Purpose. To characterize the involvement of Zip2, a zinc transporter protein, in the antioxidant functions of cultured human retinal pigment epithelial (RPE) cells. Methods. The expression of zinc transporter proteins was determined by RT-PCR. Intracellular zinc concentration was assessed by staining with a zinc-sensitive dye followed by flow cytometry. Stable overexpression of the transporter protein Zip2 was achieved by transducing ARPE-19 cells with a retroviral vector containing the open reading frame of the human Zip2 gene. Activity of nuclear factor erythroid 2-related factor 2 (Nrf2) was measured using a dual luciferase assay after transient transfection of reporter plasmids containing the antioxidant response element (ARE). Glutamate-cysteine ligase (GCL) expression was measured by quantitative real-time RT- PCR. Results. Cultured RPE cells could transport zinc with Zip2 as an influx transporter expressed in ARPE-19 cells and human RPE cells isolated from postmortem donor eyes. The mRNA level of Zip2 was influenced by intracellular and extracellular zinc concentrations. Overexpression of Zip2 resulted in increased Nrf2 activity, higher GCL expression, and increased glutathione synthesis. Conclusions. RPE cells can actively uptake zinc through the transporter Zip2, and the increased intracellular zinc upregu-lates the Nrf2-dependent antioxidant function.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience