Various roles of heme oxygenase-1 in response of bone marrow macrophages to RANKL and in the early stage of osteoclastogenesis

(1)

Published:xxxxxxxx

1

SCIentIFICREpoRtS|(2018)8:10797| DOI:10.1038/s41598-018- 29122-1

www.nature.com/scientificreports

OPE N

Received:27September2017 Accepted:5July2018

Variousrolesofhemeoxyge nase-

1inresponseofbonemarrow macrophagestoRANKLandi ntheearlystageofosteoclas togenesis

UrszulaFlorczyk-

Soluch

¹

,EwelinaJózefczuk

¹

,JacekStępniewski

¹

,KarolinaBukowska- Strakova

³

,MateuszMendel

¹

,MonikaViscardi

¹

,WitoldNorbertNowak

¹

,Alic jaJózkowicz

^1&

JózefDulak

^1,2

Hemeoxygenase-1(HO-

1;encodedbyHmox1),adownstreamtargetoftheNrf2transcriptionfactor,hasbeen postulatedtobeanegativeregulatorofosteoclasts(OCLs)differentiation.Here,wefur therexploredsuchahypothesisbyexaminingHO-

1effectsindifferentstagesofosteoclastogenesis.Weconfirmedtheinhibitionoftheex pressionofOCLsmarkersbyNrf2.Incontrast,boththelackoftheactiveHmox1geneorHO- 1silencinginOCLsprecursorcells,bonemarrowmacrophages(BMMs),decreasedth eirdifferentiationtowardsOCLs,asindicatedbytheanalysisofOCLsmarkerssuchasTR AP.However,noeffectofHO-1deficiencywasobservedwhenHO-

1expressionwassilencedinBMMsorRAW264.7macrophagecelllinepre- stimulatedwithRANKL(consideredasearly-

stageOCLs).Moreover,cobaltprotoporphyrinIX(CoPPIX)orhemin,theknownHO- 1inducers,inhibitedOCLsmarkersbothinRANKL-

stimulatedRAW264.7cellsandBMMs.Strikingly,asimilareffectoccurredinHO-1^−/

−cells,indicatingHO-1-

independentactivityofCoPPIXandhemin.Interestingly,plasmaofHO-1^−/

−micecontainedhigherTRAPlevels,whichsuggestsanincreasednumberofbone- resorbingOCLsintheabsenceofHO-1invivo.Inconclusion,ourdataindicatethatHO-

1isinvolvedintheresponseofbonemarrowmacrophagestoRANKLandtheinductionofO CLsmarkers,butitisdispensableinearly-stageOCLs.However,invivoHO-

1appearstoinhibitOCLsformation.

Osteoclasts(OCLs)aremultinucleatedmyeloidcellscrucialforconstantboneremodellingbecauseoftheirbone- resorbingactivity.ExcessiveboneresorptioncomingfromincreasednumberandactivityofOCLsliesattherootofmos tadultskeletaldiseasesincludingosteoporosis,periodontaldisease,rheumatoidarthritis,multiplemyelomaandmetast aticcancers¹.

OsteoclastogenesisisinducedbythereceptoractivatorofnuclearfactorκBligand(RANKL)afteritsbindingtotheR ANKreceptor^2–4.Inparallel,theactivationofthec-fmsreceptorbymacrophagecolony-stimulatingfactor(M- CSF)providesthesurvivalsignal^5,6.RANKLbindingenablesrecruitmentofadaptormoleculessuchasTRAF6⁷.TRAF 6activatesNF-

κB^8,9,whichisessentialfortheinitialinductionofnuclearfactorofactivatedTcells,cytoplasmic1protein(NFAT- c1)^10,11.NFAT-c1activatedbycalciumsignallingisauto-amplifiedafterbind-ingtoitsownpromoterandanactionofc- Fos¹².NFAT-c1inducestheexpressionofOCLs-specificgenessuchastartrate-

(2)

2

resistantacidphosphatase(TRAP),ca thepsinKorintegrinβ3¹³.Inaddition, RANKLactiontransientlyincreases thelevelofreactiveoxygenspecies(R OS)viacurrentlyassumedRANK/T RAF6/Rac1/Noxsignallingcascade

14–17.ROSareconsideredasintra- cellularsignallingmoleculesmostpr obablytargetingpro-

inflammatoryNF-

κBpathwayandpromotingOCLsfor mation^18,19.Ontheotherhand,oxidati vestressconditionsforcecellstoimpe lprotectivemechanisms,which,how ever,arethoughttobeattenuatedduri ngosteoclastogenesistosecureROS signalling¹⁷.Thus,suchpathwaysare expectedtobeosteoclastogenicregu latorsofpotentialtherapeuticsignifi canceforskeletaldiseases.

1DepartmentofMedicalBiotec hnology,FacultyofBiochemist ry,BiophysicsandBiotechnolo gy,JagiellonianUniversity,Kra kow,Poland.²Kardio-

MedSilesia,Zabrze,Poland.³D epartmentofClinicalImmunol ogy,InstituteofPediatrics,Jagi ellonianUniversityMedicalColle ge,Krakow,Poland.Correspond enceandrequestsformaterials shouldbeaddressedtoU.F.-S.

(email:urszula.florczyk@uj.edu .pl)orJ.D.

(email:jozef.dulak@uj.edu.pl)

(3)

www.nature.com/scientificre ports/

NuclearfactorE2-relatedfactor2(Nrf2)transcriptionfactorrepresentsoneofthecriticalcytoprotectivepath- wayscontrollingdetoxifying,antioxidantandanti-inflammatoryagentsincludinghemeoxygenase-1(HO- 1)^20,21.Nrf2/HO-1axiswasshowntoinhibitNF-

κBsignaling²².Inaddition,agrowingbodyofevidenceindicatesaroleofHO-

1incelldifferentiationasshownforendothelialprogenitors²³,myoblasts²⁴,erythroidprogenitors^25orosteoblasts^26,27.R ecentstudieshavealreadysuggestedaninhibitoryeffectofbothHO-1^{28–3 2a}ndNrf2^33,34,onosteoclastogenesis.HO- 1deficiencywasshowntodecreasebonedensityduringboneremodellinginvivomainlyduetoincreasedosteoclastogen esisandboneresorption³⁰.Importantlyhowever,deletionofHO-

1inthemyeloidlineageattenuatedtheabilityofmyeloidprogenitorstodifferentiatetowardmacrophages³⁵.Inaddition, HO-

1withitsactiveproductswasshowntoregulateactivation,proliferation,andsurvivalofmaturemacrophages³⁵.Thus,si nceHO-1seemstobeimportantformyeloidcelldifferentiationandmacrophagefunctionbuthasanti-oxidantandanti- inflammatorypotential,itsroleinosteoclastogenesismightbemorecomplexandmaydependonthestageoftheprocess.

HereweshowedthatwhileHO-

1deficiencyinOCLsprecursorsdiminishesdifferentiationinresponsetoRANKL,itisdispensableinRANKL-pre- stimulatedcellsconsideredasearly-stageOCLs.Thus,HO-

1seemstomediatetheresponseofOCLsprecursorstoRANKLandinductionofOCLsmarkersbutisdispensableinearl y-stageOCLs.Invivo,theadvantageoftheinhibitoryeffectofHO-1onosteoclastogenesismightbecon-

cluded.InhibitionoftheexpressionofOCLsmarkersbyNrf2wasverifiedandconfirmed.

MaterialsandMet

hods Reagents.R

ecombinanthumanM-CSFandrecombinanthumanRANKLwereobtainedfromSigma- Aldrichandweredissolvedinwatercontaining0.1%BSAtoaconcentrationof10μg/ml.CoPPIXandSnPPIX

werepurchasedfromFrontierScientific,whileheminwasobtainedfromCalbiochemandallwerepreparedas10mMsto cksinDMSOor100mMNaOH.SulphoraphanewaspurchasedfromSigma-

Aldrichandpreparedas5mMstockinDMSO.

Animalsandcare.A

llanimalworkwasapprovedbytheLocalEthicalCommitteeforAnimalResearchat theJagiellonianUniversity(licenseno86/2011).HO-1(Hmox1)knockout(HO-1^−/

−,C57BL/6×FVB)andwildtype(HO-1^+/+)mice,andNrf2(Nfe2l2)knockout(Nrf2^−/

−,C57BL/6)andwildtype(Nrf2^+/+)miceaged2–

4months(malesandfemales)wereusedforisolationofbonemarrowcellsorplasma.GenotypeswereverifiedbyP CR.Allexperimentswereperformedaccordingtoapprovedguidelinesandregulations.

Bonemarrowandplasmaisolation.M

iceweresacrificedwith5mg/mlketamineand2mg/mlxylazin esolution(10μlpergramofb.w.).Bloodwascollectedbydirectheartpuncturewithasyringecontaining25µlofhepa rinsolution(1000U/ml,Polfa)andcentrifuged(10min,800×g,4°C).Plasmawascollectedtonewmicro-centrifugetubes.

Bonemarrow(BM)wasisolatedfromtibialandfemoralbonesofeuthanizedmice.Themarrowcavitywasflus hedoutwithα-MEMmedium(Lonza)supplementedwith10%fetalbovineserum(FBS),100U/mlpen- icillinand100μg/mlstreptomycin(α-MEMCM)usingasterile20-

gaugeneedle.Asinglecellsuspensionofbonemarrowcells(BMCs)obtainedbypipettingwascentrifuged(5min,10 0×g,4°C),washedwithPBS,resus-

pendedinα-MEMCMandcountedusingMuse

™C

ountandViabilityAssayKitandMuseCellAnalyzer(Merck Millipore)

.

Cellcultureandtreatment.B

MCsfromH O-1^−/−,N rf2^−/

−andwildtypecounterpartsandm urineRAW264.7macrophagescelllinewereculturedinα- MEMCM,intheincubatorswithstandardizedparameters:37°C,5%CO2and95%humidity.

Bonemarrowmacrophages(BMMs)orRAW264.7cellswereusedasOCLsprecursors.RANKL- stimulatedOCLsprecursorswereconsideredasearly-

stageOCLs.Threealternativeexperimentalsettingsofthecul-

tureofprimarycellswereused(SupplementaryFig.S1).BMCs-derivedbonemarrowmacrophages(BMMs),BMCs- derivedreplatedBMMsandnonadherentBMCs(nBMCs)-

derivedreplatedBMMswereusedasOCLsprecursorsfordifferentiationtowardsOCLs.Briefly,totalBMCswerecultu redfor3daysathighdose(100ng/ml)ofM-CSF(usedtoobtainBMCs-

derivedBMMs)tostimulatetheproliferationofmacrophageswithoutgrowthofstromalcells³⁶.Alternatively,afterove rnightincubationofBMCsinthepresenceof50ng/mlM-CSF,nBMCswereharvestedtoculturestroma-

freebonemarrowcells.After3daystheadherentcellswereharvestedasnBMCs-derivedBMMs²⁹. ToinduceOCLsdifferentiationBMCs-

derivedBMMsweredirectlystimulatedwithRANKL(50ng/mlor100ng/ml)inthepresenceof100ng/mlM- CSF.Alternatively,BMCs-derivedBMMsandnBMCs-

derivedBMMswerereplated(detachedusingAccutase,centrifugedfor5minat100×g,countedandseeded)andcu lturedinthepresenceof50ng/mlRANKLand30ng/mlM-

CSF.Whereindicatedsulphoraphane(2.5μM)wasusedforNrf2activation.After3daysofincubationwithM- CSFandRANKLTRAPstainingandTRAPELISAwereperformedandOCLsmarkerswereanalyzedbyqPCR.Spe cifically,forthestainingofactinstructuresnBMCs-

derivedreplatedBMMswereculturedwith100ng/mlRANKLfor5days(inthepresenceof30ng/mlM-CSF).

(4)

www.nature.com/scientificre

ports/ Toexaminetheeffectofpharmacologicalinducers/inhibitorofHO-

1,RAW264.7wereculturedwith50ng/mlRANKLand25μMCoPPIX/hemin/SnPPIXorNaOHasavehicle.After9an d48hofincubationOCLsmarkerswereanalyzedbyqPCR.OCLsprecursors(nBMCs-

derivedBMMs)werereplatedandculturedwith30ng/mlM-

CSFand50ng/mlRANKLinthepresenceof5,15and25μMCoPPIXor5,15and25μMheminorDMSOasavehicle.Afte r3daysofincubationOCLsmarkerswereanalyzedbyqPCR.BMCs-derivedBMMswerestimulatedwith100ng/mlM- CSFand100ng/mlRANKLinthepresenceof25μMCoPPIXor25μMheminorDMSOasavehicle.After3daysofincuba tionTRAPstainingwasperformed.

(5)

FACSanalysis.T

oassesswhatisthepercentageofstrictlydefinedmonocytesandmacrophagesamongthepo pulationofBMCsandBMMs,referredtoasOCLsprecursors,FACSanalysiswasperformed.Asinglecellsuspe nsionofBMCs(1×10⁶)and/ornBMCs-

derivedBMMs(4×10⁵)werecentrifuged(5min,700×g,RT),washedwithPBS,andusedforFACSanalysis.Ford etectionofmacrophagesandmonocytesamixtureofthefol-lowinganti-mouseantibodiesagainst:CD45-APC- eFluor780,F4/80-APC,MHCII-PE-Cy7,Ly6C-PerCP-Cy5.5(ThermoFisherScientific),CD11b-PE-CF594andLy6G- BV605(BDHorizon)(0.6µgofeachantibodypersam-

ple)wasaddedfor15minat4°Cinafinalvolumeof100µlofappropriatebufferdependingonprotocolused.Fordet ectionofapoptoticcells,proliferatingcellsandROSproduction,respectively,TACSAnnexinV-

FITCApoptosisDetectionKit(Trevigen),antibodyagainstKi67-

AlexaFluor488(BDPharmingen)orCellROXGreenReagent(ThermoFisherScientific)wereusedincombination withDAPI(0.2μg/ml).Afterincubationwithdyesandantibodies,cellswerewashedwithPBS,centrifuged(5min,700

×g,RT),resuspendedin350μlof2%FBSinPBSandanalysedwiththeflowcytometer(LSRFortessa,BD)usingFACSDi vav8.1software.

OntheBDLSRFortessagreenfluorescentdyes(FITC,AlexFluor488,CellROXGreenReagent)aswellasPerCP- Cy5.5wereexcitedbya50mW488nmbluelaserandemittedlightwascollectedusing530/30BPfilterforallgreendyesan dby675/20BPfilterforthelatterdye.PE,PE-CF594andPE-Cy7wereexcitedby50mW561nmgreen-

yellowlaserandtheiremissionspectrawerecollectedwith582/15BP,610/20BPand780/60BPfilters.APCandAPC- eFluor780wereexcitedbya40mW640nmredlaserandtheiremissionwascollectedwith670/30BPand780/60BPfilter srespectively.WhereasDAPIandBV605wereexcitedbya50mW405nmvioletlaserandemittedlightwascollectedwit h450/40BPand610/20BPfilters,respectively.

Analysisandgatingstrategy.InthefirststepdoubletsexclusionwasdonebasedoneliminationofeventswithincreasedF SC-Widthvalues.ToassessspecificityofAnnexin-V,Ki67stainingandROSproductioninpopula-

tionsofinterest,FMOcontrolswereused.Asinwholebonemarrowsamplesinternalcontrolpopulationsareavailable,a positivityofremainingparameterswasdefinedbyin-

samplecellcontrolswhichdonotexpresstheantigen(i.e.,mature,cross- lineagecellsgatedinplotswithCD45and/orcross-

lineagemarkers).Thisapproachissaidtobeoneofthemostappropriatecontrolmeasuresduetotheexposureofallpopul ationstoidenticalcondition³⁷.

MonocyteswereidentifiedbasedonLy6Gnegativity,CD45positivity,highLy6CandCD11bexpression,andlowton egativeMHCIIandF4/80expression(CD45⁺Ly6G⁻Ly6C⁺CD11b⁺MHCII^low/−F4/80^low/

−).Ofnote,theterm“monocytes”isonlyusedincaseofBMCspopulationpreciselydefinedasCD45⁺Ly6G⁻Ly6C⁺CD 11b^+MHCII^low/−F4/80^low/

−.Theterm“BMCs”isreferredtothewholepopulationofcellsobtainedafterbonemarrowisolation.

MacrophageswereidentifiedbasedonpositivityforCD45,F4/80andCD11b,andnegativityforLy6G(CD45^+Ly6G

−F4/80⁺CD11b⁺)

(gatingstrategyshowninsupplementarymaterialsFig.S2).Ofnote,theterm“BMMs”isreferredtothewholepopulatio nofM-CSF-stimulatedBMCs/nBMCsand“BMMs”areconsideredasOCLsprecursors.

FlowcytometryanalysisofmacrophagesdifferentiatedinthepresenceofM- CSFareshowninsupplementarymaterials(Fig.S3).

siRNAtransfection.T

oexaminetheeffectofHO-1silencinginOCLsprecursors,nBMCs- derivedBMMswerereplatedinthepresenceof30ng/mlM-CSFandtransfectedwithsiRNAagainstHO-

1orscrambledcontrol.Onedayaftertransfectionfreshmediumcontaining50ng/mlRANKL(toinduceosteoclastogen esis)and30ng/mlM-CSFwasadded.After3daysofincubationOCLsmarkerswereanalyzedbyqPCR.

nBMCs- derivedBMMs(orRAW264.7)pretreatedwithRANKLwereusedasearlystageOCLs.ToexaminetheeffectofHO- 1silencinginearly-stageOCLs,nBMCs- derivedBMMsorRAW264.7werereplatedwith50ng/mlRANKL(and30ng/mlM- CSFincaseofBMMs)and24hlater(asearly-stageOCLs)weretransfectedwithsiRNAagainstHO- 1orscrambledcontrol.OnedayaftertransfectionproteinwascollectedforWesternblotanalysisorfreshRANKL- containingmediumwasadded.After3daysofincubationOCLsmarkerswereanalyzedbyqPCR.Cellsculturedon24- wellplatesatseedingdensityof200000cells/400μlα- MEMCM/wellweretransfectedwithSilencerSelectsiRNAagainstmurineHmox1orSilencerSelectNegativeControl (ThermoFisherScientific)usingLipofectamineRNAiMAXtransfectionreagent(ThermoFisherScientific)accordin gtothemanufacturer’sprotocol.Briefly,10pmolofsiRNA(0.5μl)wasdilutedinOptiMEMtoafinalvolumeof50μland thenmixedwith50μlofpre-dilutedlipofectamine(3μloflipofectamineto47μlOptiMEM).Thetransfectionmixture (100μl)wasincubatedfor5minatRTandthenaddeddropwiseto400μlofculturemedium(α-MEMCM).

QuantitativePCR.T

otalRNAisolationfromcellsculturedon24- wellplatesatseedingdensityof200000cells/wellwasperformedbyphenol-

chloroformextraction.Inbrief,cellswerewashedwithPBS,lysedwith400μlofFenozol(A&ABiotechnology),mixed with100μlofchloroform,vigorouslymixedbyvortexingfor60secandcentrifuged(20min,10000×g,4°C).Anupperaque ousphasewascollectedandsubjectedtoisopropanolpre-

cipitationforatleast2hat−20°C.RNApelletswereresuspendedin12–25μlofnuclease-freewater.

Reversetranscriptionreactionwascarriedouton0.5–

1μgoftotalRNAusingoligo(dT)primersandRevertAidreversetranscriptase(ThermoFisherScientific),accordingt othevendor’sinstructionsinProFlexPCRsystemthermocycler(ThermoFisherScientific)for1hourat42°Candsubse quent5minat95°C.cDNAwasstoredat−20°C.QPCRwasperformedinamixture(15μl)containingSYBRgreenJum

(6)

pstartReadyMix(Sigma),40ngcDNAandspecificprimersasfollows:Hmox-1(5′-

CCTCACTGGCAGGAAATCATC-3′and5′-CCTCGTGGAGACGCTTTACATA-3′),NFATC1(5′- CTGCGGGAGCGGAGAAACTTTG-3′and5′-CTGGCAAGGCAGAGTGTGCTGT-3′),CTSK(5′- TGCAGCAGAACGGAGGCATTGA-3′and5′-GCCACTGCTCTCTTCAGGGCTT-

3′),EF2(5′GACATCACCAAGGGTGTGCAG-3′and

5′-TCAGCACACTGGCATAGAGGC-3′)withthefollowingcyclingconditions:10minat95°C,40cycles:

(7)

30secat95°C,1minat60°C,and45secat72°C,andfinal10minincubationat72°C.QPCRwasperformedinaStepOne- Plusreal-timePCRsystem(ThermoFisherScientific).TheEF2housekeepinggenewasusedasareference.

Westernblot.C

ellswerelysedinice-

coldRIPAbuffercontainingproteinaseinhibitors,centrifuged(10min,8000×g,4°C)andresuspendedinRIPAbuff er.Proteinsamples(50µgeach)andPageRulerPrestainedProteinLadder(ThermoFisher)weresubjectedtoSDS- PAGEgelelectrophoresisfollowedbyadrytransferofproteintoanitrocellulosemembrane.Membraneswereblo ckedinblockingbuffer(TBScontaining0.1%Tween20and5%offat-

freemilk)for1hatroomtemperatureandthenincubatedovernightat4°CwithantibodiesagainstHO- 1(1:750,EnzoLifeSciences,~32kDa)andα-

tubulin(1:1000,Calbiochem,~55kDa)asaloadingcontrol.After30minwashingstepinTBScontaining0.1%Tween 20,HRP-conjugatedsecondaryantibodieswereused:anti-mouseIgG(1:5000,BDBiosciences)andanti-

rabbitIgG(1:2000,CellSignalingTechnology).Allantibodiesweredilutedintheblockingbuffer.Afterthenextwashi ngstepvisualizationwasperformedusingSuperSignalWestPicochemiluminescencesubstrate(PierceBiotech nology)accordingtothemanufacturer’sinstructions.AnalysiswasperformedusingImageJsoftware.

TRAPassessment.T

orecognizetheactiveformofTRAP,TRAP5b,TRAPstaining(assessingintracellularen zymeactivity)andTRAPELISA(measuringtheconcentrationofenzymesecretedbybone-

resorbingOCLs)weredone.

ToassesstheintracellularTRAPenzymeactivity,BMMswereculturedon96- wellplatesatseedingdensityof100000cells/well.Specifically,BMCs-

derivedBMMswerestimulatedwith100ng/mlM-CSFand100ng/mlRANKL.Alternatively,BMCs- derivedBMMsandnBMCs-derivedBMMswerereplatedandculturedinthepresenceof30ng/mlM- CSFand50ng/mlRANKL.Asacontrol,cellsstimulatedexclusivelywithM-

CSFwereused.AfterincubationcellswerefixedandattheendofexperimentTRAP(TRAcP5b)wasdetectedusingAcid Phosphatase,Leukocyte(TRAP)Kit(Sigma-Aldrich)accordingtovendor’sprotocol.TRAP-

positivecellswith3ormorenucleiwerecountedusingtheOlympusIX81microscope(Olympus).

TheconcentrationofTRAPenzymeintheplasmaofHO1^−/−andHO-1^+/

+animalsandmediacollectedfromthecells(culturedasdescribedforTRAPstaining)wasmeasuredusingaMouseT RAP

™(T

RAcP5b)ELISAImmunoassaykit(Sigma-

Aldrich)accordingtothemanufacturer’sprotocol.Briefly,100μlofanti-mouse

TRAPantibodywasaddedtotheplatecoatedwithanti-rabbitIgGantibodiesandincubatedfor1hwithshak-

ing(850rpm,RT).Theplatewasthenrinsed3timeswithawashingbuffer(250μl)followedbytheadditionofplasmaorcu lturemedia(25μl),0.9%NaCl(75μl)andthereleasereagent(25μl),andincubatedfor1hwithshaking(850rpm,TP).Af terthistimethesubstratesolution(100μl)wasaddedfor2hat37°C.Afterstopping

thereactionbyadding25μlof1MNaOH,theabsorbancewasmeasuredatλ=405nmusingtheInfinite

®200

PROreader(Tecan).TRAPconcentrationwasassessedbasedonthestandardcurve.

CTX-1ELISA.T heconcentrationofC-telopeptideoftype1collagen(CTX-

1),collagenfragmentsreleasedupondegradationofTypeIcollagenbyosteoclasts,wereassessedintheplasmaofHO1^−/

−andHO-1^+/+animalsbyRatLapsEIAaccordingtomanufacturer’sinstructions.CTX- 1concentrationwasassessedbasedonthestand-ardcurve.

Stainingofactinstructures.n B

MCs-derivedreplatedBMMswereculturedon8-

wellchamberslidesatseedingdensityof100000cells/well.SubsequentlycellswerewashedwithPBS,fixedwith3.7%

formaldehydesolutioninPBSfor10min,permeabilizedwith0.5%TritonX- 100for5minandincubatedwith100nMAlexaFluor488-

phalloidin(Cytoskeleton,Inc)for30mininthedarkness.AfterwashingwithPBS,thecellswereincu-

batedwith100nMDAPIinPBSfor30secandthencoveredwithafluorescentmountingmedium(Dako)andcoverslides .PicturesweretakenwithaNikonEclipseTimicroscope.Sinceitisdifficulttodistinguishsmalloste-

oclasts(<5nuclei)withactinstructuresbasedonimmunofluorescencestaining,onlymultinucleateOCLs(>5nuclei)w ithclustersofpodosomesintheperipheryofthecellwerereliablydistinguishedandcounted.

Statisticalanalysis.A

lldataarepresentedasmeanofindependentexperiments±standarderror(SE M).EachexperimentusingRAW264.7cellswasdoneinduplicates.Themeanofduplicatewasconsideredasonei ndependentexperiment.Incaseofexperimentsusingmice-

derivedprimarycellsorbloodplasmaeachmousewasconsideredasoneindependentexperiment.Thenumberofi ndependentexperiments(n)isindicatedintheappropriatefigureslegends.Thehighestnnumbersareshownasdotso ntheappropriategraphs.Datawereana-lyzedusingunpairedStudent’st-

testtocomparetwogroupswithnormaldistribution(checkedwithShapiro- Wilktestwhenn>6)ornonparametricMann-

Whitneytest.Differenceswereacceptedasstatisticallysignificantwhenp<0.05.

Resul

ts TheeffectofHmox1knockoutinmiceonosteoclastsprecursors.

Amongthep opula-

tionofthefreshlyisolatedBMCsandM-CSF-treatedBMCs/nBMCs(BMMs),referredtoasOCLspre-

cursors,thepercentageofstrictlydefinedmonocytesandmacrophageswasassessed.Amongthewholepopul ationofBMCsinthefreshbonemarrowofHO-1^−/

(8)

−micethepercentageofmonocytesdefinedasCD45⁺Ly6G⁻Ly6C⁺CD11b⁺MHCII^low/−F4/80^low/

−washigherincomparisontoHO-1^+/+mice(7.43±0.19vs.

5.26±0.06,respectively,Fig.1A).However,HO-1^−/−andHO-1^+/

+monocytesshowedsimilarviability(Fig.1B)andproliferation(Fig.1C).AlowerpercentageofHO-1^−/

−macrophagesdefinedasCD45⁺Ly6G⁻F4/80⁺CD11b^+wasdetectedinfreshbonemarrow(0.64±0.08vs.2.33±0.1 8ofHO-1^+/+,Fig.1D).HO-1^−/

−macrophagesweremoreviableandcontainedalowerpercentageofearlyapoptoticcells(Fig.1E)(vs.HO-1^+/

+),whilenosignificant

(9)

Figure1.TheeffectofHmox1knockoutinmiceonosteoclastsprecursorsinfreshbonemarrow.

BonemarrowwasisolatedfromHO-1^−/−andHO-1^+/+mice.

(A)Thepercentageofmonocytes(CD45⁺Ly6G⁻Ly6C⁺CD11b⁺MHCII^low/−F4/80^low/

−)amongtotalBMCs,monocytes(B)survivaland(C)proliferation(G0/G1-non-proliferatingcells,S/G2M- proliferatingcells).

(D)Thepercentageofmacrophages(CD45⁺Ly6G⁻F4/80⁺CD11b⁺)amongtotalBMCs,macrophages(E)survivalan d(F)proliferation(G0/

G1-non-proliferatingcells,S/G2M-

proliferatingcells).Flowcytometry(n=3).Eachbarrepresentsthemean±SEM.^#p<0.05,^##p<0.01vs.HO-1^+/

+.

influenceofHO-

1deficiencyonproliferationofmacrophageswasnoticed(Fig.1F).ThegatingstrategyisshowninSupplementaryFig.S2.

AftertreatmentwithM-CSFtowardsmacrophageovergrowth,nogenotype-

dependentdifferencewasobservedinthepercentageofmacrophage(Fig.2A).Amongmacrophagepopulation,thepar ticipationofalive,apoptoticcells(Fig.2B)orproliferatingcells(Fig.2C),aswellastheproductionofROS(Fig.2D),wasc ompa-rablebetweentheHO-1^−/−andHO-1^+/+groups(gatingstrategyshowninSupplementaryFig.S3).Thus,HO- 1deficiencyseemsnottoaffectmacrophagesdifferentiationinthepresenceofM-CSF.

BoththelackofHmox1geneandHO-

1silencinginosteoclastsprecursorsdecreaseRANKL-

(10)

inducedexpressionofOCLsmarkers.

ThehypothesisthatHO- 1mightbeimportantfortheRANKL-

dependentinductionofosteoclastogenesisinOCLsprecursorswasverifiedusingthreealternativeexperimentalsetti ngsofthecultureofprimarycells.Overall,thelackofHmox1geneattenuateddifferentiation

(11)

Figure2.T heeffectofHmox1knockoutonthemacrophagesdifferentiationinthepresenceofM- CSF.BonemarrowwasisolatedfromHO-1^−/−andHO-1^{+/+m i c}e.nBMCswereculturedwith50ng/mlM- CSFfor3days.(A)Thepercentageofmacrophages(CD45⁺Ly6G⁻F4/80⁺CD11b⁺)amongM-CSF- stimulatednBMCs,macrophages(B)survival,(C)proliferation(G0/G1-non-proliferatingcells,S/G2M- proliferatingcells)and

(D)ROSproduction.Flowcytometry(n=3).Eachbarrepresentsthemean±SEM.

ofBMMs(BMCs-derivedBMMs,BMCs-derivedreplatedBMMs,andnBMCs-

derivedreplatedBMMs)towardsOCLs(Fig.3).TotalBMCsHO-1^−/−culturedinthepresenceofM- CSFanddirectlystimulatedwithRANKLasBMCs-

derivedBMMsfor3daysshowedlowernumberofTRAP+cells(2.31-folddecreasevs.HO-1^+/

+,Fig.3A).SimilarresultswereobtainedwhenHO-1-deficientBMCs-derivedBMMs(1.92- folddecreasevs.HO-1^+/+,Fig.3B)ornBMCs-derivedBMMs(1.64-folddecreasevs.HO-1^+/

+,Fig.3C),werereplatedpriortoRANKLstimulation.AssessmentofTRAPconcentrationintheculturemediu mreflectedthenumberofTRAP+cellsonlytosomeextent(Fig.3D,E,F,respectively).

Moreover,theinductionofOCLsmarkers,suchasNFATc-1andcathepsinK,inRANKL- stimulatedBMCs-derivedBMMswasdiminishedintheabsenceofHO-1bothat50ng/mlRANKL(4.74- foldforNFATc-1,3.67-foldforcathepsinKvs.HO-1^+/+)or100ng/mlRANKL(7.17-foldforNFATc-1,4.32- foldforcathepsinKvs.HO-1^+/+)

(Fig.4A,B).ThelowerconcentrationofRANKLwasenoughtoinducetheexpressionofOCLsmarkersindepende ntlyofgenotype(Fig.4A,B).Interestingly,suchchangesinthelevelofNFAT-

c1andcathepsinKwerenotobservedwhenBMCs-derivedBMMsHO-1^−/

−werereplatedandstimulatedwithRANKL(vs.HO-1^+/+)(Fig.4C,D).However,thiseffectofHO-

1deficiencywasconfirmedinRANKL-treatednBMCs-derivedreplatedBMMs(1.65-folddecreaseforNFATc- 1(p<0.05),3.43-folddecreaseforcathepsinKvs.HO-1^+/

+,Fig.4E,F).Ontheotherhand,podosomes,peripheralactin-

richadhesivestructurescharacteristicforOCLs,wereformedinresponsetoRANKLstimulationofBMMsindepend entlyofHO-

1presence(Fig.5).Nosignificantdifferencesinthenumberofcellswithdetectedactinstructureswerefoundbetweent heHO-1^−/−andHO-1^+/+groups(Fig.5),whichsuggeststhatHO-

1isdispensablefortheformationofactinstructurescharacteristicforOCLs.

siRNAtransfectioneffectivelyreducedHO-1mRNAexpressioninRANKL-stimulatednBMCs- derivedBMMs(5.22-folddecreasevs.scrambled,Fig.6A).Importantly,silencingofHO-

(12)

1loweredthelevelofbothNFAT-c1(1.96-folddecreasevs.scrambled,Fig.6B)andcathepsinK(1.89- folddecreasevs.scrambled,Fig.6C).Insum,HO-1deficiencyinOCLsprecursorsreducedRANKL- induceddifferentiationandtheexpressionofOCLsmarkers.

(13)

Figure3.TheeffectofHmox1knockoutinosteoclastsprecursorsonTRAP+cellsformation.Bonemarrowwasisolated fromHO-1^−/−andHO-1^{+/+m ic}e.(A,D)BMCs-

derivedBMMswerestimulatedwith100ng/mlRANKLinthepresenceof100ng/mlM-CSFfor3days.Alternatively, (B,E)BMCs-derivedBMMsand(C,F)nBMCs-

derivedBMMswerereplatedandculturedinthepresenceof50ng/mlRANKLand30ng/mlM-CSFfor3days.

(A,B,C)Quantitativeanalysisandrepresentativepictures(magnification400x,scalebar–

50µm)ofTRAP+cells(>2nuclei).TRAPstaining(nA=18–19,nB=9–11,nC=16–19).

(D,E,F)TRAPconcentrationintheculturemedium.TRAPELISA(n=5–

6).Eachbarrepresentsthemean±SEM.^#p<0.05,^##p<0.01vs.HO-1^+/+.

HO-1isdispensableforearly-

stageosteoclasts.W

econfirmedtheinhibitoryeffectofRANKLonHO-1expression²⁹^(2.06- folddecreasevs.control)aswellasdiminishedmRNAlevelofHO-1aftersiRNAtransfection(1.94- folddecreasevs.scrambled)inearly-stageOCLs(Fig.7A).TherewasnofurtherdecreaseofHO-

1mRNAuponcombinedsiRNAandRANKLtreatment(Fig.7A).siRNAtransfectiondidnotalsoinhibittheexpression ofOCLs-specificgenessuchasNFAT-c1orcathepsinK(Fig.7B,C,respectively).AlsointheRANKL-

stimulatedRAW264.7cellline,silencingofHO-1affectedneitherNFAT-c1norcathepsinKexpression(Fig.7D–

F,SupplementaryFig.S4).Takingtogether,aninhibitoryeffectofHO-

1deficiencyontheexpressionofOCLsmarkerswasnotreportedwhenHO-1expressionwassilencedinearlystage- OCLs.

(14)

TheHO-1

^−/−

mice havehigherlevelofTRAPintheplasma.

InHO-1^−/

−mice,agreaterconcentra-

tionofanactiveformofTRAPenzyme(isoform5b)intheplasmawasdetectedincomparisontoHO-1^+/+mice 67 ±0.06vs.0.24±0.08,respectively,Fig.8A).Ontheotherhand,thelevelofCTX-

1wassimilarintheplasmaofbothgenotypes(Fig.8B).

(15)

Figure4.T heeffectofHmox1knockoutinosteoclastsprecursorsonOCLs-

specificgenesexpression.BonemarrowwasisolatedfromHO-1^−/−andHO-1^{+/+m i c}e.(A,B)BMCs- derivedBMMswerestimulatedwithRANKL(50ng/mlor100ng/ml)inthepresenceof100ng/mlM- CSFfor3days.Alternatively,(C,D)BMCs-derivedBMMsand(E,F)nBMCs-

derivedBMMswerereplatedandculturedinthepresenceof50ng/mlRANKLand30ng/mlM-CSFfor3days.

(A,C,E)NFAT-c1and(B,D,F)cathepsinKrelativeexpression(vs.EF-2).QuantitativePCR(nA,B=9–10,n_C,D=7,n_E,F=17–

20).Eachbarrepresentsthemean±SEM.*p<0,05vs.RANKL;^#p<0.05vs.HO-1^+/+.

HeminandCoPPIXinhibitOCLs-

specificgenesexpression.H

eminandCoPPIXtreatmentforeither9h(p<0.05)or48hincreasedtheleve lofHO-1inRANKL-stimulatedRAW264.7cells(Fig.9A,B).Bothpharmaco-logicalinducersofHO-

1decreasedthelevelofNFAT-c1,enhancedbyRANKLalone,withastrongereffectobservedforCoPPIX(2.06- and19.86-

folddecreaseafter9hofheminandCoPPIXtreatment,respectively,Fig.9C,D).Interestingly,nosignificanteffectwas detectedinresponsetoSnPPIX,anHO-1inhibitor(Fig.9C,D).CoPPIXandheminstimulationofwildtypeRANKL- treatednBMCs-derivedBMMsincreasedHO-

1expressionalreadyata5μMconcentration,withCoPPIXhavingastrongereffectthanhemin(11.01±1.05vs.2.25±0.33,r espec-tively,Fig.9E).Bothcompoundsconcentration-dependentlydecreasedtheexpressionofNFAT-

c1(0.83±0.18,0.42±0.16,0.17±0.03at5,15and25μMhemin,respectively;0.21±0.03,0.03±0.01,0.02±0.01at5,15and 25μMCoPPIX,respectively,Fig.9F)andcathepsinK(0.84±0.22,0.48±0.15,0.29±0.05at5,15and25μMhemin, respectively;0.21±0.01,0.11±0.02,0.07±0.01at5,15and25μMCoPPIX,respectively,Fig.9G).Moreover,thenumb erofTRAP+cellswaslowerinresponsetoheminandCoPPIX(2.86-and6.67-

folddecrease,respectively,Fig.9H).Importantly,theinhibitoryeffectoftheseagentstowardsNFAT-

(16)

c1orcathepsinKwasalsoreportedinHO-1^−/

−cells(Fig.9F,G).Strikingly,inresponsetoheminandCoPPIXnoTRAP+cellsweredetected(Fig.9H).

(17)

Figure5.TheeffectofHmox1knockoutinosteoclastsprecursorsontheformationofactinstructures.Bonemarrowwasi solatedfromHO-1^−/−andHO-1^{+/+m i c}e.nBMCs-

derivedBMMswerereplatedandculturedwith100ng/mlRANKLfor5daysinthepresenceof30ng/mlM-

CSF.Quantitativeanalysis(left)ofmultinucleated(>5nuclei)cellswithcharacterizedactinstructuresandrepresentativ epictures(right,magnification400x,scalebar–

50µm).Actinstructures(green),nuclei(blue).Whitearrowsindicaterepresentativeclustersofpodosomesintheperiph eryofmultinucleatecells.Immunofluorescencestaining(n=2–

3,eachfrom12fieldsofview).Eachbarrepresentsthemean±SEM.

Figure6.T heeffectofHO-

1silencinginosteoclastsprecursorsontheexpressionofOCLsmarkers.BonemarrowwasisolatedfromHO-1^+/

+mice.nBMCs-derivedBMMswerereplatedandtransfectedwithsiRNAagainstHO-

1orscrambledcontrol.OnedayaftertransfectionfreshmediumcontainingRANKL(50ng/ml)andM-

CSF(30ng/ml)wasaddedforthenext3days.QuantitativePCR(n=5).Eachbarrepresentsthemean±SEM.^#p<0.05,^##

p<0.01vs.scrambledcontrol.

InHO-1^−/

−cellshemintreatmentresultedinthelowerlevelofOCLsmarkersthaninthewildtypecounterparts(Fig.9F,G,H).Ofnot

(18)

e,thestrongerincreaseofNFATc-1andcathepsinKinresponsetoCoPPIXinHO-1^−/−cells(vs.HO-1^+/

+)wasnoticedbutnosuchdifferencewasdetectedinthecaseofTRAP(Fig.9F,G,H).

(19)

1 0

Figure7.TheeffectofHO-1silencinginearly-stageOCLsontheexpressionofOCLsmarkers.(A–

C)nBMCsHO-1^+/+-derivedBMMsand(D–

F)RAW264.7cellswerereplatedwithRANKL(50ng/ml)andtransfectedwithsiRNAagainstHO- 1orscrambledcontrol24hlater(asearly-

stageOCLs).OnedayaftertransfectionproteinwascollectedorfreshRANKL- containingmediumwasaddedforthenext3days.(A,D-upperpanel)HO-1,(B,E)NFAT-

c1and(C,F)cathepsinKrelativeexpression(vs.EF-2).QuantitativePCR(n=3–4).(D-lowerpanel)HO- 1proteinlevel.α-tubulinwasusedasareference.Westernblot(n=2)–the

groupingofgel/blotcroppedfromdifferentpartsofthesamegelwasperformed(full-

lengthblotpresentedinSupplementaryFigureS4).Eachbarrepresentsthemean±SEM.*p<0,05vs.RANKL;^#p<0.

05,^##p<0.01vs.scrambledcontrol.

(20)

11

Figure8.TheeffectofHmox1knockoutonosteoclastsnumberandactivityinvivo.PlasmawasisolatedfromHO-1^−/

−andHO-1^+/+mice.(A)TRAPELISA(n=5–6).(B)CTX-1ELISA(n=7–

8).Eachbarrepresentsthemean±SEM.^##p<0.01vs.HO-1^+/+.

Nrf2deficiencystimulatestheexpressionofOCLsmarkers.

A lthoughtheHO- 1effectinOCLswasshowntobemorecomplexthanpreviouslysuggested^28–

30,inaccordancewithpreviousresults^33,34,weconfirmedtheinhibitoryeffectofitsupstreamregulator,theNrf2transcrip tionfactoronOCLsmarkersexpression.

AmongBMCs-derivedBMMsobtainedfromNrf2^−/

−mice,ahighernumberofTRAP+cellsinresponsetoRANKL(49.73-foldincreasevs.Nrf2^+/+at100ng/mlRANKL) (Fig.10A)wasdetected.AdditionaltreatmentofwildtypeBMCs-

derivedBMMswithsulphoraphane,aknownNrf2activator,gavenoTRAP-positivesig-

nal(Fig.10B).AlthoughthelevelofNFAT-c1waslowerinRANKL-treatedBMCs-derivedBMMsNrf2^−/−(vs.Nrf2^+/

+)

(Fig.10C),enhancedexpressionofintegrinβ3wasreported(Fig.10D).NosignificanteffectofNrf2deficiencywa sobservedforcathepsinK(Fig.10E).Importantly,whenNrf2-deficientBMCs-

derivedBMMswerereplatedandstimulatedwithRANKL,moreTRAP+cellsweredetectedincomparisontothewi ldtypecounter-parts(1.52-foldincreasevs.Nrf2^+/+,Fig.10F)asitwasfornBMCs-derivedBMMsNrf2^{−/−(7.41-} foldincreasevs.Nrf2^+/+,Fig.10G).

Discuss

ion

Theimportanceofantioxidantsaswellastheirupstreamregulatorsinosteoclastogenesishasbeenreportedand consideredasatherapeuticapproachtowardsdestructivebonediseases.Amongthreefundamentalmechanismsregula tingtheexpressionofcytoprotectiveenzymesincludingNrf2-,FOXO-,andsirtuin-

dependentpathways,eachwasreportedtoinhibitOCLsdifferentiationandbonedestructionviaattenuationofintracell ularROSsig-nalling¹⁷.RecentstudieshavealsosuggestedaninhibitoryeffectonosteoclastogenesisofHO-

1,theNrf2down-streamtarget^28–30.However,noneofthose,aimedatacomparisonoftheeffectofHO- 1deficiencyinOCLsprecursors,i.e.,beforestimulationbyRANKL,versusearly-

stageOCLs,i.e.,afterstimulationbyRANKL.WehypothesizedthatHO- 1involvementatdifferentstagesoftheosteoclastogenesismightvary.SinceHO-

1seemstobecriticalforthedifferentiationofOCLsprogenitors,stimulatingmyeloidlineagedifferentiationandcontrol lingmacrophagecondition³⁵,itmightbeimportantnotonlyasaROSscavengerattenuatingOCLsdifferentiationbutfort heinductionofosteoclastogenesisaswell.Here,weconfirmedtheinhibitionoftheexpressionofOCLsmark-

ersbyNrf2.WeshowedthatHO-1iscrucialfortheresponseofOCLsprecursorstoRANKLbutitisdispensableinearly- stageOCLs.

Themyeloid-

monocyticlineageofthebonemarrowisrecognizedastheoriginofmacrophagesand,atalaterstage,OCLs.Weshowedt hatHO-1knockoutresultsinthelowernumberofmacrophagesinmurinebonemar-

row.ThisagreeswiththepreviousfindingsbyWegieletal.pointingattheimportanceofHO-

1/COintheabilityofmyeloidprogenitorstodifferentiatetowardmacrophages³⁵.Ontheotherhand,inourexperimental setting,HO-1deficiencyseemsnottoaffectmacrophagesdifferentiationinthepresenceofM-

CSF.Nonetheless,tocheckifthereisaroleofHO-1intheinductionofosteoclastogenesis,theeffectofHO- 1deficiencyinOCLsprecursorstowardsdifferentiationtoOCLswasexamined.

Weshowedthatboth,thelackofHmox1geneandHO-1silencinginprimarymacrophagesdecreasedRANKL- induceddifferentiationtowardsOCLs.BecausethepreliminaryresultswiththeBMCs-

derivedBMMswereunexpectedinlightwhatwasknownbeforeontheeffectofHO- 1inosteoclastogenesis,wedecidedtocheckdifferentpopulationsofBMCs-

derivedcellsandstrategiesfordifferentiationtowardsOCLs.NeitherwhenusingBMCs-derivedBMMsnorBMCs- derivedreplatedBMMsornBMCs-derivedreplatedBMMsdidweconfirmtheinhibitoryeffectofHO-

1ontheRANKL-

inducedexpressionofOCLsmarkers,whichwereobservedbyothergroups^29,30.Instead,allthreecelltypesgavesimilarr

(21)

12

esultsofTRAPstaining(inresponsetoRANKL)whichiscon-

sideredasanimportantcytochemicalmarkerofOCLs.ThisprovesthattheeffectofHO-

1deficiencyobservedbyourgroupisnotspecifictoonetypeofOCLsprecursors/experimentalsetting.Inaddition,based ontheobtainedresults,wherenoeffectofHO-1wasdetectedonthenumberandviabilityofBMCs-

derivedmacrophages,theinhib-itoryinfluenceofHO-1deficiencyontheRANKL-

inducedexpressionofOCLsmarkersmightnotberelatedtotheconditionofprecursors.Furtherinvestigationsarenecess aryinordertofindtheunderlyingmechanism.

(22)

Figure9.CoPPIXandhemineffectonosteoclasts-specificmarkers.(A–

D)RAW264.7wereculturedwith50ng/mlRANKLand25μMCoPPIX/hemin/SnPPIXorNaOHasavehiclefor(A, C)9hor(B,D)48h.(A,B)

HO-1and(C,D)NFAT-c1relativeexpression(vs.EF-2).QuantitativePCR(n=5–6).(E–

H)BonemarrowwasisolatedfromHO-1^−/−andHO-1^{+/+m i c}e.(E,F,G)nBMCs-

derivedBMMswerereplatedandculturedfor3dayswith50ng/mlRANKLand30ng/mlM- CSFinthepresenceof5,15and25μMCoPPIX/heminorDMSOasavehicleor(H)totalBMCs- derivedBMMswerestimulatedwith100ng/mlRANKLand100ng/mlM-

CSFfor3daysinthepresenceof25μMCoPPIX/heminorDMSOasavehicle.(E)HO-1(inHO-1^+/+cells),(F)NFAT- C1and(G)cathepsinKrelativeexpression(vs.EF-2).QuantitativePCR(nE,F,G=4).

(H)QuantitativeanalysisofTRAP+cells(>2nuclei).TRAPstaining(n=4).Eachbarrepresentsthemean±SEM.*p<0 .05,

**p<0.05,***p<0.01vs.vehicle;^#p<0.05,^###p<0.001vs.HO-1^+/+.

(23)

Figure10.T heeffectofNrf2deficiencyonOCLsmarkersexpression.BonemarrowwasisolatedfromNrf2^−/

−andNrf2^+/+mice.(A–E)TotalBMCs-

derivedBMMswerestimulatedwithRANKL(50or100ng/mlwhereindicated),and2.5μMsulforaphanewhereindic ated,for3daysinthepresenceof100ng/mlM-CSF.Alternatively,(F)totalBMCs-derivedBMMsor(G)nBMCs- derivedBMMswerereplatedandtreatedwithRANKL(50ng/ml)inthepresenceofM-CSF(30ng/ml).

(A,B)Quantitativeanalysisandrepresentativepictures(magnification200x-upperpaneland400x- lowerpanel,scalebar–50µm)ofTRAP+cells.TRAPstaining(n=6).(C)NFAT-c1,

(D)integrinβ3and(E)cathepsinKrelativeexpression(vs.EF-2).QuantitativePCR(n=4–6).

(F,G)QuantitativeanalysisofTRAP+cells.TRAPstaining(n=3–

4).Eachbarrepresentsthemean±SEM.*p<0.05vs.RANKL;^#p<0.05,^##p<0.01vs.control/Nrf2^+/+.

(24)

Inthestudiesofothergroups,apopulationofnBMCswasusuallyusedtoobtainBMMs.Datapublishedsofarpointed attheinhibitoryeffectofHO-1inOCLsprecursorsontheirdifferentiationtoOCLs^29,30.Namely,theinductionofHO- 1withheminorcurcumininBMMsorRAW-

DmacrophagesinhibitedosteoclastogenesisandsuppressedthereleaseofOCLscytokine,highmobilitygroupbox1(H MGB1)²⁹.Moreover,suppressionofHO-1bysiRNAinRAW-

DcellspromotedtheactivationofHMGB1²⁹.Accordingly,moreTRAP+multinucleatedcellsweredetectedinRANK L-stimulatedBMMsderivedfromHO-1^−/−mice(vs.HO-1^+/+)³⁰.TheeffectofHO-

1wascorrelatedwithhememetabolites^29,31.BothbilirubinandCORM2(aCOdonor),butnotiron,inhibitedRANKL- mediatedHO-1suppressionandconsequentlyblockedosteoclastogenesis²⁹.Specifically,HO-

1/COaxissuppressedRANKL-inducedosteoclasticdifferentiationbyinhibitingredox-sensitiveNF- κBactivation³¹.Inourhands,thedatabasedonnBMCs-

derivedBMMswasnotconvincing,sotheexperimentwasrepeatedseveraltimes(high“n”number)toverifythefinaleff ectofHO-1deficiencyinthosecells.

Thedifferentstrainofthemiceusedforisolationordetailsinexperimentalprotocolmightlayattherootofobserveddi fferencesbetweenoursandothergroups.Oneneedstoalsorememberthatsomestromalcontami-

nationofBMCsmayaffecttheresultsandtheusedpopulationsofsocalledBMMsshouldnotbeconsideredasahomogen ouscultureofmacrophages.

Inaddition,wedemonstratedthatcommonlyusedpharmacologicalinducersofHO- 1,CoPPIX,andhemin,despitetheeffectiveincreaseofHO-1level,mayevokeHO-1- independenteffectsonOCLsmarkers.Thisphenome-

nonwasnotshowninpreviousstudies.Forexample,Yashimaetal.reportedthatCoPPIXinhibitsRANKL- inducedOCLsformationinadose-

dependentmannerthroughblockingmultiplesignallingpathwayssuchasAkt,ERK,p38MAPK,JNK,andIκBα³⁸.How ever,nodirectevidencewasdemonstratedinthatstudy[20]orintheotherreportsregardinganinvolvementofHO- 1inCoPPIX-orhemin-

dependentrepressionofosteoclastogenesis^28,29,38.Inourmodel,theinhibitoryeffectofCoPPIX(5–

25μM)andhemin(5–25μM)towardsOCLsmarkerswerealsoreportedinHO-1^−/−cells.Moreover,inHO-1^−/

−cellshemintreatment(25μM)resultedinthelowerlevelofOCLsmarkers.ThismayberelatedtosensitivityofHO-1- deficientcellstocytotoxicitycausedbyhemin³⁹.TheeffectofCoPPIXseemstobemorecomplicatedandneedsfurtherin vestigation.Thus,previousconclusionsontheinhibitoryeffectofHO-

1basedonlyontheuseofthesepharmacologicalagentsshouldbereconsidered.

Interestingly,weshowedthatHO-1isdispensablewhenosteoclastogenesisisalreadyinduced(inearly-

stageOCLs),whichhasnotbeenexploredsofar.Inaddition,anincreasedplasmalevelofTRAP(TRAcP5b)wasdetect edinHO-1^−/−mice.Thismayreflecttheincreasednumberofactivebone-

resorbingOCLsinsuchanimals.However,theplasmalevelofCTX- 1,amarkerofboneresorption,wassimilarinbothgenotypes.Thus,theinhi- bitionoftheactivityofmatureOCLsinvivobyHO-

1needtobefurtherverified.Importantly,Keetal.reportedadecreaseinthebonemassandelevatedserumCTX- 1andTRACP5blevelsinHO-1^−/

−miceunderphysiologicalconditions³⁰.Atthesametime,thereappearedtobenosignificantdifferenceinboneformation asindicatedbyserumPINPandosteocalcinlevels³⁰.

TheexplanationofobserveddifferencesofHO-1inOCLsprecursorsversusearly- stageOCLs,couldberelatedtotheself-amplificationoftheNFAT-

c1transcriptionfactor¹¹.ItcouldbebasedontheobservationthatHO-

1deficiencyinOCLsprecursorsinhibitedtheexpressionofNFAT-c1andultimatelyreducedtheabundanceofdiffer- entiatedcells.Ontheotherhand,whenHO-1deficiencyoccurredafterRANKL-induceddifferentiation,NFAT- c1signallingwasalreadyinducedandprobablysustainedbyNFAT-c1auto-amplification¹¹.TheeffectofHO-1defi- ciencyinvivoonthenumberofmatureOCLsisinagreementwithpreviousstudies^30anditsantioxidantactions.

Inconclusion,theHO-

1iscrucialfortheresponseofOCLsprecursorstoRANKLandtheinductionofOCLsmarkers,butitseemstobedispensa bleinRANKL-pre-stimulatedcellsconsideredasearly-stageOCLs.However,invivoHO-

1appearstoinhibitosteoclastogenesis.ThisshowsthatcautionshouldbegiventosuggestionsontheuseofHO- 1asatargetforthetreatmentofskeletaldiseases.TheeffectsofHO-

1inboneremodellingrequiresfurtheranalysisanditsmulti-facedactioninthoseprocessesshouldbeconsidered.

References

1. Boyle,W.J.,Simonet,W.S.&Lacey,D.L.Osteoclastdifferentiationandactivation.Nature423,337–42(2003).

2. Yasuda,H.etal.Osteoclastdifferentiationfactorisaligandforosteoprotegerin/osteoclastogenesis- inhibitoryfactorandisidenticaltoTRANCE/RANKL.Proc.Natl.Acad.Sci.USA95,3597–602(1998).

3. Nakagawa,N.etal.RANKistheessentialsignalingreceptorforosteoclastdifferentiationfactorinosteoclastogenesis.Biochem.Biophys .Res.Commun.253,395–400(1998).

4. Dougall,W.C.etal.RANKisessentialforosteoclastandlymphnodedevelopment.GenesDev.13,2412–24(1999).

5. Ross,F.P.&Teitelbaum,S.L.alphavbeta3andmacrophagecolony-stimulatingfactor:partnersinosteoclastbiology.Immunol.Rev.

208,88–105(2005).

6. Ross,F.P.M-CSF,c-Fms,andsignalinginosteoclastsandtheirprecursors.Ann.NYAcad.Sci.1068,110–6(2006).

7. Naito,A.etal.Severeosteopetrosis,defectiveinterleukin-1signallingandlymphnodeorganogenesisinTRAF6-deficientmice.

GenesCells4,353–62(1999).

8. Wong,B.R.etal.TheTRAFfamilyofsignaltransducersmediatesNF-kappaBactivationbytheTRANCEreceptor.J.Biol.Chem.

273,28355–9(1998).

9. Kobayashi,N.etal.SegregationofTRAF6-mediatedsignalingpathwaysclarifiesitsroleinosteoclastogenesis.EMBOJ.20,1271–80(2001).

10. Kim,J.H.&Kim,N.RegulationofNFATc1inOsteoclastDifferentiation.J.BoneMetab.21,233–41(2014).

11. Takayanagi,H.TheroleofNFATinosteoclastformation.Ann.NYAcad.Sci.1116,227–37(2007).

(25)

12. Asagiri,M.etal.AutoamplificationofNFATc1expressiondeterminesitsessentialroleinbonehomeostasis.J.Exp.Med.202,1261–

9(2005).

13. Asagiri,M.&Takayanagi,H.Themolecularunderstandingofosteoclastdifferentiation.Bone40,251–64(2007).

14. Lee,N.K.etal.AcrucialroleforreactiveoxygenspeciesinRANKL-inducedosteoclastdifferentiation.Blood106,852–9(2005).

15. Wang,Y.etal.IdentifyingtherelativecontributionsofRac1andRac2toosteoclastogenesis.J.BoneMiner.Res.23,260–70(2008).

16. Sasaki,H.etal.NADPHoxidase-

derivedreactiveoxygenspeciesareessentialfordifferentiationofamousemacrophagecellline(RAW264.7)intoosteoclasts.J.Med.Invest .56,33–41(2009).

17. Kanzaki,H.etal.Molecularregulatorymechanismsofosteoclastogenesisthroughcytoprotectiveenzymes.RedoxBiol.8,186–91(2016).

(26)

18. Bharti,A.C.,Takada,Y.&Aggarwal,B.B.Curcumin(diferuloylmethane)inhibitsreceptoractivatorofNF-kappaBligand-inducedNF- kappaBactivationinosteoclastprecursorsandsuppressesosteoclastogenesis.J.Immunol.172,5940–7(2004).

19. Bax,B.E.etal.Stimulationofosteoclasticboneresorptionbyhydrogenperoxide.Biochem.Biophys.Res.Commun.183,1153–8(1992).

20. Alam,J.eta l.Nrf2,a Cap’n’Collartranscriptionfactor,r egulatesinductiono ftheh emeoxygenase-1gene.J.Biol.Chem.274,26071–8(1999).

21. Baird,L.&Dinkova-Kostova,A.T.ThecytoprotectiveroleoftheKeap1-Nrf2pathway.Arch.Toxicol.85,241–72(2011).

22. Yang,Y.C.etal.Docosahexaenoicacidinhibitionofinflammationispartiallyviacross-talkbetweenNrf2/hemeoxygenase1andIKK/NF- kappaBpathways.J.Nutr.Biochem.24,204–12(2013).

23. Grochot-Przeczek,A.etal.Hemeoxygenase-1isrequiredforangiogenicfunctionofbonemarrow- derivedprogenitorcells:roleintherapeuticrevascularization.Antioxid.RedoxSignal.20,1677–92(2014).

24. Kozakowska,M.etal.Hemeoxygenase-1inhibitsmyoblastdifferentiationbytargetingmyomirs.Antioxid.RedoxSignal.16,113–

27(2012).

25. Toobiak,S.,Shaklai,M.&Shaklai,N.CarbonmonoxideinducederythroiddifferentiationofK562cellsmimicsthecentralmacrophage milieuinerythroblasticislands.PLoSOne7,e33940(2012).

26. Barbagallo,I.etal.Overexpressionofhemeoxygenase-1increaseshumanosteoblaststemcelldifferentiation.J.BoneMiner.Metab.

28,276–88(2010).

27. Loboda,A.,Damulewicz,M.,Pyza,E.,Jozkowicz,A.&Dulak,J.RoleofNrf2/HO-

1systemindevelopment,oxidativestressresponseanddiseases:anevolutionarilyconservedmechanism.CellMol.LifeSci.73,3221–

47(2016).

28. Zwerina,J.etal.Hemeoxygenase1(HO-1)regulatesosteoclastogenesisandboneresorption.FASEBJ.19,2011–3(2005).

29. Sakai,E.etal.SuppressionofRANKL-dependenthemeoxygenase-

1isrequiredforhighmobilitygroupbox1releaseandosteoclastogenesis.J.CellBiochem.113,486–98(2012).

30. Ke,K.etal.Hemeoxygenase-1maintainsbonemassviaattenuatingaredoximbalanceinosteoclast.Mol.CellEndocrinol.409,11–

20(2015).

31. Bak,S.U.etal.Hemeoxygenase-1(HO-1)/carbonmonoxide(CO)axissuppressesRANKL-

inducedosteoclasticdifferentiationbyinhibitingredox-sensitiveNF-kappaBactivation.BMBRep.50,103–108(2017).

32. Lu,S.H.,Chen,T.H.&Chou,T.C.MagnololInhibitsRANKL-

inducedosteoclastdifferentiationofraw264.7macrophagesthroughhemeoxygenase-1- dependentinhibitionofNFATc1expression.J.Nat.Prod.78,61–8(2015).

33. Kanzaki,H.,Shinohara,F.,Kajiya,M.&Kodama,T.TheKeap1/Nrf2proteinaxisplaysaroleinosteoclastdifferentiationbyregulatingin tracellularreactiveoxygenspeciessignaling.J.Biol.Chem.288,23009–20(2013).

34. Rana,T.,Schultz,M.A.,Freeman,M.L.&Biswas,S.LossofNrf2acceleratesionizingradiation- inducedbonelossbyupregulatingRANKL.FreeRadic.Biol.Med.53,2298–307(2012).

35. Wegiel,B.etal.Hemeoxygenase-1derivedcarbonmonoxidepermitsmaturationofmyeloidcells.CellDeathDis.5,e1139(2014).

36. Takahashi,N.,Udagawa,N.,Tanaka,S.&Suda,T.Generatingmurineosteoclastsfrombonemarrow.MethodsMol.Med.80,129–

44(2003).

37. Dworzak,M.N.etal.AIEOP-

BFMconsensusguidelines2016forflowcytometricimmunophenotypingofPediatricacutelymphoblasticleukemia.CytometryB.Cli n.Cytom.(2017).

38. Yashima,Y.etal.Cobaltprotoporphyrinrepressesosteoclastogenesisthroughblockingmultiplesignalingpathways.Biometals28,725–

32(2015).

39. Poss,K.D.&Tonegawa,S.Reducedstressdefenseinhemeoxygenase1-deficientcells.Proc.Natl.Acad.Sci.USA94,10925–30(1997).

Acknowledgements

ThisworkwassupportedbyIuventusPlusgrantfromtheMinistryofScienceandHigherEducation(0244/

IP1/2013/72).FacultyofBiochemistry,BiophysicsandBiotechnologyofJagiellonianUniversityisapartneroftheLea dingNationalResearchCentre(KNOW)supportedbytheMinistryofScienceandHigherEducation.Wearegratefultop rof.AnupamAgarwal(UniversityofAlabamaatBirmingham,USA)forprovidingthefoundingpairsofHO-1^+/

−m i c eandProf.AntonioCuadrado(UniversidaddeAutonoma,Madrid,Spain)andProf.MasayukiYamamoto(Tohoku University,Sendai,Japan)forprovidingthefoundingpairsofNrf2KOmice.

AuthorContributions

U.F.-S.wrotethemainmanuscripttextandpreparedfigures.U.F.-S.,E.J.,M.M.andM.V.performedexperiments.

A.J.,K.B.-

S.,J.S.andW.N.N.providedmethodologicalhelp.J.D.supervisedthewholeworkandwrotethemanuscript.Allautho rsacceptedthemanuscript.

AdditionalInformation

Supplementaryinformationaccompaniesthispaperathttps://doi.org/10.1038/s41598-018-29122-1.

CompetingInterests:Theauthorsdeclarenocompetinginterests.

Publisher'snote:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffili ations.

OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense, whichpermitsuse,sharing,adaptation,distributionandreproductioninanymediumor

format,aslongasyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCre-

ativeCommonslicense,andindicateifchangesweremade.Theimagesorotherthirdpartymaterialinthisarticleareincl udedinthearticle’sCreativeCommonslicense,unlessindicatedotherwiseinacreditlinetothematerial.Ifmaterialisnotinclud edinthearticle’sCreativeCommonslicenseandyourintendeduseisnotper-

mittedbystatutoryregulationorexceedsthepermitteduse,youwillneedtoobtainpermissiondirectlyfromthecopyrightholder.To viewacopyofthislicense,visithttp://creativecommons.org/licenses/by/4.0/.