TNF-α-induced cPLA₂ expression via NADPH oxidase/reactive oxygen species-dependent NF-κB cascade on human pulmonary alveolar epithelial cells

Tumor necrosis factor-a (TNF-α) triggers activation of cytosolic phospholipase A2 (cPLA₂) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA₂ expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA₂ mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE₂ secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE₂ ELISA kit. These TNF-α-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47^phox, TRAF2, NIK, IKKa, IKKß, or p6₅. TNF-α markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47^phox. In addition, TNF-α also stimulated p47^phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47^phox complex formation in HPAEpiCs, which were attenuated by a TNF-α neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated IKKa/ß and NF-κB p6₅ phosphorylation, NF-κB (p6₅) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-α-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKα/ß pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA₂ by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway.