Intracellular Calreticulin Regulates Multiple StepsWord文档下载推荐.docx
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Intracellular Calreticulin Regulates Multiple StepsWord文档下载推荐.docx
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,1,
3.ManuelA.Pallero‡and
4.JoanneE.Murphy-Ullrich‡,2
+AuthorAffiliations
1.FromtheDepartmentsof‡Pathologyand
2.§
CellBiology,UniversityofAlabamaatBirmingham,Birmingham,Alabama35294-0019
+AuthorNotes
↵1Presentaddress:
Dept.ofAnimalBiology,SchoolofVeterinaryMedicine,UniversityofPennsylvania,Philadelphia,PA19104.
1.↵2Towhomcorrespondenceshouldbeaddressed:
VolkerHall668,1670UniversityBlvd.,Birmingham,AL35294-0019.Fax:
205-975-9340;
E-mail:
murphy@uab.edu.
Abstract
Calreticulin(CRT),achaperoneandCa2+regulator,enhanceswoundhealing,anditsexpressioncorrelateswithfibrosisinanimalmodels,suggestingthatCRTregulatesproductionoftheextracellularmatrix.However,directregulationofcollagenmatrixbyCRThasnotbeenpreviouslydemonstrated.WeinvestigatedtheroleofCRTintheregulationoffibrillarcollagenexpression,secretion,processing,anddepositionintheextracellularmatrixbyfibroblasts.MouseembryonicfibroblastsdeficientinCRT(CRT−/−MEFs)havereducedtranscriptlevelsoffibrillarcollagenIandIIIandlesssolublecollagenascomparedwithwildtypeMEFs.Correspondingly,fibroblastsengineeredtooverexpressCRThaveincreasedcollagentypeItranscriptandprotein.Collagenexpressionappearstoberegulatedbyendoplasmicreticulum(ER)calciumlevelsandintracellularCRT,becausethapsigargintreatmentreducedcollagenexpression,whereasadditionofexogenousrecombinantCRThadnoeffect.CRT−/−MEFsexhibitedincreasedERretentionofcollagen,andcollagenandCRTwereco-immunoprecipitatedfromisolatedcelllysates,suggestingthatCRTisimportantfortraffickingofcollagenthroughtheER.CRT−/−MEFsalsohavereducedtypeIprocollagenprocessinganddepositionintotheextracellularmatrix.ThereducedcollagenmatrixdepositionispartlyaconsequenceofreducedfibronectinmatrixformationintheCRT-deficientcells.Together,thesedatashowthatCRTcomplexeswithcollagenincellsandthatCRTplayscriticalrolesatmultiplestagesofcollagenexpressionandprocessing.ThesedataidentifyCRTasanimportantregulatorofcollagenandsuggestthatintracellularCRTsignalingplaysanimportantroleintissueremodelingandfibrosis.
Calcium
Cell/Trafficking
Chaperones
ExtracellularMatrix/Collagen
ExtracellularMatrix/Fibronectin
Protein/Processing
Transcription
Calreticulin
PreviousSectionNextSection
Introduction
Calreticulin(CRT),3alsoknownastheC1qreceptor,isanendoplasmicreticulum(ER)chaperone,andamodulatorofintracellularcalciumsignaling.CRTalsoisamultifunctionalproteinthatispresentinnumerouscellularcompartments,includingtheER,cytoplasm,nucleus,atthecellsurface,andasareleasedprotein(1,–,5).ThereisevidencetosuggesttheinvolvementofCRTintissueremodelingandwoundhealing.Inaporcinedermalwoundhealingmodel,topicalapplicationofpurifiedCRTincreasestherateofwoundhealingandwoundtensilestrength(6).Proteomicdatashowup-regulationofCRTexpressionwithfibrosisinaratmodelofunilateraluretericobstructionkidneyfibrosisandinamousemodelofbleomycin-inducedlungfibrosis(7).ThemechanismsbywhichCRTregulatestissueremodelingarenotwellunderstood,althoughfibronectinmatrixdepositionandmodulationofcelladhesion,motility,proliferation,andmatrixmetalloproteinaseexpressionhavebeenimplicated(6,8,–,13).
CRThaseffectsontheextracellularmatrix(ECM)andcellularresponsestotheECM.FibroblastsoverexpressingCRThaveincreasedfibronectinmRNA,protein,andmatrixdeposition,andcellslackingCRTexpresslessfibronectinthanwildtypecells(9,14).CRTintheERisthoughttoregulatethefibronectinmatrixthroughregulationofintracellularcalciumsignaling(9,14).Inaddition,CRTknock-outMEFshavedifferencesinmatrixmetalloproteinaseexpressionthatareregulatedthroughtheERKandphosphoinositide3-kinasepathways(10).CytoplasmicCRTstabilizesintegrin-mediatedadhesiontocollagenthroughbindingthecytoplasmictailoftheintegrinαsubunitandthroughcalciumsignaling(15,–,18).Finally,cellsurfaceorextracellularCRTcanpotentiallymodulateECMremodelingthroughdirectbindingtoECMmoleculesandtocelladhesionreceptors,includingcollagentypesI,III,andV,thrombospondin,α2β1integrin,andglycoproteinVI(18,–,20).
CRThasmultiplecellularfunctions.CRTisanERchaperoneforN-linkedglycoproteinsintheCRT/calnexincycle(21).Italsoactsasaclassicalchaperonefornonglycosylatedproteinsbypreventingproteinaggregationinaninvitromodel(22).CRTisanimportantregulatorofCa2+homeostasiswithintheER(23,24).TotalCRTexpressionisup-regulatedbymanyformsofcellularstress,includingaminoaciddeprivation,depletionofCa2+stores,oxidativestress,andhypoxia(25,–,29).Despiteitslackofatransmembranedomain,CRTisonthesurfaceofmanycelltypes,includingfibroblasts,endothelialcells,andapoptoticcells.Fromthecellsurface,CRTsignalsmultiplecellularprocesses,includingapoptoticclearanceofcells,focaladhesionturnover,proliferation,migration,andanoikisresistance(4,13,20,30,–,33).ManyoftheseresponsestocellsurfaceCRTaremediatedbyCRTbindingtoLRP1(lowdensitylipoproteinreceptor-relatedprotein1)(13,33,–,35).RecentstudiesfromourlaboratoryshowthatengagementoftheCRT-LRP1complexonthecellsurfacebythrombospondin-1stimulatesfibrillarcollagenexpressioninvitroandinvivo.4Invitro,exogenousCRTtreatmentcausesproliferationoffibroblasts,endothelialcells,andkeratinocytesandincreaseskeratinocyteandfibroblastmigration(6).
GivenevidenceofCRTfunctioninwoundhealinganditsexpressioninfibrotictissues,weaddressedtheroleofCRTinregulatingfibrillarcollagenexpressionbyfibroblasts.CollagenisamajorcomponentoftheECM,importantforboththeprovisionalmatrixduringwoundhealingandinpathologictissueremodelinginfibrosis(36).Collagensynthesisanddepositionareregulatedatmultiplelevels,includingtranscription,secretion,processing,incorporationintofibrils,anddegradation.Inthisstudy,weusedmouseembryonicfibroblastsdeficientinCRTandmousefibroblastsengineeredtooverexpressCRTtoexaminetheroleofCRTinregulationoffibrillarcollagenexpression,trafficking,andincorporationintotheECM.ThesestudiesshowthattypeIcollagentranscription,proteinexpression,transitthroughtheER,andincorporationofcollagenintotheECMareregulatedbyCRTexpression.Furthermore,weprovideevidenceforCRTaffectingcollagenthroughitsroleinregulationofERCa2+levelsandbymediatingproteintransitthroughtheER.Inaddition,CRThasindirecteffectsoncollagenmatrixdepositionasaconsequenceofitsroleinregulationoffibronectinexpression.ThesedataarethefirsttodemonstratetheimportanceofCRTonmultipleprocessescriticalforcollagenmatrixproduction.
EXPERIMENTALPROCEDURES
Materials
Dulbecco'
smodifiedEagle'
smedium(DMEM)with4.5g/literglucosewaspurchasedfromInvitrogen.AscorbicacidandthapsigarginwerepurchasedfromSigma.VectashieldwaspurchasedfromVectorLaboratories(Burlingame,CA),andgoatserumwaspurchasedfromMPBiomedicals(Solon,OH).RecombinantrabbitcalreticulinwasagiftfromDr.LeslieGold(NewYorkUniversity)(6).Calreticulinwasstoredin10mmTris,3mmCaCl2,pH7.0.Humanfibronectinandmouseanti-GM130antibodywerepurchasedfromBDBiosciences.Rabbitanti-CRT(SPA-600),mouseanti-CRT(SPA-601),andrabbitanti-calnexin(SPA-860)werepurchasedfromStressGen(AnnArbor,MI).Rabbitanti-mousetypeIcollagenwaspurchasedfromMDBiosciences(MD20151,Zurich,Switzerland).Rabbitanti-ERK1/2waspurchasedfromCellSignaling(p44/42MAPK,catalogno.9102,Beverly,MA).MousemonoclonalantibodytoHSP47(M16.10A1)waspurchasedfromAbcam(Cambridge,MA).Goatanti-prolyl4-hydroxylaseα-1antibodywaspurchasedfromNovusBiologicals(NB100-57852,Littleton,CO).Rabbitanti-β-tubulin,mouseanti-HA(sc-7392),andgoatanti-collagentypeI(C-18)werepurchasedfromSantaCruzBiotechnology(SantaCruz,CA).AlexaFluor488goatanti-rabbitIgG,AlexaFluor488donkeyanti-goatIgG,TexasReddonkeyanti-rabbit,AlexaFluor543goatanti-mouseIgG,andHoechst33342werepurchasedfromInvitrogen.RabbitTrueBlotTMHRPanti-rabbitandmouseTrueBlotTMHRPanti-mousesecondaryantibodieswerepurchasedfromeBioscience(SanDiego).CoomassiePlusproteinassayreagentswerepurchasedfromPierce.WesternLightningChemiluminescenceReagentPluswaspurchasedfromPerkinElmerLifeSciences,andReBlotstrongstrippingsolutionwaspurchasedfromChemicon(Billerica,MA).Peptidesusesareasfollows:
CRT-bindingTSP1peptide,hepI(ELTGAARKGSGRRLVKGPD),andcontrolpeptide,modifiedhepI,(ELTGAARAGSGRRLVAGPD)werepurchasedfromAnaSpec,Inc.(SanJose,CA).
Cells
Wildtypemouseembryonicfibroblasts(MEFs),calreticulin-null(CRT−/−MEFs),andcalreticulin-nullMEFsstablytransfectedwiththepcDNA3expressionvectortoexpressrabbitHA-taggedCRT(37)weregiftsfromDr.MarekMichalak(UniversityofAlberta,Edmonton,Alberta,Canada).TheHA-CRT-reconstitutedCRT−/−cellswereshowntoexpressHA-taggedCRTandtorespondtotheCRT-bindingpeptideofTSP1infocaladhesiondisassemblyassays(datanotshown).MouseLfibroblasts(CRTunderexpr
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