O-Linked ß-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

OBJECTIVE: Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked ß-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. APPROACH AND RESULTS: In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. CONCLUSIONS: These results suggest that O-GlcNAcylation of A20 plays a key role in the negative regulation of NF-κB signaling cascades in TNF-α-treated vascular smooth muscle cells in culture and in acutely injured arteries, thus protecting against inflammation-induced vascular injury.

Smad3-mSin3A-HDAC1 Complex is Required for TGF-ß1-Induced Transcriptional Inhibition of PPARγ in Mouse Cardiac Fibroblasts.

BACKGROUND: We have recently demonstrated that activated transforming growth factor-ß (TGF-ß) signaling suppresses myocardial peroxisome proliferator-activated receptor γ (PPARγ) expression in the pressure overloaded heart. In this study, we aim to further define the molecular mechanisms that underlie TGF-ß-induced PPARγ transcriptional inhibition. METHODS: Adult mouse cardiac fibroblasts were isolated and cultured. PPARγ promoter activity was measured by the dual-Luciferase reporter assay. Interactions between transcription factors and the target gene were identified. RESULTS: In cultured cardiac fibroblasts transfected with a plasmid containing a human PPARγ promoter, co-transfection of Smad3 and Smad4, but not Smad2, plasmids significantly enhanced TGF-ß1-induced inhibition of PPARγ promoter activity. Promoter deletion analysis and site-directed mutagenesis assays defined two Smad binding elements on the promoter of the PPARγ gene. Utilizing chromatin immunoprecipitation analysis and DNA-affinity precipitation methods, we demonstrated that the transcriptional regulatory complex consisting of Smad3, mSin3A and HDAC1 bound to the promoter of the PPARγ gene in cardiac fibroblasts in response to TGF-ß1 stimulation. Either silencing endogenous mSin3A expression by Lentivirus-mediated transduction of mSin3A shRNA or pretreatment with the specific HDAC1 inhibitor MS-275 effectively attenuated TGF-ß-induced transcriptional suppression of PPARγ. CONCLUSION: These results suggest that TGF-ß1-induced inhibition of PPARγ transcription depends on formation of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the PPARγ promoter.