Abstract 15322: O-glcnac Modification on Runx2 Promotes Vascular Calcification

Abstract

Vascular calcification is prevalent in patients with diabetes mellitus and well documented in animal models of diabetes. We have previously reported that vascular calcification in diabetes is associated with elevated vascular protein O -linked GlcNAc modification ( O -GlcNAcylation). The present studies sought to determine a causative link of O -GlcNAcylation with diabetic vascular calcification and the underlying molecular mechanisms. Using coronary arteries from diabetic subjects, we identified increased O -GlcNAcylation in the highly calcified lesions compared to those in the surrounding uncalcified areas. By generating a new mouse model with inducible SMC-specific deletion of the O -GlcNAc transferase (OGT), we demonstrated that smooth muscle cell (SMC)-specific OGT deletion significantly inhibited protein O -GlcNAcylation and calcification, and decreased aortic stiffness in the low-dose streptozotocin-induced diabetic mice. Consistent with the observations in human tissues, inhibition of vascular calcification by OGT ablation was associated with down-regulation of Runx2, the essential osteogenic regulator for vascular calcification. OGT deficiency in VSMC further attenuated Runx2-induced VSMC calcification. Immunoprecipitation analysis uncovered a direct O-GlcNAc modification on Runx2, which was abolished in the OGT-deficient VSMC. With a serial of Runx2 truncation mutants and point mutations on putative O -GlcNAcylation sites, we localized the essential Runx2 osteogenic functional domain between amino acids 391 and 432, and identified Runx2 T412 was responsible for Runx2 O -GlcNAcylation and Runx2-induced VSMC calcification. Inhibition of Runx2 O -GlcNAcylation by T412A mutation decreased Runx2 transcription activity and inhibited Runx2 binding with its key co-factors. Taken together, our studies have provided the first genetic proof demonstrating the causative effects of O -GlcNAcylation on vascular calcification in diabetes, and identified a novel mechanism underlying the essential role of O -GlcNAcylation of Runx2 in Runx2 osteogenic activity and Runx2-induced VSMC calcification. These results may shed lights on novel targets that are amenable to drug discovery for diabetic vascular calcification.