Glutathione peroxidase 4 has a major role in protecting mitochondria from oxidative damage and maintaining oxidative phosphorylation complexes in gut epithelial cells.
Intake of the micronutrient selenium, which is incorporated into 25 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 GPX4 expression was knocked down by 60% using RNA silencing and the effects were investigated using an unbiased transcriptomic analysis. Microarray analysis of the total Caco-2 cell transcriptome was carried out using Illumina HumanHT-12v3 beadchips and the data were validated by real-time PCR. Ingenuity Pathway Analysis showed that the major canonical pathways affected by GPX4 knockdown 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 knockdown affected target genes encoding components of respiratory complexes I, IV, and V as well as the protein apoptosis-inducing factor (AIF). GPX4 knockdown increased levels of mitochondrial reactive oxygen species and oxidized lipid and decreased mitochondrial adenosine triphosphate levels and mitochondrial membrane potential. Time-course experiments showed that changes in AIF expression preceded those in the respiratory complexes. We conclude that in Caco-2 gut epithelial cells GPx4, through effects on AIF, plays a major role in maintaining the oxidative phosphorylation system and protecting mitochondria from oxidative damage.
Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, UK.
Patience
P
Cole-Ezea
Dan
D
Swan
Daryl
D
Shanley
John
J
Hesketh
D000255:Adenosine Triphosphate / Q000378:metabolism; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D045744:Cell Line, Tumor; D003106:Colon / Q000473:pathology; D051546:Cyclooxygenase 2 / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology* / Q000378:metabolism; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology; D015227:Lipid Peroxidation; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D018384:Oxidative Stress; D010101:Oxygen Consumption; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D021382:Protein Sorting Signals; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D018709:Statistics, Nonparametric
C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I