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Xiaobin Liu, Christy Xavier, Hongli Wu; Glutaredoxin 2 (Grx2) Protects Retinal Pigment Epithelial Cells from Oxidative Damage by Regulating Autophagy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):248.
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© ARVO (1962-2015); The Authors (2016-present)
Glutaredoxin 2 (Grx2) is an oxidoreductase present in the mitochondria where it protects the organelle from oxidative damage and maintains its redox homeostasis. The purpose of this study is to evaluate the cytoprotective effects of Grx2 in human retinal pigment epithelial (RPE) cells and characterize its potential function in regulating autophagy.
Primary RPE cells were isolated from Grx2 knockout (KO) mice and treated with or without 400 µM H2O2 for 4 h. Human retinal pigment epithelial (ARPE-19) cells were transfected with either a human Grx2 cDNA-containing plasmid (pCR3.1-hGrx2) or an empty vector pCR3.1. Cells were treated with or without 200 µM H2O2 for 16 h. Grx2 protein expression was detected by western blot analysis. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) and autophagy pathway proteins were measured by immunoblotting.
Primary RPE cells laking Grx2 were more sensitive to oxidative damage. On the other hand, Grx2 overexpression protected RPE cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx2 were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control cells. PSSG accumulation was also attenuated by Grx2 overexpression with acute H2O2 challenge. Furthermore, the protein level of Beclin-1, a key molecule to initiate autophagy, was inhibited in Grx2 overexpressed cells with H2O2 treatment. LC3II, a protein that finalizes autophagosome maturation, was also increased. Conversely, primary Grx2 KO RPE cells showed higher levels of Beclin-1 and LC3II under oxidative stress.
Grx2 rescues RPE cells from lethal oxidative damage, possibly through alleviation of ROS-triggered autophagy and prevention of PSSG accumulation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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