Abstract
Abstract: :
Purpose: Retinal pigment epithelium (RPE) plays a critical role in the development and maintenance of photoreceptors. Oxidative stress triggers a diversity of cell–signaling alterations that may lead to cell damage or death and are involved in retinal degeneration. In this study we investigated whether NF–ΚB activation and translocation to the nucleus are intermediate steps in apoptosis induced by oxidative stress, or whether they are components of protective mechanisms employed by RPE cells to prevent apoptosis. At the same time, ERK phosphorylation and overall cell signaling cross talk were explored. Methods: ARPE–19 cells were cultured in 6–well plates up to 100% confluence, serum–starved for 18 hours, and stimulated with TNF–α/H2O2 (10 ng/ml and 800 µM, respectively) to trigger oxidative stress. For inhibition studies, cells were pretreated for 1 hour with either 50 µg/ml NF–ΚB activation inhibitor SN50 or 1µM IΚB–α inhibitor Bay 11–7082. Immunochemistry was used to study p65 activation and translocation. We also used Western blot to investigate phosphorylated caspase–9 and ERK expression. Results: SN50 augmented apoptosis induced by H2O2/TNF–α 16 hours after stimulation in ARPE–19 cells, while SN50 alone didn’t cause any apoptosis. Both IΚB–α inhibitor Bay 11–7082 and NF–ΚB inhibitor SN50 prevented H2O2/TNF–α–induced translocation of p65 to the nucleus in ARPE–19 cells. NF–ΚB inhibitor SN50 up–regulated the phosphorylation of caspase–9 in H2O2/TNF–α–treated ARPE–19 cells. Oxidative stress (H2O2/TNF–α) also caused the phosphorylation of ERK, which was down–regulated by SN50 in ARPE–19 cells. Conclusions: This study demonstrates that oxidative stress induced by H2O2/TNF–α leads to ARPE–19 cell apoptosis, which is preceded by RelA(p65) NF–ΚB subunit translocation to the nucleus and ERK activation. NF–ΚB inhibitor SN50 increased cell death and also suppressed ERK phosphorylation induced by oxidative stress. We conclude that during oxidative stress, NF–ΚB activation has a protective effect against apoptosis in ARPE–19 cells, and that phosphorylation of ERK may contribute to cell–signal integration in cell survival.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • transcription factors