The Role of Phospholipid Hydroperoxide Glutathione Peroxidase Isoforms in Murine Embryogenesis

Abstract

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.