Selective Changes in Protein Kinase C (PKC) Isoform Expression in Rabbit Corneal Epithelium During Wound Healing. Inhibition of Corneal Epithelial Repair by PKCα Antisense

Abstract

Protein kinase C (PKC) isoforms display different sensitivities to modulators, tissue specificities and subcellular localizations. PKCα 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 PKCα, γ, ϵ, μ and ι. PKCα and γ were expressed only in the cytosolic fraction, with the expression of PKCα markedly increasing 4 days after injury. Corneas cultured in the presence of rabbit-specific PKCα antisense showed a greater than 50% inhibition of wound closure, compared to controls. The PKCϵ and μ were expressed in the soluble, as well as in the membrane fraction. Additionally, 12% of PKCμ was found attached to the detergent insoluble fraction. The expression of the membrane-bound PKCϵ and μ isoforms decreased between 1 and 2 days following injury. Only 10% of the PKCι 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 PKCα is a key modulator in rabbit corneal wound repair.