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TNF-?-Induced cPLA₂ Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-?B Cascade on Human Pulmonary Alveolar Epithelial Cells.

ABSTRACT: Tumor necrosis factor-? (TNF-?) triggers activation of cytosolic phospholipase A₂ (cPLA₂) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-? induced cPLA₂ expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-?-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-?-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-?B (Bay11-7082) and transfection with siRNA of ASK1, p47 ^phox , TRAF2, NIK, IKK?, IKK?, or p65. TNF-? 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-? also stimulated p47 ^phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-? induced TNFR1, TRAF2, ASK1, and p47 ^phox complex formation in HPAEpiCs, which were attenuated by a TNF-? neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-?-stimulated IKK?/? and NF-?B p65 phosphorylation, NF-?B (p65) translocation, and NF-?B promoter activity in HPAEpiCs. Finally, we observed that TNF-?-stimulated NADPH oxidase activation and ROS generation activates NF-?B through the NIK/IKK?/? pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA₂ by TNF-? is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-?B pathway.

SUBMITTER: Lin CC

PROVIDER: S-EPMC5122718 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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TNF-α-Induced cPLA2 Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.

Lin Chih-Chung CC Lin Wei-Ning WN Cho Rou-Ling RL Wang Chen-Yu CY Hsiao Li-Der LD Yang Chuen-Mao CM

Frontiers in pharmacology 20161125

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 ...[more]

PMID: 27932980

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Project description:Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target.NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H2O2-induced carboxy-H2-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.Nigral dopaminergic neurons in situ expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.

Project description:BackgroundNADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.ResultsWe assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.ConclusionThis report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.

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TNF-?-Induced cPLA₂ Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-?B Cascade on Human Pulmonary Alveolar Epithelial Cells.

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Dataset Information

TNF-?-Induced cPLA2 Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-?B Cascade on Human Pulmonary Alveolar Epithelial Cells.

Publications

TNF-α-Induced cPLA<sub>2</sub> Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

TNF-?-Induced cPLA₂ Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-?B Cascade on Human Pulmonary Alveolar Epithelial Cells.