EGF-Receptor Phosphorylation and Signaling Are Targeted by H₂O₂ Redox Stress

Abstract

Inflammation of the respiratory tract is associated with the production of reactive oxygen species, such as hydrogen peroxide (H2O2) and superoxide (O2-), which contribute extensively to lung injury in diseases of the respiratory tract. The mechanisms and target molecules of these oxidants are mainly unknown but may involve modifications of growth-factor receptors. We have shown that H2O2 induces epidermal growth factor (EGF)-receptor tyrosine phosphorylation in intact cells as well as in membranes of A549 lung epithelial cells. On the whole, total phosphorylation of the EGF receptor induced by H2O2 was lower than that induced by the ligand EGF. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein, indicating that it was due to the activation of protein tyrosine kinase (PTK). Phosphoamino acid analysis revealed that although the ligand, EGF, enhanced the phosphorylation of serine, threonine, and tyrosine residues, H2O2 preferentially enhanced tyrosine phosphorylation of the EGF receptor. Serine and threonine phosphorylation did not occur, and the turnover rate of the EGF receptor was slower after H2O2 exposure. Selective H2O2-mediated phosphorylation of tyrosine residues on the EGF receptor was sufficient to activate phosphorylation of an SH2-group-bearing substrate, phospholipase C-gamma (PLC-gamma), but did not increase mitogen-activated protein (MAP) kinase activity. Moreover, H2O2 exposure decreased protein kinase C (PKC)-alpha activity by causing translocation of PKC-alpha from the membrane to the cytoplasm. These studies provide novel insights into the capacity of a reactive oxidant, such as H2O2, to modulate EGF-receptor function and its downstream signaling. The H2O2-induced increase in tyrosine phosphorylation of the EGF receptor, and the receptor's slower rate of turnover and altered downstream phosphorylation signals may represent a mechanism by which EGF-receptor signaling can be modulated during inflammatory processes, thereby affecting cell proliferation and thus having implications in wound repair or tumor formation.

Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells

“…Oxidative stress is believed to induce the cyclin dependent kinase inhibitor, p21, thereby inhibiting DNA replication and hence allowing additional time to repair damaged DNA. Consistent with a previous report [21] demonstrating that H 2 O 2 induced G 1 arrest in A549 cells, we have shown that a 1-h pulse of H 2 O 2 induced cell arrest at G 0 /G 1 phase and small amounts of apoptosis (only 3 %) in SV-40 transformed lung epithelial cells in the presence of growth factors.…”

Oxidative stress and regulation of glutathione in lung inflammation

“…Modulation of growth factor receptors and altered cellular signalling is proposed to occur through a redoxmediated mechanism in inflammatory and lung cells. Tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in lung epithelial cells by H 2 O 2 is thought to influence inflammatory processes in lungs [209]. H 2 O 2 is known to induce apoptosis in many cells including epithelial cells, and this response is inhibited by glutathione [3,9,210,211].…”

Stress signals for apoptosis: ceramide and c-Jun kinase

“…In this regard, ceramide generation in response to the thymidylate synthase inhibitor GW1843 which activates both NSMase and ASMase (Laethem et al, 1998), anti-IgM which signals through ASMase (Chen et al, 1998) and hypoxic damage via NSMase (Yoshimura et al, 1998) are not a ected by peptide caspase inhibition. Further, ionizing radiation (Haimovitz-Friedman et al, 1994) and H 2 O 2 (Goldkorn et al, 1998) activate sphingomyelinases in isolated membranes. Perhaps most persuasive, heat induces ceramide synthase activation and ceramide generation in Saccharomyces cerevisiae which contain no cytokine receptors or caspases (Jenkins et al, 1997;Wells et al, 1998).…”