Abstract 4880: Novel Role of P90RSK-mediated Sentrin/Sumo-specific Proteases 2 (SENP2) Phosphorylation on Disturbed Flow and H2O2-induced Apoptosis in Endothelial Cells
The atheroprotective roles of steady laminar flow (s-flow) and its inhibition against pro-inflammatory responses and endothelial cells (EC) apoptosis has been extensively studied. Emerging evidence suggested that disturbed flow (d-flow) and reactive oxygen species (ROS) are critical factors to induce EC dysfunction, which includes changes in vasoregulation, inflammatory activation, and altered barrier function with EC apoptosis. However, it remains unclear how d-flow induces EC dysfunction. We performed a survey of which kinases can be activated by d-flow, but not s-flow. We found that two kinases, p90RSK and PKCζ, were particularly activated under d-flow, but not s-flow in EC. D-flow and H2O2 (50 μM) significantly increased apoptosis in EC (5.79±0.5 fold compared with control; p<0.01). Interestingly, we found that H2O2 increased nuclear export of p53 and p53-Bcl2 binding. The contribution of p53-SUMOylation on p53 nuclear export has been reported, and cytosolic p53-Bcl2 binding inhibits Bcl2 anti-apoptotic effect and induces apoptosis. Overexpression of p90RSK increased p53-SUMOylation, and adenovirus containing dominant negative form of p90RSK (Ad-DN-p90RSK) transduction significantly inhibited H2O2-mediated p53-SUMOylation, p53 nuclear export, p53-Bcl2 binding, and apoptosis, suggesting the critical role of p90RSK in p53-SUMOylation and subsequent apoptosis. Since four isoforms of p90RSK have similar function in EC, we used DN-p90RSK instead of siRNA which may downregulated all the isoforms. In addition, we determined SENP2 as a novel target of p90RSK based on the following results;
depletion of SENP2 increased p53-SUMOylation and apoptosis in EC,
overexpression of p90RSK significantly decreased SENP2 de-sumoylation activity,
H2O2-mediated SENP2 nuclear export was significantly inhibited by Ad-DN-p90RSK transduction, and
in vitro kinase assay showed that SENP2 was a good substrate of p90RSK.
Finally, en face confocal microscopy analysis using apoE−/− mice also revealed the activation of p90RSK and the up-regulation of p53 expression at d-flow, but not at s-flow area, supporting the patho-physiological consequence of p90RSK-SENP2 and subsequent apoptosis on d-flow-mediated acceleration of atherosclerosis.