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

AutophagyinEndothelialCells

2-DG+SOD1,catalasevs2-DG+catalase,p,0.05,2-DGvs.2-DG+catalaseoverexpression).F:TheaccumulationofLC3II(green),localizationofLC3IIlysosomes(red),andDAPI(blue)stainingofnucleiinresponsetoSOD1orcatalaseoverexpressionand2-DGtreatmentwereanalyzedbyfluorescencemicroscopy.

doi:10.1371/journal.pone.0017234.g001

BAECbyWesternblotanalysis,weobservedthatbothAICARand2-DGledtothephosphorylationofAMPKaswellasitsupstreamactivatorACC(Fig.2C).Aspredicted,bothAICARand2-DGalsoattenuatedthephosphorylationofmTORanditstwotargets,p706Kand4E-BP1.Similarly,thecanonicalmTORinhibitorrapamycinincreasedofLC3-IIlevelswithoutaffectingAMPKphosphorylation(Fig.2CandFigureS2inTextS1).Thus,weconcludedthat2-DGinducedautophagyviaactivationofAMPK,whichfunctionsthroughthedownstreaminhibitionofthemTORsignalingpathway.

ROSisinvolvedin2-DG-inducedAMPKactivation,whichcanbeattenuatedbyanti-oxidanttreatment

Recentstudiesusingcancercelllines[28]andC.Elegans[35]showedthatglucosedeprivationactivatedAMPKbyproductionofROS.However,itisnotclearwhetherROSalsoregulate2-DG-inducedAMPKactivationinendothelialcells.Toevaluatethis,wefirstexaminedtheeffectsofantioxidantson2-DG-inducedAMPKactivationinBAEC.Pre-treatmentofBAECwithTempol(anO22scavenger;10mM)orNAc(athiolantioxidant;2mM)significantlyreduced2-DG-enhancedphosphorylationofAMPK.Inaddition,NAcinhibitedACCphosphorylationinducedby2-DG(Fig.4AandB,FigureS2inTextS1).WefurtherassayedAMPKactivityinBAECbyquantifyingphosphorylationoftheSAMSpeptide,anAMPKsubstrate,usinga[32P]-ATPassay.Aftertreatmentwith2-DG(5mM),AMPKactivitywas3-foldgreaterthanbaselinewithin10min,andNActreatmentcaused

AMPKactivitytodropfrom3-foldto2-foldoverbaselinelevels(Fig.4C).Finally,weoverexpressedantioxidentproteinsSOD1orcatalasetofurtherexaminetheinvolvementofROS(FigureS3inTextS1).OverexpressionofeitherSOD1orcatalase,butnotGFP,attenuated2-DG-enhancedphosphorylationofAMPKandACC(Fig.4D).IntheSAMSpeptidephosphorylationassay,2-DGtreatmentincreasedAMPKactivity7-fold,andthiswasenhanced2-foldbyoverexpressionofSOD1and2.5-foldbyoverexpressionofcatalase(Fig.4E).OveralltheseresultsindicatethatROSareinvolvedin2-DG-inducedAMPKactivationinendothelialcells,aphenomenonthatisreversibleifROSlevelsaresuppressedwithanti-oxidants.

2-DGincreasesintracellularH2O2levelsinendothelialcells

Wenextdeterminedtheeffectof2-DGonROSproductioninendothelialcellsusingCM-H2DCFDAasanintracellularH2O2probe.ItisknownthatAMPKactivationbyAICARreducesthegenerationofROS[20].TocircumventtheconfoundingpotentialofROSattenuationbyAMPKactivation,wedetectedintracel-lularH2O2in2-DG-stimulatedHUVECtransfectedwithAMPK-targetedsiRNA.Asexpected,theintracellularH2O2levelsincellsinwhichAMPKa1/2wasknockeddownweresignificantlyhigherthanincellstransfectedwiththenon-targetedcontrolsiRNAfollowinga10–min,5-mM2-DGtreatment(Fig.5A).Pre-treatmentwithacatalaseinhibitor3-amino-1,2,4-triazine(ATZ)andthioredoxinreductaseinhibitor1-chlo-2,4-dinitrobenzene

Figure2.Time-courseanddose-responsefor2-DG-inducedAMPKactivation.A:ConfluentBAECmonolayersweretreatedwith2-DG(5mM)fortheindicatedtimes.CelllysateswereanalyzedbyWesternblotusingantibodyagainstp-AMPKandAMPKa1/2(n=3;one-wayANOVA:*p,0.05vs.control).B:ConfluentBAECmonolayersweretreatedwiththeindicatedconcentrationof2-DGfor10min(n=3;one-wayANOVA:*p,0.05vs.control).C:BAECwereincubatedwith5mMof2-DGfortheindicatedtimes.AMPKwasimmunoprecipitatedfromcelllysiswithanantibodyagainstAMPKaboundtoProteinA/Gagaroseovernightat4uC.AMPKactivitywasdeterminedbySAMSphosphorylationusinga[32P]ATPassay(n=3;one-wayANOVA,*p,0.05vs.control).doi:10.1371/journal.pone.0017234.g002

PLoSONE|www.plosone.org5February2011|Volume6|Issue2|e17234

AutophagyinEndothelialCells

Figure3.2-DG-inducedautophagyisAMPK-dependent.A:BAECweretransducedwithadenovirusvectorsencodingdominantnegativeAMPK(AMPK-DN)for48hrsandthentreatedwith5mM2-DGfor24hrs(n=3;two-wayANOVA,*p,0.05,GFPvs.2-DG,p,0.05,2-DGvs.2-DG+AMPK-DN).B:HUVECweretransfectedwithAMPK-targetedsiRNAorcontrolsiRNAfor24hrsthentreatedwith5mM2-DGfor24hrs.CelllysateswereanalyzedbyWesternblotusingantibodyagainstLC3(n=3;two-wayANOVA,*p,0.05,vehiclevs.2-DG,p,0.05,ControlsiRNAvs.AMPKsiRNA).C:BAECweretreatedwith1mMAICAR,10nMrapamycin,or5mM2-DGfor24hrsandthenanalyzedbyWesternblottodeterminethelevelofphospho-AMPK-Thr172(p-AMPK),phos-ACC-Ser79(p-ACC),phos-mTOR-Ser2448(p-mTOR),mTOR,phos-p70S6K-Thr389(p-p70S6K),p70S6K,phos-4EBP1-Thr37/46(p-4EBP1),andLC3.doi:10.1371/journal.pone.0017234.g003

Figure4.Inhibitoryeffectsofantioxidantson2-DG-inducedAMPKactivationinBAEC.A,B:ConfluentBAECmonolayerswerepretreatedwith10mM4-hydroxy-Tempol(Tempol)or2mMN-Acetyl-Cysteine(NAc)for30minandthentreatedwith5mM2-DGfor10min.CelllysatesweresubjectedtoWesternblotanalysisusingantibodiesagainstp-AMPK,p-ACC,AMPKa1/2,andb-actin(n=3;two-wayANOVA,*p,0.05,2-DGvs.control,Tempol+2-DGvs.Tempol,NAc+2-DGvs.Nac,p,0.052-DGvs2-DG+TemorNAc).C:BAECwerepretreatedwithNAcandthenstimulatedwith2-DG.AMPKactivitywasdeterminedbySAMSphosphorylationusinga[32P]ATPassay(n=3;two-wayANOVA,*p,0.05,vehiclevs2-DG,Nacvs2-DG+NAc,p,0.052-DGvs.2-DG+NAc).D,E:ConfluentBAECmonolayersweretransducedwithadenovirusvectorsencodingSOD1orcatalasefor48hrsandthentreatedwith5mM2-DGfor10min.PhosphorylationofAMPKandAMPKactivityweredetected,asdescribedintheMatieralsandMethods(n=3;two-wayANOVA,*p,0.05,GFPvs.2-DG,SOD1vs2-DG+SOD1,catalasevs2-DG+catalase,p,0.05,2-DGvs.2-DG+SOD1orcatalaseoverexpression).doi:10.1371/journal.pone.0017234.g004

PLoSONE|www.plosone.org6February2011|Volume6|Issue2|e17234

AutophagyinEndothelialCells

Figure5.2-DGincreasesintracellularsynthesisofH2O2toinduceautophagy.A:HUVECweretransfectedwithAMPK-targetedsiRNAfor24hrs.ThenthecellswereincubatedinEBMmediawithoutphenolredandtreatedwith5mMof2-DGfor10min.CM-H2DCFDA(10mM;Invitrogen)wasaddedfor30minbeforequantificationoffluorescence(excitationat485nmandemissionat545nm).(n=3;two-wayANOVA,*p,0.05,2-DGvs.vehicle).B:BAECwerepre-treatedwith3-amino-1,2,3-triazine(ATZ,100mM)and1-chloro-2,4-dinitrobenzene(DNCB,100mM)for30minandthentreatedwith2-DGfor5or10min.IntracellularH2O2levelsweredetectedbyfluorescenceusingCM-H2DCFDA.(n=3;two-wayANOVA,*p,0.05,ATZ+DNCBvs.vehicle,p,0.05,2-DG+ATZ+DNCBvs.ATZ+DNCB).C:BAECweretreatedwith2-DGfor24hrs.IntracellularH2O2levelsweredetectedbyfluorescenceusingCM-H2DCFDA.(n=3;t-test,*p,0.05,2-DGvs.vehicle).D,E:HUVECweretransfectedwithAtg4-targetedsiRNAorcontrolsiRNAfor24hrsandthentreatedwith5mM2-DGfor24hrs.CelllysateswereanalyzedbyWesternblotusingantibodyagainstLC3(n=3;two-wayANOVA,*p,0.05,vehiclevs.2-DG,p,0.05,ControlsiRNAvs.Atg4siRNA).doi:10.1371/journal.pone.0017234.g005

(DNCB)ledtoasignificantlyincreasedintracellularH2O2levelin2-DG-treatedBAEC(Fig.5BandC).

SuppressionofAtg4attenuates2-DGinducedautophagy

Sherz-Shouvaletal.[16]recentlyreportedthatROSareessentialforautophagyandspecificallyregulatetheactivityofAtg4.AlthoughthesestudiesweredoneinChineseHamsterOvaryandHeLacelllines,itislikelythattheseresultscanbeextrapolatedtoendothelialcellsaswell.Basedonthesefindings,weexaminedAtg4expressioninHUVECafter2-DGtreatmentoraftertransfectionofAtg4-targetedsiRNA.AsshowninFig.5D,silencingofAtg4efficientlyblockedAtg4expressioninHUVEC.Further,2-DGtreatmentofAtg4-silencedcellsledtoanattenuatedincreaseinLC3-IIlevels.ThesefindingsareconsistentwiththeobservationthatH2O2inhibitsAtg4,whichinturnpromoteslipidationofAtg8leadingtoincreasedautophagy.

inBAECexposedto2-DGatdifferenttimepointsaftertreatment.BothAMP:ATPandADP:ATPratiossignificantlyincreasedby5minafter2-DGaddition(Fig.6A).NeitherpretreatmentofBAECwithTempolorNAc(Fig.6B)noroverexpressionofSOD1orcatalaseinhibitedtheobservedincreaseintheseratios(Fig.6C).Thisisthefirststudytodemonstratethat2-DG-inducedAMPKactivationismediatedbyROSandislikelyindependentoftheAMP:ATPnucleotideratio.

MitochondriaarethesourcesofROSin2-DG-treatedendothelialcells

Mitochondria,NAD(P)Hoxidase,andxanthineoxidase[37]cangenerateROSinbothphysiologicalandpathologicalconditions.WesoughttoidentifythemajorsourcesofROSsynthesisin2-DG-treatedendothelialcells.Mito-TempolisasyntheticTempolderivatethatpreferentiallyscavengesO22frommitochondria.Pre-treatmentofcellswith10mMmito-Tempolfor30mindramaticallydecreased2-DG-inducedphosphorylationofAMPK(Fig.7A).Further,overexpressionofMnSOD,aSODisoformlocatedinthemitochondrialmatrix(FigureS3inTextS1),attenuatedthe2-DG-inducedphosphorylationofAMPKandACC(Fig.7B).

Mitochondrialuncouplingprotein-2(UCP-2)isamitochondrialanioncarrierproteinthatmediatesmitochondrialprotonleakageanduncouplesATPsynthesisfromoxidativephosphorylation.UCP-2overexpression(FigureS3inTextS1)dramaticallydecreasedthe2-DGphosphorylationofAMPKandACC(Fig.6C).Consistently,transfectionofUCP-2-targetedsiRNA,

7

February2011|Volume6|Issue2|e17234

ROS-dependentAMPKactivationisindependentofchangesintheAMP-to-ATPratio

Scottetal.foundthatthereareregulatoryAMP-andATP-bindingsitesinthesubunitsofAMPK[36].Asaconsequence,highconcentrationsofATPantagonizeAMPKactivationthatresultsfromAMPbindingtotheheterotrimer.2-DGappearstoinduceAMPKactivationprimarilythroughthedecreaseinintracellularATPlevelsthatresultsfromblockingglycolysis.WespeculatedthatROS-mediatedAMPKactivationviaglucosedeprivationmightnotdependonachangeintheAMP-to-ATPratio.WethereforequantifiedtheADP:ATPandAMP:ATPratios

PLoSONE|www.plosone.org

AutophagyinEndothelialCells

Figure6.InhibitionofAMPKbyantioxidantsisindependentoftheAMP:ATPratio.A:ConfluentBAECmonolayersweretreatedwith5mMof2-DGfortheindicatedtimes.CellswerelysedbyperchloricacidandcentrifugedasdescribedintheMaterialsandMethods.TheultrafiltratewasinjectedintoaJascoHPLC,andATP,ADP,andAMPweremonitoredat260nm.(n=4;one-wayANOVA,*p,0.05,**p,0.01,Controlvs.2-DGtreatment).B:BAECmonolayerswerepre-treatedwith10mMTempolor2mMNACandthentreatedwith5mMof2-DGfor5min.TheAMP:ATPratioincelllysateswasmeasuredbyHPLC(n=5;two-wayANOVA,*p,0.05,**p,0.01,2-DGvs.vehicle,Tempolvs.2-DG+Tempol,NAcvs.2-DG+NAc).C:BAECoverexpressingSOD1orcatalaseweretreatedwith5mM2-DGfor5min.TheAMP:ATPratioincelllysateswasmeasuredbyHPLC.(n=3;*p,0.05,**p,0.01,two-wayANOVA,GFPvs.2-DG,SOD1vs.2-DG+SOD1,catalasevs.2-DG+catalase).doi:10.1371/journal.pone.0017234.g006

butnotcontrolsiRNA,enhancedbothbasalphospho-AMPKlevelsand2-DG-inducedAMPKphosphorylationinHUVEC(Fig.7DandFigureS3inTextS1).TofurtheridentifythesourcesofROSinthesecells,weusedinfectedcellswithadenovirusvectorscarryingdominantnegativesubunitsofNADPHoxidasetoinduceNAD(P)Hoxidasedysfunction.p47phoxandp67phoxaresubunitsoftheNADPHoxidasethatactivateNAD(P)Hoxidaseactivitybybindingwithgp91phoxonthemitochondrialmembrane[38],butexpressionofdominantnegativep47phox(p47-DN)andp67phox(p67-DN)leadtoNAD(P)Hoxidasedysfunction.p47-DNorp67-DNoverexpressiondidnotaffect2-DG-inducedAMPKandACCphosphorylation(Fig.7EandFigureS3inTextS1).Moreover,incubationofBAECwiththexanthineoxidaseinhibitors,allopurinolandoxypurinol,didnotblock2-DG-inducedAMPKandACCphosphorylation(Fig.7FandG).ThesefindingsstronglysuggestthatmitochondriaarethemainsourceofROSinducedby2-DGinBAEC.

(LDH)fromcellsasamarkerofplasmamembranedamage,reflectingthedegreeofcelldeath.LDHrelease,andthereforecelldeath,inBAECpre-treatedwith2-DG(5mM)wassignificantlylowerthanthatinuntreatedcells(Fig.8A).Intheseexperiments,wewerealsoabletodemonstratethatwhereas2-DGprotectedcellsfromhypoxia-induceddeath,pre-treatmentwith3-methyladenine(3-MA),anautophagyinhibitor,partiallyblockedthe2-DG-inducedprotection(Fig.8B).Incontrast,SOD1orcatalaseoverexpressionblockedthe2-DG-inducedprotectionandcellsurvivalunderhypoxicconditions(Fig.8C).Finally,AMPK-DNoverexpressionprevented2-DG-inducedcellsurvival(Fig.8D).Together,theseresultsshowthat2-DGinducesautophagythroughAMPKactivationinaROS-dependentmanner,therebyprotectingcellsfromhypoxia-inducedcelldeath.

Discussion

Thepresentstudyhas,forthefirsttime,demonstratedthatAMPKactivationbymitochondrialROSisrequiredfortheinductionofautophagyinendothelialcells.Mechanistically,wefoundthatROS,viaAMPKactivation,increaseautophagybyinhibitionofmTORandAtg4activityandROS-triggeredautophagyincreasesendothelialcellsurvivalunderhypoxicconditions.Thus,ourresultsrevealanovelsignalingpathwayinwhichmitochondrialROSactivateAMPKthatinturnincreasesautophagy.Inductionofautophagythenservestoincreaseendothelialcellsurvival(Fig.8E).

8

February2011|Volume6|Issue2|e17234

AMPK-regulatedautophagycontributestoendothelialcellsurvivalunderhypoxicconditions

Wenextsoughttodemonstratethephysiologicalrelevanceofourfindings.Forthispurpose,weexposedBAECtohypoxicconditionsandtreatedthemwith2-DGtodeterminetherolethatautophagyplaysunderhypoxicconditions.AsshowedinFigure8A,weobservedincreasedcelldeathinBAECafter12hrsofhypoxia.Interestingly,2-DGpre-treatmentpreventedthehypoxia-inducedcelldeath.Wequantifiedthereleaseoflactatedehydrogenase

PLoSONE|www.plosone.org