Oxidative Stress Induces Arachidonate Release from Human Lung Cells through the Epithelial Growth Factor Receptor Pathway

Abstract

Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E(2) release, cytosolic phospholipase (cPLA(2)) steady-state mRNA, cPLA(2) protein levels, cPLA(2) enzyme activity, and cPLA(2) phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E(2) release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA(2) activity at 1 h, and with a significant increase in cPLA(2) Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA(2) phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA(2) phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA(2) activity. Finally, cells stably transfected with an antisense cPLA(2) vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA(2) activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.

2,2′,4,4′-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-κB

“…Activation of PLA 2 contributed to upregulation of COX-2 in murine macrophages and human lung cells (Balsinde et al 1999;Pawliczak et al 2002Pawliczak et al ,2004. Furthermore, free AA activates a variety of proteins involved in the regulation of COX-2 expression, including p38 MAP kinase (Hii et al 1998), peroxisome proliferator-activated receptor γ (Pawliczak et al 2002(Pawliczak et al , 2004 and phosphatidyl inositol-3-kinase (Monick et al 2002).…”

“…Furthermore, free AA activates a variety of proteins involved in the regulation of COX-2 expression, including p38 MAP kinase (Hii et al 1998), peroxisome proliferator-activated receptor γ (Pawliczak et al 2002(Pawliczak et al , 2004 and phosphatidyl inositol-3-kinase (Monick et al 2002). Interestingly, expression of COX-2 was decreased in brains of PLA 2 -deficient mice relative to wild type mice (Bosetti and Weerasinghe, 2003).…”

Cytosolic Phospholipase A2 Group IVα but Not Secreted Phospholipase A2 Group IIA, V, or X Induces Interleukin-8 and Cyclooxygenase-2 Gene and Protein Expression through Peroxisome Proliferator-activated Receptors γ 1 and 2 in Human Lung Cells

“…Briefly cells grown in 6-well dishes were transfected with expression vector or empty vector as described above. After 4 h, medium was changed, and cells were labeled for 16 h with 1 Ci/ml [5,6,8,9,11,12,14 Gene Expression Measurements Using a Real Time Polymerase Chain Reaction-Cells were grown as described above and exposed to EGF, A23187, or MAFP. Total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA).…”

“…Upon activation by various stimuli (including but not limited to calcium ionophore (A23187), IL-1␤, tumor necrosis factor-␣, and interferon-␥) cPLA 2 is translocated to the cellular membranes, releasing arachidonic acid from membrane phospholipids (1)(2)(3)(4). Stimuli such as epidermal growth factor (EGF), oxidative stress, or IL-1␤ may also cause cPLA 2 activation through its phosphorylation (5,6). Previously we have reported that cPLA 2 is able to alter gene expression through PPAR-␥ activation and binding to peroxisome proliferator response elements (4).…”

“…The calcium ionophore A23187 activates cPLA 2 by increasing intracellular calcium levels, causing cPLA 2 translocation to the nuclear envelope (4,6,7). EGF is also thought to increase cytosolic phospholipase A 2 activity through activation of the mitogen-activated protein kinase pathway and phosphorylation of cPLA 2 at serine 505 (and possibly other serine residues) (5). Both stimuli are known to increase cPLA 2 enzymatic activity and arachidonate release.…”