TNF-α-Induced cPLA2 Expression 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 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, 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 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.

Keywords: ASK1; cytokines; cytosolic phospholipase A2; lung inflammation; signaling transduction.

FIGURE 1

TNF-α induces NADPH oxidase- and ROS-dependent cPLA₂ expression. (A) HPAEpiCs were pretreated with NAC, DPI, or APO for 1 h, and then incubated with TNF-α for 24 h. (B) Cells were pretreated with NAC (10 mM), DPI (1 μM), or APO (100 μM) for 1 h, and then incubated with TNF-α for 6 h. cPLA₂ mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p47^phox siRNA, and then incubated with TNF-α for 24 h. (D) HPAEpiCs were pretreated with human TNF-α neutralizing antibody (TNF-α nAb: 0.01, 0.1 and 1 μg/ml) for 1 h, and then incubated with TNF-α for 24 h. (A,C,D) The protein levels of cPLA₂ and p47^phox were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments. ^∗P < 0.05, ^#P < 0.01 as compared with the cells exposed to TNF-α alone.

FIGURE 2

TNF-α induces NADPH oxidase-dependent ROS generation. (A) HPAEpiCs were treated with TNF-α for the indicated time intervals. DHE (red) or DCF (green) fluorescence image was observed. Images shown are representative of five independent experiments with similar results. Cells were (B) treated with TNF-α for the indicated time intervals or (C) pretreated with TNFR1 neutralizing antibody (10 μg/ml), APO (100 μM), or DPI (1 μM) for 1 h, and then treated with TNF-α for 10 min. NADPH oxidase activity was measured. (D) Cells were treated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p47^phox antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-phospho-tyrosine, anti-phospho-serine, or anti-p47^phox antibody. (E) Cells were treated with TNF-α for the indicated time intervals. The cytosolic and membrane fractions were prepared and analyzed by Western blot using an anti-p47^phox, anti-G_αs, or anti-GAPDH antibody. GAPDH and Gαs were used as a marker protein for cytosolic and membrane fractions, respectively. (F) Cells were pretreated with TNFR1 neutralizing antibody, NAC, APO, or DPI for 1 h, and then incubated with TNF-α for 10 min. H₂O₂ and superoxide generation were measured. Data are expressed as mean ± SEM of three independent experiments. ^∗P < 0.05, ^#P < 0.01 as compared with the cells exposed to vesicle alone (B) or TNF-α alone (C,F).

FIGURE 3

TNF-α induces TNFR1/TRAF2/ASK1/p47^phox complex formation. (A) HPAEpiCs were transfected with scrambled or TRAF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of TRAF2 and cPLA₂ were determined. (B) Cells were pretreated without or with human TNF-α neutralizing antibody (TNF-α nAb:1 μg/ml) for 1 h and then incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-TNFR1, anti-TRAF2, anti-ASK1, or anti-p47^phox antibody, and then the immunoprecipitates were analyzed by Western blot by using an anti-TNFR1, anti-TRAF2, anti-ASK1, anti-p47^phox or anti-IgG antibody. The results of IgG were used to be the loading control. (C) Cells were transfected with scrambled or ASK1 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ASK1 and cPLA₂ were determined. (D) Cells were treated with TNF-α for the indicated time intervals. The levels of phospho-ASK1 were determined by Western blot. Data are representative of three independent experiments with similar results. ^∗P < 0.05, ^#P < 0.01 as compared with the basal group.

FIGURE 4

TNF-α induces NADPH oxidase/ROS generation and PGE₂ release via TRAF2, ASK1, and p47^phox. HPAEpiCs were transfected with scrambled, ASK1, p47^phox, or TRAF2 siRNA, and then incubated with TNF-α for (A,B) 10 min or (C) 24 h. (A) H₂O₂ and superoxide generation were measured. (B) NADPH oxidase activity was measured. (C) PGE₂ generation was measured. Data are expressed as mean ± SEM of three independent experiments. ^∗P < 0.05, ^#P < 0.01 as compared with the cells exposed to TNF-α + scrambled siRNA.

FIGURE 5

TNF-α induces cPLA₂ expression via NIK, IKKα/β, and NF-κB. (A) HPAEpiCs were transfected with scrambled, NIK, IKKα, or IKKβ siRNA, and then incubated with TNF-α for 24 h. The protein levels of NIK, IKKα, IKKβ, and cPLA₂ were determined by Western blot. (B) Cells were pretreated with Bay11-7082 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA₂ were determined by Western blot. (C) Cells were pretreated with Bay11-7082 for 1 h, and then incubated with TNF-α for 6 h. cPLA₂ mRNA levels and promoter activity were determined. (D) Cells were transfected with scrambled or p65 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p65 and cPLA₂ were determined. (E) Cells were pretreated with Bay11-7082 for 1 h, and then incubated with TNF-α for 24 h. PGE₂ generation was measured. (F) Cells were incubated with TNF-α for the indicated time intervals. Cells were fixed, labeled with an anti-p65 antibody, and then FITC-conjugated secondary antibody. Individual cells were imaged. (G) Cells were incubated with TNF-α for the indicated time intervals. NF-κB promoter activity was determined. Data are expressed as mean ± SEM of three independent experiments. ^∗P < 0.05, ^#P < 0.01 as compared with the cells exposed to TNF-α alone (C,E) or vesicle alone (G).

FIGURE 6

TNF-α induces NIK/IKKα/β-dependent NF-κB activation. (A) HPAEpiCs were pretreated with NAC (10 mM), DPI (1 μM), APO (100 μM) or Bay11-7082 (10 μM) for 1 h, and then treated with TNF-α for the indicated time intervals. The levels of p65 phosphorylation were determined by Western blot. The raw data of the GAPDH of (A) were provided with a supplementary data (Supplementary Figure S2). (B) Cells were pretreated with NAC, DPI, or APO for 1 h, and then treated with TNF-α for 2 h. NF-κB promoter activity was determined. (C) Cells were pretreated with NAC, DPI, or APO for 1 h, and then treated with TNF-α for the indicated time intervals. The nuclear fractions were prepared and analyzed by Western blot using an anti-p65 antibody. Lamin A was used as a marker protein for nuclear fractions. (D) Cells were pretreated with NAC, APO, or DPI for 1 h, and then treated with TNF-α for the indicated time intervals. The levels of IKKα/β phosphorylation were determined by Western blot. (E) Cells were transfected with scrambled, NIK, IKKα, or IKKβ siRNA, and then incubated with TNF-α for 10 min. The protein levels of phospho-IKKα/β, phospho-p65, NIK, IKKα, and IKKβ were determined. Data are expressed as mean ± SEM of three independent experiments. ^∗P < 0.05, ^#P < 0.01 as compared with the cells exposed to TNF-α alone.

FIGURE 7

Schematic representation of the signaling pathways involved in the TNF-α-induced cPLA₂ expression in HPAEpiCs. The mechanisms underlying TNF-α-mediated activation of TNFR1/TRAF2/ASK1/p47^phox-dependent NADPH oxidase is required for the expression of cPLA₂ in HPAEpiCs. Finally, the activation of ROS/NIK/IKKα/β/NF-κB pathway led to cPLA₂ gene transcription and PGE₂ release.