Abstract
We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kγ in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kγ-mediated oxidant signaling in the mechanism of NF-κB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110γ catalytic subunit of PI3Kγ. Tumor necrosis factor (TNF) α challenge of wild type ECs caused p110γ translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110γ–/– ECs. ROS production was the result of TNFα activation of Ser phosphorylation of NADPH oxidase subunit p47phox and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110γ–/– ECs. Additionally, the TNFα-activated NF-κB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kγ. TNFα challenge of p110γ–/– ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCζ. Thus, PI3Kγ lies upstream of PKCζ in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-κB activation and ICAM-1 expression.