Increased Expression of the Long Form of Glutathione Peroxidase 4 Strongly Protects Against Oxidative Damage in Cultured RPE Cells

Abstract

Purpose:: To investigate the effect of overexpression of glutathione peroxidase 1 (GPx1), GPx4 (long [L] and short [S] forms), or superoxide dysmutase (SOD) 1 and 2 in cultured RPE cells.

Methods:: ARPE 19 cells were transiently transfected with an expression plasmid carrying the gene GPx1, L-GPx4, S- L-GPx4, SOD1 or SOD2. Transfected cells were treated with various oxidants and protein carbonyl adducts were measured by ELISA.

Results:: The process of transfection caused oxidative damage demonstrated by increased protein carbonyl adducts in ARPE-19 cells transfected with empty plasmid or those containing expression constructs for SODs; this increase in carbonyl adducts was not seen in cells transfected with GPx plasmids. Despite the basal increase in carbonyl adducts, cells overexpressing one of the SODs showed reduced oxidant-induced increases in carbonyl adducts compared to control cells, but over-expression of one of the GPxs was more protective with the L-form of GPx4 having the greatest effect. After co-transfection with L-GPx4 and SOD plasmids, there was a basal increase in carbonyl adducts, but strong protection against oxidant-induced increases.

Conclusions:: Increased expression of L-GPx4 in cultured RPE cells provides good protection from oxidative damage to proteins. Additional studies are needed to test the effect of increased expression of L-GPx4 alone and in combination with SODs in animal models of oxidative damage in the retina and/or RPE.

Keywords: antioxidants • enzymes/enzyme inhibitors • retinal pigment epithelium