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M. C. McGahan, J. Harned, M. Lall; ERK 1/2 is an Important Mediator of Oxidative Stress and Iron Metabolism in Lens and Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):684.
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Activation of the ERK 1/2 pathway has been associated with protection against oxidative stress in a number of cell types. Studies indicate that oxidative stress induced by hydrogen peroxide is dependent upon iron and is mediated by the ERK 1/2 pathway. The purpose of this study was to determine how oxidative stress affects iron metabolism and to investigate if ERK 1/2 is involved in these responses.
Cultured canine lens epithelial cells (LEC) and retinal pigmented epithelial cells (RPE) were used in these studies. Hydrogen peroxide was added either as a bolus (250 µM) or generated at a steady state of 2.5 µM by glucose oxidase (GO) added to the medium. Ferritin was measured by ELISA, ERK 1/2 activation was determined by Western analysis of ERK 1/2 phosphorylation, de novo ferritin synthesis was measured by metabolic labeling with 35S-methionine.
ERK 1/2 was activated in cultured LEC and RPE treated with GO over a 24h period (constant 2.5 µM hydrogen peroxide). U-0126, a specific inhibitor of ERK 1/2 activation decreased both baseline and hydrogen peroxide induced ERK 1/2 activation. Both hydrogen peroxide (bolus and GO generated) increased ferritin concentration in LEC 1.3- and 3-fold, respectively. Interestingly, U-0126 did not inhibit the effects of hydrogen peroxide on ferritin levels. On the contrary, it was additive to hydrogen peroxide in both delivery systems, increasing ferritin concentration as much as 4 fold. Both U-0126 and hydrogen peroxide increased de novo ferritin synthesis, but their effects were not additive. Addition of the iron chelator, Dp44mT inhibited the effects of both bolus and continuous exposure to hydrogen peroxide as well as U-0126 on ferritin concentration.
Exposure of LEC and RPE to hydrogen peroxide causes an increase in ERK 1/2 activation, which was inhibited by U-0126. Both bolus and continuous addition of hydrogen peroxide caused an increase in ferritin concentration, which was not mediated by the ERK 1/2 signaling pathway as U-0126 did not inhibit this response. On the contrary U-0126 was additive to the peroxide induced increase in ferritin levels. Ferritin synthesis is increased acutely at the translational level when iron content of cells is increased. Therefore, the ERK 1/2 pathway may function to keep iron levels in the cells low since inhibiting it caused a large increase in ferritin synthesis and concentration. In addition, the effects of U-0126 and hydrogen peroxide are inhibited by the iron chelator Dp44mT. All of this evidence points to an important role of the ERK 1/2 pathway in iron metabolism.
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