Phosphatidylinositol 3-Kinase Signaling through Protein Kinase C Induces NADPH Oxidase-mediated Oxidant Generation and NF- B Activation in Endothelial Cells*

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