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P. K. Mukherjee, N. G. Bazan; Neuroprotectin D1 Is a Potent Upregulator of Oxidative-Stress-Induced Expression of Antioxidant Responsive Element (ARE) in Retinal Pigment-Epithelial (arpe-19) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4438.
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Genes that contain promoters with anti-oxidant responsive element (ARE) has an integral role in cellular responses to oxidative-stress. ARE mediates antioxidant defense, neuronal proliferation, differentiation and signal transduction. Neuroprotectin D1 (NPD1), a bioactive lipid mediator is a potent inhibitor of oxidative-stress induced neuronal cell death. Our objective in this study is to test the effect of NPD1 on the expression of ARE in ARPE-19 cells under oxidative-stress.
An ARE construct (hQR41ARE) linked to luciferase reporter gene was used to transfect ARPE-19 cells by Fugenn-6. A β-galactosidase plasmid was cotransfected as control. Transfected cells were serum starved, placed under oxidative- stress induced by TNF-α/H2O2, and then incubated with NPD1. Luciferase assays were performed using luciferin as substrate.
Our results indicated that hQR41ARE-luc (containing the ARE sequences) expression was activated in concentration dependent manner of oxidative-stress. Highest expression was detected at 400 uM and practically no expression was obtained at 600 uM oxidative-stress. Conversely, Ti-luc, lacking anti-oxidant responsive element and hQR41ARE-mut-luc, a mutant construct of ARE did not response to oxidative stress. Interestingly, NPD1 at a concentration of 1 nM and 5 nM significantly upregulates the expression of ARE fused to luciferase. Maximal induction was attained at 50-100 nM NPD1.
ARE-luc expression in ARPE-19 cells is oxidative-stress concentration dependent. Ti-luc and mutant ARE did not response to oxidative-stress. Low concentration dependent up-regulation of ARE expression by NPD1 in oxidative stressed cells suggest that this signaling is a target in NPD1-mediated cell survival. These findings further our understanding of the role of NPD1 in RPE protection in an oxidative environment.
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