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- Title
TNF-α-Induced cPLA<sub>2</sub> Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.
- Authors
Chih-Chung Lin; Wei-Ning Lin; Rou-Ling Cho; Chen-yu Wang; Li-Der Hsiao; Chuen-Mao Yang
- 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 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-a-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [Nacetyl cysteine, (NAC)], NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47phox, TRAF2, NIK, IKKa, IKKb, or p65. TNF-a 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 p47phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47phox 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-a-stimulated IKKa/b and NF-κB p65 phosphorylation, NF-κB (p65) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-a-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKa/b 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.
- Subjects
TUMOR necrosis factors; PHOSPHOLIPASE A2; PROSTAGLANDINS
- Publication
Frontiers in Pharmacology, 2016, Vol 7, p1
- ISSN
1663-9812
- Publication type
Article
- DOI
10.3389/fphar.2016.00447