Abstract 14023: P53 Thr55 Phosphorylation Mediates High Glucose-Induced Endothelial Oxidative Stress
Diabetes mellitus causes multiple vascular complications and cardiovascular dysfunction. High levels of glucose (HG) induce reactive oxygen species-mediated oxidative stress in endothelial cells (ECs), which leads to endothelial dysfunction and tissue damage. However, the precise molecular mechanisms in HG-induced endothelial oxidative stress and damage remain elusive. Here we demonstrate that HG inhibits antioxidant glutathione peroxidase 1 (GPX1) by inactivating p53, a key regulator of GPX1 expression, and that this loss of activity is mediated by phosphorylation of p53 at Thr55. Exposure of human aortic endothelial cells (HAEC) to HG (20 mM), but not high osmotic conditions (mannitol), increased cellular ATP levels and inhibited GPX1 expression, whereas 2-deoxy-glucose (2-DG), a potent inhibitor of glucose metabolism, abolished these effects. Further, HG suppressed GPX1 expression in ECs transfected with scrambled siRNA but not p53 siRNA, suggesting that the inhibitory effects of HG on GPX1 are p53 dependent. To define the molecular mechanisms, we show that HG stimulated TBP-associated factor 1 (TAF1) kinase activity and resultant p53 Thr55 phosphorylation, leading to p53 degradation and dissociation of p53 from the GPX1 promoter as determined by chromatin immunoprecipitation analysis. Inhibition of TAF1 with apigenin and TAF1 siRNA in HAEC or re-introduction of a TAF1 kinase dead mutant (A2) into TAF1 knockdown HAEC abolished HG-induced Thr55 phosphorylation, p53 protein degradation and inhibition of GPX1 expression, suggesting that TAF1 plays a role in HG-induced GPX1 inhibition. In addition, HG suppressed p53 binding to the GPX1 promoter and GPX1 expression in HAEC transfected with wild type p53 but not p53 T55A, indicating that Thr55 phosphorylation is involved in HG-induced GPX1 inhibition. Finally, HG increased intracellular H2O2 generation, cleaved caspase-3 and apoptosis, and suppressed nitric oxide (NO) bioavailability. These effects were dramatically reversed by TAF1 inhibition. We conclude that HG inhibits GPX1 function via increasing ATP levels and TAF1-mediated p53 Thr55 phosphorylation. These studies may also provide a therapeutic strategy for diabetes-associated cardiovascular diseases.
- © 2011 by American Heart Association, Inc.