Modulation of Nrf2-dependent antioxidant functions in the RPE by Zip2, a zinc transporter protein.
OBJECTIVE
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 upregulates the Nrf2-dependent antioxidant function.
Rezaei
Kasra A
KA
Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8808, USA.
Chen
Yan
Y
Cai
Jiyang
J
Sternberg
Paul
P
D000975:Antioxidants / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000502:physiology*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D006801:Humans; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015032:Zinc / Q000378:metabolism*
D000975:Antioxidants; D027682:Cation Transport Proteins; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012333:RNA, Messenger; C498424:SLC39A2 protein, human; D005721:Glutamate-Cysteine Ligase; D015032:Zinc