Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium

PURPOSE. To investigate the effect of epidermal growth factor (EGF) on the induction of phosphatidylinositol S-kinase (PI 3-kinase) gene expression du ring rabbit corneal epithelial wound repair. METHODS. Epithelial wounds (6 mm in size) were created in rabbit corneas an d EGF (2 mu g) applied every 8 hours to one eye, and the other eye served a s a control. The wound repair was monitored by staining the tissue with flu orescein followed by photography. The wound area was quantified with a comp uter program. At different time intervals, the rabbits were killed and the corneal epithelium used for estimation of PI 3-kinase activity, western blo t analysis, or reverse transcription-polymerase chain reaction (RT-PCR). Fo r in situ hybridization, the whole corneas were sectioned and the sections processed with PI 3-kinase mRNA probes. RESULTS. In the untreated eye, the epithelial wound progressively healed in a time-dependent manner, with 75% of the wound closed at is hours post wou nding. Application of EGF to the corneal epithelium further stimulated woun d repair at all time intervals, and the wound was completely closed at 48 h ours. Analysis of PI 3-kinase showed a time-dependent increase in its enzym e activity that was maximally increased at 36 hours, the time when the woun d was nearly closed. Western blot analysis revealed increased amounts of PI 3-kinase protein during the course of wound repair. Analysis of RT-PCR pro ducts from epithelial tissues, taken at different times during wound repair , showed increased PI 3-kinase expression that was maximum at 48 hours post wounding. A visible increase in PT 3-kinase gene expression was also detec ted by in situ hybridization during the course of the wound repair. This ex pression was increased maximally by EGF at 48 hours post wounding. CONCLUSIONS. The results indicate a temporal correlation between increased activation and expression of PT 3-kinase and the epithelial wound repair. T opical application of EGF further stimulates the activity and expression of PI 3-kinase. It is suggested that PI 3-kinase and its products may play a role in EGF-induced cell proliferation during corneal epithelial wound repa ir.