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.Phospholipid hydroperoxide glutathione peroxidase (phGPx or GPx4) 2 is an intracellular antioxidant enzyme (1) that directly reduces peroxidized phospholipids even if they are incorporated in biomembranes and lipoproteins (2-4). In addition, the enzyme has been implicated in sperm maturation (5, 6) and appears to be essential for regular murine embryogenesis (7,8). There are three different isoforms of GPx4 (cytosolic isoform (c-GPx4), mitochondrial isoform (m-GPx4), and nuclear isoform (n-GPx4)), but all of them derive from a single gene, which is located on human chromosome 19 (9) and in a sentential region of murine chromosome 10 (10, 11). The start codons for the m-and c-GPx4 isoforms as well as the targeting sequence that directs the mitochondrial enzyme into the mitochondria are localized in the first exon of the GPx4 gene. Expression of the 34-kDa n-GPx4 (nuclear isoform) involves transcription of an alternative first exon (12). The three phGPx isoforms are expressed at low to medium levels in most mammalian cells and have been implicated in expression regulation of redox-sensitive genes (13), in inflammation (14), in modulation of programmed cell death (15), and in oxidative injury. High concentrations of GPx4 were found in te...

Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function

“…(ii) Isoform-specific profiles of GPx4 mRNA species during murine brain development show similar amounts of m-GPx4 and c-GPx4 during early stages of embryonic development (embryonic days E6.5-E15.5). However, in later developmental stages (E16.5) and after birth, the concentration of the m-GPx4 messenger decreased, whereas c-GPx4 mRNA remained unchanged (Borchert et al, 2006). Such mRNA expression kinetics indicates transcriptional regulation.…”

“…Whether expression of n-GPx4 is driven by a separate promoter within intron 1a in vivo and whether additional mechanisms are employed in various tissues remain to be answered. This is of particular interest in light of the recent finding that expression of m-GPx4 and n-GPx4 appeared to be coupled during embryonic development and thus might be controlled by joint regulatory mechanisms (Borchert et al, 2006). In contrast, c-GPx4, which seems to share regulatory elements of transcription with m-GPx4, exhibits different expression kinetics (Borchert et al, 2006).…”

“…Expression levels of cytosolic, mitochondrial and nuclear GPx4 mRNAs in murine embryos and postnatal brains (N0-N4) are shown. These data were obtained from real-time RT-PCRs normalized for GADPH mRNA levels (from Borchert et al, 2006). Note the transient drop and overall low expression of n-GPx4 mRNA.…”

“…In brain mitochondria, GPx4 activity was mainly recovered in the inner mitochondrial membrane fraction (Panfili et al, 1991). Direct evidence of GPx4 distribution came from comparative expression analysis, revealing onset of expression as early as during gastrulation (Yant et al, 2003;Borchert et al, 2006). At E6.5, all three isoforms are expressed in extra-embryonic and embryonic structures, including the ecto-, mesoand endoderm.…”

“…Recent advances in these fields, including elucidation of the crystal structure of various GPx isoforms and novel data on regulation in response to brain injury (Borchert et al, 2006;Savaskan et al, 2007), prompted us to critically discuss the state of the art in these fields. We also address the role of GPx4 in neuronal signaling and discuss future developments in GPx4 research.…”