H₂O₂ is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes

Exposure of human keratinocytes to physiologic doses of ultraviolet B (UVB) radiation induces phosphorylation of the epidermal growth factor receptor (EGFR). We demonstrate that H₂O₂ generated by UVB mediates EGFR phosphorylation. Using dihydrorhodamine 123 as a specific fluorescent dye probe, we show that UVB irradiation (50-800 J per m²) of keratinocytes leads within minutes to concentration-dependent intracellular production of H₂O₂. A corresponding concentration-dependent increase in the release of extracellular H₂O₂ was measured by using Amplex, a derivative of dihydro- phenoxazine. The levels of intracellular H₂O₂ that are induced by UVB irradiation and that stimulate EGFR phosphorylation correlate strongly with the response induced by exogenously added H₂O₂. UVB or H₂O₂ demonstrated concentration- and time-dependent stimulation of EGFR phosphorylation that was initially observed within 1-5 min and exhibited a proportionate delay for UVB-induced production of H₂O₂. EGFR phosphorylation induced by UVB or H₂O₂ declined significantly toward baseline levels by 4 h and could be restimulated after H₂O₂ but not after UVB exposure. Phosphorylation of EGFR was inhibited by the structurally unrelated antioxidants butylated hydroxyanisole, N-acetyl-L-cysteine, and pyrrolidine dithiocarbamate, or by the H₂O₂-degrading enzyme catalase. These data indicate that generation of H₂O₂ by UVB radiation of human keratinocytes participates in the rapid, ligand-independent phosphorylation of EGFR and implicate H₂O₂ as a biologic mediator in EGFR activation and regulation of the downstream signaling cascade. UVB-induced H₂O₂ has the potential to initiate or modulate early EGFR-mediated signaling events that could play an important role in the cellular response to oxidative stress.