Contribution of Enhancer of Zeste Homolog 2 gene in Calcification Nidus Formation and Exosome Release in Arterial Smooth Muscle Cells

Abstract

Enhancer of zeste homolog 2 (Ezh2) as a histone methyltransferasehas been reported to epigenetically control gene expression, and it is known to be involved in phenotypic transition and osteogenesis, playing a critical role in multiple inflammatory diseases. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with multivesicular bodies (MVBs) and consequent exosome release play a crucial role in the SMC phenotype transition to osteogenic during arterial medial calcification (AMC). However, there is lack of evidence to show whether Ezh2 participates in the phenotypic transition and vascular calcification process. The aim of this study was to determine the role of Ezh2 in high phosphate (Pi)-induced calcification and to explore its contribution to the phenotype change and exosome secretion in vascular smooth muscle cells (SMCs) that is essential for AMC. Using aortic SMCs isolated from SMC-specific Ezh2 knockout (Ezh2fl/fl/SMCre) mice and wild-type littermates, we demonstrated that high Pi treatment increased more calcium deposition in aortic SMCs as shown by the presence of Alizarin Red-stained nodules when Ezh2 gene is deleted or its activity is inhibited by GSK126. Further, this calcium deposition was significantly decreased by MLSA-1, a TRPML-1 channel agonist in these cells. Correspondingly, Ezh2 gene deletion induced phenotypic change in Pi-treated SMCs, as depicted by decreased SM22-α (SMC marker) and increased expression of osteogenic gene osteopontin in Western blot analysis, indicating that Ezh2 contributes to phenotype change in SMCs leading to calcification nidus formation. Using nanoparticle tracking analysis, Pi-induced secretion of exosomes (100-150 nm in size) were found significantly increased in SMCs, which was augmented by Ezh2 gene deletion or GSK126, or attenuated by MLSA-1. Similarly, GSK126 significantly increased calcification and exosome secretion in Pi-treated and human aortic SMCs (HASMCs), while MLSA-1 markedly decreased calcium deposition and exosome release in these cells. These results suggest that a normal expression of Ezh2 genecritically controls phenotype transition and exosome secretion in arterial SMCs and its defect or deficiency may be an important molecular mechanism leading to AMC.