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Marie A. Shatos, Robin R. Hodges, Jeffrey A. Bair, Kameran Lashkari, Darlene A. Dartt; Stimulatory Role of PKCα in Extracellular Regulated Kinase 1/2 Pathway in Conjunctival Goblet Cell Proliferation. Invest. Ophthalmol. Vis. Sci. 2009;50(4):1619-1625. doi: 10.1167/iovs.08-2930.
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© 2016 Association for Research in Vision and Ophthalmology.
purpose. To determine whether a constitutively active protein kinase C (PKC)-α stimulates rat and human conjunctival goblet cell proliferation through activation of ERK 1/2.
methods. Conjunctivas from rat and human were minced and goblet cells were allowed to grow. Goblet cells were serum starved and incubated with an adenovirus containing a constitutively active form of PKCα (Ad-myr-PKCα, 1 × 107 pfu), EGF (10−7 M), or both. The location of myrPKCα was determined by immunofluorescence microscopy. Cultured goblet cells were preincubated with the PKC inhibitor calphostin C (10−10–10−7 M) or the ERK 1/2 inhibitor U0126 (10−9–10−6 M) before incubation with Ad-myr-PKCα. Cell proliferation was measured.
results. Transduction of rat goblet cells with Ad-myr-PKCα did not change PKC location compared with nontransduced cells. Incubation with Ad-myr-PKCα caused an increase in cell proliferation by 2.5 ± 0.3-fold, whereas EGF increased proliferation by 2.1 ± 0.2-fold. Simultaneous addition of Ad-myr-PKCα and EGF did not further increase proliferation. U0126 inhibited Ad-myr-PKCα-stimulated proliferation a maximum of 70%. In human goblet cells, incubation with Ad-myr-PKCα caused an increase in cell proliferation by 2.3 ± 0.3-fold, whereas EGF increased proliferation by 3.1 ± 0.4-fold. Simultaneous addition of Ad-myr-PKCα and EGF decreased proliferation compared with either compound alone. Ad-myr-PKCα caused ERK 1/2 to translocate to the nucleus in rat and human cells, but the translocation was blocked by U0126.
conclusions. Activation of PKCα alone by inducing phosphorylation of ERK 1/2 and translocating it to the nucleus is necessary and sufficient to cause conjunctival cell proliferation in rat, and probably human, goblet cells.
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