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N. N. Doshi, P. C. Kothary, M. A. Del Monte; The Ras/Raf Signaling Pathway Mediates PEDF Synthesis in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3422. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Abnormal proliferation and metaplasia of human retinal pigment epithelial cells (hRPE) appears to play an important role in the pathogenesis of proliferative diabetic retinopathy (PDR). The Ras/Raf signaling pathway has been shown to mediate hRPE cell proliferation. In addition, a decrease in pigment epithelial derived factor (PEDF) levels is associated with retinal neovascularization in diabetic retinopathy. Since very little is known about signaling pathways involved in intracellular PEDF synthesis in hRPE cells, we examined the role of Ras/Raf signaling in PEDF synthesis.
hRPE cells were cultured from human eyes. Cells were treated with varying concentrations of fetal bovine serum (FBS), mevastatin (a Ras inhibitor), and RAF-inhibitor (RAFI). Cell proliferation was measured by the trypan blue exclusion (T) method and 3H-thymidine incorporation (3H-thy) into hRPE cells. Intracellular PEDF synthesis was quantitated by immunoprecipitation of 14C methionine-labeled PEDF (14C-PEDF) and immunohistochemical methods. Data was analyzed using student's ‘t’ test.
FBS stimulated hRPE cell proliferation as determined by 3H-thy and T. FBS also stimulated intracellular 14C-PEDF synthesis. RAFI inhibited FBS-stimulated hRPE cell proliferation as measured by cell counting (2812.5±1372.4 vs. 113437.5±28078.1, cell number±SEM, n=8, p<.05), and FBS-stimulated 3H-thy during DNA synthesis (286±56.2 vs. 5502.8±738.4, CPM±SEM, n=9, p<.05). RAFI also inhibited FBS-stimulated intracellular 14C-PEDF synthesis (742.7±105.0 vs. 2689.2±429.8, CPM±SEM, n=14, p<.05). In addition, mevastatin inhibited FBS-stimulated hRPE cell proliferation (1875±1227.5 vs. 113437.5±28078.1, CPM±SEM,n=8, p<.05) as well as FBS-stimulated intracellular 14C-PEDF synthesis (1489.5±148.2 vs. 2793.5±169.5, CPM±SEM, n=9, p<.05). Immunohistochemical studies confirmed decreased PEDF immunoreactivity in presence of RAFI+FBS compared to FBS alone.
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