Corneal epithelial wound healing increases the expression but not long lasting activation of the p85 alpha subunit of phosphatidylinositol-3 kinase

Purpose. Corneal epithelial wound healing is a complex process involving se veral growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that prop agate and amplify growth factor-induced signals inside the cells. Phosphati dylinositol-3 kinase (PI-3K) is one such enzyme. Here we have investigated changes in PI-3K activity and expression during re-epithelialization after in vivo and in vitro corneal injury. Methods. For the in vivo model, epithelium was collected from rabbit cornea s at different stages of wound healing after complete de-epithelialization. For in vitro studies, after 7mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85 alpha antibodies were employed to determine PI-3K activ ity and expression of the p85 alpha regulatory subunit of PI-3K. Two specif ic PI-3K inhibitors, Wortmannin and LY 294002 were used to study the effect of PI-3K activity on corneal epithelial wound healing. Results. Two to four days after in vivo corneal epithelial wound healing, t here was a 6-8 fold increase in the expression of the p85 alpha subunit of PI-3K. By 8 days, the expression of p85a was similar to non-injured tissue. Increased expression of the 85kDa protein was observed mainly in the membr ane fraction. Similarly, the expression of PI-3K was increased 24h after in jured corneas were maintained in organ culture. Increase of p85a was confin ed to the wound region and surrounding area. No concomitant increase in PI- 3K activity was observed in any of the wound models. Forty-eight hours afte r the central injury, Wortmannin and LY294002 inhibited wound healing by ab out 50%. Conclusions. Association of most of the increased p85 alpha with the membra ne fraction and no detectable increase in PI-3K activity during corneal re- epithelialization indicates that PI-3K activation is transitory. The result s also suggest a mechanism of down regulation of the enzyme to avoid uncont rollable growth and cellular hypertrophy after growth factor stimulation du ring wound healing.