Abstract 603: O-GlcNAcylation of Runx2 is Essential for Vascular Calcification

Abstract

Osteogenic differentiation of vascular smooth muscle cells (VSMC) contribute predominantly to vascular calcification, a characteristic of advanced atherosclerosis, chronic kidney disease, and diabetes. Using Runx2 shRNA and SMC-specific Runx2 deletion mice, we have demonstrated an essential role of Runx2 in regulating vascular calcification in vitro and in vivo. The present studies aim to pinpoint the Runx2 functional domain that is critical for its osteogenic function in VSMC and to elucidate the underlying molecular mechanisms. A series of Runx2 deletion mutants were generated and stably transfected into VSMC. Full-length Runx2 protein and Runx2 deletion mutants containing amino acids 1-495 or 1-432, but not Runx2 1-391, induced VMC calcification, indicating that the Runx2 osteogenic functional domain is located between amino acids 391-432. Site-directed mutagenesis analysis identified that mutations at the amino acids 412, 413, 425 and 426-428 markedly reduced Runx2 transactivity and inhibited Runx2-induced VSMC calcification. Mutations at amino acids 412, 413 and 425 were found to inhibit Runx2 post-translational modification by O-GlcNAcylation. Immunoprecipitation revealed that Runx2 O-GlcNAcylation at 412, at 413, and proximal to 425 was critical for its binding to Smad 1/5/8 and Smad 4, but not Smad 2/3. Using lentivirus-mediated shRNA, we determined that knockdown of Smad1/5/8, but not Smad 2/3, inhibited Runx2-induced VSMC calcification, suggesting that BMP but not TGF-beta signaling is critical for the osteogenic function of Runx2 in VSMC. Taken together, these data demonstrate that Runx2 amino acids 412, 413, 425 and 426-428 are key for its osteogenic function in VSMC, and Runx2 O-GlcNAcylation at 412, 413 and 425 is essential for its interaction with Smad proteins to determine Runx2 transactivity and VSMC calcification. We have identified a novel Runx2 post-translational modification by O-GlcNAcylation at key residues that are essential for its osteogenic function. Our studies provide molecular insights into the function of Runx2 in regulating VSMC calcification, which may shed light on novel targets that are amenable to drug discovery for vascular calcification.