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

Abstract

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 γ (PI3Kγ), AS 605240, PI3Kγ 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 PI3Kγ 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 PI3Kγ significantly reduced LPS-induced ROS generation in A549 cells. Treatment of A549 cells with LPS or hydrogen peroxide increased the nuclear factor-κB (NF-κB) in the nucleus, accompanying an increase in phosphorylation of inhibitory κB-α, degradation of the protein, and reduction of cytosolic NF-κB. Pretreatment with AS 605240 reduced these LPS-induced changes. In addition, pretreatment with PM or N-acetyl cysteine resulted in inhibition of nuclear NF-κB activation. These results suggest that PI3Kγ plays a key role in LPS-induced ROS generation in alveolar epithelial cells, thereby activating NF-κB.