Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration

Abstract

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCα activation to promote epithelial wound healing [Sharma, G.D., Ottino, P., Bazan, H.E.P., 2005. Epidermal and hepatocyte growth factors, but not keratinocyte growth factor, modulate protein kinase C alpha translocation to the plasma membrane through 15( S)-hydroxyeicosatetraenoic acid synthesis. J. Biol. Chem. 280, 7917—7924]. Protein kinase C alpha (PKCα) and protein kinase C epsilon (PKCɛ) are two differentially regulated isoenzymes. While PKCα requires Ca 2+ for its activation, PKEɛ is Ca 2+ independent. However, growth factor-induced activation of these enzymes and their specific regulation of epithelial migration and proliferation have not been explored. In the present study, we overexpressed PKCɛ fused to green fluorescent protein to examine its translocation in real-time to the plasma membrane in living human corneal epithelial cells. Stimulation with HGF and KGF demonstrated translocation of PKCɛ to the plasma membrane. Because HGF activates both PKCs, this growth factor was used to stimulate wound healing. PKCα or PKCɛ-genes were knocked down individually without affecting the basal expression of the other PKC isoforms. Gene knockdown of PKCα significantly inhibited HGF-stimulated proliferation of human corneal epithelial cells. In contrast, PKCɛ-gene-silencing severely impaired the HGF-stimulated migratory ability of human corneal epithelial cells. When migrating epithelial cells in the cornea wound bed after injury were transfected with specific PKCα- or PKCɛ-siRNA, there was a significant delay in wound healing. Corneal wound healing stimulated with HGF in similar conditions was also inhibited. On the other hand, overexpression of PKCα or PKCɛ-genes fused with green fluorescent protein in migrating corneal epithelium accelerated repair of the epithelial defect. Our findings demonstrate that PKCα and PKCɛ modulate different stages of wound healing stimulated by HGF and contribute to epithelial repair by playing selective regulatory roles in epithelial proliferation and migration, both crucial to corneal wound healing.