H₂O₂ mediates oxidative stress-induced epidermal growth factor receptor phosphorylation

We used a well-established thiol-alkylating agent, N-ethylmaleimide (NEM), to oxidatively stress human keratinocytes. Time course studies revealed that NEM rapidly depleted keratinocytes of reduced glutathione (GSH), which was followed by rapidly increasing levels of intracellular reactive oxygen species (ROS) and subsequently by phosphorylation of epidermal growth factor receptor (EGFR). Pretreatment with antioxidants or enhanced catalase activity in keratinocytes inhibited ROS/H₂O₂ accumulation and EGFR phosphorylation, demonstrating that H₂O₂ production is a mediator required for EGFR phosphorylation. Collectively, these results suggest a sequence of events leading to EGFR phosphorylation which is likely shared by oxidative stress-inducing agents, namely: (1) GSH depletion; (2) H₂O₂ accumulation; and (3) EGFR phosphorylation. We propose that depletion of GSH and accumulation of H₂O₂ are upstream events and critical mediators required for ligand-independent phosphorylation of growth factor receptors in response to oxidative stress.