Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE: Implication for protection against oxidative stress

PURPOSE. To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS. Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS. Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 μM, zinc increased the GSH level by 70%. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS. Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).