Role of cPKCα and nPKCε in EGF-Stimulated Goblet Cell Proliferation

PURPOSE. The authors determined the role of the protein kinase C (PKC) isoforms cPKC zeta and nPKC epsilon in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells. METHODS. Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Go 6983 (10(-10)-10(-7) M) for 20 minutes before stimulation with EGF (10(-7) M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKC alpha (Ad DNPKC alpha, 10(4) pfu), dominant-negative nPKC epsilon (Ad DNPKC epsilon, 104 pfu), and wild-type cPKC alpha (Ad WTPKC alpha, 10(7) pfu), and proliferation was measured. RESULTS. In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Go 6983 inhibited proliferation by 53% (+/-) 15%. In human goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C. PKC alpha, -beta I, -beta II, -delta, -epsilon, -iota/lambda, -theta, -gamma, and -zeta were found in cultured rat goblet cells. Ad DNPKC alpha and Ad DNPKC epsilon inhibited EGF-stimulated proliferation in rat goblet cells by 78% +/- 6% and 92% +/- 8%, respectively. Incubation with Ad WTPKC alpha alone significantly increased proliferation. CONCLUSIONS. cPKC alpha and nPKC epsilon play key roles in conjunctival goblet cell proliferation. (Invest Ophthalmol Vis Sci. 2009;50:614-620) DOI:10.1167/iovs.08-2467