title: Corneal epithelial wound healing increases the expression but not long lasting activation of the p85alpha subunit of phosphatidylinositol-3 kinase.
abstract: OBJECTIVE
  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 7 mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85alpha antibodies were employed to determine PI-3K activity and expression of the p85alpha 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 p85alpha subunit of PI-3K. By 8 days, the expression of p85alpha 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 p85alpha 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 p85alpha 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.
authors.affiliation: LSU Eye Center and Neuroscience Center of Excellence, Louisiana State University Medical Center, New Orleans 70112, USA.
authors.forename: G
authors.initials: G
authors.lastname: Chandrasekher
authors.forename: H E
authors.initials: HE
authors.lastname: Bazan
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002867:Chromones / Q000494:pharmacology; D065306:Corneal Injuries; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000201:enzymology*; D005131:Eye Injuries / Q000201:enzymology*; D009025:Morpholines / Q000494:pharmacology; D009924:Organ Culture Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011233:Precipitin Tests; D011817:Rabbits; D000077191:Wortmannin; D014945:Wound Healing
chemical_list: D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one