NigoraMukhamedova,*GenevieveEscher,†*WilissaDʼSouza,*UrbainTchoua,*AngelaGrant,†ZigmundKrozowski,*MichaelBukrinsky,andDmitriSviridov1,*
BakerIDIHeart&DiabetesInstitute,*Melbourne,Victoria,Australia;andDepartmentofMicrobiology,ImmunologyandTropicalMedicine,†GeorgeWashingtonUniversity,WashingtonDC
AbstracteffluxEightproteinspotentiallyinvolvedincholesterolCholesteroleffluxisthefirstandmostlikelytherate-teinlimitingstepofreversecholesteroltransport(RCT).RCTcholesteryl(PLTP),[ABCA1,scavengerABCG1,receptorCYP27A1,typephospholipidtransferpro-isapathwayremovingexcesscholesterolfromextrahe-(apoA-I)]paticcells,mostimportantlyvascularcells,thusprotectingRAWwereestertransferprotein,andBI(SR-BI),apolipoproteincaveolin-1,A-Iagainstdevelopmentofatherosclerosis.Severalpathwaystor,ofcholesteroleffluxhavebeendescribed.Oneisadiffu-PLTP,overexpression264.7macrophages.overexpressedoftheWhenalonecombinationapoA-IorwasincombinationinofABCA1,usedasanaccep-sionpathway,whichdoesnotdependonanyspecificcel-4.3-fold.andlularprotein.Severalspecificpathwaysinvolveanumberwasofcellularproteins.FirstaretheABCtransportersABCA1apoA-IduetoItSR-BIwas(CombinationI)enhancedtheCYP27A1,effluxbyincreasedestablishedabundancethatthestimulationofeffluxcombinationbinding(1)andABCG1(2),mediatingcholesteroleffluxtolipid-latedcausedtonon-ABCA1onlyasmallsitesofincreaseonABCA1macrophages.andincreasedoftheeffluxtoThisiso-freeapolipoproteinA-I(apoA-I)andmatureHDL,re-sionspectively.ScavengerreceptortypeBI(SR-BI)isanother(CombinationofHDL.caveolin-1WhenHDLwasusedasanacceptor,overexpres-cellularproteininvolvedincholesteroleffluxforwhichHDL,II)wasorathecombinationofcaveolin-1andSR-BIHDLisanacceptor(3).WehaverecentlydemonstratedthethatCYP27A1(4)andcaveolin-1(5)arealsoinvolvedinsioninwithoutvivomouseaffectingmodelthemostofeffluxactive,doublingtheeffluxtocholesteroltoapoA-I.Whentestedincholesterolefflux.AnumberofotherproteinshavebeenportofABCA1andCombinationIelevatedefflux,cholesteroloverexpres-ex-demonstratedtocontributetocholesterolefflux,includ-overexpressionfrommacrophagesanofcaveolin-1toplasma,orliver,andfeces,whereasingintracellularapoA-I(6),phospholipidtransferproteinusingeffect.(PLTP)(7),andcholesterylestertransferprotein(CETP)tionapoA-IWeasconcludeanacceptorthatmakepathwaysCombinationIIdidnothaveapredominantofcholesterolcontribu-efflux(8);themechanismsoftheirinvolvementincholesterolMukhamedova,tocholesteroleffluxaremostlyunknown.
Grant,AlsounknownishowthedifferentpathwaysandtheiringZ.Krozowski,N.,G.exportfrommacrophagesinvivo.—M.Escher,W.DʼSouza,U.Tchoua,A.componentsinteractwitheachother,whethertheyarecholesterolapolipoproteinA-I-dependentBukrinsky,cholesterolandD.Sviridov.effluxEnhanc-elevatessynergistic,competing,orredundant,andwhattherelative2008.49:2312export–2322.
frommacrophagesinvivo.J.LipidRes.contributionofeachpathwayistotheoverallexportofcholesterolfromcells.IthasbeensuggestedthatABCA1Supplementarykeywordsreversecholesteroltransport•atherosclero-andABCG1workinsequenceandinsynergy,leadingtosis•lipoproteins•lipids•ABCtransporters
theformationofcholesterol-richHDL(9–11),whereasSR-BIcompeteswithABCA1andABCG1forcholesteroldestinedforefflux(12,13);interactionsbetweenothercomponentsofthecholesteroleffluxpathwayhavenotbeeninvestigated.Easilytransfectablecelllineswereusedinthemajorityofthestudies;itis,however,unclearThisAustraliaworkwassupportedbyawhethertheseobservationsaccuratelydescribecholesterol
FoundationtoD.S.ofAustralia.
D.S.istoD.S.andZ.K.(Ggrant04Mfrom1536),theaNationalgrantfromHearttheSwissFoundationNationalofaG.E.,FellowandofthebyNationalagrantfromHealththeandPerpetualMedicalCharitableResearchTrustCounciltoManuscriptAbbreviations:apo,apolipoprotein;CETP,cholesterylestertrans-re-revisedformreceived11July212008.
February2008andinrevisedform16June2008andinferprotein;PLTP,phospholipidtransferprotein;RCT,reversecholes-terolPublished,1
transport;SR-BI,scavengerreceptortypeBI.Towhomcorrespondenceshouldbeaddressed.DOI10.1194/jlr.M800095-JLR200
JLRPapersinPress,July12,2008.e-mail:Dmitri.Sviridov@Bakeridi.edu.au
Copyright©2008bytheAmericanSocietyforBiochemistryandMolecularBiology,Inc.
2312JournalofLipidResearchVolume49,2008
Thisarticleisavailableonlineathttp://www.jlr.org
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014effluxpathwaysincellsofthevesselwall,mostimportantly,macrophages.Thereareconsiderabledifferencesinthepathwaysofcholesteroleffluxandtheirregulationindif-ferentcellsandtissues(14).Variouseffluxpathwaysusedifferentcholesterolacceptors,andthecontributionoftheseacceptors,andconsequentlyofthecellularproteinsinteractingwiththeseacceptors,tooverallcholesterolex-portisunclear.ThemajorityofapoA-Iinplasmaispresentinthelipidatedform,andyetinactivationofABCA1,whichuseslipid-freeapoA-Iasacceptor,resultsinsevereatherosclerosis(15,16),whereasinactivationofABCG1,whichuseslipidatedapoA-I,hasasmallandinconsistenteffectonatherosclerosis(17–19).
Inthisstudy,wetookadvantageofanewmethodofhigh-efficiencytransfectionofmacrophages(20)toselec-tivelyenhancevariouspathwaysofcholesteroleffluxandtoinvestigatethecontributionsofthepathwaysusingapoA-IversusthoseusingHDLasacceptorstooverallcho-lesterolexportfrommacrophagesinvitroandinvivo.
MATERIALSANDMETHODS
Cellsandplasmids
RAW264.7mousemacrophagecellsweremaintainedandtransfectedasdescribedpreviously(20).THP-1cellsweremain-tainedinRPMIsupplementedwith10%FBS.Cellsweredif-ferentiatedintomacrophage-likecellsbyincubationinRPMIsupplementedwith10%FBS,100nMphorbol12-myristate13-acetate(PMA)and100nMvitaminD3for48h.DifferentiatedcellsweretransfectedusingMetafectene(Biontex,Munich,Germany)andmanufacturerʼsprotocolandculturedinRPMIsupplementedwith10%FBS,100nMPMA,100nMvitaminD3,and1mM5-azacytidine.PlasmidscontaininghumanABCA1andhumanABCG1-GFPwereakindgiftfromDr.A.Remaley,plasmidcontainingABCG1-mycwasagiftfromDr.W.Jessup.PlasmidcontainingmouseSR-BIwasagiftfromDrs.Z.ChenandA.Bocharov,andplasmidcontaininghumanPLTPwasagiftfromDr.C.Ehnholm.GenesforhumanapoA-I,caveolin-1,andCYP27A1havebeendescribedpreviously(4,5,21).AllgeneswereclonedintopcDNA3.1plasmid.pCMV-b-Galplasmidwasusedformocktransfectionsandtomonitorefficiencyoftransfection.
Lipoproteins
HDLwasisolatedfromhumanplasma(pooledplasmasup-pliedbytheRedCross)bysequentialcentrifugation.ApoA-IwasisolatedfromHDLasdescribedpreviously(22).LDLwaspurifiedfromhumanplasmabysequentialcentrifugationandacetylatedasdescribedbyBasuetal.(23).ApoB-deficientplasmawasobtainedfromnormolipidemicplasmabyprecipitat-ingtheapoB-containinglipoproteinswith0.9g/ldextransulfate/45mMMgCl2.
Immunoblotting
Cellsweretreatedasindicated,washed,andharvested.Pro-teinswereseparatedonSDS-PAGE,followedbyimmunoblotting.Inbrief,PolyVinyliDieneFluoridemembraneswereblockedwith2.5%skimmilksolution,washed,andincubatedfor1hatroomtemperaturewiththeantibodiesagainstABCA1[mono-clonal,NDF4C2(24)],ABCG1(polyclonal,Abcamab52617),PLTP(polyclonal,Abcamab7735),SR-BI(polyclonal,Abcamab369),caveolin-1(polyclonal,BDTransductionLaboratories),
ormyc(monoclonal,clone9E10)andfor1hwithanti-rabbitoranti-mouseIgGsecondaryantibodiesorbiotin-anti-mouseIgMandstreptavidin-HRPconjugate.BandswerevisualizedbySuperSignalWestPicoChemiluminescentSubstratekit(Pierce),andtherelativeintensitiesofthebandswerequantitatedbydensitometry.Theabundanceofb-actinwasusedasloadingcontrol(monoclonalantibodyfromSigma).
Cholesterolefflux
Cellularcholesterolwaslabeledbyincubatingcellsinserum-containingmediumwith[1a,2a(n)-3H]cholesterol(Amersham,finalradioactivity0.5MBq/ml)for48hinaCO2incubator.Cellswerethenwashedandincubatedfor18hat37°Cinserum-freemedium,andwashedandincubatedforanother3hat37°Cinserum-freemediumcontaining30mg/mloflipid-freeapoA-IorHDL,2%humanapoB-depletedplasma,or5mMmethyl-b-cyclodextrin.Themediumwascollected,centrifugedfor15minat4°Cat10,000g,andaliquotsofsupernatantwerecountedinab-counter.Cellswereharvested,andcell-associatedradioactivitywascounted.Cholesteroleffluxwasexpressedastheproportionof[3H]cholesteroltransferredfromcellstomedium.Cholesterolmasswasmeasuredusingthecolorimetrictechnique.
Cholesteroltrafficking
Theabundanceofcholesterolincholesterol-richdomainsandtraffickingofcholesteroltothesedomainswereassessedasdescribedpreviously(25).Inbrief,cellswerelabeledwith[14C]cholesterol(finalradioactivity0.5MBq/ml)for48hat37°C,andwashedandlabeledwith[3H]acetate(finalradioactivity5MBq/ml)for3hat15°C.Cellswerethenwarmedto37°Cfor20min,cooledto4°C,washed,andtreatedwithcholesteroloxidase(1U/ml)for3hat4°C.CellularlipidswereisolatedandseparatedonTLCasdescribed(25).
ApoA-Ibinding
TomeasureapoA-IbindingspecificallytoABCA1,across-linkingassaywasusedasdescribedbyWangetal.(26).Briefly,transfectedcellswereincubatedwithapoA-I(50mg/ml)for1hat37°Candwashed.Dithiobis(succinimidyl)propionate(DSP,Pierce)wasaddedtothefinalconcentrationof250mMandincubatedfor1hatroomtemperature.CellswerethenlysedwithRIPAbufferandincubatedwithpolyclonalanti-ABCA1antibody(Novus)overnightat4°C.ComplexofABCA1andantibodieswasimmunoprecipitatedwithProteinGSepharosebycoincubationfor4hat4°C.Immunoprecipitatedandun-boundfractionsweredilutedwithloadingbuffercontaining5%2-mercaptoethanol,boiledfor5min,andsubjectedto12%SDS-PAGEandWesternblot;apoA-Iwasdetectedusingmono-clonalanti-apoA-IantibodyAI-4.1.
Invivostudies
CholesteroleffluxinvivowasmeasuredasdescribedbyWangetal.(10)withmodifications.Briefly,RAW264.7macrophagecellsweretransientlytransfectedwithindicatedplasmidsasdescribedabove.Cellsweresimultaneouslyradiolabeledandloaded3withcholesterolbyincubationfor48hwith1.1GBq/ml[H]cholesterolandacetylatedLDL(50mg/ml)for48h.Cellswerewashed,incubatedfor24hinserum-freemedium,har-vested,and7resuspendedin0.15Msterilesalineataconcentra-tionof10cells/ml.CellswereinjectedintraperitoneallyintomaleC57BL/6mice(23106cellscontaining53106dpmpermouse).Eachgroupconsistedofsixanimals.After24h,micewereeuthanized,andblood,liver,andfeceswerecollected.Aliquotsofplasmawerecounted,andcholesterolfromliverandfeceswasextractedaccordingtoFolch,Lees,andSloane-
Pathwaysofcholesteroleffluxinvivo2313
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014Stanley(27)AnimalexperimentationwasapprovedbytheAlfredMedicalResearchandEducationPrecinctAnimalEthicsCom-sionofABCA1didnotaffectcholesteroleffluxtoHDL.Amittee;experimentswereconductedincompliancewiththemoderateeffectofABCA1overexpressionontheeffluxto“Principlesoflaboratoryanimalcare”(NIHpublicationNoHDLobservedbyOkuhiraetal.(29)wasattributedtotheef-85-23)andAustralianlaws.
fluxtolipid-freeapoA-IdissociatedfromHDL.Theamountoflipid-freeapoA-IinfreshpreparationsofHDLusedinthisStatisticalanalysis
studywasundetectablebynativePAGEandWesternblot;ifaAllexperimentswerereproducedtwotofourtimes,andrep-smallamountwasstillpresent,thisamountwasinsufficienttoresentativeexperimentsareshown.Unlessindicatedotherwise,contributetotheoveralleffluxtoHDL.
experimentalgroupsconsistedofquadruplicates;means6SEMThesmalleffectofABCG1overexpressiononcholes-arepresented.TheStudentʼst-testwasusedtodeterminestatis-teroleffluxtoHDLwasunexpectedandinconsistentwithticalsignificanceofthedifferences.
theresultsofstudiesoverexpressingABCG1inothercelltypes(30),butwasnotdramaticallydifferentfromtheonlystudyoverexpressingABCG1inmurinemacrophagesRESULTS
(10).Onereasonforthisresultcouldbethefactthatinthepresentstudy,weusedABCG1-GFPconjugate,whichOverexpressiontoapoA-IorHDLofinvariousvitro
proteinsandcholesteroleffluxmayhavepropertiesdifferentfromthoseofABCG1.Wethereforetestedanotherconstruct,ABCG1-myc.ABCG1-Eightproteinspreviouslyimplicatedintheirinvolve-mycalsocausedonlyasmallstimulationofcholesterolmentincholesteroleffluxwereoverexpressedinmouseeffluxfromRAW264.7macrophages(notshown).
macrophagesRAW264.7usingapreviouslydescribedpro-AnotherreasonforthemodesteffectofABCG1overex-tocol(20),whichprovidedan80%efficiencyoftransfec-pressioninmacrophagescouldbeahighlevelofABCG1tionanduniformexpressionoftheheterologousgeneininRAW264.7cells(asreflectedbythehighlevelofeffluxtheentirecellularpopulation.Unlessindicated,cellsweretoHDLfromnonactivatedRAW264.7cells),reducingnotloadedwithcholesterol,becauseloadingwouldhavepossibleeffectsofitsoverexpression.Indeed,arelativelyupregulatedseveralcholesteroleffluxpathways,includinghighlevelofABCG1wasfoundinnonactivatedRAWABCtransporters.Whenlipid-freeapoA-Iwasusedasan264.7cells(Fig.2A),andalthoughtransfectionincreasedacceptor,overexpressionofABCA1elevatedcholesteroltheABCG1levelby70%(6.1vs.3.6relativeunits,Fig.2A),effluxby2.4-fold,whereasoverexpressionofCYP27A1,theconcentrationofendogenousABCG1mayalreadyPLTP,andSR-BIelevatedcholesteroleffluxbyabouthavebeenapproachingafunctionallysaturatinglevel.To2-fold(Fig.1A).OverexpressionofABCG1,caveolin-1,andfurthertestthispossibility,weexaminedtheoverexpres-apoA-IdidnotaffectcholesteroleffluxtoapoA-I(Fig.1A);sionofABCG1plasmidsinthreeothercelltypes.Twoaswedemonstratedpreviously,overexpressionofCETPalsohumancelllines,HeLacells,acellmodellackingABCdidnotaffectcholesterolefflux(28).WhenHDLwasusedtransporters,andTHP-1humanmacrophages,knowntoasanacceptor,overexpressionofcaveolin-1increasedcho-expresslowlevelsofABCtransportersunlessactivatedwithlesteroleffluxby1.8-fold;smallbutstatisticallysignificantin-LXRagonist,weretested.ComparedwithRAW264.7cells,creasesofcholesteroleffluxwerealsoobservedaftercholesteroleffluxtoHDLfrommock-transfectedHeLaoverexpressionofABCG1andSR-BI(Fig.1B).Overexpres-
andTHP-1cellswas1.6-foldand3.6-foldlower,respec-
Fig.1.Overexpressionofvariousgenesandcholesteroleffluxfrommacrophages.RAW264.7cellsweretransientlytransfectedwiththeindicatedgenes.Cellswerelabeledwith[3H]cholesterol,andcholesteroleffluxtolipid-freeapolipoproteinA-I(apoA-I)(A)orHDL(B)(finalconcentrationofboth,30mg/ml)was3assessedasdescribedinMaterialsandMethods.Cholesteroleffluxwasexpressedasaproportionof[H]cholesteroltransferredfromcellstomedium.Means6SEMofquadruplicatedeterminationsareshown.*P,0.05versusmock-transfectedcells.
2314JournalofLipidResearchVolume49,2008
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014Fig.2.TheeffectofoverexpressionofABCG1onABCG1abundanceinRAW264.7cells(A)andcholes-teroleffluxtoHDLfromHeLacells(B),THP-1macroophages(C),and3T3fibroblasts(D).A:RAW264.7cellsweretransientlytransfectedwithABCG1-GFPandtheabundanceofABCG1wasassessedbyWesternblot.B–D:HeLacells(B),THP-1humanmacrophages(C),or3T3murinefibroblasts(D)weretransfectedasindicatedandlabeledwith[3H]cholesterol,andcholesteroleffluxtoHDL(finalconcentration30mg/ml)wasassessedasdescribedinMaterialsandMethods.Cholesteroleffluxwasexpressedasaproportionof[3H]cholesteroltransferredfromcellstomedium.Means6SEMofquadruplicatedeterminationsareshown.*P,0.05versusmock-transfectedcells.
tively.OverexpressionofbothABCG1-mycandABCG1-Theresultsoftheanalysisoftheeffectsofoverexpres-GFPelevatedcholesteroleffluxbyapproximately1.5-foldsionofcombinationsofproteinsarepresentedinFig.3.and7.5-foldinHeLaandTHP-1cells,respectively.(Fig.2B,WhenapoA-Iwasusedasanacceptor,overexpressionofC).Thethirdcelllinetestedwasmouse3T3fibroblasts.SR-BIorCYP27A1togetherwithABCA1addedtothestim-CholesteroleffluxtoHDLfromthesecellswas1.8-foldulationofcholesterolefflux,whereasadditionofPLTPdidhighercomparedwithRAW264.7cells,andoverexpressionnot(Fig.3A).ThecombinationofproteinsthatcausedofABCG1hadnoeffectoncholesteroleffluxtoHDLthebiggeststimulationofcholesteroleffluxwasABCA11(Fig.2D).Thus,itappearsthatoverexpressionofABCG1SR-BI1PLTP1CYP27A1;thiscombinationcausedastimulatedcholesteroleffluxtoHDLfromcellswithalow4.3-foldstimulationofcholesterolefflux(Fig.3A).Thebasalleveloftheefflux,butnotfromcellswithahighbasalcombinationofSR-BI1PLTP1CYP27A1withoutABCA1levelofcholesterolefflux,pointingtoapossibilitythatthewaslesseffective,causinga2-foldincreaseofcholesterolef-ABCG1levelinthelatterwasfunctionallysaturated.How-flux.WhenHDLwasusedasanacceptor,overexpressionever,wecannotexcludeapossibilityofspecies-specificdif-ofSR-BItogetherwithcaveolin-1slightlyincreasedcholes-ferences,inasmuchasoverexpressionofABCG1stimulatedterolefflux;thedifferencewithoverexpressionofcaveolin-1cholesteroleffluxfromhumancellsbutnotmurinecells.alone,however,wasnotstatisticallysignificant(Fig.3B).AllTotesttheeffectofoverexpressionofcombinationsofothercombinationsonlyminimallyaffectedcholesterolef-theproteins,thefollowingapproachwasused.ThecellsfluxtoHDL.Importantly,overexpressionofacombinationwerecotransfectedwiththeoneoftwoproteinsthathadofABCA11SR-BI1PLTP1CYP27A1thatcausedaconsid-thebiggesteffectoncholesterolefflux,i.e.,ABCA1orerableelevationofcholesteroleffluxtoapoA-IcausedonlyaCYP27A1fortheeffluxtoapoA-Iandcaveolin-1orSR-BIsmall(30%)elevationofcholesteroleffluxtoHDL(Fig.3B).fortheeffluxtoHDL,andoneoftheremainingsevenpro-Althoughtheeffluxoflabeledcholesteroltolipid-freeteins.ThecombinationoftwoproteinsthatcausedtheapoA-Ireflectsthemovementofcholesterolmass,thebiggesteffectwasthencotransfectedwithoneofthere-effluxtoHDLisaccompaniedbycholesterolexchangemainingsixproteins,andsoon.ThemaximumnumberbetweencellsandHDL,thereforenotnecessarilyreflect-ofproteinswewereabletocoexpresswasfour;coex-ingthemovementofcholesterolmass.Whentotalcholes-pressionofthefifthproteincausedasharpreductionofterolmasswasmeasuredinthecellsandthemedium,cholesteroleffluxindependentlyofwhatthecombina-incubationwithHDLresultedin4%ofcellularcholesteroltionwas.movedtoHDLafter2h.However,therewasnostatistically
Pathwaysofcholesteroleffluxinvivo
2315
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014Fig.3.Overexpressionofacombinationofgenes3andcholesteroleffluxfrommacrophages.RAW264.7cellsweretransientlytransfectedwiththeindicatedgenes.Cellswerelabeledwith[H]cholesterol,andcholesteroleffluxtolipid-freeapoA-I(A)orHDL(B)(finalconcen-trationofboth,30mg/ml)wasassessedasdescribedinMaterialsandMethods.Cholesteroleffluxwasexpressedasaproportionof[3H]cholesteroltransferredfromcellstomedium.Means6SEMofquadruplicatedeterminationsareshown.*P,0.05versusmock-transfectedcells.#P,0.01versusABCA1.
significantdifferencebetweenmock-transfectedcellsandsignificanteffect(Fig.4A).WhenHDLwasusedasancellstransfectedwithABCA1,ABCA11SR-BI1PLTP1acceptor,onlytransfectionwithcaveolin-1increasedcho-CYP27A1,caveolin-1orcaveolin-11SR-BI(notshown).lesterolefflux(Fig.4B).Theeffectofcaveolin-1wasat-Tofurthercharacterizetheeffectofoverexpressionoftenuatedincholesterol-loadedcells,suggestingthatthecombinationsofproteinsoncholesterolefflux,theabovecontributionofthispathwaytocholesteroleffluxtoHDLexperimentswererepeatedwithcholesterol-loadedcells.mayhavechangedaftercholesterolloading,i.e.,duetoTransfectedcellswereloadedwithcholesterolbyincu-upregulationofABCtransporters.
bationwithacetylatedLDL(50mg/ml)for24h.WhenToassesstheincrementofabundanceofeachpro-apoA-Iwasusedasanacceptor,transfectionwithABCA1teinafteroverexpressionofthecombinationofgenes,andwithABCA11SR-BI1PLTP1CYP27A1elevatedcellswerecotransfectedwithABCA11SR-BI1PLTP1cholesteroleffluxby6-and11-fold,respectively(Fig.4A).CYP27A1ortransfectedwithcaveolin-1alone,andexpres-Transfectionofcellswithcaveolin-1marginallyincreasedsionofeachproteinwasassessedbyWesternblottingandcholesterolefflux(P50.04),whereastransfectionwithcomparedwithitsexpressioninmock-transfectedcellsacombinationofcaveolin-11SR-BIhadnostatistically(Fig.5).TheabundancesofABCA1,SR-BI,andPLTPwere
Fig.4.Overexpressionofacombinationofgenesandcholesteroleffluxfromcholesterol-loadedmacro-phages.RAW264.7cellsweretransientlytransfectedwiththeindicatedgenes.Cellswereloadedwithcholes-terolbyincubationwithacetylatedLDL(50mg/ml)for24handsimultaneouslylabeledwith[3H]cholesterol.Cholesteroleffluxtolipid-freeapoA-I(A)orHDL(B)(finalconcentrationofboth,30mg/ml)wasas-sessedasdescribedinMaterialsandMethods.Cho-lesteroleffluxwasexpressedasaproportionof[3H]cholesteroltransferredfromcellstomedium.Means6SEMofquadruplicatedeterminationsareshown.*P,0.05,**P,0.001(versusmock-transfectedcells).
2316JournalofLipidResearchVolume49,2008
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014Fig.5.Theeffectofoverexpressionofacombinationofgenesontheirabundance.RAW264.7cellsweretransientlytransfectedwithABCA11scavengerreceptortypeBI(SR-BI)1phospholipidtransferprotein(PLTP)1CYP27A1(A)orcaveolin-1(B).TheabundanceofindividualproteinswasassessedbyWesternblotandquantitatedbydensitometryasdescribedinMaterialsandMethods.Numbersundertheblotsindicateincreaseofabundanceoftheindicatedprotein(fold,relativetomock-transfectedcells).
increasedby1.9-,3.5-,and1.8-fold,respectively(Fig.5A).alwaysamajordeterminantofplasmacholesteroleffluxThesevaluesrepresentacombinationofabundancesofcapacity(31,32).Wehypothesizethattheincreaseofcho-endogenousandheterologousproteins,andalthoughitlesteroleffluxwasmainlyduetotheeffluxtoplasmalipid-cannotberuledoutthatexpressionofendogenouspro-freeapoA-I.Thesedataindicatethatcholesteroleffluxteinswasaffected,itismorelikelythatmostoftheincre-frommacrophagesmaybeenhancedbymanipulatingcel-mentwasduetoheterologousexpression.TheexceptionlularpathwaysresponsiblefortheeffluxtoapoA-I.istheabundanceofendogenousABCA1,whichcouldbeaffectedbyCYP27A1andPLTP;thisissueisexaminedCholesteroleffluxinvivo
below.OverexpressionofCYP27A1wasassayedusingTofurthertesttheeffectofstimulationofcholesterolanti-mycantibody,preventingacomparisonwiththebasaleffluxtoapoA-IorHDLoncholesterolexportfromexpressionofCYP27A.Overexpressionofcaveolin-1re-macrophages,theinvivomodeldescribedbyWangetal.sultedina1.6-foldincreaseofitsabundance(Fig.5B).(10)wasused.Labeledcholesterol-loadedmacrophagesTransfectionofcellsdidnotaffectabundanceofb-actinoverexpressingindicatedproteinswereimplantedinto(Fig.5A,B).
theperitonealcavityofmice;theappearanceoflabeled
Cholesteroleffluxtoplasmainvitro
Testingtheeffectsofoverexpressionofvariousproteinsoncholesteroleffluxresultedinestablishingtwodistinctcellularmodels.Onemodel,RAW264.7cellscotransfectedwithABCA1orABCA11SR-BI1PLTP1CYP27A1,hadelevatedeffluxtolipid-freeapoA-I,butnottoHDL.An-othermodel,RAW264.7cellstransfectedwithcaveolin-1orcaveolin-11SR-BIhadelevatedeffluxtoHDL,butnottolipid-freeapoA-I.Thesemodelspresentedanoppor-tunitytocomparethecontributionofthesetwoacceptorstocholesterolexportinvitroandinvivo.Totesttheeffluxinvitro,weremovedapoB-containinglipoproteinsfromplasmabyprecipitationwithdextran-sulfate,thusminimiz-ingnonspecific(diffusional)efflux.Theplasmawasusedataconcentrationof2%,theconcentrationwithinthelinearpartofthedose-dependentcurve(notshown).WhenapoB-depletedhumanplasmawasusedasanacceptor,overexpressionofABCA1orABCA11SR-BI1PLTP1Fig.6.OverexpressionofvariousgenesandcholesteroleffluxtoCYP27A1stimulatedcholesteroleffluxby30%and40%,re-humanplasma.RAW264.7cellsweretransientlytransfectedwithspectively,whereasoverexpressionofcaveolin-1orcaveolin-theindicatedgenes.Cellswerelabeledwith[3H]cholesterol,and11SR-BIfailedtoaffectcholesterolefflux(Fig.6).Thelattercholesteroleffluxto2%apoB-depletedhumanplasmawasassessedfindingwasunexpected,inasmuchaslipidatedHDLrepre-asdescribedinMaterialsand3Methods.CholesteroleffluxwassentsthemajorityofplasmaapoA-I;thisisconsistent,how-expressedasaproportionof[H]cholesteroltransferredfromcellsever,withfindingsshowingthatplasmaHDLlevelisnot
tomedium.Means6SEMofquadruplicatedeterminationsareshown.*P,0.05versusmock-transfectedcells.
Pathwaysofcholesteroleffluxinvivo2317
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014cholesterolinplasma,liver,andfeceswasfollowedafterPLTP,andCYP27A1increasedtheabundanceofABCA1.24h.Overexpressionofvariousgenesortheircombina-Wehavepreviouslydemonstratedthatoverexpressionoftionshadnoeffectoncholesterolloadingofcells,inas-CYP27A1increasestheabundanceofABCA1inRAWmuchasthecellularcholesterolcontentandthespecific246.7macrophages(20).Wefurtherfoundthattransfec-activityofcellular[3H]cholesterolwerenotaffectedbytionofRAW246.7cellswithPLTPalsoincreasedtheabun-transfections.OverexpressionofABCA1(“A”)onlyslightlydanceofABCA1inthecells(Fig.8A),afindingconsistentincreasedtheexportoflabeledcholesteroltoplasma,butwiththatofOrametal.(7).Incontrast,overexpressionofincreasedtheamountoflabeledcholesterolintheliverSR-BI,ifanything,decreasedtheabundanceofABCA1,aandfecesby2.5-and1.6-fold,respectively(Fig.7A–C).findingconsistentwiththeobservationsofChenetal.ThisfindingisconsistentwiththatofCalpe-Berdieletal.(12).However,iftheabundanceofABCA1wascompared(33)andWangetal.(10),whodemonstratedimpairedinthecellstransfectedwithABCA1aloneorwiththecom-cholesteroltransportfromABCA1-deficientmacrophagesbinationABCA11SR-BI1PLTP1CYP27A1,theabun-invivo.OverexpressionofABCA11SR-BI1PLTP1danceofABCA1wassimilarinsingletransfectionsandinCYP27A1(“A1”)increasedtheexportofcholesteroltransfectionswithacombinationofgenes(Fig.8B).Thus,toplasma,liver,andfecesby1.8-,3.0-,and1.8-fold,re-althoughenhancementofABCA1abundanceexplainsspectively(Fig.7A–C).OverexpressionofABCG1(“G”),stimulationofcholesteroleffluxbyPLTPandCYP27A1caveolin-1(“C”),orcaveolin-11SR-BI(“C1”)didnotalone(Fig.1A)andincombinationwitheachother(Fig.3),affectcholesterolexportfrommacrophagestoplasma,itdoesnotexplainfurtherstimulationofcholesteroleffluxliver,orfeces.AppearanceoflabeledcholesterolinthebytheABCA11SR-BI1PLTP1CYP27A1combination.bileacidfractionoffeceswasnotaffectedbyanyoftheWenextinvestigatedwhetherthedifferenceincholes-transfections(Fig.7C,leftpanel).
teroleffluxwasduetoincreasedamountofcholesterolMechanismsofcholesteroleffluxstimulation
ontheplasmamembraneorenhancedtraffickingofcho-lesteroltotheplasmamembrane.Labeledcellswerein-TheeffectofoverexpressionofABCA1oncholesterolcubatedfor1hwith5mMmethyl-b-cyclodextrinateithereffluxwasexpected,butthemechanismofhowSR-BI,4°Cor37°C.RapidnonspecificremovalofcholesterolbyPLTP,andCYP27A1furtherstimulatedtheeffluxwasun-cyclodextrinat4°C,whencholesteroltraffickingisblocked,clear.ThefirstpossibilityweconsideredwasthatSR-BI,wasusedtoassesstheamountofcholesterolontheplasma
Fig.7.Overexpressionofvariousgenesandcholesteroleffluxinvivo.RAW264.7macrophagesweretransfectedwithmock(M),ABCA1(A),ABCA131SR-BI1PLTP1CYP27A1(A1),ABCG1(G),caveolin-1(C),orcaveolin-11SR-BI(C1),loadedwithcholesterol,labeledwith[H]cholesterol,andinjectedintraperitoneallyintomice.Theappearanceoflabelinplasma(A),liver(B),andfeces(C)wasassessedafter24hasdescribedinMaterialsandMethods.Means6SEM(n56)areshown;*P,0.01versusmock-transfected.
2318JournalofLipidResearchVolume49,2008
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014Fig.8.Possiblemechanismsofstimulationofcholesterolefflux.A,B:RAW264.7cellsweretransientlytrans-fectedwithmock(M),SR-BI,PLTP,ABCA1(A),orABCA11SR-BI1PLTP1CYP27A1(A1).Theabun-danceofABCA1incellswasassessedbyWesternblotting.C:RAW264.7cellsweretransientlytransfectedwithmock(M),ABCA1(A),ABCA11SR-BI1PLTP1CYP27A1(A1),caveolin-1(C),orcaveolin-11SR-BI(C1).Cellswerelabeledwith[3H]cholesterol,andcholesteroleffluxtomethyl-b-cyclodextrin(finalconcen-tration5mM)at4°C(left)or37°C(right)wasassessedasdescribedinMaterialsandMethods.Cholesteroleffluxwasexpressedasaproportionof[3H]cholesteroltransferredfromcellstomedium.Means6SEMofquadruplicatedeterminationsareshown.D:RAW264.7cellsweretransientlytransfectedwithmock(M),ABCA1(A),orABCA11SR-BI1PLTP1CYP27A1(A1).Cellswerelabeledwith[14C]cholesterolfor48hat37°C,washed,andlabeledwith[3H]acetatefor3hat15°C.Cellswerethenwarmedto37°Cfor20min,cooledto4°C,washed,andtreatedwithcholesteroloxidase(finalconcentration1U/ml)for3hat4°C.CellularlipidswereisolatedandseparatedonTLCasdescribedinMaterialsandMethods.Theratioofoxidizedcholesteroltocholesterolisshown.*P,0.01(versusmock-transfectedcells).E:RAW264.7cellsweretransientlytransfectedwithmock(M),ABCA1(A),orABCA11SR-BI1PLTP1CYP27A1(A1).TheabundanceofABCA1oncellsurfacewasassessedasaccessibilityofABCA1tobiotinilationasdescribedinMaterialsandMethods.F,G:RAW264.7cellsweretransientlytransfectedwithmock(M),ABCA1(A),orABCA11SR-BI1PLTP1CYP27A1(A1).ApoA-Iwasaddedtothefinalconcentrationof50mg/ml,incubatedfor1hat37°C,andwashedout.Dithiobis(succinimidyl)propionatewasaddedtothefinalcon-centrationof250mMandincubatedfor1hatroomtemperature.Cellswerethenlysed,andABCA1wasimmunoprecipitatedwithpolyclonalanti-ABCA1antibodies.Precipitated(F)andunbound(G)fractionsweredilutedwithloadingbuffercontaining5%2-mercaptoethanol,boiledfor5min,andsubjectedtoSDS-PAGEandWesternblotusingmonoclonalantibodyagainstapoA-I(F,G)orb-actin(H).
membrane(34).At37°C,cholesteroleffluxtocyclodextrinisABCA11SR-BI1PLTP1CYP27A1hadanyeffectontheareflectionoftheamountofbothpreexistingcholesterolandeffluxtocyclodextrinat4°Cand37°C(Fig.8C),indicatingcholesteroldeliveredtotheplasmamembraneduringthe1hthatneithertheamountofcholesterollocatedontheplasmaincubation(34).OverexpressionofneitherABCA1normembranenoritsdeliverytotheplasmamembranewasaf-Pathwaysofcholesteroleffluxinvivo
2319
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014fectedbythetransfections.Overexpressionofcaveolin-1orthecaveolin-11SR-BIcombinationalsodidnotaffectcholes-teroleffluxtocyclodextrinat37°C(Fig.8C).Thus,theob-servedeffectsoncholesteroleffluxtobothlipid-freeapoA-IandHDLwerespecific.
Toassesstheamountandtherateofdeliveryofcholes-teroltospecificallyexposedcholesterol-richdomains,wein-vestigatedtheeffectoftransfectionsonthesusceptibilityoftotalandnewlysynthesizedcholesteroltooxidationbycholesteroloxidase,amethodweemployedpreviouslytoin-vestigatetraffickingofcholesteroltocholesterol-richdo-mains(25).Cellswerelabeledwith[14C]cholesterolfor48hat37°Ctolabeltheentirecellularcholesterolpool,andwith[3H]acetatefor3hat15°Ctolabeltheintracel-lularnewlysynthesizedcholesterolpool,followedby20minincubationat37°Ctoallowsomeofthenewlysynthesizedcholesteroltomovetotheplasmamembrane.Thetotalamountofcholesterolinthecholesterol-richdomainsincreasedby30–60%afterbothABCA1andABCA11SR-BI1PLTP1CYP27A1transfections(Fig.8D),whereastherateoftransferofcholesteroltocholesterol-richdomainswasnotstatisticallydifferentintransfectedversusmock-transfectedcells.Thus,consistentwithfindingsofVaughanandOram(35),transfectionwithABCA1in-creasedtheabundanceofcholesterolincholesterol-richdo-mains,butthiswasnotthemechanismfortheadditionalincreaseofcholesteroleffluxaftercotransfectionwithSR-BI1PLTP1CYP27A1.
Totestwhetheroverexpressionofthecombinationofgenesincreasedtheabundanceofcell-surfaceABCA1,weassessedtheaccessibilityofABCA1tobiotinylationaftertransfectionwithABCA1aloneandwithABCA11SR-BI1PLTP1CYP27A1.Asexpected,theabundanceofABCA1onthecellsurfacewasincreasedinbothcases,butthedifferencebetweenthetwotransfectionswasminimal(Fig.8E).Further,weanalyzedbindingofapoA-Itocells,andmorespecifically,theamountofapoA-IboundtoABCA1.ApoA-Iwasincubatedwithcells,followedbycross-linkingofboundapoA-Itocellsurfaceproteins.ABCA1wasthenprecipitatedwithanti-ABCA1antibody(cross-reactingwithhumanandmouseABCA1)(24),andtheamountofapoA-IboundtoABCA1(precipitated)andtheamountboundtootherproteins(supernatant)wereassessedbyWesternblot.Transfectionsdidnotaf-fecttheamountofapoA-IboundtoABCA1(Fig.8F),con-sistentwithourpreviousfindingthatapoA-IbindingtoABCA1andABCA1-dependentcholesteroleffluxmaybedissociated(24).However,therewastwiceasmuchapoA-Iinthesupernatantfraction(Fig.8G),indicatingthatthereisanenhancedbindingofapoA-ItocellsaftertransfectionwithABCA11SR-BI1PLTP1CYP27A1,butthebindingsitewasnotABCA1.Thisfractionwasthenimmunoprecipitatedwithanti-apoA-Iantibodiesandde-velopedwithanti-PLTP,anti-SR-BI,oranti-mycantibodies.NoneofthethreeproteinscoprecipitatedwithapoA-I(datanotshown).Thus,coexpressionofSR-BI,PLTP,andCYP27AwithABCA1mayenhancecholesteroleffluxbyincreasingbindingofapoA-Itothecells.Theexactnatureofthebindingsitesandtheircompositionarenot
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Volume49,2008
clear;theymayincludebindingtoproteinand/orlipidcomponentsoftheplasmamembrane,i.e.,phospholipid-richdomains.
DISCUSSION
Anumberofcholesteroleffluxpathwaysweredescribed,buttherelativecontributionofeachofthemtocholesterolexportfromcellsremainsunclear.Inactivationofaspe-cificeffluxpathwayinsomecellsresultsinsignificantim-pairmentofcholesterolexportandaccumulationofcholesterol,whereasinothercells,thesameinactivationhasminimaleffectoncholesteroleffluxandcholesterolaccumulation.Forexample,ABCA1knockoutinmousemacrophages,resultinginasevereimpairmentofcholes-terolefflux,leadstoanaccumulationofcholesterolinthesecells(36),butthereisnoaccumulationofcholes-terolinTangierfibroblastsorendothelialcellslackingABCA1(11).Further,overexpressionofCYP27A1(4,20),caveolin-1(5),andABCG1(30)leadstoasignificanten-hancementofcholesteroleffluxinvitro,andyetknockoutofthesegenesinvivohasonlyamodesteffectoncholes-terolaccumulationinmacrophages(19,37,38).Theseandotherresultsindicatethatdifferentcellsmayusedifferentpathwaysforcholesteroleffluxtomaintaincholesterolhomeostasisand/orthereisaconsiderableredundancyinthepathwaysofcholesterolexport.Anotherpossibilityisthatutilizationofdifferentpathwaysdependsonprevailingmetaboliccircumstancesandintracellularlocationoftheexcesscholesterol.Thus,cellularcholesterolpoolsinmacro-phagesoverloadedbyexposuretoLDLweremoreeffec-tivelyreducedbytheABCG1-dependentpathway,whereascellularpoolsincellsoverloadedbyexposuretoacety-latedLDLwerereducedmainlybytheABCA1-dependentpathway(39).
Themainfindingofthisstudyisthatenhancingcholes-teroleffluxpathwaysthatuseapoA-Iasacceptorleadstoenhancedcholesterolexportfrommacrophagesinvivo,whereasenhancingpathwaysthatuseHDLasacceptorisineffective.ThefactthatcholesteroleffluxinvivowasenhancedcoordinatelywiththeincreaseofcholesteroleffluxthroughpathwaysutilizingapoA-I,butnotHDL,indicatesthattheformermayhaveapredominantcontri-butiontooverallcholesterolexportoratleasttoitsup-regulatedcomponent,whichcanbepotentiallytargetedfortreatment.Thisisconsistentwithfindingsthatinactiva-tionofmacrophageABCA1,themainelementofthecho-lesteroleffluxpathwayusingapoA-I,promotesatherosclerosis(36),whereasoverexpressionofABCA1protectsagainstatherosclerosis(15).ItisalsoconsistentwiththerecentfindingsofAdornietal.(40)thatABCA1-dependenteffluxtohumanserumexvivoisapredominantcholesteroleffluxpathway.InactivationoroverexpressionofkeyelementsoftheeffluxpathwaythatusesHDLasprimaryacceptor,suchasABCG1,hasbeenshowntohavealimitedeffectonthedevelopmentofatherosclerosis(19).Itmustberecognized,however,thattheeffectsofoverexpressionandinactivationofproteinsarenotnecessarilycomplementary.Forexam-
Downloaded from www.jlr.org at Shanghai Information Center for Life Sciences, CAS, on August 12, 2014ple,overexpressionofaproteinmaynotaffectthefunctionifthestepitisinvolvedinisnotrate-limiting,orifthepro-teinisalreadyatafunctionallysaturatingconcentration,asmightbethecaseforABCG1.Thus,ourfindingsareindic-ativeofpossibleapproachestoenhanceRCTratherthanofpossiblecausesofitsimpairment.
AnotherfindingofthisstudyisthatCYP27A1,PLTP,andSR-BIsupplementABCA1-dependentcholesteroleffluxtoapoA-I.Theexactmechanismofinteractionbe-tweentheseproteinsandtheroleofeachindividualpro-teinremainunclear.ComparisonofcholesteroleffluxaftervarioustransfectionsclearlyshowstherequirementforABCA1inthiscombination,inasmuchasonlyamodestincreaseincholesteroleffluxwasachievedwhenABCA1wasomitted,andeventhatwasprobablyduetotheeffectofoverexpressedproteinsonABCA1abundance.However,theabundanceofABCA1inthecellsandtheabundanceofcell-surfaceABCA1didnotincreaseaftercoexpressionofABCA11CYP27A11PLTP1SR-BIovertheamountfoundafteroverexpressionofABCA1alone.Theamountofcholesterolontheplasmamembranealsodidnotchange,andalthoughABCA1increasedtheamountofcholesterolincholesterol-richdomains,coexpressionofthreeothergenesdidnotfurtherincreaseit.Neithercho-lesteroltraffickingtotheplasmamembranenorbindingofapoA-ItoABCA1wasaffectedbythecotransfections.How-ever,thetotalamountofapoA-IboundtocellstransfectedwithABCA11CYP27A11PLTP1SR-BIdoubled,com-paredwithmock-andABCA1-transfectedcells,whichmayexplaintheenhancedcholesterolefflux.Theaddi-tionalapoA-Ibindingsiteswerenotonthefouroverex-pressedproteins,andtheirnatureremainsunclear.Ithasbeensuggestedrecentlythateffectivecholesteroleffluxrequiresformationofphospholipid-richcurvedmembranedomainscapableofhigh-affinitybindingtoapoA-I(41,42).ItispossiblethatoverexpressionofthecombinationofproteinsstimulatestheformationofsuchdomainsandenhancesassociationofapoA-Iwiththesedomainsortheirproteinand/orlipidconstituents.Theroleofindi-vidualproteins,especiallythatofSR-BI,whichwasmainlyimplicatedincholesteroleffluxtoHDL,remainsunclear,andthemechanismisnotyetdefined.
Inconclusion,wedemonstratedthatcholesteroleffluxtoapoA-Imaymakeapredominantcontributiontocholes-terolexportfrommacrophages.ThekeyelementofthispathwayseemstobeABCA1,withCYP27A1,PLTP,andSR-BIplayingasupplementaryrole.
TheauthorsacknowledgethetechnicalassistanceofMs.S.PenfoldandMs.A.Gatt.
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