Abstract 10624: Proteasomal Degradation of O-GlcNAc Transferase Enhances Hypoxia-mediated Vascular Endothelial Inflammatory Response
Hypoxia enhances vascular endothelial inflammatory response by mechanism not fully understood. Emerging evidence implicates O-GlcNAc transferase (OGT) in both hypoxia and vascular inflammation. We sought to identify the role of OGT in hypoxia-mediated vascular inflammation. Hypoxia was either mimicked by cobalt chloride (CoCl2) exposure (200 μM, 12h) or induced (hypoxia chamber with 1% O2, 36h) in cultured endothelial cells. Surprisingly, hypoxia significantly reduced the cellular levels of OGT protein but not mRNA, which was associated with elevated gene expression of inflammatory cytokines IL-6, MCP-1, and E-Selectin (n=3, p<0.05). However, adenoviral overexpression of OGT but not green fluorescent protein (GFP) blocked the elevation in expression of these genes. Hypoxia only failed to reduce OGT in cells respectively pretreated with structurally unrelated proteasome inhibitors MG132, epoxomicin, and lactacystin, suggesting the involvement of proteasomes. Indeed, the 26S proteasome reporter (UbG76V-GFP) cells showed less reporter proteins when CoCl2 was present, indicating an enhancement of 26S proteasome functionality, which was attributable to the increase in both chymotrypsin-like activities and stabilized association of proteasome sub-complexes detected in the cells. Mechanistically, the ubiquitin E3 ligase β-TrCP1 mediated OGT degradation since siRNA knocking down of the ligase abrogated the degradation. Further, superoxide had an upstream key role because pre-incubation of superoxide scavenger mTempol (1mM, 1h) prevented proteasome-mediated degradation of OGT induced by CoCl2. Consistently, in vivo administration of CoCl2 (10mg/kg/d, i.p., 2d) in mice (C57BL/6J) increased mRNA levels of IL-6, MCP-1, and E-Selectin (n=5/group, CoCl2 vs vehicle, p<0.05, lung tissue); however, pre-treatment with UDP-GlcNAc (25 μM in drinking water, 7d), an endogenous activator of OGT, obliterated theses changes (n= 5/group, the treated vs untreated CoCl2 groups, p<0.05). Taken together, 26S proteasome-mediated loss of OGT contributes to hypoxia-induced vascular endothelial inflammatory response. Modulation of OGT may represent a new approach to treat inflammation in patients with hypoxia complications.
- © 2013 by American Heart Association, Inc.