Abstract 21191: Disturbed Flow-Mediated Reactive Oxygen Species Production and Subsequent Protein Kinase Cζ Activation Plays a Key Role in Endothelial Apoptosis via PIASy (SUMO E3 ligase)-Mediated p53-SUMOylation and p53 Protein Stability
Atherosclerosis formation is readily observed in certain areas of the arterial tree such as branch points, which experience disturbed flow (d-flow). A positive correlation between protein kinase C (PKC)ζ activation and d-flow has been reported, but the exact mechanism how d-flow-mediated PKCζ activation accelerates atherosclerosis remains unclear. A critical role of endothelial cells (EC) apoptosis for this process has been suggested, but the relationship between d-flow and a pro-apoptotoic factor p53 in EC remains largely unknown. In this study, we found that d-flow, but not steady laminar flow (s-flow), increased apoptosis in EC, which was inhibited by depletion of PKCζ with siRNA and adenovirus vector containing dominant negative form of PKCζ (Ad-DN-PKCζ). D-flow also significantly increased p53 expression and nuclear export with p53-Bcl2 association, which leads to apoptosis by inhibiting Bcl2 anti-apoptotic function. We also found the increase of p53 expression and nuclear translocation, and TUNEL positive EC in d-flow area by en face confocal microscopy analysis in mice aortae in vivo. The key role of p53-SUMOylation was confirmed by PIASy siRNA in EC, which inhibited p53-SUMOylation, p53 nuclear export, and p53-Bcl2 binding as well as subsequent p53 protein stability induced by d-flow. D-flow-mediated PKCζ activation, p53-SUMOylation and p53 expression were inhibited by ebselen and Mn(III)TBAP, suggesting the critical role of ROS production. Although PKCζ could not phosphorylate PIASy, the RING finger-like zinc domain of PIASy (aa 301–400) associates with PKCζ and inhibition of this binding with a PIASy (aa 301–400) fragment abolished p53-SUMOylation. Finally, we found the decrease of p53 expression and nuclear export compared with s-flow area in the aorta of PIASy knockout mice in vivo. These data strongly suggests the key role of PKCζ-PIASy binding on p53-SUMOylation and p53 protein stability in d-flow-mediated EC apoptosis both in vitro and in vivo.
- © 2010 by American Heart Association, Inc.