Corneal epithelial wound healing increases the expression but not long lasting activation of the p85α subunit of phosphatidylinositol-3 kinase

Purpose. Corneal epithelial wound healing is a complex process involving several growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that propagate and amplify growth factor-induced signals inside the cells. Phosphatidylinositol-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 corneas 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 activity and expression of the p85 alpha regulatory subunit of PI-3K. Two specific 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, there 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 membrane fraction. Similarly, the expression of PI-3K was increased 24h after injured corneas were maintained in organ culture. Increase of p85a was confined 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 after the central injury, Wortmannin and LY294002 inhibited wound healing by about 50%. Conclusions. Association of most of the increased p85 alpha with the membrane fraction and no detectable increase in PI-3K activity during corneal re-epithelialization indicates that PI-3K activation is transitory. The results also suggest a mechanism of down regulation of the enzyme to avoid uncontrollable growth and cellular hypertrophy after growth factor stimulation during wound healing.