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TNF-α-Induced cPLA(2) Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells
Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A(2) (cPLA(2)) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA(2) expression were not fully defined in human pulmonary alveolar epith...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122718/ https://www.ncbi.nlm.nih.gov/pubmed/27932980 http://dx.doi.org/10.3389/fphar.2016.00447 |
Sumario: | Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A(2) (cPLA(2)) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA(2) expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA(2) mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE(2) secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE(2) 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, 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 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 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 cPLA(2) 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. |
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