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Piyush C Kothary, Monte A Del Monte; Mechanism of Activation of Hypoxia-Inducible Factor 1α by Insulin-like Growth Factor 1 in Cultured Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):225. doi: https://doi.org/.
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Human retinal pigment epithelial cells produce Hypoxia-inducible Factor 1α (HIF1α) which plays a major role in the pathogenesis of proliferative diabetic retinopathy (PDR). Insulin-like growth factor1 (IGF1) also plays an important role in the pathogenesis of diabetic complications but the pathogenic mechanism is not completely understood. IGF1 is known to induce HIF1 alpha, however, little is known about the mechanism if this interaction. In this study we investigated the role of RAS and ERK1/2 protein in IGF1 induced HIF1α production in hRPE cells.
hRPE cultures were established from normal human eyes obtained from the Michigan Eye Bank. Cell proliferation and viability were quantitated by 3H-thymidine (3H-thy) incorporation and by the trypan blue exclusion (T) method. HIF1α syntheses was measured by immunoprecipitation in hRPE cells exposed to increasing concentrations of IGF1 in the presence and absence of mevastatin (a RAS inhibitor) and PD98059 (PD) (an ERK1/2 inhibitor). Phase contrast microscopy and immunocytochemistry were also performed. Statistical significance was determined by Student “t” test.
Fetal bovine serum stimulates hRPE cell proliferation in a dose dependent manner as determined by T and 3H-thy. IGF1 exposure increased this proliferation and also induces 14C-HIF 1α synthesis, also in a dose dependent manner. Mevastatin and PD both inhibit IGF1 stimulated hRPE proliferation as determined by T. Mevastatin (30 µM) inhibits IGF1 stimulated HIF 1α protein synthesis (161.71±19.76 vs. 331.43±82.54, (CPM±SEM), n=5, p≤0.05). PD (25 µM) also inhibits IGF1 stimulated HIF 1α protein synthesis (249.18±60.14 vs. 331.43±82.54, (CPM±SEM), n=5, p≤0.05). Phase contrast microscopy confirms that IGF1 increases hRPE cell number and IGF1+PD had only minor effect on hRPE cell morphology. Immunocytochemical analysis showed increase expression HIF1α in presence of IGF1 that was decreased by PD.
This study demonstrates that IGF1 stimulate hRPE cell proliferation and HIF 1α synthesis. Mevastatin (a RAS inhibitor) and PD (an ERK1/2 inhibitor) block IGF1 stimulated HIF 1α synthesis. We can therefore conclude that RAS and ERK1/2 kinase signaling may play a role in IGF1 stimulated HIF1α synthesis. Our data suggest a potential role for RAS and ERK1/2 signaling targeted therapies in the prevention or treatment of PDR.
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