FoxC2 Enhances BMP7-Mediated Anabolism in Nucleus Pulposus Cells of the Intervertebral Disc

17 Pages·2016·1.93 MB·English

RESEARCHARTICLE
FoxC2 Enhances BMP7-Mediated Anabolism
in Nucleus Pulposus Cells of the Intervertebral
Disc
ZhengWang1,ChangfengFu1,YongChen2,FengXu1,ZhenyuWang1,ZhigangQu1*,
YiLiu1*
1 DepartmentofSpinalSurgery,TheFirstHospitalofJilinUniversity,No.71,XinminAvenue,Chaoyang
District,Changchun,JilinProvince130021,China,2 DepartmentofNeurosurgery,TheFirstHospitalofJilin
University,No.71,XinminAvenue,ChaoyangDistrict,Changchun,JilinProvince130021,China
*[email protected](YL);[email protected](ZQ)
Abstract
a11111
Bone-morphogeneticprotein-7(BMP-7)isagrowthfactorthatplaysamajorroleinmediat-
inganabolismandanti-catabolismoftheintervertebraldiscmatrixandcellhomeostasis.In
osteoblasts,ForkheadboxproteinC2(FoxC2)isadownstreamtargetofBMPsandpro-
motescellproliferationanddifferentiation.However,theroleFoxC2mayplayindegenera-
tivehumanintervertebraldisctissueandtherelationshipbetweenFoxC2andBMP-7in
OPENACCESS nucleuspulposus(NP)cellsremaintobeelucidated.Thisstudyaimstoinvestigatethe
Citation:WangZ,FuC,ChenY,XuF,WangZ,Qu presenceandsignalingmechanismsofFoxC2indegenerativehumanintervertebraldisc
Z,etal.(2016)FoxC2EnhancesBMP7-Mediated tissueandNPcells.Westernblotandreal-timequantitativereversetranscriptionpolymer-
AnabolisminNucleusPulposusCellsofthe
asechainreaction(qRT-PCR)analyseswereusedtomeasureFoxC2expressionintheNP
IntervertebralDisc.PLoSONE11(1):e0147764.
tissueandcells.TransfectionswerecarriedouttomeasuretheeffectofFoxC2onBMP-7-
doi:10.1371/journal.pone.0147764
mediatedextracellularmatrixupregulation.Adenoviralknock-downofSmad1wasper-
Editor:DraganaNikitovic-Tzanakaki,Universityof
formedtoinvestigatethemechanismofBMP-7-inducedFoxC2expression.Indegenerative
Crete,GREECE
NPtissue,FoxC2wasmarkedlyupregulatedandpositivelycorrelatedwithincreaseddisc
Received:August28,2015
degeneration.InductionofNPcellproliferationwasconfirmedbyusingcellcountingkit-8
Accepted:January7,2016
assay,immunocytochemistryandreal-timeqRT-PCRforKi67.FoxC2ledtodecreased
Published:January29,2016 nogginexpressionandincreasedSmad1/5/8phosphorylation.Duringcombinedtreatment
Copyright:©2016Wangetal.Thisisanopen withBMP-7,FoxC2greatlypotentiatedanabolismthroughsynergisticmechanismsonECM
accessarticledistributedunderthetermsofthe formation.CombinationtherapyusingBMP-7andFoxC2maybebeneficialtothetreatment
CreativeCommonsAttributionLicense,whichpermits
ofintervertebraldiscdegeneration.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Theminimaldatafor
Table1andFigs1–7hasbeenuploadedtoFigshare
Introduction
(http://dx.doi.org/10.6084/m9.figshare.2027862).
Funding:Theauthorsreceivednospecificfunding Degenerativediscdisease(DDD)isthoughttobeamajorcauseoflowerbackpainwhichinter-
forthiswork. fereswithnormalactivitiesandimpactstheabilitytowork[1].AlthoughtheprocessofDDD
remainsunclear,progressivebreakdownoftheextracellularmatrix(ECM)isthoughttobeone
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. ofthemostimportantcausativefactors.ChangesinthestructureoftheECMareassociated
PLOSONE|DOI:10.1371/journal.pone.0147764 January29,2016 1/17
FoxC2PromotesAnabolisminDiscDegeneration
withadversegenetic,biomechanicalandnutritionalfactors[2].InthecourseofDDD,the
breakdownoftheECMfromagechangesandadversebiomechanicalloadingcanbeenhanced
byageneticallyinferiormatrix[3].Theinterplayamongdiversifiedcytokines,growthfactors,
matrix-degradingenzymesandtheirinhibitorsformsanintricatebalancebetweenanabolic
andcatabolicprocesses[4,5].Amongthese,matrixmetalloproteases(MMPs)andaggreca-
nasesincreaseexpressionofmatrix-degradingenzymes,andbothareinducedbyvarious
proinflammatorycytokines[6–9].Hiyamaetal.foundactivationofWNT/β-cateninsignaling
promotesthebreakdownofthematrixandmaymodulateMMPandTGFβsignalinginNP
cells[10].Inaddition,previousreportssuggestthatTGF-βsignalingpathwayisimportantin
theregulationofdegenerativeprocessesinIVDs[11,12].Indegenerativediscs,aggrecanand
collagenIIareobservedtobemostnotablydownregulatedintheECM[13].Toamelioratepro-
gressionofdiscdegeneration,growthfactorshavebeenusedtoshiftthemetabolicstatusfrom
catabolictoanabolictoreestablishtheirbalance[14].Furtherunderstandingofthepathophys-
iologicalmechanismsofDDDmayhelpverifybiologicaltreatments,capableofpromoting
ECMrepairandregeneration[15].
Bone-morphogeneticproteins(BMPs),whichconstitutethelargestsubgroupofthetrans-
forminggrowthfactor-β(TGF-β)superfamily,areoftenusedastherapeuticfactorsintreating
discdegeneration[16,17].BMP-7isexpressedinNPcellswherebyitexertspotenteffectson
cellanabolismanddifferentiation[18].BMP-7canstimulatetheproductionandformationof
aggrecanandCollagenIIviatheSmad1/5/8signalingpathways[19].Recently,Zhangetal.
foundBMP-7hasasimilaranaboliceffectonECMmetabolisminbovineintervertebraldisc
(IVD)cells[20].Furthermore,similarfindingsarereportedbyMasudaandImaiinrabbitor
humanIVDcells,providingfurtherevidencesforthefunctionalroleBMP-7playsindecreas-
ingintervertebraldisc(IVD)degeneration[21,22].Takegamietal.reportthatafterECM
depletioninresponsetoIL-1,BMP-7caneffectivelyincreasetheexpressionofIVDECM
genesbyrabbitNPcells[23].TheseresultsdemonstratetheimportanceofBMP-7signaling
pathwaysduringtheoccurrenceanddevelopmentofDDD,andsupportusingBMP-7forthe
treatmentofDDDisafeasiblebiologicalapproach.
FoxC2isamemberoftheforkheadbox(FOX)familyoftranscriptionfactors.Whenthe
abbreviationofFOXC2containsalluppercaseletters,thisreferstohumanform.WhenFoxc2
hasonlythefirstlettercapitalized,thisreferstomouse,andwhenFoxC2hasthefirstandsub-
classletterscapitalized,thisreferstoallchordates[24].Foxc2hasbeenshowntobeinvolvedin
cancermetastases,epithelial-mesenchymaltransition(EMT)andlymphedema-distichiasis
syndrome[25,26].
Recently,Nifujietal.haveshownthatfollowingtreatmentofskeletalprecursorswith
BMPs,Foxc2mayplayaroleintheearlystageofchondrogenicdifferentiation,because
increasedexpressionofFoxc2isobservedinrelationtoSOX9,nogginandaggrecan[27].Fur-
thermore,Parketal.havefoundthatFoxc2isadownstreamtargetofBMP-2andpromotes
thesurvival,proliferationanddifferentiationofosteoblasts[28].Recentstudiesimplythat
BMPsignalingisrequiredforFoxc2expressionandthatFoxc2actsasamodulatorofthepath-
way[29].AsimilarstudyhasfoundthatforcedexpressionofFOXC2inC2C12cellsresultsin
upregulationofBMP-4[30].Overall,theseresultssuggestthatFoxc2isavitalelementofECM
secretionviafacilitationofBMPsignalingpathwaysandfailureofproperFoxc2transcriptreg-
ulationmayresultinDDD.BMPsplayanimportantroleinregulatinganabolicprocessesof
theNPcellsinwhichFoxC2mayhavesignificanteffectinDDDpathogenesis.Thekeyques-
tionweaddresshereistodemonstratetherelationshipbetweenFoxC2anddegenerative
humanintervertebraldisc,aswellasthefunctionalroleofFoxC2ontheproliferationand
anabolismofprimaryculturedratNPcells.Specifically,wealsoelucidatetheunderlying
molecularmechanismbetweenFoxC2andBMPsignalingpathways.
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FoxC2PromotesAnabolisminDiscDegeneration
MaterialsandMethods
Ethicsstatement
ThestudyprotocolwasapprovedbytheInstitutionalEthicsCommitteeoftheFirstHospitalof
JilinUniversity.HumanlumbarandcervicalIVDsampleswereobtainedfrompatientsunder-
goingspinalsurgeriesduringMarch5th,2014toMarch8th,2015,followingapprovalfromthe
InstitutionalReviewBoardoftheFirstHospitalofJilinUniversity.Patientsconsentedtothe
collectionanduseoftissuesamplesforresearchandprovidedwritteninformedconsentinall
cases.Allthehumantissuesamplesusedforthisstudywereobtainedfrompatientsasastan-
dardpartofthepatients'spinalsurgerieswithoutadditionalbiopsies.Allanimalexperiments
werecarriedoutinahumanemannerafterreceivingapprovalfromtheInstitutionalAnimal
ExperimentCommitteeofJilinUniversityandinaccordancewiththeRegulationforAnimal
ExperimentsandFundamentalGuidelineforProperConductofAnimalExperimentsofour
university.
Reagents
BMP-7inhibitornogginwaspurchasedfromProtect(RockyHill,NJ,USA).Allotherchemi-
calsandreagentswerepurchasedfromSigma(Oakville,ON,Canada)unlessotherwise
specified.
Patientsandsamples
PatientsampleswerecollectedandisolatedaspreviouslydescribedbyYuetal[31].Wecol-
lectedhumanlumbarNPspecimensfrom36patientswithlumbardiscdegeneration(average
age47.36±6.651,range33–57years)and4patientswithidiopathicscoliosisasacontrol(aver-
ageage19.75±1.258,range18–21years).Allthesepatientshadtakenroutinemagneticreso-
nanceimaging(MRI)scansofthelumbarspinebeforetheoperation.AmodifiedPfirrmann
classificationwasusedtogradethedegreeofdiscdegenerationfromT2-weightedimages.
AccordingtothemodifiedclassificationsystemoftheInternationalSocietyfortheStudyofthe
LumbarSpine[32],7ofthesepatientsweresequestration(S),10weretransligamentousextru-
sion(TE),8weresubligamentousextrusion(SE)and11wereprotrusions(p).Fourofthe36
sampleswereobtainedfromthelevelofL3–L4,17fromL4–L5,and14fromL5–S1.After
excludingtheherniationandgranulationtissue,wewashedthesetissuespecimensthreetimes
withphosphate-bufferedsaline(PBS)andseparatedNPfromtheannulusfibrosus(AF)using
astereotaxicmicroscopeandfinallyfrozentheminliquidnitrogen(Table1).
RatNPcellsisolationandculture
DisctissuewasharvestedfrommaleWistarrats(200–250g).Alltheratswerepurchasedfrom
AnimalExperimentalCenterofJilinUniversityandwerekilledwithalethaldoseof10%chlo-
ralhydrate.LumbarandtailIVDswereseparatedfromthespinalcolumnunderasepticcondi-
tions.AfterseparationoftheNP,thetissuewascutinto1-mm3blocks,whichweretreated
separatelywith0.1%collagenaseIIfor4‒6hours[33].NPcellswerereleasedbyenzymatic
digestioninDulbecco’smodifiedEagle’smedium(DMEM)and10%fetalbovineserum(FBS)
supplementedwith50U/mLpenicillinand50μg/mLstreptomycininahumidifiedatmo-
spherecontaining5%CO at37°C.Cellswerethenpassagedandfurtherculturedinthepres-
2
enceofBMP-7(10‒100ng/ml).Onreachingapproximately80%confluence,cellswere
subculturedin60-mmculturedishes(4×105cells/well).Allexperimentsinthisstudyused
first-orsecond-generationcells.
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FoxC2PromotesAnabolisminDiscDegeneration
Table1. ClinicalFindingin50PatientswithLDH.
PatientNo. Sex Age(yr) Level TypeofLDH MRIScores
1 F 48 L5/S1 TE 4
2 F 33 L4/L5 TE 2
3 M 43 L5/S1 S 5
4 F 51 L4/L5 SE 4
5 F 39 L4/L5 TE 3
6 M 45 L5/S1 P 4
7 F 55 L4/L5 SE 5
8 M 52 L5/S1 S 5
9 M 43 L4/L5 P 4
10 M 38 L4/L5 SE 2
11 F 53 L4/L5 SE 5
12 M 47 L3/L4 P 4
13 F 41 L5/S1 P 3
14 F 52 L5/S1 SE 4
15 F 49 L4/L5 TE 5
16 F 55 L3/L4 SE 4
17 M 39 L4/L5 P 3
18 M 51 L4/L5 S 5
19 F 53 L5/S1 S 4
20 M 51 L5/S1 TE 4
21 M 43 L4/L5 P 3
22 M 48 L5/S1 TE 5
23 F 56 L5/S1 TE 3
24 F 36 L3/L4 P 4
25 M 57 L4/L5 TE 4
26 M 46 L5/S1 P 5
27 F 52 L3/L4 S 5
28 M 54 L4/L5 TE 3
29 M 43 L5/S1 P 5
30 M 51 L4/L5 SE 5
31 M 53 L5/S1 S 4
32 F 46 L5/S1 P 3
33 F 42 L4/L5 S 5
34 F 52 L4/L5 TE 4
35 M 55 L4/L5 SE 3
36 F 33 L4/L5 P 3
LDH,lumbardischerniation;F,indicatesfemale;M,male;P,protrusion;S,sequestration;SE,subligamentousextrusion;TE,transligamentousextrusion;
L,lumbar;S,sacral;MRI,magneticresonanceimaging
doi:10.1371/journal.pone.0147764.t001
Celltransfection
Accordingtothemanufacturer’sinstructions,Lenti-FoxC2,Lenti-GFPorscramblewastrans-
fectedintothecellsbyDharmaFECT1Reagent(Dharmacon,TX,USA)atafinaloligonucleo-
tideconcentrationof10nmol/L.Foreachcelltransfection,tworeplicationexperimentswere
performed.
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FoxC2PromotesAnabolisminDiscDegeneration
Westernblotanalysis
Cellandtissuelysateswerepreparedusingmodifiedradioimmunoprecipitationassay(RIPA)
bufferaspreviouslydescribed[34].Totalproteinconcentrationsofcelllysatesweredetermined
usingthebicinchoninicacid(BCA)proteinassay(Pierce,Rockford,IL,USA).Equalamounts
ofprotein(35μgperlane)wereresolvedby10%SDS-PAGEandtransferredtoanitrocellulose
membraneforwesternblotanalysesasdescribedpreviously[35].Immunoreactivitywasvisual-
izedusingtheECLsystem(AmershamBiosciences,Piscataway,NJ,USA).
Real-timePCR
PCRamplificationwascarriedoutinaccordancewiththepreviouslydescribedmethod.Total
RNAwasisolatedusingTRIzolreagent(Invitrogen,Carlsbad,CA)followingthemanufactur-
er’sinstructions.cDNAwasthenreversetranscribedaccordingtothemanufacturer’sinstruc-
tions.QuantitativePCRwascarriedoutusingaSYBRgreenJumpstartTaqReadyMix
(TaKaRa)onaRocheLightCycler480.AthresholdcycleforeachPCRamplificationwassub-
jectedto35cyclesof95°Cfor15sand54°Cfor2min.Theprimersequencesandtheircondi-
tionsforuseweresummarizedinTable2.Glyceraldehyde3-phosphatedehydrogenase
(GAPDH)wasusedtonormalizeexpressionlevels.
Immunofluorescencemicroscopy
Cellswereplatedinflatbottom96-wellplates(4×103/well)andtreatedwithBMP-7for1‒24
h.Afterincubation,cellswerefixedwith4%paraformaldehyde,permeabilizedwith0.2%tri-
ton-X100inPBSfor10min,blockedwithPBScontaining5%FBS,andincubatedwithanti-
bodiesagainstFoxC2(1:200),aggrecan(1:100),SOX9(1:200),andMMP-2(1:200)at4°C
overnight.Asanegativecontrol,cellswerereactedwithisotypeIgGundersimilarconditions.
Afterwashing,thecellswereincubatedwithDyLightTM488-conjugatedgoatanti-rabbitanti-
bodyandfluorescein-orDyLightTM594-conjugatedgoatanti-mouseantibody(JacksonLabo-
ratory),atadilutionof1:200for45minatroomtemperature.Fluorescencesignalswere
capturedusinganOlympusFluoviewFV1000confocalmicroscopeandanalyzedbythe
FV10-ASW1.6Viewerprogram(Olympus,Japan).
CellCountingKit-8(CCK8)assay
NPcellswereseededin96-wellplatesatadensityof1,000cellsperwellwith100μlofDMEM/
F12medium.Cellswerethenculturedforanother0,24,48,72or96h.Thesupernatantswere
removedand100μlofDMEM/FBSmediumcontaining10μlofCCK-8wasaddedtoeachwell
priortoincubatingfor3hat37°C.Thecultureplateswerethenshakenfor10minandthe
opticaldensity(OD)valueswerereadat450nm.
Statisticalanalysis
Allmeasurementswereperformedintriplicateanddatawerepresentedasmeans±standarddevi-
ation(SD).DifferencesbetweengroupswereanalyzedbyStudent’sttest;(cid:1)p<0.05,(cid:1)(cid:1)p<0.01.
Results
ExpressionofFOXC2indegenerativeNPtissueandcorrelationwith
degenerationgrade
Ofthe36DDDpatients,19femalesand17males,theaverageageofthesepatientswas47.36
±6.651(range33–57years).Theclinicalfindingsforthesepatientsweresummarizedin
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Table2. PrimerSequencesforPCR.
Primers Forward Reverse Tm(°C)
FoxC2 5’-CGCCTAAGGACCTGGTGAAG-3’ 5’-GGAAGCGGTCCATGATGA-3 56
BMP-7 5’-GGTGGCAGGACTGGATCATC-3’ 5’-GCGCATTCTCCTTCACAGTAATAC-3’ 56
Aggrecan 5’-AAGGACTGTCTATCTGCACGCCAA-3’ 5’-TCACCACCCACTCCGAAGAAGTTT-3’ 56
CollagenTypeII 5’-TCATAAGGATGTGTGGAAGCCCGA-3’ 5’-GGCTGAGGCAGTCTTTCATGTCTT-3’ 56
Noggin 5’-AGCGAGATCAAAGCGCTGGAGTT-3’ 5’-TCTGTAACTTCCTCCGCAGCTTCT-3’ 56
Ki-67 5’-TCCTTTGGTGGGCACCTAAGACCTG-3 5’-TGATGGTTGAGGTCGTTCCTTGATG-3 56
doi:10.1371/journal.pone.0147764.t002
Table1.FOXC2expressioninthedegenerativeNPtissuewassignificantlyhighercomparedto
theidiopathicscoliosisNPtissueasdeterminedbyreal-timePCR(Fig1A).Withrespecttothe
expressionofFOXC2inthe36DDDpatients(Fig1Band1C),nosignificantdifferencewas
observedbetweensamplesfromdifferentherniationtypesorgenders.However,expressionof
FOXC2waspositivelycorrelatedwiththediscdegenerationgrade(n=36,r=0.881,p<0.05).
ImmunofluorescenceandReal-TimePCRvalidationofprimarycultured
ratNPcells
Afterapproximately7‒10days,thePrimaryNPcellsreachedcompleteconfluence.Thecells
wereroundorpolygonalwithagranularcytoplasm.CollagenIIandaggrecanexpressionin
thesecellswereobservedbyimmunofluorescence(Fig2Aand2B).Asearlierstudiesindicated,
collagentypeIhadsignificantlylowerexpressionlevelsinNPcellsandNPtissuethaninAF
tissue(Fig2C),whilstCollagenIIwasfoundtohavesignificantlyhigherexpressionlevelsin
NPcellsandNPtissuethaninAFtissue(Fig2D).
FoxC2inducedNPcellsproliferation
Todeterminewhetherup-regulationofFoxC2inNPcellsinducesurvival,proliferationand
differentiationofNPcells,wetransfectedLenti-FoxC2inNPcellsandtransfectedLenti-GFP
asthecontrol.TransfectingLenti-FoxC2inNPcellssignificantlyincreasedtheexpressionof
Ki-67,afamousproliferativemarker,inimmunofluorescencestaining(Fig3A).Compared
withthecontroloruntreatedgroups,thepercentageofKi-67-positiveNPcellswasobviously
increasedinthegrouptransfectedwithLenti-FoxC2.Also,transfectingLenti-FoxC2inNP
cellsinducedtheup-regulationofKi-67mRNAlevelasatime-dependentmanner,andtheKi-
67mRNAlevelwassignificantlyincreasedafter48hand72hwhencomparedtocontrol(Fig
3B).WeusedCCK-8proliferationassaytocomparetheeffectsofProliferationbetweenLenti-
FoxC2andLenti-GFPtransfectedtoNPcells.AsshowninFig3Ctherewasasignificant
increaseofcellproliferationintheLenti-FoxC2transfectedNPcellscomparedwiththe
control.
BMP-7inducesFoxC2expressioninNPcells
Asmentionedabove,BMPslikelyplayedanimportantroleduringtheprocessofFoxC2
expression[25].WeinvestigatedwhetherBMP-7couldinduceFoxC2expressioninNPcells.
Immunofluorescenceanalysisconfirmedthisresult,showingthatBMP-7treatment(50‒100
ng/ml,2h)promotedtheexpressionofFoxC2protein(Fig4A),andhigherconcentrationsof
BMP-7hadamoresignificanteffectcomparedwithlowerconcentrationsandthecontrol.
FoxC2proteinwasinducedbyaddingBMP-7totheculturemediuminadose-dependentand
time-dependentmanner.Thereal-timeqPCRresultsdemonstratedthatstimulationofratNP
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FoxC2PromotesAnabolisminDiscDegeneration
Fig1.TheexpressionofFOXC2inhumannucleuspulposustissue.(A)TheexpressionofFOXC2exhibitedsignificantlyhigherlevelsin4degenerative
nucleuspulposustissuecomparedto4idiopathicscoliosisnucleuspulposustissue.(B)ThecorrelationbetweentheexpressionofFOXC2andPfirrmann
scoresofdiscdegenerationgrade.n=36,r=0.881,p<0.05.(C)RT-PCRanalysisofFOXC2inthehumannucleuspulposustissueof36patients.Errorbars
representSD.Ascomparedwithcontrol,**indicatesp<0.01.
doi:10.1371/journal.pone.0147764.g001
cellsculturedinmonolayerwithBMP-7(100ng/ml)inducedaprominentincreaseafter2
hoursinFoxC2geneexpression(Fig4B).Westernblotsoverthe60-mintimeframeshowed
thatFoxC2wassignificantlyup-regulatedattheoptimalconcentrationof100ng/mlBMP-7
(Fig4C).
TodeterminewhetherincreasedFoxC2byBMP-7isrelatedtotheBMPsignalingpathway,
wequantifiedFoxC2mRNAexpressioninthepresenceandabsenceofBMP-7,SiRNA-Smad1,
ScrambleandtheBMPinhibitornoggin(Fig5Aand5B).Concordantwithourhypothesis,
whentheBMPsignalingpathwaywasinhibitedbySiRNA-smad1,thisledtoasignificant
downregulationoftheanabolismeffectthatFoxC2increasedinNPcells,comparedtointhe
presenceofBMP-7andScrambleoronlyinthepresenceofBMP-7.Also,theupregulationof
FoxC2byBMP-7wassignificantlyinhibitedbynoggin.Furthermore,wefoundthatFoxC2
overexpressioninducedthephosphorylationofSmad/1/5/8obviously,whichwereknownto
beactivatedbyBMPs(Fig5C).TofurtherunderstandtherelationshipbetweenFoxC2and
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FoxC2PromotesAnabolisminDiscDegeneration
Fig2.Immunofluorescencecharacterizationandreal-timequantitativePCRvalidationofprimaryculturedhumanNPcells.(A)Fluorescence
microscopyimagesshowingcollagenasetypeIIwasobservedinNPcells.NucleiwerestainedwithDAPI,showninblue.Imageswereacquiredusinglaser
scanningconfocalmicroscopyundera40×objective.(B)FluorescencemicroscopyimagesshowingaggrecanwasobservedinNPcells.Nucleiwerestained
withDAPI,showninblue.Imageswereacquiredusinglaserscanningconfocalmicroscopyundera40×objective.(C)Real-timeRT-PCRanalysisofnegative
NPcellmarkergenecollagentypeIinNPcells,NPandAF.Real-timeRT-PCRanalysiswasperformedintriplicateandtheexpressionlevelsofcollagenase
typeImRNAswerenormalizedtoGAPDHmRNAs.(D)Real-timeRT-PCRanalysisofNPcellmarkergenecollagentypeIIinNPcells,NPandAF.Real-time
RT-PCRanalysiswasperformedintriplicateandtheexpressionlevelsofcollagenasetypeIImRNAswerenormalizedtoGAPDHmRNAs.Errorbars
representSD.Ascomparedwithcontrol,**indicatesp<0.01.
doi:10.1371/journal.pone.0147764.g002
BMP-7,wemeasuredmRNAlevelsofBMP-7followingFoxC2overexpression.Underthis
condition,BMP-7waspositivelyupregulatedwhilstnogginwassuppressedbyFoxC2(Fig5D).
FoxC2enhancesthematrixgormationdynergisticeffectfromtheBMP-7
inNPCells
WenextexaminedtheeffectsofFoxC2ontheexpressionofanabolicgenes.Asexpected,the
real-timeqPCRresultsdemonstratedBMP-7stimulatedtheexpressionofECMgenes(Fig6A
and6B).Specifically,FoxC2hadcomplementaryeffectsonCollagenIIandaggrecanexpres-
sion,withFoxC2respectivelyinducinga2.4-fold(p<0.05)and2.8-fold(p<0.01)increasein
CollagenIIandaggrecangeneexpressionwhencomparedwithcontrols.Moreover,the
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Fig3.OverexpressionofFoxC2promotesNPcellsgrowth.(A)ImmunohistochemicalstainingofNPcellsagainstKi-67.NucleiwerestainedwithDAPI,
showninblue.Imageswereacquiredusinglaserscanningconfocalmicroscopyundera40×objective,andhadKi-67-positivepercentagesinculturedNP
cells48haftertransfectionwithLentiFoxC2orLentiGFPornotransfection.(B)Real-timeRT-PCRanalysisofKi-67mRNAexpressioninNPcellsafter
transfectionofLentiFoxC2for0h,24h,48hor72h.(C)GrowthofNPcellswasshownaftertransfectionwith50nmol/LofLentiFoxC2orLentiGFPorno
transfection.Thegrowthindexwasassessedat1,2,3,4,and5days.ErrorbarsrepresentSD.Ascomparedwithcontrol,*indicatesp<0.05,**indicates
p<0.01.
doi:10.1371/journal.pone.0147764.g003
combinationofFoxC2withBMP-7furtherincreasedCollagenIIandaggrecangeneexpression
abovelevelsinducedbyBMP-7alone.Inaddition,wefoundthatFoxC2waseffectiveagainst
increasedlevelsofnogginexpressioninducedbyBMP-7(Fig6Cand6D).Theseresultssuggest
thatFoxC2enhancesBMP7-mediatedupregulationofanabolicgenes.Takingtogether,wepro-
posethatFoxC2blocksthepositivefeedbackregulationofBMP-7bynoggininNPcells,likely
throughthecrosstalkbetweenothersignalingpathways.
Discussion
Discdegenerationisacomplexprocesscausedwhenthedelicatebalancebetweenanabolicand
catabolicprocessesisbroken.Frompreviousresearchesatthemolecularlevel,weknowthat
genesinvolvedinNPcelldegenerationaretightlyregulatedandcoordinatedbyseveralsignal-
ingpathways[10,36–38].ItisknownthatactivationofthetranscriptionfactorSmadthrough
theBMP/SmadsignalingpathwaysisessentialforenhanceECMrepairandregenerationinNP
PLOSONE|DOI:10.1371/journal.pone.0147764 January29,2016 9/17
FoxC2PromotesAnabolisminDiscDegeneration
Fig4.ImmunohistochemicalstainingofNPcellsagainstFoxC2.(A)After1-dayserumdeprivation,NPcellsweretreated50ng/mlor100ng/mlBMP-7
inserum-freemediumfor2h.NPcellswereImmunohistochemicalstainedagainstFoxC2.NucleiwerestainedwithDAPI,showninblue.Imageswere
acquiredusinglaserscanningconfocalmicroscopyundera40×objective.FoxC2-positivepercentagesinculturedNPcells2haftertreatedwith50ng/mlor
100ng/mlBMP-7orcontrolwasunderanalysis.(B)NPcellswerestimulatedbyBMP-7(100ng/ml)fortheindicatedperiodsoftime.Andthenexpression
levelofFoxC2wasmeasuredbyreal-timeRT-PCR.(C)NPcellswerestimulatedby10,50,and100ng/mlBMP-7for2horNPcellswerestimulatedbyBMP-
7(100ng/ml)fortheindicatedperiodsoftime.Celllysateswerethenpreparedandanalyzedbywesternblottingwithspecificanti-FoxC2antibody.β-actin
wasusedasacontrolfornormalization.ErrorbarsrepresentSD.Ascomparedwithcontrol,*indicatesp<0.05,**indicatesp<0.01.
doi:10.1371/journal.pone.0147764.g004
cells[39,40].However,duringdiscdegeneration,themolecularmechanismsfortheregulation
andcoordinationbetweenthesepathwaysremainunclear.Inpreviousstudies,FoxC2is
reportedtoserveasakeyregulatorindiversebiologicalandpathologicalprocesses,including
cellproliferation,differentiationandapoptosis[41–43].However,whetherFoxC2haspatho-
genicsignificanceindegenerativeNPtissueandthesignificanceofFoxC2duringtheprocess
ofDDDareyettobeelucidated.Inthisstudy,weprovideevidenceforanovelmechanism
wherebyFoxC2playsanimportantroleinpromotingNPcellproliferationandstimulatessyn-
thesisoftheECMbyregulatingtheBMPsignalingpathways.
PLOSONE|DOI:10.1371/journal.pone.0147764 January29,2016 10/17