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Marie A. Shatos, Robin R. Hodges, Yoshia Oshi, Jeffrey A. Bair, Driss Zoukhri, Claire Kublin, Kameran Lashkari, Darlene A. Dartt; Role of cPKCα and nPKCε in EGF-Stimulated Goblet Cell Proliferation. Invest. Ophthalmol. Vis. Sci. 2009;50(2):614-620. doi: 10.1167/iovs.08-2467.
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purpose. The authors determined the role of the protein kinase C (PKC) isoforms cPKCα and nPKCε 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 (10−10-10−7 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 cPKCα (Ad DNPKCα, 104 pfu), dominant-negative nPKCε (Ad DNPKCε, 104 pfu), and wild-type cPKCα (Ad WTPKCα, 107 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% ± 15%. In human goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C. PKCα, -βI, -βII, -δ, -ε, -ι/λ, -θ, -γ, and -ζ were found in cultured rat goblet cells. Ad DNPKCα and Ad DNPKCε inhibited EGF-stimulated proliferation in rat goblet cells by 78% ± 6% and 92% ± 8%, respectively. Incubation with Ad WTPKCα alone significantly increased proliferation.
conclusions. cPKCα and nPKCε play key roles in conjunctival goblet cell proliferation.
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