Multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) 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 CaMKIIγ isoform in medial wall VSM. Smooth muscle knockout of CaMKIIγ enhanced injury-induced VSM neointimal hyperplasia, whereas CaMKIIγ 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 CaMKIIγ expression in cultured VSM cells and inhibited CaMKIIγ 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 CaMKIIγ and differentiation markers, including myocardin, and inhibited VSM cell proliferation and injury-induced neointima formation. This study indicates that CaMKIIγ 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-γ isoform (CaMKIIγ) is associated with differentiated vascular smooth muscle (VSM) and negatively regulates proliferation in VSM synthetic phenotype (VSM_Syn) cells. This study demonstrates that thymine DNA glycosylase (TDG) plays a key role in regulating CaMKIIγ expression in VSM through promoter cytosine methylation/demethylation. TDG expression is strongly induced in VSM_Syn cells and plays key roles in negatively regulating CaMKIIγ expression and more broadly VSM phenotype switching.

Keywords: CaM kinase II; CaMKIIγ; cytosine methylation/demethylation; thymine DNA glycosylase; vascular injury; vascular smooth muscle.