A study of glutathione peroxidase 4 function in human intestinal epithelial cells

Abstract

Intake of the micronutrient selenium, which is incorporated into selenoproteins in humans, has been implicated in affecting risk of colorectal cancer. A genetic variant in the gene encoding the selenoprotein glutathione peroxidase 4 (GPx4) has been reported to influence colorectal cancer risk. In this study the role of GPx4 was investigated in the Caco-2 intestinal cell line using RNA silencing. GPX4 expression was knocked–down by ~60% and an unbiased gene microarray analysis of the total Caco-2 cell transcriptome was carried out using Illumina HumanHT-12v3 beadchips. The data were validated by real-time PCR. Ingenuity Pathway analysis showed that the major canonical pathways affected by GPX4 knock-down were oxidative phosphorylation, ubiquinone biosynthesis and mitochondrial dysfunction and the top two toxicological lists were mitochondrial dysfunction and oxidative stress. Western blotting and real-time PCR confirmed that knock-down affected target genes encoding components of respiratory complexes I, IV and V as well as the protein apoptosis-inducing factor (AIF). GPX4 knock-down increased levels of mitochondrial reactive oxygen species and oxidised lipid, and decreased mitochondrial adenosine triphosphate (ATP) levels and mitochondrial membrane potential. Time course experiments showed changes in AIF expression preceded those in the respiratory complexes. GPX4 knock-down increased apoptosis and changed protein expression of Caspase-9, Bax and Bcl-2. Treatment of cells with the antioxidant mitoquinone prevented the effects of GPX4 knockdown on mitochondrial reactive oxygen species, oxidised lipid and mitochondrial membrane potential but not the effect on AIF. These data suggest that in intestinal epithelial cells GPx4, through effects on lipid peroxidation and AIF, plays a complex role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage and apoptosis.