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F.X. Yu, K.–P. Xu; Wounding–Induced Epidermal Growth Factor Receptor Activation Is Mediated by Src Tyrosine Kinase in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2126.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:We previously demonstrated that corneal epithelial wound healing is mediated by activation of epidermal growth factor receptor (EGFR). In this study, we sought to identify the underlying mechanism for EGFR activation in response to wounding in cultured human corneal epithelial cells (HCECs). Methods: SV40–immortalized HCECs were wounded and allowed to heal in the presence or absence of PP2, a selective inhibitor of Src kinase and methyl–ß–cyclodextrin (MßCD), a cholesterol sequestering drug. The activation of EGFR was analyzed by immunoprecipitation of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylations of extracellular signal–regulated kinase (ERK) and phosphatidylinositol 3'–kinase (PI3K) were analyzed by Western blotting with antibodies specific to phosphorylated proteins. Lipid rafts were isolated from Triton–insoluble material using equilibrium density gradient centrifugation. The presence of phosphorylated Src and EGFR in raft fractions was assessed using Western blotting. Results: Wounding of HCECs induced phosphorylation of Src at tyrosine 418. The Src kinase inhibitor PP2 blocked wound–induced Src phosphorylation and attenuated epithelial migration and wound closure with or without exogenously added heparin–binding EGF–like growth factor. Concomitant with the inhibition of epithelial wound healing, presence of PP2 in the culture media also impaired the wound–induced EGFR activation and its downstream signaling pathways, ERK and PI3K, in a concentration–dependent manner. Phosphorylated Src at tyrosine 418 and EGFR at tyrosine 845 (a Src dependent phosphorylation site) were preferential distributed in the lipid raft fractions. Depletion of cholesterol by MßCD retarded wound–induced activation of Src and EGFR and disrupted their localization in lipid rafts. Conclusions: Non–receptor tyrosine kinase Src plays an important role in corneal epithelial wound healing via regulating EGFR phosphorylation. Membrane microdomains are important for the formation of signaling complexes including Src and EGFR in response to wounding in HCECs.
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