The apelinergic system in the developing lung: expression and signaling

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Peptides

jo u rn al h om epa g e:w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

The

apelinergic

system

in

the

developing

lung:

Expression

and

signaling

夽

Paulina

Piairo

a,b

_{, Rute}

_S.

_Moura

a,b

_,

_Cristina

_{Nogueira-Silva}

a,b,c

_,

_Jorge

_{Correia-Pinto}

a,b,d,∗ a_Life_and_Health_Sciences_Research_Institute_(ICVS),_School_of_Health_Sciences,_University_of_Minho,_Braga,_Portugal

b_ICVS/3B’s_–_PT_Government_Associate_Laboratory,_{Braga/Guimarães,}_Portugal c_Department_of_Obstetrics_and_Gynecology,_Hospital_of_Braga,_Portugal d_Department_of_Pediatric_Surgery,_Hospital_of_Braga,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22August2011

Receivedinrevisedform4October2011 Accepted5October2011

Availableonline12October2011 Keywords: Apelin APJ Lungdevelopment MAPkinases

a

b

s

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ApelinanditsreceptorAPJconstituteasignalingpathwaybestrecognizedasanimportantregulatorof cardiovascularhomeostasis.Thismultifunctionalpeptidergicsystemiscurrentlybeingdescribedtobe involvedinembryoniceventswhichextendintovascular,ocularandheartdevelopment.Additionally, itishighlyexpressedinpulmonarytissue.Therefore,theaimofthisstudywastoinvestigatetherole ofapelinergicsystemduringfetallungdevelopment.ImmunohistochemistryandWesternblotanalysis wereusedtocharacterizeapelinandAPJexpressionlevelsandcellularlocalizationinnormalfetalrat lungs,atfivedifferentgestationalagesaswellasintheadult.Fetalratlungexplantswereculturedinvitro withincreasingdosesofapelin.Treatedlungexplantsweremorphometricallyanalyzedandassessedfor MAPKsignalingmodifications.Bothcomponentsoftheapelinergicsystemareconstitutivelyexpressed inthedevelopinglung,withAPJexhibitingmonomeric,dimericandoligomericformsinthepulmonary tissue.Pulmonaryepitheliumalsodisplayedconstitutivenuclearlocalizationofthereceptor.Fetalapelin expressionishigherthanadultexpression.Apelinsupplementationinhibitoryeffectonbranching mor-phogenesiswasassociatedwithadosedependentdecreaseinp38andJNKphosphorylation.Theresults presentedprovidethefirstevidenceofthepresenceofanapelinergicsystemoperatinginthedeveloping lung.Ourfindingsalsosuggestthatapelininhibitsfetallunggrowthbysuppressingp38andJNKsignaling pathways.

1. Introduction

Theexistenceoftheapelinergicsystembegantounfoldin1998 whenAPJ(angiotensinIIreceptor-like1),a formerlyorphan G-proteincoupledreceptor(GPCR),anditsligandwerefinallypaired byTatemotoetal.[45].Apelinwasidentifiedasanendogenous ligandforAPJreceptor.Thispeptideistranslatedasa77-amino acidprecursor,whichundergoesproteolyticmaturation generat-ingshorteractiveapelinpeptides.Apelin-36wasthefirstofthese shorterC-terminalsequencesbeingdescribedtobindandactivate APJ[45].AlsoApelin-17,Apelin-13anditspyroglutamylisoform whichisresistanttodegradation,(Pyr1)-Apelin-13,wereprovento actasfunctionalligandsofAPJandinsomecasesexhibitedmuch higherbiologicalactivitythanApelin-36[45,17,18,25,46].

夽 Grants:ThisprojectwasfundedbyFundaçãoparaaCiênciaeaTecnologia (PTDC/SAU-OBD/108051/2008),PPwassupportedbyFundaçãoparaaCiênciaea Tecnologia(referenceSFRH/BD/33410/2008).RSMwassupportedbyFundaçãopara aCiênciaeaTecnologia(referenceSFRH/BPD/15408/2005).

∗ Correspondingauthorat:EscoladeCiênciasdaSaúde,UniversidadedoMinho, CampusdeGualtar,4710-057Braga,Portugal.Tel.:+351253604910;

fax:+351253604862.

E-mailaddress:[email protected](J.Correia-Pinto).

Apelinreceptorwasoriginallyidentiﬁedasareceptorrelatedto angiotensinIIreceptor1,duetohighhomologybetweenthetwo receptorproteins[38].Thisreceptoris380aminoacidslong, con-sistsofseventransmembranedomainsandalsoincludesasignal sequencethatallowsagonist-independentnuclearlocalization,a featurethatmaybecell-speciﬁc[27].GPCRsaretheestimated tar-getsofnearlyhalfofallcurrentlyavailableclinicallyuseddrugs [11]andarekeycomponentsofthesignaltransductionmachinery [35].BindingofapelintoAPJactivatessecondmessengersignaling cascadesaftercouplingtoGproteins,whichresultsinactivation ofcentralsignalingmoleculessuchasmitogen-activatedprotein kinases(MAPKs)andthePI3K/AKTpathwaythatareresponsibleto instigatemultiplebiologicalresponses[1,28–31].

Theapelinergicsystemhasa widespreadpatternof distribu-tioninhumanandanimaltissuesanditsestablishedphysiological actionsareextensive.Overall,apelinandAPJmRNAtranscriptsand respectivepeptides,areabundantlypresentincentralnervous sys-temandalsoinperipheraltissuessuchasvascularendothelium, heart,lung,kidneyandmammarygland[18,25,40,32,10,21], sug-gestingafunctionalroleofapelin/APJinthesetissues.Remarkably thebulkofthestudiesreportcardiovascularactionsofapelin/APJ. Moreover,thispeptidergicsystemhasbeenproposedtohavearole inbodyﬂuidhomeostasis,immunologicmodulation,diabetesand

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obesity.[39,12,26,22,4].Recentlytheapelinergicsystemhasbeen describedtopromoteembryonicandtumorangiogenesis[19,9,43]. Growingevidencesofapelin/APJinvolvementinembryonicevents currentlyextendbeyondvasculardevelopment,intoocular[20] andheartdevelopment[42,49,14].

Interesting ﬁndings regarding theapelinergic systemclearly revealthatthelungisoneoftheorganswithstrongestexpressionof bothligandandreceptor.Furthermore,emergingevidencesofthis system’simplicationsinembryonicdevelopmentpromptthe spec-ulationthattheremightbeanunderlyingroleinembryoniclung development.However,theexpressionproﬁleofthispairof pro-teinsandtheirfunctionalroleduringnormallungdevelopmentis hithertounknown.Sofar,theeffectsofapelinonlungdevelopment havebeendescribed onlyinone studythatreports attenuation oflung injury in neonatalratsexposed toprolongedhyperoxia [47]. Therefore we proposed toinvestigate the apelin–APJ sys-tem during fetal lung development. In this report a thorough characterizationofbothcomponentsoftheapelinergicsystemby immunohistochemistryandWesternblotinseveralstagesoffetal lungdevelopmentisprovided.Wefurtherinvestigatedtheroleof thissysteminbranchingmorphogenesisofthelungandthe intra-cellulareffectorsimplicated.

2. Materialsandmethods

2.1. Animalmodelandexperimentaldesign

Animal experiments were performed according to the Por-tuguese law for animal welfare. Animals were housed in an accreditedmouse house and treated as specified in the‘Guide fortheCareandUseofLaboratoryAnimalspublishedbytheUS NationalInstitutesofHealth’s(NationalInstitutesofHealth Pub-lication No. 85-23, revised 1996). Sprague–Dawleyfemale rats (225g,Charles-River,Spain)weremaintainedinappropriatecages undercontrolledconditions,fedwithcommercialsolidfoodand after mating they were checked for vaginal plug. The average pregnancy length in normal adult rat is of 21.5 days and the dayofpluggingwasdefinedasgestationalday0.5fortime dat-ingpurposes.Pregnancieswereconfirmedbyregularweightings. At different time-points [13.5, 15.5, 17.5, 19.5, and 21.5 days postconception (dpc)], pregnant female rats were sacrificed by rapiddecapitationandfetuseswereharvestedbycesareansection. Fetuseswerealsosacrificedbydecapitation,fetalandadultlungs wereexcised,processedandcollectedforimmunohistochemistry (IHC)orWesternblotanalysis.Regardinglung explantcultures, fetuseswereharvestedat13.5dpcandtheirlungsweredissected, culturedinvitroforfourdaysandthencollectedforWesternblot analysis.

2.2. Westernblotanalysis

Proteinlysatesof13.5and15.5dpcexcisedlungsandprotein lysates ofpooled lung explants wereobtainedby homogeniza-tionofthefetaltissue witha pelletpestle motor(Kontes,USA) onice;asforlungsoflatergestationalagesandadultsamplesa minibeadbeater(BiospekProductsInc.,USA)wasusedfor homog-enization.Differentpooledlungsamplesforeachgestationalage andalsotheadultwereusedandthreeindependentexperiments wereperformed.ProteinswereobtainedaccordingtoKlingetal. [24].WholeproteinconcentrationwasquantiﬁedbytheBradford method[3].Eithertwenty-ﬁveortenmicrogramsofproteinwere loadedonto12.5%or10%acrylamideminigelsunderdenaturing andreducingconditions,electrophoresedforapproximately2hat

100Vatroomtemperatureandthentransferredontonitrocellulose membranes(HybondTM_-C_Extra_GE_Healthcare_Life_Sciences,_UK)

inawettransfersystemfor1h.Blotswereprobedwith antibod-iestoapelin[1:500;Apelin(FL-77),SantaCruzBiotechnologyInc., USA],apelinreceptor[1:500;APLNR(H-300),SantaCruz Biotech-nologyInc.]andnon-phosphorylatedand phosphorylatedforms of p38, p44/42(ERK1/2)and JNK (1:1000; Cell Signaling Tech-nology Inc., USA) according tothe manufacturer’s instructions. Forloadingcontrol,blotswerereprobedwith_␤-tubulinantibody (1:150000Abcam, UK), which were previously incubated with EzWayTM_Antibody_Erasing_Buffer_(Komabiotech _Inc.,_Korea)_for

primaryandsecondaryantibodyremoval.Afterwardsblotswere incubatedwithasecondaryhorseradishperoxidaseconjugateand developedwithSuperSignalWestFemtoSubstrate(Pierce Biotech-nologyInc.,USA).Thechemiluminescentsignalwascapturedusing a Chemidoc XRS(BioRad, USA) apparatusand subsequent den-sitometricanalysisofnonsaturatingbandswasperformedusing QuantityOnesoftware(BioRad).

2.3. Immunohistochemistry

Immunostaining was performed on paraformaldehyde-fixed andparaffin-embeddedexcisedlungsandembryos.Five microm-eterssectionswereplacedontoglassmicroscopeslides.Primary antibodies for apelin [1:50; Apelin (M-77), Santa Cruz Biote-chology Inc.]and apelin receptor (APJ, Abcam Inc.) were used. Tissuesectionsweredeparaffinizedinxyleneandrehydratedin ethanol,boiledin10mMcitratebufferforantigenretrievaland cooleddownatroomtemperature.Incubationwiththeprimary antibody occurredat 4◦C overnight. Negativecontrol reactions included omission of the primary antibody and immunoreac-tive apelin and APJ staining were not observed in thesecases. Sectionswereincubatedwithalabeledstreptavidin–biotin immu-noenzymaticantigendetectionsystem(UltraVisionLargeVolume Detection System Anti-Polyvalent, Horseradish Peroxidase, Lab VisionCorporation,USA)accordingtomanufacturer’sinstructions. For visualization of the immune reaction, a diaminobenzidine tetrahydrochloridesolution(Dako,Denmark)wasused.Sections were finally counterstained with hematoxylin. The slides were observed and photographed with Olympus BX61 microscope (Olympus,Japan).Thepicturespresentedarerepresentativeofsix animals(N=6),twelvesampleswereexaminedforeachgestational ageaswellastheadultandthreeindependentexperimentswere performed.

2.4. Fetallungexplantcultures

Lungswereremovedfrom13.5dpcembryos,harvestedand dis-sectedunderastereomicroscope(SZX16,Olympus).Thesewere transferred toporousmembranes (IsoporeTM _membrane _ﬁlters,

Millipore,USA)previouslypresoakedin DMEM(Invitrogen,UK) andincubatedintwenty-four-wellcultureplates(Nunc,Denmark). Threeexplantspermembranewerepositionedinawell-separated arrangementandtheseﬂoatingcultureswereincubatedatan air-mediuminterfaceinachemicallydeﬁnedmediumcontaining50% DMEM,50%F-12nutrientmixture(Invitrogen)andsupplemented with 100mg/mL penicillin, 100units/mL streptomycin (Invitro-gen),0.25mg/mLascorbicacid(Sigma–Aldrich,USA)and10%FCS (Invitrogen).Fetallungexplantswereincubatedina5%CO2

incu-batorat37◦Cfor96h,andthemediumwasreplacedevery48h. Culturesweresupplementeddailywithseveraldosesof (Pyr1)-Apelin-13(Bachem,Switzerland)rangingfrom10−11to10−5M.

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2.5. Morphometricanalysis

Branchingmorphogenesiswasmonitoreddailyby photograph-ingtheexplantsusingastereomicroscopeequippedwithacamera (DP71,Olympus).Atday0(D0:0h)andday4(D4:96h)ofculture,

thetotal numberofperipheralairwaybudsinalllungexplants wasdetermined. Also, epithelial perimeter, epithelial area and totalexplantareaweremeasuredusingImageJimage process-ingandanalysissoftware(version1.44,USA).Forallexperimental conditions, the results of the above mentioned morphometric parameterswereexpressedasD4/D0ratio.

2.6. Statisticalanalysis

Allquantitative data are presented as mean±SEM. Statisti-calanalysiswasperformedbyone-wayANOVAandsubsequently Student–Newman–Keulstestwasusedforpost-testanalysis,using SigmaStat3.5(SystatSoftwareInc.,USA). Statisticalsigniﬁcance wassetatp<0.05.

3. Results

3.1. ApelinandAPJproteinexpressionproﬁleduringratlung development

This study aimed to characterize apelin and APJ protein expressionprofileinthedeveloping ratlung. Westernblotand immunohistochemicalstudieswereperformedinorderto char-acterizeapelinandAPJproteinexpressionlevelsaswellastheir localizationanddistributionduringratpulmonarydevelopment. Bothproteinswereassessedat fiveprenatal stages,specifically 13.5,15.5,17.5,19.5and21.5dpcandalsointheadult.

3.1.1. Apelinproteinexpressionproﬁleduringratlung development

Westernblotanalysisrevealedthatapelinisexpressed through-outallstudiedgestationalagesinfetallungaswellasintheadult lung(Fig.1).Apelinexpressionlevelsweremaximalattheearliest gestationalagestudied,13.5dpc,earlypseudoglandularstagein lungdevelopment,andthendeclinedirregularlytowardtheendof gestation.Lowestapelinexpressionlevelsinprenatalperiodwere detectableat17.5dpc,latepseudoglandularstage.Intheadultrat lung,apelinexpressionlevelsdeclinetoastatisticallysigniﬁcant remarkableminimum.

Regardingimmunohistochemical studies(Fig.2), atthe ear-liest gestational age studied, 13.5dpc, apelin-positive cells are detectedinbothmajortissuesshapingtheembryoniclung, epithe-liumandmesenchyme,inanextensivefashion(Fig.2AandA). At15.5dpc,immunostainingwasobserved intheepitheliumof thedevelopingairwaysaswellasinthemesenchyme surround-ingtheairwaysandalsointheendothelium(Fig.2BandB).At late pseudoglandular stage, 17.5dpc, apelin immunostaining is observedinthewholeorgan,moreintenselyintheepitheliallining oftheairways,alsoinsurroundingmesenchymeandthe vascu-larendothelium(Fig.2CandC).Thesameexpressionpatternis detectedincanalicularstageofdevelopment,19.5dpc(Fig.2Dand D).Atthelatergestationalage,21.5dpc,apelinimmunostaining isprimarilyassociated withbothbronchialand alveolar epithe-liumandthevascularendothelium(Fig.2EandE).Theadultrat lungseemstoexhibitadissimilarexpressionproﬁlefromtheone observedduringgestational stages,bronchial epithelialstaining andendothelialexpressionsarenolongerpredominant,alveolar epitheliumhasmoresigniﬁcantexpression,andthereisalso posi-tiveimmunostainingdetectableinblood(Fig.2FandF).

Fig.1.Proteinlevelsofapelinduringfetalratlungdevelopment.Westernblot anal-ysisofapelinperformedinseveralgestationalages,from13.5to21.5dpcandalso intheadult.Representativeblotexamplesareshown.Allproteinlevelswere nor-malizedto␤-tubulin,whichwasusedasloadingcontrol.Densitometricanalyses ofthreeblots(N=3)areplottedabovetherepresentativeblots.Valuesrepresent mean±SE.p<0.05:*vs.adult;#vs.17.5dpc;§vs.15.5dpc.

3.1.2. APJproteinexpressionproﬁleduringratlungdevelopment Western blot analysis of the apelin receptor, APJ, revealed immunoreactivity for speciﬁc bands correlatingtoan expected molecular mass of ∼42kDa for the unglycosylated monomeric receptoraswellasbandsofhighermolecularmassconsistentwith glycosylatedmonomericreceptor,_∼45kDa.Additionalbands con-sistentinmolecularweighttomultiplesofthemonomerwerealso observed,dimericreceptorspeciesat90–95kDa,andoligomeric receptorspeciesat125–135kDa.

Bothmonomericandmultimericformsoftheapelinreceptor wereexpressedthroughoutallstudiedgestationalagesinfetallung aswellasintheadult,althoughexhibiting differentexpression proﬁles(Fig.3).Asfor themonomericprotein(Fig.3A),highest expressionlevelsweredetectedattheearliestgestationalage stud-ied,13.5dpc,decreasingprogressivelyat15.5and 17.5dpc,the latterbeingthestageinwhichexpressionlevelwaslowestduring theprenatalperiod.Stages19.5and21.5dpcshowaslightincrease intheproteinexpressionlevelscomparingto17.5dpc,nonetheless thesameprogressivelydecreasingtendencyinAPJmonomer pro-teinexpressionduringdevelopmentprevails.Lowestexpression levelswithstatisticallysigniﬁcanceweredetectedintheadultrat lung.Regardingthedimericformofthereceptor(Fig.3B),despite nostatisticaldifferenceestablished,adultratlunghasthehighest expressionleveldetected.APJdimericexpressionlevelsthroughout allgestationalagesstudiedremainedmostlyunvarying.Regarding theoligomericformofthereceptor(Fig.3C),againhighest expres-sionlevelsweredetectedattheearliestgestationalagestudied, 13.5dpc,decreasingprogressivelyintothelatergestational age. Alsoforthisformofthereceptor,lowestexpressionlevelswere detectedintheadultratlung.

Considering immunohistochemistrydata,atthe earliest ges-tational stages studied, 13.5 and 15.5dpc, apelin receptor immunoreactivityobservedhasaverysimilardistributionpattern tothatof apelinimmunoreactivity (Fig.4).In theﬁrst,there is APJ-positivecellsinbothepithelialandmesenchymaltissuewith comparableintensity.Atthelatterstage,positivityisdetectedin theepitheliumoftheairways,inthemesenchyme surrounding

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Fig.2. Apelinexpressionpatternduringfetalratlungdevelopment.Apelinispresentthroughoutallstagesoflungdevelopmentfromearly13.5dpcuntillate21.5dpcand alsointheadult.Representativeimmunohistochemistrystainingof(A–E)developinglung(F)adultlung.(A)Originalmagniﬁcation200×and(A₎_original_{magniﬁcation} 400×.aw:developingairwayandbv:bloodvessel.

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Fig.3.ProteinlevelsofAPJduringfetalratlungdevelopment.WesternblotanalysisofAPJproteinlevelsindevelopingratlungatvariousfetalagesandalsointheadult.(A) Monomeric,(B)dimericand(C)oligomericformsofthereceptorweredetected.Representativeblotsexamplesareshown.Allproteinlevelswerenormalizedto␤-tubulin, whichwasusedasloadingcontrol.Densitometricanalysesofthreeblots(N=3)areplottedabovetherepresentativeblots.Valuesrepresentmean±SE.p<0,05:*vs.adult; #vs.17.5dpc,vs.19.5dpc,vs.21.5dpc,and§vs.15.5dpc.

theairwaysandalsointheendothelium(Fig.4A,B,AandB).At stage17.5dpc,although thereiswidespreadstainingdetectable intheorgan,itismoreintenseinthecells oftheepithelial lin-ingoftheairwaysandstainingispredominantlynuclear,thereis alsoweakerstaininginthevascularendotheliumandsurrounding areas(Fig.4C andC).Atoneofthelatergestational ages stud-ied,19.5dpc,itisalsoobservedabroaddistributionoftheapelin receptorinthefetallung,butimmunoreactivityismoreintense intheairwayepithelialcells,inwhichpositivityisdetectedinthe nucleus,thecytoplasmandthemembrane(Fig.4DandD).Bythe endofgestationat21.5dpc,APJexpressionisalsorelatedmainly tothebronchialand alveolarepithelium,which isthe predomi-nanttissueatthis stage,weakerexpressionisdetectableinthe endothelialliningofthevasculature(Fig.4EandE).Intheadultrat lung,aweakAPJexpressionisdetectableinthealveolithat com-prisesmostoftherespiratorytissue,andalsointheendothelial liningofthevesselsandthesmoothmusclesurroundingbronchi andlargepulmonaryvessels.Moreintensestainingisdetectedin thenucleusandcytoplasmoftheepithelialcellsoftherespiratory bronchi(Fig.4FandF).

3.2. Effectsofapelinsupplementationinfetallungexplant cultures

Thisstudyalsoaimedtoelucidatetheroleofapelininthe devel-oping ratlung. Therefore, apelin supplementation studieswere carriedoutinthefetallungexplantculturemodel.Normalfetallung explantsweretreatedwithdifferentdosesofrecombinant (Pyr1)-Apelin-13.InFig.5,representativeexamplesofnormalfetallung explantstreatedwithincreasingApelin-13dosesareillustrated. Apelin-13appearstohaveaninhibitoryeffectonlungexplants growth,exceptwiththelowestdosetestedasshownbythe mor-phometricanalysisdatasummarizedinFig.6.Infetallungexplants, regardlessofaslightincreaseingrowthverifiedinallthe mor-phometricparameters inducedbythelowestdoseofApelin-13 tested,nostatisticalsignificanceagainsttheabsenceofapelin treat-mentwasobserved.Incontrast,supplementationwiththehighest dose,10−5M,significantlyreducedwholeexplantgrowthwhen comparedwiththelowestdoseofApelin-13tested,demonstrated bybothepithelialandexternalexplantarea.Also,consideringthe epithelialperimeter,asignificantinhibitoryeffectisinducedbythe highestdosetestedwhencomparedtolackofsupplementationor treatmentwithlowApelin-13doses,10−11and10−9M. Consider-ingthenumberofperipheralairwaybuds,nosignificantstatistical differencewasobserved.

3.3. ApelinsupplementationeffectsonMAPKsignalingpathway Inordertofurtherinvestigatetheunderlyingmolecular mech-anismsresponsiblefortheeffectofapelinonfetallunggrowth,the doseeffectofapelinonmitogen-activatedproteinkinases signal-ingwasdetermined.Expressionlevelsofp38,JNK1/2,andERK1/2 wereassessedbyWesternblotinpooledsamples(N=12)of Apelin-13treatedfetallungexplants.ActivationofMAPKsisregulatedby phosphorylation,thusbothunphosphorylatedandphosphorylated proteinstateswereassessed,asshowninFig.7.Foreachproteinthe ratioofphosphorylatedtounphosphorylatedformwasdetermined (Fig.8)revealingthatapelintreatmentinducedadosedependent decreaseinactivationofbothp38andJNK,sinceincreasingdoses of apelin graduallyreduced phosphorylationof thesetwo MAP kinases(Fig.8AandB).Incontrast,thehighestdoseofapelintested inducedactivationofERK1/2,whereassupplementationwithlower doses of apelin reduced phosphorylationwhen compared with absenceofapelintreatment(Fig.8C).

4. Discussion

Thisstudyanalysesforthefirsttime,fetalpulmonaryprotein expressionprofileofbothcomponentsoftheapelinergicsystem, apelinandAPJ,byWesternblotandimmunohistochemistry, paral-lelcomparisonwiththeadultprofileisalsopresented.Additionally, invitroapelinsupplementationstudieswereperformedin fetal lung explantsin order toelucidate itsputativerole in branch-ingmorphogenesisandwe furtherinvestigated MAPKsignaling contribution tothe effects of apelin on fetal lung growth. We demonstrated that both components of the apelinergic system areconstitutively expressedinthelung during allstudied ges-tationalagesand alsointheadult.Westernblotanalysisyields importantdataregardingthecharacterizationofapelin andAPJ in lung development.Apelin immunoreactivity hasbeen previ-ously detectedas a band of approximately 16kDa in different tissue lysates [28], as it is observed in our sample lysates. In fetal lung,apelin proteinexpressionlevelshave a maximumat anearlystageindevelopment(13.5dpc),inalltheother gesta-tionalagesstudiedexpressionlevelsarelower,anditsminimum isreachedintheadulttissue.Thisfact,pointstoward,possibly,a morerelevantroleofthispeptideduringearlystagesoflung devel-opment.Regardingapelinreceptor,Westernblotanalysismadeit possibletoobservethatthisreceptorexitsinavarietyof presen-tationsinthepulmonarytissue,whichincludepost-translational modifications and multimeric arrangements. At lower molecu-larweight,bothunglycolysated andglycolysatedforms (42and

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Fig.4. APJexpressionpatternduringfetalratlungdevelopment.APJispresentthroughoutallstagesoflungdevelopmentfromearly13.5dpcuntillate21.5dpcandalsoin theadult.Representativeimmunohistochemistrystainingof(A–E)developinglung(F)adultlung.(A)Originalmagniﬁcation200×and(A)originalmagniﬁcation400×.aw: developingairwayandbv:bloodvessel.

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Fig.5. Branchingmorphogenesisinratlungexplantculturesystem.RepresentativeexamplesofcontrolandfetallungexplantstreatedwithincreasingApelin-13 concen-trations.Upperpanelshowsrepresentativephotographoflungexplantat13.5dpcatcultureday0(D0).Bottompanelshowsrepresentativephotographoflungexplantat 13.5dpc,treatedwithseveralApelin-13concentrations,atday4inculture(D4).Originalmagniﬁcation40×.

45kDa)ofthemonomericreceptorspeciesarepresentinall stud-iedgestationalages.Athighermolecularweights,correspondingto multiplesofthemonomer,dimeric(∼90–95kDa)andoligomeric (_∼125–135kDa)receptorspecieswereobserved.Accordingtothe literaturemanyGPCRs,besidesthemonomericform,areexpressed asdimersandoligomers,butitisalsocommonforGPCRtoappear ashomo-andhetero-dimersandoligomers[5,41,33].WhetherAPJ

multimericspeciesobservedinthefetalandadultpulmonary tis-suesinourstudycorrespondtohomo-orhetero-multiplesofthe receptorisstillnotknownandcouldonlybeclariﬁedbyfurther studies.Nonetheless,itwasalreadydemonstratedinvitrothatAPJ canformheterodimerswithangiotensinIIAT1receptor[7]. More-overimmunohistochemicalstudiesperformedinourlabrevealed thatallrenin–angiotensinsystemcomponents,includingAT1and

Fig.6. Morphometricanalysisoffetalratlungexplantssupplementedwithincreasingapelinconcentrations.Severalparametersincontrolandtreatedexplantswere analyzed.(A)Epithelialperimeter,(B)epithelialarea,(C)numberoftotalairwaybudsand(D)totalexplantarea.Measuresoftenlungexplants(N=10)perconditionare plottedabove.ResultsareexpressedasD4/D0ratio.Valuesrepresentmean±SE.p<0,05:*vs.10−11M,#vs.0M(control),and§vs.10−9M.

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Fig.7.MAPkinasesactivityinfetalratlungexplantstreatedwithincreasingapelinconcentrations.Westernblotanalysisofp38,JNK1/2,andERK1/2inpooledsamples (N=12)ofApelin-13treatedfetallungexplants.Bothunphosphorylatedanddiphosphorylatedformsofp38(dp-p38),p44/42(dp-p44/42),JNK1/2(dp-JNK1/2)wereassessed. ␤-Tubulinwasusedasloadingcontrol.

AT2receptors,areexpressedinfetallungthroughoutthe gesta-tionalagesalsocharacterizedinthisstudy[36].

Theimmunohistochemicalstudieswereperformedtofurther characterizethepresenceofapelinandAPJinthepulmonary tis-sue.Thesestudiesallowustoconcludethatapelinlungexpression ismainlyrelatedtobothairwaysandvasculatureduringthe devel-opmentalstagesstudied.Apelinimmunostainingwasdetectedin theairwayslining,eitherinbronchialepitheliumthroughout ges-tation,aswellasinthealveolarepitheliumasitgraduallyappeared. Regardingthevasculature,positiveimmunostainingwasdetected in the endothelial lining of the blood vessels from 15.5dpcto 21.5dpc.Whereasintheadultratlung,apelinpresenceisstrongest inthealveolarepithelium,lessintenseinthebronchialepithelium andnearlyabsentinthevasculature.Interestinglythereisapelin immunoreactivityinthecontentofthepulmonarybloodvessels whichisnotassociatedwithbloodcells,whichmaypossiblybe inaccordancewithpreviousreportsthatdetectedplasmalevels ofapelininhumansandconsequentlyshowedthatapelinisa cir-culatingpeptide[6,13,16].Fewstudiesreportapelinlocalization inpulmonarytissuebyimmunohistochemistry,showingintense stainingintherespiratoryepitheliumandsmoothmusclein nor-maladulthumantissues[10],andalsopresenceinbronchialand alveolarepithelialcellsinneonatalratlung[47].

Immunohistochemistryforthereceptorproteinrevealedthat APJlocalizationispredominantlyassociatedwiththepulmonary airwaysinthedevelopmentalstagesstudied.Moststrikingisthe

distributionofthereceptorwithinthebronchialepithelium,where APJcanbedetectedeitherinthecytoplasm,themembraneand alsothenucleusof theseepithelialcells, nuclearlocalizationof thereceptorappearsasearlyas15.5dpcandisdetectableinall subsequentstages.Apelinreceptornuclearlocalizationis there-foreindicativeofnucleartranslocationofAPJoccurringprobably onlyincertaincellpopulationswithinpulmonarytissue.Todate, therehavebeenmanyreportsoftheintercellularlocalizationand traffickingofGPCRs,althoughGPCRsarebestknownascellsurface mediatorsofsignaltransductionthereisanincreasingnumberof reportsofGPCRscapableofnucleartranslocation [15].Thusfar, besidesourfindingsinpulmonarytissuethereisonlyonereport showingthatapelinreceptorexhibitsnuclearlocalization occur-ringinbraintissue[27].Thisnuclearlocalizationofapelinreceptor pointsouttopreviouslyunforeseenfunctionsofthisreceptoras amodulatorofnucleartranscriptionthatareworthinvestigating, inadditiontothewell-establishedroleofthisreceptorfamilyat thecellsurface.Intheadultratlung,APJimmunostainingismost intense in thebronchial epithelium and respiratory epithelium where itisobservedthat expressioniscytoplasmatic, membra-nousandnuclear.Additionally, lowerintensityimmunostaining was detected in the alveolar epithelium, the endothelium and alsothesmoothmusclearoundthebronchi.Theseresultsmatch previousfindingsofimmunoreactivityinhumanlungtissuethat showedthatAPJisexpressedinendothelialcellsandsmooth mus-cleofsmallpulmonary vessels,andalsodemonstratedreceptor

Fig.8.EffectsofapelinonMAPKsignalinginfetalratlungexplants.Ratioofphosphorylatedtounphosphorylatedformwasdeterminedfor(A)p38,(B)JNK1/2and(C) ERK1/2.Allproteinlevelswerenormalizedto␤-tubulin,whichwasusedasloadingcontrol.Densitometricanalysisresultsarepresentedasarbitraryunits.

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immunoreactivityin bronchial epithelial cells aswell asin the endotheliumandinsmoothmusclecellsinrattissue[23].Thefetal lungdisplaysthenecessarymachineryforapelin/APJsignaling,and theepitheliummightactasboththephysiologicalsourceofapelin andalsoasatargettissueforapelintoexertitsphysiologicalaction duringfetallungdevelopment.

Ourfindingsonthepresenceandsignificantfeatures regard-ingthecharacterizationofboth componentsofthis peptidergic systemonthepulmonary tissue during developmentled usto hypothesizethatitmighthaveanunderlyingroleinlungbranching morphogenesis.Hence,inordertobeginexploringthis assump-tion,invitroapelinsupplementationstudieswereperformedin thefetallungexplantculturemodel.Themorphometricanalysis onthefetal lungexplantsexposed toincreasingdosesofapelin in culture,showed a steady tendency for aninhibition oflung explantgrowth,especiallysignificantinthehighestdosetested. Itiswellknownthatnegativemolecularmediatorsareessentialin pulmonarydevelopmentgiventhatthere isastrictdependence onthebalancebetweeninductiveandsuppressivemechanisms occurringin thisprocess.Therefore, orderlybranching morpho-genesisalsodependsonmultipleantagonists[34,48].Interestingly, itwasrecentlydemonstratedinthesameinvitroculturemodel, thatangiotensinII(AngII)supplementationinducedanincreasein lungexplantsgrowth[36].Moststrikingaboutthesefindingsisits straightassociationwithanemergingkeyfeatureoftheapelin/APJ system,itsinteractionwiththe renin–angiotensinsystem. Cur-rentlytherelationbetweenthesetwosystemsisdescribedtobea directinteractionofthetwosystemsatbothmolecularand tran-scriptionallevelstomediateopposingphysiologicalactions[2].APJ andAT1receptor–receptorinteractionsarepresentlyaccountedas thepotentialmolecularmechanismofcrosstalkresponsibleforthis reciprocalcounter-regulationbetweenapelinandAngIIpathways, sinceitwasobservedinvitrothattherespectivereceptorscanform heterodimersandthatthisheterodimerisationinfluences down-streamsignaling[7].Ourobservationsonfetallunggrowtharein accordancewithothersreportingthattheproposedeffectsofthe apelin–APJsystemareoppositetothoseoftheAngII–AT1receptor pathway[7,44].

Ononehand,couplingofapelintoitsreceptorAPJhasbeen reportedtoactivateMAPkinasessignalingpathways[1,28]. On theotherhand,MAPKwereshowntobeimportantsignaling path-wayformurinelungdevelopmentthroughregulationofbranching morphogenesis,epithelialcellproliferation,cellsurvivaland dif-ferentiation[24].Hence,evaluatingMAPKsignalinginthecontext ofelucidatingapelin’seffectsonfetallunggrowthwasregarded pertinent.Ourresultsclearly showdosedependentdecrease in activationofboth p38and JNK inducedbyapelin treatmentas wellasactivationofERK1/2inducedbythehighestdoseofapelin tested.Inpreviousstudiesenhancedfetallungbranchingwas asso-ciatedwithstimulationofp38phosphorylation[37]likewise,lung growthinhibitioninducedbyAT1antagonistwasreportedtobe mediatedbyadecreaseofp38andJNKphosphorylation[36]. There-fore,apelinsupplementationinhibitoryeffectonlunggrowthis mostlikelymediatedbydecreasedp38andJNKphosphorylation. ItiscommonforMAPkinasestosharesubstratesandtointegrate signalsfromindividualcascadesnotonlyduringtheearlysignal transductioneventsand withinthekinasecascades,butalsoat thelevel ofsubstrate phosphorylation[8]. In viewof this, it is onlyreasonabletoassumethat theseapparentdiscrepancies in theexpressionofp38,JNKandERK1/2mightreﬂectcross-cascade interactionstakingplaceinordertorestoreintracellularsignaling balance.

Inconclusion,thedataherepresentedprovidestheﬁrst evi-denceofthepresenceofanapelinergicsystemoperatinginthe developing lung. Our resultsalsodemonstrated that the apelin receptorexhibitsnuclearlocalizationinlungepithelialcells,which

unveilsanimportantandnovelfeatureofthis systemthat sug-gestunanticipatedfunctionsforthisG-proteincoupledreceptorin nucleartranscriptionregulation.Moreover,itisalsodemonstrated thatapelin’sinhibitoryeffectonfetallung growthisassociated withinhibitionofp38andJNKsignalingpathways.Wehavefurther characterizedthisnovelpeptidergicsysteminthedevelopinglung.

Acknowledgement

TheauthorswishtothankMiguelCarvalhoandLuísMartinsfor skillfulassistanceinanimaleuthanasia.

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