Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.
Nomura
K
K
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan.
Imai
H
H
Koumura
T
T
Arai
M
M
Nakagawa
Y
Y
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D003847:Deoxyglucose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000494:pharmacology*; D008293:Malates / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D018384:Oxidative Stress; D010545:Peroxides / Q000378:metabolism; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051381:Rats; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D008293:Malates; D010545:Peroxides; D019253:Proto-Oncogene Proteins c-bcl-2; D013481:Superoxides; C058705:diethyl malate; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005947:Glucose