High-phosphate-induced calcification is related to SM22α promoter methylation in vascular smooth muscle cells

Abstract

Hyperphosphatemia is closely related to vascular calcification in patients with chronic kidney disease. Vascular smooth muscle cells (VSMCs) exposed to high phosphate concentrations in vitro undergo phenotypic transition to osteoblast-like cells. Mechanisms underlying this transdifferentiation are not clear. In this study we used two in vitro models, human aortic smooth muscle cells and rat aortic rings, to investigate the phenotypic transition of VSMCs induced by high phosphate. We found that high phosphate concentration (3.3 mmol/L) in the medium was associated with increased DNA methyltransferase activity and methylation of the promoter region of SM22a. This was accompanied by loss of the smooth muscle cell-specific protein SM22a, gain of the osteoblast transcription factor Cbfa1, and increased alkaline phosphatase activity with the subsequent in vitro calcification. The addition of a demethylating agent (procaine) to the high-phosphate medium reduced DNA methyltransferase activity and prevented methylation of the SM22a promoter, which was accompanied by an increase in SM22a expression and less calcification. Additionally, downregulation of SM22a, either by siRNA or by a methyl group donor (S-adenosyl methionine), resulted in overexpression of Cbfa1.In conclusion, we demonstrate that methylation of SM22a promoter is an important event in vascular smooth muscle cell calcification and that high phosphate induces this epigenetic modification. These findings uncover a new insight into mechanisms by which high phosphate concentration promotes vascular calcification. ß

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1

“…Several studies provide support for the idea that SMC differentiation marker gene expression is regulated by DNA methylation. For example, the decrease in SM22 expression observed in human SMCs treated with high phosphate was associated with increased SM22 promoter methylation 44 while SM22 methylation was decreased in multipotential adventitial cells that were induced to differentiate into SMC 45 . Hu et al has also shown that SM α-actin expression in fibroblasts inversely correlated with DNA methylation near the SM α-actin transcription start site 46 .…”

Calcifying Human Aortic Smooth Muscle Cells Express Different Bone Alkaline Phosphatase Isoforms, Including the Novel B1x Isoform

“…β-Glycerophosphate seems to induce ALP consistently when used as a component of calcification medium for VSMCs, and this induction is potentiated by the addition of other procalcific agents [6,8,30,31,41,42]. Experimental setups with other phosphate donors have demonstrated both stimulatory, [43,44] absent [45] or even inhibitory [46] effects on ALP activity in calcifying VSMCs. Prosdocimo et al [45] found a rapid, ALP-independent reduction of extracellular pyrophosphate in calcifying rat VSMCs stimulated with cAMP and phosphate salts, which was associated with the downregulation of the pyrophosphate membrane transporter ANK.…”

Arterial Calcification in Chronic Kidney Disease: Key Roles for Calcium and Phosphate

“…The increased methylation resulted in reduced expression of SM22α, increased Runx2 expression, and increased ALP activity, which together resulted in an increased level of calcification. 67 Inhibition of DNA methylation by procaine was able to prevent these transcriptional changes when VSMCs were grown in high P media, and this resulted in reduced calcification.…”