Reactive Oxygen Species and Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Mediate Hyperoxia-Induced Cell Death in Lung Epithelium

Abstract

Therapy with high oxygen concentrations (hyperoxia) is often necessary to treat patients with respiratory failure. However, hyperoxia may exacerbate the development of acute lung injury, perhaps by increasing lung epithelial cell death. Therefore, interrupting lung epithelial cell death is an important protective and therapeutic strategy. In the present study, hyperoxia (95% O(2)) results in murine lung epithelium cell death by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end labeling, and Annexin V-fluorescein isothiocyanate flow cytometry assay. We show that hyperoxia increases superoxide production, as assessed by nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity and flow cytometric assay, and increases phospho-extracellular signal-regulated kinase (ERK)1/2 by Western blot analysis. These processes are inhibited by a reactive oxygen species inhibitor, diphenylene iodonium (DPI), and by an inhibitor of the mitogen-activated protein (MAP) or ERK kinase (MEK)/ERK1/2 pathway, PD98059. ERK1/2 activation in hyperoxia is also inhibited by DPI. Hyperoxia-induced cell death is associated with cytochrome c release, subsequent caspase 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, which can all be suppressed by DPI and PD98059. However, the broad caspase inhibitor z-VAD-FMK protects cells from death without affecting superoxide generation and ERK1/2 activation. Taken together, our data suggest that hyperoxia, by virtue of activating NADPH oxidase, generates reactive oxygen species (ROS), which mediates cell death of lung epithelium via ERK1/2 MAPK activation, and functions upstream of caspase activation in lung epithelial cells.

Hyperoxia-induced signal transduction pathways in pulmonary epithelial cells☆

“…The presence of apoptosis of hyperoxic epithelium was evaluated by the presence of oligonucleosomal DNA fragments as assessed by terminal dUTP nick end labeling (TUNEL) analysis, and specific internucleosomal DNA laddering, immunohistochemical and biochemical assays for apoptotic gene expression and activities [12,25,26,[35][36][37][38][39]. In addition, increased caspase activities have also been observed in hyperoxic lungs by several investigators [12,38,40]. Caspase activation is considered to represent the onset of irreversible apoptotic pathway.…”

“…Caspase-1, an important mediator in both apoptosis and inflammation, is activated in fractionated lysates of hyperoxic mouse lungs [34]. Exposure of caspase-3 deficient mice to hyperoxia for 72 hours induced less apoptotic cell death of lung epithelial cells, compared to that of the wildtype mice [12]. This suggests that caspase-3 activation is one of the critical steps in hyperoxia induced cell death.…”

“…at levels exceeding the capacity of the lung antioxidant defense mechanisms [7][8][9]. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, in conjunction with mitochondria, are the major sites of ROS generation during hyperoxia [10][11][12][13].…”

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

“…22,23 MEK and MKK are the upstream kinases for ERK1/2 and p38 MAPK, respectively. Recently, the MAP kinase pathways have been linked to hyperoxic toxicity in both cell cultures and animal models; 24,25 hyperoxia primarily activates ERK1/2 but less so p38. 25 Buckley et al have demonstrated that increased ERK1/2 may correlate with protection against hyperoxic toxicity in rats.…”

“…25 Buckley et al have demonstrated that increased ERK1/2 may correlate with protection against hyperoxic toxicity in rats. 24,26,27 p38 MAPK is linked to mismatch repair DNA response via the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents. 28 The only study relating BER to cell cycle regulators demonstrates the link of long-patch BER to p21 in mouse embryonic fibroblast cell line.…”