Role of cPKC␣ and nPKC in EGF-Stimulated Goblet Cell Proliferation

Abstract

PURPOSE. The authors determined the role of the protein kinase C (PKC) isoforms cPKC␣ and nPKC 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 Gö 6983 (10 Ϫ10 -10 Ϫ7 M) for 20 minutes before stimulation with EGF (10 Ϫ7 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␣ (Ad DNPKC␣, 10 4 pfu), dominant-negative nPKC (Ad DNPKC, 10 4 pfu), and wild-type cPKC␣ (Ad WTPKC␣, 10 7 pfu), and proliferation was measured. RESULTS. In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53% Ϯ 15%. In human goblet cells, EGFstimulated proliferation was completely inhibited by calphostin C. PKC␣, -␤I, -␤II, -␦, -, -/, -, -␥, and -were found in cultured rat goblet cells. Ad DNPKC␣ and Ad DNPKC inhibited EGF-stimulated proliferation in rat goblet cells by 78% Ϯ 6% and 92% Ϯ 8%, respectively. Incubation with Ad WTPKC␣ alone significantly increased proliferation. CONCLUSIONS. cPKC␣ and nPKC play key roles in conjunctival goblet cell proliferation. (Invest Ophthalmol Vis Sci. 2009;50: 614 -620