title: TNF-α-Induced cPLA2Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.
abstract: Tumor necrosis factor-α (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 cPLA2expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA2mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE2secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE2ELISA 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, p47phox, TRAF2, NIK, IKKα, IKKβ, 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 p47phox. In addition, TNF-α also stimulated p47phoxphosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47phoxcomplex 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 IKKα/β 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/β pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA2by 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.
authors.affiliation: Department of Anesthetics, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University Tao-Yuan, Taiwan.
authors.forename: Chih-Chung
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Graduate Institute of Basic Medicine, Fu Jen Catholic University New Taipei City, Taiwan.
authors.forename: Wei-Ning
authors.initials: WN
authors.lastname: Lin
authors.affiliation: Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University Tao-Yuan, Taiwan.
authors.forename: Rou-Ling
authors.initials: RL
authors.lastname: Cho
authors.affiliation: Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University Tao-Yuan, Taiwan.
authors.forename: Chen-Yu
authors.initials: CY
authors.lastname: Wang
authors.affiliation: Department of Anesthetics, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University Tao-Yuan, Taiwan.
authors.forename: Li-Der
authors.initials: LD
authors.lastname: Hsiao
authors.affiliation: Department of Anesthetics, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung UniversityTao-Yuan, Taiwan; Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung UniversityTao-Yuan, Taiwan; Research Center for Industry of Human Ecology, Research Center for Chinese Herbal Medicine, and Graduate Institute of Health Industry Technology, Chang Gung University of Science and TechnologyTao-Yuan, Taiwan.
authors.forename: Chuen-Mao
authors.initials: CM
authors.lastname: Yang
keywords: ASK1; cytokines; cytosolic phospholipase A2; lung inflammation; signaling transduction