Activation and induction of cytosolic phospholipase A₂ by TNF‐α mediated through Nox2, MAPKs, NF‐κB, and p300 in human tracheal smooth muscle cells

Abstract

Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA(2) expression and PGE(2) synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we report that TNF-α-induced cPLA(2) protein and mRNA expression, PGE(2) production, and phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which were attenuated by pretreatment with a ROS scavenger [N-acetyl-L-cysteine, (NAC)] and the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNA of Nox2, p47(phox) , MEK1, p42, p38, or JNK2. TNF-α-induced cPLA(2) expression was also inhibited by pretreatment with a selective NF-κB inhibitor [helenalin (HLN)] or transfection with dominant negative mutants of NF-κB inducing kinase (NIK) or IκB kinase (IKK)α/β. TNF-α-induced NF-κB translocation was blocked by pretreatment with NAC, DPI, APO, or HLN, but not by U0126, SB202190, or SP600125. In addition, pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA blocked cPLA(2) expression and PGE(2) synthesis induced by TNF-α. We further confirmed that p300 was associated with the cPLA(2) promoter which was dynamically linked to histone H4 acetylation stimulated by TNF-α, determined by chromatin immunoprecipitation assay. Association of p300 and histone H4 to cPLA(2) promoter was inhibited by U0126, SB202190, and SP600125. These results suggested that in HTSMCs, activation of p47(phox) , MAPKs, NF-κB, and p300 is essential for TNF-α-induced cPLA(2) expression and PGE(2) release.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression

“…We and others have clearly demonstrated that the human cPLA 2 ␣ promoter (in our studies, extending to approximately Ϫ 6.8 kb) is not responsive to cytokine activation (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). This discrepancy may result from these investigators' use of diseased synovial fi broblasts in conjunction with IL-1 ␤ concentrations 15 times (30 ng/ml) higher than our dosage and well beyond physiologically relevant levels.…”

“…cPLA 2 ␣ expression and activity through the proximal promoter, including nuclear factor (NF)-B ( 22,23 ), Krüppel-like factor (KLF) ( 24,25 ), hypoxia-inducible factor (HIF-1) ( 26 ), and specifi city protein 1 (Sp1) ( 27 ). A 48 bp CA dinucleotide repeat at approximately Ϫ 200 bp has been shown to confer a negative regulatory effect on cPLA 2 ␣ basal promoter activity ( 16,28 ).…”

Quantitative Proteomics Reveals the Induction of Mitophagy in Tumor Necrosis Factor-α-activated (TNFα) Macrophages

“…These analyses also confirmed the identification of several proteins known to be modulated by TNF-␣, such as cPLA 2 and TRAP1. Previous reports have described the activation of cPLA 2 upon TNF-␣ activation of macrophages (48,49). This protein is activated by calcium and comprises both lysophospholipase and transacylase activities.…”

Inhibition of NF-κB prevents high glucose-induced proliferation and plasminogen activator inhibitor-1 expression in vascular smooth muscle cells

“…NF-κB is one of the key factors of atherosclerosis detected in VSMCs, macrophages, and endothelial cells from human atherosclerotic lesions (Brand et al, 1996). Even though inflammatory cytokines such as TNF-α (Lee et al, 2011) or advanced glycation endproducts (Yoon et al, 2009) promoted the progression of atherosclerosis through activation of NF-κB pathway, that is not adequate evidences which explain about the direct effect of hyperglycemia itself in diabetic vasculopathy. Therefore, in this study, we observed that activation of NF-κB was stimulated by high concentrations of glucose as evidenced by translocation of the p65 subunit, increased luciferase activity, and degradation of IκB-α.…”