Abstract 241: O-GlcNAcylation of Runx2 in Nuclear Matrix Targeting Signal Modulates Vascular Calcification

Abstract

Vascular calcification is prevalent in patients with atherosclerosis, diabetes, and chronic kidney diseases and increases the mortality and morbidity of those patients. Osteogenic differentiation of vascular smooth muscle cells (VSMC) has been recognized as a key feature of the calcification process. Using smooth muscle-specific deletion mouse model, we have demonstrated an essential role of SMC-derived Runx2 in regulating vascular calcification in atherosclerosis in vivo. The present study further defined Runx2 regulation and its functional domains that control osteogenesis of VSMC. Using a serial of Runx2 truncation mutants, we located the domain between amino acids 391 and 432 as responsible region for Runx2 osteogenic function in VSMC. This region contains the nuclear matrix targeting signal, which has been shown to interact with Smad proteins. Accordingly, we determined the contribution of Smad proteins in Runx2-regulated VSMC calcification. By knockdown individual Smad in VSMC, we demonstrated that Smads 1/5/8, but not Smads 2/3, were required for Runx2 osteogenic function and VSMC calcification. In addition, we found the osteogenic function of Runx2 was abolished by inhibition of protein O-GlcNAcylation, a unique posttranslational modification that we have recently reported to be critical in regulating VSMC calcification. Runx2 O-GlcNAc modification was further demonstrated by immunoprecipitation. Based on bioinformatics analysis, we found several putative O-GlcNAcylation sites within Runx2 osteogenic domain. Using site-directed mutagenesis, we demonstrated that mutation on S385, S387, T404, T406, T412, S413, or T427 decreased Runx2 O-GlcNAcylation and Runx2 binding to Smads 1/5/8, and thus decreasing Runx2 activity and VSMC calcification. In summary, we have identified the Runx2 osteogenic functional domain in VSMC; and demonstrated that O-GlcNAc modification of Runx2 in its osteogenic functional domain is critical for Runx2-regualted VSMC calcification. Our studies provide molecular insights into the regulation and function of Runx2 in VSMC calcification, which may shed lights on novel targets that are amenable to drug discovery for vascular calcification.