Chandrasekher G, Bazan N G, Bazan H E
Louisiana State University Medical Center, Department of Ophthalmology and Neuroscience Center of Excellence, 2020 Gravier St. , Suite D, New Orleans, LA, 70112, USA.
Exp Eye Res. 1998 Nov;67(5):603-10. doi: 10.1006/exer.1998.0555.
Protein kinase C (PKC) isoforms display different sensitivities to modulators, tissue specificities and subcellular localizations. PKCalpha increases during rabbit corneal epithelial wound healing. Here we report differential expression of PKC isoforms in the cornea of rabbits at 1, 2, 4 and 8 days during re-epithelization. Cytosolic, membrane and detergent-insoluble fractions from epithelium were analysed by Western blot using monoclonal antibodies against the different PKC isoforms. We have identified PKCalpha, gamma, epsilon, mu and iota. PKCalpha and gamma were expressed only in the cytosolic fraction, with the expression of PKCalpha markedly increasing 4 days after injury. Corneas cultured in the presence of rabbit-specific PKCalpha antisense showed a greater than 50% inhibition of wound closure, compared to controls. The PKCepsilon and mu were expressed in the soluble, as well as in the membrane fraction. Additionally, 12% of PKCmu was found attached to the detergent insoluble fraction. The expression of the membrane-bound PKCepsilon and mu isoforms decreased between 1 and 2 days following injury. Only 10% of the PKCiota expressed in corneal epithelium was membrane bound, but between 4 and 8 days after de-epithelization, the expression in this fraction increased three-fold. Our results suggest that changes in the expression and distribution within the various fractions of selective isoforms of PKC after injury could be involved in events leading to wound healing and that PKCalpha is a key modulator in rabbit corneal wound repair.
蛋白激酶C（PKC）亚型对调节剂表现出不同的敏感性、组织特异性和亚细胞定位。兔角膜上皮伤口愈合过程中PKCα增加。在此我们报告了兔角膜在再上皮化过程中1、2、4和8天时PKC亚型的差异表达。使用针对不同PKC亚型的单克隆抗体，通过蛋白质印迹法分析上皮细胞的胞质、膜和去污剂不溶性组分。我们鉴定出了PKCα、γ、ε、μ和ι。PKCα和γ仅在胞质组分中表达，损伤后4天PKCα的表达显著增加。与对照组相比，在兔特异性PKCα反义寡核苷酸存在下培养的角膜伤口闭合抑制率超过50%。PKCε和μ在可溶性组分以及膜组分中表达。此外，发现12%的PKCμ与去污剂不溶性组分结合。损伤后1至2天，膜结合的PKCε和μ亚型的表达下降。角膜上皮中表达的PKCι仅有10%是膜结合的，但在上皮剥脱后4至8天，该组分中的表达增加了三倍。我们的结果表明，损伤后PKC选择性亚型在各组分中的表达和分布变化可能参与导致伤口愈合的事件，并且PKCα是兔角膜伤口修复中的关键调节剂。
