1、ARTICLESInactivation of the ferroptosis regulator Gpx4 triggersacute renal failure in miceJose Pedro Friedmann Angeli1,Manuela Schneider2,Bettina Proneth1,Yulia Y.Tyurina3,Vladimir A.Tyurin3,Victoria J.Hammond4,Nadja Herbach5,Michaela Aichler6,Axel Walch6,Elke Eggenhofer7,Devaraj Basavarajappa8,Olof
2、 Rdmark8,Sho Kobayashi1,9,Tobias Seibt1,Heike Beck10,Frauke Neff6,Irene Esposito11,Rdiger Wanke5,Heidi Frster1,Olena Yefremova1,Marc Heinrichmeyer1,Georg W.Bornkamm12,Edward K.Geissler7,Stephen B.Thomas13,Brent R.Stockwell13,Valerie B.ODonnell4,Valerian E.Kagan3,Joel A.Schick1and Marcus Conrad1,14Fe
3、rroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types,and in engineered cellsoverexpressing oncogenic RAS.Yet,its relevance in non-transformed cells and tissues is unexplored and remains enigmatic.Here,we provide direct genetic evidence that the knockout
4、 of glutathione peroxidase 4(Gpx4)causes cell death in a pathologicallyrelevant form of ferroptosis.Using inducible Gpx4/mice,we elucidate an essential role for the glutathione/Gpx4 axis inpreventing lipid-oxidation-induced acute renal failure and associated death.We furthermore systematically evalu
5、ated a library ofsmall molecules for possible ferroptosis inhibitors,leading to the discovery of a potent spiroquinoxalinamine derivative calledLiproxstatin-1,which is able to suppress ferroptosis in cells,in Gpx4/mice,and in a pre-clinical model ofischaemia/reperfusion-induced hepatic damage.In sum
6、,we demonstrate that ferroptosis is a pervasive and dynamic form of celldeath,which,when impeded,promises substantial cytoprotection.Previously it was assumed that apoptosis was the only regulated formof cell death.Yet,recent years have shown that non-apoptotic celldeath pathways are highly heteroge
7、neous processes characterized bymorphologically and biochemically distinct events1.Non-apoptoticcell death pathways have gained special attention particularly dueto their role in inflammation and as potential therapeutic target inapoptosis-resistant tumours2,3.In this context,at least two cell death
8、pathways are executed in response to detrimental concentrations ofpartially reduced forms of oxygen(that is,reactive oxygen species4).They include regulated necrosis(that is,necroptosis)5,and an iron-dependent form of cell death,named ferroptosis6.Ferroptosis is anewly discovered cell death pathway
9、that can be induced in a subsetof tumour cell types,including cell lines engineered to overexpressoncogenic RAS,by a number of small molecules named ferroptosis-inducing agents7(FINs).It is characterized by increased levels of lipidhydroperoxidesandironoverload,leadingtocaspase-andnecrosome-independ
10、ent cell death.FINs have been classified into class I FINsthat involve cellular glutathione(GSH)depletion and class II FINsthat lack this characteristic.Recently,it has been shown that class IIFINstriggerferroptosisthroughinhibitionofglutathioneperoxidase4(Gpx4;ref.8).So far,exploitation of ferropto
11、sis has been proposed for killingtumour cells in response to specific compounds,and as a potentialmechanism involved in oxidative glutamate toxicity in cells and brain1Helmholtz Zentrum Mnchen,Institute of Developmental Genetics,Ingolstdter Landstr.1,85764 Neuherberg,Germany.2Institute for Stroke an
12、d DementiaResearch,Klinikum der Universitt Mnchen,Ludwig-Maximilians-University Munich,Marchioninistr.15,81377 Munich,Germany.3Department of Environmentaland Occupational Health,University of Pittsburgh,Pittsburgh,Pennsylvania 15219,USA.4Department of Infection,Immunity and Biochemistry,School of Me
13、dicine,Cardiff University,Heath Park,Cardiff CF14 4XN,UK.5Institute of Veterinary Pathology,Center for Clinical Veterinary Medicine,Ludwig-Maximilians-UniversityMunich,Veterinaerstr.13,80539 Munich,Germany.6Institute of Pathology,Helmholtz Zentrum Mnchen,Ingolstdter Landstr.1,85764 Neuherberg,German
14、y.7Department of Surgery,University Medical Center of Regensburg,Franz-Josef-Strauss Allee 11,93053 Regensburg,Germany.8Department of Medical Biochemistryand Biophysics,Division of Physiological Chemistry II,Karolinska Institutet,S-17177 Stockholm,Sweden.9Department of Food and Applied Life Sciences
15、,Faculty ofAgriculture,Yamagata University,Tsuruoka,Yamagata 997-8555,Japan.10Walter Brendel Centre of Experimental Medicine,Munich Heart Alliance,Ludwig-Maximilians-University,Marchioninistr.15,81377 Munich,Germany.11Institute of Pathology,Technische Universitt Mnchen,Ismaningerstr.22,81675Munich,G
16、ermany.12Institute of Clinical Molecular Biology and Tumor Genetics,Helmholtz Zentrum Mnchen,Marchioninistr.25,81377 Munich,Germany.13Department of Biological Sciences and Department of Chemistry,Howard Hughes Medical Institute,Columbia University,550 West 120th Street,Northwest CornerBuilding,MC 48
17、46,New York,New York 10027,USA.14Correspondence should be addressed to M.C.(e-mail:marcus.conradhelmholtz-muenchen.de)Received 13 August 2014;accepted 14 October 2014;published online 17 November 2014;DOI:10.1038/ncb3064NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION1 2014 Macmillan Publishers Limite
18、d.All rights reserved.ARTICLESslices9,10.We report here direct in vivo evidence that this type of celldeath is not only limited to specific tumours treated with FIN agents,butthatGpx4preventsprematuredeathofmicebyactivelyrestrainingthe ferroptotic machinery in kidney tubular cells.In light of the ye
19、t-uncharacterized role of lipid hydroperoxides in ferroptosis,we nowprovide a detailed picture of a complex lipid oxidation signature inresponsetoGpx4inactivationandferroptosisinduction.Furthermore,we identified an in vivo ferroptosis inhibitor shown to be efficaciousin inducible Gpx4/cells and mice
20、,as well as tissues subjected toischaemia/reperfusion.This class of small-molecule inhibitors willnot only serve to define the involvement of ferroptotic cell death inpathological conditions,but will also facilitate characterization of theunderlying molecular paradigms that regulate ferroptosis.RESU
21、LTSInducible disruption of Gpx4 causes acute renal failure andearly death in an Alox15-independent mannerWe previously showed that the inducible disruption of Gpx4 in mouseembryonic fibroblast(MEF)cells(Pfa1 cells)leads to a form of celldeath with necrotic-like features that involves 12/15-lipoxygen
22、ase(Alox15)and apoptosis inducing factor(AIF)activation11.Accord-ingly,Alox15/cells were found to be resistant to the effects of Gpx4inhibition through depletion of GSH by L-buthionine sulphoximine11(BSO;Fig.1a).To extend this initial analysis,inducible Gpx4/cellswere treated with a series of inhibi
23、tors including MJ33,a competi-tive and reversible inhibitor of calcium-independent phospholipaseA2(aiPLA2),the lipoxygenase inhibitor zileuton,MK886(Alox5),PD146176(Alox15)and the dual inhibitor BWA4C(Alox5 and 15),which all prevented cell death in response to Gpx4 deletion(Fig.1b).In light of these
24、 and our previous data,we sought to challenge thein vivo relevance of the Gpx4Alox15 axis in this cell death pathway.We generated and characterized mouse models based on mice withloxP-flanked Gpx4 alleles,along with mice proficient or deficient inAlox15(ref.12).After23breedingstepsCreERT2;Gpx4fl/fl/
25、Alox15+/+and CreERT2;Gpx4fl/fl/Alox15/mice were generated,which did notshow any abnormalities in the absence of tamoxifen(TAM).OnTAM feeding,the overall behaviour of CreERT2;Gpx4fl/fl/Alox15+/+changed markedly,as indicated by catatonia,hunched postureand loss of body weight,whereupon mice had to be
26、euthanized.Unexpectedly,CreERT2;Gpx4fl/fl/Alox15/revealed exactly the samebehaviour and had to be euthanized at the same stage as inducedCreERT2;Gpx4fl/fl/Alox15+/+mice(Fig.1c).To define the role of Alox15 in cell death,cellular systems wereestablished from CreERT2;Gpx4fl/fl/Alox15/mice(PZL cells)an
27、dfrom CreERT2;Gpx4fl/fl/Alox15+/+mice as control(PZ cells).WhenGpx4 deficiency was induced by TAM(Supplementary Fig.1A),PZLcells died like PZ cells(Supplementary Fig.1b),which could notbe rescued by N-acetylcysteine(NAC)but by-tocopherol(Toc)as previously reported11(Supplementary Fig.1b).As 5-lipoxy
28、genase(Alox5)might compensate for loss of Alox15(ref.13),Alox5 wasdepleted by short hairpin RNA(shRNA)in PZL and PZ cells(Supplementary Fig.1c,d).Whereas the knockdown of Alox5 in PZLcells partially delayed the cell death process,it did not impact on celldeath in PZ cells.From these findings we conc
29、lude that no individuallipoxygenase could be identified that is solely responsible for the celldeath processes downstream of Gpx4 inactivation.Inferring that more than one lipid oxygenase is involved in celldeath,we endeavoured to characterize in more detail why animalsdie on Gpx4 disruption.On diss
30、ection,grossly,slightly enlarged andpale kidneys were evident in the Gpx4/mice(Fig.1d).In rare cases(5%),signs of focal liver necrosis were evident.Analysis of Gpx4expression showed a marked decrease of Gpx4 in the kidney alreadyseven days after TAM treatment(Fig.1e),which was paralleled byprogressi
31、ve,unselective proteinuria(Fig.1f)and altered biochemicalkidney parameters(Supplementary Table 1).Immunohistochemistry demonstrated a strong Gpx4 staining inthe proximal tubules of wild-type kidney that was lost on TAMadministration(Fig.1g).Histological and ultrastructural analysis aswell as termina
32、l deoxynucleotidyl transferase(TdT)-mediated dUTPnick end labelling(TUNEL)revealed widespread cell death of prox-imal tubular epithelia with progression to effacement of all tubularepithelia and of the parietal epithelial cells of the Bowmans capsule(Fig.1h,i and Supplementary Fig.1f).Proteinaceous
33、casts and cellulardebris were observed in affected tubuli(Fig.1h),and focal signs oftubular regeneration were evident(Fig.1i).Mononuclear interstitialinfiltrationandedemawereobservedinadvancedlesions(Fig.1handSupplementary Fig.1g).Notably,CreERT2;Gpx4fl/fl/Alox15/micedevelopedcomparablelesionstothos
34、eofCreERT2;Gpx4fl/fl/Alox15+/+mice(Supplementary Fig.1e).Immunohistochemical staining againstcleaved caspase-3 revealed faint staining,mainly confined to the lu-menofrenaltubules,possiblyresultingfromsecondaryinflammatoryprocesses(Supplementary Fig.1g).Inducible Gpx4 disruption induces Nec1-sensitiv
35、e ferroptoticcell deathTo define the modality of cell death,we used inducible Gpx4/cells and chemical inducers for the subsequent studies.Using thepan-caspase inhibitor z-VAD-FMK(zvad)and determining caspaseactivation,we ruled out an involvement of caspases in cell deathinduced by Gpx4 loss and FINs
36、,in contrast to parental cellstreated with staurosporine or tumour necrosis factor (TNF)(Supplementary Fig.2a-c).This indicates that a non-apoptotic formof cell death is triggered in Gpx4/cells.Therefore,we next addressed whether necroptosis or ferroptosis isactivatedonGpx4inactivation.Indeed,therec
37、eptorinteractingkinase1(Rip1)inhibitor necrostatin-1(Nec1)and ferrostatin-1(Fer1),thefirst described inhibitors of necroptosis and ferroptosis,respectively,rescued Gpx4/cells from cell death(Fig.2a).As ferroptotic celldeath entails cellular iron accumulation and lipid peroxidation6,celldeathcouldals
38、obeinhibitedbytheironchelatordeferoxamine(DFO)and by Toc(Fig.2a;ref.11).Likewise,depletion of the Gpx4 substrate GSH through BSO orerastin(Fig.1a and Supplementary Fig.2d)efficiently triggered rapidcell death(Supplementary Fig.2e)that could be prevented by thesame inhibitors(Fig.2b and Supplementary
39、 Fig.2f).Furthermore,cell death induced by(1S,3R)-RSL3(hereafter RSL3),the firstdescribed Gpx4 inhibitor8,could also be prevented by Fer1 andNec1(Fig.2b),indicating that availability of cellular GSH andproper Gpx4 function tightly control ferroptosis.Consistently,allcompounds failed to induce caspas
40、e activation(SupplementaryFig.2c),whereas Gpx4 overexpression conferred resistance to FINs(Supplementary Fig.2g,h;ref.14).2NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 2014 Macmillan Publishers Limited.All rights reserved.ARTICLESZileutonMK866PD146176MJ33BWA4CdeMr(K)A2501501001575503725f+/fl,Crefl
41、/fl,CreGpx4-tubulin0Mr(K)7 13 14DaysGpx4-tubulinControlKnockoutgCreERT2;Gpx4+/flhH&EH&EiPH-3TUNELLipid peroxidationCys-SHGSHGCSGpx4GSSGSystem xCErastinBSORSL3aCreERT2;Gpx4fl/flCreERT2;Gpx4+/flCreERT2;Gpx4fl/flGpx4Gpx4CreERT2;Gpx4+/flCreERT2;Gpx4fl/fl5 mmKOWTWT/Alox15/WT/Alox15+/+KO/Alox15+/+KO/Alox1
42、5/cPercentage of cell viability0306090120(M)b010203040Percentage of survival0204060100080520251510Days after tamoxifen treatment19195555Cys-SS-CysFigure 1 Inducible Gpx4 disruption causes ARF and death in mice.(a)Ascheme showing the most important steps of glutathione(GSH)biosynthesis.Toc,-tocophero
43、l;BSO,L-buthionine sulphoximine;GSSG,oxidizedglutathione;GCS,-glutamylcysteine-synthase.(b)Inhibitorsagainstenzymes of arachidonic acid metabolism prevent Gpx4-deletion-induced celldeath in a dose-dependent manner.Gpx4 was disrupted in Pfa1 cells bythe addition of 1M TAM in the presence of increasin
44、g concentrations ofinhibitors.Cell viability was assessed by using AquaBluer 72h after knockoutinduction.Data shown represent the mean s.d.of n=4 wells of a 96-wellplate from a representative experiment performed independently four times.(c)Mouse survival after TAM feeding.All induced Gpx4/(KO)mice
45、diedafter approximately 2 weeks of TAM feeding regardless of Alox15 expression.Noneofthecontrolmice(CreERT2;Gpx4+/fl/Alox15+/+(WT/Alox15+/+),CreERT2;Gpx4+/fl/Alox15/(WT/Alox15/)died in the period investigated.Data are percentage of live animals;mean survival of Gpx4-null miceis13.5daysaftertheonseto
46、fTAMfeeding(n=8animalsforKO/Alox15/and WT/Alox15/and n=19 animals for KO/Alox15+/+and WT/Alox15+/+).GehanBreslowWilcoxon test:P 25CYP450 inhibition2C9 IC50(M)252D6 IC50(M)=4.13A4 IC50(M)251A IC50(M)252C19 IC50(M)25Mouse PK(i.v.)1mgkg1t1/2(h)4.6V(ml)849C(gml1)0.4Total Cl(mlh1)128Total Cl(mlmin1kg1)66
47、MRTINF(h)4.9Vss(ml)629Mouse PK(p.o.)5mgkg1Cmax(gml1)0.08Tmax(h)2Bioavailability(%)52An initial ADME-Tox profile for Liproxstatin-1 is provided,including cytotoxicity inHepG2 cells and inhibition of hERG and cytochrome P450(CYP450)enzymes,as wellas basic pharmacokinetic parameters.hERG,ether-go-go-re
48、lated gene;ADME-Tox,absorption,distribution,metabolism and excretiontoxicity;PK,pharmacokinetics;Cl,clearance;MRTINF,mean residence time extrapolated to infinity.RSL3-induced cell death(Fig.7a).Similar findings were obtained inthe immortalized human renal proximal tubule epithelial cell line,HK-2(Su
49、pplementary Fig.7b).Next,we knocked down Gpx4 inHK-2 cells using a pool of siRNAs,revealing a small yet significantdecrease in cell viability sensitive to Toc treatment(SupplementaryFig.7c).Inducing cell death through Gpx4 knockdown,however,turned out to be challenging for the high expression levels
50、 of Gpx4 inkidney tubular epithelial cells(Supplementary Fig.7d).Nonetheless,the Gpx4 knockdown rendered cells more sensitive to ferroptosis-inducing agents(Supplementary Fig.7e),indicating a Gpx4-regulatedferroptotic machinery in human proximal tubular epithelial cells.Moreover,RSL3-induced BODIPY