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Piyush C. Kothary, Monte A. Del Monte; Map Kinase Signaling Pathway Is Involved In Erythropoietin Production In Cultured Human Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3341.
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© ARVO (1962-2015); The Authors (2016-present)
Acute intensive insulin therapy may worsen proliferative diabetic retinopathy. Since erythropoietin (Epo) plays an important role in neovascularization, we investigated the role of the RAS/Raf/ERK kinase signaling pathways in regulating Epo synthesis in cultured human retinal pigment epithelial (hRPE) cells.
hRPE cultures were established from three human eyes. hRPE cell proliferation was quantitated by 3H-thymidine incorporation (3H-thy). hRPE cells were treated with varying concentrations of insulin. Cells were labeled with 14C-methionine. Anti-Epo was used for immuno-precipitation of 14C-Epo and immuno-histochemical analysis. The insulin treated cells were also exposed to PD98059, a selective inhibitor of MAP kinase (also known as MAPK/ERK kinase), to mevastatin, an inhibitor of RAS signaling pathway, and to a RAF inhibitor (RafI). Data were analyzed by Student 't' test.
Increasing concentrations of insulin stimulate hRPE proliferation as determined by 3H-thy. Insulin (0-5 µg/ml) stimulates 14C-Epo synthesis in a dose-dependent manner. Insulin (5 µg/ml) stimulated 14C-Epo synthesis was inhibited by mevastatin (30µM) (1215.38±370.87 vs.180.12±47.41, CPM±SEM, p<0.006, n=7), by RafI (1215.38±370.87 vs.611.64±220.18, CPM±SEM, p<0.04, n=7) and by PD98059 (25 µM) (3258.31±1547.15 vs.2622.30 ±1641.29, CPM±SEM, p<0.04, n=4). Immuno-histochemical studies showed an increase in Epo immuno-reactive cells in presence of insulin (5 µg/ml) when compared to controls and to cells exposed to insulin supplemented with PD98059 (25 µM).
Insulin stimulates Epo synthesis in hRPE cells and this synthesis appears to be mediated by the RAS/Raf/ERK kinase signaling pathway. Understanding of the complex regulation of Epo synthesis may provide useful information that will contribute to the development of therapeutic pharmaco-therapy to treat or prevent proliferative eye diseases.
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