Transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase, mitochondrial thioredoxin reductase and glutathione peroxidase 4

Biochimica et Biophysica Acta 1790 (2009) 1575–1585

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Review

Transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase, mitochondrial thioredoxin reductase and glutathione peroxidase 4 Marcus Conrad ⁎ Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Marchioninistr. 25, 81377 Munich, Germany

a r t i c l e

i n f o

Article history: Received 4 April 2009 Received in revised form 23 April 2009 Accepted 5 May 2009 Available online 9 May 2009 Keywords: Apoptosis inducing factor (AIF) Cre/loxP 12/15-lipoxygenase Oxidative stress PHGPx Redox regulation

a b s t r a c t Selenium, as an integral part of selenoproteins, is essential for mammals. Unequivocal evidence had been provided more than a decade ago when it was proven that mice incapable of producing any of the 24 selenoproteins failed to develop beyond the gastrulation stage (E6.5). Since then, more specific attempts have been made to unmask novel and essential functions of individual selenoproteins in mice. Genetic disruption of glutathione peroxidase 4 (GPx4; also referred to as phospholipid hydroperoxide glutathione peroxidase, PHGPx) in mice showed for the first time that a specific selenoenzyme is in fact required for early embryonic development. Later on, systemic ablation of cytosolic thioredoxin reductase (Txnrd1) or mitochondrial thioredoxin reductase (Txnrd2) yielded embryonic lethal phenotypes. Beside those three, no other selenoproteins have been found being indispensable for murine development so far. This review aims at summarizing mainly the in vivo findings on these important mammalian selenoenzymes, which have not only common attributes of being required for embryogenesis, but that they are also instrumental in the regulation of cellular redox metabolism. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Compelling evidence established the redox regulation of protein functions as an additional regulatory mechanism of normal cell physiology [1,2]. For instance, dithiol–disulfide exchange reactions have been identified as molecular switches participating in the regulation of many cellular processes. Redox-sensitive cysteine residues in these proteins exploit the unique chemistry of sulfur to flip from one oxidation state to the other on exposure to oxidizing conditions. On the other hand perturbation of cellular redox balance may cause oxidative stress, which has been frequently linked with ageing and many pathophysiological processes, e.g. neurodegeneration, atherosclerosis, diabetes and cancer [3,4]. Since selenoproteins carry at least one catalytically active and highly reactive selenocysteine (Sec) moiety, many of the known selenoproteins are predestined enzymes to decisively contribute to proper redox control [5]. The fact that selenium is an essential trace element in mammals was corroborated by the selective removal of Trsp, the gene encoding the Sec-specific tRNA, in mice [6,7]. It is interesting enough that mice lacking γ-glutamylcysteine synthetase (γ-GCS), the enzyme catalyzing the first and rate-limiting step in glutathione (GSH) synthesis, die just a bit later than Trsp−/− mice during murine embryogenesis (E7.5 versus E6.5) [8]. As four of the eight glutathione-dependent peroxidases in mice are selenoproteins, it could be expected someway that one of these might be limiting for murine development. But ⁎ Tel.: +49 89 7099 525; fax: +49 89 7099 500. E-mail address: [email protected]. 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.05.001

individual disruption of GPx1 and GPx2, alone or in combination, only induced rather mild phenotypes [9,10]; mice lacking GPx3 appear to be viable as mentioned in a presentation at the 8th International Symposium on Selenium and Medicine, 2006 [11]. By contrast, GPx4deficient mice die at the same embryonic stage (E7.5) as γ-GCS knockout mice, indicating that GPx4 is not only a very important glutathione-dependent enzyme, but also a vitally important selenoprotein (see Section 4) (Table 1). Besides the substantial roles of cytosolic (Txnrd1) and mitochondrial thioredoxin reductases (Txnrd2) in embryogenesis and brain and heart development (Table 1), other selenoproteins, including selenoprotein P [12,13], type 2 iodothyronine deiodinase [14] and methionine-R-sulfoxide reductase 1 (MsrB1, also designated as SelR or SelX) [15], are dispensable for embryonic development, even though they may feature significant functions in adult mice. Thus, the specific inactivation and in vivo analysis of individual selenoproteins in mice is mandatory to assign crucial and relevant functions in physiology and under stress conditions. 2. Mice lacking selenoprotein expression in embryos and adult tissues: insights into the importance of the selenoprotein pool in tissue function Selenoproteins are characterized by carrying one or several selenocysteine(s) (Sec), and for some selenoproteins the essential role of Sec for enzyme catalysis has been confirmed already. Selenium labeling experiments in rodents had previously suggested the presence of at least 30 to 50 selenoproteins [16]. However,

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Table 1 Major findings obtained from Txnrd1, Txnrd2 and GPx4 knockout studies in mice. Gene

Authors

Knockout approach

Major phenotype

Ref.

Txnrd1

Jakupoglu et al.

Conditional/ubiquitous (Cre-deleter)

[61]

Bondareva et al. Soerensen et al.

Heart-specific (MLC2α-Cre) Conditional/ubiquitous Nervous system-specific (Nestin-Cre)

Soerensen et al. Conrad et al.

Neuron-specific (Tα1-Cre) Conditional/ubiquitous (Cre-deleter)

GPx4

Geisberger et al. Kiermeyer et al. Soerensen et al. Yant et al. Imai et al. Garry et al. Seiler et al.

Heart-specific (MLC2α-Cre) T cell (CD4-Cre)- and B cell (CD19-Cre)-specific Inducible heart-specific (α-MHC-MerCreMer) Nervous system-specific (Nestin-Cre) Constitutive knockout Constitutive knockout Constitutive knockout Conditional/ubiquitous (Cre-deleter) Neuron-specific (CamKIIα-Cre)

nGPx4

Conrad et al.

Constitutive knockout

Embryonic lethality between E8.5 and E10.5 due to severe proliferation defects and overall growth and developmental retardation No obvious phenotype Embryonic lethality at E8.5 Cerebellar hypoplasia due to severe proliferation defects of external granular layer No obvious phenotype Embryonic lethality between E13.5 and E15.5 due to perturbed heart development and fetal hematopoiesis Congestive heart failure and postnatal death No obvious phenotype No obvious phenotype No obvious phenotype Embryonic lethality at E7.5 Embryonic lethality at E7.5 Embryonic lethality at E7.5 Embryonic lethality at E7.5 Marked neurodegeneration in hippocampus and cortex due to lipid peroxidation Fully viable/delayed sperm chromatin condensation due to reduced protamine oxidation

Txnrd2

computational genome-wide analysis identified only 25 genes for selenoproteins in human and 24 in mouse, indicating that alternative splicing, as evident for some selenoproteins, contributes to the selenoprotein make-up [5]. The sole difference in the selenoprotein make-up between mouse and human is olfactory glutathione peroxidase (GPx6), which is a Cys-containing variant in mice. Some of the selenoproteins can be classified in the thioredoxin reductase, glutathione peroxidase and deiodinase family of proteins, whereas others are unique with respect to their primary structure and function. On the basis of the complexity and similarity of the Sec insertion mechanism, it has been postulated that Sec utilization arose before the division into the three domains, but was then lost in some organisms during evolution. It is, however, still somewhat difficult to understand why some organisms lost selenoprotein expression and why many orthologs of selenoproteins with similar function, including glutathione peroxidases and thioredoxin reductases, exist as Cys variants in yeast, plants and protozoan. A recent study by Gladyshev's laboratory proposed that selenoprotein expression correlates with the organism's life environment — the more obligate to terrestrial life the smaller the likelihood to express selenoproteins [17]. Yet, selenoproteins are indispensable for embryonic development in mammals. Mice with targeted deletion of the gene encoding the Sec-specific tRNA (tRNAsec(ser), Trsp) fail to develop shortly after implantation and knockout embryos are resorbed lately by day 6.5 post-coitum [6]. These initial findings provided first insight that at least one or several selenoproteins might be vital for early development. It is noteworthy, that trophoblast outgrowth and propagation of inner cell mass cells ex vivo are only partially compromised by Trspdeficiency. This suggests that early embryonic development does either not rely on the intrinsic selenoprotein expression, or that essential factors are provided by maternal tissues, or that in the absence of Trsp Cys incorporation at the UGA codon in one or several of the putative “limiting” selenoproteins may cope for the Sec requirement in mammals in a manner similar to Euplotes crassus selenoproteins as recently reported [18]. Embryonic lethality of Trsp−/− mice was further confirmed by Hatfield's laboratory by using mice carrying a loxP-flanked (floxed) Trsp allele [7]. Ubiquitous Cre-mediated removal of the floxed Trsp allele and subsequent intercross of Trsp+/− mice never yielded viable offspring. In subsequent studies it was shown by the same laboratory that mice lacking selenoproteins specifically in the liver die within 1 to 3 months after birth due to severe hepatocellular degeneration and necrosis [19]. Interestingly,

[61] [62] [70] [70] [63]

[63] [69] [68] [70] [91] [124] [92] [93] [93] [108]

Suzuki et al. demonstrated recently that tissue-specific deletion of Trsp in macrophages and liver causes increased oxidative stress and the induction of genes involved in the detoxification of oxygen radicals, e.g. NAD(P)H:quinone oxidoreductase, glutathione S-transferase P1, the catalytic subunit of γ-GCS, and heme oxygenase [20]. All these antioxidant enzymes are direct target genes of the oxidative stressactivated transcription factor NF-E2-related factor 2 (Nrf2). Accordingly, simultaneous disruption of Trsp and Nrf2 clearly aggravates the sensitivity of ex vivo cultured macrophages towards H2O2 treatment. Concomitant loss of Trsp and Nrf2 in liver induces early hepatic degeneration and compound mutant mice die within 3 to 7 weeks after birth, whereas more than 70% of liver-specific Trsp knockout mice on an Nrf2 wild-type background survive during the observation period of 24 weeks. Thus, these studies suggest that Nrf2-dependent gene induction compensates for the lack of selenoprotein synthesis in Trsp null mice to some extent [20]. Endothelial-restricted Trsp−/− mice show an overall poorly developed vascular system, develop severe developmental abnormalities in limbs, tails and head from E12.5 onwards and eventually die before E18.5 [21]. In the same study, it was also shown that musclecell-specific Trsp ablation causes acute myocardial failure around 12 days after birth [21]. T cell-specific Trsp disruption is associated with partial atrophy of thymus, spleen and lymph nodes and an overall reduction of splenic CD3+ and CD4+ T cells. Also, TCR-induced proliferation of T cells is strongly impaired due to decreased IL-2 receptor expression and impaired ERK signaling. T cell-dependent immunization experiments further revealed that T cell-specific Trsp−/− mice are compromised in the generation of antigen-specific antibodies, indicating that selenoproteins have non-compensatory functions in T cell-mediated immunity [22]. Many of the selenoproteins, including thioredoxin reductases and glutathione peroxidases (see also below), are involved in oxidative stress defense. Although increased oxidative stress has been linked to the pathogenesis of diabetic nephropathy, podocyte-specific ablation of Trsp, which essentially disrupts all selenoproteins, along with streptozotocin-induced β-cell loss in pancreas does not lead to any increase in oxidative stress nor does it impinge on body weight, blood glucose levels and urinary albumin/creatinine ratios in the diabetic mouse model [23]. Also, markers for oxidative stress were found to be similar in the glomeruli of Trsp−/− and control mice. Conclusively, tissue-specific Trsp knockout mice represent the most valuable tool to unravel selenoprotein insertion mechanism in

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vivo and to define and identify tissues which are obligate for one or several selenoproteins. Despite all the strengths of such a system, it is obvious that more specific investigations with targeted disruption of individual selenoproteins or even specific isoforms of a given selenoprotein are still needed. 3. Mice with targeted disruption of cytosolic and mitochondrial thioredoxin reductase Mammals harbor three distinct genes for thioredoxin reductases. Cytosolic thioredoxin reductase (Txnrd1) was the first thioredoxin reductase to be discovered [24], and shown to contain Sec as the penultimate amino acid [25]. Txnrd1 is ubiquitously expressed, and it primarily localizes to the cytosol [24]. Complex transcriptional regulation and alternative splicing at the 5′ end have been reported for human thioredoxin reductase 1, which may lead to five different potential N-terminal variants [26]. Mitochondrial thioredoxin reductase (Txnrd2; also known as TR3)) was first purified and cloned in rats [27,28]. Due to an N-terminal mitochondrial leader sequence, Txnrd2 mainly localizes to mitochondria. Various N-terminal splicing variants have been identified also for Txnrd2, which may direct Txnrd2 to other subcellular compartments [29]. Yet, further experimental evidence is required to substantiate the biological implications of these variants. A third thioredoxin reductase was discovered in 2001. With its N-terminal glutaredoxin-like domain extension and combined thioredoxin and glutathione reductase activities, it was named thioredoxin-glutathione-reductase (TGR or Txnrd3). Txnrd3 is mainly expressed in testis [30], and later on it was shown that the N-terminal glutaredoxin-like domain confers an additional protein-disulfide isomerase function in sperm cells [31]. Thioredoxin reductases are flavoproteins belonging to the pyridine nucleotide-disulfide oxidoreductase protein family. They exist as homodimers and each subunit of approximately 54–58 kDa in size contains one FAD binding domain, one NAD(P)H binding domain, and one interface domain required for dimerization. These homodimeric selenoproteins are arranged in a head to tail fashion so that the two Nand C-terminally located redox-active centers can functionally interact and transfer electrons from NADPH/H+ eventually to their substrates [32,33]. The N-terminal redox center lies within the FAD binding domain and consists of -Cys-Val-Asn-Val-Gly-Cys-, while the C-terminal redox-active catalytic site is made up of -Cys-Sec- with Sec as the penultimate amino acid. Thioredoxin reductase catalysis follows a ping-pong mechanism: electrons are delivered from NADPH/H+ to the prosthetic group FAD, transferred via the Nterminal catalytically active cysteines to the C-terminal redox-active site of the adjacent monomer from where they are finally passed on to the substrate [34–38]. This mechanism was further confirmed very recently by crystal structure analysis of thioredoxin reductase 1 by Arnér's laboratory [39]. Sec was previously shown by various groups to be critical for thioredoxin reductase activity [37,38,40]. Mammalian thioredoxin reductases are highly versatile with regard to their substrates, which most likely relies in their easily accessible Cterminal catalytic center and the Sec moiety (the reader is referred to a recent review provided by E.S. Arnér in this journal [41]). In addition to their respective major substrates, cytosolic thioredoxin (Txn1) and mitochondrial thioredoxin (Txn2), thioredoxin reductases reduce a wide range of substrates including H2O2, lipid peroxides, vitamin C, selenite, α-lipoic acid, α-Tocopherol, ubiquinone, insulin, NK-lysin, Lcystine, alloxan, and vitamin K [42–44]. The presence of Sec instead of Cys, the latter is found in other organisms including Drosophila melanogaster, may account for the broad substrate specificity in mammals [45]. First indication why mammalians thioredoxin reductases use Sec instead of Cys in their active site was provided by an elegant study by Gromer et al. [45]. In this study, the catalytic properties of human thioredoxin reductase 1 were compared with the non-selenium containing homologue of D.

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melanogaster. Due to the easily oxidizable selenolate anion, the Seccontaining enzyme has been considered to be more active than Cys variants by two or three orders of magnitude. There is, however, no significant difference between human and fly thioredoxin reductase regarding enzyme activity parameters. Mutational and functional studies of both enzymes revealed that the pKa of the non-selenium catalytic site in the fly enzyme is lowered by surrounding acidic amino acid residues, leading to an equal activation of Cys compared to the selenium-containing human counterpart. While the thioredoxin reductase activity of the human Sec-containing thioredoxin reductase could not be increased by changing the amino acids surrounding the active site in a manner resembling the D. melanogaster homologue, it was, however, possible to generate a Sec/Cys variant with the aforementioned changes, which is equally active as the wild-type enzyme when assayed with the model substrate DTNB or thioredoxin itself. The only difference is that the Sec/Cys variant has a limited substrate spectrum. Hence, Sec is essential for conferring full versatility to thioredoxin reductases [45]. In this context it is noteworthy that apparently there is much more complexity in thioredoxin reductase catalysis with regard to the N-terminal and Cterminal catalytic sites and different substrates. Lothrop et al. showed very recently that the Sec/Cys mutant or the N-terminal reactive center alone is sufficient to reduce selenium-containing substrates and lipoic acid, while there is a sharp drop of kcat towards thioredoxin [46]. Besides the above-named low molecular compounds, thioredoxin reductases maintain thioredoxins in their active reduced state. Cytosolic and mitochondrial thioredoxins are small redox-active proteins with two redox-active cysteinyl residues organized in a characteristic thioredoxin fold with significant functions in cell–cell communication, DNA metabolism and repair, transcription regulation, protein folding and degradation, and signal transduction [43,47,48]. Thioredoxin was initially described in E. coli as an essential co-factor for ribonucleotide reductase which converts ribonucleotides to deoxyribonucleotides [49]. Human thioredoxin was originally cloned as a cytokine-like factor in human T cell leukemia virus type Itransformed cells [50]. Acting as electron donors for some peroxiredoxins [51], thioredoxins also efficiently protect cells from oxidative damage. Thioredoxin may also act as molecular switches using dithiol/disulfide-dependent structural changes. This may lead to activation/inactivation of target proteins, and thus affects downstream signaling cascades, including the inhibitory binding of reduced Trx1 to ASK1 [52]. For instance, oxidation of thioredoxin 1 was shown to liberate ASK1 and to activate the MAP kinase p38 dependent apoptosis pathway [53]. Recently, it was also demonstrated that thioredoxin 1 interferes with the binding of the CD30 ligand (CD30L) to its receptor and prevents the CD30-induced decrease in cytotoxic effector functions of effector cells [54]. Cytosolic and mitochondrial thioredoxins have also been shown to harbor denitrosylase activity towards caspase-3, thus impinging on specific signaling pathways regulated by S-nitrosylation [55]. A plethora of other thioredoxin functions have been reported to date and needless to say many functions await to be discovered in the near future. Several gene targeting approaches in mice corroborated the importance of the thioredoxin/thioredoxin reductase system in development and adult physiology. Deletion of either Txn1 or Txn2 revealed that both genes are essential for murine embryonic development [56,57]. Loss of Txn1 in mice causes early embryonic death (E3.5) due to severely compromised proliferation of the inner mass cells. Txn2-deficiency is associated with marked exencephaly, strongly increased apoptosis, and embryonic death at around E10.5. Conversely, overexpression of human thioredoxin 1 in transgenic mice was shown to confer high resistance to a multitude of stress-inducing conditions [58,59]. To investigate the functions of Txnrd1 and Txnrd2 in embryogenesis and various adult tissues, the Cre/loxP technology was exploited to achieve spatio-temporal disruption of gene function of Txnrd1 and

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Txnrd2 in mice. The ubiquitous inactivation of Txnrd1 and Txnrd2 unmasked that either of the genes is indispensable for embryonic development, albeit in different ways [60]. Txnrd1−/− embryos display overall growth and developmental retardation with the exception of the embryonic heart [61]. A significant drop in cell proliferation throughout the embryo, and not an increase in the number of apoptotic cells, is the underlying cause of embryonic death between E8.5 and E10.5. Apparently, the time of embryonic death of Txnrd1 knockout embryos very well correlates with the genetic background of the mice (unpublished observation). The higher the C57BL/6 background the earlier the embryos are resorbed during embryonic development. In line with the severe proliferation defects of Txnrd1−/− embryos, mouse embryonic fibroblast cultures could never be established directly from Txnrd1−/− embryos (see also below). Recently, Bondareva et al. reported similar findings obtained also with a conditional Txnrd1 knockout mouse model [62]. Ubiquitous disruption of Txnrd1 allele leads to embryonic death at E8.5, and the authors never observed embryos, which developed beyond gastrulation. Whole mount in situ hybridization analysis indicated that the development of primitive streak mesoderm is defective in the null mutants. Moreover, transcriptome analysis of single embryos revealed that, while the transcript levels of Txnrd2, glutathione reductase and peroxiredoxins are indifferent between wild-type and knockout embryos, IGF-binding protein-1, sulfiredoxin 1, glutathione-Stransferases and others are up-regulated in response to Txnrd1 inactivation. The major difference between both knockout mouse models is that in the study by Jakupoglu et al. the last exon, encoding the C-terminal catalytic center and the SECIS element, was specifically removed, whereas Bondareva et al. targeted the first two exons, encoding all functional ATGs and the N-terminal catalytic center including Cys59 and Cys64 [62]. Like Txnrd1 null embryos, Txnrd2−/− embryos are embryonic lethal [63]. In contrast to Txnrd1 knockout embryos, Txnrd2-deficient embryos develop somewhat further, but die around E13.5 to E15.5. Reduced proliferation of cardiac cells and increased apoptosis of fetal blood cells in the liver are the major reasons for the severe anemic phenotype and partial growth retardation of Txnrd2−/− embryos. Hematopoietic stem cell differentiation is not affected by Txnrd2 inactivation. Although MEF cultures could be established from Txnrd2−/− embryos, proliferation and survival of ex vivo cultured mouse embryonic fibroblasts (MEFs) is clearly compromised. The difference between wild-type and knockout embryonic fibroblasts is even more pronounced when the de-novo synthesis of GSH is inhibited by L-buthionine sulfoximine (BSO), a highly specific inhibitor of γ-GCS. GSH depletion rapidly induces apoptosis in Txnrd2−/− cells, but not in wild-type cells. High ROS accumulation accounts for the rapid onset of cell death, which can be prevented by N-acetylcysteine (NAC), a thiol-containing antioxidant [63]. These investigations provided the first hint that there must be some kind of partial overlap between the thioredoxin and glutathione-dependent systems in mammalian cells as later shown also in plants [64]. The embryonic phenotypes of Txnrd1 and Txnrd2 knockout mice, however, are less pronounced than those of the respective knockouts for Txn1 and Txn2. This implies that there is either functional overlap between Txnrd1 and Txnrd2 during embryogenesis to some extent or that there are other crucial functions of Txn1 and Txn2, which do not require functional thioredoxin reductase. Along this line, a Cterminally truncated form of Txn, which lacks redox activity, was shown to exert potent mitogenic activity on resting human peripheral mononuclear cells [65]. It might also well be that in terms of maintaining thioredoxin activity, different tissue expression, different subcellular localization and yet-unknown functions of Txnrd3 may substitute for each other to some degree. For instance, it was shown by Turanov et al. that Txnrd1 and Txnrd2 lack preferences towards Txn1 and Txn2, respectively, and that both thioredoxins are equally good substrates for both Txnrd1 and Txnrd2 [29].

Various tissue-specific knockout approaches for Txnrd1 and Txnrd2 were subsequently undertaken to better understand the compromised function of embryonic tissues to the lethal phenotypes of both mutant lines and to gain first insights into the role of both selenoproteins in adult mice. Heart-specific disruption of Txnrd2 induces dilated cardiomyopathy and death within a couple hours after birth, highlighting the essential role for Txnrd2 in cardiac tissue [63]. This implicates that the perturbed cardiac development as observed in the Txnrd2 null embryos is not the sole reason, but rather one contributory factor in embryonic death of Txnrd2-deficient embryos. Ultrastructural analysis of newborn hearts revealed severe structural abnormalities of mitochondria, indicating that mitochondrial dysfunction, possibly due to the sudden work overload induced by the separation of the maternal circulation, causes acute heart failure. In this context it is noteworthy that Txnrd2-deficient newborn hearts display a similar phenotype as patients suffering from KeshanDisease, an endemic disease in China caused by severe seleniumdeficiency in conjunction with an enteroviral infection [66,67]. In contrast to these findings, heart-specific Txnrd1 knockout mice are born and are grossly normal [61], which is in accordance with the embryonic observations. To bypass postnatal lethality of heart-specific Txnrd2 knockout mice, mice with tamoxifen (Tam)-inducible, cardiac tissue-restricted disruption of Txnrd2 were generated [68]. Interestingly, inducible adult heart-specific Txnrd2 null mice are fully viable, and cardiac tissue does not display any signs of severe histopathological abnormalities in induced knockout mice. Hence, those mice will prove most suitable to address whether Txnrd1 and Txnrd2 confer essential functions in the stressed heart. Further investigations using T cell- and B cell-specific disruption of Txnrd2 failed to phenocopy some of the embryonic defects in fetal hematopoiesis [69]; however, it does not rule out that Txnrd2 inactivation in the myeloid compartment may provoke yet-unrecognized abnormalities in adult myelopoiesis. Mice with nervous-system (NS)-specific removal of Txnrd1 are born at the Mendelian ratio, but they are clearly smaller in overall size compared to control siblings [70]. The NS-specific Txnrd1 null mice can be easily distinguished from their wild-type counterparts as they are ataxic and display tremor. Only when the diet is provided in the bedding, knockout mice survive weaning. Later on the knockout mice cope with their movement defects, and knockout mice are even able to generate and foster viable offspring, albeit at decreased success rates. Knockout mice show a striking cerebellar hypoplasia from E18.5 onwards, while the development of all other regions of the brain appears to be normal. Progressive differences in the foliation and formation of the molecular-, Purkinje cell- and granular layers are evident during cerebellar development (Fig. 1). Already within the first week after birth, the external granular layer (EGL), which is the origin of cells for the granular layer, was shown to be reduced in thickness in the anterior cerebellar region. Purkinje cells are ectopically localized, and the otherwise well-organized trilaminarstructured cerebellum is hardly discernible. Three weeks after birth, the time when cerebellar development is normally completed in mice, lobules V to I are widely absent in knockout mice. In the dysmorphic region, the internal granular layer is missing, the Purkinje cells are still ectopic, though the total number of Purkinje cells is not altered, and their dendritic arborisation is severely affected. Strongly impaired proliferation in the granular layer of the anterior cerebellum, and not an increase in the number of apoptotic cell number is the underlying reason for the striking cerebellar abnormalities, which also fits with the proliferation defects of Txnrd1 null embryos [61,62]. The posterior part of cerebellum is only merely affected. The Bergmann glia, required for neuronal migration during pre- and postnatal cerebellar development, shows clew-like alignment, disorientation, shortening and a reduced density in the anterior cerebellum. Most interestingly, NS-specific ablation of Txnrd2 or neuron-specific ablation of Txnrd1 does not cause any apparent histopathological abnormalities in any of the brain regions, which strongly argues for an essential and

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Fig. 1. Txnrd1 but not Txnrd2 is required for cerebellar development. (A) Brain-specific inactivation of Txnrd1 by utilizing the Nestin-Cre (Nes-Cre) causes marked cerebellar hypoplasia from E18.5 onwards. Note the cerebellar foliation, as indicated with Roman numerals, is dramatically perturbed in brain-specific Txnrd1 knockout mice. The posterior lobules X to VII of knockout mice appear relatively well developed, while the remaining lobules are widely absent. Shown are sagittal sections of the cerebellum of 21 days old mice. (B) Neuron-specific disruption of Txnrd1 allows normal development of cerebellum. This indicates that non-neuronal cells, such as Bergmann glia and/or granule cells, and not neurons depend on functional Txnrd1. (C) Txnrd2 is not needed for brain development as shown by brain-specific inactivation of Txnrd2 (adopted from [70]).

distinctive role for Txnrd1 in the rapid expansion of non-neuronal cells, particularly that of radial glial (Bergmann cells and/or granule precursor cells) [70]. As cerebellar development requires a complex, well-coordinated series of intricately connected developmental processes, including proliferation/death of granule cell, migration along Bergmann glial fibers and outgrowth of Purkinje cells, one may conclude that defective postnatal proliferation of non-neuronal cells triggers cerebellar hypoplasia of Txnrd1 knockout mice. It is noteworthy that brain-specific inactivation of the transcription factor N-myc, by using the same Cre mouse line, is associated with similar cerebellar alterations as found in NS-specific Txnrd1 knockout mice [71]. Since Txnrd1 is a target gene of the proto-oncogene C-myc as shown by nuclear run-on experiments in a human B cell line [72], and N-myc and C-myc functions are largely interchangeable, one may speculate that Txnrd1 is an important gene involved in cell cycle progression. The reciprocal findings obtained by heart-specific and brain-specific deletion of Txnrd1 and Txnrd2 identify both enzymes as

not mere housekeeping genes, but they emerge to play important tissue- and organ-specific functions. These knockout studies provide conclusive evidence that Txnrd1 is critically involved in the rapid proliferation of embryonic and brain tissues. In fact, multiple reports have linked the thioredoxin 1/ thioredoxin reductase 1 system to cell proliferation, cancer development, angiogenesis, invasiveness, and drug resistance of many malignant cells [73,74], and thus Txnrd1 has been proposed as a highly promising target for cancer therapy. Experimental evidence was provided by Hatfield's laboratory, which showed that siRNA-mediated knockdown of Txnrd1 in the Lewis Lung Carcinoma cell line (LLC1) reverses the anchorage-independent growth properties and reduces tumor progression and metastasis [75]. While many of the aforementioned studies have been descriptive, the conditional knockout mouse models for Txnrd1 and Txnrd2 and cellular systems derived thereof will be powerful tools to study the molecular and cellular mechanisms in cell cycle progression and redox-regulated apoptosis signaling.

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4. Transgenic mouse models for glutathione peroxidase 4 4.1. Systemic ablation of GPx4 and mice transgenic for GPx4 Mammals express eight glutathione peroxidases (GPx). GPx1 to GPx4 and GPx6 are selenoproteins in humans, while the latter is a Cyscontaining variant in mice [5]. GPx5, GPx7 and GPx8 are Cyscontaining glutathione peroxidases, and GPx7 and GPx8 share significant structural similarities to GPx4. GPx4, frequently referred to as phospholipid hydroperoxide glutathione peroxidase (PHGPx) due to its unique biochemical functions towards phospholipid hydroperoxides, was first purified by rigorous biochemical techniques from the “cell sap” of pig liver to 535-fold homogeneity [76]. This lipid peroxidation-inhibiting protein with a molecular weight of around 20 kDa was shown to protect phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. Preferred substrates of this glutathione peroxidase were found to be hydroperoxide groups of phosphatidylcholine and on cumene and t-butyl hydroperoxides. After its cloning, it became apparent that it is related to the previously identified classical or cytosolic glutathione peroxidase [77]. Despite its structural and biochemical similarities with other glutathione peroxidases, GPx4 is special — hence it has attracted considerable interest in the past, and several knockout studies for GPx4 have been performed in mice. Since this review aims at summarizing very recent findings on GPx4, mainly obtained by in vivo studies, I refer the reader to previous comprehensive review articles, which thoroughly reviewed the biochemical traits and the molecular and cellular functions of GPx4 [78–81]. GPx4 is monomeric and, despite its smaller size, has a broad substrate specificity not only for its above-named substrates, but also with regard to its co-factor (see also below). Under physiological conditions glutathione (GSH), present in somatic cells up to 10 mM, is the favored electron source (Fig. 2). But under very low GSH concentrations, as physiologically occurring in developing sperm cells, GPx4 also accepts electrons from protein thiols. Initially it was believed that GPx4 consists of 7 exons, whereby exon 1 encodes either the mitochondrial or the cytosolic variant [82]. Exon 1 is separated from exons 2, 3, and 4 by a larger intron, and this cluster is again separated from exons 5 to 7 by another larger intron. Exon 3 encodes the Sec-encoding UGA codon and exon 7 the SECIS element. Purification of a sperm-nuclei-specific selenoprotein, Nterminal sequencing and in silico analyses with then rudimentary protein/nucleotide databases revealed the existence of a novel exon in the first intron of the GPx4 gene, which is expressed exclusively in testis [83] — apparently it was hidden for almost 20 years after the discovery of GPx4. This finding resulted to change the first conditional knockout strategy for GPx4 (unpublished), as the first strategy aimed to insert the one loxP site and the positive selection marker for homologous recombination in embryonic stem cells exactly where the alternative exon was found (see below). Immunogold studies provided initial evidence that GPx4 is also present in sperm nuclei, though it was not clear at that time whether the positive staining was due to the presence of cytosolic GPx4 or a possible nuclear variant [84]. Subsequent studies confirmed that the nuclear form must be expressed from its own promoter located just upstream from the alternative exon [85,86], whereas alternative transcription initiation at exon 1 determines if the longer (mitochondrial) form or the shorter (cytosolic) form is expressed. Moreover, expression of the three different GPx4 forms follows a peculiar pattern. Multiple studies showed that cytosolic GPx4 is expressed in most tissues including during embryogenesis to variable extent, whereas the nuclear form is almost exclusively found in testis [87]. S1 nuclease experiments reported already in 1995 that the mitochondrial isoform is the prevailing form in testis [88], which was confirmed by RT-PCR analyses [87]. Yet, there are controversial results pointing to

Fig. 2. Glutathione peroxidase 4 catalysis. (A) Like in other glutathione peroxidases the selenolate anion is most likely oxidized by a hydroperoxide to selenenic acid and subsequently reduced in its active state with two molecules of GSH, involving a selenadisulfide intermediate step. Normally, oxidized GSH (GSSG) is recycled by glutathione reductase at the expense of NADPH/H+ [125]. (B) At low GSH concentrations (e.g. in mature sperm cells) GPx4 converts into a protein thiol peroxidase and introduces disulfide bridges into proteins. If free thiols are no longer available, GPx4 may also become cross-linked via selenenylsulfide bridges (⁎) or disulfide bridges (cytosolic GPx4 harbors 8 cysteine residues in its peptide chain) to other proteins.

significant expression of the mitochondrial variant also during embryogenesis, postnatal brain and cardiac tissue [89]. The same group additionally showed that the guanine-rich sequence-binding factor 1 (Grsf1), which binds specifically to the 5′ untranslated region of mitochondrial GPx4 mRNA, is responsible for expression regulation on the post-transcriptional level, which appears to be important for embryonic brain development [90]. In the mean time GPx4 has been disrupted by four different laboratories independently, which certainly reflects the strongly growing interest in GPx4. Almost simultaneously, Yant et al. and Imai et al. disrupted GPx4 expression by removing either exons 2 to 7 [91] or the entire GPx4 gene [80], which both causes early embryonic death at E7.5. A third group generated constitutive GPx4 knockout mice and they detected reduced GPx4 activity in various tissues of GPx4+/− mice [92]. They found that lung fibroblasts with only one GPx4 allele are more sensitive towards hydrogen peroxide, cadmium chloride and cumene hydroperoxide, but somewhat surprisingly, they also found that heterozygous cells are equally resistant to phosphatidylcholine hydroperoxide as wild-type cells [92]. However, the strategy was designed as such that only a part of exon 1 (including mitochondrial and cytosolic ATGs), intron 1 and a small part of exon 2 is replaced by a lacZ/neo cassette. Thus, it still needs to be ruled out that minor amounts of truncated GPx4 products may be generated in

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these mice. Finally, ubiquitous, Cre-mediated disruption of a loxPflanked GPx4 allele causes early embryonic death just after gastrulation (see Section 4.3) [93]. Since the constitutive knockout approaches for GPx4 impedes to explore GPx4 functions in adult tissue in homozygous mutant mice, various studies have been conducted with heterozygous GPx4+/− mice and cells. For instance, hemizygous mice are more sensitive to γirradiation [91], but surprisingly also live slightly longer than wildtype mice due to altered pathologies, including delayed occurrence of fatal lymphoma and reduced severity of glomerulonephritis [94]. Compared to wild-type cells, hemizygous mouse embryonic fibroblasts (MEFs) were also shown to be more sensitive to t-butyl hydroperoxide and culturing at 20% oxygen [95]. Conversely, transgenic mouse models have been created, which express GPx4 either in the entire body or in specific tissues. For instance, mice expressing human GPx4 from a genomic clone are more resistant to diquat-induced liver damage [96], and GPx4 overexpression sustains mitochondrial ATP production in liver from diquatinduced oxidative stress [97]. In addition, cultured primary cortical neurons from these mice are more resistant to hydrogen peroxide, tbutyl hydroperoxide, and β-amyloid-induced cytotoxicity [98]. Hence, all these studies are confirmatory for the numerous cell culture studies, which indicated that GPx4 protects cells against various apoptotic triggers, such as pro-oxidants, DNA-damaging agents, glucose depletion, and irradiation (reviewed in [81]). In addition to this, isolated hearts from mice expressing rat mitochondrial GPx4 under the control of the CMV-chicken β-actin promoter were found to have better cardiac functions, including higher rates of contraction and relaxation, an increase in the developed pressure and peak systolic pressure following global ischemia/reperfusion [99]. 4.2. Distinctive functions of GPx4 variants in sperm maturation Another peculiarity of GPx4 was uncovered by Ursini et al. in 1999. In search of a specific selenoprotein, whose absence may account for the structural defects and male infertility induced by severe seleniumdeficiency in rodents, they came across with GPx4 [100]. It is known for more than four decades that selenium is essential for male fertility [101,102]. Spermatozoa from selenium-deprived animals were found to be immotile and to display major morphological lesions. These include giant heads, breaks in the neck, hairpins between the principal piece and the mid-piece in addition to irregularly-shaped mitochondria around the axoneme [103,104]. Ursini et al. impressively demonstrated that GPx4 is the major structural component – in this form it lacks glutathione peroxidase activity – of the mitochondrial capsule, which embeds sperm mitochondria and is thus required for structural sperm stability [100]. In its catalytically inactive form GPx4 is cross-linked to high molecular mass complexes with other capsular proteins [105]. The mechanism of inactivation is thought to be related to GSH depletion, occurring during germ cell maturation [106]. Mice expressing rat mitochondrial GPx4 exclusively in seminiferous epithelium, achieved by utilizing the mouse synaptonemal complex protein 1 promoter, display impaired fertility due to an increase in the number of apoptotic spermatocytes in transgenic animals [107]. Hence, it seems that tight regulation of mitochondrial GPx4 expression in testis is vital for proper sperm development. As the nuclear form of GPx4 (nGPx4) is primarily (or even almost exclusively) expressed in testicular tissue, mice with constitutive inactivation of nGPx4 were created [108]. To this end, the nuclear/ alternative exon was replaced by the eGFP gene. This strategy leads to disruption of the nuclear form without touching the expression of mitochondrial and cytosolic isoforms; it was hypothesized that mitochondrial and/or cytosolic GPx4 are essential for the survival of mice. In accordance with the expression pattern, nGPx4 null mice are born at the Mendelian ratio, ruling out any substantial contribution of nGPx4 to murine development. Quite surprisingly it turned out that

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male (and female) nGPx4 knockout mice are fully fertile, leaving the question unanswered why mammals express a selenoprotein specifically in testis which is apparently dispensable for fertility? Despite this rather disappointing result, various efforts were undertaken to address the possible role of GPx4 as a protein thiol peroxidase, which had been unraveled already by in vitro biochemical techniques [109]. In fact, the assessment of sperm chromatin condensation and labeling of free thiols with monobromobimane provided concluding data that (n)GPx4 works as protein thiol peroxidase also in vivo (Fig. 2B). Yet, it remains to be shown whether these thiol peroxidase functions may also happen in somatic cells or under pathophysiological conditions. 4.3. GPx4 is a key regulator of a novel oxidative stress-induced signaling pathway Due to early embryonic lethality of GPx4 null mice, a mouse model was developed with spatio-temporal inactivation of GPx4. This model allows to study GPx4 functions in adult tissue and to establish cells with inducible disruption of GPx4. To this end, the last three exons of GPx4 including the SECIS element were flanked by loxP sites [93]. This strategy aims at removing the last three exons by Cre recombinase, leading to a non-functional GPx4 due to premature translational STOP at the Sec codon on exon 3. Intercross of GPx4+/− mice never yielded viable GPx4−/− offspring. Embryonic analyses at various gestational days revealed that GPx4 null embryos die shortly after gastrulation (E7.5), which is in line with the above described systemic knockout approaches [80,91,92]. Therefore removal of the last three exons is actually sufficient to fully abrogate GPx4 function. Since it is almost impossible to study the molecular and cellular mechanisms of (embryonic) death in early embryos and adult tissues, a 4-OH-tamoxifen (Tam)-inducible GPx4 ex vivo knockout system from conditional GPx4 knockout mice was developed [93]. Moreover, this cellular system provides a suitable tool to address a putative superior role for GPx4 in redox-regulated cell death signaling pathways. To this end, mouse embryonic fibroblast (MEFs) cultures were isolated from GPx4lox/lox (lox = loxP) embryos and stably transfected with a plasmid encoding MERCreMER (mutated estrogen receptor). Tam treatment of cells causes a drop of GPx4 mRNA and GPx4 protein levels after 24 h and 48 h, respectively, followed by massive cell death in GPx4lox/lox [MERCreMER] cells, but not in control cells. The add-back of wild-type GPx4 is capable to fully rescue cell death induced by inducible deletion of endogenous GPx4. Interestingly, only the lipophilic antioxidant α-Tocopherol (α-Toc, vitamin E) prevents cell death induced by GPx4 disruption, while water-soluble antioxidants are ineffective. Since this cell death is sensitive to α-Toc administration, lipid peroxidation was regarded being a major trigger of cell death downstream of GPx4 deletion. In fact, lipid peroxidation, and not accumulation of soluble oxygen radicals, is a very early and critical event in GPx4 knockout cells. Needless to say, this is not really surprising since it fits very well with the initial discovery of GPx4 as a “lipid peroxidation-inhibiting protein” [76]. Yet, it had remained opaque how lipid peroxides are generated and accumulate in GPx4 knockout cells. Previously, GPx4, similar to other glutathione peroxidases, was described to regulate arachidonic acid metabolizing enzymes, including lipoxygenases (LOX) and cyclooxygenases (COX). For instance, GPx4 knockdown in a human carcinoma cell line leads to up-regulation of 12-lipoxygenase and cyclooxygenase 1 (COX1) [110], whereas overexpression of GPx4 impairs arachidonic acid metabolism and leukotriene secretion [111–113]. Furthermore, GPx4 controls 5-, 12-, and 15-lipoxygenase activities in vitro [114–116]. Despite these reports concerning the regulatory role for GPx4 towards arachidonic acid metabolizing enzymes, it was unclear whether GPx4 shows any specificity towards individual COX and LOX isoenzymes in vivo. Treatment of GPx4 knockout cells with increasing concentrations of arachidonic acid or linoleic acid accelerates cell death induced by GPx4 disruption, implying that at least one or several polyunsaturated fatty

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acid metabolizing enzyme(s) must be involved in the execution of cell death. By using an array of LOX- and COX-specific inhibitors it was finally possible to demonstrate that the 12/15-LOX-specific inhibitor PD146176 and not inhibitors against other LOX isoforms or cyclooxygenases fully antagonize lipid peroxidation-mediated cell death induced by GPx4 depletion [93]. Since GSH is supposed to be the major reductant for GPx4 in somatic cells, it was investigated whether experimental depletion of endogenous GSH – a condition frequently found in many degenerative diseases – may induce a phenotype similar to that induced by GPx4 disruption. GSH-deprivation causes massive cell death in wildtype cells, which can be inhibited again by α-Toc or PD146176 like in GPx4−/− cells. To corroborate that 12/15-LOX is downstream of GPx4 inactivation or GSH depletion for the execution of cell death, MEFs were isolated from 12/15-LOX−/− embryos [117]. 12/15-LOX−/− cells are indeed highly resistant to GSH depletion, demonstrating that the cell death progression downstream of GSH depletion or GPx4 inactivation requires functional 12/15-LOX. Apparently, cell death

progression does not involve the classical apoptotic cell death machinery, as shown by AnnexinV/propidium iodide staining, caspase activation, and thus cannot be prevented by forced Bcl-2 expression. It rather involves the activation of apoptosis inducing factor (AIF) as an alternative cell death pathway [118,119]. Finally, this novel oxidative stress-induced cell death pathway is not only active in fibroblasts but also acts in neurons as neuron-specific inactivation of GPx4 in the brain or in ex vivo cultured neurons causes massive neurodegeneration in vivo and ex vivo [93,120]. Aberrant cell death due to the accumulation of detrimental amounts of ROS is common to the etiology of many complex (agerelated) diseases, such as neurodegeneration, ischemia/reperfusioninduced tissue detriment, atherosclerosis and diabetes. Previously, it was believed that oxidative stress causes overall oxidation of essential biomolecules in cells and eventually cell death — in this event it would have been extremely difficult to combat oxidative stress-related diseases specifically. Hence, the identification of a distinct cell death signaling cascade, sensing oxidative stress via the

Fig. 3. GPx4 along with GSH sense and translate oxidative stress into a distinct cell death signaling pathway. GPx4 and GSH regulate an oxidative-stress-induced cell death pathway, which consists of (i) the release of arachidonic acid from membranes by phospolipase A2 (PLA2); (ii) the specific oxygenation of arachidonic acid (or linoleic acid) by 12/15-LOX, yielding for instance 15-HPETE (13-HPODE), which may further spark the lipid peroxidation chain reaction; (iii) the relocation (= activation) of apoptosis inducing factor (AIF) from mitochondria to the nucleus, and (iv) the AIF-mediated large-scale DNA fragmentation causing cell death. In the absence of GPx4 or at very low cellular GSH concentrations due to high accumulation of reactive oxygen species (ROS), which may occur under various acute or chronic disease conditions, increased 12/15-LOX activity triggers this cell death signaling pathway. At each single step this cascade can be interrupted either by the 12/15-LOX-specific inhibitor PD146176, α-Tocopherol (a lipid-soluble antioxidant belonging to vitamin E compounds) or RNAi-mediated depletion of AIF. It is not clear at present what the exact nature of lipid peroxide generated by 12/15-LOX is, nor is known how GPx4 interferes with the 12/15-LOX metabolic pathway (indicated by question marks).

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GSH/GPx4 system and translating it into a 12/15-lipoxygenasedependent lipid peroxide signal that finally activates AIF (Fig. 3), opens promising cues to systematically explore the benefit of redoxtargeted therapeutic interventions in the cure of degenerative conditions. Along this line, 12/15-LOX-deficient mice are highly resistant to experimental brain ischemia in a manner similar to wildtype mice pre-treated with a 12/15-LOX inhibitor [121,122]. On the other hand, the same knockout mice develop myeloproliferative disease due to increased activation of the phosphatidylinositol 3kinase and Akt pathways, subsequent up-regulation of Bcl-2 and prolonged survival of leukemic cells [123]. Thus, 12/15-LOX is a double-edged sword: it is required for maintenance of tissue homeostasis, however, when accidentally activated it is deleterious and causes profound cell and tissue damage. Conclusively, GPx4 has emerged not only as one of the most important selenoproteins, but also as a key glutathione peroxidase and a major player involved in cellular redox control. 5. Perspectives Without doubt, knockout mouse models for Trsp and in particular for individual selenoproteins are powerful tools not only to unmask their multifaceted roles in embryo and tissue development, but in combination with (genetic) stress models to decipher their functional relevance in disease development, which ultimately leads to a better understanding of the often reported beneficial effects of selenium. Equally important, (inducible) knockout models for individual selenoproteins represent a perfect reservoir for the development of ex vivo cell culture systems in order to unravel the cellular and biochemical mechanism of fundamental cellular processes, like programmed cell death and cell cycle progression. Acknowledgments The author would like to thank Pankaj Kumar Mandal for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (CO 291/2-1) and the DFG-Priority Programme SPP1190. References [1] E.A. Veal, A.M. Day, B.A. Morgan, Hydrogen peroxide sensing and signaling, Mol. Cell 26 (2007) 1–14. [2] B. D'Autreaux, M.B. Toledano, ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis, Nat. Rev., Mol. Cell Biol. 8 (2007) 813–824. [3] M.T. Lin, M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, Nature 443 (2006) 787–795. [4] S.G. Rhee, Cell signaling. H2O2, a necessary evil for cell signaling, Science 312 (2006) 1882–1883. [5] G.V. Kryukov, S. Castellano, S.V. Novoselov, A.V. Lobanov, O. Zehtab, R. Guigo, V.N. Gladyshev, Characterization of mammalian selenoproteomes, Science 300 (2003) 1439–1443. [6] M.R. Bosl, K. Takaku, M. Oshima, S. Nishimura, M.M. Taketo, Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp), Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 5531–5534. [7] E. Kumaraswamy, B.A. Carlson, F. Morgan, K. Miyoshi, G.W. Robinson, D. Su, S. Wang, E. Southon, L. Tessarollo, B.J. Lee, V.N. Gladyshev, L. Hennighausen, D.L. Hatfield, Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium, Mol. Cell. Biol. 23 (2003) 1477–1488. [8] Z.Z. Shi, J. Osei-Frimpong, G. Kala, S.V. Kala, R.J. Barrios, G.M. Habib, D.J. Lukin, C.M. Danney, M.M. Matzuk, M.W. Lieberman, Glutathione synthesis is essential for mouse development but not for cell growth in culture, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 5101–5106. [9] W.H. Cheng, Y.S. Ho, D.A. Ross, B.A. Valentine, G.F. Combs, X.G. Lei, Cellular glutathione peroxidase knockout mice express normal levels of seleniumdependent plasma and phospholipid hydroperoxide glutathione peroxidases in various tissues, J. Nutr. 127 (1997) 1445–1450. [10] R.S. Esworthy, R. Aranda, M.G. Martin, J.H. Doroshow, S.W. Binder, F.F. Chu, Mice with combined disruption of Gpx1 and Gpx2 genes have colitis, Am. J. Physiol., Gastrointest. Liver Physiol. 281 (2001) G848–855. [11] K.E. Hill, J. Whitin, A.K. Motley, L. Austin, R.F. Burk, H. Cohen, Gpx3 deletion affects gluttahione metabolism, Abstracts of 8th International Symposium on Selenium in Biology and Medicine, Wisconsin, Madison, USA, 2006.

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