Abstract 777: Caveolin-1 Regulates NADPH Oxidase Subcellular Location And Activity In Endothelial Cells.

Abstract

Background: The biological setting or location where reactive oxygen species (ROS) are produced is a new concept which may provide a mechanistic explanation for redox signaling specificity. In this regard, we recently reported that NADPH oxidase, a major superoxide producing enzyme system, is localized and functional in endothelial cell caveolae. Here, we investigate the role of caveolin-1, the main structural protein of caveolae, in the organization and regulation of NADPH oxidase activity and ROS mediated responses to cytokine challenge. Methods and Results: Superoxide production was measured by DHE assay in endothelial cells derived from wild type (wt) and caveolin-1 knockout (Cav−/ −) mouse lungs (MLEC). TNFα failed to induced superoxide production in Cav−/ − endothelial cell cultures (MOI = 0). However, infection of knockout cells with full-length caveolin-1 containing Adenovirus at MOI of 10 and 50 progressively restored TNFα effect on superoxide production with MOI 50 nearly reaching those observed in wt cells. The distribution of NADPH oxidase within the plasma membrane showed localization to membrane rafts fractions in wt cells which shifted to the non-raft membrane fractions in Cav-1−/ − MLEC. Re-expression of caveolin-1 (MOI 50) restored raft targeting of NADPH oxidase and both gp91 and p22phox co-precipitated with newly expressed caveolin-1 immunoprecipitated from raft fractions. To evaluate the cellular consequences of a potential redox signaling relay involving caveolin-1, we examined an NADPH oxidase dependent pathway involving activation of JNK1/2 MAP kinases and downstream upregulation of ICAM. We found that phosphorylation of JNK1/2 observed in wt MLEC in response to TNFα was significantly attenuated in Cav−/ − MLEC. In addition, TNFα did not induce upregulation of ICAM expression in caveolin-1 knockout MLEC or wt cells pretreated with DPI. Conclusions: Collectively, these results indicate that caveolin-1 is a necessary component of the molecular machinery for proper subcellular positioning required for cytokine activation of NADPH oxidase and superoxide production in endothelial cells. Thus, caveolin-1 may constitute a potential therapeutic target for altering ROS associated with cardiovascular pathology.