REVIEW
Transcriptionalregulationbycalcium,calcineurin,andNFAT
PatrickG.Hogan,1LinChen,3JulieNardone,1andAnjanaRao1,2,41TheCenterforBloodResearchand2DepartmentofPathology,HarvardMedicalSchool,Boston,Massachusetts02115,USA;3DepartmentofChemistryandBiochemistry,UniversityofColoradoatBoulder,Boulder,Colorado80309-0215,USA
TheNFATfamilyoftranscriptionfactorsencompassesfiveproteinsevolutionarilyrelatedtotheRel/NFBfam-ily(ChytilandVerdine1996;Graefetal.2001b).TheprimordialfamilymemberisNFAT5,theonlyNFAT-relatedproteinrepresentedintheDrosophilagenome.NFAT5isidenticaltoTonEBP(tonicityelementbindingprotein),atranscriptionfactorcrucialforcellularre-sponsestohypertonicstress(López-Rodríguezetal.1999;Miyakawaetal.1999).Wefocushereonthere-mainingfourNFATproteins(NFAT1–NFAT4,alsoknownasNFATc1–c4;seeTable1),referringtothemcollectivelyasNFAT.
ThedistinguishingfeatureofNFATisitsregulationbyCa2+andtheCa2+/calmodulin-dependentserinephos-phatasecalcineurin.NFATproteinsarephosphorylatedandresideinthecytoplasminrestingcells;uponstimu-lation,theyaredephosphorylatedbycalcineurin,trans-locatetothenucleus,andbecometranscriptionallyac-tive,thusprovidingadirectlinkbetweenintracellularCa2+signalingandgeneexpression.NFATactivityisfurthermodulatedbyadditionalinputsfromdiversesignalingpathways,whichaffectNFATkinasesandnuclearpartnerproteins.Inthefirstpartofthisreview,wedescribetheinfluenceofthesemultipleinputsonthenuclear–cytoplasmicdistributionandtranscriptionalfunctionofNFAT.
RecentstructuraldataemphasizetheremarkableversatilityofNFATbindingtoDNA.AtcompositeNFAT:AP-1elementsfoundintheregulatoryregionsofmanytargetgenes,NFATproteinsbindcooperativelywithanunrelatedtranscriptionfactor,AP-1(Fos–Jun;Chenetal.1998).AtDNAelementsthatresembleNFBsites,NFATproteinsbindDNAasdimers(Giffinetal.2003;Jinetal.2003).Inthesecondsectionofthisreview,wedescribethesetwomodesofDNAbindingbyNFAT.NFATalsoactssynergisticallywithtranscriptionfactorsotherthanFosandJun,butthestructuralbasisforsyn-ergyremainsunknown.Drawingonpublishedstruc-tures,wediscussthepotentialcooperationofNFATwithotherclassesofDNA-bindingproteins.
4ItisclearthatNFATactivatestranscriptionofalargenumberofgenesduringaneffectiveimmuneresponse(Raoetal.1997;Kianietal.2000;Serflingetal.2000;Maciánetal.2001).Inthethirdpartofthisreview,wepresentinformationobtainedfromthesestudies,high-lightingexperimentalandbioinformaticsapproachestoidentifyingNFATtargetgenes.WediscussthefindingthatNFATandNFAT–Fos–Juncomplexesactivatedis-tinctsubsetsoftargetgenesinlymphocytes(Maciánetal.2002).Wealsodescribeanovelaspectofgeneregu-lationbyNFAT,inwhichthistranscriptionfactorpar-ticipatesinanearlyphaseofchromatinremodelingthatoccursatspecificgeneticlociindifferentiatingTcells(Avnietal.2002).
ThereisevidencethatNFATregulatescelldifferen-tiationprogramsincelltypesotherthanimmunecells(CrabtreeandOlson2002;HorsleyandPavlath2002;Graefetal.2003;Hill-Eubanksetal.2003).Inthelastsectionofthisreview,weselectthreedifferentiationprograms—fiber-typespecificationindifferentiatedskel-etalmuscle,cardiacvalvedevelopment,andosteoclastdifferentiation—fordetailedconsideration.WeevaluatetheevidenceforNFATinvolvement,pointoutnovelcel-lularandmolecularmechanismsthatmightregulatethisfamiliartranscriptionfactor,anddiscusshowNFATexertsitsbiologicaleffects.BecausethephenotypesofNFATknockoutmicehavebeenreviewedelsewhere(CrabtreeandOlson2002;HorsleyandPavlath2002),werefertothemonlyasnecessarytoillustratespecificpoints.
CellularinputsaffectingNFATCa2+andcalcineurin
NFATisactivatedbycell-surfacereceptorscoupledto“store-operated”Ca2+entryviaphospholipaseC(Fig.1).Theimportanceofthisprocessisillustratedbypatientswitharareformofhereditaryseverecombinedimmu-nodeficiency(Feskeetal.2000,2001).Tcellsfromthesepatientsshowedmultiplecytokinedeficiencyandase-lectiveinabilitytoactivateNFAT;NFBandAP-1acti-vationwerenormal.Theprimarydefectwastracedtoapronouncedreductioninstore-operatedCa2+entry,whichledtoamarkedimpairmentinactivationofallNFATs.
Correspondingauthor.
E-MAILarao@cbr.med.harvard.edu;FAX(617)278-3280.
Articleandpublicationareathttp://www.genesdev.org/cgi/doi/10.1101/gad.1102703.
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Table1.ProteinNFAT1NFAT2NFAT3NFAT4NFAT5
ListofNFATproteins
OthernamesNFAT,NFATc2NFATc,NFATc1NFATc4
NFATx,NFATc3TonEBP
Regulation
Ca2+/calcineurinCa2+/calcineurinCa2+/calcineurinCa2+/calcineurin
Osmoticstress
Integrin(␣64)activation
References
McCaffreyetal.1993Northropetal.1994Hoeyetal.1995
Hoeyetal.1995;Masudaetal.1995;Hoetal.1995
Miyakawaetal.1999;Lo´pez-Rodrı´guezetal.1999;Jauliacetal.2002
NFATactivationisinitiatedbydephosphorylationof
theNFATregulatorydomain,aconserved∼300-amino-acidregionlocatedN-terminaltotheDNA-bindingdo-main(Fig.2).ThisdomainisencodedinasingleexoninallfourNFATproteinsfromallvertebratespeciesforwhichsequencedataareavailable(Graefetal.2001b).Thedomainisheavilyphosphorylatedinrestingcells,withthephosphorylatedresidues(serines)distributedamongfourclassesofconservedserine-richsequencemotifs(SRR-1,SPxxrepeat,SRR-2,andKTSmotifs;Fig.2;Bealsetal.1997a;Okamuraetal.2000).Calcineurindephosphorylatesthreeofthefourtypesofmotifs,thustriggeringNFATnuclearaccumulationandincreasingtheaffinityofNFATforitstargetsitesinDNA(Shawetal.1995;Okamuraetal.2000;Porteretal.2000;NealandClipstone2001).Itisnotformallyknownwhetherdephosphorylationisanorderedprocess,butmassspec-trometryexperimentssuggestthattheSRR1region,whichisimmediatelyadjacenttothemajorcalcineurindockingsite(PxIxIT;seebelow),ispreferentiallydephos-phorylatedatlowcalcineurinactivity(Okamuraetal.2000).NFATmutantswithsmalldeletionsorSAsubstitutionsintheSRR1regionaremoresusceptiblethanwild-typeNFATtodephosphorylationoftheSPxxrepeatsbycalcineurin(Zhuetal.1998;Aramburuetal.1999).
Efficientdephosphorylationrequiresadockinginter-actionbetweenNFATandcalcineurin(Aramburuetal.1998,1999;Chowetal.1999;J.Liuetal.2001).ThemajordockingsiteforcalcineurinislocatedattheNterminusoftheNFATregulatorydomain,andhastheconsensussequencePxIxIT(SPRIEITinNFAT1;Fig.2).TheindividualNFATproteinspossesscharacteristicPxIxITsequenceswithalowaffinityforcalcineurin(Kd=10–30µM),neededtomaintainsensitivitytoenvi-ronmentalsignalsandpreventconstitutiveactivationofNFAT.SubstitutionoftheSPRIEITsequenceofNFAT1withHPVIVIT,ahigher-affinityversionobtainedbypep-tideselection,increasedthebasalcalcineurinsensitivityoftheproteinandresultedinpartialnuclearlocalization(Aramburuetal.1999).TheA238LproteinofAfricanswinefevervirus,apotentviralinhibitorofNFAT,containsaPKIIITsequencethatislikelytobeinterme-diateinaffinitybetweentheSPRIEITandHPVIVITse-quences(Miskinetal.2000).ThesurfaceofNFAT–cal-cineurininteractionislikelytobemoreextensivethanthePxIxITmotif,however,becauseasecondinteractingsequencehasbeenidentifiedinNFAT2andinNFAT4
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(Liuetal.1999;Parketal.2000).Thissequence(calci-neurin-bindingsequenceBinFig.2)ismoderatelycon-servedintheNFATproteinsandresemblesahighlycon-servedsequenceinthecalcineurininhibitorsDSCR1/MCIP1(Fuentesetal.2000;KingsburyandCunningham2000;Rothermeletal.2000).Structuralandcell-biologi-calstudiesshouldestablishwhetherNFATandDSCR1/MCIP1usethisregionforcalcineurinbindingandinhi-bition,andwhethertheycompeteforcalcineurinbind-ingincells.
Inoverexpressionexperiments,NFATnuclearlocal-izationisaccompaniedbysomerelocalizationofcalci-neurinfromthecytoplasmintothenucleus(Shibasakietal.1996).Thisprocesshasbeendocumentedforendog-enouscalcineurinandNFAT1inprimarykeratinocytes(Al-Darajietal.2002),butisnotreadilyobservedfortheendogenousproteinsinothercelltypes.Nevertheless,itisclearthatcalcineurinispresentinthenucleusofstimulatedcells,whereitmaintainsthedephosphory-latedstatusandnuclearlocalizationofNFAT.WhenCa2+entryispreventedorcalcineurinactivityisinhib-ited,NFATisrephosphorylatedbyNFATkinasesandrapidlyleavesthenucleus(t1/2∼15min),andNFAT-dependentgeneexpressionisterminated(Garrityetal.1994;Lohetal.1996a,b;Timmermanetal.1996).AsaresultofthisabsolutedependenceonCa2+/calcineurinsignaling,NFAThasaremarkableabilitytosensedy-namicchangesinintracellularCa2+levels([Ca2+]I)andfrequenciesofCa2+oscillationsincells(Dolmetschetal.1997,1998;Lietal.1998).
TheparametersofCa2+/calcineurinsignalingcanbemodulatedindiverseways.TheTNFfamilymemberRANKLelicitsCa2+oscillationsindifferentiatingosteo-clasts(Takayanagietal.2002).InTcells,TGFinhibitsTeckinaseactivityandtherebyCa2+influx(Chenetal.2003).Calcineurinactivitymaybecontrolledinde-pendentlyof[Ca2+]IbymodulatingtheexpressionofmembersoftheDSCR/MCIPfamilyofendogenouscal-cineurininhibitors.Thisprocessincorporatesanegativefeedbackloop:Calcineurin/NFATsignalsup-regulateDSCR1/MCIP1expression,whichthenfeedsbacktodown-regulatecalcineurinactivity(J.Yangetal.2000).NFATkinases
The13serineresiduesthatcontrolNFAT1nuclearlo-calizationarelocatedindiversesequencecontextsthatareunlikelytoberecognizedbyasinglekinase(Fig.2).
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TranscriptionalregulationbyNFAT
Figure1.SchematicviewoftheNFATactivationcycle.NFATisactivatedbycell-surfacereceptorscoupledtoCa2+mobilization:Immunoreceptorsandreceptortyrosinekinases(RTKs)activatephosphatidylinositol-specificphospholipaseC(PLC)-␥,whileG-protein-coupledreceptors(GPCR)activatePLC.PLCactivationraisesintracellularfreeCa2+levels([Ca2+]I)inseveralsequentialsteps:Hydrolysisofphosphatidylinositol(PI)-4,5-bisphosphate(PIP2)byPLCresultsingenerationofinositol-1,4,5-trisphosphate(IP3);IP3bindstoIP3receptors(IP3R)intheendoplasmicreticulum(bluecompartment)andpromotesabriefspikeof[Ca2+]IincreasebydepletingERCa2+stores;andstoredepletionissensedbyanas-yet-uncharacterizedsignalingmechanismthattriggersasustainedprocessof“store-operated”Ca2+entrythroughCRACchannelsintheplasmamembrane.[Ca2+]Iincreasesresultinactivationofmanycalmodulin(CaM)-dependentenzymes,includingthephosphatasecalcineurinandtheCaM-dependentkinasesCaMKIIandCaMKIV.CalcineurindephosphorylatesmultiplephosphoserinesonNFAT,leadingtoitsnucleartranslocationandactivation.CalcineurinactivityisinhibitedbytheimmunosuppressivedrugscyclosporinA(CsA)andFK506,whichactascomplexeswiththeirintracellularimmunophilinreceptorscyclophilinandFKBP12,respectively.Inparallel,hydrolysisofPIP2byPLCresultsinproductionofdiacyl-glycerols(DAG),whichactivateRasGRPandproteinkinaseC(PKC).Receptoractivationiscoupledtoactivationofproteintyrosinekinases(PTKs),Ras,MAPkinases(MAPK),andPI-3kinase(PI3K).MAPkinaseactivationleadstosynthesisandactivationofFosandJun,thecomponentsoftheheterodimerictranscriptionfactorAP-1,whichthenbindscooperativelywithNFATtocompositeNFAT:AP-1sitesfoundintheregulatoryregionsofmanyNFATtargetgenes.Ca2+mobilizationisterminatedbyCa2+-bindingproteinsandbyCa2+ATPasesintheERandplasmamembranes,whichpumpCa2+backintoERstoresandoutofthecell,respectively.TheERenzymeisinhibitedbythapsigargin,whichdepletesERCa2+storesandactivatesCRACchannelsintheabsenceofreceptorstimulation.Inneuronsandvascularsmoothmusclecells,NFATisselectivelyactivatedbyCa2+influxthroughL-typeCa2+channels(LTCC;Graefetal.1999;Stevensonetal.2001).Theunderlyingmechanismisnotunderstood.Potentially,calcineurinandNFATcouldbelocalizedtothevicinityofplasmamembranesignalingcomplexescontainingLTCC,NMDAreceptors,andscaffold/adapterproteinssuchasPSD95andAKAP-79.Alternatively,assuggestedforCREBactivation,themechanismcouldinvolveselectiveactivationofMAPKpathwaysviacalmodulinboundtoLTCC(Dolmetschetal.2001).
Theimplicationisthatseveralconstitutivekinasesco-operatetomaintaintheinactive,phosphorylatedstateofNFATinrestingcells;similarly,severalinducibleand/orconstitutivekinasesmayacttorephosphorylateNFATthathasbeendephosphorylatedduringcellactivation.SequencecomparisonsshowthattheSRR-1regionisextendedinNFAT2–4,whereastheSP-2andSP-3motifsaretruncatedinNFAT3(Okamuraetal.2000);thus,differentNFATproteinsmayberegulatedbyoverlap-pingbutdistinctsetsofconstitutiveandinducibleki-nases.Becausephosphorylationspecifiesthesubcellulardistribution,DNAbindingaffinity,andtranscriptionalactivityofNFAT(Shawetal.1995;Bealsetal.1997a;Okamuraetal.2000;Porteretal.2000;NealandClip-stone2001),regulationofeachNFATproteinbymul-tiplekinasesshouldintheorypermitanalmostcontinu-ousmodulationofthelevelofNFATactivation.
AlthoughseveralNFATkinaseshavebeenidentified,anintegratedpictureofNFATphosphorylationhasnotyetemerged.CK1andGSK3areconstitutiveNFATki-nasesthatpromoteNFATnuclearexport(Bealsetal.1997b;Zhuetal.1998);phosphorylationbyGSK3re-quirespriorphosphorylationbyaprimingkinasesuchasPKA(Sheridanetal.2002).Consistentwithitsknownsequencepreference(Harwood2001),GSK3phosphory-latestheSPxxmotifsofNFAT2(Bealsetal.1997b).TheMAPkinasesp38andJNKareinduciblekinasesthatpromoteNFATnuclearexport,byselectivelyphos-phorylatingNFATproteinsattheSer–Pro(SP)sequencesatthebeginningoftheirSRR-1regions:JNK1phosphory-
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Figure2.TheNFATregulatorydomain:sitesofphosphorylationandinteractionwithcalcineurin.(Top)OverallstructureofNFATproteins.ThediagramisbasedonmurineNFAT1(Okamuraetal.2000).Regulatory-domainphosphorylationsinconservedsequencemotifsareshownascirclesbelowthemotif.Redcirclesindicatephosphategroupsthatareremovedbycalcineurin,whereastheblackcircleintheSRR-2regionisnotsusceptibletodephosphorylationbycalcineurin.Thetworegionsinvolvedincontactingcalcineurinareindicated.RegionAisthePxIxITsequencefoundinallNFATproteins,andregionBhasbeendefinedinNFAT2andinNFAT4.(AD)Activationdomains;(NLS)nuclearlocalizationsignalintheregulatorydomain.AsubsidiaryNLSislocatedintheRHR-Cdomain(Bealsetal.1997a).(Middle)SequencesofphosphorylatedmotifsinmurineNFAT1,withphosphorylatedresiduesindicatedinblueboxes,otherconservedresiduesshadedinyellow,andthecoreofthenuclearlocalizationsignal(KRR)showninbold.ThephosphorylatedresiduesintheSPxx,SRR-2/NLS,andKTSmotifswereidentifiedbymassspectrometry;thoseshownasphosphory-latedintheSRR-1regionindicateonepossiblearrangementofthefiveresiduesknowntobephosphorylated,amongthesevenserinespresentinthemotif(Okamuraetal.2000).(Bottom)Sequencesofthecalcineurin-bindingregionBofNFAT2andNFAT4alignedwiththecorrespondingsequencesofotherNFATproteinsandthesimilarsequenceinDSCR1.Allsequencesarefromthehumanproteins.
latesNFAT2andNFAT4,whereasp38selectivelytar-getsNFAT1andNFAT3(Chowetal.1997;GómezdelArcoetal.2000;Yangetal.2002).ForJNK1,aproposedmechanismisthatphosphorylationoftheSPRIEITcal-cineurin-dockingsiteofNFAT2blockstheinteractionofNFAT2withcalcineurin(Chowetal.2000).
SelectiveactivationofexportkinasesexplainshowindividualNFATproteinsmightbedifferentiallyreg-ulatedinasinglecelltype.Forinstance,developingskeletalmusclecellsshowselectivenuclearlocaliza-tionofNFAT2inconjunctionwithcytoplasmiclocal-izationofNFAT1andNFAT4(Abbottetal.1998).ThisbehaviorisalsoobservedinTcellsstimulatedfor5–6hthroughtheT-cellreceptor(TCR;Lohetal.1996a);itcouldoccurthroughactivationofexportkinasesselectiveforNFAT1and/orinhibitionofex-portkinasesselectiveforNFAT2.Inanotherillustra-tion,NFAT3inhippocampalneuronsremainednuclearfor60–90minafterbriefdepolarizationwithhighK+,reflectingeitherinhibitionofanNFAT3kinaseorun-usuallyprolongedcalcineurinactivation(Graefetal.1999).InTcells,T-cellreceptorstimulationiscoupledtoCa2+/calcineurinsignalingandNFATnuclearimport,whereasstimulationthroughthecostimulatoryrecep-torCD28potentiatesactivationofthePI-3kinasepath-wayandhencethekinaseAkt/PKB(Parryetal.1997;Cantrell2002).Aktpromotesaninhibitoryphosphory-lationofGSK3(Crossetal.1995).ThenetresultisthatCD28costimulation,byactivatingAkt,inhibitsGSK3andthereforeNFATnuclearexport,prolongingthedurationofNFATnuclearresidenceinTcellsstimu-latedthroughboththeTCRandCD28,relativetoTcellsstimulatedthroughtheTCRalone(Diehnetal.2002).
Aswithothertranscriptionfactors,NFATtranscrip-tionalactivitymayberegulatedbymodificationofthetransactivationdomains.Thisaspecthasnotbeenstud-iedextensively,however.InduciblephosphorylationofthetransactivationdomainhasbeenobservedforNFAT1,andmutationofthemodifiedserineseliminates
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transcriptionalactivity(Okamuraetal.2000).TheCot1andPim1kinasespotentiatetransactivationbyNFAT1andNFAT2,respectively(deGregorioetal.2001;Rainioetal.2002),butitisnotknownwhetherthisoccursthroughdirectphosphorylationoftheNFATtransacti-vationdomain.Otherconsiderations
Multisitephosphorylation:ThephosphorylationstatusoftheregulatorydomainspecifiesDNA-bindingaffinityaswellasrelativeexposureofnuclearlocalization(NLS)andnuclearexport(NES)sequences(Shawetal.1995;Bealsetal.1997a;Okamuraetal.2000;Porteretal.2000;NealandClipstone2001),buthowthisoccurshasnotyetbeenelucidatedatastructurallevel.ThemostlikelyhypothesisisthatdephosphorylationfacilitatesaglobalconformationalswitchofNFATfromaninactivetoanactiveconformation(Okamuraetal.2000),possiblybyinterruptingmultipleinteractionsofphosphorylatedmotifswithcomplementarysequencesonNFATitselfand/oronpartnerproteins(Bealsetal.1997a;Porteretal.2000;NealandClipstone2001).Multisitephosphorylationisknowntoincreasethesensitivityofbiochemicalprocessestotheirsignal-inginputs(e.g.,seeNashetal.2001;Orlickyetal.2003).InamathematicalmodeloftheNFATconforma-tionalswitch,SalazarandHöfer(2003)addressedthelongstandingpuzzleofwhytherearesomanyphos-phorylationsitesonNFAT,bycalculatingthesensi-tivityofNFATtochangesincalcineurinactivityasafunctionofthenumberofdephosphorylationsrequiredtoinducetheactiveconformation.Ifthenumberofde-phosphorylationswassetatthe13sitesactuallyob-servedforNFAT1(Okamuraetal.2000),thedose-re-sponsecurvewassteepandhighlycooperative,definingathresholdofcalcineurinactivitybelowwhichNFATremainedinactiveandabovewhichfullactivationwasobtained.Incontrast,ifdephosphorylationatonly1–2siteswaspostulatedtobesufficientforfullactivation,averyshallowdose-responsecurvewasobtained(i.e.,evenalargeincreaseincalcineurinactivityinducedonlyasmallincreaseinNFATactivity;SalazarandHöfer2003).
Nucleartransport:Thenuclear–cytoplasmicratioattainedbyanNFATproteinreflectsnotonlythebal-anceofcalcineurinandNFATkinaseactivities,butalsothesummationofimportandexportratesofNLS-exposedandNES-exposedforms.Theexportkinet-icsofNFATlagbehindthekineticsofitsrephos-phorylationinthenucleus(Lohetal.1996a,b),possi-blybecausethephosphorylatedSPmotifsneedtoberestoredtotheproperconfigurationbythephos-phoSP-selectiveprolylisomerasePin1(W.Liuetal.2001).TheimportreceptorforNFAThasnotbeendefinitivelyidentified,althoughapotentialcandidateisRch1(Torgersonetal.1998);theexportreceptorismostlikelyCrm1,thetargetofthenuclearexportinhibitorleptomycinB(Klemmetal.1997;Kehlen-bachetal.1998),whichpreferentiallybindsphos-TranscriptionalregulationbyNFAT
phorylatedNFAT1(Okamuraetal.2000).ItisnotclearwhetherphosphorylatedNFATexposesanintrin-sicNES(assuggestedforNFAT2;Klemmetal.1997),orwhetherNESfunctionisconferredbyproteinssuchas14.3.3thatbindtothephosphorylatedform(assuggestedforNFAT3;ChowandDavis2000).TheputativeNESof14.3.3wasshowntofunctiongloballyinligandbindingratherthandirectlymediatingnu-cleartransport(Brunetetal.2002),indicatingthatadif-ferentNESisneeded.Whateverthespecificmechan-ism,import/exportkineticswillcontributetoaverag-ingNFATactivityunderconditionsofintermittentstimulation.
AutoregulationofNFAT2:TheshortestisoformofNFAT2(NFATc/A)isinducedinaCsA-sensitiveman-nerbyNFATitself,inaprocesssuggestedtoconstituteapositiveautoregulatoryloop(Zhouetal.2002).Thisproteinisgeneratedthroughutilizationofadistinctinduciblepromoterthatispreferentiallycoupledtothemostproximalpolyadenylationsite(Chuvpiloetal.1999).Asaresult,NFATc/AlackstheentireC-terminaldomainandcontainsanalternateN-ter-minaldomain,whichdiffersfromthatofotherNFATisoformsinthatitisnothighlyacidic.Positiveauto-regulationofoneisoformofatranscriptionfactorisafamiliarstrategyincelllineagecommitment,becauseitensuresthathighlevelsofthefactorareavailabletomaintainthecommittedstate(Davidson2001).ThefactthatanNFAT2isoformistheonlyNFATpro-teinsubjecttopositiveautoregulationexplainswhyNFAT2ismostoftenidentifiedasthemajorNFATproteinparticipatinginabiologicalresponse(e.g.,Th2andosteoclastdifferentiation;GlimcherandMurphy2000;Takayanagietal.2002).ThisimportantaspectofgeneregulationbyNFATisdiscussedmoreextensivelybelow.
SignalingpathwaysthataffectAP-1activity:Asnotedbelow,theAP-1transcriptionfactorisamajortrans-criptionalpartnerofNFAT(Maciánetal.2001).Clas-sically,AP-1consistsofheterodimersofFos-andJun-familyproteins:Fosproteinsdonotdimerizewithoneanother,andJundimersbindDNAwithloweraffinitythanFos–Jun(ChinenovandKerppola2001;Jochumetal.2001;Mechta-Grigoriouetal.2001).BecauseAP-1activityisalsomodulatedbydiversesignalinginputs(Davis2000),NFAT:AP-1-dependenttranscriptioninte-gratesaverylargenumberofsignalingpathwaysandprocesses:Themagnitudeandkineticsof[Ca2+]Iin-creases;thelevelofcalcineurinactivity;theactivitiesofkinasesthatmodulateNFATnuclearexport,DNA-binding,orintrinsictranscriptionalactivity;theactivi-tiesofPKC/MAPkinasepathwaysandothersignal-ingpathwaysthatinfluenceFos–Junsynthesisandactivation;andtheintegrityofcytoskeletalinteractionsandotherintracellularprocessesthathavemoreglo-baleffects.Inconsequence,NFAT:AP-1-basedreporterassaysareidealforoverallevaluationsofcellularsignal-ing:InTcellsstimulatedthroughantigenandcostimu-latoryreceptors,NFAT:AP-1reporteractivityisinflu-encedbyoverexpressionofwild-typeormutantversions
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ofamultitudeofsignalingproteins(e.g.,seeSosinowskietal.2000;Kaminumaetal.2001).Inothercelltypes,Ca2+mobilizationiscoupledtostimulationthroughreceptortyrosinekinasesandG-protein-coupledre-ceptors,andNFATreporterswouldbeusefulinanun-biasedquestforsignalingmoleculesinthesepathwaysaswell.
DiversemodesofDNAbindingbyNFATStructuralrelationtotheRel/NFBfamily
AllfiveproteinsoftheNFATfamily,NFAT1–NFAT4andNFAT5/TonEBP,maybeclassifiedasmembersoftheextendedNFB/Relfamily(Fig.3).TheclassificationisbasedonthestructuralsimilaritiesoftheirDNA-bind-ingdomains,alsoknownasRelhomologyregions(RHR).ThecanonicalmodeofDNAbindingbytheRHRhasbeenwellcharacterizedinthestructuresofseveralNFB–DNAcomplexes(Ghoshetal.1995;Mulleretal.1995):TheRHRcontainstwofunctionallydistinctdo-mains,anN-terminalspecificitydomain(RHR-N)thatmakesbase-specificDNAcontacts,andaC-terminaldo-maininvolvedindimerformationandIBbinding(RHR-C;Fig.4;Huxfordetal.1998;JacobsandHarrison1998).OfthefiveNFATproteins,NFAT5/TonEBPshowsthehighestdegreeofstructuralsimilaritytoNFB,formingasymmetricdimerwithastrikingresemblancetotheNFB–DNAcomplex(Fig.4,cf.AandB;Stroudetal.2002).ThedetaileddimerizationinteractionsmediatedbytheRHR-CofNFAT5areverysimilartothoseob-servedinNFB,buttheuniquefeatureoftheNFAT5dimer–DNAcomplexisaseconddimerinterfaceformedbytheEЈFloopoftheRHR-Ndomain(Fig.4B).Asaresult,NFAT5completelyencirclesitsDNA(Fig.4B),explainingtheunusuallyslowdissociationkineticsoftheNFAT5–DNAcomplex(Stroudetal.2002).
TwoNFAT1dimercomplexes,boundtoBsitesfromtheIL-8promoterandtheHIV-1LTR,haverecentlybeencharacterizedatthestructurallevel(Giffinetal.2003;Jinetal.2003).AsintheNFAT5andReldimercomplexes,theNFAT1dimerinterfaceresidesintheC-terminalregionoftheRHR(Fig.4C,D).However,thisinterfaceissurprisinglydifferentfromthesymmetricandhydrophobicRHR-CdimerinterfaceseeninNFAT5andNFB:Itisasymmetricandlargelyhydrophilic,andinvolvesresiduesnotusedinRelorNFAT5.Theinter-faceisessentiallyidenticalintheNFAT1dimersboundtotheIL-8andHIV-1LTRBsites,butthesetwoNFAT1dimercomplexesdiffersignificantlyintheirRHR-Ninteractionsandhencetheiroverallconforma-tion(Fig.4,cf.CandD).Specifically,theNFAT1dimerontheHIV-1LTRBsitecompletelyencirclestheDNAthroughEЈF-loopinteractionsintheRHR-Ndomain,inamannersimilartothatseenintheNFAT5–DNAcom-plex(Fig.4,cf.BandC).IntheternaryNFAT/Fos–Jun/DNAcomplex(seeFig.5;Chenetal.1998),theEЈFloopconstitutesthemajorbindingsiteforFos–Jun,empha-sizingtheversatilityofthisproteinsurfaceinpromotingassemblyofdistincttranscriptioncomplexescontainingNFAT.
DNAbindingbyNFATdimersisaversatilemecha-nismpermittinghomo-andheterodimerformationatresponseelementswithvaryingsequencesandspacing.ManyoftheRHR-CinterfaceresiduesobservedintheNFAT1dimerareconservedinNFAT2andNFAT4;incaseswheretheresiduesdiffer,interfacecomplemen-tarityappearstobemaintainedbycovariationofthein-teractingresidues.NFAT3isapotentialexceptionbe-causeithassignificantlydifferentresiduesatpositions
Figure3.AlignmentofNFB/RelproteinswithNFAT5andNFAT1(RelalignmentmodifiedfromRothwarfandKarin1999).TheDNA-bindingdomainsarealigned,andtheN-andC-terminalportionsoftheRelhomologyregionareindicated(RHR-NandRHR-C,respectively).AleucinezippermotifinRelBisshownasaredbox.Inp100andp105,serine-richregionsandankyrinrepeatsareshownasgrayboxesandbluecircles,respectively,andthesiteofcleavagetop52orp50isindicatedbyaverticalline.TheNFATregulatorydomainispatternedasinFigure2.
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TranscriptionalregulationbyNFAT
Figure4.CrystalstructuresofNFATandReldimericprotein/DNAcomplexes.Theproteinsareshowninribbonstyle,withtheRHR-NdomainingreenandtheRHR-Cdomaininyellow,andtheDNAisdrawninstickmodel.(A)NFBp50homodimerboundtoaBsite.(B)NFAT5/TonEBPboundtoatonicityresponseelement.(C)TheNFAT1dimerboundtotheBsitefromtheHIV-1LTR(identicalinsequencetothemurineIgBsite).(D)TheNFAT1dimerboundtoaBsitesimilartothatinthehumanIL-8promoter.InBandC,theEЈFloopofeachproteinpartnerextendstowardthemidlinebelowtheDNAhelixandformsaseconddimerinterface.TheDNAsequenceofeachcomplexisshownbelowthestructure.
correspondingtothoseintheNFAT1dimerinterface.ModelingstudiessuggestthatNFAT1,NFAT2,andNFAT4wouldbecapableofformingbothhomo-andheterodimersonB-likeDNAsites.TheseinteractionsarelikelytobeconfinedtotheNFATfamily,however:AlthoughNFAT1–4,NFAT5/TonEBP,andNFB/ReldimerscanallbindsimilarDNAelements,theirmodesofdimerizationaredifferent(Fig.4)andtheyrespondtodistinctintracellularsignals(López-Rodríguezetal.2001),suggestingthatmixedNFAT/NFAT5/NFBdimersdonotnormallyoccur.
The3siteoftheTNF␣promoter(TGGAGAAACCC)isagoodexampleofaphysiologicalB-likesitetowhichNFATbindsasadimer(Goldfeldetal.1993;McCaffreyetal.1994).Itresemblesthe10-bpBsiteusedforstruc-turedeterminationbyGiffinetal.(2003).Bindingisnucleatedatthehalf-siteshowninbold,buttheotherhalf-site(underlined)isquicklyoccupiedatlowNFATconcentrations(McCaffreyetal.1994).MutationofNFATcontactresiduesimpairsTNF␣promoteractivityinreporterassaysinTandBcells(Goldfeldetal.1993).SitesthatcanbindbothNFATandNFBhavebeeniden-tifiedintheHIV-1LTRandintheIL-8,IL-13,andGM-CSFpromoters(forreview,seeRaoetal.1997;Maciánetal.2001);whichfactorsactuallyoccupythesiteinvivodependsonthecelltypebeingexaminedandthespecific
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conditionsofstimulation.BecauseNFATisnotanobli-gatedimerandthecooperativityoftheDNA-bounddimerislessthanthatobservedfortheNFAT:AP-1com-plex,itisplausiblethatsomedimericelementsmaybindNFATeitherasamonomerorasadimerdependingontheavailableconcentrationofthistranscriptionfac-torinthenucleus.
CooperativeinteractionswithFos–Junproteinsinthenucleus
NFAT1–4proteinsformstrongcooperativecomplexesonDNAwiththeunrelatedtranscriptionfactorAP-1(Fos–Jundimers;Fig.5;Chenetal.1998;Maciánetal.2001).TheternaryNFAT:Fos:Juncomplexesserveassig-nalintegratorsfortwodiametricallydifferentsignalingpathways,thecalcium/calcineurinpathwaythatacti-vatesNFATandthephorbolester-responsiveMAPki-nasepathwaythatpromotesthesynthesisandactivationofFosandJunfamilyproteins.TheNFAT:Fos:Juncom-plexescontactan∼15-bpstretchofDNA,inwhichtheNFATandAP-1elementsarepreciselyapposedtocreateatrue“composite”site(Fig.5;Chenetal.1998).TheresiduesinvolvedinFos–JuncontactarelocatedlargelyintheN-terminalRHRdomainsofNFAT,andformanextensivenetworkofmostlypolarinteractionswithresi-duesinthebasicleucinezipperregionsofFosandJun;theyarenotstrongenoughtostabilizetheNFAT:AP-1complexintheabsenceofDNA.Theinteractingresi-duesarealmostcompletelyconservedinNFAT1–4butareabsentfromNFAT5(López-Rodríguezetal.1999),
Table2.
indicatingthattheabilitytocooperatewithFosandJunwasalateevolutionarydevelopment.
AwiderangeofcompositebindingsitesisobservedintheregulatoryregionsofNFATtargetgenes(Keletal.1999;forreview,seeRaoetal.1997;Maciánetal.2001).Byanalyzingasetof11experimentallyverifiedNFAT:AP-1compositesites,Keletal.(1999)demonstratedthatthefreeenergyofNFATandAP-1bindingtotheirre-spectivesubsitesshowedastrikinginversecorrelation,inthatstrongNFATbindingtendedtobepairedwithweakAP-1bindingandviceversa(seeTable2).Presum-ably,thetotalfreeenergyofbindingismaintainedwithinanarrowrangebecauseofbiologicalconstraints,suchasaneedforrapiddisassemblyofNFAT:AP-1-con-tainingtranscriptioncomplexeswhentheinitiatingstimulusdecays.
Acommonfeatureofnaturalpromotersandenhancersisthepresenceofmultiplebindingsitesforcriticaltran-scriptionfactors(Davidson2001).ThisisespeciallytrueforNFATtargetgenes,whosepromotersanddistalen-hancersgenerallycontainmultipleNFATorNFAT:AP-1sites(Raoetal.1997;Maciánetal.2001).CouldNFAT:AP-1complexesseparatedbytens,hundreds,orthou-sandsofbasepairsinteractthroughtheirRHR-Cdimerinterfaces?IntheternaryNFAT:AP-1/DNAcomplex,theRHR-CresiduesinvolvedinNFATdimerizationareexposedtosolventbutlieinadeepgroovebetweentheRHR-NandRHR-C,stericallyprecludingsuchinterac-tions.However,giventhemultipleorientationsofRHR-CobservedindifferentNFATcomplexes,theRHR-CintheNFAT:AP-1complexcouldconceivably
SitesshowingcooperativebindingofNFATandAP-1inbindingassaysinvitroaIndependentbindingdRegulatoryelementIL2ARRE2(−180)
SpbCompositeSitecAGGAAAaacTGttTCAAGGAAAattTGttTCATGGAAAcatTtAGTttTGGAAAcccTGAGTttcGGAGccccTGAGTCATGagGccctTGAGTCATGGAAAgatgacaTCATaGAAAgatgacaTCAAGGAAAgcaaGAGTCA
AGGAAAgcagaggccccaGAGTCATGGAAAttttcgTtACaCcTGGAAAattttaTtACaCc
NFATyesyesnonoyesno
AP-1nononoweakweakno
Reference
Jainetal.1993b
Jainetal.1992,1993a,bLeeetal.1995
Cockerilletal.1995Cockerilletal.1995Cockerilletal.1995
hm
IL5Ph
m
GGM-CSFM330h
m
GGM-CSFM420h
m
ehGM-CSFGM550
m
ehIL4(−88to−61)
m
ayesnoRooneyetal.1995
CooperativebindingisdefinedassignificantlyincreasedbindingofNFATorAP-1totheregulatoryelementinthepresence,comparedwiththeabsence,ofitspartnerprotein.Thebindingassaysusedwereinvitrofootprinting(Rooneyetal.1995)andelectrophoreticmobilityshiftassays(allothers).bSpecies:(h)human;(m)mouse.cBoldfaceindicatesoligonucleotideusedinreferencedstudy.SequencesincapitallettersdenoteidentitywithconsensusNFAT(WGGARA)orAP-1(TGASTCA)sites.dIndependentbinding:bindingintheabsenceofpartner.eThemouseGM550element,andthehumanandmouseIL-4elements,donotshowthestrictspacingbetweentheNFATandAP-1sitesassumedfromthecrystalstructuretobeessentialforoptimalNFAT:AP-1cooperation(Fig.5).CooperativebindingontheIL-4elementwasdemonstratedbyfootprintingwithrecombinantFos,Jun,andNFAT.InthecaseofGM-CSF,theGM330,GM420,andGM550elementsarepartofasingleenhancerregioninwhichtheymostlikelyhaveredundantfunctions;thereisalsoafourthelementthatdoesnotshowcooperativebinding.Thus,thedifferenceinspacingbetweenthehumanandmouseGM550elementscouldreflectthefactthatthiselementisnotessentialforenhancerfunction,eveninhumans.
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Figure5.ThecrystalstructureoftheNFAT/Fos–Jun/DNAcomplexboundtotheARRE-2siteoftheIL-2promoter(Chenetal.1998).Thestructureofthiscomplexisastrikingillustra-tionofsignalintegrationbyNFATandAP-1.Twobifurcatingsignaltransductionpathwaysconvergeonthecooperativecom-plex:TheNFATcomponentisactivatedbyCa2+/calcineurinsignaling,whiletheAP-1component(Fos–Jun)isactivatedbyPKC/MAPkinasesignals.
rearrangetoallowRHR-Cinterfaceformation,thuspro-motingtheassemblyoflong-range,higher-orderNFAT:AP-1complexes.FurtherversatilitywouldarisefromutilizationofdifferentNFAT–Fos–Juncombinations.Withtheadventofnewreagents(“tetheredAP-1”dimers;Bakirietal.2002),constitutivelyactiveNFATproteins(Maciánetal.2002;MonticelliandRao2002;PorterandClipstone2002),andmutantNFATproteinsimpairedforRHR-NorRHR-C-mediateddimerforma-tion(Giffinetal.2003;Jinetal.2003),thefunctionsofdifferentNFAT:AP-1combinationsandhigher-orderNFAT:AP-1complexesmaybeexplicitlyassessed.Interactionswithothertranscriptionfactors
InadditiontoitsinteractionwithAP-1,NFATengagesindirectprotein–proteininteractionsand/orinfluencestranscriptionsynergisticallywithseveralfamiliesoftranscriptionfactors:proteinssuchasMaf,ICER,andp21SNFTthatbelongtothesamebasicregion-leucinezipper(bZIP)familyasAP-1(Hoetal.1996;Bodoretal.2000;Boweretal.2002);thezincfingerproteinsGATA(seebelow),andEGR(Deckeretal.1998,2003);thehe-lix–turn–helixdomainproteinsOct,HNF3,andIRF-4(Fürstenauetal.1999;Bertetal.2000;Huetal.2002;Rengarajanetal.2002);theMADS-boxproteinMEF2(forreview,seeOlsonandWilliams2000;CrabtreeandOl-son2002;McKinseyetal.2002);andthenuclearreceptor
TranscriptionalregulationbyNFAT
PPAR-␥(X.Y.Yangetal.2000).Fortranscriptionalpart-nersotherthanAP-1,itisnotknownwhetherthesyn-ergywithNFAToccursinthecontextoftrue“compos-ite”regulatoryelementsthathaveadefinedgeometryandspatialorientationforcooperativebindingofNFATandthesepartnerproteins.
bZIPproteinsotherthanFosandJun:TheinteractionofNFATwithFos–JunproteinscannotbedetectedinsolutionbutisremarkablycooperativeonDNA(Jainetal.1992,1993a,b;Chenetal.1995;Cockerilletal.1995).Incontrast,anotherbZIPprotein,Maf,wasidentifiedasatranscriptionalpartnerforNFATbasedonapurelyprotein–proteininteractioninayeasttwo-hybridscreen(Hoetal.1996).Incotransfectionassays,NFATandMafsynergisticallyactivatedareporterplasmiddrivenbytheIL-4promoter.AlthoughcooperativeNFAT:Mafbindingwasnotobservedonthesiteoriginallyinvestigatedbe-causeoftheinverseorientationoftheNFATsiterelativetotheMafbindingsite(Hoetal.1996),MafandNFATcouldconceivablycooperateatclassicalNFAT:AP-1sites(Fig.6).ThekeyNFAT-bindingresiduesinJunarenotallconservedinMaf,butthemodeledNFAT–Mafinterfacehasgoodshapeandchemicalcomplementarity.MutagenesisstudiesindicatethatArg285ofJuniscriti-calforNFATbinding(Petersonetal.1996);inthemod-eledMaf/NFATinterface,aGlnresidueinthispositioncanmakesimilarcontactswithNFAT.
TwoothersmallbZIPproteins,ICERandp21SNFT,havebeenreportedtobindNFATandinhibitNFAT-dependenttranscription(Bodoretal.2000;Boweretal.2002).Again,ICERdoesnotconserveallthecriticalNFAT-bindingresiduesinJun,buttheNFAT:ICERin-terfacehasaplausiblelevelofcomplementarity.Infact,theTyrresidueinICERthatcorrespondstoArg285inJuncouldpotentiallyinteractwithNFATevenmorestrongly(Fig.6).p21SNFTbindsNFATmainlyasahet-erodimerwithJun(Boweretal.2002),anditisthereforeexpectedthatthebindinginteractionsaremostlymedi-atedbyJunandwillbesimilartothatseenintheNFAT/Fos–Jun/DNAcomplex.
IRFandEGRproteins:NFATcooperateswithIRF4attheIL-2andIL-4promoters(Huetal.2002;Rengarajanetal.2002)andwithEGR-1attheIL-2andTNF-␣pro-moters(Deckeretal.1998,2003).Theseinteractionsarenotwellcharacterized—theinteractingdomainshavenotbeendelineated,andcompositeDNAelementshavenotbeendefined.InanIRF-4/NFATcomplexmodeledonaputativecompositesitefromtheIL-4promoter(Huetal.2002),IRF-4andNFATcanpotentiallybindDNAsimultaneously,andtheirDNAbindingdomainscanin-teract;similarly,anEGR-1/NFATcomplexmodeledonasitefromtheTNF-␣promoter(Deckeretal.2003)allowssimultaneousDNAbindingwithdirectcontactbetweentheRHRofNFATandthezincfingerdomainofEGR-1(LC;datanotshown).Althoughtheflexibleconforma-tionoftheRHR-Cposesuncertaintyinmodelingthesehigher-ordertranscriptioncomplexes,italsoincreasesthelikelihoodthatNFATcouldinteractwithavarietyofpartnersondifferentcompositesites.
GATAproteins:CooperationofNFATwithGATA
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Figure6.ThebZIPdomainsofMafandICERmaybindtheEЈFloopofNFAT.(Top)DetailedinteractionsbetweenNFATandJun.(Bottom)Sequencealign-mentbetweenhumanc-Jun,c-Maf,andICER.Thecoloredblocksindicateresi-duesinJunthatbindNFAT.Thesubsti-tutionsofE284,R285,andK292ofJunwithGln,Gln,andGluinMafdonotdis-rupttheinterfacehydrogen-bondingpat-ternsignificantly.ThereplacementofR285ofJunbyTyrinICERmayincreasetheinterfacevanderWaalscontacts.R288ofJunmakesseveralhydrogenbondstoNFAT,whereasthecorrespondingValofMafandCysofICERcouldmakevanderWaalscontactstoT533ofNFAT.
familymembershasbeenobservedinmanysystems(seeTable3).NFAT:GATAcooperationhasbeenestablishedbysynergisticactivationofreporterplasmids(Molkentinetal.1998;Morimotoetal.2001;Avnietal.2002;NemerandNemer2002;Wadaetal.2002)aswellasbydirectbindingofRHR-CofNFAT3toaDNA-binding“bait”fragmentofGATA4inayeasttwo-hybridassay(Molkentinetal.1998).Thereisevidenceforpreferentialinteractionsinvolvingtheendogenousproteins.Incoim-munoprecipitationexperimentsusingaskeletalmusclecellline,GATA2boundNFAT2butnotNFAT1(Musaroetal.1999).ThereisnooverlapbetweenNFAT:GATAandNFAT-AP-1interactionsurfaces,consistentwithin-volvementofRHR-CandRHR-N,respectively.AnNFAT1proteinbearingmutationsinthreekeyFos–Jun-interactingresiduesthatabrogatedtheabilityofNFAT1tocooperatewithAP-1wasaseffectiveasormoreeffec-tivethanwild-typeNFAT1initsabilitytosynergizefunctionallywithGATA3inatransientreporterassayinT-cells(Avnietal.2002).However,sequenceinspec-tionofregulatoryregionshasnotledtounambiguousidentificationofacompositeNFAT:GATAelementwithspecificspacingandorientation,andcooperativeNFAT:GATAbindingonDNAhasnotbeenreported(Table3).
Biologicalimplications
NFAT1–4proteinsbindDNAasmonomersatcognate(GGAA)sites(L.Chen,unpubl.),asdimersatB-like
responseelements(Fig.4),andascooperativecomplexeswithFosandJunatNFAT:AP-1compositesites(Fig.5).ThegeneralfeatureofthesevariouscomplexesisthattheDNA-bindinginteractionsandconformationsoftheNFATRHR-Narehighlyconserved,buttheconforma-tionoftheRHR-Cisquitevariable.Thisdiversityofbindingmodesarises,atleastinpart,fromthefactthatNFATisnotanobligatedimer;thus,incontrasttoRelandNFAT5proteins,whicharedimersunderallcircum-stances,theRHR-NandRHR-CofNFATarefreetoadoptavarietyoforientationsindifferenttranscriptioncomplexes.ThisremarkableconformationalflexibilityofNFATondifferentDNAsitesislikelytofacilitateassemblyofNFATintodistincthigher-ordercomplexescontainingdiverseDNA-bindingpartners,andwouldbeexpectedtomodulaterecruitmentofspecifictranscrip-tionalcoregulatorsindifferentpromotercontexts(Lefs-tinandYamamoto1998;Agaliotietal.2000;Escalanteetal.2002),asillustratedforthePOU-domainproteinPit-1(Scullyetal.2000).
TranscriptionalregulationbyNFAT
StrategiesforidentificationofdistalregulatoryelementsthatbindNFAT
Althoughgeneexpressionisultimatelyregulatedattheproximalpromoter,whichbindsRNApolymeraseandthecoretranscriptionalmachinery,distalregulatoryre-gionshaveprofoundeffectsontheexpressionofnearly
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allgenes(Davidson2001).Thisisillustratedbythefactthattheproximalpromoteralonerarelysupportscorrect,cell-type-specificexpressionofalinkedre-portergeneintransgenicmice—verylargeregions,amountingtohundredsofkilobasesofsurroundingDNA,areoftenneeded(Lakshmananetal.1999;Yuietal.2001).ThepresenceofdistalregulatoryelementsisageneralfeatureofNFATtargetgenesinTcells;inparticular,distalenhancersarepresentinallcytokinegenesthathavebeenexaminedinsufficientdetail,in-cludingtheIL-3,GM-CSF,IL-4,IL-10,andIFN-␥genes(Cockerilletal.1993,1995;Duncliffeetal.1997;Agar-waletal.2000;Avnietal.2002;Hawwarietal.2002;S.-H.Im,D.U.Lee,andA.Rao,unpubl.).DistalNFAT-dependentgeneregulatoryelementshavebeenlocatedexperimentallybyhuntingforinducibleDNaseIhyper-sensitivesiteswhoseinductionisblockedbyCsAorFK506(Cockerilletal.1993,1995;Duncliffeetal.1997;Agarwaletal.2000;Hawwarietal.2002).Theseregionsoften(butnotalways)correspondtohighlyconservednoncodingsequences(CNS)identifiedbycomparingthesequencesofgenelociintwoormoremammaliange-nomesusingWeb-basedprogramssuchasVISTA(Mayoretal.2000;Lootsetal.2002;Brayetal.2003)orPip-Maker(Schwartzetal.2000).ThispointisillustratedinFigure7fortheIL2,IL-3,andGM-CSFgenes.Theen-hancersmaybelocated5Јoftheproximalpromoter,withinintronicregions,or3Јofthegene;theytendtocontainbindingsitesnotonlyforNFAT,butalsoforconstitutiveorlineage-specifictranscriptionfactorsthatconfercell-typespecificityofgeneexpression(Agarwaletal.2000;Avnietal.2002;D.U.LeeandA.Rao,un-publ.).
Althoughbioinformaticscomparisonofgenomicse-quencesisausefulstrategyforidentifyingregulatoryregions,itdoesnotbyitselfdelimitindividualtranscrip-tion-factor-bindingsites;pattern-matchingprogramsorvisualinspectionmustbeusedtosearchwithinthecon-servedareaforknownbindingmotifs.Recognitionsitesforfactorswithpoorlydefinedbindingmotifswillnotbeidentifiedinthisway,although,ifbiologicalexperi-mentsimplicatesuchafactor,aCNScorrelatedwithaDNaseIhypersensitivesiteisanexcellentcandidatere-gioninwhichtosearchforaDNAtarget.CompositeNFAT:AP-1elements,andindeedallcompositeDNAelements,presentaparticularproblemforbioinformat-icsrecognitionofbindingsitesonDNA.Becausepro-tein–proteininteractionscontributetothestabilityofthecooperativeNFAT:AP-1complex,eitherNFATorAP-1mayattachtoasuboptimalsequenceiftheotherpartnerhasboundtoahigh-affinitysite.Thesesubopti-malsequencesareoftenrecognizablewhenNFAT/AP-1bindinghasbeenshownexperimentally(seeTable2),butaretoodegeneratetobeusefulinlong-rangescreensofextendedgenomicregions.Itisimportanttonote,however,thatevenbioinformaticspredictionsforeasily
Table3.
Regulatoryregion
FunctionalNFAT:GATAbindingsitesingenesandNFAT:GATAinteractionsincells
Celltype(stimulus)Th2cells(TCR)Th2cells(TCR)Th2cells(TCR)
Location/arrangementof
bindingsitesa−180 −114>N:A>28 NFAT−927;GATA−27 NFATNFAT1,2NFAT1,2NFAT1,2 GATAGATA3GATA3GATA3 SynergyinreporterassayY2H,synergyinreporterassay; mutationinNFATelementabrogatesreporteractivityMutationsinbindingelementsabrogatereporteractivityCo-ip,synergyinreporterassaySynergyinreporterassay Co-ip,colocalisationinnucleiof differentiatingcellsCo-ip,synergyinreporterassay Avnietal.2002 Assayforinteraction Reference IL-4promoterIL-5promoterIL-4enhancer BNPpromoter Cardiomyocytes(angiotensinII) NFAT3GATA4 Molkentinetal.1998 Adss-1promoter(−1.9kb)EDN-1promoter(−204bp)EDN-1promoter(−1.4kb) SkeletalmusclemyosinSm-MyHC aCardiomyocytes(angiotensinIIorelectricalpacing)Cardiomyocytes(-adrenergicstimuli)Endocardial differentiationIGF-1-inducedskeletalmusclehypertrophy DifferentiatingVSMC(lowserum) NFAT−556NFAT3GATA4 Xiaetal.2000;Wenetal.2002Morimotoetal.2001NemerandNemer2002Musaroetal.1999Wadaetal.2002 GATA−136NFAT2GATA4 −280 NFAT2NFAT2 GATA5GATA2 SitesnotidentifiedNFAT2GATA6 N:AorA:N,compositeNFAT:AP-1site(underliningindicatesbindingelement);>N:A>, GENES&DEVELOPMENT2215 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. Figure7.Correlationofconservednoncodingsequences(CNS)withDNaseIhypersensitive(DH)sites.MouseandhumansequencescontainingIL2andthelinkedIL3/GM-CSFlociweresubjectedtoVISTAcomparisontoidentifyCNSregions,followedbyFindpat-ternsanalysistoidentifyconservedNFATsites.Weconsidered(AorT)GGAAAtobeoptimal,butalsosearchedwith(AorT)GGAGAandtheminimalmotifGGAAinordertodetectsuboptimalNFATbindingsites.IneachVISTAgraph,theextentofsequenceidentityisplottedontheY-axisagainsttheindicatedreferencesequence(humanormouse);exonsareshowninblue;CNS,with>75%conservation,inpink.NotethattheCNSflankingthetranscriptionalunitshavehighersequencesimilaritybetweenhumanandmousethandotheexons(thisislikelytoreflectstrongevolutionarypressurefrompathogensonthecytokinegenes).Adiagramofpredicted,conservedNFATbindingsitesinonebiologicallyinterestingCNSisgivenbeloweachgraph.(N)NFAT;(A)AP-1.(A)IL-2.ThebifurcatedCNSimmediatelyupstreamofthestartoftranscriptioncorrelateswithDHsitesII(constitutive)andIII(inducible)observedbySiebenlistetal.(1986).Thetwo5ЈCNSregionsarenothypersensitiveundertheirconditions.TheARRE2compositeNFAT:AP-1site(Fig.5)isindicated.(B)IL-3.TheindicatedCNScorrespondstoboththeinducible−4.5kbandtheconstitutive−4.1kbDHsitesobservedbyHawwarietal.(2002);thesecondandthirdCNScorrelatewiththeconstitutivesitesat−1.5and−0.1kb(promoter).(C)GM-CSF.ThesecondandfourthCNScorrelatewiththeinducibleDNaseIhypersensitivesitesat−2.0and−0.1kb(promoter)describedbyOsborneetal.(1995);thefirstandthirdCNSarenothypersensitiveundertheirconditions.AccessionnumbersforthesequencesusedareNT_007072(humanIL3andGM-CSF),NT_039520(murineIL3andGM-CSF),NT_0163(humanIL2),andNT_039228(murineIL2). recognizedsitesneedtobevalidatedbybiologicalexperi-ments. RedundantversusnonoverlappingfunctionsofindividualNFATproteins ThedifferentNFATproteinsappeartohaverelativelyredundantfunctionsincells,asjudgedbythegenerallymildphenotypesofmicelackingindividualNFATpro-teins(forreview,seeCrabtreeandOlson2002).Inthreenotableexceptions,deletionofNFAT2resultsinembry-oniclethalitybecauseofdefectsincardiacvalveforma-tion(delaPompaetal.1998;Rangeretal.1998a),thesmallestisoformofNFAT2isselectivelyunabletopro-moteapoptosisinTcells(Chuvpiloetal.2002),anddeletionofNFAT1alonesubstantiallyreducescytokineproductionbymastcells(TsytsykovaandGoldfeld2000;Solymaretal.2002).Inmostcases,however,pronouncedfunctionalimpairmentsarenotobservedunlesstwoormoreNFATproteinsarelacking.Forinstance,deletionofbothNFAT1andNFAT2isrequiredforeffectivelossofcytokineproductioninTcells(Pengetal.2001);de-letionofbothNFAT1andNFAT4isrequiredtoproduceamajorbiastowardTh2cytokineproductioninmice 2216GENES&DEVELOPMENT Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press (Rangeretal.1998b);deletionofbothNFAT3andNFAT4isrequiredtoproducelethaldefectsinvascularpatterningintheembryo(Graefetal.2001a);anddele-tionofthreemembers—NFAT1,NFAT3,andNFAT4—isrequiredtoobservestrikingdefectsinaxonalout-growthinthecentralandperipheralnervoussystems(Graefetal.2003).ThedisparatephenotypesofNFAT-deficientmice(CrabtreeandOlson2002)maythereforereflectdifferencesinintracellularregulation(seeabove)and/ordifferentexpressionlevelsindifferentcelltypes.InfactconstitutivelyactiveversionsofNFAT1andNFAT2,inwhichalargefractionofthephosphorylatedserineshavebeenmutatedtoalaninetomimicthede-phosphorylatedform,havebeengenerated(Maciánetal.2002;MonticelliandRao2002;PorterandClipstone2002);whenexpressedatlowlevelsinTcells,underconditionsinwhichtheactivitiesofendogenousNFATproteinsareblockedwithCsA,thetwoproteinsaresimilarintheirabilitytoelicitexpressionofmostcyto-kinegenes(MonticelliandRao2002). DespitetheapparentlyinterchangeableactivitiesofNFATproteinsinacuteassaysforgeneexpression,NFAT2isreportedtohaveapredominantroleinatleasttwocellulardifferentiationprogramsinvivo:differentia-tionofosteoclastsfrommonocyteprecursors(Takay-anagietal.2002)anddifferentiationofTh2cellsfromantigen-“naive”Tcells(forreview,seeGlimcherandMurphy2000).ThiscouldreflectselectiveregulationofNFAT2asreportedfordevelopingskeletalmusclecells(Abbottetal.1998).Alternatively,iftheprecursorscon-tainmultiplemembersoftheNFATfamilyasshownfornaiveTcells,thebiascouldstemfromthefactthatthesmallestisoformofNFAT2istheonlyNFATproteinsubjecttopositiveautoregulation(Chuvpiloetal.1999;Zhouetal.2002).Basedonprecedentsinothersystems(Davidson2001),alikelyscenarioisthattheNFATpro-teinspresentinrestingnaiveTcellsbecomeactivatedatthestartoftheT-celldifferentiationprogram,andup-regulateexpressionofthesmallisoformofNFAT2.Thisproteinthenmaintainshighlevelsofitsownexpressioninapositivefeedbackloop.If,asislikely,high-levelexpressionisessentialtomaintainthedifferentiatedstate,lossoftheredundantNFATproteinspresentatlowlevelsintherestingprecursorcellswouldhavelittleornodiscernibleeffectonthedifferentiationprogram,whereasgeneticmanipulationsthatresultedinlossoftheshortNFAT2isoformwouldhaveamajoreffect.Thisdistinctionmaybeexacerbatedbythefactthat,asdiscussedabove,theshortNFAT2isoformhasatrans-activationdomainthatdifferssignificantlyfromthoseoftheotherNFATproteins,andsomayassembleintodif-ferenttypesoftranscriptionalcomplexesinthenucleus.NFAT-regulatedtargetgenesindifferentiatedTcellsStudyofNFATfunctionintheimmunesystemhasthemajoradvantagethatverymanyNFATtargetgenesareknown.ManysuchgeneswereidentifiedbyindividualanalysisofTcells,Bcells,NKcells,andmastcellsac-tivatedthroughtheirantigenandFcreceptors(forre- TranscriptionalregulationbyNFAT view,seeRaoetal.1997;Kianietal.2000;Serflingetal.2000;Maciánetal.2001).OtherswerefoundbyanalysisofcellslackingoneormoremembersoftheNFATfam-ily:Forinstance,matureTcellsexpresspredominantlytwoNFATproteins,NFAT1andNFAT2,andTcellslackingbothproteinsproducedalmostnocytokinesuponstimulation(Pengetal.2001),indicatingthatNFATisessentialforactivatingtranscriptionofmostT-cellcytokinegenes.YetothershavebeenfoundbyDNAarrayanalyses:Forinstance,transcriptionalprofil-ingofTcellsfromcontrolindividualsandfrompatientswithaprimarydefectinstore-operatedCa2+entryre-vealedthatalmostasmanygenesarerepressedasareactivatedbyCa2+/calcineurinsignaling(Feskeetal.2001).Itislikelythatmanyofthecalcineurin-regulatedgenesarealsoNFATtargetgenes.NFATcouldrepressgenetranscriptionbyrecruitingcorepressorsorbypart-neringwithknowntranscriptionalrepressorsonDNA;ithasbeensuggestedthatCDK4transcriptioncanbere-pressedbyrecruitinghistonedeacetylasestoasitejust3ЈofthetranscriptionstartsiteoftheCDK4gene(Bakshetal.2002). TranscriptionalprofilingofdifferentiallystimulatedTcellsrevealedtwodistinctclassesofNFATtargetgenes,oneclasscontrolledbyNFATwithAP-1andtheotherclassbyNFATwithoutAP-1(Maciánetal.2002).TheNFAT:AP-1complexregulatesaverylargesetofactiva-tion-associatedgenes,classicallyassociatedwithanon-goingimmuneresponse;itisformedthroughactivationofbothCa2+andPKC/MAPkinasesignalingpathwaysasdiscussedpreviously.Incontrast,Ca2+signalingwithoutPKC/MAPkinasesignalingactivatesNFATbutnotAP-1,inducingamuchsmallersetofgenesthatencodepu-tativenegativeregulatorsoftheimmuneresponse.Itisstrikingthatthesametranscriptionfactor,NFAT,canimposethesetwoopposingbiologicalprogramsinthesamecells.ThegenescontrolledbyNFATwithoutAP-1mayberegulatedthroughB-likeDNAelementsthatbindNFATdimers(seeabove);orthroughelementstowhichNFATbindscooperativelywithpartnersthatarepresentinrestingcellsorareactivatedbyCa2+signaling.InvolvementofNFATinchromatin-basedprocessesunderlyingcelldifferentiation NotonlydoesNFATregulategeneexpressionprogramsinfullydifferentiatedTcells,butalsoitplaysacriticalroleinthedifferentiationprocessitself(Avnietal.2002;forreview,seeAvniandRao2000;Anseletal.2003).NaiveTcellsareTcellsthathavematuredinthethy-musandemergedintotheperipherybuthavenotyetencounteredantigen.Whenthesecellsarefirstexposedtoantigen,theydifferentiateintoeffectorTcellswiththeabilitytotranscribespecificsubsetsofcytokinegenesinresponsetosecondarystimulation.EffectorTh1cellstranscribetheIFN-␥genebutsilencethelinkedIL-4,IL-5,andIL-13genes,whereaseffectorTh2cellsdisplaytheconverseexpressionpattern.TheprocessofTh1/Th2differentiationinvolvesearlyantigen-inducedchangesinchromatinstructureatallthesecytokine GENES&DEVELOPMENT2217 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. genes;thesechangesaretransientunlessmaintainedbysimultaneousstimulationwithcytokines(IL-12andIL-4forTh1andTh2differentiation,respectively).Mechanis-tically,thefirststimulationwithantigenandcytokineelicitstransientactivationofNFATandSTATtranscrip-tionfactors(STAT4andSTAT6inresponsetoIL-12andIL-4stimulation,respectively).NFATcooperatesfunc-tionallywithSTATproteinsatcytokineregulatoryre-gions,initiatinglong-rangechangesinDNaseIhyper-sensitivityandhistonemodificationthroughoutthelo-cus.Atthesametimeantigenandcytokinestimulationsynergizetoinduce(presumablyviaNFATandSTATfactors)theexpressionoflineage-specifictranscriptionfactorsthatmaintainthetranscriptionallycompetentstatusofthecytokinegenes.LikeNFAT2,thelineage-specificfactors(T-betandGATA3inTh1andTh2cells,respectively)autoregulatetheirownexpression,thuscontributingtomaintenanceofthedifferentiatedstate;uponrestimulationwithantigen,theycooperatewithNFATtoinducerapid,high-levelexpressionofthecy-tokinegenes. LikeTh1/Th2differentiation,osteoclastdifferentiationrepresentsadevelopmentalchoiceinwhichNFATdrivesacommonprecursorcelltowardoneoftwodis-tinctsiblinglineages(macrophagesorosteoclasts)bycontrollingtheexpressionofaspecificsubsetoftargetgenes.Incardiacvalvedevelopment,NFATiscalledupontomakeaspatiallyandtemporallyprecisecontri-butiontoacomplexmorphogeneticprogram,inwhichNFATactivationinaverysmallsubsetofcellsataveryspecifictimeisessentialfortissueremodeling.Inslow-twitchfiberdifferentiationinskeletalmuscle,NFATparticipatesinactivity-dependentreprogrammingofmyosinheavychain(MyHC)geneexpression,sensingaCa2+signalthatreflectsaprolongedchangeinthepat-ternofcontractileactivity,andeffectingachangeinphe-notypeofafullydifferentiatedcell. Osteoclastdifferentiation Osteoclastsarekeycellularparticipantsinboneresorp-tionandremodeling.Theydifferentiatefromprecursorsbelongingtothemonocyte/macrophagelineageundertheinfluenceofsignalsfromosteoblastsorbonemarrowstromalcells(Chambers2000;Teitelbaum2000).Se-cretedM-CSFandRANKLpresentedonthesurfaceofosteoblastsorstromalcellsaretheessentialsignalsinvivo,withotherextracellularsignalingmoleculesandsubstrateadhesionalsoplayingarole.Thetranscriptionfactorsc-FosandNFBhavebeenrecognizedasessentialforosteoclastdifferentiationbasedontheosteopetroticphenotypeofc-Fos-andNFB-deficientmice(Grigoria-disetal.1994;Franzosoetal.1997).Osteoclastdifferen-tiationisaprocessoccurringoverseveraldays(Fig.8),withsequentialexpressionofmarkersand,usually,fu-sionintomultinucleatedcells.Fullymatureosteoclasticcellsaremarkedbytheexpressionofthecalcitoninre- OtherbiologicalsystemsinwhichNFATisimplicatedNFATisexpressedinmanycelltypesandcontributestodiversecellularfunctions(CrabtreeandOlson2002;HorsleyandPavlath2002;Graefetal.2003;Hill-Eu-banksetal.2003).Asdiscussedintheprevioussections,thecellularcontext,thesourceandtimingoftheCa2+/calcineurinsignal,thenatureofothersignalsthatareactivatedatthesametime,andtheflexibilityofNFATinformingtranscriptionalcomplexeswithdifferentpart-ners,allmakeimportantcontributionstothefinalbio-logicaloutput.InthissectionwediscussthreeselectedsystemsofcelldifferentiationinwhichCa2+/calcineurinsignalingandNFATactivationhavebeenimplicated. Figure8.Osteoclastogenesis.Osteoclastdifferentiationisamultistepprocessoccurringoverseveraldays.Itsstageshavebeendefinedgeneticallybythepointatwhichosteoclastdevelopmentisarrestedinanimalswhenthefunctionofspecificgenesisabsent(yellowboxes).Theearlystagesofcommitmenttothemonocyte/macrophage/osteoclastlineagesandthesurvivalandproliferationofprecursorcellsrequirethetranscriptionfactorPU.1andthesignalprovidedbyM-CSF.DifferentiationrequiresthefurthersignalsuppliedbyRANKL,andsignalingviathetranscriptionfactorsc-FosandNFB.RecentworkindicatesthatsignalingviaNFATisalsoessential.Cellularpolarizationandproductiveattachmenttothebonesubstrate,aprerequisiteforboneresorption,dependon␣v3integrinandtheintracellularsignalingproteinsTRAF6andc-Src.BoneresorptionbyfullydifferentiatedosteoclastsiscarriedoutbyeffectorproteinsincludingcathepsinK,carbonicanhydraseII,andaspecificH+ATPase.FigurereprintedwithpermissionfromTeitelbaum(2000).©2000AmericanAssociationfortheAdvancementofScience. 2218GENES&DEVELOPMENT Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Figure9.Bonemarrowcellscanbeinducedtodifferentiateintoosteoclastsinvitro.MousebonemarrowmacrophagesweretreatedwithM-CSFandRANKLinvitrofor6d.Thecellspicturedareattachedtowhaledentin,analternativetargetforresorptionbyosteoclasts.Fullexpressionoftheosteoclastdif-ferentiationprograminthesecellsisdemonstratedbytheirstrongtartrate-resistantacidphosphatasestaining(purple)andbytheresorptionpits(arrows)visibleadjacenttosomeofthecells.Bar,50µm.MicrographcreditedtoDeborahNovack,re-printedwithpermissionfromTeitelbaum(2000).©2000AmericanAssociationfortheAdvancementofScience. ceptor(HattersleyandChambers19)andtheabilitytoresorbbone. Variousexperimentalmodelsofosteoclastdifferentia-tionhavebeendeveloped.ThecombinationofM-CSFandsolubleRANKLwillsupportdifferentiationofosteo-clastsfromamixedpopulationofbonemarrowcellsorspleencells(Fig.9;Laceyetal.1998;Yasudaetal.1998),andinclusionofTGFincreasestheyieldofdifferenti-atedcells(Chambers2000).ThecellularcomplexityofthemodelisreducedinstudiesusingtheRAW2.7monocyte/macrophagecellline,whichundergoesdiffer-entiationwhenstimulatedwithsolubleRANKLaloneorwithRANKLtogetherwithadditionalsignals(Hsuetal.1999;Meiyantoetal.2001;Ishidaetal.2002;Shuietal.2002).Withsomeprotocols,RAW2.7cellsprogresstomultinucleatedosteoclast-likecellsthatexpresscalcito-ninreceptor(Shuietal.2002)andareabletoresorbboneinvitro(Hsuetal.1999;Meiyantoetal.2001;Shuietal.2002). BothcalcineurinsignalingandNFAThavebeenim-plicatedinthedifferentiationofosteoclasts.Inanearlyreportusinglimbbonesculturedfromfetalrats,CsAdidnotinitiallyalterboneresorption,butdecreasedboneresorptionafterseveraldaysinculture,afindingattrib-utedtoitsinhibitionofosteoclastformation(Orceletal.1991).Amorecompellingconnectionbetweencalcineu-rinandosteoclastdifferentiationhasbeenmadeinabonemarrowculturemodel,inwhicheitherCsAorFK506causedaconcentration-dependentinhibitionof TranscriptionalregulationbyNFAT differentiationoftartrate-resistantacidphosphatase(TRAP)-positivemultinucleatedcells(Takayanagietal.2002).Similarly,intheRAW2.7cellmodelofosteo-clastogenesis,CsAblockedRANKL-inducedformationofmultinucleatedcells(Ishidaetal.2002),andeitherCsAorFK506largelyinhibitedformationofmultinucle-atedcellsandup-regulationofcalcitoninreceptormRNAbythecombinationofM-CSF,RANKL,andTGF(Shuietal.2002).TwoseparatelinesofevidenceimplicateNFAT2intheprocess.First,NFAT2−/−EScellsfailtodifferentiateintoosteoclastsundercondi-tionsinwhichwild-typecellsdifferentiate(Takayanagietal.2002).Second,inRAW2.7cells,reductionofNFAT2proteinlevelsbyintroductionofanantisenseNFAT2constructmimickedtheinhibitoryeffectofCsAonformationofmultinucleatedcells(Ishidaetal.2002).Thelatterconclusionisprovisional,becauseunexpect-edlytheeffectdidnotrequireinductionoftheantisensemRNAwithdoxycycline,andbecausenocontrolanti-senseconstructsweretested. Boththeexperimentswithbonemarrowcells(Takay-anagietal.2002)andthosewithRAW2.7cells(Ishidaetal.2002)haveprovidedevidenceofatwo-stagediffer-entiationprocess,inwhichfirstNFAT2expressionisswitchedfromarelativelylowtoarelativelyhighlevelbyinducibletranscriptionfactorsincludingc-FosandNFB,andthenNFAT2cooperateswithsomeofthesameproteinstoactivatetranscriptionfrompromotersofgenescharacteristicofterminaldifferentiation.De-pendingontheexperimentalmodel,therearedifferencesindetailatthefirststage.Inbonemarrowosteoclasts,FK506blockedNFAT2mRNAandproteininduction,andoverexpressionofNFAT2forcedexpressionoftheendogenousNFAT2gene.Moreover,c-FoswasnecessaryforNFAT2expression.Thesefindingsarecircumstantialevidencethatthecalcineurin-NFATpathwaypartici-patesinNFAT2inductionthroughcompositeNFAT-AP1sitesintheNFAT2promoter,amechanismthathasbeendocumentedpreviouslyinTcells(Chuvpiloetal.2002;Zhouetal.2002).InRAW2.7cells,incontrast,CsAdidnotblockNFAT2proteininductionorTRAPexpression,eventhoughitpreventedprogressiontomul-tinucleatedcells(Ishidaetal.2002).Thedifferenceisnotamajordiscrepancy,butrathersuggeststhatitispos-sibletobypasstherequirementforNFATwhencertainothertranscriptionfactorsarepresentinsufficientamounts.Itisclearfromotherevidencethatthesignal-ingcontextinRAW2.7cellsdivergesfromthatinthebonemarrowosteoclastprecursors,becauseRAW2.7cellsneedonlyRANKLstimulationtotriggerdifferen-tiation. PointsthathavenotbeenclarifiedfordifferentiatingbonemarrowcellsarethesourceoftheCa2+signalthatinitiatesup-regulationoftheNFAT2geneandwhetherNFAT2itselforanotherNFAT-familyproteintransmitstheCa2+signalattheearliesttimes.NFAT4mRNAhasbeendetectedbyDNAmicroarrayanalysisinabonemarrowcellpopulationthatcontainsprecursorsofos-teoclasts(M.Suˇsaˇ,pers.comm.),andNFAT1proteinandNFAT4mRNAhavebeendocumentedinRAW2.7 GENES&DEVELOPMENT2219 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. cells(Wangetal.1995;Ishidaetal.2002).Itcanbepre-sumedthat,beyondtheearliesttimesofinduction,NFAT2expressionbecomesself-reinforcingasthepro-teinaccumulatesandisactivated. Inthesecondstageofdifferentiation,whenNFATco-operateswithotherfactorstocontrolacollectionofdif-ferentiationgenes,thebasisforitstranscriptionalsignal-ingisclear.NFATproteinlevelsarehigh.Newlyaccu-mulatedNFAT2proteinisatfirstmainlycytoplasmic,bothindifferentiatingbonemarrowcellsandinRAW2.7cells(Ishidaetal.2002;Takayanagietal.2002).Aspartofthedifferentiationprocess,RANKLleadstocytoplasmicCa2+oscillationsafter∼24h(Takay-anagietal.2002),andNFAT2becomesmainlynuclearby72hindifferentiatingbonemarrowosteoclasts,andbyday2ofstimulationwithRANKLinRAW2.7cells(Ishidaetal.2002;Takayanagietal.2002). TargetgenesforNFATmayincludemanyofthede-finitivedifferentiationmarkersofosteoclasts.SequencestypicalofcompositeNFAT-AP1sitescanberecognizedinthepromotersofseveralosteoclastgenes,includingthegenesencodingTRAPandcalcitoninreceptor(Anusaksathienetal.2001;J.Nardone,unpubl.).NFAT2andc-FosactsynergisticallyattheTRAPpromoter,andthiscooperationrequiresNFAT2residuesthathavebeenshowntostabilizetheNFAT–AP1interaction(Takay-anagietal.2002),consistentwithNFAT–AP1coopera-tionatcompositesitesintheTRAPpromoter.Overex-pressionofNFAT2inosteoclastprecursorcellscausesdifferentiationintheabsenceofRANKL(Takayanagietal.2002).Aphysicalinterpretation,totheextentthatcompositeNFAT–AP1sitesareused,isthatahighlevelofNFATshiftstheequilibriumforcomplexformationonDNAsothatthelowerlevelsofAP1proteinspresentwithoutRANKLstimulationaresufficient.Itisplau-siblethatasimilarshiftoccursintherequirementfor Figure10.Schematicviewofcardiacvalveformation.Theprimitivehearttubeconsistsoftwocellularlayers,anouterlayerofdevelopingmyocardiumandaninnerlayerofdevelop-ingendocardium.Initially,thecardiaccushionsformaslocallythickenedextracellularmatrixbetweenthemyocardialanden-docardialcelllayers.Latersomecellsoftheendocardiallayerdetachfromtheirneighbors,undergoanendothelialmesen-chymaltransition,andmigrateintotheunderlyingcardiaccushion.Thismigrationisfollowedbysecretionofadditionalcomponentsofextracellularmatrix,cellproliferation,furtherdifferentiation,andremodelingofthevalveasawholeintoitsdefinitivemorphology.Intheschematicdiagram,labelsindi-catetheendothelial(E)andmyocardial(M)layersfororienta-tion,theacellularcardiaccushionprimordiumorthecardiaccushion(C),andthefullyformedvalve(V). 2220GENES&DEVELOPMENT Figure11.DevelopmentofthecardiacvalvesisabnormalinNFAT2-nullmouseembryos.MicrographsofsectionsfrommouseheartatE13.5.Atthisstageinwild-typeembryos,theatrioventricularvalves(arrows)haveformedandhavebeenex-tensivelyremodeled.InNFAT2-nullmice,theatrioventricularvalves(arrows)arepoorlydeveloped.Figurereprintedwithper-missionfromdelaPompaetal.(1998).©1998NaturePublish-ingGroup. othercooperatingtranscriptionfactorswhenNFAT2isoverexpressed. ThemodelsofosteoclastdifferentiationareattractiveexperimentalpreparationsforstudyinghowNFATsig-nalingisintegratedwithotherinputsinaprogressiveprocessofdifferentiation.Theexperimentssofarhaveuncoveredadiversesetofpotentialtargetgenesinclud-ingpreviouslycharacterizedmarkersofosteoclastdiffer-entiationandthegenesshownbymicroarrayanalysistobeinducedindifferentiatingosteoclasts(Cappellenetal.2002;Takayanagietal.2002)andinRAW2.7cells(Ishidaetal.2002).Thesewell-definedinvitromodelswillpermitincreasinglyfinedissectionoftheroleofNFATindifferentiationofosteoclastsfromtheirprecur-sorcells,intermsofwhichgenesarecontrolled,thetimingoftheirinduction,thepromotersiteswhereNFATbinds,andthepartnerproteinsthatcooperateingeneinduction. Cardiacvalvedevelopment Vertebrateheartdevelopmentisachoreographedpro-gression—intricatelypatternedintimeandspace—fromspecificationofcardiogenicmesodermtoananatomi-callyorganizedandfunctioningheart(FishmanandChien1997;Sucov1998;Bruneau2002;McFaddenandOlson2002).Onecrucialelementofcardiogenesisistheformationofvalvesbetweenthedevelopingatrialandventricularchambersandintheoutflowtract(EisenbergandMarkwald1995).Inoutline,thisprocessrequiresspecificationofthelocationsatwhichthevalveprimor-diawillform;theinitialelaborationofanacellularma-trixatthesesites;thepopulationofthismatrixbycellsthatoriginateintheearlyendothelium;andsubsequentcellproliferation,productionofmatrixmaterials,andremodeling(Fig.10). GenetargetinghasimplicatedNFAT2inthisdevelop-mentalprocess.Micehomozygousfortwodifferent Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press single-exondeletionsintheNFAT2genedisplaydefectsincardiacvalveandseptumformation(Fig.11;delaPompaetal.1998;Rangeretal.1998a).Consistentwiththeobserveddevelopmentaldeficits,NFAT2mRNAandproteinarepresentinmurinecardiacendotheliumthroughouttheperiodofcardiacmorphogenesisfromasimpletubetoafour-chamberedheart(delaPompaetal.1998;Rangeretal.1998a;Kimetal.2001).NFAT2isnotdetectedinthetwootherclassesofcellsdirectlyin-volvedinvalvedevelopment,myocardialcellsandcar-diaccushionmesenchymalcells. NFATisactivatedinthecardiacendotheliumofwild-typeembryos.TheNFAT2proteininendocardialcellsliningthecardiaccushionsismostlylocalizedtothecellnucleiintherelevantperiodfromembryonicday8.5(E8.5)toE12.5,anditsnuclearlocalizationisreversedbyahighconcentrationofCsAorFK506appliedtothewholeembryo(delaPompaetal.1998;Rangeretal.1998a).Theseobservationsareconsistentwithanongo-inglocalsignal,frommyocardiumorfromendocardiumitself,thatactsintracellularlythroughtheCa2+/calci-neurinpathwaytoactivateNFAT.Fluidshearforcesarealsolikelytotriggerormodulatetranscriptionlocallyintheembryonicheart(Hoveetal.2003),buttheirconnec-tiontoactivationofNFAThasnotbeeninvestigated.Alessconventionalproposal,basedonthereducednuclearlocalizationofNFAT2inatrioventricularcanalendothe-liumofconnexin-45-nullmouseembryosatE9.5,isthatasignaltoactivatetheCa2+/calcineurinpathwaypropa-gateslaterallybetweenendocardialcellsviagapjunc-tions(Kumaietal.2000).However,connexin-45ispre-dominantlyexpressedinmyocardiumratherthaninen-dothelium(Alcoléaetal.1999),andalreadyatE9–E9.5,themyocardiumofconnexin-45-nullmouseembryosshowsimpairedconductionandcontractileactivityintheatrioventricularcanal(Kumaietal.2000).Thustheobservationsinconnexin-45-nullembryosmightequallybeexplainedinmoreconventionalterms,eitherbyre-ducedsignalingfrommyocardiumorbyreducedshearstresses. ApossiblelocalsignalforNFATactivationisVEGF,whichtriggersnuclearimportofNFAT2inpulmonaryvalveendothelialcellsculturedfrompostnatalhumanheart(Johnsonetal.2003),justasVEGFtriggersactiva-tionofNFAT1invascularendothelialcells(Armesillaetal.1999;Hernándezetal.2001).VEGFanditsreceptorVEGF-R2/Flk-1arepresentintheheartrudiment,anditisknownthatincreasedexpressionofVEGFatE10ter-minatestheendothelialmesenchymaltransforma-tioninthevalveprimordiaandpossiblypromotesex-pansionanddifferentiationoftheendothelialcelllayer(Doretal.2001,2003).GiventhatNFATproteinisnuclearasearlyasE8.5,if,infact,VEGFistheprincipaltriggerforNFATactivation,eventhelowlevelsofVEGFpresentbeforeE10haveadirectroleincardiacvalveformation. Ithasnotbeenresolvedwhetherthecrucialrequire-mentforNFAT2incardiacvalvedevelopmentisfortranscriptionofagene(orgenes)duringremodelingofthecardiaccushionsintothedefinitivevalvemorphol- TranscriptionalregulationbyNFAT ogy,orfortranscriptioninearlyendotheliumthatsetsthecellularcontextforlaterdevelopmentaldecisionswhenthesecellscompletedifferentiationasendothe-liumorleavetheendotheliumandbecomecardiaccush-ionmesenchymalcells.Theprominentnuclearlocaliza-tionofNFAT2bothearlyintheendotheliumofthehearttubeandlaterinendotheliumoverlyingthecardiaccushionsisconsistentwitheitherscenario.Italsore-mainstobedeterminedwhethertheabnormalitiesinmicedeficientinNFAT2arecausedbyanalteredlevelandtimingofexpressionofendothelialgenesthatarecommontocardiacandvascularendothelium,orresultfromfailuretoreadoutgenesthatarespecifictocardiacendothelium.ThepreferentialexpressionofNFAT2andGATA5incardiacendothelialcellshintsatthelatterpossibility. InsightintothetranscriptionaltargetsdownstreamofNFAT2inlaterstagesofcardiacendothelialdevelop-menthascomefromexperimentswiththeTC13cellline(alMoustafaandChalifour1993),whichservesasamodelofdifferentiationofcardiacmesodermintoendo-cardium.Inthesecells,NFAT2andGATA5synergisti-callyactivatealuciferasereporterlinkedtotheendothe-lin-1(EDN1)promoter(NemerandNemer2002).Re-ducedexpressionofendothelin-1wouldnotbyitselfexplainthedefectsinNFAT2-nullmice,becausethecar-diacphenotypeofEDN1-nullmicehasratherlowpen-etranceandisbasicallyrestrictedtotheoutflowtract(Kuriharaetal.1995).However,GATA5isakeytran-scriptionfactorinexpressionoftheendothelial/endocar-dialrepertoireinTC13cells(NemerandNemer2002),andislikelytoplayasimilarroleintheembryothroughitsexpressioninprecardiacmesodermanditsprogres-siverestriction,withintheheart,toendotheliumduringearlycardiogenesis(Morriseyetal.1997).InestablishingthatNFAT2cancooperatewithGATA5,theexampleoftheEDN1promoterhighlightsthepossibilitythatendo-cardialgenepromoterswillbecomeacasestudyforfunc-tionalandbiochemicalinteractionsofNFAT-andGATA-familyproteins. PhysiologicalcontrolofmyosinheavychainI(MyHCI)expression Mammalianskeletalmusclesareamosaicofmusclefi-bersthatdifferintheircomplementofmyofibrillarpro-teinsandofmetabolicenzymes,andthereforeintheircontractileproperties(Fig.12).ThecontractilepropertiesofindividualmusclefiberscorrelatewiththeMyHCiso-formexpressed.Thus,ausefulclassificationdividesmusclefibersintofourprincipaltypes:slow-twitchfi-bersthatexpressMyHCI,andthreesubtypesoffast-twitchfibersthatexpressMyHCIIa,IId,orIIb,respec-tively(PetteandStaron2000).ProlongedchangesinthecontractileactivityofadifferentiatedmusclefibercanaltertheexpressionofMyHCandothergenes,andthuscancauseatransitionofanexistingmusclefiberfromoneclassintoanother.Completionofsuchatransitionrequiresweeks,owingtotheslowturnoverofmyofibril-larproteins. GENES&DEVELOPMENT2221 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. Figure12.Musclefibercontractilepropertiescorrelatewiththeisoformofmyosinpresent.Thispointwasfirstestablishedbyobservingthecontractilepropertiesofindividualmotorunits,thesubsetofmusclefibersthatcontractwhenasinglemotornerveaxonisstimulated.Intheexperimentillustrated,threeclassesofmechanicalresponsewereobservedincatgastrocnemius,anddesignatedFF,FR,andS.(Intheclassificationadoptedinthisreview,FFmotorunitsconsistoftypeIIborIIdfibers,FRunitsoftypeIIafibers,andSunitsoftypeIfibers.)Theresponsesaremostreliablyclassifiedbytherateofdeclineinpeaktensionduringrepeatedtrainsofstimulation(FATIGUE)andbythepresenceorabsenceofaslight“sag”intheresponsetoasingletrainofstimuli(TETANUS).Afterthecontractilepropertiesweredetermined,musclefibersbelongingtothemotorunitweremarkedbyprolongedstimulationtodepletetheirstoresofglycogen,andserialcross-sectionsweremadefromthemuscle.Setsofcross-sectionsfromthesamemuscleswhosemechanicalresponsesaredepictedattheleftshowthefibersmarkedbydepletionofglycogen(arrows)andthestainingformyofibrillarATPaseunderdifferentconditions.IndividualmyosinisoformshavedifferingintrinsicATPaseactivityanddifferingsensitivitytopreincubationatpH4.65,andthisfeatureisapparentinthemosaicpatternofATPasestaining.However,allthemusclefibersinnervatedbyasingleaxonsharedacommonhistoryofactivitypriortotheexperiment,andhavesimilarmyofibrillarATPasestaining.ThisexperimentwasnotdesignedtoanswerwhetherindividualmusclefibersexpressasingleisoformofMyHCoramixtureofisoforms.However,morerecentstudiesofdissectedsinglemusclefibershavedocumentedthetightcorrelationbetweenmusclecontractileproperties,specificallyspeedofcontraction,andthespecificmyosinisoformexpressed(forreview,seePetteandStaron2000).FiguremodifiedwithpermissionfromBurkeetal.(1971).©1971AmericanAssociationfortheAdvancementofScience. Calcineurin,NFAT,andinductionofMyHCIgeneexpression:Calcineurinisclearlyimplicatedinthetran-sitioninwhichfullydifferentiatedfast-twitchmusclefibersareinducedbychronicactivitytoexpressslow-twitchMyHCI.Inoneexperimentalmodel,myotubesfromnewbornrabbithindlimbmusclesweregrownincultureongelatinbeadmicrocarriers,whichprovidefirmanchorageforthemyotubesandpreventthedetach-mentofmaturecontractingmyotubesthatusuallyoc-cursinculture(Kubisetal.1997).Overaperiodofweeks,theculturesmaturedtocontainamixtureofthefast-twitchMyHCisoformsIIa,IId,andIIb,andnegli-giblelevelsoftheslow-twitchMyHCI(Kubisetal.1997;Meißneretal.2000,2001;Kubisetal.2002).Electricalstimulationinapatterncharacteristicoftheactivityofslow-twitchmusclealteredthecourseofmaturation,re-sultinginelevatedexpressionofMyHCImRNAandprotein(Meißneretal.2001;Kubisetal.2002).TheCa2+ionophoreA23187mimickedthiseffectofstimulation(Kubisetal.1997;Meißneretal.2000,2001),andCsApreventedtheup-regulationofMyHCImRNAandpro-teininbothcases(Meißneretal.2001),thusimplicatingaCa2+/calcineurinpathwayintheprocess.Asecondmodelexaminedisolatedindividualskeletalmusclefi-bersfrommouseflexordigitorumbrevis(LiuandSchneider1998).Thismuscleiscomposedmainlyoffast-twitchfibers,andMyHCIexpressionisnotdetect-ableinthemajorityoffibersbysingle-fiberreversetran-scription–PCR(RT–PCR;LiuandSchneider1998).Whentheisolatedmusclefiberswerestimulatedinapatterncharacteristicofslow-twitchmuscle,MyHCImRNAbecamedetectableinnearlyalltheindividualfiberswithin6d(LiuandSchneider1998).Theup-regulationofMyHCImRNAintheseexperimentsinvitrodirectlyparallelsthatobserveduponcomparableelectricalstimulationoffast-twitchmuscleinvivo(Kirschbaumetal.1990;Brownsonetal.1992;Windischetal.1998).Bothinintactmusclestimulatedexvivoandintherab-bitmyotubes,anincreaseinMyHCImRNAisdetectedasearlyas24haftertheonsetofstimulation(Barton-Davisetal.1996;Kubisetal.2002,2003). Severalotherexperimentalprotocolshaveimplicatedcalcineurininactivity-dependentreplacementofMyHCIIisoformsbyMyHCIinvivo(Dunnetal.1999;Serrano 2222GENES&DEVELOPMENT Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press TranscriptionalregulationbyNFAT Figure13.CsApreventsexpressionofMyHCIinresponsetoactivity.Ratsoleusmusclesweredenervated,damagedwithbupivacaine,andallowedtoregenerate.Cross-sectionsweretakenafterregenera-tionfromamusclethathadnotbeenstimulated(left),amusclethathadbeenstimulatedelectricallyinafiringpatternre-semblingslow-twitchmuscleactivity(cen-ter),andamusclethathadbeenelectricallystimulatedandtreatedwithCsA(right).StainingwithanantibodytoMyHCIdem-onstratedthatMyHCIproteinwaspresentinthevastmajorityoffibersinthestimu-latedmuscle,butnotinfibersoftheunstimulatedmuscle,andthattheappearanceofMyHCIwaspreventedbyCsA.FigurereprintedwithpermissionfromSerranoetal.(2001).©2001AmericanAssociationfortheAdvancementofScience. etal.2001;Irintchevetal.2002;Pallafacchinaetal.2002;forreview,seeSchiaffinoandSerrano2002).Forinstance,CsA,FK506,oroverexpressionofafragmentofthecalcineurininhibitorCain/Cabin1blockstheup-regulationofMyHCIelicitedinregeneratingratormousesoleusmusclebyphysiologicalsignalingfromitsnerve(Serranoetal.2001;Irintchevetal.2002;Pallafac-chinaetal.2002).CsAsimilarlyblockstheup-regulationtriggeredindenervatedregeneratingsoleusbyelectricalstimulation(Fig.13;Serranoetal.2001).Intheseproto-cols,musclesatellitecellsrecapitulatethedevelopmen-talprogressionmyoblastmyotubemusclefiberandthesequentialexpressionofmyosinisoformsthatoccursduringsoleusdevelopment,andthesignalingpathwaysandpromoterelementscalledintoplayarelikelytobemorecomplexthaninthesimplecaseofelectricalstimulationoffullydifferentiatedfast-twitchmusclefi-bers.CsAblockstheincreaseinMyHCImRNAandMyHCI-positivefibersbroughtaboutbyexperimentallyinducedoverloadofmouseplantarisinvivo(Dunnetal.1999).Thisexperimentalmodelalsotriggersadditionalsignalingpathways,becauseitleadstoaprominentcom-pensatoryhypertrophythatinitiallyincreasestheex-pressionofseveralmyosinisoforms.Finally,calcineurinmaycontributetothemaintenanceofMyHCIexpres-sioninslow-twitchmusclefibersinvivo,becausechronicCsAtreatmentresultsinthepartialreplacementofMyHCIproteinbyfast-twitchmyosinisoformsinthesoleusmuscleofmiceandrats(Chinetal.1998;Bigardetal.2000;Irintchevetal.2002).Althoughtheinvivomodelsdifferindetailfromtheinvitromodelsdescribedabove,collectivelythesefindingsreinforcethemessagethatcalcineurinconveysasignalforexpressionofMyHCI. TheevidencethatNFATisatranscriptionaleffectorforMyHCIgeneexpressionismorecircumstantial.NFATmRNAsandproteinsarepresentinskeletalmuscle,includingtherabbitmyotubesgrownongelatinbeads(Hoeyetal.1995;Abbottetal.1998;Dunnetal.2000;Swoapetal.2000;Meißneretal.2001;Kubisetal.2002).NFAT2,orNFAT2-GFPexpressedfromanadeno-virusvector,isactivatedandimportedintothenucleuswhenthemyotubesormusclefibersareelectricallystimulated,andthisresponseispreventedbyCsA(Y.Liuetal.2001;Kubisetal.2002,2003).NuclearimportisobservedinresponsetospecificpatternsofstimulationthatproduceMyHCIinduction,butnotinresponsetoapatternthatfailstoinduceMyHCI(Kubisetal.2002).Thus,empirically,NFATisasuitabletranscriptionalef-fectortorelatemuscleactivitytoMyHCIgeneexpres-sion,eitherdirectlybyitsbindingtotheMyHCIpro-moterorindirectlythroughregulatinggenesthatcontrolMyHCIinduction. NFATactivationbymuscleactivity:Howdoesintra-cellularCa2+signalinginskeletalmusclefibersactivateNFAT?ContractingskeletalmusclecellsgeneratespikesincytoplasmicCa2+concentrationthatareex-ceedinglybriefandfrequentcomparedwithCa2+oscil-lationsinTcells.Theconventionalinterpretationhasbeenthatthebriefspikescontrolcontraction,whereasasustainedelevationofbaselineCa2+controlstranscrip-tionalactivation.Thisinterpretationmaybeanoversim-plification.Clearly,NFATintheexperimentalmodelscanrespondtoelevatedbasalCa2+,andMyHCImRNAcanbeinduced,asshowninexperimentsusingCa2+ionophores(Kubisetal.1997;Meißneretal.2000,2001;Y.Liuetal.2001).ThereisalsoevidencethatbasalCa2+infullydifferentiatedslow-twitchfibersismodestlyhigherthanthatinfast-twitchfibers(Carrolletal.1997),asituationthatcouldcontributetothemaintenanceofMyHCIgeneexpressioninslow-twitchfibers.However,stimulationthatinitiatedtheswitchtoMyHCIexpres-sionintheexperimentalmodelsinvitroproducednochangeinthefastCa2+transientsandnochangeinbasalCa2+(Liuetal.1999;Kubisetal.2003).Thus,althoughcontinuouselectricalstimulationleadsundersomecon-ditionstomoderateincreasesinbasalCa2+(Sreteretal.1987;Carrolletal.1999),elevatedbasalCa2+isnotanobligatorypartofthecalcineurin–MyHCIpathwaynorofNFATactivation.Rather,NFATisrespondingeithertothepatternofrapidCa2+transientsortohighlylocal-izedchangesinCa2+concentrationthatarenotdetectedasgloballyelevatedbasalCa2+.Ineithercase,themodeofactivationdiffersfromtheclassicalmodeofNFATactivationbyasimplesustainedelevationofcytoplas-micCa2+. OnceNFATisactivated,moreover,itshowsanovelabilitytodeliverasignaltothenucleusthataverages GENES&DEVELOPMENT2223 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. muscleactivityoverlengthyperiods,includingperiodswhenactivityisinterruptedbyintervalsofrest.Inbothexperimentalmodelstherewasaprogressiveaccumula-tionofnuclearNFAT2duringongoingintermittentstimulation,andonlyasloworveryslowexitofNFAT2fromthenucleusduringtheintervalsbetweenperiodsofstimulation(Y.Liuetal.2001;Kubisetal.2002,2003).Strikingly,inrabbitmyotubesafter24hofintermittentstimulation,NFATremainedfullynuclearinthemajor-ityofcellseven30minafterstimulationceased(Kubisetal.2003),andintheisolatedindividualmusclefibers,exportofNFAT2wasincompleteeven2hafterstimu-lationended(Y.Liuetal.2001).Theslownuclearaccu-mulationandslowexportofNFAT2inskeletalmusclestandincontrasttotherapidimportandexportofNFATproteinsinTcells.Theunderlyingmechanismisnotknown:Calcineurinsignalingcouldbeprolongedinthenucleusoffiberssubjectedtochroniclow-frequencystimulation,thecapacityforrephosphorylationofNFATcouldbelimitedbylowlevelsorbyinhibitionoftherelevantkinases,orexportofNFATfromthenucleuscouldlagbehindrephosphorylation. TheeffectiveconfinementofintracellularCa2+signal-ingtorestrictedspatialdomainsisincreasinglyrecog-nized(Zaccoloetal.2002).Skeletalmusclefibers,inparticular,withtheirstereotypedultrastructuralorgani-zation,offervastopportunitiesforspatialcompartmen-talizationofsignaling,anditisconceivablethatcom-partmentalizationshapestheresponseofNFATtomuscleactivity.Intheexperimentsonisolatedsinglemusclefibers,NFAT2-GFPwaspresentattheZ-disks(Y.Liuetal.2001).Calsarcin-familyproteinsmediatebindingofaproportionofskeletalmusclecalcineurintotheZ-disks(Freyetal.2000;FreyandOlson2002),plac-ingbothcalcineurinandNFAT2-GFPnearthesitesofintracellularCa2+release—junctionaltriads—whichflanktheZ-disks.Ontheotherhand,overexpressedNFAT2-GFPwasnotdetectablydepletedfromthesesitesbyphysiologicalstimulationthatproducedmaxi-malnuclearimport(Y.Liuetal.2001),whereasendog-enousNFAT2indifferentiatedmyotubescouldbeessen-tiallyquantitativelyrecruitedtothenucleus(Kubisetal.2003).PossiblytheZ-linesitesarenotthesourceofNFAT2thatisrecruitedtothenucleus;alternatively,itmaybethatthesesitesarethesourceunderphysiologi-calconditions,butthatoverexpressionofNFAT2-GFPprovidesmuchhigherlevelsofproteinthancanbepro-cessedbythesignalingandnuclearimportmachinery.Itwillbeimportanttoknowwhethertheobservedlocal-izationofNFAT2-GFPreflectsthelocalizationofendog-enousNFAT2orotherendogenousNFATisoformsinmusclecells,and,ifso,whetherthisspatialdistributionismandatoryforefficientNFATactivation. PromoterelementscontrollingMyHCIexpression:Thekeystepsinactivity-dependentinductionofMyHCIinfast-twitchfibershavenotbeendelineated,butitislikelythatlong-rangechangesinchromatinstructureareinvolved.TheMyHCIgene(termedthe-MyHCgeneinthecardiacliterature)isatalocusdirectlyadjacenttothecardiac␣-MyHCgene.OnlyMyHCIisnormally2224GENES&DEVELOPMENT expressedinskeletalmuscleofadultmammals.Bothinvivoandinvitro,however,thetransitionfromfast-twitchtoslow-twitchmyosininskeletalmuscleisac-companiedbythetransientexpressionofcardiac␣-MyHC(Peukeretal.1995,1999;Kubisetal.1997),suggestingchangesinchromatinstructurethroughouttheregioncontainingthelinkedgenes.Conversely,-MyHC(MyHCI)isexpressedindevelopinghamstercardiacmuscleandisdown-regulatedintheheartshortlyafterbirth,andcorrespondingalterationsinchro-matinaccessibilityaredetectableinprominentDNaseIhypersensitivesites2.3kbupstreamofthetranscriptionstartsiteandintheproximalpromoterregion(Huangetal.1997;HuangandLiew1998).Initialexpressionofgenesindevelopmentalprogramsisoftencontrolledbysignalinginputsaddressedtoonepromotermodule,whereascontinuingstableexpressionislockedinbytransferringcontroltoanothermodulewithoverlappingbutnotidenticalinputs(Davidson2001).Todeterminewhethertherearededicatedpromoterelementsthatini-tiatethefast-twitchtoslow-twitchtransitioninskeletalmuscle,itwouldbeusefultomapDNAseIhypersensi-tivesitesintheentirelocusinfullydifferentiatedfast-twitchandslow-twitchskeletalmuscle,aswellasinfast-twitchskeletalmuscleduringtheearlystagesoftheMyHCtransition. MaintenanceofMyHCIexpressionindifferentiatedslow-twitchmusclefibersinvolvesaproximal∼600-bppromoterelementthathasbeenshowntoconferexpres-sioninslow-twitchskeletalmuscleoftransgenicani-mals(Rindtetal.1993)andadditionalregulatoryregionsfurtherupstreamofthetranscriptionstartsite(Rindtetal.1993;Gigeretal.2000).Therequirementforup-streamelementsisdisplayedvividlyinthehigherex-pressionlevelofareportertransgenedrivenbyan∼5.6-kbfragmentoftheMyHCIpromoter,comparedwiththeleveldrivenbythe600-bppromoterfragment(Rindtetal.1993).Althoughthe5.6-kbpromoterfragmentgavewell-regulatedexpressionofitsreportertransgeneinmousesoleus(Rindtetal.1993;Knottsetal.1996),twooffourtransgeniclinesalsoshowedunanticipatedstrongexpressioninmousemasseter(Rindtetal.1993),amusclethathasnoslow-twitchfibers,indicatingthatevenmoredistalregionscontainingadditionaltranscrip-tionalcontrolelementsremaintobeidentified.Com-parativesequenceanalysis,discussedinaprevioussec-tion,showsthatthereareconserveduntranslatedse-quencesscatteredtoatleast12.5kbupstreamofthetranscriptionstartsiteoftheMyHCIgene(J.Nardone,unpubl.).TheseobservationscallforarenewedanalysisofwhichelementsoftheMyHCIpromoterconferex-pressioninslow-twitchmusclefibersinvivo. Eventhoughinformationonthecis-actingelementslinkingmuscleactivitytoMyHCIexpressionisincom-plete,itisusefultoconsiderbrieflyhowNFATsignalscouldconvergewithothersignalstocontroltheexpres-sionofmuscle-specificgenes.Cellularidentityasskel-etalmuscleislikelytoberegisteredatthegenepromoterbymuscle-specifictranscriptionfactorssuchasMyoD-familyproteins.ThefactthatMyHCexpressionissen- Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press sitivetodiverseinputs,includingpatternofactivity,me-chanicalloading,hormonalsignals,anddevelopmentalsignals,impliesthattherearemanyrelevantinduciblepathways,butamongthemtwocallforspecialcom-ment.MEF2proteinsareprominentinskeletalmuscleandrepresentasecondpathwayfromcalcineurintoDNA(Wuetal.2000;McKinseyetal.2002).MEF2isactiveinbothtypeIslow-twitchfibersandtypeIIafast-twitchfibers,andisdownstreamofcalcineurininasignalingpathwaythatcontrolsthetransitionfromtypeIIbandIIdfast-twitchfiberstotypeIIafast-twitchfibersinresponsetomuscleactivity(Wuetal.2000,2001;AllenandLeinwand2002).Inprinciple,MEF2couldbindinconjunctionwithNFATatsomepromoters(Chinetal.1998)andconveyitssignalindependentlyatotherpromoters.ThereisalsothefamiliarpossibilitythatAP1couldcooperatewithNFATattheMyHCIpromoterasitdoesatcytokinegenepromoters.Heretheevidenceiscircumstantial:First,overexpressionofanactivatedRasprotein,actingviatheRas–MAPkinasepathway,causedexpressionofMyHCIindenervatedregeneratingsoleus(Murgiaetal.2000);second,overex-pressionofadominant-negativeformofRaspreventedinductionofMyHCIbyreinnervation(Murgiaetal.2000),provingtheplaceofRasinnormalphysiologicalsignaling;third,AP1proteinsaredownstreameffectorsoftheRas–MAPkinasepathway;andfinally,plausiblecompositeNFAT–AP1sitesarepresentintheproximalpromoterregionoftheMyHCIgeneinseveralmam-malianspecies(Keletal.1999;J.Nardone,unpubl.).Theseobservationsareopentootherinterpretations,ofcourse,becausetheRas–MAPkinasepathwayhasabun-danteffectsonothersignalingproteinsandtranscriptionfactors. Transcriptionalcontrolofslow-twitchfiberdifferen-tiation:MyHCIgeneexpressionduringthetransitionfromfast-twitchfibertoslow-twitchfiberisonlyonecomponentofaglobalreprogrammingthatincludeschangesinothermyofibrillarproteins;inproteinsthatrelease,bind,orsequesterCa2+;andinmetabolicen-zymes.ItisnaturaltoaskwhetherNFATlinksCa2+signalinginskeletalmuscletoabatteryofgeneschar-acteristicoftheslow-twitchfiberdifferentiationpro-gram,butthelimitedexperimentalevidenceonthispointhasledtodivergentinterpretations(Chinetal.1998;Calvoetal.1999;Swoapetal.2000;Wuetal.2000).BroaderparticipationofNFATincontrolofslow-twitchfibergeneexpressionwouldnotnecessarilyimplythatNFATisthepreeminentinputtoeachofthegenesorthatNFATbyitselftriggersthedifferentiationpro-gram.Targetsforfurtherresearchwillbetoidentifytheparticularpromotersintheslow-twitchfiberdifferentia-tionprogramatwhichNFATcontributestoexpression,andtoexplorehowitscontributionatthesepromotersisshapedbycellidentityasreflectedinchromatinstruc-ture,bycellhistoryreflectedinthecomplementofavail-abletranscriptionfactorsandcofactors,andbythespe-cificcontext—normaldevelopment,alteredactivity,orregeneration—inwhichtheslow-twitchfiberdifferentia-tionprogramisinvoked. TranscriptionalregulationbyNFAT Conclusion Inthisreview,wehavedevelopedtwocorethemes:thattheactivationofNFATproteinsisdeterminedbytheirphosphorylationstate,whichreflectstheintensityofCa2+/calcineurinsignalingandtheactivitiesofseveralkinases;andthattranscriptionallyactiveNFATinte-gratesinputsfrommultiplepathwaysthroughitsinter-actionswithpartnerproteinsonDNA.Thefourcal-cium-regulatedNFATproteinsdivergedfromNFAT5/TonEBP,theirnearestsiblingintheRelfamily,lateinevolutionaryhistory,byacquiringaregulatoryregionthatprovidedanewchannelforcommunicationofin-tracellularCa2+signalingtoDNAandbyacquiringanewphysicalflexibilityandnovelproteincontactsur-facesthatenabledthemtocooperatewithavarietyofpartnerproteinsinthenucleus.Thismoleculardiversi-ficationcoincidedwiththeappearanceofbiologicalspe-cializationsthatinitiatedthevertebratelineage,andthenewNFATproteinstookonasurprisinglyvariedsetofspecializedtranscriptionalroles.ThedetailedstudiesofNFATfunctioninTcellsareanimportantstartingpoint,butaffordonlyaglimpseofthevariedinputsandproteinpartnersthatareutilizedbythefourNFAT-fam-ilyproteinsinotherbiologicalcontexts.Acknowledgments WethankStefanFeskeforanoriginalversionofFigure1,andStephenC.Harrison,ThomasHo¨fer,RikoNishimura,StefanoSchiaffino,MartinF.Schneider,MiraSuˇsaˇ,TatsuoTakeya,andErwinF.Wagnerfordiscussionsandunpublisheddata.A.R.acknowledgesthesupportoftheNationalInstitutesofHealthandtheSandlerProgramforAsthmaResearch.L.C.acknowl-edgesthesupportoftheNationalInstitutesofHealthandtheW.M.KeckFoundation. References Abbott,K.L.,Friday,B.B.,Thaloor,D.,Murphy,T.J.,andPav-lath,G.K.1998.ActivationandcellularlocalizationofthecyclosporineA-sensitivetranscriptionfactorNF-ATinskel-etalmusclecells.Mol.Biol.Cell9:2905–2916. Agalioti,T.,Lomvardas,S.,Parekh,B.,Yie,J.,Maniatis,T.,andThanos,D.2000.Orderedrecruitmentofchromatinmodi-fyingandgeneraltranscriptionfactorstotheIFN-pro-moter.Cell103:667–678. Agarwal,S.,Avni,O.,andRao,A.2000.Cell-type-restrictedbindingofthetranscriptionfactorNFATtoadistalIL-4enhancerinvivo.Immunity12:3–652. Al-Daraji,W.I.,Grant,K.R.,Ryan,K.,Saxton,A.,andReynolds,N.J.2002.Localizationofcalcineurin/NFATinhumanskinandpsoriasisandinhibitionofcalcineurin/NFATactivationinhumankeratinocytesbycyclosporinA.J.Invest.Derma-tol.118:779–788. Alcoléa,S.,Thevéniau-Ruissy,M.,Jarry-Guichard,T.,Marics,I.,Tzouanacou,E.,Chauvin,J.P.,Briand,J.P.,Moorman,A.F.,Lamers,W.H.,andGros,D.B.1999.Downregulationofconnexin45geneproductsduringmouseheartdevelop-ment.Circ.Res.84:1365–1379. Allen,D.L.andLeinwand,L.A.2002.Intracellularcalciumandmyosinisoformtransitions.Calcineurinandcalcium-calmodulinkinasepathwaysregulatepreferentialactivationoftheIIamyosinheavychainpromoter.J.Biol.Chem.277:45323–45330. GENES&DEVELOPMENT2225 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. alMoustafa,A.E.andChalifour,L.E.1993.Immortalcelllinesisolatedfromheartdifferentiatetoanendothelialcelllin-eageinthepresenceofretinoicacid.CellGrowthDiffer.4:841–847. Ansel,M.K.,Lee,D.U.,andRao,A.2003.AnepigeneticviewofhelperTcelldifferentiation.Nat.Immunol.4:616–623.Anusaksathien,O.,Laplace,C.,Li,X.,Ren,Y.,Peng,L.,Gol-dring,S.R.,andGalson,D.L.2001.Tissue-specificandubiq-uitouspromotersdirecttheexpressionofalternativelysplicedtranscriptsfromthecalcitoninreceptorgene.J.Biol.Chem.276:22663–22674. Aramburu,J.,Garcia-Cózar,F.,Raghavan,A.,Okamura,H.,Rao,A.,andHogan,P.G.1998.SelectiveinhibitionofNFATactivationbyapeptidespanningthecalcineurintargetingsiteofNFAT.Mol.Cell1:627–637. Aramburu,J.,Yaffe,M.B.,López-Rodríguez,C.,Cantley,L.C., Hogan,P.G.,andRao,A.1999.Affinity-drivenpeptidese-lectionofanNFATinhibitormoreselectivethancyclospo-rinA.Science285:2129–2133. Armesilla,A.L.,Lorenzo,E.,GómezdelArco,P.,Martínez-Mar-tínez,S.,Alfranca,A.,andRedondo,J.M.1999.Vascularen-dothelialgrowthfactoractivatesnuclearfactorofactivatedTcellsinhumanendothelialcells:Arolefortissuefactorgeneexpression.Mol.Cell.Biol.19:2032–2043. Avni,O.andRao,A.2000.Tcelldifferentiation:Amechanistic view.Curr.Opin.Immunol.12:6–659. Avni,O.,Lee,D.,Macián,F.,Szabo,S.J.,Glimcher,L.H.,and Rao,A.2002.T(H)celldifferentiationisaccompaniedbydynamicchangesinhistoneacetylationofcytokinegenes.Nat.Immunol.3:3–651. Bakiri,L.,Matsuo,K.,Wisniewska,M.,Wagner,E.F.,andYaniv, M.2002.Promoterspecificityandbiologicalactivityofteth-eredAP-1dimers.Mol.Cell.Biol.22:4952–49. Baksh,S.,Widlund,H.R.,Frazer-Abel,A.A.,Du,J.,Fosmire,S., Fisher,D.E.,DeCaprio,J.A.,Modiano,J.F.,andBurakoff,S.J.2002.NFATc2-mediatedrepressionofcyclin-dependentki-nase4expression.Mol.Cell10:1071–1081. Barton-Davis,E.R.,LaFramboise,W.A.,andKushmerick,M.J.1996.Activity-dependentinductionofslowmyosingeneex-pressioninisolatedfast-twitchmousemuscle.Am.J.Physiol.271:C1409–C1414. Beals,C.R.,Clipstone,N.A.,Ho,S.N.,andCrabtree,G.R.1997a.NuclearlocalizationofNF-ATcbyacalcineurin-de-pendent,cyclosporin-sensitiveintramolecularinteraction.Genes&Dev.11:824–834. Beals,C.R.,Sheridan,C.M.,Turck,C.W.,Gardner,P.,andCrab-tree,G.R.1997b.NuclearexportofNF-ATcenhancedbyglycogensynthasekinase-3.Science275:1930–1934. Bert,A.G.,Burrows,J.,Hawwari,A.,Vadas,M.A.,andCock-erill,P.N.2000.ReconstitutionofTcell-specifictranscrip-tiondirectedbycompositeNFAT/Octelements.J.Immu-nol.165:56–5655. Bigard,X.,Sanchez,H.,Zoll,J.,Mateo,P.,Rousseau,V.,Vek-sler,V.,andVentura-Clapier,R.2000.Calcineurinco-regu-latescontractileandmetaboliccomponentsofslowmusclephenotype.J.Biol.Chem.275:19653–19660. Bodor,J.,Bodorova,J.,andGress,R.E.2000.SuppressionofT cellfunction:ApotentialrolefortranscriptionalrepressorICER.J.LeukocyteBiol.67:774–779. Bower,K.E.,Zeller,R.W.,Wachsman,W.,Martinez,T.,andMcGuire,K.L.2002.Correlationoftranscriptionalrepres-sionbyp21SNFTwithchangesinDNA.NF-ATcomplexin-teractions.J.Biol.Chem.277:34967–34977. Bray,N.,Dubchak,I.,andPachter,L.2003.AVID:Aglobalalignmentprogram.GenomeRes.13:97–102. Brownson,C.,Little,P.,Mayne,C.,Jarvis,J.C.,andSalmons,S.2226GENES&DEVELOPMENT 1992.Reciprocalchangesinmyosinisoformexpressioninrabbitfastskeletalmuscleresultingfromtheapplicationandremovalofchronicelectricalstimulation.Symp.Soc.Exp.Biol.46:301–310. Bruneau,B.G.2002.Transcriptionalregulationofvertebratecardiacmorphogenesis.Circ.Res.90:509–519. Brunet,A.,Kanai,F.,Stehn,J.,Xu,J.,Sarbassova,D.,Frangioni,J.V.,Dalal,S.N.,DeCaprio,J.A.,Greenberg,M.E.,andYaffe,M.B.2002.14-3-3transitstothenucleusandparticipatesindynamicnucleocytoplasmictransport.J.CellBiol.156:817–828;erratum157:533. Burke,R.E.,Levine,D.N.,ZajacIII,F.E.,Tsairis,P.,andEngel,W.K.1971.Mammalianmotorunits:Physiological–histo-chemicalcorrelationinthreetypesincatgastrocnemius.Science174:709–712. Calvo,S.,Venepally,P.,Cheng,J.,andBuonanno,A.1999.Fi-ber-type-specifictranscriptionofthetroponinIslowgeneisregulatedbymultipleelements.Mol.Cell.Biol.19:515–525. Cantrell,D.2002.ProteinkinaseB(Akt)regulationandfunc-tioninTlymphocytes.Sem.Immunol.14:19–26. Cappellen,D.,Luong-Nguyen,N.H.,Bongiovanni,S.,Grenet,O.,Wanke,C.,andSuˇsaˇ,M.2002.Transcriptionalprogramofmouseosteoclastdifferentiationgovernedbythemacro-phagecolony-stimulatingfactorandtheligandfortherecep-toractivatorofNFB.J.Biol.Chem.277:21971–21982.Carroll,S.L.,Klein,M.G.,andSchneider,M.F.1997.Decayofcalciumtransientsafterelectricalstimulationinratfast-andslow-twitchskeletalmusclefibres.J.Physiol.501:573–588. Carroll,S.,Nicotera,P.,andPette,D.1999.Calciumtransientsinsinglefibersoflow-frequencystimulatedfast-twitchmuscleofrat.Am.J.Physiol.277:C1122–C1129. Chambers,T.J.2000.Regulationofthedifferentiationandfunc-tionofosteoclasts.J.Pathol.192:4–13. Chen,C.H.,Seguin-Devaux,C.,Burke,N.A.,Oriss,T.B.,Wat-kins,S.C.,Clipstone,N.,andRay,A.2003.TransforminggrowthfactorblocksTeekinasephosphorylation,Ca2+in-flux,andNFATctranslocationcausinginhibitionofTcelldifferentiation.J.Exp.Med.197:16–1699. Chen,L.,Oakley,M.G.,Glover,J.N.,Jain,J.,Dervan,P.B.,Hogan,P.G.,Rao,A.,andVerdine,G.L.1995.OnlyoneofthetwoDNA-boundorientationsofAP-1foundinsolutioncooperateswithNFATp.Curr.Biol.5:882–8. Chen,L.,Glover,J.N.,Hogan,P.G.,Rao,A.,andHarrison,S.C.1998.StructureoftheDNA-bindingdomainsfromNFAT,FosandJunboundspecificallytoDNA.Nature392:42–48.Chin,E.R.,Olson,E.N.,Richardson,J.A.,Yang,Q.,Humphries,C.,Shelton,J.M.,Wu,H.,Zhu,W.,Bassel-Duby,R.,andWilliams,R.S.1998.Acalcineurin-dependenttranscrip-tionalpathwaycontrolsskeletalmusclefibertype.Genes&Dev.12:2499–2509. Chinenov,Y.andKerppola,T.K.2001.Closeencountersofmanykinds:Fos–Juninteractionsthatmediatetranscriptionregulatoryspecificity.Oncogene20:2438–2452. Chow,C.W.andDavis,R.J.2000.IntegrationofcalciumandcyclicAMPsignalingpathwaysby14-3-3.Mol.Cell.Biol.20:702–712. Chow,C.W.,Rincón,M.,Cavanagh,J.,Dickens,M.,andDavis,R.J.1997.NuclearaccumulationofNFAT4opposedbytheJNKsignaltransductionpathway.Science278:1638–11. Chow,C.W.,Rincón,M.,andDavis,R.J.1999.RequirementfortranscriptionfactorNFATininterleukin-2expression.Mol.Cell.Biol.19:2300–2307. Chow,C.W.,Dong,C.,Flavell,R.A.,andDavis,R.J.2000.c-Jun Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press NH2-terminalkinaseinhibitstargetingoftheproteinphos-phatasecalcineurintoNFATc1.Mol.Cell.Biol.20:5227–5234. Chuvpilo,S.,Zimmer,M.,Kerstan,A.,Glöckner,J.,Avots,A.,Escher,C.,Fischer,C.,Inashkina,I.,Jankevics,E.,Berberich-Siebelt,F,.etal.1999.AlternativepolyadenylationeventscontributetotheinductionofNF-ATcineffectorTcells.Immunity10:261–269. Chuvpilo,S.,Jankevics,E.,Tyrsin,D.,Akimzhanov,A.,Moroz,D.,Jha,M.K.,Schulze-Luehrmann,J.,Santner-Nanan,B.,Fe-oktistova,E.,König,T.,etal.2002.AutoregulationofNFATc1/AexpressionfacilitateseffectorTcellstoescapefromrapidapoptosis.Immunity16:881–5. Chytil,M.andVerdine,G.L.1996.TheRelfamilyofeukaryotictranscriptionfactors.Curr.Opin.Struct.Biol.6:91–100.Cockerill,P.N.,Shannon,M.F.,Bert,A.G.,Ryan,G.R.,andVa-das,M.A.1993.Thegranulocyte-macrophagecolony-stimu-latingfactor/interleukin3locusisregulatedbyaninduciblecyclosporinA-sensitiveenhancer.Proc.Natl.Acad.Sci.90:2466–2470. Cockerill,P.N.,Bert,A.G.,Jenkins,F.,Ryan,G.R.,Shannon,M.F.,andVadas,M.A.1995.Humangranulocyte-macro-phagecolony-stimulatingfactorenhancerfunctionisasso-ciatedwithcooperativeinteractionsbetweenAP-1andNFATp/c.Mol.Cell.Biol.15:2071–2079. Crabtree,G.R.andOlson,E.N.2002.NFATsignaling:Choreo-graphingthesociallivesofcells.Cell109Suppl:S67–S79.Cross,D.A.,Alessi,D.R.,Cohen,P.,Andjelkovich,M.,and Hemmings,B.A.1995.Inhibitionofglycogensynthaseki-nase-3byinsulinmediatedbyproteinkinaseB.Nature378:785–7. Davidson,E.H.2001.Genomicregulatorysystems:Develop-mentandevolution.AcademicPress,Boston,MA. Davis,R.J.2000.SignaltransductionbytheJNKgroupofMAP kinases.Cell103:239–252. Decker,E.L.,Skerka,C.,andZipfel,P.F.1998.Theearlygrowth responseprotein(EGR-1)regulatesinterleukin-2transcrip-tionbysynergisticinteractionwiththenuclearfactorofactivatedTcells.J.Biol.Chem.273:26923–26930. Decker,E.L.,Nehmann,N.,Kampen,E.,Eibel,H.,Zipfel,P.F.,andSkerka,C.2003.Earlygrowthresponseproteins(EGR)andnuclearfactorsofactivatedTcells(NFAT)formhet-erodimersandregulateproinflammatorycytokinegeneex-pression.NucleicAcidsRes.31:911–921. deGregorio,R.,Iñiguez,M.A.,Fresno,M.,andAlemany,S.2001.Cotkinaseinducescyclooxygenase-2expressioninTcellsthroughactivationofthenuclearfactorofactivatedTcells.J.Biol.Chem.276:27003–27009. delaPompa,J.L.,Timmerman,L.A.,Takimoto,H.,Yoshida,H.,Elia,A.J.,Samper,E.,Potter,J.,Wakeham,A.,Marengere,L.,Langille,B.L.,etal.1998.RoleoftheNF-ATctranscriptionfactorinmorphogenesisofcardiacvalvesandseptum.Na-ture392:182–186. Diehn,M.,Alizadeh,A.A.,Rando,O.J.,Liu,C.L.,Stankunas,K.,Botstein,D.,Crabtree,G.R.,andBrown,P.O.2002.GenomicexpressionprogramsandtheintegrationoftheCD28co-stimulatorysignalinTcellactivation.Proc.Natl.Acad.Sci.99:11796–11801;erratum99:15245. Dolmetsch,R.E.,Lewis,R.S.,Goodnow,C.C.,andHealy,J.I.1997.DifferentialactivationoftranscriptionfactorsinducedbyCa2+responseamplitudeandduration.Nature386:855–858;erratum388:308. Dolmetsch,R.E.,Xu,K.,andLewis,R.S.1998.Calciumoscil-lationsincreasetheefficiencyandspecificityofgeneexpres-sion.Nature392:933–936. Dolmetsch,R.E.,Pajvani,U.,Fife,K.,Spotts,J.M.,andGreen-TranscriptionalregulationbyNFAT berg,M.E.2001.SignalingtothenucleusbyanL-typecal-ciumchannel–calmodulincomplexthroughtheMAPkinasepathway.Science294:333–339. Dor,Y.,Camenisch,T.D.,Itin,A.,Fishman,G.I.,McDonald,J.A.,Carmeliet,P.,andKeshet,E.2001.AnovelroleforVEGFinendocardialcushionformationanditspotentialcontributiontocongenitalheartdefects.Development128:1531–1538. Dor,Y.,Klewer,S.E.,McDonald,J.A.,Keshet,E.,andCameni-sch,T.D.2003.VEGFmodulatesearlyheartvalveformation.Anat.Rec.271A:202–208. Duncliffe,K.N.,Bert,A.G.,Vadas,M.A.,andCockerill,P.N.1997.ATcell-specificenhancerintheinterleukin-3locusisactivatedcooperativelybyOctandNFATelementswithinaDNaseI-hypersensitivesite.Immunity6:175–185. Dunn,S.E.,Burns,J.L.,andMichel,R.N.1999.Calcineurinisrequiredforskeletalmusclehypertrophy.J.Biol.Chem.274:21908–21912. Dunn,S.E.,Chin,E.R.,andMichel,R.N.2000.Matchingofcalcineurinactivitytoupstreameffectorsiscriticalforskel-etalmusclefibergrowth.J.CellBiol.151:663–672. Eisenberg,L.M.andMarkwald,R.R.1995.Molecularregulationofatrioventricularvalvuloseptalmorphogenesis.Circ.Res.77:1–6. Escalante,C.R.,Shen,L.,Thanos,D.,andAggarwal,A.K.2002.StructureofNF-Bp50/p65heterodimerboundtothePRDIIDNAelementfromtheinterferon-promoter.Structure10:383–391. Feske,S.,Draeger,R.,Peter,H.H.,Eichmann,K.,andRao,A.2000.ThedurationofnuclearresidenceofNFATdeterminesthepatternofcytokineexpressioninhumanSCIDTcells.J.Immunol.165:297–305. Feske,S.,Giltnane,J.,Dolmetsch,R.,Staudt,L.M.,andRao,A.2001.GeneregulationmediatedbycalciumsignalsinTlym-phocytes.Nat.Immunol.2:316–324. Fishman,M.C.andChien,K.R.1997.Fashioningthevertebrateheart:Earliestembryonicdecisions.Development124:2099–2117. Franzoso,G.,Carlson,L.,Xing,L.,Poljak,L.,Shores,E.W.,Brown,K.D.,Leonardi,A.,Tran,T.,Boyce,B.F.,andSieben-list,U.1997.RequirementforNf-BinosteoclastandB-celldevelopment.Genes&Dev.11:3482–3496. Frey,N.andOlson,E.N.2002.Calsarcin-3,anovelskeletalmuscle-specificmemberofthecalsarcinfamily,interactswithmultipleZ-discproteins.J.Biol.Chem.277:13998–14004. Frey,N.,Richardson,J.A.,andOlson,E.N.2000.Calsarcins,anovelfamilyofsarcomericcalcineurin-bindingproteins.Proc.Natl.Acad.Sci.97:14632–14637. Fuentes,J.J.,Genescà,L.,Kingsbury,T.J.,Cunningham,K.W.,Pérez-Riba,M.,Estivill,X.,anddelaLuna,S.2000.DSCR1,overexpressedinDownsyndrome,isaninhibitorofcalci-neurin-mediatedsignalingpathways.Hum.Mol.Genet.9:1681–1690. Fürstenau,U.,Schwaninger,M.,Blume,R.,Jendrusch,E.M.,andKnepel,W.1999.Characterizationofanovelcalciumresponseelementintheglucagongene.J.Biol.Chem.274:5851–5860. Garrity,P.A.,Chen,D.,Rothenberg,E.V.,andWold,B.J.1994.Interleukin-2transcriptionisregulatedinvivoatthelevelofcoordinatedbindingofbothconstitutiveandregulatedfac-tors.Mol.Cell.Biol.14:2159–2169. Ghosh,G.,vanDuyne,G.,Ghosh,S.,andSigler,P.B.1995.StructureofNF-Bp50homodimerboundtoaBsite.Na-ture373:303–310. Giffin,M.J.,Stroud,J.C.,Bates,D.,vonKoenig,K.D.,Hardin,J., GENES&DEVELOPMENT2227 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. andChen,L.2003.StructureofNFAT1boundasadimertotheHIV-1LTRBelement.Nat.Struct.Biol.(inpress).Giger,J.M.,Haddad,F.,Qin,A.X.,andBaldwin,K.M.2000.In vivoregulationofthe-myosinheavychaingeneinsoleusmuscleofsuspendedandweight-bearingrats.Am.J.Physiol.CellPhysiol.278:C1153–C1161. Glimcher,L.H.andMurphy,K.M.2000.Lineagecommitment intheimmunesystem:TheThelperlymphocytegrowsup.Genes&Dev.14:1693–1711. Goldfeld,A.E.,McCaffrey,P.G.,Strominger,J.L.,andRao,A.1993.Identificationofanovelcyclosporin-sensitiveelementinthehumantumornecrosisfactor␣genepromoter.J.Exp.Med.178:1365–1379. GómezdelArco,P.,Martínez-Martínez,S.,Maldonado,J.L.,Ortega-Pérez,I.,andRedondo,J.M.2000.Aroleforthep38MAPkinasepathwayinthenuclearshuttlingofNFATp.J.Biol.Chem.275:13872–13878. Graef,I.A.,Mermelstein,P.G.,Stankunas,K.,Neilson,J.R., Deisseroth,K.,Tsien,R.W.,andCrabtree,G.R.1999.L-typecalciumchannelsandGSK-3regulatetheactivi-tyofNF-ATc4inhippocampalneurons.Nature401:703–708. Graef,I.A.,Chen,F.,Chen,L.,Kuo,A.,andCrabtree, G.R.2001a.SignalstransducedbyCa2+/calcineurinandNFATc3/c4patternthedevelopingvasculature.Cell105:863–875. Graef,I.A.,Gastier,J.M.,Francke,U.,andCrabtree,G.R.2001b. EvolutionaryrelationshipsamongReldomainsindicatefunctionaldiversificationbyrecombination.Proc.Natl.Acad.Sci.98:5740–5745. Graef,I.A.,Wang,F.,Frederic,C.,Chen,L.,Neilson,J.,Tessier-Lavigne,M.,andCrabtree,G.R.2003.Neurotrophinsandnetrinsrequirecalcineurin/NFATsignalingtostimulateoutgrowthofembryonicaxons.Cell113:657–670. Grigoriadis,A.E.,Wang,Z.Q.,Cecchini,M.G.,Hofstetter,W., Felix,R.,Fleisch,H.A.,andWagner,E.F.1994.c-Fos:Akeyregulatorofosteoclast-macrophagelineagedeterminationandboneremodeling.Science266:443–448. Harwood,A.J.2001.RegulationofGSK-3:Acellularmultipro-cessor.Cell105:821–824. Hattersley,G.andChambers,T.J.19.Calcitoninreceptorsasmarkersforosteoclasticdifferentiation:Correlationbe-tweengenerationofbone-resorptivecellsandcellsthatex-presscalcitoninreceptorsinmousebonemarrowcultures.Endocrinology125:1606–1612. Hawwari,A.,Burrows,J.,Vadas,M.A.,andCockerill,P.N. 2002.ThehumanIL-3locusisregulatedcooperativelybytwoNFAT-dependentenhancersthathavedistincttissue-specificactivities.J.Immunol.169:1876–1886. Hernández,G.L.,Volpert,O.V.,Iñiguez,M.A.,Lorenzo,E.,Mar-tínez-Martínez,S.,Grau,R.,Fresno,M.,andRedondo,J.M.2001.Selectiveinhibitionofvascularendothelialgrowthfac-tor-mediatedangiogenesisbycyclosporinA:RolesofthenuclearfactorofactivatedTcellsandcyclooxygenase2.J.Exp.Med.193:607–620. Hill-Eubanks,D.C.,Gomez,M.F.,Stevenson,A.S.,andNelson, M.T.2003.NFATregulationinsmoothmuscle.TrendsCar-diovasc.Med.13:56–62. Ho,I.C.,Hodge,M.R.,Rooney,J.W.,andGlimcher,L.H.1996.Theproto-oncogenec-mafisresponsiblefortissue-specificexpressionofinterleukin-4.Cell85:973–983. Ho,S.N.,Thomas,D.J.,Timmerman,L.A.,Li,X.,Francke,U.,andCrabtree,G.R.1995.NFATc3,alymphoid-specificNFATcfamilymemberthatiscalcium-regulatedandexhib-itsdistinctDNAbindingspecificity.J.Biol.Chem.270:198–19907.2228GENES&DEVELOPMENT Hoey,T.,Sun,Y.L.,Williamson,K.,andXu,X.1995.IsolationoftwonewmembersoftheNF-ATgenefamilyandfunc-tionalcharacterizationoftheNF-ATproteins.Immunity2:461–472. Horsley,V.andPavlath,G.K.2002.NFAT:Ubiquitousregula-torofcelldifferentiationandadaptation.J.CellBiol.156:771–774. Hove,J.R.,Köster,R.W.,Forouhar,A.S,Acevedo-Bolton,G.,Fraser,S.E.,andGharib,M.2003.Intracardiacfluidforcesareanessentialepigeneticfactorforembryoniccardiogenesis.Nature421:172–177. Hsu,H.,Lacey,D.L.,Dunstan,C.R.,Solovyev,I.,Colombero,A.,Timms,E.,Tan,H.L.,Elliott,G.,Kelley,M.J.,Sarosi,I.,etal.1999.TumornecrosisfactorreceptorfamilymemberRANKmediatesosteoclastdifferentiationandactivationin-ducedbyosteoprotegerinligand.Proc.Natl.Acad.Sci.96:30–35. Hu,C.M.,Jang,S.Y.,Fanzo,J.C.,andPernis,A.B.2002.Modu-lationofTcellcytokineproductionbyinterferonregulatoryfactor-4.J.Biol.Chem.277:49238–49246. Huang,W.Y.andLiew,C.C.1998.Chromatinremodellingofthecardiac-myosinheavychaingene.Biochem.J.330:871–876. Huang,W.Y.,Chen,J.J.,Shih,N.,andLiew,C.C.1997.Multiplemuscle-specificregulatoryelementsareassociatedwithaDNaseIhypersensitivesiteofthecardiac-myosinheavy-chaingene.Biochem.J.327:507–512. Huxford,T.,Huang,D.B.,Malek,S.,andGhosh,G.1998.ThecrystalstructureoftheIB␣/NF-Bcomplexrevealsmecha-nismsofNF-Binactivation.Cell95:759–770. Irintchev,A.,Zweyer,M.,Cooper,R.N.,Butler-Browne,G.S.,andWernig,A.2002.Contractileproperties,structureandfiberphenotypeofintactandregeneratingslow-twitchmusclesofmicetreatedwithcyclosporinA.Cell&TissueRes.308:143–156. Ishida,N.,Hayashi,K.,Hoshijima,M.,Ogawa,T.,Koga,S.,Miyatake,Y.,Kumegawa,M.,Kimura,T.,andTakeya,T.2002.Largescalegeneexpressionanalysisofosteoclastogen-esisinvitroandelucidationofNFAT2asakeyregulator.J.Biol.Chem.277:41147–41156. Jacobs,M.D.andHarrison,S.C.1998.StructureofanIB␣/NF-Bcomplex.Cell95:749–758. Jain,J.,McCaffrey,P.G.,Valge-Archer,V.E.,andRao,A.1992.NuclearfactorofactivatedTcellscontainsFosandJun.Nature356:801–804. Jain,J.,McCaffrey,P.G.,Miner,Z.,Kerppola,T.K.,Lambert,J.N.,Verdine,G.L.,Curran,T.,andRao,A.1993a.TheT-celltranscriptionfactorNFATpisasubstrateforcalcineurinandinteractswithFosandJun.Nature365:352–355. Jain,J.N.,Miner,Z.,andRao,A.1993b.Analysisofthepreex-istingandnuclearformsofnuclearfactorofactivatedT-cells.J.Immunol.151:837–848. Jauliac,S.,López-Rodríguez,C.,Shaw,L.M.,Brown,L.F.,Rao,A.,andToker,A.2002.TheroleofNFATtranscriptionfac-torsinintegrin-mediatedcarcinomainvasion.Nat.CellBiol.4:0–4. Jin,L.,Sliz,P.,Chen,L.,Macián,F.,Rao,A.,Hogan,P.G.,andHarrison,S.C.2003.AnasymmetricNFAT1dimeronapseudo-palindromicB-likeDNAsite.Nat.Struct.Biol.(inpress). Jochum,W.,Passegue,E.,andWagner,E.F.2001.AP-1inmousedevelopmentandtumorigenesis.Oncogene20:2401–2412. Johnson,E.N.,Lee,Y.M.,Sander,T.L.,Rabkin,E.,Schoen,F.J.,Kaushal,S.,andBischoff,J.2003.NFATc1mediatesvascularendothelialgrowthfactor-inducedproliferationofhuman Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press pulmonaryvalveendothelialcells.J.Biol.Chem.278:1686–1692. Kaminuma,O.,Deckert,M.,Elly,C.,Liu,Y.C.,andAltman,A.2001.Vav-Rac1-mediatedactivationofthec-JunN-terminalkinase/c-Jun/AP-1pathwayplaysamajorroleinstimulationofthedistalNFATsiteintheinterleukin-2genepromoter.Mol.Cell.Biol.21:3126–3136. Kehlenbach,R.H.,Dickmanns,A.,andGerace,L.1998.Nucleo-cytoplasmicshuttlingfactorsincludingRanandCRM1medi-atenuclearexportofNFATinvitro.J.CellBiol.141:863–874.Kel,A.,Kel-Margoulis,O.,Babenko,V.,andWingender,E.1999.RecognitionofNFATp/AP-1compositeelementswithingenesinducedupontheactivationofimmunecells.J.Mol.Biol.288:353–376. Kiani,A.,Rao,A.,andAramburu,J.2000.Manipulatingim-muneresponseswithimmunosuppressiveagentsthattargetNFAT.Immunity12:359–372. Kim,R.Y.,Robertson,E.J.,andSolloway,M.J.2001.Bmp6andBmp7arerequiredforcushionformationandseptationinthedevelopingmouseheart.Dev.Biol.235:449–466. Kingsbury,T.J.andCunningham,K.W.2000.Aconservedfamilyofcalcineurinregulators.Genes&Dev.14:1595–1604. Kirschbaum,B.J.,Schneider,S.,Izumo,S.,Mahdavi,V.,Nadal-Ginard,B.,andPette,D.1990.Rapidandreversiblechangesinmyosinheavychainexpressioninresponsetoincreasedneuromuscularactivityofratfast-twitchmuscle.FEBSLett.268:75–78. Klemm,J.D.,Beals,C.R.,andCrabtree,G.R.1997.Rapidtar-getingofnuclearproteinstothecytoplasm.Curr.Biol.7:638–4. Knotts,S.,Sanchez,A.,Rindt,H.,andRobbins,J.1996.Devel-opmentalmodulationofamyosinheavychainpromoter-driventransgene.Dev.Dyn.206:182–192. Kubis,H.P.,Haller,E.A.,Wetzel,P.,andGros,G.1997.Adult fastmyosinpatternandCa2+-inducedslowmyosinpatterninprimaryskeletalmuscleculture.Proc.Natl.Acad.Sci.94:4205–4210. Kubis,H.P.,Scheibe,R.J.,Meißner,J.D.,Hornung,G.,andGros,G.2002.Fast-to-slowtransformationandnuclearimport/exportkineticsofthetranscriptionfactorNFATc1duringelectrostimulationofrabbitmusclecellsinculture.J.Physiol.1:835–847. Kubis,H.P.,Hanke,N.,Scheibe,R.J.,Meißner,J.D.,andGros, G.2003.Ca2+transientsactivatecalcineurin/NFATc1andinitiatefast-to-slowtransformationinaprimaryskeletalmuscleculture.Am.J.Physiol.CellPhysiol.285:C56–C63. Kumai,M.,Nishii,K.,Nakamura,K.,Takeda,N.,Suzuki,M.,andShibata,Y.2000.Lossofconnexin45causesacushiondefectinearlycardiogenesis.Development127:3501–3512. Kurihara,Y.,Kurihara,H.,Oda,H.,Maemura,K.,Nagai,R.,Ishikawa,T.,andYazaki,Y.1995.Aorticarchmalforma-tionsandventricularseptaldefectinmicedeficientinen-dothelin-1.J.Clin.Invest.96:293–300. Lacey,D.L.,Timms,E.,Tan,H.L.,Kelley,M.J.,Dunstan,C.R.,Burgess,T.,Elliott,R.,Colombero,A.,Elliott,G.,Scully,S.,etal.1998.Osteoprotegerinligandisacytokinethatregu-latesosteoclastdifferentiationandactivation.Cell93:165–176. Lakshmanan,G.,Lieuw,K.H.,Lim,K.C.,Gu,Y.,Grosveld,F.,Engel,J.D.,andKaris,A.1999.Localizationofdistanturo-genitalsystem-,centralnervoussystem-,andendocardium-specifictranscriptionalregulatoryelementsintheGATA-3locus.Mol.Cell.Biol.19:1558–1568.TranscriptionalregulationbyNFAT Lee,H.J.,Masuda,E.S.,Arai,N.,Arai,K.,andYokota,T.1995.Definitionofcis-regulatoryelementsofthemouseinterleu-kin-5genepromoter.Involvementofnuclearfactorofacti-vatedTcell-relatedfactorsininterleukin-5expression.J.Biol.Chem.270:171–17550. Lefstin,J.A.andYamamoto,K.R.1998.AllostericeffectsofDNAontranscriptionalregulators.Nature392:885–888.Li,W.,Llopis,J.,Whitney,M.,Zlokarnik,G.,andTsien,R.Y.1998.Cell-permeantcagedInsP3estershowsthatCa2+spikefrequencycanoptimizegeneexpression.Nature392:936–941. Liu,J.,Masuda,E.S.,Tsuruta,L.,Arai,N.,andArai,K.1999.Twoindependentcalcineurin-bindingregionsintheN-ter-minaldomainofmurineNF-ATx1recruitcalcineurintomurineNF-ATx1.J.Immunol.162:4755–4761. Liu,J.,Arai,K.,andArai,N.2001.InhibitionofNFATxactiva-tionbyanoligopeptide:DisruptingtheinteractionofNFATxwithcalcineurin.J.Immunol.167:2677–2687. Liu,W.,Youn,H.D.,Zhou,X.Z.,Lu,K.P.,andLiu,J.O.2001.BindingandregulationofthetranscriptionfactorNFATbythepeptidylprolylcis–transisomerasePin1.FEBSLett.496:105–108. Liu,Y.andSchneider,M.F.1998.Fibretype-specificgeneex-pressionactivatedbychronicelectricalstimulationofadultmouseskeletalmusclefibresinculture.J.Physiol.512:337–344. Liu,Y.,Cseresnyes,Z.,Randall,W.R.,andSchneider,M.F.2001.Activity-dependentnucleartranslocationandintra-nucleardistributionofNFATcinadultskeletalmusclefi-bers.J.CellBiol.155:27–39. Loh,C.,Carew,J.A.,Kim,J.,Hogan,P.G.,andRao,A.1996a.T-cellreceptorstimulationelicitsanearlyphaseofactiva-tionandalaterphaseofdeactivationofthetranscriptionfactorNFAT1.Mol.Cell.Biol.16:3945–39. Loh,C.,Shaw,K.T.,Carew,J.,Viola,J.P.,Luo,C.,Perrino,B.A.,andRao,A.1996b.Calcineurinbindsthetranscriptionfac-torNFAT1andreversiblyregulatesitsactivity.J.Biol.Chem.271:10884–101. Loots,G.G.,Ovcharenko,I.,Pachter,L.,Dubchak,I.,andRubin,E.M.2002.rVistaforcomparativesequence-baseddiscoveryoffunctionaltranscriptionfactorbindingsites.GenomeRes.12:832–839. López-Rodríguez,C.,Aramburu,J.,Rakeman,A.S.,andRao,A.1999.NFAT5,aconstitutivelynuclearNFATproteinthatdoesnotcooperatewithFosandJun.Proc.Natl.Acad.Sci.96:7214–7219. López-Rodríguez,C.,Aramburu,J.,Jin,L.,Rakeman,A.S.,Michino,M.,andRao,A.2001.BridgingtheNFATandNF-Bfamilies:NFAT5dimerizationregulatescytokinegenetranscriptioninresponsetoosmoticstress.Immunity15:47–58. Macián,F.,López-Rodríguez,C.,andRao,A.2001.Partnersintranscription:NFATandAP-1.Oncogene20:2476–24.Macián,F.,Garciá-Cózar,F.,Im,S.H.,Horton,H.F.,Byrne,M.C.,andRao,A.2002.Transcriptionalmechanismsunder-lyinglymphocytetolerance.Cell109:719–731. Masuda,E.S.,Naito,Y.,Tokumitsu,H.,Campbell,D.,Saito,F.,Hannum,C.,Arai,K.I.,andArai,N.1995.Nfatx,anovelmemberofthenuclearfactorofactivatedTcellsfamilythatisexpressedpredominantlyinthethymus.Mol.Cell.Biol.15:2697–2706. Mayor,C.,Brudno,M.,Schwartz,J.R.,Poliakov,A.,Rubin,E.M.,Frazer,K.A.,Pachter,L.S.,andDubchak,I.2000.VISTA:VisualizingglobalDNAsequencealignmentsofar-bitrarylength.Bioinformatics16:1046–1047. McCaffrey,P.G.,Luo,C.,Kerppola,T.K.,Jain,J.,Badalian,T.M., GENES&DEVELOPMENT2229 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. Ho,A.M.,Burgeon,E.,Lane,W.S.,Lambert,J.N.,Curran,T.,etal.1993.Isolationofthecyclosporin-sensitiveTcelltran-scriptionfactorNFATp.Science262:750–7. McCaffrey,P.G.,Goldfeld,A.E.,andRao,A.1994.TheroleofNFATpincyclosporinA-sensitivetumornecrosisfactor-␣genetranscription.J.Biol.Chem.269:30445–30450. McFadden,D.G.andOlson,E.N.2002.Heartdevelopment:Learningfrommistakes.Curr.Opin.Genet.Dev.12:328–335. McKinsey,T.A.,Zhang,C.L.,andOlson,E.N.2002.MEF2:Acalcium-dependentregulatorofcelldivision,differentiationanddeath.TrendsBiochem.Sci.27:40–47. Mechta-Grigoriou,F.,Gerald,D.,andYaniv,M.2001.ThemammalianJunproteins:Redundancyandspecificity.On-cogene20:2378–23. Meißner,J.D.,Kubis,H.P.,Scheibe,R.J.,andGros,G.2000.ReversibleCa2+-inducedfast-to-slowtransitioninprimaryskeletalmuscleculturecellsatthemRNAlevel.J.Physiol.523:19–28. Meißner,J.D.,Gros,G.,Scheibe,R.J.,Scholz,M.,andKubis, H.P.2001.Calcineurinregulatesslowmyosin,butnotfastmyosinormetabolicenzymes,duringfast-to-slowtransfor-mationinrabbitskeletalmusclecellculture.J.Physiol.533:215–226. Meiyanto,E.,Hoshijima,M.,Ogawa,T.,Ishida,N.,andTakeya, T.2001.OsteoclastdifferentiationfactormodulatescellcyclemachineryandcausesadelayinSphaseprogressioninRAW2cells.Biochem.Biophys.Res.Commun.282:278–283. Miskin,J.E.,Abrams,C.C.,andDixon,L.K.2000.AfricanswinefevervirusproteinA238Linteractswiththecellularphos-phatasecalcineurinviaabindingdomainsimilartothatofNFAT.J.Virology74:9412–9420. Miyakawa,H.,Woo,S.K.,Dahl,S.C.,Handler,J.S.,andKwon,H.M.1999.Tonicity-responsiveenhancerbindingprotein,arel-likeproteinthatstimulatestranscriptioninresponsetohypertonicity.Proc.Natl.Acad.Sci.96:2538–22. Molkentin,J.D.,Lu,J.R.,Antos,C.L.,Markham,B.,Richardson,J.,Robbins,J.,Grant,S.R.,andOlson,E.N.1998.Acalcineu-rin-dependenttranscriptionalpathwayforcardiachypertro-phy.Cell93:215–228. Monticelli,S.andRao,A.2002.NFAT1andNFAT2arepositive regulatorsofIL-4genetranscription.Eur.J.Immunol.32:2971–2978. Morimoto,T.,Hasegawa,K.,Wada,H.,Kakita,T.,Kaburagi,S., Yanazume,T.,andSasayama,S.2001.Calcineurin–GATA4pathwayisinvolvedin-adrenergicagonist-responsiveen-dothelin-1transcriptionincardiacmyocytes.J.Biol.Chem.276:34983–349. Morrisey,E.E.,Ip,H.S.,Tang,Z.,Lu,M.M.,andParmacek,M.S.1997.GATA-5:Atranscriptionalactivatorexpressedinanoveltemporallyandspatially-restrictedpatternduringem-bryonicdevelopment.Dev.Biol.183:21–36. Muller,C.W.,Rey,F.A.,Sodeoka,M.,Verdine,G.L.,andHarri-son,S.C.1995.StructureoftheNF-Bp50homodimerboundtoDNA.Nature373:311–317. Murgia,M.,Serrano,A.L.,Calabria,E.,Pallafacchina,G.,Lømo, T.,andSchiaffino,S.2000.Rasisinvolvedinnerve-activity-dependentregulationofmusclegenes.Nat.CellBiol.2:142–147. Musaro,A.,McCullagh,K.J.,Naya,F.J.,Olson,E.N.,andRosen-thal,N.1999.IGF-1inducesskeletalmyocytehypertrophythroughcalcineurininassociationwithGATA-2andNF-ATc1.Nature400:581–585. Nash,P.,Tang,X.,Orlicky,S.,Chen,Q.,Gertler,F.B.,Menden-hall,M.D.,Sicheri,F.,Pawson,T.,andTyers,M.2001.Mul-2230GENES&DEVELOPMENT tisitephosphorylationofaCDKinhibitorsetsathresholdfortheonsetofDNAreplication.Nature414:514–521. Neal,J.W.andClipstone,N.A.2001.Glycogensynthaseki-nase-3inhibitstheDNAbindingactivityofNFATc.J.Biol.Chem.276:3666–3673. Nemer,G.andNemer,M.2002.Cooperativeinteractionbe-tweenGATA5andNF-ATcregulatesendothelial–endocar-dialdifferentiationofcardiogeniccells.Development129:4045–4055. Northrop,J.P.,Ho,S.N.,Chen,L.,Thomas,D.J.,Timmerman,L.A.,Nolan,G.P.,Admon,A.,andCrabtree,G.R.1994.NF-ATcomponentsdefineafamilyoftranscriptionfactorstar-getedinT-cellactivation.Nature369:497–502. Okamura,H.,Aramburu,J.,García-Rodríguez,C.,Viola,J.P.,Raghavan,A.,Tahiliani,M.,Zhang,X.,Qin,J.,Hogan,P.G.,andRao,A.2000.Concerteddephosphorylationofthetran-scriptionfactorNFAT1inducesaconformationalswitchthatregulatestranscriptionalactivity.Mol.Cell6:539–550. Olson,E.N.andWilliams,R.S.2000.Remodelingmuscleswithcalcineurin.Bioessays22:510–519;erratum22:1049. Orcel,P.,Denne,M.A.,anddeVernejoul,M.C.1991.Cyclospo-rin-Ainvitrodecreasesboneresorption,osteoclastforma-tion,andthefusionofcellsofthemonocyte-macrophagelineage.Endocrinology128:1638–16. Orlicky,S.,Tang,X.,Willems,A.,Tyers,M.,andSicheri,F.2003.Structuralbasisforphosphodependentsubstrateselec-tionandorientationbytheSCFCdc4ubiquitinligase.Cell112:243–256. Osborne,C.S.,Vadas,M.A.,andCockerill,P.N.1995.Tran-scriptionalregulationofmousegranulocyte-macrophagecolony-stimulatingfactor/IL-3locus.J.Immunol.155:226–235. Pallafacchina,G.,Calabria,E.,Serrano,A.L.,Kalhovde,J.M.,andSchiaffino,S.2002.AproteinkinaseB-dependentandrapamycin-sensitivepathwaycontrolsskeletalmusclegrowthbutnotfibertypespecification.Proc.Natl.Acad.Sci.99:9213–9218. Park,S.,Uesugi,M.,andVerdine,G.L.2000.Asecondcalcineu-rinbindingsiteontheNFATregulatorydomain.Proc.Natl.Acad.Sci.97:7130–7135. Parry,R.V.,Reif,K.,Smith,G.,Sansom,D.M.,Hemmings,B.A.,andWard,S.G.1997.LigationoftheTcellco-stimulatoryreceptorCD28activatestheserine–threonineproteinkinaseproteinkinaseB.Eur.J.Immunol.27:2495–2501. Peng,S.L.,Gerth,A.J.,Ranger,A.M.,andGlimcher,L.H.2001.NFATc1andNFATc2togethercontrolbothTandBcellactivationanddifferentiation.Immunity14:13–20. Peterson,B.R.,Sun,L.J.,andVerdine,G.L.1996.Acriticalar-ginineresiduemediatescooperativityinthecontactinter-facebetweentranscriptionfactorsNFATandAP-1.Proc.Natl.Acad.Sci.93:13671–13676. Pette,D.andStaron,R.S.2000.Myosinisoforms,musclefibertypes,andtransitions.MicroscopyRes.Tech.50:500–509.Peuker,H.andPette,D.1995.Reversetranscriptase-polymer-asechainreactiondetectsinductionofcardiac-like␣myosinheavychainmRNAinlowfrequencystimulatedrabbitfast-twitchmuscle.FEBSLett.367:132–136. Peuker,H.,Conjard,A.,Putman,C.T.,andPette,D.1999.Tran-sientexpressionofmyosinheavychainMHCI␣inrabbitmuscleduringfast-to-slowtransition.J.MuscleRes.CellMotility20:147–1. Porter,C.M.andClipstone,N.A.2002.SustainedNFATsignal-ingpromotesaTh1-likepatternofgeneexpressioninpri-marymurineCD4+Tcells.J.Immunol.168:4936–4945.Porter,C.M.,Havens,M.A.,andClipstone,N.A.2000.Identi- Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press ficationofaminoacidresiduesandproteinkinasesinvolvedintheregulationofNFATcsubcellularlocalization.J.Biol.Chem.275:33–3551. Rainio,E.M.,Sandholm,J.,andKoskinen,P.J.2002.Cuttingedge:TranscriptionalactivityofNFATc1isenhancedbythePim-1kinase.J.Immunol.168:1524–1527. Ranger,A.M.,Grusby,M.J.,Hodge,M.R.,Gravallese,E.M.,dela Brousse,F.C.,Hoey,T.,Mickanin,C.,Baldwin,H.S.,andGlimcher,L.H.1998a.ThetranscriptionfactorNF-ATcisessentialforcardiacvalveformation.Nature392:186–190. Ranger,A.M.,Oukka,M.,Rengarajan,J.,andGlimcherLH. 1998b.InhibitoryfunctionoftwoNFATfamilymembersinlymphoidhomeostasisandTh2development.Immunity9:627–635. Rao,A.,Luo,C.,andHogan,P.G.1997.Transcriptionfactorsof theNFATfamily:Regulationandfunction.Annu.Rev.Im-munol.15:707–747. Rengarajan,J.,Mowen,K.A.,McBride,K.D.,Smith,E.D.,Singh, H.,andGlimcher,L.H.2002.Interferonregulatoryfactor4(IRF4)interactswithNFATc2tomodulateinterleukin4geneexpression.J.Exp.Med.195:1003–1012. Rindt,H.,Gulick,J.,Knotts,S.,Neumann,J.,andRobbins,J.1993.Invivoanalysisofthemurine-myosinheavychaingenepromoter.J.Biol.Chem.268:5332–5338. Rooney,J.W.,Hoey,T.,andGlimcher,L.H.1995.CoordinateandcooperativerolesforNF-ATandAP-1intheregulationofthemurineIL-4gene.Immunity2:473–483. Rothermel,B.,Vega,R.B.,Yang,J.,Wu,H.,Bassel-Duby,R.,andWilliams,R.S.2000.AproteinencodedwithintheDownsyndromecriticalregionisenrichedinstriatedmusclesandinhibitscalcineurinsignaling.J.Biol.Chem.275:8719–8725. Rothwarf,D.M.andKarin,M.1999.TheNF-Bactivationpath-way:Aparadigmininformationtransferfrommembranetonucleus.Science’sStke:SignalTransductionKnowledgeEn-vironment1999:RE1. Salazar,C.andHöfer,T.2003.Allostericregulationofthetran-scriptionfactorNFAT1bymultiplephosphorylationsites:Amathematicalanalysis.J.Mol.Biol.327:31–45. Schiaffino,S.andSerrano,A.2002.Calcineurinsignalingandneuralcontrolofskeletalmusclefibertypeandsize.TrendsPharmacol.Sci.23:569–575. Schwartz,S.,Zhang,Z.,Frazer,K.A.,Smit,A.,Riemer,C.,Bouck,J.,Gibbs,R.,Hardison,R.,andMiller,W.2000.Pip-Maker—AWebserverforaligningtwogenomicDNAse-quences.GenomeRes.10:577–586. Scully,K.M.,Jacobson,E.M.,Jepsen,K.,Lunyak,V.,Viadiu,H.,Carrière,C.,Rose,D.W.,Hooshmand,F.,Aggarwal,A.K.,andRosenfeld,M.G.2000.AllostericeffectsofPit-1DNAsitesonlong-termrepressionincelltypespecification.Sci-ence290:1127–1131. Serfling,E.,Berberich-Siebelt,F.,Chuvpilo,S.,Jankevics,E.,Klein-Hessling,S.,Twardzik,T.,andAvots,A.2000.TheroleofNF-ATtranscriptionfactorsinTcellactivationanddifferentiation.Biochim.Biophys.Acta.1498:1–18. Serrano,A.L.,Murgia,M.,Pallafacchina,G.,Calabria,E.,Coni-glio,P.,Lømo,T.,andSchiaffino,S.2001.Calcineurincon-trolsnerveactivity-dependentspecificationofslowskeletalmusclefibersbutnotmusclegrowth.Proc.Natl.Acad.Sci.98:13108–13113. Shaw,K.T.,Ho,A.M.,Raghavan,A.,Kim,J.,Jain,J.,Park,J.,Sharma,S.,Rao,A.,andHogan,P.G.1995.Immunosuppres-sivedrugspreventarapiddephosphorylationoftranscriptionfactorNFAT1instimulatedimmunecells.Proc.Natl.Acad.Sci.92:11205–11209.TranscriptionalregulationbyNFAT Sheridan,C.M.,Heist,E.K.,Beals,C.R.,Crabtree,G.R.,andGardner,P.2002.ProteinkinaseAnegativelymodulatesthenuclearaccumulationofNF-ATc1byprimingforsubse-quentphosphorylationbyglycogensynthasekinase-3.J.Biol.Chem.277:486–48676. Shibasaki,F.,Price,E.R.,Milan,D.,andMcKeon,F.1996.RoleofkinasesandthephosphatasecalcineurininthenuclearshuttlingoftranscriptionfactorNF-AT4.Nature382:370–373. Shui,C.,Riggs,B.L.,andKhosla,S.2002.Theimmunosuppres-santrapamycin,aloneorwithtransforminggrowthfactor-,enhancesosteoclastdifferentiationofRAW2.7monocyte-macrophagecellsinthepresenceofRANK-ligand.CalcifiedTissueIntl.71:437–446. Siebenlist,U.,Durand,D.B.,Bressler,P.,Holbrook,N.J.,Norris,C.A.,Kamoun,M.,Kant,J.A.,andCrabtree,G.R.1986.Pro-moterregionofinterleukin-2geneundergoeschromatinstructurechangesandconfersinducibilityonchlorampheni-colacetyltransferasegeneduringactivationofTcells.Mol.Cell.Biol.6:3042–3049. Solymar,D.C.,Agarwal,S.,Bassing,C.H.,Alt,F.W.,andRao,A.2002.A3ЈenhancerintheIL-4generegulatescytokinepro-ductionbyTh2cellsandmastcells.Immunity17:41–50.Sosinowski,T.,Pandey,A.,Dixit,V.M.,andWeiss,A.2000.Src-likeadaptorprotein(SLAP)isanegativeregulatorofTcellreceptorsignaling.J.Exp.Med.191:463–474. Sreter,F.A.,Lopez,J.R.,Alamo,L.,Mabuchi,K.,andGergely,J.1987.ChangesinintracellularionizedCaconcentrationas-sociatedwithmusclefibertypetransformation.Am.J.Physiol.253:C296–C300. Stevenson,A.S.,Gomez,M.F.,Hill-Eubanks,D.C.,andNelson,M.T.2001.NFAT4movementinnativesmoothmuscle.ArolefordifferentialCa2+signaling.J.Biol.Chem.276:15018–15024. Stroud,J.C.,López-Rodríguez,C.,Rao,A.,andChen,L.2002.StructureofaTonEBP–DNAcomplexrevealsDNAen-circledbyatranscriptionfactor.Nat.Struct.Biol.9:90–94. Sucov,H.M.1998.Molecularinsightsintocardiacdevelop-ment.Ann.Rev.Physiol.60:287–308. Swoap,S.J.,Hunter,R.B.,Stevenson,E.J.,Felton,H.M.,Kan-sagra,N.V.,Lang,J.M.,Esser,K.A.,andKandarian,S.C.2000.Thecalcineurin–NFATpathwayandmusclefiber-typegeneexpression.Am.J.Physiol.CellPhysiol.279:C915–C924. Takayanagi,H.,Kim,S.,Koga,T.,Nishina,H.,Isshiki,M.,Yoshida,H.,Saiura,A.,Isobe,M.,Yokochi,T.,Inoue,J.,etal.2002.InductionandactivationofthetranscriptionfactorNFATc1(NFAT2)integrateRANKLsignalinginterminaldifferentiationofosteoclasts.Dev.Cell3:8–901. Teitelbaum,S.L.2000.Boneresorptionbyosteoclasts.Science2:1504–1508. Timmerman,L.A.,Clipstone,N.A.,Ho,S.N.,Northrop,J.P.,andCrabtree,G.R.1996.RapidshuttlingofNF-ATindis-criminationofCa2+signalsandimmunosuppression.Nature383:837–840. Torgerson,T.R.,Colosia,A.D.,Donahue,J.P.,Lin,Y.Z.,andHawiger,J.1998.RegulationofNF-B,AP-1,NFAT,andSTAT1nuclearimportinTlymphocytesbynoninvasivedeliveryofpeptidecarryingthenuclearlocalizationse-quenceofNF-Bp50.J.Immunol.161:6084–6092. Tsytsykova,A.V.andGoldfeld,A.E.2000.NuclearfactorofactivatedTcellstranscriptionfactorNFATpcontrolssuperantigen-inducedlethalshock.J.Exp.Med.192:581–586. Wada,H.,Hasegawa,K.,Morimoto,T.,Kakita,T.,Yanazume, GENES&DEVELOPMENT2231 Downloaded from genesdev.cshlp.org on November 14, 2013 - Published by Cold Spring Harbor Laboratory Press Hoganetal. T.,Abe,M.,andSasayama,S.2002.Calcineurin–GATA-6pathwayisinvolvedinsmoothmuscle-specifictranscrip-tion.J.CellBiol.156:983–991. Wang,D.Z.,McCaffrey,P.G.,andRao,A.1995.Thecyclospo-rin-sensitivetranscriptionfactorNFATpisexpressedinsev-eralclassesofcellsintheimmunesystem.Ann.NYAcad.Sci.766:182–194. Wen,H.Y.,Xia,Y.,Young,M.E.,Taegtmeyer,H.,andKellems,R.E.2002.Theadenylosuccinatesynthetase-1geneisacti-vatedinthehypertrophiedheart.J.Cell.Mol.Med.6:235–243. Windisch,A.,Gundersen,K.,Szabolcs,M.J.,Gruber,H.,and Lømo,T.1998.Fasttoslowtransformationofdenervatedandelectricallystimulatedratmuscle.J.Physiol.510:623–632. Wu,H.,Naya,F.J.,McKinsey,T.A.,Mercer,B.,Shelton,J.M.,Chin,E.R.,Simard,A.R.,Michel,R.N.,Bassel-Duby,R.,Ol-son,E.N.,etal.2000.MEF2respondstomultiplecalcium-regulatedsignalsinthecontrolofskeletalmusclefibertype.EMBOJ.19:1963–1973. Wu,H.,Rothermel,B.,Kanatous,S.,Rosenberg,P.,Naya,F.J., Shelton,J.M.,Hutcheson,K.A.,DiMaio,J.M.,Olson,E.N.,Bassel-Duby,R.,etal.2001.ActivationofMEF2bymuscleactivityismediatedthroughacalcineurin-dependentpath-way.EMBOJ.20:14–23. Xia,Y.,McMillin,J.B.,Lewis,A.,Moore,M.,Zhu,W.G.,Willi-ams,R.S.,andKellems,R.E.2000.ElectricalstimulationofneonatalcardiacmyocytesactivatestheNFAT3andGATA4pathwaysandup-regulatestheadenylosuccinatesynthetase1gene.J.Biol.Chem.275:1855–1863. Yang,J.,Rothermel,B.,Vega,R.B.,Frey,N.,McKinsey,T.A.,Olson,E.N.,Bassel-Duby,R.,andWilliams,R.S.2000.Inde-pendentsignalscontrolexpressionofthecalcineurininhibi-toryproteinsMCIP1andMCIP2instriatedmuscles.Circ.Res.87:E61–E68. Yang,T.T.,Xiong,Q.,Enslen,H.,Davis,R.J.,andChow,C.W. 2002.PhosphorylationofNFATc4byp38mitogen-activatedproteinkinases.Mol.Cell.Biol.22:32–3904. Yang,X.Y.,Wang,L.H.,Chen,T.,Hodge,D.R.,Resau,J.H., DaSilva,L.,andFarrar,W.L.2000.ActivationofhumanTlymphocytesisinhibitedbyperoxisomeproliferator-acti-vatedreceptor␥(PPAR␥)agonists.PPAR␥co-associationwithtranscriptionfactorNFAT.J.Biol.Chem.275:41–44. Yasuda,H.,Shima,N.,Nakagawa,N.,Yamaguchi,K.,Kinosaki, M.,Mochizuki,S.,Tomoyasu,A.,Yano,K.,Goto,M.,Mu-rakami,A.,etal.1998.Osteoclastdifferentiationfactorisaligandforosteoprotegerin/osteoclastogenesis-inhibitoryfac-torandisidenticaltoTRANCE/RANKL.Proc.Natl.Acad.Sci.95:3597–3602. Yui,M.A.,Hernandez-Hoyos,G.,andRothenberg,E.V.2001.A newregulatoryregionoftheIL-2locusthatconfersposition-independenttransgeneexpression.J.Immunol.166:1730–1739. Zaccolo,M.,Magalhaes,P.,andPozzan,T.2002.Compartmen-talisationofcAMPandCa2+signals.Curr.Opin.CellBiol.14:160–166. Zhou,B.,Cron,R.Q.,Wu,B.,Genin,A.,Wang,Z.,Liu,S.,Rob-son,P.,andBaldwin,H.S.2002.RegulationofthemurineNfatc1genebyNFATc2.J.Biol.Chem.277:10704–10711.Zhu,J.,Shibasaki,F.,Price,R.,Guillemot,J.C.,Yano,T.,Dötsch,V.,Wagner,G.,Ferrara,P.,andMcKeon,F.1998.IntramolecularmaskingofnuclearimportsignalonNF-AT4bycaseinkinaseIandMEKK1.Cell93:851–861. 2232GENES&DEVELOPMENT 因篇幅问题不能全部显示,请点此查看更多更全内容
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