PI3Kγ activation is required for LPS-induced reactive oxygen species generation in respiratory epithelial cells

In this study, we investigated the molecular basis of reactive oxygen species (ROS) generation induced by lipopolysaccharide (LPS) in A549 cells-an alveolar epithelial cell line. A549 cells or normal human bronchial epithelial (NHBE) cells were stimulated with LPS. ROS generation was measured in A549 cells or NHBE cells pre-treated with a selective inhibitor of phosphatidylinositol 3-kinase gamma (PI3K gamma), AS 605240, PI3K gamma siRNA, or a ROS scavenger, pyridoxamine (PM). Treatment of A549 cells or NHBE cells with LPS caused a significant increase in intracellular ROS generation. Pretreatment with the PI3K gamma inhibitor, AS 605240 decreased the LPS-induced increase of ROS generation, phosphorylation of Akt, and production of phosphatidyl 3,4,5-trisphosphate in A549 cells. In addition, interference with siRNA for PI3K gamma significantly reduced LPS-induced ROS generation in A549 cells. Treatment of A549 cells with LPS or hydrogen peroxide increased the nuclear factor-kappa B (NF-kappa B) in the nucleus, accompanying an increase in phosphorylation of inhibitory kappa B-alpha, degradation of the protein, and reduction of cytosolic NF-kappa B. Pretreatment with AS 605240 reduced these LPS-induced changes. In addition, pretreatment with PM or N-acetyl cysteine resulted in inhibition of nuclear NF-kappa B activation. These results suggest that PI3K gamma plays a key role in LPS-induced ROS generation in alveolar epithelial cells, thereby activating NF-kappa B.