IdentificationofaNovelTransport-independentFunctionofPiT1/SLC20A1intheRegulationofTNF-inducedApoptosis*Receivedforpublication,April6,2010,andinrevisedform,July30,2010Published,JBCPapersinPress,September3,2010,DOI10.1074/jbc.M110.130989
ChristineSalau¨n‡§1,ChristineLeroy‡§,AliceRousseau‡§,Vale´rieBoitez‡§,LaurentBeck‡§,andGe´rardFriedlander‡§Fromthe‡GrowthandSignalingResearchCenter,INSERMU845,F-75015Paris,Franceandthe§Faculte´deMe´decine,Universite´ParisDescartes,F-75015Paris,France
PiT1/SLC20A1isasodium-dependentPitransporterex-pressedbymostmammaliancells.Interestingly,PiT1transcrip-tionhasbeenshowntobeup-regulatedbythetumornecrosisfactor␣(TNF),andwehavenowinvestigatedthepossibleinvolvementofPiT1inTNF-inducedapoptosis.WeshowthatPiT1-depletedcellsaremoresensitivetotheproapoptoticactiv-ityofTNF(i.e.whentheantiapoptoticNFBpathwayisinacti-vated).TheseobservationsweremadeinthehumanHeLacan-cercelllineeithertransientlyorstablydepletedinPiT1byRNAinterferenceandinimmortalizedmouseembryonicfibroblastsisolatedfromPiT1knock-outembryos.DepletionofthecloselyrelatedfamilymemberPiT2hadnoeffectonTNF-inducedapo-ptosis,showingthatthiseffectwasspecifictoPiT1.TheincreasedsensitivityofPiT1-depletedcellswasevidentregard-lessofthepresenceorabsenceofextracellularPi,suggestingthatadefectinPiuptakewasnotinvolvedintheobservedphe-notype.Importantly,weshowthatthere-expressionofaP/؊iuptakemutantofPiT1inPiT1؊mouseembryonicfibroblastsdelaysapoptosisasefficientlyastheWTprotein,showingthatthisfunctionofPiT1isunrelatedtoitstransportactivity.Caspase-8ismoreactivatedinPiT1-depletedcells,andourdatarevealthatthesustainedactivationoftheMAPKJNKisup-reg-ulatedinresponsetoTNF.JNKactivityisactuallyinvolvedinPiT1-depletedcelldeathbecausespecificJNKinhibitorsdelayapoptosis.
PiT1/SLC20A1andPiT2/SLC20A2aretheuniquemembersofthemammalianPitransporterfamilySLC20A(1).Bothwerefirstidentifiedasretrovirusreceptors(2,3)beforebeingrecog-nizedassodium-dependentPitransporters(PiTs)2(4).Theyalsobelongtothelargefamilyoftransporters2A.20(TransportClassificationDatabase(5)),whichcomprisessymportersthatuseeitheraprotonorsodiumgradienttoimportPiandarefoundthroughoutallkingdomsoflife.ThebroadmRNAdis-tributionofPiT1andPiT2inmammaliantissuesandorganshasledtotheproposalthatPiT1andPiT2couldserveahouse-
*ThisstudywassupportedbygrantsfromINSERM,theUniversite´ParisDes-cartes,andtheAssociationLaboratoiresdeRecherchesPhysiologiques.1Towhomcorrespondenceshouldbeaddressed:CentredeRechercheINSERMU845,Universite´ParisDescartes,Faculte´deMe´decine,156RuedeVaugirard,75015Paris,France.Tel.:33-140615492;Fax:33-143060443;E-mail:christine.salaun@inserm.fr.2Theabbreviationsusedare:PiT,inorganicphosphatetransporter;NFB,nuclearfactorB;TNFR,TNFreceptor;TNFϩC,TNFpluscycloheximide;MEF,mouseembryonicfibroblast;AICAR,5-amino-4-imidazolecarboxam-ideriboside;AMPK,AMP-activatedproteinkinase;PARP,poly(ADP-ribose)polymerase;JNKi,JNKinhibitor.
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keepingroleinPihomeostasis(4,6)andprovidecellswiththeirbasicPineeds.However,theknock-outofPiT1inmice(bytwodifferentgroups,includingours(7,8))revealedanunexpectedphenotype.HomozygousdeletionofPiT1resultedinembry-oniclethalityatembryonicday12.5(7).FurtherinvestigationsshowedthatPiT1isanessentialgeneforliverdevelopment(7).PiT1-deficientembryosdisplayedhypoplasticfetalliversandsubsequentreducedhematopoiesis,resultinginsevereanemiaanddeath.Embryonicday12.5livercellswerereducedinnum-berandshowedsignsofmassiveapoptosis(7).Inanotherstudypublishedrecently,wehavealsorevealedtheinvolvementofPiT1intheregulationofcancercellproliferation(9).InvitrodepletionofPiT1inHeLaorHepG2cellsimpairstheirprolif-eration.Importantly,weshowedthatthispropertywasnotsharedwithPiT2,whosedepletionhadnoeffectonprolifera-tion.Finally,weprovideddirectevidencethatthemodulationofcellproliferationbyPiT1isindependentofitstransportfunctionbecausetheproliferationofPiT1-depletedcellscouldberescuedbynon-transportingPiT1mutants(9).
Althoughnotformallydemonstrated,severalstudieshavesuggestedthatPiT1expressioncouldberegulatedbytheinducedorbasalactivityofthetranscriptionfactorNFB(nuclearfactorB)(10–12).TheNFBpathwayisawelldescribedantiapoptoticpathwaythatisinducedbyvariouscytokinesorchemicals,suchastumornecrosisfactor␣(TNF),interleukin-1␣,orphorbol12-myristate13-acetateforexam-ple.Interestingly,theseagentsalsoincreasetheexpressionofPiT1indiversecelltypes(10,12).Theup-regulationofPiT1expressioninducedbytheNFBpathwaypromptedustoinvestigatewhetherPiT1couldbeinvolvedinprovidingsomeprotectionagainstcellapoptosis.WechosetoinvestigatetheTNF-inducedapoptosismodelbecauseofthephysiologicalimportanceofTNF(13,14)andbecauseTNFup-regulatesPiT1mRNA(12).
TNFisapleiotropicproinflammatorycytokinethathasimportantrolesindiversecellularevents,suchascellprolifer-ation,differentiation,andapoptosis(14).TNFcanbindtotwodifferentreceptors(TNFRs),TNFR1andTNFR2.TNFR1isbroadlyexpressedinmosttissues,whereastheexpressionofTNFR2ishighlyregulatedandistypicallyfoundincellsoftheimmunesystem.TNFbindingtoTNFR1promotesitscluster-ingandtheformationofseveralsequentialintracellularcom-plexes(15,16).Thefirstcomplex(termedcomplexI)(15)isproposedtobemainlyinvolvedinsignalingpathways,inducingtheactivationofseveralkinases,suchasIBkinase,JNK,p38,ERK,andothers.EarlysignalingthroughJNK(17,18)andespe-ciallythroughIBkinaseandtheNFBpathway(14,19,20)
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constitutesthemainantiapoptoticsignalstriggeredinresponsetoTNF.Subsequently,complexIdissociatesfromthemem-brane-boundTNFR1andrelocatestothecytoplasmtoformseveraldistinctproapoptoticcomplexes,allofthemcontainingcaspase-8astheinitiatorcaspase(15,16).Apoptosisoccur-renceismainlyregulatedbytheinterplaybetweentheprosur-vivalNFBpathwaymentionedaboveandtheproapoptoticsustainedphaseofJNKactivation(21–24).NFBinducesthesynthesisofimportantantiapoptoticproteinsregulatingcaspase-8activationandalsolimitsthedurationofJNKactivityviaseveralmechanisms(21,25–28).JNKisastress-activatedMAPK,andtherearethreemammalianJNKgeneswithsplicingvariantsp46andp54(29).JNK1and-2areubiquitouslyex-pressed,whereasJNK3isrestrictedtospecifictissues.BothJNK1andJNK2havebeenshowntobeinvolvedinTNF-in-ducedapoptosisindifferentcelltypes,andthesustainedphaseofJNKsignalingisemergingasacentralactivatorofapoptosis(18).JNKinducestheaccelerateddegradationoftheantiapo-ptoticproteincFLIP(cellularFLICE-interactingprotein)(30,31)andalsomediatesthereleaseofSmac(smallmitochondrialactivatorofcaspase)fromthemitochondria,whichisessentialforcaspase-8activation(32).
OurresultsdemonstratethatPiT1depletion(butnotPiT2)sensitizesbothhumanandmouseimmortalizedcellstotheproapoptoticactivityofTNF.ThisphenotypeisindependentofthepresenceorabsenceofPiintheextracellularmedium.There-expressionofPiT1inPiT1knock-outmousefibroblastsdelaysTNF-inducedapoptosis.Importantly,thesameprotec-tionisprovidedbytheexpressionofatransport-incompetentmutantofPiT1,showingthattheinvolvementofPiT1inTNF-inducedapoptosisisindependentofitsPitransportfunction.Finally,weshowthatthesustainedphaseofJNKactivityisenhancedinPiT1-depletedcellsandthatJNKactivityisinstru-mentalintheirdeath.
EXPERIMENTALPROCEDURES
ChemicalsandAntibodies—HumanTNFwaspurchasedfromGentaur(Brussels,Belgium),JNKifromMerck.DNase-freeRNase,cycloheximide,propidiumiodide,BI-78D3,andSP600125werefromSigma.LipofectamineLTXwasobtainedfromInvitrogen.ThefollowingantibodieswerepurchasedfromCellSignalingTechnology:anti-phospho-SAPK/JNK(Thr183–Tyr185)(catalogno.9251),IB␣(catalogno.4814),RIP1(recep-tor-interactingprotein1)(catalogno.3493),cleavedcaspase-3(catalogno.9661),poly(ADP-ribose)polymerase(PARP)(cat-alogno.9532),caspase-8(catalogno.9746),AMPK␣(catalogno.2532),andphospho-AMPK␣Thr172(catalogno.2535).ThehumanPiT1antibodywasproducedinthelaboratoryanddescribedpreviously(9).Monoclonalanti--actincloneAC-74(catalogno.A5316)andanti--tubulincloneTUB2.1(catalogno.T4026)werefromSigma.
Cells—HeLacellsandimmortalizedfibroblastsweregrowninDulbecco’sminimumessentialmedium(Invitrogen)supple-mentedwith5%fetalbovineserum(Hyclone)andgentamicin(Invitrogen).Isolationofembryonicday12.5mouseembryonicfibroblasts(MEFs)wasperformedusingestablishedprocedures(33).Theywereimmortalizedatpassage2bythetransfection(withLipofectamineLTX)ofaplasmidencodingthethermo-NOVEMBER5,2010•VOLUME285•NUMBER45
sensitiveSV40largeTantigenunderthehumanvimentinpro-moter(34)andthengrownat33°C.HeLacellclonesstablyexpressingshRNAsweredescribed(9).
LentiviralVectorStocks—TheintroductionoftheS621AmutationintoPiT1wasdescribedpreviously(9).TheDNAconstructscontainingeitherWTPiT1orPiT1S621AwereinsertedinthelentiviralvectorpHAGE-CMV-MCS-IZsGreenW.Twoindependentpopulationsofcellswereobtainedforeachconstructbyinfectingcellswithtwodistinctviralstocks.GFP-positivecellswereenrichedbyfluorescentcellsorting.
PiUptake—Thetransportofphosphatewasmeasuredasdescribedpreviously(9).
ApoptosisInductiononHumanandMouseCells—HeLacellsweretransfectedwithsmallinterferingRNAasdescribed(9)48–72hbeforetreatment.MEFsweresplit48hbeforetreat-ment.Apoptosiswasinducedbythetreatmentofcellswith50ng/mlhumanTNFand10–100g/mlcycloheximideincom-pletemedium.WhenJNKinhibitorswereused,thecellswerepretreatedfor30minwiththedrugandthentreatedwithTNFpluscycloheximide(TNFϩC)togetherwiththedrug.WhenexperimentsindefinedPimediumwereperformed,thecellswerepreparedasusualandwashedthreetimeswith0.9%NaClbeforetreatmenttoremovealltracesofPi.Pi-free/pyruvate-freeDulbecco’sminimumessentialmedium(Invitrogencata-logno.11971)(supplementedwith1mMsodiumpyruvate,Invitrogencatalogno.11360)orthecorrespondingregularmedium(Invitrogencatalogno.41966)(containing0.9mMPiand1mMsodiumpyruvate)wereusedwithoutanyserumaddedbecauseserumcontainsPi.
PropidiumIodideStainingforDNAAnalysisbyFlowCytometry—CellsweretransfectedwiththecorrespondingsiRNAs72hbeforetreatment.Allcellswerecollected(float-ingandattached),resuspendedinPBS,andfixedincold70%ethanolovernight(orlonger)at4°C.TheywerethenwashedinPBSandresuspendedinasolutioncontaining0.02mg/mlpropidiumiodideinPBScontaining0.1%TritonX-100,0.2mg/mlDNase-freeRNasefor30minatroomtemperaturebeforeFACSanalysis.TheDNAcontentwasanalyzedbyflowcytometryonaBDBiosciencesFACSCaliburflowcytometerwithCELLQuestsoftware.
ImmunoblotAnalysis—Cellswereplacedonice,andfloatingcellswerecollected.Attachedcellswererinsedoncewith0.9%NaCl,andthissolutionwasusedtowashthepelletofdetachedcells.Cellswerethenlysedbytheadditionofice-coldlysisbuffer(150mMNaCl,10mMTris-HCl,5mMEDTA,1%Non-idetP-40,0.5%deoxycholate,0.1%SDS,andcompleteproteaseinhibitormix(RocheAppliedScience),2mMNa3VO4,2mMNaF,5mMsodiumpyrophosphate,20mMN-ethylmaleimide)oniceanddetachedwithcellscrapers.Thelysatewasusedtoresuspendthepelletofdetachedcells,andthecombinedlysatewasleftonicefor30min.Lysateswerethencentrifugedfor15minat16,000ϫg(4°C)toremoveinsolublematerials,andthesupernatantswereusedforWesternblotanalysis,whichwasperformedasdescribed(9).ThenumberstotheleftofallWest-ernblotfiguresrepresentthemolecularweightofproteinsfromtheFull-RangeRainbowmarker(catalogno.RPN800E,Amer-shamBiosciences),andnumberstotherightarethetheoreticalsizesoftheproteinsrecognizedbythespecificantibodies.
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Statistics—BlotswerequantifiedwiththeImageJsoftware.AllgraphsareplottedasmeanϮS.E.StatisticsfordualcomparisonweregeneratedusingStudent’sttests(notsignifi-cant(ns),pՆ0.05;*,pϽ0.05;**,pϽ0.005;***,pϽ0.0005).
RESULTS
AcuteorStablePiT1DepletionSensitizesImmortalizedHumanandMouseCellstoTNF-inducedApoptosis—PiT1wastransientlyde-pletedinthehumancarcinomaHeLacelllinewithansiRNAoligo-nucleotide.TheefficiencyofthesiRNAsusedinthisstudyatdown-regulatingtheexpressionoftheendogenousPiT1inHeLacellshasbeendemonstratedpreviously(9).Whentreatedwiththeproinflam-matorycytokineTNFalone,mostcellsresistitsproapoptoticactivity,unlesstheyaresensitizedwiththeadditionoftheproteinsynthesisinhibitorcycloheximide.TNFisapowerfulinduceroftheantiapo-ptoticNFBpathway,andtheproductionofNFB-dependentproteinsneedstobeinhibitedforapoptosistoproceed(19).HeLacellsweretreatedwithacombina-tionofTNFandcycloheximidefor4h.Apoptosisprogressionwasfol-lowedbydetectionofthespecificcleavageofPARP,aproteinwhoseprocessingisunderthecontrolofFIGURE1.PiT1-depletedcellsaremoresensitivetoTNF-inducedapoptosis.A–C,HeLacellsweretransientlythe“terminalcaspase”caspase3.transfectedwithansiRNAcontrol(Ct)oransiRNAdirectedagainstPiT1.48haftertransfection,theywereeitherleftFig.1AshowsthatPiT1-depleteduntreatedHeLacellsweremoresensitivetotheagainstg/mlcycloheximideortreatedwithPARPandtubulin(TNF10ϩg/mlcycloheximide(C)alone,50ng/mlTNFalone(TNF),or50ng/mlTNFplus10asCa)loadingfordifferentcontrol.times.TheA,bandscellswerecorrespondingtreatedfor4tohfull-lengthandthenanalyzedPARP(p116)byWesternandcleavedblotproapoptoticactivityofTNFthanPARP(p89)werequantified,andthepercentageofcleavedPARPisgivenbelowthepanelsofblots.B,cellswerethecontrolcells(66%ofcleavedtreatedwithTNFϩCfor0,2,or4handanalyzedbyWesternblotagainstPARPandtubulin.Thebandscorrespond-ingtofull-lengthPARP(p116)andcleavedPARP(p89)werequantified,andthepercentageofcleavedPARPisgivenPARPversus43%).Thedepletionofunderthepanelsofblots.C,cellswerestainedwithpropidiumiodidefortheanalysisoftheirDNAcontent,andthePiT1aloneortheadditionofcyclo-percentageofcellswithasub-G(clones1and2)were1stainingwascalculated.D,HeLacellclonesstablyexpressingeitheracontrol(Ct)oraPiT1shRNAtreatedwith50ng/mlTNFand10g/mlcycloheximide(TNFϩC)for0,3,or4h.heximidealonecouldnotinduceApoptosisinductionwasdetectedbyWesternblotshowingthecleavageofPARP.Thedetectionoftubulinwasapoptosisinthesecells,showingtheusedasaloadingcontrol.ThepercentageofcleavedPARPwasquantifiedandisgivenbelow.E,WesternblotspecificactivityofTNFinapoptosisshowingthetotalamountofPiT1proteinexpressedbythedifferentclones.ThePiT1antibody(9)isspecificforthehumanproteinanddetectsmainlytwoformsoftheproteininHeLacellsindicatedbyanasteriskandadoubleinduction.PARPprocessingcanbeasterisk,whichmaydifferbytheglycosylationstatus.ThenumbersgivenbelowaretheratiooftheintensityofPiT1detectedearlier(at2hpost-treat-bandsovertubulin,usedasaloadingcontrol.F,cells(shCtcloneorshPiT1clone2)weretreatedwithTNFϩCfor4handanalyzedforPARPcleavagebyWesternblotasinD.ValuesarethemeanϮS.E.(errorbars)ofsixindependentment)andprogressesfaster(at4hexperiments.***,pϽ0.0005.G,shRNAcontrolorPiT1cellswereeitherleftuntreated(Ct)ortreatedwith10g/mlpost-treatment)inPiT1-depletedcycloheximidealone(C)or50ng/mlTNFplus10g/mlcycloheximide(TNFϩC)for4h.ApoptosiswasdetectedbycellsthaninWTcells(Fig.1B).thecleavageofPARP,andtubulinwasusedasaloadingcontrol.Theintensityofthebandswasquantified,andthepercentageofcleavedPARPisgivenbelowtheWesternblotpanels.H,TSV40immortalizedMEFsfromaknock-outWealsofollowedlatereventsinPiT1embryo(Ϫ/Ϫ)oraWTlittermate(ϩ/ϩ)wereincubatedwith50ng/mlhumanTNFand100g/mlcyclo-apoptosisbydetectingtheappear-heximide(TNFϩC)or100g/mlcycloheximidealone(C)for7.5h.CellswerelysedandanalyzedbyWesternanceofasub-Gblotfortheappearanceofthecleavedformofcaspase-3andtubulinasaloadingcontrolaswellastheamount1DNApeakbypro-ofIB␣.ThenumbersgivenbelowaretheratiooftheintensityofIB␣signaloveractin,usedasaloadingpidiumiodidestainingofthecellscontrol.I,quantificationoftheapoptosisinductionforfourindependentexperimentsperformedasinH.andFACSanalysis(Fig.1C).Wild-ValuesarethemeanϮS.E.***,pϽ0.0005.
type(WT)cellsstartedshowing
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someapoptoticstainingby8h,reaching30%at50hpost-TheRoleofPiT1inTNF-inducedApoptosisIsNotSharedtreatment.PiT1-depletedcellsweremoresensitivetoapopto-withPiT2andDoesNotDependonthePresenceofExtracellularsis,andthepercentageofapoptoticcellswasdoublethatofWTPhosphate—HeLacellsalsoexpressPiT2/SLC20A2,thesecondcellsat25and50hpost-treatment.WhentreatedwithTNFPitransporterencodedbythePiT/SLC20Afamily(9).PiT1andalone,HeLacells(withorwithoutPiT1)didnotshowanymas-PiT2depletionreducePiuptakeinHeLacells(9).However,assiveapoptosis,confirmingthatcycloheximidewasrequiredtoopposedtoPiT1depletion,siRNA-mediatedPiT2depletion(9)revealtheproapoptoticactivityofTNF.
hadnoeffectonthesensitivityofHeLacellstoTNF-inducedWehavepreviouslygeneratedHeLacelllineswithastableapoptosis,showingthatthiseffectisspecifictoPiT1depletionreducedexpressionofPiT1byusinganshRNAtransfection(9).(Fig.2A).ThisspecificitywasfurtherensuredbytheuseoftwoFig.1DshowsthattwodifferentshRNAPiT1clonesweremoredifferentsiRNAoligonucleotidestargetingdistinctregionsofsensitivetoapoptosisthanHeLacellsexpressingacontrolthePiT1mRNAintheexperimentshowninFig.2A.BothwereshRNA,asshownbytheirincreasedPARPprocessingat3andequallyefficientatreducingtheamountofendogenousPiT14hpost-TNFϩCtreatment.ThisshowsthatTNF-mediatedexpressedbyHeLacells(9).CellstransfectedwitheitherPiT1apoptosiswasaffectedbyeithertransientorstablePiT1deple-siRNApresentedanenhancedcleavageofPARPascomparedtion.Moreover,thiseffectmaybedependentontheamountofwithWTcells,strengtheningtheinvolvementofPiT1inTNF-PiT1stillexpressedbythecellsbecauseclone2,whichinducedapoptosis.AsopposedtocellstransfectedwithPiT1expressesalesseramountofPiT1thanclone1(Fig.1E),wassiRNAs,cellstransfectedwithaPiT2siRNAdidnotshowanymoresensitivetoapoptosisthanclone1.Clone2waschosenforincreaseinPARPcleavageandapoptosis,showingthatthisfurtherexperiments,andFig.1FrepresentstheresultsgatheredeffectisspecifictoPiT1depletion.Fig.2Brepresentstheaver-fromsixindependentexperimentsperformedwiththiscellageddatagatheredfromthreeindependentexperimentsandclone.Onaverage,86Ϯ3%ofPARPwascleavedinPiT1-de-showsthattheamountofapoptosisincellstreatedwithsiRNAspletedHeLacellsafter4hofTNFϩCtreatmentcomparedwithdirectedagainstPiT1wasalmosttwicetheamountshownby40Ϯ6%incontrolcells.Consistentwithresultsobtainedincellstreatedeitherwithacontrol(89Ϯ4%ofcleavedPARPHeLacellstransientlydepletedinPiT1bysiRNA,thiscloneversus50Ϯ5%)oraPiT2(38Ϯ5%ofcleavedPARP)siRNAwasnotsensitivetocycloheximidealone,shownasacontrolafter4hofTNFϩCtreatment.
inFig.1G.
ThesedataalsosuggestedthatadeficiencyinPitransportInordertostrengthenourresults,wealsotestedwhetherwasnotlikelytoberesponsiblefortheobservedphenotype.wecouldreproducetheminadifferentcellularsystem.WeInordertoinvestigatefurthertheimpactofPionTNF-immortalizedMEFsisolatedfromWTandPiT1knock-outinducedapoptosis,weperformedexperimentsinthepres-embryonicday12.5embryos(7)withSV40largeTantigenenceorabsenceofPiintheincubationmedium.Asshowninexpression.AswithHeLacells,wetreatedthesecellswithTNFFig.2,CandD,theimpactofPiT1depletiononTNF-inducedand10g/mlcycloheximideinpreliminaryexperiments(notapoptosisinHeLacellswasobservedregardlessofthepresenceshown).WechosetousethehumanTNFcytokinebecauseitorabsenceofPiinthemedium.Moreover,thesensitivityofWTexclusivelybindstomouseTNFR1(andnotTNFR2)(35).TheHeLaandMEFcells(thereforeexpressingPiT1)toTNF-in-amountofcycloheximideusedsofar(10g/ml)wasactuallyducedapoptosiswasnotdifferentwhethercellswereincubatednotenoughforacompleteinhibitionofNFBdependentpro-withorwithoutPi(Fig.2,C–E),whichstronglysuggeststhatateinsynthesisinresponsetoTNF(36).BecauseMEFs(asdefectinPiuptakeviaPiT1wasnotresponsiblefortheopposedtoHeLacells)canwithstandanincreasedamountofincreasedsensitivityofPiT1-depletedcellstoTNF-inducedcycloheximidewithoutadverseshorttermeffects,weincreasedapoptosis.Finally,becausePiisastructuralcomponentofribo-theconcentrationofcycloheximideto100g/mlinsubsequentnucleotides,wereasonedthatPiT1depletioncouldaffecttheexperiments.CycloheximidealonedidnotinduceapoptosiscellularATPavailability.TheAMP-activatedproteinkinase(Fig.1H)butwasindeedabletofullyblockproteinsynthesis(AMPK)isanextremelysensitiveindicatorofcellmetabolicinducedbytheactivationoftheNFBpathwayinresponsetostressandbecomesphosphorylatedwhentheAMP/ATPratioTNF,asshownbythecompletelackofIB␣denovosynthesisisincreased(37).Wethereforedeterminedthebasalphosphor-severalhafterTNFstimulation(Fig.1H).IB␣isknowntobeylationstatusofAMPKinPiT1-depletedcells.Fig.2FshowsrapidlydegradedinresponsetoTNFstimulation,anditsthatthiskinasewasphosphorylatedneitherinWTnorinresynthesisnormallystartswithin1h,strictlyunderthecon-PiT1Ϫ/ϪMEFs,despitethepresenceofafunctionalenzyme,astroloftheNFBtranscriptionfactor(36).Fig.1,HandI,demonstratedbyitsphosphorylationfollowingtheadditionofthedrug5-amino-4-imidazolecarboxamideriboside(AICAR),showsthatPiT1geneknock-outincreasedthesensitivitytoaspecificactivator(37).Therefore,thisobservationsuggestedapoptosisofimmortalizedmousefibroblasts,asdetectedbythethatPiT1depletiondidnotcreateanybasalenergeticstressincreasedcleavageofcaspase3.Therewasanaveraged6.5-foldwithinthecell.
increaseincaspase-3cleavageinPiT1Ϫ/ϪMEFscomparedwithPiT1InvolvementinTNF-mediatedApoptosisIsUncoupledPiT1ϩ/ϩMEFsafter7hofTNFϩCtreatment(Fig.1I).
fromItsPTakentogether,thesedatashowedthatPiT1depletioniTransportActivity—TotestmoredirectlywhetherPiT1involvementinTNF-mediatedapoptosiswaslinkedtoits(eitherbytransientorstableRNAinterferenceorgeneknock-Piuptakeactivity,wegeneratedatransport-incompetentout)increasedthesensitivitytoTNF-mediatedapoptosisinmutantofPiT1inwhichtheserineatposition621wasmutatedbothhumanandmouseimmortalizedcells.
toanalanine(9).TheV5-taggedconstruct,encodingeitherWT
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PiT1ortheS621A-mutatedprotein,wasinsertedintoalenti-detectedby3h30mininthesecells.Onaverage,caspase-8viralvectorandstablytransducedintoPiT1Ϫ/ϪMEFs.MEFcleavagewas3.5moreintenseinPiT1-depletedcellscomparedcellswerechosenoverHeLacellsbecausetheyprovidethewithcontrolcells(Fig.3B).Consistentwiththis,wecouldalso“cleanest”backgroundforarescueexperiment,havingnodetectanimportantcleavageofRIP1(receptorinteractingpro-expressionatallofendogenousPiT1duetogenedeletion.Foreachconstruct,wecharacterizedtwoindependentlytrans-tein1),acaspase-8substrate(38),atearlytimepointsinHeLaducedpopulations(T1andT2)ofPiT1Ϫ/ϪMEFsexpressingcellsdepletedofPiT1(Fig.3C).TheamountofRIP1cleavagetherecombinantproteins,detectedwasmorethandoubledinPiT1-depletedcellscomparedwith
withtheanti-PiT1antibody(Fig.2G).Asexpected,PiT1Ϫ/ϪMEFsre-expressingtheWTPiT1proteindisplayedasignificantlyincreasedPiuptakecomparedwiththeparentalcells(Fig.2H),whereasPiuptakewasunaffectedbytheexpressionofthetransport-incompetentmu-tantPiT1S621A.Fig.2Ishowedthatthere-expressionofWTPiT1inPiT1Ϫ/Ϫcellsdelayedtheappear-anceofthecleavedproductofcaspase-3,showingthatPiT1deple-tionwasindeedinvolvedintheincreasedsensitivityofthecellstoapoptosis.Importantly,theexpres-sionofthePiT1S621Amutantpro-teinalsodelayedapoptosis,asshownbythereducedcleavageofcaspase-3(Fig.2J).Bothproteinswereequallyefficientatreducingtheappearanceofcaspase-3to50%ofthatdisplayedbythecontrolPiT1Ϫ/ϪMEFs(Fig.2K).Thisdem-onstratesthatPiT1involvementinTNF-inducedapoptosiscanthere-forebeuncoupledfromitsPiuptakefunction.
Caspase-8ActivationIsIncreasedbyPiT1DepletioninTNF-inducedApoptosis—Wenextwishedtochar-acterizethemolecularmechanismunderlyingtheincreasedsensitivityofPiT1-depletedcellstothepro-apoptoticactivityofTNF.OneofthefirstcaspasestobeactivatedduringTNF-inducedapoptosisistheinitiatorcaspasecaspase-8(15).Wethereforeinvestigatedwhethercaspase-8wasmoreactivatedinPiT1-depletedcells.Westernblotanalysiscoulddetectthecleavageofcaspase-8initsfirstprocessedform(p43)3h30minafterTNFtreat-mentinHeLacellsexpressingacon-trolshRNA(Fig.3A).However,caspase-8processingwasevidentasearlyas2hpost-TNFϩCtreatmentinshRNAPiT1HeLacells,andthefullyactivatedform(p18)couldbe
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FIGURE3.Theinitiatorcaspase,caspase-8,isactivatedearlyinPiT1-depletedcells.A–D,HeLacellclonesstablyexpressingeitheracontrol(Ct)oraPiT1shRNA(PiT1)weretreatedwith50ng/mlTNFand10g/mlcycloheximide(TNFϩC)for0,2,or3.5h.CellswerelysedandanalyzedbyWesternblotwithanantibodyrecognizingallformsofcaspase-8(full-length(p57)andcleaved(p43andp18))(A)andRIP1(full-lengthp78andcleavedp36)(C).Tubulinwasusedasaloadingcontrol.BandD,cellswereincubatedwithTNFϩCfor3.5handanalyzedasdescribedinAandC,respectively.ValuesarethemeanϮS.E.(errorbars)ofthreeindependentexperiments.***,pϽ0.0005;**,pϽ0.005.EandF,immortalizedPiT1Ϫ/Ϫ(Ϫ/Ϫ)andWT(ϩ/ϩ)MEFswereincubatedwith50ng/mlTNFand100g/mlcycloheximide(TNFϩC)for0,4,or7h.Cellswerelysed,andsampleswereanalyzedbyWesternblotwithanantibodyagainstRIP1,cleavedcaspase-3,ortubulinasaloadingcontrol.F,MEFsweretreatedwithTNFϩCfor7handanalyzedasdescribedinE.ValuesarethemeanϮS.E.offiveindependentexperiments.*,pϽ0.05.
controlcellsaftera3h30mintreatmentwithTNFϩC(Fig.earlyeventsassociatedwithTNF-triggeredapoptosisbothin3D).Similarresultswereobtainedwithmousecells.RIP1washumanandmouseimmortalizedcells.
cleavedearlierinPiT1Ϫ/ϪMEFsthaninWTcells,showingthatSustainedJNKActivityIsIncreasedinPiT1-depletedCellsincaspase-8activationwas,asinhumancells,increasedbyPiT1ResponsetoTNFϩC—Ithasbecomeincreasinglyclearthatthedepletion(Fig.3,EandF).PiT1depletionthereforeimpairsMAPKJNKisamajorproapoptoticeffectorofTNF-induced
FIGURE2.PiT1involvementinTNF-inducedapoptosisis(i)notsharedwithPiT2,(ii)independentofthepresenceofextracellularPactivity.A,HeLacellsweretransientlytransfectedwithasiRNAcontrol(Ct),twodifferentsiRNAsagainstPiT1(P1aandP1b),iand(iii)unrelatedtoitstransportoransiRNAagainstPiT2(P2).48hpost-transfection,cellswereeithermock-treated(0)ortreatedwith50ng/mlTNFand10g/mlcycloheximidefor4h.CellswerelysedandanalyzedbyWesternblotwithantibodiesdirectedagainstPARPandactin(asaloadingcontrol).B,valuesarethemeanϮS.E.ofthreeindependentexperimentsperformedasinA.**,pϽ0.005.C,shCtorshPiT1HeLacellswereincubatedwithorwithoutacombinationof50ng/mlTNFand10g/mlcycloheximide(TNFϩC)inmediumcontainingeither0or0.9mMPthreeindependentexperimentswereperformedifor4h.ApoptosiswasdetectedbyWesternblotwithantibodiesagainstPARPandactinϩasloadingϪ/Ϫcontrol.D,asinCandquantified.ValuesarethemeanϮS.E.(errorbars).*,pϽ0.05.E,PiT1ϩ/andPiT1MEFswereincubatedwithorwithout50ng/mlTNFplus100g/mlcycloheximide(TNFϩC)for7hinmediumcontainingeither0or0.9mMPApoptosiswasdetectedbytheappearanceofcleavedcaspase-3,andactinwasusedasaloadingcontrol.Thisrepresentativeblotwasquantified,andthei.correspondingvaluesaregivenbelowtheWesternblotpanel.F,PiT1ϩ/ϩandPiT1Ϫ/ϪMEFswereeitherleftuntreated(Ϫ)ortreated(ϩ)with2mMAICARfor7h.CelllysateswereanalyzedϪbyWesternblotforthephosphorylationofAMPK(pAMPK)andthetotalamountofAMPK.Thedetectionoftubulinwasusedasaloadingcontrol.G–K,PiT1Ϫ/MEFsweretransducedwithlentiviralvectorsexpressingeitherthehumanWTPiT1proteinortheS621AmutantandtheGFPproteinfromaninternalribosomeentrysite.Twoindependentinfections(withtwodifferentvirusstocks)wereperformedtoobtaintwoindependentpopulationsineachcase(T1andT2).GFP-positivecellswereenrichedbyfluorescentcellsorting.G,PiT1proteinsweredetectedwiththeanti-PiT1antibody.H,sodium-dependentPiuptakewasmeasuredinthedifferenttransducedpopulations.*,pϽ0.05;**,pϽ0.005;ns,nonsignificant.IandJ,thedifferentpopulationsweretreated(ϩ)ornot(Ϫ)with50ng/mlTNFand100g/mlcycloheximidefor3.5h.Celllysateswereanalyzedfortheappearanceofcleavedcaspase-3,andthedetectionofactinwasusedasaloadingcontrol.K,quantificationoftheblotsshowninIandJ.Threeindependentexperiments(withthetwodifferentcellpopulationsforeachconstructs)wereanalyzed.Theratiooftheintensitiesofthecleavedcaspase-3band/actinbandwasquantifiedandpooledforeachconstructandispresentedastherelativepercentageoftheintensityofcaspase-3cleavagequantifiedinGFPexpressingcontrolPiT1Ϫ/ϪMEFs.***,pϽ0.0005.
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wereoverphosphorylatedinshRNAPiT1cells,onlyp54pJNKwassig-nificantlyincreased(Fig.4B).
WeconfirmedtheseresultsbytransientlyknockingdownPiT1inHeLacells.ThetransientdepletionofPiT1alsoresultedinanincreasedsensitivitytoTNF-mediatedapo-ptosis(asshownbyPARPcleavage),whichcorrelatedwithanincreasedphosphorylationofp46andp54JNK(Fig.4,CandD).ThisshowsthatJNKoveractivationwasnotduetoanadaptationofthecellclonestothelongtermdepletionofPiT1.Onceagain,p54JNKwassignifi-cantlyoveractivated(Fig.4D).More-over,siRNA-mediatedPiT2deple-tiondidnotincreaseapoptosisanddidnotenhancesustainedJNKphosphorylationascomparedwithsiRNAcontrol-treatedcells,show-ingthespecificityoftheeffectmedi-atedbyPiT1depletion(Fig.4,CandD).Finally,Fig.4,EandF,showsthattheseobservationsarealsovalidinmousecells.Immor-talizedPiT1Ϫ/ϪMEFsdisplayedanenhancedphosphorylationofJNKcomparedwithPiT1ϩ/ϩcells,whichcorrelatedwellwiththeincreasedapoptosisdetectedbytheFIGURE4.JNKsustainedactivityisincreasedinPiT1-depletedcells.AandB,HeLacellclonesstablyexpress-appearanceofcleavedcaspase-3.
ingeitheracontrol(Ct)oraPiT1shRNA(PiT1)weretreatedwith50ng/mlTNFand10g/mlcycloheximideBothJNKisoformsweresignifi-(TNFϩC)for0,2,or3.5h.CellswerelysedandanalyzedbyWesternblotwithanantibodyrecognizingthecantlyoveractivatedinthiscellphosphorylatedforms(pJNK)ofallJNKisoformsandgenes(p46andp54).TheapoptosisprogressionwasfollowedbytheappearanceofthecleavedformofPARP.B,cellsweretreatedornotwithTNFϩCfor3.5hand
type(Fig.4F).Importantly,theen-analyzedasdescribedinA.ValuesarethemeanϮS.E.(errorbars)ofsixindependentexperiments.***,pϽhancedactivationofJNKthere-0.0005.CandD,HeLacellswereeithertransientlytransfectedwithansiRNAcontrolordirectedagainstPiT1orPiT2.48hpost-transfection,thecellswereeitherleftuntreated(0)ortreatedwith50ng/mlTNFand10g/mlforeoccurredinPiT1-depletedcycloheximide(TNFϩC)for5h.ApoptosisprogressionwasfollowedbythecleavageofPARP,andphosphor-cellswhethertheNFBpathwayylatedJNKwasdetectedbyWesternblot(pJNK).D,valuesarethemeanϮS.E.offourindependentexperimentswaspartly(HeLacells)orfullyperformedasdescribedinC.**,pϽ0.005.EandF,PiT1Ϫ/ϪandPiT1ϩ/ϩMEFswereincubatedwith50ng/mlTNFand100g/mlcycloheximide(TNFϩC)for0,3.5,or7h.Celllysateswereanalyzedwithantibodiesagainst
(MEFs)inactivated.phosphorylatedJNKandcleavedcaspase-3.Tubulinoractinwasusedasaloadingcontrol.F,cellsweretreated
JNKActivityIsInstrumentalinwithTNFϩCfor7handanalyzedasdescribedinE.ValuesarethemeanϮS.E.offourindependentexperi-TNF-inducedApoptosisinPiT1-de-ments.*,pϽ0.05;**,pϽ0.005.pletedCells—TotestwhetherJNK
activitycouldberesponsibleforthe
apoptosis.TNFR1stimulationinducesaveryrapidandtran-observedincreasedsensitivitytoapoptosisinPiT1-depletedsientactivationofJNK(30min)inviablecells,whereasincells,wetreatedHeLacellswiththeJNKinhibitorSP600125,NFB-inhibitedcells,whichareprimedtoundergocelldeath,whichactsasanATPcompetitiveinhibitor(39).Figs.5,AandJNKispersistentlyactivatedandremainssoforseveralh(18).B,showsthatSP600125significantlyreducedtheappearanceofSustainedJNKactivation(Ͼ1h)isrequiredforcaspase8acti-cleavedPARPinHeLacellstransientlytransfectedwithaPiT1vation(30,32),andwethereforeinvestigatedwhethersus-siRNA(from81Ϯ5to57Ϯ4%).InordertoruleoutanyofftainedJNKactivationcouldbemodulatedbyPiT1depletion.targeteffectofthedrugused,wenexttestedwhethertwootherFig.4Ashowsthatbothp46andp54isoformsofJNKweremoreJNKinhibitors,JNKi(40)andBI-78D3(41)couldalsopreventphosphorylatedinPiT1shRNAHeLacellsthanincontrolTNF-inducedapoptosisonMEFs.Theseinhibitorsactassub-shRNAcellsinresponsetoTNFϩCat2or3.5hpost-treat-stratecompetitiveinhibitors.Fig.5(C–F)showsthatbothJNKment.Thiscorrelateswellwithanincreaseinapoptosis,de-inhibitorsgreatlyreducedthecleavageofcaspase-3inPiT1Ϫ/ϪtectedbyPARPcleavage.ThequantificationofsixindependentMEFs(Fig.5D,morethan3timesforJNKi;Fig.5F,2.5timesforexperimentssuggestedthatalthoughbothisoformsofJNKBI-78D3).Theseresultsindicatethatthehyperactivationof
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reactstofluctuationsintheAMP/ATPratio(37).Furthermore,PiT2depletioninHeLacells,whichalsoresultsinadecreaseinPiuptake,doesnothaveanyeffectonapopto-sis,suggestingthattheincreasedsensitivitytoTNF-inducedapopto-sisrevealedinPiT1-deficientcellsisindependentofPiuptake.ThishypothesisisstrengthenedbythefactthattheincreasedsensitivitytoTNF-inducedapoptosisthatismediatedbyPiT1depletioniscon-servedregardlessofthepresenceorabsenceofPiinthemedium,argu-ingforaPi-independentroleofPiT1intheseeffects.Finally,weshowthattheexpressionofaPiT1mutantthatcannottransportPiisequallyefficientatdelayingapoptosisinPiT1Ϫ/Ϫcellsasthewildtypetrans-porter.ThisdemonstratesthatPiT1involvementinTNF-inducedapo-ptosisisuncoupledfromitsPitrans-portactivity.Inaccordancewiththishypothesis,wehavepreviouslyshownthatPiT1hadanothertrans-port-independentfunctionintheregulationofcancerandnormalcell
FIGURE5.JNKactivityisinstrumentalintheapoptosisofPiT1-depletedcells.AandB,HeLacellsweretransientlytransfectedwithacontrolsiRNA(Ct)oransiRNAagainstPiT1(PiT1)orPiT2(PiT2).48hpost-proliferation(7,9).transfection,cellswereeithermock-treated(0)ortreatedwith50ng/mlTNFand10g/mlcycloheximide
Wehaveinitiatedthedissection(TNFϩC)for5h.Cellswerepreincubated(ϩSP)(ornot)for30minwiththeJNKinhibitorSP600125(30M),ofthemolecularmechanismunder-andtheinhibitorwaskeptinthemediumforthewholetimecourseoftheexperiment.A,cellswerelysedandanalyzedbyWesternblotwithantibodiesdirectedagainstPARPandthephosphorylatedformsofJNK
lyingtheincreasedsensitivityof(pJNK)andtubulin(asloadingcontrols).B,valuesarethemeanϮS.E.(errorbars)ofthreeindependentPiT1-depletedcellstoTNF-inducedexperimentsperformedasdescribedinA.*,pϽ0.05.CandD,PiT1knock-out(Ϫ/Ϫ),orWTMEFs(ϩ/ϩ)werepreincubated(TNFϩCϩJNKi)ornot(TNFϩC)withthespecificJNKinhibitorJNKi(5apoptosis.ThetreatmentofPiT1-M)for30min.50
ng/mlTNFand100g/mlcycloheximidewerethenaddedtothemediumfor6h.C,totalcelllysateswere
depletedcellswithTNFinproapo-analyzedbyWesternblotwithantibodiesagainstcleavedcaspase-3,phosphorylatedJNK,andtubulinasaptoticconditionsleadstotheearlyloadingcontrol.D,valuesarethemeanϮS.E.ofthreeindependentexperimentsperformedasdescribedinC.*,pϽ0.05.EandF,PiT1Ϫ/ϪandPiT1ϩ/ϩMEFswerepreincubated(TNFϩCϩBI),ornot(TNFϩC),for30minwith
activationoftheinitiatorcaspasetheJNKinhibitorBI-78D3(5M)andthentreated(ornot;BI)with50ng/mlTNFand100g/mlcycloheximidecaspase-8.TheabsenceofPiT1for9h.E,celllysateswereanalyzedwithantibodiesagainstphosphorylatedJNKandcleavedcaspase-3.Actinwasusedasaloadingcontrol.F,valuesarethemeanϮS.E.offourindependentexperimentsperformedasthereforeimpactsonearlyeventsdescribedinE.**,pϽ0.005.
inducedbyTNF.Theactivationofcaspase-8appearstooccurthroughtheformationoftwodistinctcom-JNKisindeedinvolvedintheincreasedsensitivitytoapoptosisplexesinresponsetoTNF(16,42).ComplexIIAisformedvia
ofPiT1-depletedcells.
theassociationofTNFR-associateddeathdomain,Fas-associ-DISCUSSIONateddeathdomain,andcaspase-8,whereascomplexIIBcon-tainsFas-associateddeathdomain,RIP1,andcaspase-8.OurThedatapresentedhereshowthatPiT1depletionsensitizesresultssuggestthatcomplexIIBmayconstitutethemainapo-cellstoTNFϩC-mediatedapoptosis.ThiscanbereproducedptoticcomplexthatarisesfromTNFstimulationinourcondi-eitherbyacuteorstableRNAinterferenceinhumancancertions.Inagreementwiththishypothesis,Fig.3showsthatRIP1cellsorbygeneknock-outinmouseimmortalizedfibroblasts.iscleavedduringthecourseofapoptosisinPiT1-depletedcells,WeshowthatthiseffectisspecifictothedepletionofPiT1suggestingthatcomplexIIBindeedformsandtherebyallowsbecausePiT2depletionhasnoeffectonthistypeofapoptosis.forthecloseassociationofcaspase-8withRIP1thatisnecessaryBecausePiT1functionsasasodium-Pisymporter(4),PiT1forRIP1processing(38).RegardlessofthetypeofproapoptoticdepletionreducesPiuptakeincells,bothinHeLacellsandcomplexthatmayforminPiT1-depletedcells,wehaverevealedimmortalizedMEFs.However,PiT1Ϫ/ϪmousefibroblastsdoanincreasedphosphorylationofJNK,whichcouldbedirectlynotshowanysignofmajorenergeticstress,asshownbytheinvolvedincaspase-8activation.JNKhaspreviouslybeenabsenceofphosphorylationofthekinaseAMPK,whichrapidlyshowntobenecessaryforcaspase-8activation(30,32),proba-NOVEMBER5,2010•VOLUME285•NUMBER45
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blywithinbothcomplexes(16).Ourdataalsorevealedthatalthoughbothp46andp54JNKisoformswereoverphosphory-latedinPiT1-depletedcells,p54increasewasmorepro-nounced(Fig.4).Thereareconflictingresultsregardingwhichgeneand/orisoformismoreimportantforTNF-inducedapo-ptosis(43,44).Importantly,weshowthatJNKactivityisinstru-mentalintheapoptosisofPiT1-depletedcells,andthreediffer-entJNKinhibitorsdelayapoptosisinPiT1-depletedhumanandmousecells.TheregulationofJNKsustainedactivityinresponsetoTNFisstillincompletelyunderstood.Theproduc-tionofantiapoptoticproteinsunderthecontrolofNFBisthemainprotectivemechanismthatisinducedbyTNFinmostcells(45),andNFBcontrolsthedurationofJNKactivity(21,25–28).WeshowherethatPiT1-depletedcellsdisplayanincreaseinsustainedJNKactivationandthatthisistruewhethertheNFBpathwayisonlypartly(inHeLacells)orfullyinhibited(inMEFs)byincreasingconcentrationsofcyclohexi-mide.ThisthereforesuggeststhatPiT1absencederegulatesanantiapoptoticpathwayinvolvedinthedampeningofsustainedJNKactivity.ThispathwaywouldthenbeadditionaltothemainNFBpathwaybecauseitprotectsWTMEFswhencyclo-heximidefullyinhibitsNFB-dependentproteinsynthesis.Alternatively,astrongerproapoptoticsignal(resultinginanenhancedactivationofJNK)maybetriggeredbyTNFincellsdepletedforPiT1.WearecurrentlyinvestigatingtheupstreamkinasesthatareinvolvedinJNKactivationinPiT1-depletedcells,andpreliminarydatashowthatbothMKK4andMKK7(JNKdirectupstreamkinases)maybeoveractivatedinresponsetoproapoptoticTNF(datanotshown).ItisconceivablethatPiT1couldbindtosomeintracellularproteins(viaitslargecentralhydrophilicdomain(9))involvedinthecompositionofoneofthemultiplesignalingcomplexesformedinresponsetoTNF,anditsabsencewouldthereforeaffecttheresponseofthecell.
Thereareonlysparseexamplesintheliteratureoftransport-ersorchannelsthathavebeenshowntopossessadditionalfunctionsindependentlyoftheirtransportfunction.Afirstclasswouldbeconstitutedbytheso-called“transceptors”thatareactingasexternalsensorsofthecompoundstheyaretrans-portingandthensignaltothecell,independentlyoftheactualtransportactivity(reviewedinRef.46).TheyeastPho84phos-phatetransporterisonesuchexample(46),andbindingofPitoPho84inducesaconformationalchangethatmediatestherapidactivationoftheproteinkinaseApathway.Thecompletetrans-portcycleisnotrequiredforsignaling(46).HumanPiT2hasbeenshowntomodifyitsoligomerizationindependentlyofPiuptakeinresponsetochangesintheextracellularPiconcentra-tion,suggestingapossibleroleofPiT2inPisensing(47,48).However,itisnotknownwhetherPiT2(orPiT1)hasanydirectsignalingactivityinresponsetoPi.Nevertheless,experimentsperformedinthepresentstudyshowthatthepresenceofPiisnotrequiredforTNF-inducedapoptosistoproceed.ThisarguesagainstaroleofPiT1(orPiT2)inPisensingandofextracellularPiitselfduringTNF-inducedapoptosis.Apoten-tialroleofPiTproteinsinPisensingmaybeimportantforothercellularfunctions,suchasproliferation,andthisinterestinghypothesisrequiresfurtherinvestigation.
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MembersoftheNa,K-ATPasefamilycouldconstituteasec-ondclassoftransportershavingfunctionsinadditiontothetransportoftheirspecificsubstrate.Theyareinvolvedinmor-phogenesisandoncogenesis,andthesefunctionsareindepen-dentoftheirtransportactivity(49–51).InCaenorhabditiselegans,thecation-transportingATPaseCATP-1interactswiththeinsulin/IGFandRas-MAPKpathwaystocontrolseveralpostembryonicevents,independentlyofitstransportfunction(52).Apotentialextracellularligandhasnotbeenidentifiedinthiscase.Incontrast,itisnowwellknownthatmammalianNa,K-ATPasesbindtocardiotonicsteroidslikeouabainandrelaytheextracellularsignaltointracellularcompartmentsviaseveralkinasecascades,somemediatedbyadirectbindingofthepumptoSrc(53,54).ItispossiblethatPiT1hasligandsinadditiontoitstransportedsubstrates,inorganicphosphateandsodium.Inlinewiththis,itisnoteworthytopointoutthatPiT1andPiT2alsooperateasretrovirusreceptors.Itwouldthere-forebepossiblethatspecificenvelopeproteinsproducedfromendogenousretroviralelementscouldserveasadditionalligandsforbothtransporters.Interestingly,suchaligand(namedFeLIX)hasbeenfoundinthegenomeofcats(55).Itpresentsaveryhighhomologytotheenvelopeofthefelineleukemiavirusandplaysacrucialroleinhelpingtheinfectionofthesecells(viaPiT1)bytheTcell-tropicfelineleukemiavirus,whichisotherwisefusion-deficientonitsown.Thepos-siblemodulationofthefelinePiT1cellularfunctionsbyFeLIXhasnotyetbeeninvestigated,andthehumangenomedoesnotencodeahomologofFeLIX.However,twohumanendogenousretroviralenvelopes(syncytin1/HERV-Wand2/HERV-FRD)havebeenfoundtoperformimportantphysiologicalfunctions(inthemorphogenesisoftheplacenta,reviewedinRef.56)throughtheirspecificinteractionwiththeircognatereceptor(57,58),whereastheexpressionofothersmaybeinvolvedinthedevelopmentofseveralpathologies,includingcancer(reviewedinRef.59).Sixteensequencesencodingfull-lengthendogenousretroviralenvelopeshavenowbeenidentifiedinthehumangenome(60),andsomeofthemmaythereforepotentiallyactasligandsforPiTproteinsandmodulatecellularfunctions.
Athirdfamilyofreceptorsseemtohavetransport-inde-pendentandligand-independentfunctions,suchassomepotassiumchlorideco-transporters.Forexample,theover-expressionofNKCC1/SLC12A2transporterimpairstheembryogenesisofXenopuslaevis(61),whereastheneuronalKCC2isassociatedwithneuraldevelopmentinmammals(62,63).KCC2playsastructuralroleandinteractswiththeunder-lyingcytoskeletontopromotedendriticspinedevelopment,independentlyofitsClϪtransportactivity(63).Theinteractionoftransporterswiththecytoskeletonbeneathisthoughttobeanotherimportantpropertyofthesemultitransmembranemolecules,inadditiontoorindependentoftheirtransportfunction(64).Thisanchoringcanregulatecellmigration,adhe-sion,andshape(64).AscaffoldingroleisalsorevealedbytheDrosophilaNa,K-ATPase,whichseemstohaveevolvedfromhavingprimarilyapumpingfunctiontoamorespecialized,pump-independentroleasascaffoldproteinfortheformationandfunctioningofepithelialjunctions(50).PiT1hasbeenshowntoco-localizewiththeactincytoskeletoninmurine
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Downloaded from www.jbc.org by guest, on April 30, 2012osteoclasts(65),anditsabsencecouldthereforeaffectthewholecouplingoftheplasmamembranetothecytoskeletonandindirectlymodifythecellresponsetoexogenousstimuli.PiT1couldalsoplayaroleasascaffoldingprotein,viaitslargeintracellulardomain,andgathertheproteinsinvolvedinsignal-ingorthedampeningofthissignaling.
Inconclusion,consideringthatPiT1(butnotPiT2)mRNAseemstobetranscribedinresponsetoanactivationoftheNFBpathway(10–12),3itispossiblethatPiT1expressioncouldbeincreasedduringcancercellprogression,whichmayrelyonelevatedNFBactivity(19,66).ResultspresentedheresuggestthatPiT1mayprovidesomeprotectiontocancercellsagainstTNF-inducedapoptosis.TakentogetherwithourrecentresultsrevealingtheimpairedproliferationofPiT1-de-pletedcancercells(9),thismaysuggestthatPiT1couldplayaroleduringcancerpathogenesis.
Acknowledgments—WearegratefultoDr.S.Fabrega(Plateforme“TransfertdeGe`nesa`l’AidedeVecteursViraux,”IFR94,Universite´ParisDescartes,Paris)forthegenerationoflentiviralvectorstocksandtoC.CordierandJ.Megret(PlateformeTriCellulaire,IFR94,Paris)forthecellsorting.WethankProf.D.Paulin(Universite´PierreetMarieCurie,Paris)forthegiftoftheTSV40plasmidandDr.F.Jaisser(CentredeRecherchedesCordeliers,Paris)forhelpfuldiscus-sionontheimmortalizationofMEFs.Finally,wethankDr.S.Sullivanforhelpfuldiscussionandforeditingofthemanuscript.REFERENCES
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