Abstract 176: O-GlcNAc Transferase Promotes Vascular Smooth Muscle Cell De-differentiation

Vascular smooth muscle cells (VSMCs) in mature vessels have a differentiated, contractile phenotype but retain the ability to reversibly de-differentiate in response to environmental cues. Plasticity is necessary for development and wound healing, but also contributes to pathologies such as atherosclerosis and intimal hyperplasia. O- linked N- acetylglucosamine ( O -GlcNAc) transferase (OGT) mediates nutrient-sensitive post-translational modification of serine and threonine residues and has been implicated in multiple diseases, but its role in VSMC phenotypic modulation is not known. In human coronary SMCs, we found that OGT knockdown significantly increased contractile marker expression, including MYH11 and ACTA2, at the mRNA and protein levels. OGT knockdown also decreased proliferation rates after serum treatment in VSMCs. In addition, rapamycin, a differentiating agent, decreased OGT protein expression and O- GlcNAc levels. Conversely, de-differentiation with PDGF treatment increased OGT expression and global O- GlcNAc. Neither treatment affected OGT mRNA levels. OGT has been shown to increase the protein stability of EZH2, the enzyme that generates the inhibitory histone epigenetic mark H3K27me3. We found that OGT knockdown did not affect EZH2 mRNA levels but significantly decreased EZH2 protein expression as well as global H3K27me3 in VSMCs. Similar to OGT, EZH2 expression was decreased upon rapamycin treatment and increased with PDGF. A decrease in ubiquitination of EZH2 was observed after PDGF treatment, suggesting an increase in protein stability. Importantly, EZH2 knockdown increased protein expression of SMC contractile markers while EZH2 overexpression repressed these markers. Wire injury of mouse femoral arteries increased H3K27me3 levels after 21 days. OGT expression was elevated in the innermost layer of the neointima at the same time point. These data demonstrate that OGT inhibits VSMC differentiation and that OGT and its target EZH2 are both dynamically regulated during phenotypic modulation. Future work will illuminate if OGT promotes VSMC de-differentiation through EZH2-mediated histone modification at contractile gene promoters.