Inflammatory Macrophages_Induce_Transcription_Factor-dependent_Proteasome_Activity_in_Colonic_N1

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Supplemental Material can be found at:http://wendang.chazidian.com/content/suppl/2011/10/11/M111.274902.DC1.html

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.286,NO.47,pp.40911–40921,November25,2011

©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedintheU.S.A.

InflammatoryFactor-dependentMacrophages

CellsandTherebyConferProteasomeInduceNrf2Transcription

Anti-apoptoticActivityinProtectionColonicNCM460*□S

Receivedforpublication,June23,2011,andinrevisedform,October7,2011Published,JBCPapersinPress,October11,2011,DOI10.1074/jbc.M111.274902

SusanneSebens?§,IrisBauer?,ClaudiaGeismann?,EvelinGrage-Griebenow?§,StefanEhlers¶,Marie-LuiseKruse?,AlexanderArlt?,andHeinerSchäfer?1

Fromthe?DepartmentofInternalMedicineI,LaboratoryofMolecularGastroenterology&Hepatologyand§Institutefor

ExperimentalMedicine,Universita¨tsklinikumSchleswigHolstein-CampusKiel,Kiel,Germanyandthe¶DivisionofMolecularInflammationMedicine,ResearchCenterBorstel,LeibnizCenterforMedicine&Biosciences,Borstel,Germany

Adaptationofepithelialcellstopersistentoxidativestresstoapoptosis.Thismechanismmightcontributetoinflamma-playsanimportantroleininflammation-associatedcarcinogen-tion-associatedcarcinogenesis,e.g.ofthecolon.

esis.ThisadaptationprocessinvolvesactivationofNrf2whichhasbeenrecentlyshownto

contributetocarcinogenesisthroughtheinductionofprotea-Asanimportantmechanismininflammation-associatedcar-somalgeneexpressionandproteasomeactivity.Toverifythiscinogenesis,epithelialcellsundergoapermanentadaptationtopossiblelinkbetweeninflammation,oxidativestress,andNrf2-oxidativestressconditionscomingalongwiththechronicdependentproteasomeactivation,weexploredtheimpactofexposuretoinflammatorycellsandtheirreleasedmediators.inflammatory(M1)macrophagesonthehumancolonepithelialAlongwiththisadaptation,thestressedepitheliumacquiresacelllineNCM460.Transwellcocultureswithmacrophagesdif-cellularphenotypepavingthewayforthedevelopmentofcan-ferentiatedfromgranulocytemonocyte-colony-stimulatingfac-cer(1–3).Onemechanismofstressadaptationistheactivationtor-treatedmonocytesledtoanincreasedactivityofNrf2inoftheoxidativestress-responsivecap’ncollar-bZIPtranscrip-NCM460cellsalongwithanelevatedproteasomeactivity.ThistionfactorNrf2UnderhigherproteasomeactivityresultedfromNrf2-dependentconditionsofoxidativeandelectrophilicstress,Nrf2isacti-inductionofproteasomalgeneexpression,asshownforthe19vatedthroughitsreleasefromtheinhibitoryproteinKeap1and20SsubunitproteinsS5aand?5,respectively.Theseeffectsandsubsequentnuclearofmacrophagecoculturewereprecededbyanincreaseofreac-accumulation(4–8).

tiveoxygenspeciesincoculturedNCM460cellsandcouldbeNrf2hasbeenprimarilyrecognizedasmodulatorofananti-blockedbycatalaseorbythereactiveoxygenspeciesscavengeroxidativeresponsebyinducingtranscriptionofphaseIITiron,whereastransienttreatmentofNCM460cellswithH2O2enzymessuchasGST,hemeoxygenase-1,orNAD(P)H-qui-similarlyledtoNrf2-dependentproteasomeactivation.noneoxidoreductase1,whichprotectthecellfromreactiveThroughtheNrf2-dependentincreaseofproteasomalgeneoxygenspecies(ROS)-induced2damage(7).Thisprotectionexpressionandproteasomeactivity,thesensitivityofNCM460includesthepotentialofNrf2-inducedphaseIIenzymeexpres-cellstotumornecrosisfactor-relatedapoptosis-inducingsiontopreventgenotoxicinsultsfromoxidativestress,thusligand-oririnotecan-inducedapoptosisdeclined.Thesefind-makingNrf2activationapromisingtoolinchemopreventioningsindicatethatinflammatoryconditionssuchasthepresenceofcancer(6).Electrophilsandanti-oxidantssuchastert-butyl-ofM1macrophagesandtheresultingoxidativestressarehydroquinone(tBHQ)andthephytochemicalsulforaphaneareinvolvedintheNrf2-dependentgainofproteasomeactivityinwellknowntoinduceNrf2andtherebytostimulatephaseIIepithelialcells,e.g.colonocytes,givingriseofgreaterresistanceenzymeexpression.Thus,Nrf2inducingagentshavebeensug-

gestedtobeofsubstantialbenefitincancerprevention(5,6,9).

*ThisworkwassupportedbyDeutscheForschungsgemeinschaftGrant2Theabbreviationsusedare:ROS,reactiveoxygenspecies;c-H2DCF-DA,□SCHA677/9-1andtheClusterofExcellence“InflammationatInterfaces.”5-carboxy-2?,7?-dichlorodihydro-fluoresceinediacetate;GM-CSF,granulo-STheon-lineversionofthisarticle(availableathttp://wendang.chazidian.com)containscytemonocyte-colonystimulatingfactor;Suc-LLVY-AMC,N-succinyl-L-leu-supplementaltextandFigs.S1–S3.cyl-L-leucyl-L-valyl-L-tyrosyl-7-amido-4-methylcumarin;M-CSF,monocyte-1Towhomcorrespondenceshouldbeaddressed:Dept.ofInternalMedicine,colonystimulatingfactor;M-g,GM-CSFmacrophage;M-m,M-CSFIArnold-Heller-Strasse3,Bldg.6,24105Kiel,Germany.Fax:431-5971427;macrophage;tBHQ,tert-butyl-hydroquinone;TRAIL,tumornecrosisfac-E-mail:hschaef@1med.uni-kiel.de.tor-relatedapoptosis-inducingligand;ARE,antioxidantresponseelement.NOVEMBER25,2011?VOLUME286?NUMBER

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MacrophagesandNrf2-dependentProteasomeActivation

However,evidenceaccumulatesthatNrf2canalsopromotetumorigenesis(10–13)andresistanceoftumorstochemother-apy,e.g.byinducingdetoxificationofanti-cancerdrugsinaphaseII-dependentfashion(14–16).Moreover,Nrf2isinvolvedinthegainofanti-apoptoticprotectionbytumorcells(11,17,18).Tosomeextent,thisprotectiveeffectagainstapo-ptosiscouldbeattributedtotheNrf2-inducedexpressionofproteasomalgenesleadingtoanelevatedproteasomeactivityintumorcells(11,19–24).WhereasthisproteasomeinducingeffectofNrf2inresponsetooxidativeandelectrophilicstresshasbeenwidelyreportedalready(11,25–30),recentdatarevealedthatthecloselyrelatedtranscriptionfactorNrf1(TCF11)playsaparticularroleinthemaintenanceofconstitu-tiveproteasomeactivity(31).Moreover,Nrf1mediatestheinducingeffectofproteasomeinhibitorsonproteasomalgeneexpressiontoagreaterextentthanNrf2(31,32).Thus,Nrf1isregardedasmodulatorofbasalproteasomalgeneexpressionandproteasomeactivity,whereasNrf2isbelievedtomediatetheinducedproteasomalgeneexpressionandproteasomeactivity(33),particularlyinresponsetoelectrophilicandoxi-dativestress(34).

Throughanenhancedproteasomeactivityintumorcells,suchascoloncancercells(11),Nrf2activationconfersresist-ancenotonlytoanti-cancerdrugs,heretogetherwiththedetoxificationbyphaseIIenzymes,butalsotodeathligandssuchasTRAIL.Inthelattercase,wehaverecentlyshownthatNrf2-dependentproteasomeactivationpromotesNF-?Bacti-vationbyTRAIL,aneffectthatmainlyreliesontheforcedturn-overofI?B?asaresultofthegainofproteasomeactivity(11).FromTRAIL-inducedNF-?Bactivation,increasedexpressionlevelofcertainanti-apoptoticgenes(e.g.cIAP1)emerged(11)throughwhichTRAIL-inducedapoptosisisinhibited.Inthisway,Nrf2activationandsubsequentinductionoftheprotea-somemightcontributetotheacquisitionofatumorigenicphe-notypeofepithelialcellsduringinflammationassociatedcarci-nogenesis,e.g.ofthecolon.

Thepresentstudythereforeaimedatelucidatingwhetherthepresenceofinflammatorycells,inparticularmacrophagesthatareamajorconstituentofimmunecellinfiltratesandasourceofROSinchronicallyinflamedtissues(35–38),altersprotea-somalgeneexpressionandproteasomeactivityincolonicNCM460cellsinaNrf2-dependentfashion.Moreover,itwasinvestigatedwhetherundertheseconditionstheprotectionofNCM460cellsagainstapoptosisisenhanced.Ourdatademon-stratethattheexposuretoinflammatorymacrophagesleadstoaNrf2-dependentincreaseofproteasomalgeneexpressionandproteasomeactivityinNCM460cellsandasaconsequenceleadstoapoptosisresistance.ItwasalsofoundthatROSplayaparticularroleinthisresponseofNCM460cellsexposedtoinflammatorymacrophages.

EXPERIMENTALPROCEDURES

Materials—IL-1?andIL-6werefromCalbiochem(Mann-heim,Germany).TNF?,Tiron,andcatalasewerepurchasedfromSigma.TNF?,IL-6,TGF?,andIL-10ELISAswerefromEBioscience(Frankfurt,Germany),andtheIL-1?ELISAwasfromR&DSystems(Wiesbaden,Germany),andN-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-7-amido-4-methylcumarin

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(Suc-LLVY-AMC)andMg132werefromBiomol(Taunusstein,Germany).Killer-TRAILwasfromEnzoLifeScience/Alexis(Lörrach,Germany),andirinotecanwasfromPfizer(Berlin,Germany).5-Carboxy-2?,7?-dichlorodihydro-fluoresceindi-acetate(c-H2DCF-DA)andMitoSOXTMRedwerepurchasedfromMolecularProbesviaInvitrogen.

CellCulture—HumanNCM460colonocytes(39)werepro-videdbyINCELL(SanAntonio,TX)andculturedinM3Amedium(INCELL)containing10%FCS.Thecellswerecul-turedat37°C,5%CO2,and85%humidity.Forcoculture,NCM460cellswereseededonto12-wellplatesatadensityof1?105cells/well,incubatedfor24h,andtheninsertswith5?105macrophageswereaddedfor6–96h.ForthetimeofsiRNAtreatmentofNCM460cells(seebelow),theinsertswereplacedinaseparate12-wellplate;afterward,thecocultureofsiRNA-treatedNCM460cellsandmacrophagescontinued.TransientexposureofNCM460cellstoH2O2(200?M)wasconductedinprewarmedPBSfor30minatstandardcultureconditions,andthentheculturemediumwasreplaced.

GenerationandCultureofMacrophages—Fifteen?106monocytes(95%purity)generatedfromhumanperipheralbloodmononuclearcellsbycounterflowcentrifugationweretransferredintoTeflonbags(VueLifeTeflonbags;Süd-Laborbedarf,Gauting,Germany)andculturedinRPMI1640(Biochrom,Berlin,Germany)supplementedwith1%L-gluta-mine,10%FCS,and100?g/mlpenicillin/streptomycin(PAALaboratories,Cölbe,Germany)inthepresenceof50ng/mlGM-CSForM-CSF(RELIATech,Wolfenbüttel,Germany)for7daysasdescribed(40,41).MacrophagesweredetachedbyplacingtheTeflonbagsonicefor1h,andthenthecellswereresuspendedinice-coldPBS.Thephenotypeofthemacro-phageswasverified(supplementarymaterial)byrealtimeRT-PCRandELISAfordeterminationoftheexpressionofpro-inflammatory(IL-1?,TNF?,andIL-6)versusanti-inflam-matory(IL-10andTGF?)mediatorsaswellasbyCD11b,CD14,andCD163immunoflowcytometry.

FluorometricProteasomeAssay—Forthedeterminationofproteasomalactivityinlivingcells,afluorometricassayusingtheproteasomesubstrateSuc-LLVY-AMCwasperformedasdescribedrecently(27).Allofthemeasurementswerecarriedoutinduplicate.

DeterminationofIntracellularROS—ThemediumofNCM460cellswasreplacedby1mlofprewarmedPBS,andthecellsweretreatedwith10?MofthecellularROSindicatorc-H2DCF-DAdissolvedinMe2SOorwiththevehiclealonefor20minat37°C.ThenthelabeledNCM460cellswereincubatedinOPTIMEM(PAALaboratories)for5minat37°Cfollowedbymono-orcocultureinOPTIMEMfor1,3,and6h.Fluores-cenceinresponsetooxidationofc-H2DCF-DAwasmeasuredwiththeTechan200infinitemicroplatereader.Fluorescenceunitswerenormalizedtotheproteincontentdeterminedinparallel.Todiscriminateendogenousgenerationofmitochon-drialROS,NCM460cellswerestainedwith250nMMitoSOXTMRedfor30minfollowingthemanufacturer’sinstructions.Thencellsweremono-orculturedasabove,andstainedcellswereanalyzedwiththeTechan200infinite,aswell.WesternBlotting—Preparationofnuclearandcytoplasmicextractsortotalcelllysateswascarriedoutasdescribedbefore

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(11,42).Afterelectrophoresisandsemi-dryelectroblottingontoPVDFmembranes,thefollowingprimaryantibodieswereusedforimmunodetectionata1000-folddilutionin5%(w/v)nonfatmilkpowder,0.05%Tween20inTBS(50mMTris/HCl,pH7.6,and150mMNaCl):Nrf2(EpitomicsviaBiomol),Hsp90andNrf1(SantaCruz,Heidelberg,Germany),tubulin(Sigma),S5aand?5(bothfromAffiniti/Biomol,Hamburg,Germany),andcleavedcaspase-3andPARP1(CellSignalingTechnology,Frankfurt,Germany).Afterincubationovernightat4°C,theblotswereexposedtotheappropriatehorseradishperoxidase-conjugatedsecondaryantibody(CellSignalingTechnology)diluted(1:1000)inblockingbufferanddevelopedusingtheDuradetectionkit(PerbioSciences,Bonn,Germany).DataacquisitionwasdonewiththeChemidoc-XRSTMgeldocumen-tationsystem(Bio-Rad)usingtheQuantityOne?software(Bio-Rad).Hsp90servedasaloadingcontrolandwasusedforbanddensitynormalization.

RNAPreparationandRealTimePCR—IsolationoftotalRNAandreversetranscriptionintosingle-strandedcDNAwascarriedoutasdescribed(27).cDNAwassubjectedtorealtimePCR(iCycler;Bio-Rad)usingtheSYBR-Greenassaywithgene-specificprimersatafinalconcentrationof0.2?M.TheprimersequencesandPCRconditionsforthedetectionofS5aand?5,aswellas?-actin,havebeendescribedrecently(27).Forampli-ficationofNrf1andNrf2,theforward/reverseprimers5?-cgg-tatgcaacaggacattg-3?/5?-actggttggggtcttctgtg-3?and5?-ttccct-gcacaggtgcctagtg3?/5?-cttccatggcctgcatttccatg-3?wereused,respectively,andamplificationwascarriedoutat95°Cfor15s,56°Cfor30s,and72°Cfor20s.ThedatawerecollectedduringannealingstepsandwerefurtheranalyzedbyusingtheiCycleriQopticalsystemsoftware(Bio-Rad).Allofthesampleswereanalyzedinduplicate,andthedataareexpressedastheamountsofmRNAnormalizedto?-actinmRNA.

siRNATreatment—ForsiRNAtransfection,NCM460cellsgrownin12-wellplatesweresubmittedtolipofectionusing6?loftheHiperFectreagent(Qiagen)and150ng/wellofeithernegativecontrolsiRNA(Qiagen)orNrf2(SI03246614;Qiagen),Nrf1(SI00657909;Qiagen),S5a(SI03019331;Qiagen),or?5(SI100043316;Qiagen)siRNA.After8h,themediumwaschanged,andcellsweresubjectedtofurthermono-orcoculture.

DualLuciferaseAssay—NCM460cellsgrownin12-wellplatesweresubjectedtolipofectionusingEffectene(Qiagen)andeither0.2?gofpAREandptkRLortheemptyvectorandptk-RL(allvectorsfromQiagen/SABioscience).After8h,themediumwaschangedfollowedbymono-orcocultureforupto72horbytreatmentsasindicated.Afterward,thecellswerewashedwithPBSandlysedin150?l/wellpassivelysisbuffer(Promega,Mannheim,Germany).Thelysateswerecentrifugedfor2minat4°Cat13,000rpm.20?lofthesupernatantwereusedforthedualluciferaseassayprocedure,usingDual-GlowluciferaseassaysystemfromPromega.Fireflyluciferaseexpres-sionwasnormalizedtoconstitutiveRenillaluciferaseexpres-sion.Allofthemeasurementswerecarriedoutinduplicate.LivingCellMicroscopy—NCM460cellsgrownontocover-slipswerestainedwith2?Mc-H2DCF-DAand100nMMitoSOXTMRedfor5minat37°CinprewarmedPBS.ThencellswereincubatedwithOPTIMEMfor5minat37°Cfol-NOVEMBER25,2011?VOLUME286?NUMBER

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lowedbymono-andcocultureasspecified.AnalysiswasdonewithanAxioScanfluorescencemicroscope(Zeiss,Jena,Ger-many)equippedwithadip-inobjective(40?magnification).MeasurementofApoptosis—ApoptosisinducedbyKiller-TRAILoririnotecanwasdeterminedbymeasurementofcaspase-3/7activity(Promega)andofthegenerationofthecytokeratin18neoepitope(M30-Apoptosense?ELISAkit;PEVIVA,Alexis,Grünwald,Germany)accordingtothemanu-facturer’sinstructionsandasdescribed(42).Alloftheassaysweredoneinduplicate.Caspase-3/7activityandM30immu-noreactivitywerenormalizedtotheproteincontentoftheana-lyzedcelllysatesandexpressedasactivity/mgprotein.

Statistics—Thedatarepresentthemeans?standarddevia-tion.SignificanceswerecalculatedbyStudent’sttestwithdatafromatleastfourindependentexperiments,apvalue?0.05wasconsideredasstatisticallysignificant.

RESULTS

Nrf2IsActivatedinNCM460ColonocytesExposedtoInflam-matoryMacrophages—ToinvestigatewhethertheexposuretomacrophagesaffectstheactivityofNrf2inNCM460colono-cytes,Transwellcocultureexperimentswereconductedusinginflammatorymacrophages(M1polarized)derivedfromelutri-atedmonocytesanddifferentiatedinthepresenceofGM-CSFfor7days(43).VerificationoftheinflammatoryphenotypewasdonebyrealtimePCRanalysisandELISAdetectinghighlevelexpressionofTNF?,IL-1?,andIL-6andlowlevelsofIL-10andTGF?(supplementalFig.S1).Inaddition,immunoflowcytom-etryanalysis(supplementalFig.S2)revealedhighCD11bandCD14expressionbuttheabsenceofCD163inthesecellsindi-catingM1polarization(41).After2daysofcoculturewiththesemacrophages(M-g),nuclearproteinlevelsofNrf2inNCM460cells,indicativeofitsactivation,wereanalyzedbyWesternblotting.AsshowninFig.1A,higherNrf2proteinlevelsweredetectableinnuclearextractsfromM-gcoculturedNCM460cellsthaninnuclearextractsfrommonoculturedNCM460cells.Conversely,theNrf2proteinlevelincytoplasmicextractsdecreased,thusindicatingenhancednuclearaccumulationofNrf2.Next,welookedwhethertheincreasednuclearNrf2pro-teinlevelscorrespondtohigherNrf2transcriptionalactivity.Forthispurpose,reportergeneassaysusingfireflyluciferaseunderthecontroloftheantioxidantresponseelement(ARE)wereconducted.AsshowninFig.1B,M-gcoculturedNCM460cellsexhibiteda2–3-foldincreaseofARE-drivenreportergeneexpressionincomparisonwithmonoculturedNCM460cells.ToelucidatewhethertheinductionofNrf2activationinNCM460cellsdependsonthepro-inflammatoryphenotypeofthemacrophages,cocultureexperimentswerealsosetupusinganti-inflammatorymacrophagesthatwereobtainedfrommonocytestreatedwithM-CSF(41).RealtimePCRanalysisandELISA(supplementalFig.S1)revealedlowerexpressionofTNF?,IL-1?,andIL-6intheseM-CSF-derivedmacrophages(M-m),alongwithhigherexpressionofIL-10andTGF?com-paredwithGM-CSFmacrophages.Immunoflowcytometryanalysis(supplementalFig.S2)furtherdetectedCD163positiv-ityofM-CSFmacrophagesincontrasttoGM-CSFmacro-phageslackingthisM2marker(41).AsshowninFig.1,aftercoculturewiththesemacrophages(M-m)boththenuclearand

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FIGURE1.CoculturewithinflammatorymacrophagesinducesNrf2activityinNCM460cells.MacrophagesderivingfrommonocytestreatedwithGM-CSF(M-g)orM-CSF(M-m)wereusedforcoculturewithNCM460cells.A,nuclear(n.e.)andcytoplasmic(c.e.)extractsfromNCM460cellsculturedfor2dayseitherintheabsence(mono)orpresenceofthesemacrophagesweresubmittedtoNrf2immunoblotting,usingHsp90andtubulinasloadingcontrol.Arepresent-ativeblot(leftpanel)andthenormalizedNrf2bandintensities(rightpanel)asdeterminedbybanddensitometryofblotsfromfourindependentexperimentsareshown(means?S.D.).*,p?0.05.B,NCM460cellsweretransfectedwiththeNrf2-responsivepAREorthecontrol(pGL3)fireflyluciferasereportervectortogetherwiththeptkRLreferencevectorexpressingRenillaluciferase.Afterward,transfectantsweremono-orcoculturedfor2days,andNCM460celllysateswereanalyzedforfireflyandRenillaluciferaseexpression.LuminescenceinducedbyfireflyluciferasewasnormalizedtotheluminescenceinducedbyRenillaluciferase;thedatarepresentthemeans?S.D.offourindependentexperimentsperformedinduplicate.RLU,relativelightunits.

cytoplasmicNrf2proteinlevelsaswellasNrf2transcriptionalefficacyofbothsiRNAswasconfirmedbyrealtimeRT-PCRactivitywerenotsignificantlyalteredinNCM460cells.(Fig.2B,lowerpanel)andWesternblotanalysis(supplementalExposureofNCM460CellstoInflammatoryMacrophagesFig.S3).BecausetheNrf1proteinisalmostundetectableintheElevatesProteasomeActivityandProteasomalGeneExpressionabsenceofproteasomeinhibitors(32),lysatesfordetectionofinaNrf2-dependentManner—ToinvestigatewhethertheNrf2Nrf1proteinweregeneratedfromNCM460cellstreatedwithinductionbyinflammatorymacrophages(M-g)isfollowedbytheproteasomeinhibitorMg132(supplementalFig.S3).analteredproteasomeactivity,NCM460cellswerecoculturedBecausetheNrf2-dependentincreaseofproteasomeactivityfor3daysandthensubmittedtotheAMC-Suc-LLVYprotea-reliesontheinductionofproteasomalgeneexpression(11,25,someassay.Itcouldbeshownthattheproteasomeactivitywas26,29),realtimeRT-PCRanalysesoftheproteasomalgenessignificantlyincreasedinM-gcoculturedNCM460cellswhenS5a/psmd4/Rpn10and?5/psma5wereperformed.AsshowncomparedwithmonoculturedNCM460cells(Fig.2A).Bycon-inFig.2C,S5aand?5mRNAsweredetectedat2–2.8-foldtrast,coculturewithM-CSFderivedmacrophages(M-m)didhigherlevelinM-gcoculturedNCM460cellswhencomparednotsignificantlyinduceproteasomeactivityinNCM460cellswithmonoculturedNCM460cells(Fig.2C).UponNrf2siRNA(Fig.2A).treatment,theincreaseofS5aand?5mRNAlevelinThesignificantinductionofproteasomeactivityaftercocul-NCM460cellscoculturedwithinflammatorymacrophagesturewithinflammatorymacrophages(M-g),exclusivelyusedin(M-g)couldnotbedetectedanymore,thusconfirmingtheallfurthercocultureexperiments,wasnearlyabrogatedinNrf2dependence.Asimilareffectof0cocultureonprotea-NCM460cellsifNrf2expressionwasspecificallyknockedsomalgeneexpressioninNCM460cellswasdemonstrateddownbyNrf2-siRNAtreatment(Fig.2B,upperpanel).Bycon-byWesternblotanalysisdetectinghigherproteinlevelofS5atrast,knockdownofNrf1onlymoderatelyalteredproteasomeand?5(Fig.2D).Again,theincreasingeffectbythecocul-activityincoculturedNCM460cellsindicatingthattheinducedturewasmarkedlyreducedafterknockdownofNrf2expres-proteasomeactivitydependsonNrf2,butnotonNrf1.ThesioninNCM460cells.Underthesameconditions,the40914JOURNALOFBIOLOGICALCHEMISTRYVOLUME286?NUMBER47?NOVEMBER25,2011Downloaded from http://wendang.chazidian.com at Biomedical Library, UCSD, on March 2, 2012

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FIGURE2.Nrf2-dependentinductionofproteasomeactivityinNCM460cellsbycoculturewithinflammatorymacrophages.AandB,NCM460cellseithermonocultured(mono)orcocultured(3days)withmacrophages(M-gandM-m)weresubmittedtotheSuc-LLVY-AMCassay(A)orNCM460cellstreatedwithcontrol,Nrf1,orNrf2siRNAandculturedalone(mono)orwithGM-CSFmacrophages(M-g)weresubmittedtotheSuc-LLVY-AMCassay(B,upperpanel)orrealtimeRT-PCR(B,lowerpanels).Specificproteasomeactivity(AandB,upperpanel)wasdeterminedbythereleaseoffluorogenicAMCnormalizedtotheproteincontent;thedatarepresentthemeans?S.D.ofsixindependentexperiments.*,p?0.05.RealtimeRT-PCRwasconductedusingNrf1,Nrf2,and?-actinprimers(B,lowerpanels),anddatafromthreeindependentexperiments(means?S.D.)performedinduplicateareshown.CandD,NCM460cellstreatedwithcontrol,Nrf1,orNrf2siRNAandcultured(3days)alone(mono)orwithG-MCSFmacrophages(M-g)weresubmittedtorealtimePCRusingS5a,?5,and?-actinprimers(C)ortoimmunoblottingusingS5a,?5,andHsp90antibodies(D).Datafromthreeindependentexperiments(means?S.D.)performedinduplicateareshowninC,andDshowsrepresentativeblotsandthenormalizedS5aand?5bandintensitiesasdeterminedbybanddensitometryofblotsfromthreeindependentexperiments(means?S.D.).

knockdownofNrf1didnotalterthecoculture-inducedresultedinadecreasedfluorescencecausedbythedecayofexpressionofS5aand?5(Fig.2,CandD).Thesefindingsthedye,butatthistimethedifferencesbetweenmono-andpointtoaninvolvementofNrf2butnotNrf1intheenhancedcoculturedNCM460cellsturnedtobelesspronounced,proteasomeactivationincoloniccellsinthepresenceofindicatingcompensationoftheoxidativestressincocul-inflammatorymacrophages.turedNCM460cells.Underthesameconditions,TheInducingEffectofCoculturewithInflammatoryMacro-MitoSOXTMstainingformitochondrialROSrevealednophagesonProteasomeActivityandProteasomalGeneExpres-significantdifferencesoffluorescenceintensitiesbetweensionDependsonROS—Todeterminewhethercoculturewithmono-andM-gcoculturedNCM460cellsovertheentireinflammatorymacrophages(M-g)increasesoxidativestressinperiod(Fig.3B).Livingcellfluorescencemicroscopy(Fig.NCM460cells,stainingswiththecellularROSindicator3C)http://wendang.chazidian.comparedwithmonoculturedcocultured(1h)thaninmonoculturedNCM460cells.Whencells,NCM460cellsexhibitedsignificantlygreaterfluorescencestainingthecellswithMitoSOXTM(Fig.3D),stainingpat-afterM-gcoculturefor1and3h(89and62%greaterfluores-ternswerealmostsimilar,indicatingnodifferencesinthecence,respectively),resultingfromhigheroxidationofthemitochondrialROSactivitybetweencoculturedandmon-incorporateddye(Fig.3A).Longermono-andcoculture(6h)oculturedNCM460cells.

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