Phosphatidylinositol 3-Kinase γ Signaling through Protein Kinase Cζ Induces NADPH Oxidase-mediated Oxidant Generation and NF-κB Activation in Endothelial Cells

Randall S Frey,Xiaopei Gao,Kamran Javaid,Shahid S Siddiqui,Arshad Rahman,Asrar B Malik

doi:10.1074/jbc.m508810200

Abstract

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.

Highlights

phosphatidylinositol 3-kinase (PI3K) catalyze the conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is involved in the recruitment and activation of a variety of regulatory proteins via interactions with their pleckstrin homology and phox homology domains [21]. phox domains, present in two subunits of the NADPH oxidase complex, p47phox and p40phox, bind to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate [21, 22]
We addressed a possible role for PI3K␥ as an upstream regulator of PKC␨ activation and thereby in mediating NADPH oxidase assembly and generating the oxidant signaling required for NF-␬B activation and intercellular adhesion molecule (ICAM)-1 expression in endothelial cells (ECs)
Because the described role of PI3K␥ in immunity is largely restricted to leukocytes [15], in the present study we investigated the possibility that PI3K␥ plays a potentially important role in regulating the immune response in the vessel wall, the vessel wall lining ECs

Summary

Introduction

PI3Ks catalyze the conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is involved in the recruitment and activation of a variety of regulatory proteins via interactions with their pleckstrin homology and phox homology domains [21]. phox domains, present in two subunits of the NADPH oxidase complex, p47phox and p40phox, bind to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate (both breakdown products of PIP3) [21, 22]. We showed that PKC␨ activation of NADPH oxidase was required for TNF␣-induced oxidant generation in ECs [28].

Results

Conclusion