内皮细胞的葡萄糖处理诱导自噬活性氧介导激活腺苷酸活化蛋白激酶 - 图文

2-Deoxy-D-GlucoseTreatmentofEndothelialCellsInducesAutophagybyReactiveOxygenSpecies-MediatedActivationoftheAMP-ActivatedProteinKinase

QilongWang,BinLiang,NajeebA.Shirwany,Ming-HuiZou*

SectionofMolecularMedicine,DepartmentofMedicine,DepartmentofBiochemistryandMolecularBiology,UniversityofOklahomaHealthSciencesCenter,OklahomaCity,Oklahoma,UnitedStatesofAmerica

Abstract

Autophagyisacellularself-digestionprocessactivatedinresponsetostressessuchasenergydeprivationandoxidativestress.However,themechanismsbywhichenergydeprivationandoxidativestresstriggerautophagyremainundefined.Here,wereportthatactivationofAMP-activatedproteinkinase(AMPK)bymitochondria-derivedreactiveoxygenspecies(ROS)isrequiredforautophagyinculturedendothelialcells.AMPKactivity,ROSlevels,andthemarkersofautophagyweremonitoredinconfluentbovineaorticendothelialcells(BAEC)treatedwiththeglycolysisblocker2-deoxy-D-glucose(2-DG).TreatmentofBAECwith2-DG(5mM)for24hoursorwithlowconcentrationsofH2O2(100mM)inducedautophagy,includingincreasedconversionofmicrotubule-associatedproteinlightchain3(LC3)-ItoLC3-II,accumulationofGFP-taggedLC3positiveintracellularvacuoles,andincreasedfusionofautophagosomeswithlysosomes.2-DG-treatmentalsoinducedAMPKphosphorylation,whichwasblockedbyeitherco-administrationoftwopotentanti-oxidants(TempolandN-Acetyl-L-cysteine)oroverexpressionofsuperoxidedismutase1orcatalaseinBAEC.Further,2-DG-inducedautophagyinBAECwasblockedbyoverexpressingcatalaseorsiRNA-mediatedknockdownofAMPK.Finally,pretreatmentofBAECwith2-DGincreasedendothelialcellviabilityafterexposuretohypoxicstress.Thus,AMPKisrequiredforROS-triggeredautophagyinendothelialcells,whichincreasesendothelialcellsurvivalinresponsetocellstress.

Citation:WangQ,LiangB,ShirwanyNA,ZouM-H(2011)2-Deoxy-D-GlucoseTreatmentofEndothelialCellsInducesAutophagybyReactiveOxygenSpecies-MediatedActivationoftheAMP-ActivatedProteinKinase.PLoSONE6(2):e17234.doi:10.1371/journal.pone.0017234Editor:AiminXu,UniversityofHongKong,China

ReceivedSeptember17,2010;AcceptedJanuary26,2011;PublishedFebruary28,2011

Copyright:?2011Wangetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

Funding:ThisworkwassupportedbyNIHgrants(HL079584,HL080499,HL074399,HL089920,HL096032,andHL105157),aresearchawardfromtheAmericanDiabetesAssociation,andthefundsfromPaulH.DorisEatonTravisChairinEndocrinologyfromtheUniversityofOklahomaHealthSciencesCenter.Dr.ZouisarecipientoftheNationalEstablishedInvestigatorAwardofAmericanHeartAssociation.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:ming-hui-zou@ouhsc.edu

Introduction

Autophagyisatightlyregulatedcatabolicprocessinvolvingthedegradationofcellularcomponentsusinglysosomalmachinery.Thisprocessplaysanimportantroleincellgrowth,development,andhomeostasisbymaintainingabalancebetweenthesynthesis,degradation,andsubsequentrecyclingofcellularproducts.Autophagyisamajormechanismbywhichastarvingorstressedcellreallocatesnutrientsfromancillaryprocessestomoreessentialones[1–2].Forexample,autophagycanbeinducedbyhypoxia[3],energydeprivation[4],starvation[5]andischemia[6].Mechanistically,autophagyisinitiatedwhentheautophagosome,adouble-membranestructure,isformedtosurroundcertaintargetedcytoplasmicproteinsandorganelles.Thisprocessandthedouble-membranestructuresareassociatedwiththeconversionofthemicrotubule-associatedproteinlightchain3B-I(LC3-I)toLC3B-II.Theprotein/organellecontainingautophagosomefuseswithalysosometodegradeitsinnercontents[1].Lyso-somescanbedisruptedbychloroquineorbafilomycinAtoblockautophagosomedegradationandprovokeautophagosomeaccumulation,whichismarkedbyanincreaseinLC3-II[7].

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Increasingevidencesuggeststhatautophagyplaysanimportantroleinthecardiovascularsystemunderphysiologicalandpathologicalconditionsincludingischemia-reperfusioninjuryintheheartandotherorgans[8],cardiomyopathy[9],myocardialinjury,atherosclerosis[10,11],andvascularpathologyinAlzhei-mer’sdisease[12].

Reactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS)arereportedtobeimportantinmediatingautophagy[13,14].ROShavealsobeenreportedtostabilizeautophagosomesduringperiodsofnutrientdeprivation,hypoxia,ischemia-reperfusioninjury,andgeneralcellstress[15].Forexample,duringcellularstarvationornutrientdeprivation,increasedgenerationofmitochondrial-derivedhydrogenperoxide(H2O2)inducesoxidationandconsequentinhibitionofAtg4,thecysteineproteases(autophagins)whichplaycrucialrolesinautophagybyproteolyticactivationofAtg8paralogsfortargetingtoautophagicvesiclesbylipidconjugation,aswellasinsubsequentdeconjuga-tionreactions[16].DespiteofgrowingevidencethattheredoxregulationofthecysteineproteaseAtg4byROScorrelateswiththeoccurrenceofautophagy,themechanisticdetailsofhowROS/RNSinitiatesautophagyremaintobeelucidated.

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AMPKisaserine/threoninekinase,whichoperatesasametabolicswitchthatisengagedinconditionswhencellularATPisbecomingdepleted.Uponactivation,AMPKinducesformationofthetuberoussclerosiscomplextoinhibitphosphorylationofthemammaliantargetofrapamycin(mTOR),whichtriggersautophagythroughtwodownstreamsignalingpartners,ribosomalproteinS6kinaseand4E-bindingprotein1(4-eBP1)[17].SomerecentreportshaveimplicatedAMPKwithregulationofautophagy.Forexample,aminoimidazolecarboxamideribonucleotide(AICAR)treatmentandglucosedepri-vationofhumanmammarycancerderivedcells(MCF-7s)inhibitautophagy[18].Matsuiandcolleaguesalsoreportedthatincardiacmyocytes,autophagyisinducedbyinhibitionofmTOR,aphenomenonthatprotectsagainstcelldeath[19].

PublishedstudiesfromourlaboratoryandothershaveestablishedanintricatebalancebetweenAMPKsignalingandtheredoxstateofvascularendothelialcells.ROSandRNSmediateAMPKactivationinducedbyawiderangeofstimuli,includinghyperglycemia[20],hypoxia[21],treatmentwithmetformin[22],nicotine[23],andtherapywithstatindrugs[24].Conversely,AMPKactivationinhibitstheformationofROSbyNADPHoxidaseandstimulatesnitricoxide(NO)byendothelialNOsynthase(eNOS)[25].Further,AMPKhasalsobeenimplicatedinc-JunN-terminalkinase(JNK)activation,nuclearfactor(NF)-kBmediatedtranscription,E-selectinandvascularcelladhesionmolecule-1(VCAM-1)expressioninendothelialcells[26].However,whetherornotAMPKisinvolvedinROS-triggeredautophagyisunknown.

2-Deoxy-D-glucose(2-DG)isarelativelyspecificblockerforglycolysisbecauseitcannotbefurthermetabolizedbyhexokinase.Therefore,2-DGtriggersglucosedeprivationwithoutalteringothernutrientsormetabolicpathways[27].Inaddition,2-DGisreportedtoactivateAMPK,increaseROSincancercells[28],andtriggerautophagy[29].Thus,2-DGappearstobeanidealtooltodissecttheinteractionsbetweenautophagy,ROS,andAMPK.However,while2-DGinducesautophagyincancercellsandmouseembryonicfibroblasts[30,31],itsabilitytotriggerautophagyinendothelialcellshasnotbeendemonstrated.Inthisstudy,weprovidethefirstdemonstrationthatAMPKisrequiredforROS-triggeredautophagyinendothelialcellsexposedto2-DG.

factors(Lonza)and1%antibiotics(Invitrogen).BAECandHUVECwerebothculturedat37uCina5%CO2humidifiedatmosphere.ConfluentBAECandHUVECwerestudiedbetweenpassages3and9.

AdenoviralInfection

ProliferatingBAECat60%confluenceina6-wellplatewereinflectedwithadenovirusencodingsuperoxidedismutase1(SOD1),catalase,MnSOD,AMPK-dominantnegativemutants(AMPK-DN),p47phox-domitantnegativemutants(p47-DN),orp67phox-dominantnegativemutants(p67-DN).Adenovirusexpres-sionofgreenfluorescenceprotein(GFP)wasusedasnegativecontrol.After48hrs,theinfectionefficiencyexceeded80%asdeterminedbythenumberofGFP-expressingcells.

siRNATransfection

Controlnon-targetedsiRNA,AMPKa,orUCP-2targetedsiRNA(10mM)wereaddedtoOPTI-MEMreducedserummedia(GIBCO,Invitrogen)withLipofectamineTM2000.ProliferatingHUVECat60%confluenceina6-wellplatewereincubatedin1.0mLEBMsupplementedwithsiRNAfor6hrs.Then1.0mLEBMcontaining2Xfetalbovineserumandantibioticswasadded,andthecellsculturedfor24hrs.

DeterminationofROS

BAECwereincubatedinEBMwithoutphenolredcontainingCM-H2DCFDA(5mM)asamembrane-permeableprobetodetectintracellularROS,undervariousstimulationconditions.After30minincubation,fluorescencewasdetectedusingaSynergyHTmicroplatereader(BIO-TEK)withexcitationsetat490nmandemissiondetectedat520nm.

MeasurementofAMP,ADP,andATP

AMP,ADP,andATPinBAECwereassessedbyhighperformanceliquidchromatography(HPLC)asdescribedpreviously[32].

AMPKActivityAssay

AMPKactivitywasdeterminedusingtheSAMSpeptideasanAMPKsubstrate,aspreviouslydescribed[22].

MaterialsandMethodsMaterials

Bovineaorticendothelialcells(BAEC)wereobtainedfromLonzaBiologics,Inc.(Walkersville,MD).Humanumbilicalveinendothelialcells(HUVEC)werepurchasedfromCascadeBiologics(Portland,OR).[c-32P]waspurchasedfromPerkinElmerLifeSciences(Waltham,MA).2-DG,4-hydroxy-Tempo,N-Acetyl-L-cysteine(NAc),ATP,ADP,AMP,allopurinol,andoxypurinolwerepurchasedfromSigma-Aldrich(St.Louis,MO).ProteinA/G-agarose,AMPKasiRNA,andUCP-2siRNAwereobtainedfromSantaCruzBiotechnology,Inc.(SantaCruz,CA).5(and-6)-chloromethyl-29,79-dichlorofluoresceindiacetate,acetylester(CM-H2DCFDA),andLipofectamine2000reagentwereobtainedfromInvitrogenCorp.(Carlsbad,CA).Antibodiesagainstphospho-acetyl-CoAcarboxygenase(ACC;Ser79),phos-pho-AMPK(Thr172),phosphor-mTOR(Ser2448),AMPK,LC3B,andb-actinwerepurchasedfromCellsignalingTechnol-ogy,Inc.(Beverly,MA).AntibodyagainstUCP-2wasobtainedfromSantaCruzBiotechnology,Inc.

WesternBlottingandQuantification

Westernblotwasperformedasdescribedpreviously[22].Bandintensity(area6density)onWesternblotswasanalyzedbydensitometry(modelGS-700,ImagingDensitometer;Bio-Rad,Hercules,CA).Backgroundwassubtractedfromthequantificationarea.

LightMicroscopyandImmunofluorescence

ConfluentBAEConglassslidesweretransducedwithLC3B-GFPencodingBacMamvirus(Invitrogen)for24hrs.BAECwerethenincubatedfor0.5hrwith1mMLysoTrackerRed(Invitro-gen)andrinsedseveraltimeswithPBS.Thecellsontheslideswerefixedwith4%formaldehydefor10minandpermeablizedwith0.2%tritonX-100for20min.TheslidesweremountedandobservedwithOLYMPUSIX71microscope(Olympus,Tokyo,Japan).

StatisticalAnalysis

Dataarepresentedasmean6standarderrorofthemean(SEM).Differenceswereanalyzedbyusingaone-wayanalysisofvariance(ANOVA)followedbyaTukeypost-hocanalysis.pvalueslessthan0.05wereconsideredstatisticallysignificant.

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CellCulture

BAECandHUVECweregrowninendothelialcellbasalmedium(EBM)containingfetalbovineserum(5%,v/v),growth

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Results

2-DGinducesautophagyinendothelialcells

Toestablishwhether2-DGexposuretriggeredautophagyinendothelialcells,BAECwereexposedfor2to24hrsto5mM2-DG,adosecomparabletothephysiologicconcentrationofD-Glucose,andtheconversionofLC3-ItoLC3-IIwasthendetermined.ExposureofBAECto2-DGfor24hrsmarkedlyincreasedtheconversionofthecytoplasmicLC3-Itotheautophagosomalmembrane-boundLC3-II,indicating2-DGmightinduceautophagy(Fig.1A).However,ratherthaninducingautophagy,anincreaseinLC3-IIcouldinsteadbedueto2-DGrepressingautophagosomefusionwithlysosomesanddegradationofLC3-II.Todeterminethemechanismof2-DGaction,weexaminedtheeffectofdisruptinglysosomalfunctionusingchloroquineandbafilomycinA.ThesecompoundsincreaselysosomalpHandinterferewiththefunctionoflysosomalenzymes[7],therebyincreasingautophagosomeaccumulationinthecell.BAECstreatedwith2-DGandchloroquineorbafilomycinAhadhigherlevelsofLC3-IIthancellstreatedwith2-DGalone(Fig.1B),whichindicates2-DGactsbyinducingautophagosomeformationanddoesnotdisrupttheirdownstreammaturationintoautophagolysosomes.

Toconfirmtheeffectof2-DGonautophagosomeformation,immunofluorescenceanalysisofBAECexpressingGFP-LC3andstainedwithLysotracker(anorganelle-selective,fluorescentprobethatlabelsandtracksacidicorganelles,suchaslysosomes)waspreformed.2-DGincreasedformationofautophagosomes,indicatedbyincreaseinthenumberanddistributionofGFP-taggedLC3spots(Fig.1C).Treatmentofcellwithboth2-DGandchloroquinefurtherincreasedthenumberanddistributionofLC3-GFPspotsinthesecells.Further,2-DGincreasedfusionofGFP-LC3taggedvacuoleswithlysosomes,whichisindicatedbyco-localizationofGFP-LC3withLysoTrackerstainedvesicles(Fig.1C,mergedimage).Thisdemonstratedthat2-DGtreatmentincreasedfusionofautophagosomeswithlysosomes,adefinitiveeventintheinductionofcellularautophagy.Together,thesedatashowthat2-DGinducesconversionofLC3-ItoLC3-IIandtriggersautophagyinendothelialcells.

spots)comparedtonon-2-DGtreatedcells(Fig.1F).However,SOD1andcatalaseoverexpressionattenuatedthe2-DG-mediatedformationofautophagosomes.OveralltheseresultsindicatethatROSarecriticallyinvolvedin2-DG-inducedautophagyandthatH2O2treatmentofendothelialcellsinducesautophagy.

2-DGinducedAMPKactivationinadose-andtime-dependentmanner

TofurtherestablishifROSisresponsiblefor2-DG-inducedAMPKactivation,weperformedatime-coursestudytodetermineifROSproductionproceedsAMPKactivation.ConfluentBAECwerestimulatedwith5mM2-DGfor5to120min,andAMPKactivationwasmeasuredbymonitoringphosphorylationofAMPKa-Thr172.AsdepictedinFig.2A,2-DGtreatmentincreasedthephosphorylationofAMPKa-Thr172within5min,andthisreachedamaximumof2.8-foldgreaterthanthatin2-DGuntreatedcellsby10minaftertreatmentwithoutaffectingthetotalAMPKlevels.After10min,phosphorylatedAMPKa-Thr172levelsdecreasedandreturnedtobasallevelsby60minaftertreatment.ACCisawell-establisheddownstreamtargetofAMPKandisactivatedbyphosphorylationofSer79.WemeasuredACC-Ser79phosphorylationandobservedasimilartimecourseforACCactivationasthatforAMPK(FigureS1inTextS1).Further,2-DGincreasedthephosphorylationofAMPK-Thr172(Fig.2B)andACC-Ser79(FigureS1inTextS1)inadose-dependentmanner.2-DGalsoincreasedAMPKactivityto4-foldafter5mincomparedtobasalactivityandreachedapeak5.5-foldhigherthanbasalactivityafter10min(Fig.2C).Thesedataestablishthat2-DGinducesAMPKactivationinbothatime-anddose-dependentmanner.

AMPKisrequiredfor2-DG-inducedautophagy

Incancercells,AMPKisreportedtobeakeyregulatorofautophagyviaamechanismthatinvolvesinactivationofmTOR[30]andcardiacmyocytes[31].WethereforeexploredwhetherAMPKwasalsoinvolvedin2-DG-inducedautophagyinendothelialcells.BAECweretransducedwithanadenovirusvectorencodingadominant-negativeAMPK(AMPK-DN)toblockcellularAMPKfunctions.2-DGtreatmentofcellsexpressingtheGFPnegativecontrolfor24hrsinduceda2-foldincreaseinLC3-IIlevels,butAMPK-DNoverexpressionblockedthe2-DG-inducedincreaseinLC3-IIlevels(Fig.3A).WeconfirmedtheseresultsbysiRNA-mediatedsilencingoftheAMPKagene,whichisthecriticalcatalyticsubunitofAMPKinHUVEC(Fig.3B).AsobservedinBAEC,2-DGtreatmentincreasedLC3-IIlevels2-foldby24hrsposttreatmentincellstransfectedwithnon-targetedcontrolsiRNA,buttransfectionwithAMPKa1/2-targetedsiRNAdramaticallyinhibitedAMPKexpressionandreducedthe2-DG-inducedincreaseintheLC3-IIlevelsto1.3-fold(Fig.3B).Takentogether,thesedatashowthatAMPKisrequiredfor2-DG-inducedautophagyinendothelialcells.

H2O2mediates2-DG-inducedautophagy

CurrentliteratureindicatesthatH2O2inducesautophagyinHeLacellsandHEK293cells[14,33].Thesefindingsareintriguingbecausenutrientstarvation(suchas2-DGtreatment)isknowntogenerateROSinavarietyofcelltypes.WethereforetestedwhetherH2O2couldalsotriggerautophagyinendothelialcells.TreatmentofBAECfor30minwithH2O2(100mM)increasedtheLC3-II-to-actinratio1.6-fold(Fig.1D).WealsoreasonedthatblockingtheeffectsofROSshouldmitigatethephenotypeof2-DG-inducedautophagy.Wetestedthishypothesisbyassessingtheeffectof2-DGontheconversionofLC3ItoLC3IIinBAECoverexpressingeitherSOD1orcatalasefromanadenovirusvector.SOD1isanenzymethatbuffersO22byconvertingittoH2O2andcatalasethenscavengesH2O2(FigureS1inTextS1).BothSOD1andcatalaseoverexpressiondecreased2-DG-enhancedLC3-IIconversion.2-DGtreatmentofcellsexpressingtheGFPnegativecontrolhada2.0-foldincreaseinLC3-IIconversion,whichwashigherthanthe1.5-foldincreaseobservedinSOD1-overexpressingcellsand1.4-foldincreaseincatalase-overexpressingcells(Fig.1E).Tofurtherelucidatetheeffectofantioxidantenzymeoverexpressionon2-DG-inducedautophagosomeformation,immunofluorescenceanalysisofBAECexpressingGFP-LC3waspreformed.2-DGtreatment(lowerthreepanels)increasedformationofautophagosomes(greenfluorescent

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AMPKinducesautophagyviadownstreammTORsignaling

ThemTORintegratestheinputfromseveralupstreampathwaysbysensingthenutrientlevels,bioenergeticstatus,andredoxstateofthecell[34].WehypothesizedthatmTORsignalingwasinvolvedinthe2-DGinducedAMPK-mediatedinductionofautophagy.WethereforeexaminedphosphorylationstatusofAMPKandmTORafterAICAR,2-DG,andrapamycintreatment.The70-kDaribosomalproteinS6kinase(p70S6K)andeukaryoteinitiationfactor4Ebindingprotein1(4E-BP1)arethetwowell-characterizedtargetsofmTORC.Whenweprobed

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Figure1.2-DG-inducedautophagyisROS-dependent.A:ConfluentmonolayersofBAECweretreatedwith5mM2-DGfortheindicatedtimes.CelllysateswereanalyzedbyWesternblotusingantibodyagainstLC3B.(n=3;one-wayANOVA:*p,0.05vscontrol).B:ConfluentmonolayersofBAECweretreatedwith5mM2-DGfor24hrsinthepresenceorabsenceofchloroquine(3mM)orbafilomycinA(10nM).CelllysateswereanalyzedbyWesternblotfordetectionofLC3.(n=3;two-wayANOVA:*p,0.052-DGvsvehicle,2-DG+chloroquinevs.chloroquine,2-DG+bafilomycinAvs.bafilomycinA.p,0.05,2-DGvs.2-DG+bafilomycinAor2-DG+chloroquine).C:BAECexpressingGFP-LC3weretreatedwithchloroquineor2-DG,andtheaccumulationofLC3II(green),localizationofLC3IIwithlysosomes(red),andDAPI(blue)stainingofnucleiinresponsetotreatmentwereanalyzedbyfluorescencemicroscopy.Scalebars,5mm.D:ConfluentmonolayersofBAECweretreatedwith100mMH2O2fortheindicatedtimes.CelllysatesanalyzedbyWesternblotusingantibodyagainstLC3(n=3;one-wayANOVA:*p,0.05vscontrol).E:BAECweretransducedadenovirusvectorsencodingSOD1orcatalasefor48hrsandthenexposedto5mM2-DGfor24hrs(n=3;two-wayANOVA,*p,0.05,GFPvs.2-DG,SOD1vs

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