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2003 review transcriptional regulation by calcium calcineurin and NFAT

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REVIEW

Transcriptionalregulationbycalcium,calcineurin,andNFAT

PatrickG.Hogan,1LinChen,3JulieNardone,1andAnjanaRao1,2,41TheCenterforBloodResearchand2DepartmentofPathology,HarvardMedicalSchool,Boston,Massachusetts02115,USA;3DepartmentofChemistryandBiochemistry,UniversityofColoradoatBoulder,Boulder,Colorado80309-0215,USA

TheNFATfamilyoftranscriptionfactorsencompassesfiveproteinsevolutionarilyrelatedtotheRel/NF␬Bfam-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).AtDNAelementsthatresembleNF␬Bsites,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;NF␬BandAP-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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Hoganetal.

Table1.ProteinNFAT1NFAT2NFAT3NFAT4NFAT5

ListofNFATproteins

OthernamesNFAT␳,NFATc2NFATc,NFATc1NFATc4

NFATx,NFATc3TonEBP

Regulation

Ca2+/calcineurinCa2+/calcineurinCa2+/calcineurinCa2+/calcineurin

Osmoticstress

Integrin(␣6␤4)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,TGF␤inhibitsTeckinaseactivityandtherebyCa2+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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Hoganetal.

ofamultitudeofsignalingproteins(e.g.,seeSosinowskietal.2000;Kaminumaetal.2001).Inothercelltypes,Ca2+mobilizationiscoupledtostimulationthroughreceptortyrosinekinasesandG-protein-coupledre-ceptors,andNFATreporterswouldbeusefulinanun-biasedquestforsignalingmoleculesinthesepathwaysaswell.

DiversemodesofDNAbindingbyNFATStructuralrelationtotheRel/NF␬Bfamily

AllfiveproteinsoftheNFATfamily,NFAT1–NFAT4andNFAT5/TonEBP,maybeclassifiedasmembersoftheextendedNF␬B/Relfamily(Fig.3).TheclassificationisbasedonthestructuralsimilaritiesoftheirDNA-bind-ingdomains,alsoknownasRelhomologyregions(RHR).ThecanonicalmodeofDNAbindingbytheRHRhasbeenwellcharacterizedinthestructuresofseveralNF␬B–DNAcomplexes(Ghoshetal.1995;Mulleretal.1995):TheRHRcontainstwofunctionallydistinctdo-mains,anN-terminalspecificitydomain(RHR-N)thatmakesbase-specificDNAcontacts,andaC-terminaldo-maininvolvedindimerformationandI␬Bbinding(RHR-C;Fig.4;Huxfordetal.1998;JacobsandHarrison1998).OfthefiveNFATproteins,NFAT5/TonEBPshowsthehighestdegreeofstructuralsimilaritytoNF␬B,formingasymmetricdimerwithastrikingresemblancetotheNF␬B–DNAcomplex(Fig.4,cf.AandB;Stroudetal.2002).ThedetaileddimerizationinteractionsmediatedbytheRHR-CofNFAT5areverysimilartothoseob-servedinNF␬B,buttheuniquefeatureoftheNFAT5dimer–DNAcomplexisaseconddimerinterfaceformedbytheEЈFloopoftheRHR-Ndomain(Fig.4B).Asaresult,NFAT5completelyencirclesitsDNA(Fig.4B),explainingtheunusuallyslowdissociationkineticsoftheNFAT5–DNAcomplex(Stroudetal.2002).

TwoNFAT1dimercomplexes,boundto␬BsitesfromtheIL-8promoterandtheHIV-1LTR,haverecentlybeencharacterizedatthestructurallevel(Giffinetal.2003;Jinetal.2003).AsintheNFAT5andReldimercomplexes,theNFAT1dimerinterfaceresidesintheC-terminalregionoftheRHR(Fig.4C,D).However,thisinterfaceissurprisinglydifferentfromthesymmetricandhydrophobicRHR-CdimerinterfaceseeninNFAT5andNF␬B:Itisasymmetricandlargelyhydrophilic,andinvolvesresiduesnotusedinRelorNFAT5.Theinter-faceisessentiallyidenticalintheNFAT1dimersboundtotheIL-8andHIV-1LTR␬Bsites,butthesetwoNFAT1dimercomplexesdiffersignificantlyintheirRHR-Ninteractionsandhencetheiroverallconforma-tion(Fig.4,cf.CandD).Specifically,theNFAT1dimerontheHIV-1LTR␬BsitecompletelyencirclestheDNAthroughEЈF-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.AlignmentofNF␬B/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)NF␬Bp50homodimerboundtoa␬Bsite.(B)NFAT5/TonEBPboundtoatonicityresponseelement.(C)TheNFAT1dimerboundtothe␬BsitefromtheHIV-1LTR(identicalinsequencetothemurineIg␬Bsite).(D)TheNFAT1dimerboundtoa␬BsitesimilartothatinthehumanIL-8promoter.InBandC,theEЈFloopofeachproteinpartnerextendstowardthemidlinebelowtheDNAhelixandformsaseconddimerinterface.TheDNAsequenceofeachcomplexisshownbelowthestructure.

correspondingtothoseintheNFAT1dimerinterface.ModelingstudiessuggestthatNFAT1,NFAT2,andNFAT4wouldbecapableofformingbothhomo-andheterodimerson␬B-likeDNAsites.TheseinteractionsarelikelytobeconfinedtotheNFATfamily,however:AlthoughNFAT1–4,NFAT5/TonEBP,andNF␬B/ReldimerscanallbindsimilarDNAelements,theirmodesofdimerizationaredifferent(Fig.4)andtheyrespondtodistinctintracellularsignals(López-Rodríguezetal.2001),suggestingthatmixedNFAT/NFAT5/NF␬Bdimersdonotnormallyoccur.

The␬3siteoftheTNF␣promoter(TGGAGAAACCC)isagoodexampleofaphysiological␬B-likesitetowhichNFATbindsasadimer(Goldfeldetal.1993;McCaffreyetal.1994).Itresemblesthe10-bp␬Bsiteusedforstruc-turedeterminationbyGiffinetal.(2003).Bindingisnucleatedatthehalf-siteshowninbold,buttheotherhalf-site(underlined)isquicklyoccupiedatlowNFATconcentrations(McCaffreyetal.1994).MutationofNFATcontactresiduesimpairsTNF␣promoteractivityinreporterassaysinTandBcells(Goldfeldetal.1993).SitesthatcanbindbothNFATandNF␬Bhavebeeniden-tifiedintheHIV-1LTRandintheIL-8,IL-13,andGM-CSFpromoters(forreview,seeRaoetal.1997;Maciánetal.2001);whichfactorsactuallyoccupythesiteinvivodependsonthecelltypebeingexaminedandthespecific

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Hoganetal.

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

IL5Ph

GGM-CSFM330h

GGM-CSFM420h

ehGM-CSFGM550

ehIL4(−88to−61)

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.),asdimersat␬B-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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TranscriptionalregulationbyNFAT

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−180GATA>3>N:A>−143

−114>N:A>28N>1>GATA>2>GATA>24>N>

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

−280Sitesnotidentified

NFAT2NFAT2

GATA5GATA2

SitesnotidentifiedNFAT2GATA6

N:AorA:N,compositeNFAT:AP-1site(underliningindicatesbindingelement);>N:A>,Abbreviations:(Adss-1)adenylosuccinatesynthetase-1;(BNP)b-typenatriureticpeptide;(EDN-1)endothelin-1;(Sm-MyHC)smoothmusclemyosinheavychain;(TCR)T-cellreceptor;(VSMC)vascularsmoothmusclecells;(Y2H)yeasttwo-hybridscreen,(co-ip)coimmunoprecipitation.

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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

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(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-1mayberegulatedthrough␬B-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

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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-FosandNF␬Bhavebeenrecognizedasessentialforosteoclastdifferentiationbasedontheosteopetroticphenotypeofc-Fos-andNF␬B-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-FosandNF␬B.RecentworkindicatesthatsignalingviaNFATisalsoessential.Cellularpolarizationandproductiveattachmenttothebonesubstrate,aprerequisiteforboneresorption,dependon␣v␤3integrinandtheintracellularsignalingproteinsTRAF6andc-Src.BoneresorptionbyfullydifferentiatedosteoclastsiscarriedoutbyeffectorproteinsincludingcathepsinK,carbonicanhydraseII,andaspecificH+ATPase.FigurereprintedwithpermissionfromTeitelbaum(2000).©2000AmericanAssociationfortheAdvancementofScience.

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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),andinclusionofTGF␤increasestheyieldofdifferenti-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-FosandNF␬B,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

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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).

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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

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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.

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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

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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

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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-

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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.

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