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The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults

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Abstract

Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4−/− embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4+/− mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4+/− mice are markedly sensitive to inducers of oxidative stress, including γ-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4+/− mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.

Introduction

Aerobic metabolism results in the generation of reactive oxygen species (ROS) that can damage DNA, proteins, and cellular membranes [1]. In response to this challenge, aerobic organisms use a network of antioxidant enzymatic systems to minimize damage induced by ROS, including superoxide dismutase, glutathione peroxidase, and catalase. Four selenium-containing glutathione peroxidases that catalyze the reduction of peroxides at the expense of glutathione have been identified [2]: GPX1, the classical form which was the first mammalian selenoprotein to be identified; GPX2, which is restricted in expression to the GI tract; GPX3, a plasma isoform; and GPX4, which reduces lipid hydroperoxides. Importantly, GPX4 is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with α-tocopherol (vitamin E) to inhibit lipid peroxidation [3].

Mice lacking classical glutathione peroxidase (GPX1), which can reduce hydrogen peroxide to water, do not display an obvious increase of basal levels of oxidative damage [4], indicating that there is redundancy with other peroxidases for at least some of its biochemical function in vivo. In vitro overexpression studies have shown that GPX4 inhibits the proliferative effect of oxidized LDL in aortic smooth muscle cells [5]. Further, its overexpression in various cell lines strongly modulates many markers of apoptosis, such as the release of cytochrome c, DNA fragmentation [6], and inhibition of NF-κB [7]. In addition to being the only major antioxidant to eliminate destructive lipid peroxides, GPX4 also reduces thymidine peroxides, suggesting a possible role for GPX4 in the repair of DNA damage [8].

To help clarify the role of GPX4 at the organismal level as a component of the mammalian antioxidant system, we used homologous recombination in embryonic stem (ES) cells to disrupt the gene encoding GPX4 (Gpx4) in mice. Additionally, cell lines derived from Gpx4+/− embryos were used to evaluate the role of GPX4 in protecting cells against oxidative insults.

Section snippets

Generation of Gpx4-deficient mice

A Gpx4 genomic clone was isolated from a 129/SVJ mouse genomic library by hybridization screening using a rat Gpx4 cDNA clone. The targeting vector was designed to delete exons 3, 4, 5, 6, and 7 of Gpx4 (Fig. 1A). Targeted ES cell clones were generated and chosen for blastocyst injection. Briefly, 20μg of Asp718-linearized targeting vector were electroporated into AB2.2 ES cells, which were then cultured in HAT (hypoxanthine-aminopterin-thymidine) medium with 0.2 mM FIAU [1-

Results and discussion

While Gpx4+/− mice displayed no gross morphological or behavioral abnormalities and were fertile, no Gpx4−/− animals were detected among 284 pups from heterozygote intercrosses (chi-square = 1.74 × 10−21). Litters from Gpx4+/− intercrosses were collected in their decidual swellings, embedded in paraffin, and sectioned serially. Morphologic abnormalities could not be distinguished in E5.5–6.5 embryos examined from Gpx4+/− intercrosses, but by E7.5, 11 of 45 examined implanted embryos from

Acknowledgements

We thank Thomas Pugh and Karen Downs for technical help with interpretation of histological findings and Karin Panzer for technical help in animal colony maintenance. This work was supported by a grant from the National Cancer Institute to T.A.P., a Merit Review grant from the Department of Veteran Affairs to H.V.R., a NIH Program Project Grant 1 PO1 AG19316 to A.R., and the Nathan Shock Center of Excellence in Basic Biology of Aging (PO3 AG13319) at the University of Texas Health Science

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