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C. Zhang, N.J. Caplen, N. Strunnikova, J. Baffi, C.C. Chan, S.W. Cousins, K.G. Csaky; Regulation of c-FLIP Prevents Tumor Necrosis Factor – Alpha (TNF-a) Induced Programmed Cell Death in Human Retinal Pigment Epithelium (hRPE) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):473.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Even though macrophages are detected in the sub-RPE space in the early stages of age-related macular degeneration and are capable of producing TNF- a, hRPE cell death, seen clinically as geographic atrophy, is only seen in the later stages of the diseases. Previous studies have demonstrated that resistance of hRPE to TNF-a induced cell death is associated with an increased ratio of a survival factor, c-FLIP (a primary inhibitor of the death receptor signaling pathway) to the pro-apoptotic protein caspase-8. The present study aims to extend this observation by directly determining the role of c-FLIP in hRPE resistance to TNF-a. Methods: Cell viability of ARPE-19 cells was determined by XTT assay. Activity of PI3 kinase was determined by phosphorylation of AKT and the role of PI3 kinase was investigated using specific PI3 kinase inhibitors. c-FLIP protein expression was monitored by western blotting. Highly specific suppression of c-FLIP was achieved using RNA interference. Immuno-localization of proapoptotic factors was performed with organelle fractionation from both ARPE-19 cells and in U937 cells that are known to be sensitive to TNF-a induced cell death. Mitochondrial and lysosomal morphology was monitored by mitotracker or lysotracker fluorescence and electron microscopy.Results: In ARPE-19 cells, exogenous TNF-a induces an increase in c-FLIP expression. This increase was coordinate with an upregulation of Akt phosphorylation/ PI3 kinase activity and was disrupted by applying the specific inhibitor, LY294002. The downregulation of the PI3 kinase activity by LY294002 was accompanied by the appearance of nuclear shrinkage and extensive microorganelles vacuolization and the release of mitochondrial cytochrome C following TNF-a addition. ARPE-19 cells in which c-FLIP RNA and protein was eliminated by RNA interference showed similar morphological changes in response to TNF-a. Conclusions: These findings provides additional evidence that TNF-a modulates hRPE survival/death by affecting c-FLIP expression via signaling cross talk with the PI3 kinase pathway and that c-FLIP may be a critical determinant in governing hRPE fate in response to TNF-a exposure.
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