Rapamycin Induces MAP Kinase Phosphatase (MKP)2-

RapamycinInducesMAPKinasePhosphatase(MKP)-1ExpressionThroughActivationofProteinKinaseBandMitogen-activatedProteinKinaseKinasePathways•Background:MAPkinasephosphatase(MKP)-1playsacriticalroleinregulatinginflammationininnateandadaptiveimmunity.•Results:mTORinhibitionleadstoinductionofMKP-1throughtheactivationofAKT1andMEK1/MEK2pathways.RapamycinpretreatmentofmacrophagesinhibitsLPS-mediatedp38activationandIL-6andnitricoxideproduction.•Conclusion:BothAKT1andMEK1/2regulaterapamycin-mediatedMKP-1induction.•Significance:mTORC1inhibitionregulatesimmunitythroughMKP-1induction•ABSTRACT•(MKP)-1,alsoknownasdualspecificityphosphatase(DUSP)-1,playsacrucialroleinthedeactivationofMAPkinases•theeffectofmTORinhibitionthroughrapamycinandadualmTORinhibitor(AZD2014)onMKP-1expression.LowdoserapamycinledtoarapidactivationofbothAKTandERKpathwayswithasub-sequentincreaseinMKP-1expression.RapamycintreatmentledtophosphorylationofCREB,ATF1andATF2,threetranscription•factorsthatbindtothecyclicAMPresponsiveelements(CRE)ontheMkp-1promoter.InhibitionofeithertheMEK/ERKortheAKTpathwayattenuatedrapamycin-mediatedMKP-1induction.•AZD2014didnotactivateAKTbutactivatedtheERKpathway,leadingtoamoderateMKP-1induction•rapamycintreatmentledtoanincreasedMKP1expressionin•BMDMfromWT,butfailedtodosoinBMDMlackingAKT1isoformorMEK1andMEK2•INTRODUCTION•Mammaliantargetofrapamycin(mTOR)isamultifunctionalkinasecomplexthatplaysacentralroleincellgrowthandmetabolism•mTORC1:mToR-Raptor复合物作用主要是通过调节蛋白质的合成影响细胞生长和增殖。其活性能够被雷帕霉素抑制。•mTORC2:mTOR-Rictor复合物的活性不能被雷帕霉素抑制，主要参与细胞骨架蛋白的构造•mTORC1andmTORC2exerttheiractionsbyregulatingseveralimportantkinases,suchasS6KandAKT.WhileAKTactivatesmTORC1,theTORC2complexdirectlyphosphorylatesAKTatSer473•Rapamycinhasemergedasapotentantiproliferativemedicationwithimmunosuppressiveproperties•However,mTORplaysanimportantroleinterminallydifferentiatedmacrophagesandinregulationofinnateimmuneresponses•ThreeprincipalMAPKsubfamilies,includingtheextracellularsignal-regulatedkinases(ERK)1and2,c-Junamino-terminalkinases(JNKs),andp38,areessentialforinitiationofacuteinflammationinresponsetomitogensorstresssignals•activationofMAPKsisimportantincellularprocesses,failuretoterminatethisactivationmayleadtodetrimentalsystemiceffectsandchronicinflammatorydiseases•MAPkinasephosphatases(MKPs)areagroupofdualspecificityphosphatases(DUSPs)thatdephosphorylateboththreonineandtyrosineintheTXYmotifonMAPKs,andtherebymodulateinflammation.MKP-1preferentiallydephosphorylatesp38andJNKwhileMKP-3(DUSP-6)andDUSP-5preferERKsassubstrates•TheMKP-1(DUSP-1)geneisrapidlyinducedinresponsetogrowthfactors,stress,andseveralcytokines(TNF-αandTGF-β)•Theeffectsofseveralantiinflammatorydrugs,suchascorticosteroidsandcyclicAMPanalogs,havebeenshowntodepend,atleastinpart,onMKP-1induction(17).Forexample,theanti-inflammatoryeffectofcorticosteroidsisimpairedinMKP-1-/-macrophagesandMKP-1knockoutmice•Wehypothesizedthattheimmunosupp-ressiveeffectofrapamycinis,atleastinpart,duetoup-regulationofMKP-1expression•WeshowthatrapamycinrapidlyactivatesbothAKTandERKpathways(bothinmurinemacrophagesandBMDMs),enhancestheactivitiesofseveraltranscriptionfactorsthatdirectlybindtotheMkp-1promoterand•upregulateMKP-1expressioninmacrophages.Moreover,blockingeithertheMEK/ERKpathwayortheAKTpathwayattenuatesrapamycinmediatedMKP-1inductioninresponsetomTORC1inhibition.Finally,weexploretheroleofAKT1and2isoform,MEK1and2inrapamycinmediatedMKP-1inductionusingmacrophagesdeficientinthecorrespondinggenesEXPERIMENTALPROCEDURES•Chemicalsandantibodies:•Cellculture•MiceandIsolationofBoneMarrow•ProteinExtractionandImmunoblotting•CellViability•Enzyme-LinkedImmunosorbentAssay•RNAExtractionandQuantitativeReverseTranscriptase/RealTime-PCR•SeparationofCytoplasmicandNuclearFractions•StatisticalAnalyses-StatisticalRESULTSRapamycinpotentlyinducesMKP-1expression•Figure1.Time-dependenteffectofrapamycinonMKP-1expression.A.RapamycinincreasesMKP-1proteinlevels.Murinemacrophages(B6-MCL)weretreatedwithrapamycin(Rapa,10ng/mL)fordifferenttimeperiods.WholecellextractsweresubjectedtoWesternblotanalysisusingantibodiesagainstMKP-1andβactin.B.DensitometricquantificationofthreeblotsforMKP-1plottedagainsttime,errorbars,means±SEM(ANOVAMann-WhitneyUtest;*=p0.05;**=p0.01).C.andD.•RapamycinincreasesMKP-1butnotMKP-3mRNAexpression.B6-MCLcellsweretreatedwithrapamycin(10ng/mL)for5,15,30,and60min.RNAwasisolatedfromcellsandsubjectedtoRT-PCRassessingMKP-1(C)andMKP-3(D)expression.Valueswerenormalizedtoβ-actinandareshownasfoldchange.Thedatarepresentmeanvalues±SEMof4independentexperiments,eachperformedintriplicates.*=p0.05;**=p0.01.EandF.InductionanddecayofMKP-1inresponsetorapamycin•andCHX.Cellsweretreatedwithrapamycin(10ng/mL)for60min.Cellswerethentreatedwith100ng/mLcycloheximide(CHX)for10,30,60,and90min.CelllysatesweresubjectedtoWesternblotanalysisusingantibodiesagainstMKP-landβ-actin.F.DensitometricquantificationofthreeblotsforMKP-1plottedagainsttime.•Figure2.RapamycintreatmentactivatesERKbutnotJNKandp38.Murinemacrophages(B6-MCL)weretreatedwithrapamycin(A,B,C)fordifferentt