Abstract 17015: Increased Protein O-GlcNAcylation Inhibits TNF-α-Induced NFkB Signaling in Rat Aortic Smooth Muscle Cells
We have previously shown that acutely augmenting O-linked-N-acetylglucosamine (O-GlcNAc) protein modification with glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) attenuates inflammatory responses and neointima formation following endoluminal injury of the rat carotid artery and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response process. Our current study tested the hypothesis that GlcN or PUGNAc treatment can protect vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress in vitro by inhibiting NFkB signaling. Quiescent cultured rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (0.1 mM) or vehicle for 1 hr and then stimulated with TNF-α (10 ng/ml) for another 1 or 6 hrs. Both treatments increased global O-GlcNAc modified protein levels [Western blot (WB) with the selective CTD110.6 antibody] (6 hr sample); inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1and P-Selectin](6 hr sample) and NFkB DNA binding activity (−40%, TransAM ELISA)(1 hr sample) . Using immunoprecipitation and WB techniques (Fig, 1 hr sample), we demonstrated that GlcN and PUGNAc treatments inhibited TNF-α induced nuclear phosphorylation of NFκB p65 (- 50%) and promoted the binding of IkBα and NFκB p65. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased TNF-α induced binding of NFkB p65 to CINC-2β and MCP-1 promoters in GlcN and PUGNAc treated cells (1 hr sample). In conclusion, increased protein O-GlcNAc modification inhibited TNF-α-Induced expression of inflammatory mediators in RASMCs through inhibition of NFκB signaling. Ongoing studies are directed toward identifying the specific protein targets for O-GlcNAcylation in the NFkB pathway.
- © 2010 by American Heart Association, Inc.