Abstract 14601: Increased O-Linked N-Acetylglucosamine Modification Induces Vascular Calcification by Activation of AKT
Objective Vascular calcification is prevalent in patients with diabetes mellitus and increases morbidity and mortality of these patients. Vascular calcification has been demonstrated in several experimental diabetic models, including the streptozotocin-induced mouse model of diabetes. We found that low-dose streptozotocin induced protein O-linked GlcNAc (O-GlcNAc) modification in mouse vasculature. Therefore, we determined the role of O-GlcNAc modification in regulating vascular calcification. Methods and Results –Thiamet-G, an inhibitor of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc, significantly increased O-GlcNAc modification and induced calcification of cultured primary mouse vascular smooth muscle cells (VSMC) in a dose-dependent manner (0.1-10 μM). Thiamet-G-induced VSMC calcification was associated with increased expression of osteogenic protein osteocalcin (fold=4.85±0.10, n=4, p<0.05). Additionally, Thiamet-G induced the expression (fold=2.97±0.31) and transactivity (fold=1.48±0.17, n=4, p<0.05) of Runx2, the key osteogenic transcription factor for VSMC calcification. Knockdown of OGA by short-hairpin RNA dramatically increased O-GlcNAc modification and induced calcification of VSMC. In addition to enhanced Runx2 transactivity (fold=1.85±0.18, n=4, p<0.05), increased O-GlcNAc modification by OGA knockdown enhanced and sustained activation of AKT (phosphorylation at S473), an upstream signal that we have previously reported regulates Runx2 activity. Mutation of AKT at predicted O-GlcNAc modification sites, T430 and T479, but not S122, significantly decreased O-GlcNAc modification and phosphorylation of AKT at S473. Decreased O-GlcNAc modification decreased Runx2 transactivity (T430=38%, T479=33% of control, n=3, p<0.05) and reduced VSMC calcification as indicated by reduced total calcium content (T430=68%, T479=53% of control). Conclusion – We have demonstrated that increased O-GlcNAc modification enhances AKT activation via modification at T430 and T479, which results in increased Runx2 transactivity and VSMC calcification. These observations provide novel mechanistic insights into the role of protein O-GlcNAc modification in regulating diabetic vascular calcification.
- © 2012 by American Heart Association, Inc.