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 dihydrophenoxazine. 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.