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Differential responses of SOD1-deficient mouse embryonic fibroblasts to oxygen concentrations
Satoshi Tsunoda1, Noriko Kibe, Toshihiro Kurahashi
1Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, Iidanishi, Japan.
Abstract:
Superoxide dismutase (SOD) plays a role in antioxidation, and SOD1-knockout (KO) mice show moderate phenotypes. Primary cultured mouse embryonic fibroblasts (MEFs) lead to growth failure and eventual death under normoxic culture (20% oxygen). We attempted to elucidate the molecular mechanisms responsible for the oxygen toxicity in SOD1-KO MEFs. Increases in reactive oxygen species, lipid peroxidation products, and senescence-associated β-galactosidase activity were observed in SOD1-KO MEFs. Hypoxic culture (2% oxygen) averted immediate cell death but could not recover the proliferative ability of the SOD1-KO cells. The cell cycles of SOD1-deficient MEFs were arrested at the G2 and M phases, leading to the accumulation of tetraploid cells under hypoxic culture. The suppressed expression of cyclin A2 and B1 and the concomitant induction of p21(Waf1) were evident in SOD1-KO cells. The phosphorylation of p53 and histone H2Ax and the induction of the two proapoptotic genes Bax and Noxa were evident in SOD1-deficient MEFs and more enhanced under normoxic culture than under hypoxic culture. We concluded that low levels of oxygen consumption moderately activates the p53 pathway, and leads to cellular senescence, but that high levels of oxygen consumption hyperactivates the p53 pathway, which results in cell death in SOD1-deficient MEFs.
Insights
Superoxide dismutase 1 (SOD1) deficiency causes oxygen toxicity in mouse cells, leading to senescence or death. Reduced oxygen levels trigger cell cycle arrest and senescence, while high oxygen hyperactivates cell death pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Superoxide dismutase (SOD) enzymes are critical for cellular antioxidant defense.
- SOD1-knockout (KO) mice exhibit moderate phenotypes, but their primary cultured mouse embryonic fibroblasts (MEFs) show severe growth defects and death under normoxia.
- The precise molecular mechanisms underlying oxygen toxicity in SOD1-deficient MEFs remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanisms responsible for oxygen toxicity in SOD1-knockout mouse embryonic fibroblasts.
- To investigate the impact of varying oxygen concentrations on cellular processes in SOD1-deficient MEFs.
Main Methods:
- Culture of SOD1-knockout (KO) mouse embryonic fibroblasts (MEFs) under normoxic (20% O2) and hypoxic (2% O2) conditions.
- Assessment of reactive oxygen species (ROS), lipid peroxidation, senescence markers, cell cycle progression, and gene/protein expression (including p53 pathway components and apoptosis-related genes).
Main Results:
- SOD1-KO MEFs exhibited increased ROS, lipid peroxidation, and senescence under normoxia.
- Hypoxia prevented immediate cell death but did not restore proliferation, causing G2/M cell cycle arrest and tetraploid cell accumulation.
- SOD1-deficient cells showed suppressed cyclin A2/B1 and induced p21(Waf1), alongside p53 and H2Ax phosphorylation.
- Apoptotic genes Bax and Noxa were induced, with greater enhancement under normoxia than hypoxia.
Conclusions:
- Oxygen toxicity in SOD1-deficient MEFs is mediated by differential activation of the p53 pathway.
- Low oxygen consumption leads to moderate p53 activation, resulting in cellular senescence.
- High oxygen consumption hyperactivates the p53 pathway, causing cell death in SOD1-deficient MEFs.
