Oxidative Stress Causes Activation of the ERK Signaling Pathway in Cultured Human Retinal Pigment Epithelial Cells

Abstract

Purpose: : Introduction: Human retinal pigment epithelial cells (hRPE) have been implicated in the pathogenesis of age related macular degeneration (AMD). Hydrogen peroxide (H2O2) induces hRPE cell death in AMD by apoptosis and necrosis. Activation of extracellular signal-regulated protein kinases (ERK1-2) have been shown to play a role in hRPE cell oxidative damage by H2O2. Since very little is known about the role of RAS/RAF kinases in activation of ERK1-2 in hRPE cells, we investigated their role in the activation of ERK1-2 synthesis in hRPE cells.

Methods: : Methods: Human RPE specimens were cultured from postmortem non-pathological eyes. hRPE proliferation and viability in the presence of increasing concentrations of H2O2 (0-1 mM) and FBS were measured by [3H]thymidine (3H-thy) incorporation and the trypan blue exclusion (T) method respectively. The effect of increasing concentrations of H2O2 on intracellular synthesis of pERK in presence and absence of RAS (mevastatin), RAF inhibitors (RAFI) and PD98059 (PD), an inhibitor of 14C-pERK1-2, were measured by immunoprecipitating 14C-methionine-pERK (14C-pERK). We also performed western blot analysis of pERK synthesis using anti-pERK. Statistical significance was determined by Student "t" test.

Results: : Results: FBS (0-10%) increased hRPE cell proliferation in a dose-dependent manner as measured by T and 3H-thy. In contrast, H2O2 (0.1mM-0.5mM) decreased hRPE cell viability and proliferation as measured by 3H-thy and T. Increasing concentrations of H2O2 increased intracellular synthesis of 14C-pERK in a dose dependent manner. Mevastatin (30 µM) inhibited H2O2 stimulated 14C-pERK synthesis (671.02± 122.03 vs 1253.14±274.32, n=6, CPM±SEM, p<0.05), RAFI also inhibited H2O2 stimulated 14C-pERK synthesis (394.32± 113.96 vs 1253.14±274.32, n=6, CPM±SEM, p<0.05). PD inhibited H2O2 stimulated 14C-pERK synthesis (313.36± 104.45 vs 1253.14±274.32, n=6, CPM±SEM, p<0.05). Furthermore, DAPI (4',6-diamidino-2-phenylindole) studies showed hRPE cell damage in presence of H2O2 and western blot analysis confirmed an increase production of pERK1-2 in H2O2-induced hRPE cells which was inhibited by PD inhibits.

Keywords: age-related macular degeneration • signal transduction • retinal pigment epithelium