Evaluation of lipid rafts, caveolae and endocytosis in regulation of redox signaling in endothelial cells

Caveolae serve as assembly platforms for NADPH oxidase subunits and can undergo internalization via a dynamin‐2 mediated process. Here, primary endothelial cells were pretreated with either methyl‐β‐cyclodextrin (CD), to disrupt rafts and caveolae compartments, cav‐1 siRNA, to deplete cells of caveolae or with Dyn‐2 siRNA, to block caveolae mediated endocytosis in order to understand how these plasma membrane microdomains might participate in redox signaling. We found that all pretreatments blocked NADPH‐dependent ROS production in response to TNF‐α. Similarly, In vivo studies in Cav1KO mice also showed lack of ROS production in the vessel wall compared to WT mice upon systemic administration of TNF‐α. Next, redox signaling pathways that contribute to induction of an endothelial inflammatory phenotype were examined. Blockade of NADPH oxidase mediated ROS production (gp91‐ds‐tat), loss of caveolae organelles, and inhibition of endocytosis attenuated redox sensitive phosphorylation of IkB‐α, NFkB activation and ICAM‐1 expression, but not activation of SAPK's. In contrast, pretreatment with CD attenuated all signaling events. These findings suggest that caveolae regulate NADPH oxidase dependent ROS generation and redox signaling events via a process that involves endocytosis. Further, lipid‐rafts appear to mediate pathways that link TNF‐α to SAPK's, independent of caveolae or redox signaling.