Phosphatidylinositol 3-kinase γ signaling through protein kinase Cζ induces NADPH oxidase-mediated oxidant generation and NF-κB activation in endothelial cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3K gamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3K gamma-mediated oxidant signaling in the mechanism of NF-kappa B activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110 gamma catalytic subunit of PI3K gamma. Tumor necrosis factor (TNF)alpha challenge of wild type ECs caused p110 gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5- trisphosphate production coupled to ROS production; however, this response was blocked in p110 gamma (-)/(-) ECs. ROS production was the result of TNF alpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110 gamma(-/-) ECs. Additionally, the TNF alpha- activated NF-kappa B binding to the ICAM-1 promoter, ICAM- 1 protein expression, and PMN adhesion to ECs required functional PI3K gamma. TNF alpha challenge of p110 gamma(-/-) ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKC xi. Thus, PI3K gamma lies upstream of PKC xi in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappa B activation and ICAM-1 expression.