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Dinusha Rajapakse, Mei Chen, Tim Curtis, Heping Xu; Photoreceptor outer segments (POS) induce Retinal pigment epithelial (RPE) cell multinucleation through modulating the Protein Kinase C pathway. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Multinuclear RPE cells have been frequently observed in the aging eye. RPE multinucleation can be induced in vitro by feeding cells with oxidised photoreceptor outer segments (oxPOS). We hypothesize that multinucleation is a mechanism of RPE cells to repair damage in the aging eye, although the underlying mechanism remains to be elucidated. The enzymes of Protein kinase C (PKC) family are critically involved in various cell activation pathways including cytokinesis. The aim of this study was to understand whether the PCK pathway is involved in oxPOS-induced RPE multinucleation.
The expression of total PKC proteins in RPE cells with/without oxPOS treatment was determined by Western blotting and the active PKC levels were determined by ELISA. The expression of different members of the PCK family in RPE cells was examined by RT-PCR and immunofluorescence investigations. The contribution of different PKC members to RPE multinucleation was determined by using target-specific PKC inhibitors. The involvement of Ca2+ in the activation of PKC was determined using the Fluo-4 calcium indicators and Ca2+ inhibitors.
RT-PCR study revealed that under normal culture conditions RPE cells express PKCα, PKCg, PKC ζ, PKCµ, and PKCε. oxPOS treatment increased the expression of PKCα, PKC ζ, PKCµ, and PKCε, but decreased the expression of PKCg. The expression of PKC i was below detectable level in naïve RPE cells and the expression was significantly upregulated by oxPOS treatment. The expression levels of total PKC protein was not affected by oxPOS treatment in Western blot analysis, however the active PKC levels were significantly increased following oxPOS treatment (p<0.001). In line with the PKC activation, phosphorylation of p27 kip 1, but not p21 protein was observed in oxPOS treated cells. A 10% reduction in RPE multinucleation was observed when intracellular Ca2+ was chelated (p<0.05). Pharmacological inhibition of PKC ζ, PKCα and PKC i all significantly suppressed oxPOS-induced RPE multinucleation, although the strongest suppressive effect was observed in PKC ζ inhibitor treated cells.
oxPOS induces RPE cell multinucleation through activating the PKC, in particular PKCζ, pathway. Further understanding the molecular mechanism of RPE multinucleation may shed light on the pathogenesis of age-related macular degeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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