Activation of epidermal growth factor receptor during corneal epithelial migration

PURPOSE. Epidermal growth factor (EGF) and related growth factors: transfor ming growth factor (TGF)-alpha, heparin-binding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to 1 common EGF receptor ( EGFR), tyrosine kinase. We have used in vivo and organ culture wound-heatin g models to examine the kinetics and extent of EGFR activation during corne al epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process. METHODS. In the in vivo model. 3-mm debridement wounds were made in rat cor neas and allowed to heal in situ. Activation of EGFR was analyzed by 1) ind irect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF-fluorescein isothiocyanate (FITC). Relative levels of mRNA fur EGF. TG F-alpha, HB-EGF, and AR were determined using reverse transcription-polymer ase chain reaction. To determine whether inhibiting EGFR activation slows e pithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0-50 mu M), a specific inhibitor of E GFR kinase activity. RESULTS. In unwounded corneas, EGFR was localized in basal cells and appear ed to be membranous. Within 1 hour after wounding, EGFR was no longer immun olocalized in the membranes of cells migrating into the wound area. EGF-FIT C-binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyr osine residues within 30 minutes after wounding and that phosphorylation le vels increased after wounding. Levels of mRNA for TGF-alpha. HB-EGF, and AR all appeared to increase after wounding. In organ culture experiments, tyr phostin AG1478 inhibited migration rates in a dose-dependent manner. CONCLUSIONS. These data indicate that EGFR was activated during corneal epi thelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signali ng cascade significantly slowed migration rates.