Increased expression of mitochondrial glutaredoxin 2 (Grx2) protects human retinal pigment epithelial cells from oxidative stress-induced cell death

Abstract

Purpose: Glutaredoxin 2 (Grx2) is a mitochondrial isozyme of thioltransferase in the oxidoreductase family that is a key regulator of redox homeostasis through dethiolation of protein-glutathione mixed disulfides (PSSG). Grx2 is expressed in various human and rodent tissues, including the retina, where its functional role is unknown. The purpose of this study is to investigate the presence of Grx2 in human retinal pigment epithelial cells and evaluate its potential anti-apoptotic function.

Methods: Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx2-containing plasmid or an empty vector. Normal ARPE-19 cells and transfected cells were treated with or without 200 µM H₂O₂for 24 h. Grx2 protein expression was detected by Western blots. Grx2 enzyme activity was assayed by spectrophotometric method in the mitochondrial fraction. 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. Mitochondrial membrane potential was evaluated by using JC-1 assay kit. The level of mitochondrial PSSG was measured by immunoblotting using anti-PSSG antibody.

Results: Grx2 protein level and enzyme activity in Grx2 transfected cells were significantly increased as compared to non-transfected and vector transfected cells. Grx2 overexpression protected ARPE-19 cells from H₂O₂-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx2 were relatively more resistant to H₂O₂ with fewer cells undergoing apoptosis as compared to vector control or non-transfected cells. Furthermore, Grx2 overexpressed cells were more resistant to the loss of mitochondrial membrane potential induced by H₂O₂. PSSG accumulation in mitochondria was also dramatically attenuated by Grx2 overexpression.

Conclusions: Grx2 can protect human retinal pigment epithelial cells against H₂O₂-induced cell death. The mechanism of this protection is likely associated with its ability to prevent lethal accumulation of PSSG in mitochondria.

Keywords: 412 age-related macular degeneration • 634 oxidation/oxidative or free radical damage • 701 retinal pigment epithelium