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G.D. Sharma, H.E. P. Bazan; Protein Kinase C Alpha and Epsilon Gene–Knockdown Differentially Affects HGF–Induced Cell Proliferation and Migration in Human Corneal Epithelial Cells (HCEC) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2127.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies have shown that hepatocyte growth factor (HGF) translocates PKCα and PKCε to the plasma membrane, inducing their activation in HCEC, and that inhibition of PKCα by Go6976 decreases cell proliferation (Sharma et al. ARVO. 2003). Since there are no available PKC inhibitors specific for one isoform, in the present study we have used PKCα and PKCε–siRNA to knockdown these genes and investigate more precisely their roles in HGF–induced cell proliferation and migration in HCEC. Methods: HCEC were seeded in 60–mm dishes (5 x 105 cells/well) or 96–well plates (10,000 cells/well), allowed to grow to 50–60% confluence, and then transfected with PKCα– and PKCε–siRNA overnight in KBM (without growth supplements). Cells were then fed with fresh KGM medium before they were serum–starved and stimulated with HGF for migration studies using a scrape–wound method (Sharma et al. J. Biol. Chem. 21989, 2003). For proliferation, transfected cells were treated with HGF and allowed to grow for 48 h before being analyzed for proliferation using a DNA–binding fluorescent dye. Results: PKCα and PKCε genes were knocked down up to 60–70% with more than 80% of the cells transfected. HCEC transfected with PKCα–siRNA showed a significant (p<0.005) inhibition in cell proliferation compared to non–transfected control at 48 h. Similarly, when transfected cells were incubated with HGF, there was a significant (p<0.001) reduction in cell proliferation compared to HGF–stimulated non–transfected cells. On the other hand, cells transfected with PKCε–siRNA showed no significant inhibition in cell proliferation with or without HGF stimulation. Proliferation was unaffected by transfection reagent alone. Cell migration was inhibited by more than 80% in PKCε–siRNA–transfected cells compared to controls, with or without HGF stimulation. In contrast, PKCα–siRNA–transfection did not affect the migration of cells stimulated by HGF. Conclusions: Our results suggest that the two isoenzymes, PKCα and PKCε, had functional selectivity in proliferation and migration stimulated by HGF. This elucidates a mechanism involving both PKCs to regulate different stages of corneal epithelial repair (Supported by NIH–NE1 EY06635).
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