Chandrasekher G, Bazan H E
LSU Eye Center and Neuroscience Center of Excellence, Louisiana State University Medical Center, New Orleans 70112, USA.
Curr Eye Res. 1999 Mar;18(3):168-76. doi: 10.1076/ceyr.18.3.168.5372.
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.
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.
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%.
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.
角膜上皮伤口愈合是一个复杂的过程，涉及多种生长因子，这些生长因子与酪氨酸激酶受体（RTK）相互作用，导致与第二信使偶联的酶的募集，从而在细胞内传播和放大生长因子诱导的信号。磷脂酰肌醇-3激酶（PI-3K）就是这样一种酶。在此，我们研究了体内和体外角膜损伤后再上皮化过程中PI-3K活性和表达的变化。
对于体内模型，在完全去上皮化后的伤口愈合不同阶段，从兔角膜收集上皮组织。对于体外研究，在施加7mm中央刮伤后，将兔角膜维持在器官培养中。使用抗p85α抗体进行免疫沉淀和蛋白质印迹，以确定PI-3K活性和PI-3K的p85α调节亚基的表达。使用两种特异性PI-3K抑制剂渥曼青霉素和LY 294002来研究PI-3K活性对角膜上皮伤口愈合的影响。
体内角膜上皮伤口愈合后2至4天，PI-3K的p85α亚基表达增加了6至8倍。到第8天，p85α的表达与未受伤组织相似。观察到85kDa蛋白的表达增加主要在膜部分。同样，在器官培养中维持受伤角膜24小时后，PI-3K的表达增加。p85α增加局限于伤口区域和周围区域。在任何伤口模型中均未观察到PI-3K活性的相应增加。中央损伤48小时后，渥曼青霉素和LY294002将伤口愈合抑制了约50%。
角膜再上皮化过程中，大部分增加的p85α与膜部分相关，且未检测到PI-3K活性增加，这表明PI-3K激活是短暂的。结果还提示了一种酶下调机制，以避免伤口愈合期间生长因子刺激后不可控的生长和细胞肥大。
