G. Chandrasekher et Hep. Bazan, Corneal epithelial wound healing increases the expression but not long lasting activation of the p85 alpha subunit of phosphatidylinositol-3 kinase, CURR EYE R, 18(3), 1999, pp. 168-176
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.