Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Carlson, Bradley A., Ryuta Tobe, Elena Yefremova, Petra A. Tsuji, Victoria J. Hoffmann, Ulrich Schweizer, Vadim N. Gladyshev, Dolph L. Hatfield, and Marcus Conrad. 2016. “Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration.” Redox Biology 9 (1): 22-31. doi:10.1016/j.redox.2016.05.003. http:// dx.doi.org/10.1016/j.redox.2016.05.003. Published Version doi:10.1016/j.redox.2016.05.003 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:27662198 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Redox Biology 9 (2016) 22–31 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research paper Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration Bradley A. Carlson a, Ryuta Tobe a, Elena Yefremova b, Petra A. Tsuji c, Victoria J. Hoffmann d, Ulrich Schweizer e, Vadim N. Gladyshev f, Dolph L. Hatfield a,n, Marcus Conrad b,nn a Molecular Biology of Selenium Section, Mouse Cancer Genetics Program, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA b Helmholtz Zentrum München, Institute for Developmental Genetics, Neuherberg, Germany c Department of Biological Sciences, Towson University, Towson, MD, USA d Office of the Director Diagnostic and Research Services Branch, National Institutes of Health, Bethesda, MD, USA e Institut für Biochemie und Molekularbiologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany f Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA article info abstract Article history: The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, Received 17 May 2016 which protects cells against detrimental lipid peroxidation and governs a novel form of regulated ne- Accepted 18 May 2016 crotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important Available online 26 May 2016 selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion Keywords: of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA À/À Selenoprotein (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4 mice died shortly after birth and À À Ferroptosis presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4 / livers mani- Lipid peroxidation fested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4À/À pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was re- versible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice 4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4À/À mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study de- monstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver func- tion, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid perox- idation. Published by Elsevier B.V. 1. Introduction eight glutathione peroxidases due to its broad substrate specificity, the fact that it functions as a phospholipid hydroperoxidase pro- Glutathione peroxidase 4 (Gpx4) is unique among a family of tecting cells from lipid peroxidation and serves a structural func- tion in mature sperm cells [1]. Moreover, Gpx4 was recently de- scribed to control a novel, iron-dependent form of regulated non- apoptotic cell death, ferroptosis [2], which is characterized by Keywords: Gpx4, glutathione peroxidase 4; Txnrd1, cytosolic thioredoxin re- ductase; Sec, selenocysteine; Trsp, Sec tRNA gene; Nrf2, nuclear factor (erythroid- generation of high levels of lipid hydroperoxides [3,4]. Ferroptotic derived)-like 2; Alb-Cre, Albumin-Cre; H&E, hematoxylin and eosin; qPCR, Quan- cell death was found not only to be relevant for cell death of titative PCR; GSH, glutathione; SEM, standard error of the mean; Gpx1, glutathione malignant cells, but emerged to play an important role in cells and peroxidase 1; SelR, Selenoprotein R; SepW, Selenoprotein W; Gsta1, glutathione tissue demise in kidney tubule cells [5], viral immunity [6], and S-transferase alpha 1; Srxn1, sulfiredoxin 1; Cbr3, carbonyl reductase 3; Gclc, glutamate-cysteine ligase; Gsr, glutathione reductase; TBARS, thiobarbituric acid motor neuron degeneration [7]. Among the proteins encoded by reactive substances 25 selenoprotein genes in mammals, Gpx4 enzyme appears to be nn Corresponding author. particularly critical for murine survival in that its knockout in mice n Correspondence to: Molecular Biology of Selenium Section, Mouse Cancer Ge- is early embryonic lethal [8], and it can only be partially knocked netics Program, National Cancer Institute, National Institutes of Health, 37 Convent down in cultured cells [9,10]. Furthermore, its targeted removal in Dr, Building 37, Room 5016, Bethesda, MD 20892, USA. E-mail addresses: hatfi[email protected] (D.L. Hatfield), neural and skin tissues virtually mimics the knockout of the gene [email protected] (M. Conrad). encoding the selenocysteine (Sec)-specific tRNA, Trsp (see [11,12], http://dx.doi.org/10.1016/j.redox.2016.05.003 2213-2317/Published by Elsevier B.V. B.A. Carlson et al. / Redox Biology 9 (2016) 22–31 23 respectively). manner for further analysis. Tissues used for pathological analysis A remarkable interrelationship exists between Gpx4 and vita- were immediately placed in 10% neutral-buffered formalin and min E, which is a lipid soluble antioxidant responsible for pro- paraffin embedded. Tissue sections were routinely stained with tecting membranes from oxidative damage [5,13]. Inducible in- hematoxylin and eosin (H&E) and examined by light microscopy activation of Gpx4 in cells has been shown to result in lipid per- by a board certified veterinary pathologist. oxidation-mediated cell death, which could be prevented by vi- tamin E supplementation [10]. In addition, this relationship was 2.4. RNA isolation and qPCR analysis also demonstrated by disrupting Gpx4 in mouse endothelium, which had no apparent effect on normal vascular homeostasis, Total RNA was isolated from tissues or cells using TriPure iso- unless the mice were restricted from dietary vitamin E [13]. Vi- lation reagent following the manufacturer's instructions and re- tamin E removal from the diets of these animals resulted in en- verse transcribed using an iScript cDNA synthesis kit. Quantitative dothelial cell death and subsequent thrombus formation in mul- PCR (qPCR) was performed in triplicate using iTaq Universal SYBR tiple organs and consequently early death of mice. Green Supermix according to the manufacturer's instructions. Knockout of either Trsp (TrspÀ/À) [14,15] or Txnrd1 (Txnrd1À/À) Results were normalized to mouse Gapdh. Primers sequences used [16] was found to be embryonic lethal; however, the targeted for analyses are shown in Supplementary Table 3. knockout of either gene in liver was not embryonic lethal [17–19]. The incidence of hepatocarcinogenesis in hepatocyte-specific 2.5. Protein isolation and western blotting conditional knockout mice carrying the Albumin-Cre (Alb-Cre) transgene (genotype Alb-Cre; Txnrd1fl/fl) was dramatically in- Tissues were washed twice with DPBS and then harvested in creased compared to control mice expressing normal levels of ice cold lysis buffer (50 mM Tris; pH 7.5, 150 mM NaCl, 1 mM Txnrd1 (genotype Alb-Cre; Txnrd1þ/ þ), suggesting that this major EDTA, 0.1% Igepal and protease inhibitors). Western blot analysis redox regulator selenoprotein has a role in protecting hepatocytes was carried out as described in Supplementary Information. from cancer [18]. Since mice carrying hepatocyte-specific knockout of Trsp sur- 2.6. Thioredoxin reductase activity and lipid peroxidation assays and vived for several months and their death appeared to be due to quantification of GSH and GSSG severe hepatocellular degeneration and necrosis [17], while mice carrying hepatocyte-specific knockout of Txnrd1 exhibited no ap- Txnrd1 activity was determined spectrophotometrically based parent phenotype [18], we interrogated whether the ferroptosis on the method of Holmgren [21] and lipid peroxidation assessed regulator Gpx4 could be the critical selenoprotein responsible for as described in Supplementary Information. Total glutathione the effect observed in the Trsp knockout mice. To this end, we (GSH) levels in liver were quantified using a glutathione assay kit generated a series of mice with hepatocyte-specific deletion of according to the manufacturer's instructions (Sigma-Aldrich). Gpx4 alone or in combination with Trsp, and as controls hepato- cyte-specific knockout of Txnrd1 in combination with Trsp. Find- 2.7.