Msx2 Exerts Bone Anabolism via Canonical Wnt Signaling*DS

18 Pages·2008·1.01 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.283,NO.29,pp.20505–20522,July18,2008
©2008byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Msx2 Exerts Bone Anabolism via Canonical Wnt Signaling*□S
Receivedforpublication,February1,2008,andinrevisedform,May7,2008 Published,JBCPapersinPress,May15,2008,DOI10.1074/jbc.M800851200
Su-LiCheng1,Jian-SuShao1,JunCai,OscarL.Sierra,andDwightA.Towler2
FromtheDepartmentofMedicine,DivisionofBoneandMineralDiseases,WashingtonUniversitySchoolofMedicine,
St.Louis,Missouri63110
Msx2isahomeodomaintranscriptionfactorfirstidentifiedin calcitriol-regulated transcript in osteoprogenitors isolated
craniofacialboneandhumanfemoralosteoblasts.Wehypothe- from human femur, and subsequently shown to be highly
sized that Msx2 might activate skeletal Wnt signaling. There- expressed in the murine embryonic craniofacial skeleton (2).
fore,weanalyzedtheeffectsofCMV-Msx2transgene(Msx2Tg) Soon thereafter, Msx2 was identified as a transcriptional
expression on skeletal physiology and composition. Skeletal repressoroftheosteoblast-specificosteocalcin(OC)3promoter
Msx2 expression was increased 2–3-fold by Msx2Tg, with (3,4).StudiesofMsx2-regulatedgeneexpressioninbonehave
expandedproteinaccumulationinmarrow,secondaryossifica- emphasized its role as a transcriptional repressor of the late
tion centers, and periosteum. Microcomputed tomography osteogenic phenotype (5–7). For example, in the developing
established increased bone volume in Msx2Tg mice, with tooth,wherestage-specificosteogenicgeneexpressionprofiles
increasednumbersofplate-liketrabeculae.Histomorphometry arespatiallyresolved,Msx2andOCexhibitreciprocalpatterns
revealed increased bone formation in Msx2Tg mice versus ofmRNAaccumulation(8).Msx2suppressesOCgeneexpres-
non-Tg siblings, arising from increased osteoblast numbers. sion in a cell-autonomous fashion, mediated via antagonistic
While decreasing adipogenesis, Msx2Tg increased osteogenic protein-protein interactions between Msx2 and Runx2-con-
differentiation via mechanisms inhibited by Dkk1, an antago- tainingcomplexesthatsupportOCpromoteractivity(6,7).
nistofWntreceptorsLRP5andLRP6.BonefromMsx2Tgmice However, elegant genetic studies have demonstrated that
elaborated higher levels of Wnt7 canonical agonists, with Msx2 also promotes craniofacial bone mineralization and is
diminished Dkk1, changes that augment canonical signaling. necessaryforrobusttrabecularandcorticalboneformation(9,
Analysis of non-Tg and Msx2Tg siblings possessing the TOP- 10).Msx2(cid:1)/(cid:1)miceexhibitparietalforamina,characterizedby
GAL reporter confirmed this; Msx2Tg up-regulated skeletal reducedmineralizationincalvarialfieldsthatgiverisetomem-
(cid:1)-galactosidase expression (p < 0.01), along with Wnt7a and branous bone (9). Moreover, Msx2(cid:1)/(cid:1) mice exhibit a global,
Wnt7b, and reduced circulating Dkk1. To better understand low turnover osteopenia (9). Simultaneous deletion of both
molecularmechanisms,westudiedC3H10T1/2osteoprogeni- Msx2 and Msx1, a homologous Msx gene family member,
tor cells. As in bone, Msx2 increased Wnt7 genes and down- results in the complete absence of craniofacial bone (9, 11).
regulated Dkk1, while inducing the osteoblast gene alkaline Furthermore,anonsynonymousCCCtoCACmutationatthe
phosphatase.Msx2-directedRNAinterferenceincreasedDkk1 7thcodonoftheMsx2homeodomaincausesBoston-typeauto-
expressionandpromoteractivity,whilereducingWnt7a,Wnt7b, somaldominantcraniosynostosis,characterizedbyprecocious
and alkaline phosphatase. Moreover, Msx2 inhibited Dkk1 pro- mineralizationanddifferentiationofosteoblastsinthecalvarial
moteractivityandreducedRNApolymeraseassociationwithDkk1 suture (12). In vivo and in vitro data indicate that this
chromatin. RNA interference-mediated knockdown of Wnt7a, Msx2(P148H)variantismostlikelyagain-of-functionvariant;
Wnt7b, and LRP6 significantly reduced Msx2-induced alkaline Msx2(P148H) exhibits increased DNA binding to the Msx
phosphatase. Msx2 exerts bone anabolism in part by reducing CTGAATTRGbindingcognate(13,14).Intriguingly,however,
Dkk1 expression and enhancing Wnt signaling, thus promoting the frequency of transgene-induced craniosynostotic bone is
osteogenicdifferentiationofskeletalprogenitors. threetimemoreprevalentinwild-typeMsx2transgenicmice
than Msx2(P148H) transgenic mice (71 versus 27%, respec-
Msx2,alsoknownasHox-8,isahomeodomaintranscription
3The abbreviations used are: OC, osteocalcin; ALP, alkaline phosphatase;
factor first characterized by Sharpe and co-workers (1) as a
BMD,bonemineraldensity;BSP,bonesialoprotein;C3H10T1/2,murine
multipotentmesenchymalcellline;ChIP,chromatinimmunoprecipitation;
CMV,cytomegalovirusimmediatelyearlyenhancer/promoter;CTX,typeI
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth collagenC-terminaltelopeptide;FBS,fetalbovineserum;Fzd,frizzledWnt
GrantsAR43731andHL81138(toD.A.T.)andtheBarnes-JewishHospital co-receptor; micro-CT, microcomputed tomography; Msx, muscle seg-
Foundation.Thecostsofpublicationofthisarticleweredefrayedinpartby menthomeoboxgene;Msx2Tg,transgenicmouseexpressingMsx2cDNA
the payment of page charges. This article must therefore be hereby fromtheCMVpromoter;Osx,osterix;PBS,phosphate-bufferedsaline;PKC,
marked“advertisement”inaccordancewith18U.S.C.Section1734solelyto proteinkinaseC;polII,RNApolymeraseII;RT-qPCR,reversetranscription-
indicatethisfact. quantitativePCR;siRNA,smallinterferingRNA;Tg,transgenicmouse;TCF,
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains T-cellfactor;LEF,lymphoidenhancerfactor;TOPGAL,TCF/LEFoptimalpro-
supplementalFigs.S1–S6. moter-galactosidasereportertransgenicmouse;TRAP5c,osteoclasttar-
1Bothauthorscontributedequallytothiswork. trate-resistantacidphosphatase;Wnt,Wingless/intgenefamily;WT,wild-
2Towhomcorrespondenceshouldbeaddressed:InternalMedicine,Bone type sibling mouse; sibs, siblings; DMEM, Dulbecco’s modified Eagle’s
andMineralDiseases,WashingtonUniversitySchoolofMedicine,Campus medium; E3, ubiquitin-protein isopeptide ligase; ELISA, enzyme-linked
Box8301,660SouthEuclidAve.,St.Louis,MO63110.Fax:[phone]; immunosorbentassay;BFR,boneformationrate;RNAi,RNAinterference;
E-mail:[email protected]. TV,tissuevolume.
This is an Open Access article under the CC BY license.
JULY18,2008•VOLUME283•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 20505
WntSignalsMediateMsx2BoneAnabolism
tively)(13).ThissuggeststhatinvivotheP148Hmutationper- Cruz Biotechnology (Santa Cruz, CA). The Wnt1 antibody
turbsotherimportantMsx2functions(13).Indeed,theregion (ab15251-500, lot 315275, rabbit polyclonal antibody) pur-
of the homeodomain N-terminal arm altered by the P148H chasedfromAbcamwasalsousedandprovedtobesuperiorfor
substitutionparticipatesinregulatoryprotein-proteininterac- Wnt1immunohistochemistry.Thealkalinephosphatase-con-
tions with Dlx5 (5, 15) and other transcription factors (5–7). jugatedgoatanti-rabbitsecondaryantibodyusedforWestern
ConsistentwiththeMsx2gain-of-functionmodelarisingfrom blots with rabbit primary immunoreagents was sc-2057 (lot
CMV-Msx2transgenicmice,distaltrisomyofchromosome5q, I12503,SantaCruzBiotechnology).Wnt10bantibody(ratanti-
the physical region encompassing the Msx2 gene, has been mouse, monoclonal antibody 2110, clone 254206) was pur-
identifiedinsixpatientswithcraniosynostosis(16);thisdem- chased from R & D Systems (Minneapolis, MN). Goat poly-
onstrates the exquisite sensitivity of the developing human clonal antibody to LRP5 (sc-21390) and the donkey anti-goat
craniofacial skeleton to Msx2 gene dosage (10, 17). Although secondaryantibody(sc-2022)werebothpurchasedfromSanta
detailshaveemergedastohowMsx2inhibitsosteoblasttermi- CruzBiotechnology.VECTASTAINEliteABCperoxidasesec-
nal differentiation via cell-autonomous actions (5–7), little is ondary antibody immunovisualization kits directed toward
knownofthemechanismswherebyMsx2promotesosteoblast- goat (PK-6105) or rabbit (PK-6101) primary antibodies were
mediated bone formation. The mechanistic underpinnings of purchasedfromVectorLaboratories(Burlingame,CA).Anti-
the low turnover osteopenia demonstrated in Msx2(cid:1)/(cid:1) long bodies to pol II (N-20, sc-899, SantaCruzBiotechnology)and
boneduringpostnatalgrowthhaveyettobedetermined(9).In phospho-pol II (H5, MMS-129R, Covance) were used in ChIP
seminal studies of calvarial bone formation, Maxson and co- assaysasdescribedpreviously(6,24).Theamplimerpairs5(cid:2)-CGG
workers(10,11)showedthatMsx2promotestheproliferative AGGTCCCGAAGTTGAG-3(cid:2)and5(cid:2)-AAAGGTCAGGAA
expansionandsurvivalofneuralcrest-derivedosteoblasts. AAGAGAGGTCACT-3(cid:2)and5(cid:2)-ATTATTGGTGACTTG
Recently,weidentifiedthatMsx2participatesintheectopic GTGGTGATCT-3(cid:2)and5(cid:2)-ATTTTATGAGGCACAGTT
medialarterycalcificationcharacteristicoftypeIIdiabetes(18, GATGTCTT-3(cid:2)wereusedforquantifyingDNAinprecipitates
19). Murine models of diabetic medial calcification spatially byfluorescenceqPCRfortheDkk1andosteopontingenes,respec-
resolved adventitial Msx2-expressing cells and the ALP-posi- tively.Primerpairsweredirectedtoregionsjustdownstreamof
tivemedialcalcifyingvascularcellsthatdirectmatrixmineral- thetranscriptioninitiationsite,withintheearlyregionsencoding
ization;therefore,wededucedthatMsx2-expressingcellselab- thepolII-dependentprimarytranscripts.Asecondamplimerpair
orateparacrinesignalsthatcontrolosteogenicdifferentiation directed(cid:3)1.5kbintotheDkk1gene(5(cid:2)-GAAAGCATCATT
ofneighboringprogenitors(19).Indeed,weshowedthatcon- GAA AAC CTT GGT-3(cid:2) and 5(cid:2)-GCC TTC CCC GCA GTA
ditioned media from Msx2-expressing C3H10T1/2 cells pos- ACA-3(cid:2))wasalsousedtoconfirmtheeffectsofMsx2onpol
sessed a pro-osteogenic, adipostatic activity (19) resembling II-Dkk1 chromatin interactions. For Western blot assays,
thatofcanonicalWntligandssuchasWnt3a(20),Wnt7a(21), antigen-antibodycomplexesweredetectedbyalkalinephos-
andWnt10b(22,23).MolecularprofilingbyquantitativeRT- phatase-dependent disodium 3-(4-methoxyspiro[1,2-diox-
qPCRdemonstratedup-regulationofcanonicalWntagonists etane-3,2(cid:2)-(5(cid:2)-chloro)-tricyclo[3.3.13,7]decan]-4-yl)phenyl
Wnt3aandWnt7ainaortictissues(19).Bycontrast,expression phosphate chemiluminescence (Tropix, Applied Biosystems,
ofaorticDkk1,theprototypicvertebrateinhibitorofcanonical Foster City, CA). Conjugated secondary antibodies, blocking
Wnt signaling, was concomitantly down-regulated by Msx2 reagents,andperoxidase-basedimmunovisualizationkitswere
(19).ExogenousrecombinantDkk1antagonizedMsx2regula- purchased from Tropix or Vector Laboratories (Burlingame,
tion of osteogenesis and adipogenesis (19). Thus, Msx2-pro- CA).Custom-synthesizedoligodeoxynucleotidesweresynthe-
ducingvascularadventitialcellsenhancedaorticWntsignaling sized and purchased from Invitrogen. TOPGLOW (catalog
andpromotedectopicmineraldepositionviaosteogenicmech- number 21-204) and FOPGLOW (catalog number 21-205)
anisms(19). luciferase reporter plasmids were purchased from Millipore.
To better understand the mechanisms whereby Msx2 aug- PCRwasusedtogeneratecDNAinsertsforexpressionplas-
mentsorthotopiclongboneformation,wehaveevaluatedthe midsforpcDNA3-Wnt7aandpcDNA3-Wnt7busingmeth-
skeletal phenotype of adult CMV-Msx2 (Msx2Tg) transgenic ods described previously (8). Dkk1 promoter-luciferase
mice(19).WeshowthataugmentingMsx2expressioninlong reporter constructs 1141 DKK1LUC ((cid:1)1141 to (cid:1)1), 451
bonedirectsbonemesenchymalprogenitorstoosteogeniclin- DKK1LUC((cid:1)451to(cid:1)1),170DKK1LUC((cid:1)170to(cid:1)1),120
eage,enhancingskeletalosteoblastnumbers,mineralizingsur- DKK1LUC ((cid:1)120 to (cid:1)1), and 70 DKK1LUC ((cid:1)70 to (cid:1)1)
face, trabecular number, and trabecular bone formation via were generated by ligating the indicated segments of the
canonicalWntsignals. mouseDkk1promoterintopGL2Basic(Promega);methods
have been detailed previously (25). The numbering of base
EXPERIMENTALPROCEDURES
pairsisrelativetothestartsiteofDkk1exon1(physicalbase
Antibodies, ChIP Assays, Luciferase Assays, Plasmids, and pair position 30,620,374 of mouse chromosome 19, RefSeq
Other Reagents—Antibodies for Msx2 (sc-15396, lot C0404, GeneNM_010051).Thecontrolminimalpromoter-reporter
polyclonal antibody), Wnt7a/b (sc-32865, lot F2006, rabbit RSVLUC has been described previously (25). Transient
polyclonal), Wnt1 (sc-5630, lot E2605, goat polyclonal anti- transfections and luciferase assays were carried out as
body),Wnt3a(sc-28824,lotK0205,rabbitpolyclonalantibody), detailedpreviously(6).Custom-synthesizedsiRNAreagents
Dkk1, (sc-25516, lot J1404, rabbit polyclonal antibody), actin werepurchasedfromQiagen(Valencia,CA).Commercially
(sc-8432), and eIF2(cid:1)(sc-11386) were purchased from Santa availablesiRNAswerepurchasedfromSantaCruzBiotech-
20506 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER29•JULY18,2008
WntSignalsMediateMsx2BoneAnabolism
nologyasindicated.SYBRGreenwaspurchasedfromeither ImmunohistochemistryandWesternBlotAnalysis—Multiple
RocheAppliedScienceorAppliedBiosytems.Total(cid:2)-cate- commerciallyavailableimmunoreagentsweretestedforsensi-
ninELISAkit(catalognumber900-135)waspurchasedfrom tivityandspecificity,andonlythosethatprovedsensitiveand
AssayDesigns(AnnArbor,MI).Allotherreagentswerepur- specificarereportedhere.Priortoimmunohistochemistry,the
chasedeitherfromInvitrogen,Fisher,orSigma. specificityoftheantibodyreagentswasfirstgaugedbyWestern
Assays of Osteogenic and Adipogenic Potential of Primary blotanalysisasdetailedpreviously(30),usingproteinextracts
Mouse Bone Cell Cultures—Calvarial osteoblasts were pre- preparedfromthefollowing:(a)C3H10T1/2cellstransduced
paredfromneonatalmousecalvariabytimedcollagenasediges- witheithercontrolSFG-LacZvirusorwithSFG-Msx2andthen
tionusingmethodsdescribedpreviously(26).Bonemesenchy- (b)mouseprimarycalvarialosteoblastcellextractsfromeither
malcellswereisolatedfromthemarrowcompartmentoflong WTorMsx2Tgmice.Onlythosereagentsthatgeneratedsingle
bones of 4-month-old Msx2Tg and WT mice using methods immunoreactive bands of the appropriate relative mass fol-
described previously (27) and then tested for the capacity to lowingSDS-PAGEandWesternblotanalysiswereutilizedfor
undergo osteogenic or adipogenic differentiation. For osteo- immunoperoxidasestaining.Forneonatallongbones,follow-
genicdifferentiation,cellswereseededin96-welltissueculture ing48hoffixationin10%neutralbufferedformalin,samples
platesatadensityof1(cid:4)105cells/well,leftundisturbedfor7 wereembeddedinparaffinwithoutdecalcification,and5-(cid:3)m
longitudinal sections were prepared for Msx2, Wnt7a/b, and
days,andthenmaintainedinmineralizationmedium(modified
Eagle’s medium containing 10% FBS, 50 (cid:3)g/ml ascorbic acid, Wnt1 immunohistochemistry. For adult long bones, ossicles
and 10 mM (cid:2)-glycerophosphate) for 3 additional weeks. Cul- were fixed for 48–72 h in 10% neutral buffered formalin and
tures were subsequently stained for calcium deposition using thendecalcifiedfor3weeksat4°Cin0.375MEDTA,pH8,with
constant gentle stirring. Decalcified adult long bones were
Alizarinred.Wellscontainingrednoduleswith15ormorecells
embeddedinparaffin,and5-(cid:3)msectionswerecutforsubse-
were considered positive. The osteogenic potential was deter-
quentimmunohistochemistryusingmethodsdescribedprevi-
minedasthepercentofpositivewellsintotalwells.Foradipogenic
differentiationassays,bonemesenchymalcellswereseededat1(cid:4) ously(19).Briefly,afterdeparaffinizationofthesectionswith
xylene and rehydration via graded aqueous ethanolic baths,
107cellsper10-cmculturedish.Sevendayslater,cellsweretreated
withadipogenicmediumcontaininginsulin(5(cid:3)g/ml),dexameth- endogenousperoxidaseswerequenchedbytreatmentwith1%
asone(10(cid:1)7M),andindomethacin(50(cid:3)M)for9additionaldays. H2O2 in methanol for 20 min, rinsed with water for 1 min,
followedby10moreminofrinsinginPBS,andthenincubated
Cultures were stained with the lipotrophic dye Oil Red O as
inblockingsolution(10%normalserumcognatetothesecond-
detailedpreviously(28,29).Thenumbersofadipocytecolonies
aryantibodyand5%bovineserumalbumininPBS)for30min
wereenumerated,anddatawereexpressedasthepercentoftotal
at 25°C. Sections were then incubated with 2 (cid:3)g/ml primary
cellcoloniesthatstainedpositivewithOilRedO.
antibody in blocking solution overnight ((cid:5)18 h) at 4°C. The
CMV-Msx2(Msx2Tg)andTOPGALTransgenicMice—The
nextmorning,afterwashingthreetimesfor10minwithPBSat
generationandmaintenanceofCMV-Msx2Tgmice(Msx2Tg)
room temperature, antigen-antibody complexes were tagged
ontheC57Bl/6murinebackgroundhavebeendetailedprevi-
withVECTASTAINEliteABCperoxidasekitsdirectedtoward
ously (19). Msx2Tg mice were generated essentially as
eithergoat(PK6105)orrabbit(PK6101)primaryantibodiesas
described by Maxson and co-workers (13). The eukaryotic
appropriate(seeabove),byincubatingsectionswith1:200dilu-
pcDNA3-Msx2 expression construct DT21.14 (30) was
tion of the biotinylated secondary antibody for 30 min. After
digested with BglII and NruI to release the 3.2-kb fragment
washing and incubation with ABC anti-biotin reagent, speci-
encoding the CMV immediate early promoter, mouse Msx2
menswereagainwashedtwotimesfor10minwithsterilePBS,
cDNA (with N-terminal Met-FLAG tag), and the bovine
and complexes were visualized by diaminobenzidine staining
growth hormone 3(cid:2)-untranslated region and polyadenylation
(SK-4100),withstainintensitymonitoredbymicroscopy.Reac-
signal from pcDNA3 (Invitrogen). This 3.2-kb fragment was
tionswerestoppedbyrinsingwithtapwater.Equaltimeofstain
usedtogeneratetransgenicmicemadebymaleC57Bl/6pro-
developmentwasutilizedforbothWTandMsxTgtissuesec-
nuclearinjectionattheWashingtonUniversityMouseGenet- tionsmonitoredinparallel.Afterdehydrationingradedetha-
ics Core. PCR genotyping for the CMV-Msx2-bGHpoly(A) noltoxylene,samplesweremountedusingVectaMountper-
transgene was directed toward uniquely juxtaposed 3(cid:2)-cDNA manent mounting media and digital images were captured
Msx2 (5(cid:2)-TGT GCT CCC CAT CCC GCC TGT TGG ACT using a Leica 4100 DM microscope coupled with a DFC 420
CTA-3(cid:2))andvector3(cid:2)-untranslatedregionsequences(5(cid:2)-AAG digitalcamera,operatedwithFW4000software(LeicaMicro-
GACAGTGGGAGTGGCACCTTCCAGGGT-3(cid:2))usingthe systems,Bannockburn,IL)asdetailedpreviously(19).
indicated primers, following the protocol of Stratman et al. Histochemistry and Histomorphometric Methods—Histo-
(31).TOPGALmice(32)(TCF/LEFOptimalPromoter/Galac- chemistryforLacZactivityinMsx2Tg;TOPGALandTOPGAL
tosidase reporter transgenic mice) were purchased from The mice was carried out essentially as detailed previously (19).
Jackson Laboratories (mouse stock 004623; genotyping Long bones from Msx2Tg;TOPGAL and TOPGAL siblings
amplimers, 5(cid:2)-GAG TGA CGG CAG TTA TCT GGA AGA wereexcised,rinsedtwiceinPBS,andthenembeddedinOCT
TCA GGA-3(cid:2) and 5(cid:2)-GGA AAC CGA CAT CGC AGG CTT (Sakura Tissue-Tek). Subsequently, 25-(cid:3)m-thick longitudinal
CTG CTT CAA TCA-3(cid:2)). TOPGAL mice were bred with frozensectionsofnondecalcifiedspecimenswereanalyzedby
Msx2TgmicetogenerateMsx2Tg;TOPGALanimalsonamixed histochemistry for (cid:2)-galactosidase (LacZ) enzyme activity as
C57Bl/6:CD1background. detailed previously for mouse aortas (19), comparing LacZ
JULY18,2008•VOLUME283•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 20507
WntSignalsMediateMsx2BoneAnabolism
staining of proximal tibiae in Msx2Tg;TOPGAL versus TOP- ingDkk1,Msx2,Msx2(P148H),andMsx2(T147A)weregener-
GALanimals. ated using methods detailed previously (28). Transduction of
For dynamic and static histomorphometric studies, C3H10T1/2 cells and primary mouse mesenchymal cells was
6-month-old male mice (7 Msx2Tg, 11 WT siblings) were performed as detailed (28). Cells transduced with SFG-LacZ
sequentiallydosedat8and3dayspriortosacrifice,firstwith encoding(cid:2)-galactosidasewereusedasanegativecontrol.The
subcutaneoustetracycline(1mg/0.1mlper30-gbodyweight) expression of Msx2 and Dkk1 in SFG-Msx2- and SFG-Dkk1-
andthenwithsubcutaneouscalcein(1mg/0.1mlper30-gbody transducedcellswasverifiedviarealtimeRT-qPCRanalysisof
weight),respectively.Followingeuthanasiaviaexsanguination mRNA(seebelow).
underketamine-xylazineanesthesia,righthindlimbsweredis- Gene Expression by Fluorescence RT-qPCR—Two-step RT-
sectedenbloc,andthecombinedfemur,genu,andtibiawere qPCR was used to quantify gene expression in total RNA
dissectedfreeofadherentmuscleandfascia.Carefullyliberated extracted from either mouse bone (44) or cultured cells (28)
rightfemurswerefixedin10%neutralbufferedformalinat4°C usingtherespectivemethodsdetailedpreviously.Amixtureof
for2days,washedtwicefor15mininPBS,andthenstoredin randomhexamersandoligo(dT)wasusedtoprimecDNAsyn-
70%ethanoluntilembeddedinOsteo-BedResinforprepara- thesis,sothatrelativemRNAaccumulationcouldbenormal-
tionoflongitudinalplasticsections.Dynamicandstatichisto- izedtothesignalobtainedfor18SribosomalRNAinparallel.
morphometrywasperformedunderafee-for-servicecontract AmplimerpairsusedforquantitativeRT-qPCRwereasfollows:
withMDSPharmaServices,formerlySkeleTech,Inc.(Bothell, Msx2, 5(cid:2)-CCG CCG CCC AGA CAT A-3(cid:2) and 5(cid:2)-CTT CCG
WA),usingOsteomeasuresoftware(33),andfollowingnomen- GTT GGT CTT GTG TTT C-3(cid:2); Wnt1, 5(cid:2)-AAA TGG CAA
clature and recommendations of the American Society for TTC CGA AAC CG-3(cid:2) and 5(cid:2)-CGA AGA TGA ACG CTG
Bone and Mineral Research Histomorphometry Committee TTTCTCG-3(cid:2);Wnt3a,5(cid:2)-CATGCACCTCAAGTGCAA
(34).Forboneformationrate(BFR),thetissuevolumereferent ATG-3(cid:2)and5(cid:2)-TGAGGAAATCCCCGATGGT-3(cid:2);Wnt5a,
(TV), i.e. BFR/TV (%), was chosen to facilitate comparisons 5(cid:2)-CCA CGC TAA GGG TTC CTA TGA G-3(cid:2) and 5(cid:2)-TGT
withcontemporaryreportsofWnt-regulated(35),Dkk1-regu- CCTACGGCCTGCTTCA-3(cid:2);Wnt7a,5(cid:2)-TGGATGCCC
lated(36),andotheranabolicresponses(37–41)inthemouse GGGAGATC-3(cid:2)and5(cid:2)-CCGACCCGCCTCGTTATT-3(cid:2);
skeleton. Wnt7b,5(cid:2)-TTCTGGAGGACCGCATGAA-3(cid:2)and5(cid:2)-GGT
ConebeamMicro-CTImaging—Thelefttibiaeobtainedfrom CCAGCAAGTTTTGGTGGTA-3(cid:2);Wnt10b,5(cid:2)-CGAGAA
6-month-oldmicedescribedabovewereusedtoassesstrabec- TGCGGATCCACAA-3(cid:2)and5(cid:2)-CCGCTTCAGGTTTTC
ularbonesusingScancomicro-CT40(ScancoUSAInc.,South- CGT TA-3(cid:2); bacterial (cid:2)-galactosidase/LacZ, 5(cid:2)-GGT TAC
eastern,PA)atanenergysettingof55kV,currentsettingof145 GAT GCG CCT ATC TAC AC-3(cid:2) and 5(cid:2)-CTC CGC GGG
mA,andwithanintegrationtimeof300ms.Afixedthreshold AAC AA CG-3(cid:2); ALP, 5(cid:2)-ACA CCA ATG TAG CCA AGA
of270Hounsfieldunitswasusedtoseparatebonefromback- ATG TCA-3(cid:2) and 5(cid:2)-GAT TCG GGC AGC GGT TAC T-3(cid:2);
ground.Atotalof300–350transverseCTsliceswasobtained, Dkk1, 5(cid:2)-GCT GCA TGA CGC ACG CTA T-3(cid:2) and 5(cid:2)-AGA
and three-dimensional analysis was performed on trabecular GGGCATGCATATTCCATTT-3(cid:2);Runx2,5(cid:2)-AGGAGG
bonesinthe50slices(16(cid:3)mthick/slice)startingatabout0.1 GAC TAT GGC GTC AA-3(cid:2) and 5(cid:2)-TCG GAT CCC AAA
mm below the lowest point of the growth plate. Trabecular AGAAGCTTT-3(cid:2);Osx,5(cid:2)-CCCTTCTCAAGCACCAAT
morphometric parameters, including the percentage of bone GG-3(cid:2)and5(cid:2)-AAGGGTGGGTAGTCATTTGCATA-3(cid:2);
volumepertotalvolume(%BV/TV),trabecularthickness(Tb BSP,5(cid:2)-CAGAGGAGGCAAGCGTCACT-3(cid:2)and5(cid:2)-GCT
Th, (cid:3)m), trabecular number (per mm3), trabecular spacing GTCTGGGTGCCAACACT-3(cid:2);LRP5,5(cid:2)-GAGCGAGGA
((cid:3)m),structuremodelindex,andconnectivitydensity(connec- GGC CAT CAA-3(cid:2) and 5(cid:2)-GCC CGA GAT GAC AAT GTT
tionspermm3)wereacquiredbydirectmethodofcalculation CT-3(cid:2); LRP6, 5(cid:2)-TGT GGG CCT GAC CGT GTT-3(cid:2) and
(42). 5(cid:2)-TTCGAGCCTGGACCTTGGT-3(cid:2);18SrRNA,5(cid:2)-CGG
Serum ELISAs for Tartrate-resistant Acid Phosphatase CTACCACATCCAAGGAA-3(cid:2)and5(cid:2)-GCTGGAATTACC
(TRAP5c), CTX, and Dkk1—After overnight fasting, mice GCGGCT-3(cid:2).
were anesthetized and blood was withdrawn from the infe- KnockdownofMsx2-WntSignalingComponentsbysiRNAin
rior vena cava in heparinized syringes. Blood samples were C3H10T1/2MouseMesenchymalOsteoprogenitors—Commer-
layered on top of Microtainer Serum Separator tubes cially available siRNAs directed to murine Wnt1 (sc-36840),
(365956; BD Biosciences) and incubated overnight at 4°C. Wnt5a (sc-41113), Wnt7a (sc-41115), Wnt7b (sc-41117),
Sera were obtained after centrifugation at 2,000 (cid:4) g for 20 Wnt10b (sc-37186), and control siRNA (sc-44230) were pur-
min at room temperature. Serum concentration of oste- chased from Santa Cruz Biotechnology. Sequences for these
oclast-derived tartrate-resistant acid phosphatase form 5b reagentscanbeobtaineduponrequestfromthemanufacturer.
was determined by using MouseTRAP assay kit (Code CustomsiRNAsusedforknockdownofLRP5,LRP6,andMsx2
TR103; IDS Inc., Fountain Hills, AZ). Commercial ELISAs wereorderedfromQiagen(Valencia,CA)ashighperformance
for CTX (RatLaps, 1RTL4000, Nordic Bioscience Diagnos- purity-grade reagents. The siRNA designed for Msx2 knock-
ticsviaIDSInc.)andDkk1(DuoSetDY1765;R&DSystems) down was derived from the viral RNAi strategy previously
werecarriedoutperthemanufacturer’sinstructions. describedbyCossuandco-workers(45).Customsequencesfor
Transfection and Retroviral Transduction—The pseudo- mouseLRP5andLRP6knockdownweredesignedusingEasy
typedretrovirusSFG-LacZ(43)wasthekindgiftofDr.DanOry siRNAsoftware(ProteinLounge,SanDiego).Specificreagents
(WashingtonUniversity,St.Louis).SFGretrovirusesexpress- used for Msx2, LRP5, and LRP6 knockdown were as follows:
20508 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER29•JULY18,2008
WntSignalsMediateMsx2BoneAnabolism
Msx2,5(cid:2)-r(CAGUACCUGUCCAUAGCAG)dTdT-3(cid:2)and Msx2mRNAwassignificantlyincreasedincalvarialosteoblasts
5(cid:2)-r(CUG CUA UGG ACA GGU ACU G)dTdT-3(cid:2); LRP5, (2.57(cid:6)0.07-fold)andbonemarrowcellcultures(1.81(cid:6)0.17-
5(cid:2)-r(GGA GAU CCU UAG UGC UCU G)dTdT-3(cid:2) and 5(cid:2)- fold)inMsx2TgmiceascomparedwithWTlittermates(p(cid:7)
r(CAG AGC ACU AAG GAU CUC C)dTdT-3(cid:2); LRP6, 0.05forboth).Westernblotanddigitalimageanalysisofosteo-
5(cid:2)-r(GAG AAU GCA ACG AUU GUA G)dTdT-3(cid:2) and blastextractsisolatedfromlongboneandcalvariaconfirmed
5(cid:2)-r(CUACAAUCGUUGCAUUCUC)dTdT-3(cid:2)andordered that Msx2 protein levels paralleled the transgene-mediated
fromQiagen(Valencia,CA). increases in mRNA accumulation (Fig. 1A). We localized the
Targeted mRNA degradation using siRNA technology was spatialpatternsofMsx2expressioninlongbonefromMsx2Tg
performed essentially as described by Brazas and Hagstrom micebyimmunoperoxidasestainingin3-day-oldmice.Overall
(46),accordingtotheprotocolprovidedbySantaCruzBiotech- signal intensity was increased in Msx2Tg versus WT siblings,
nology.Briefly,C3H10T1/2cellswereseededat2(cid:4)105/wellin becauseofexpandedMsx2immunoreactivityinthebonemar-
6-well cluster plates (35-mm diameter wells) in DMEM con- rowcompartment,theperiosteum,andsecondaryossification
taining 10% FBS the day before lipofection. To prepare lipid- centers(Fig.1Bandsupplementalmaterial).Secondary(epiph-
siRNAcomplexes,80pmoloftheindicatesiRNAduplexin100 yseal) ossification centers begin to form in mice in the first
(cid:3)l of Transfection Medium (sc-36868) and 6 (cid:3)l of siRNA postnatalweek(47,48);inlongbonesectionswheresecondary
Transfection Reagent (sc-29528) in 100 (cid:3)l of Transfection ossificationcenterswerevisualized,Msx2expressioninassoci-
Medium were combined, incubated for 30 min at 25°C, and atedhypertrophicchondrocyteswasalsoexpandedinMsx2Tg
thendilutedwith800(cid:3)lofpre-warmedTransfectionMedium. mice (Fig. 1B). The recently described accumulation of Msx2
Cellwererinsedoncewithserum-freeDMEM,and1000(cid:3)lof proteininasubsetofearlyhypertrophiczonechondrocyteswas
lipid-siRNA admixture described above was applied per well. also noted (49) and was enhanced in Msx2Tg versus WT sibs
Afterincubationfor6hat37°Cinahumidified5%CO cell (Fig.1B).Noimmunoreactivitywasobservedintheabsenceof
2
culturechamber,anadditional1mlof20%FBSinDMEMwas primaryantibody(Fig.1B,rightpanel).Intheepiphysealossi-
addedperwell,andlipofectionwasallowedtocontinuedover- fication center, Msx2 appeared to localize primarily to chon-
night.Thenextmorning,thelipofectionmediawasaspirated, drocyte nuclei (Fig. 1C). However, in marrow and periosteal
andtransfectedmonolayerscellsre-fedwithfreshC3H10T1/2 venues,bothnuclearandcytoplasmicMsx2immunoreactivity
growthmedia(10%FBSinDMEM).Twentyfourhourslater, wereclearlynoted(Fig.1D),presumablyreflectingstage-spe-
total cellular RNA was harvested as detailed previously (19) cificre-localizationofMsx2inbonemesenchymalprogenitors
using Ambion Turbo DNase treatment and removal kit asdescribedrecently(50).
(AM1907;AppliedBiosystems)toremovealltracesofgenomic ToexaminetheeffectsofMsx2onbonecontentandstruc-
DNA.QuantitativeRT-qPCRwascarriedoutintriplicateusing ture,weperformedconebeammicro-CTanalysisoftheproxi-
methodsdescribedpreviously(28).EfficiencyoftargetmRNA maltibia(51),comparingMsx2TgversusWTsiblings.Consist-
knockdown of (cid:5)30% (range 30–50%) was observed for all ent with increased BMD (see below), Msx2Tg mice exhibited
siRNAreagentspresented,usingRT-qPCRfortargetedmRNA significant increases in trabecular bone volume (Tb.BV/TV;
accumulationasanassay.ForRNAiusingprimarybonemar- 40%increase),trabecularnumber(Tb.N;20%),andtrabecular
row cell cultures from Msx2Tg mice, the above protocol was connectivitydensity(80%increase;Fig.2,A–C).Thestructure
alsoimplemented,exceptthatTransIT-TKOtransfectionrea- model index (SMI), an index that diminishes as trabeculae
gent (Mirus, Madison, WI) was used to introduce the siRNA become more sturdy and plate-like (52), was 40% lower in
reagents. Msx2Tgmice(Fig.2D;p(cid:8)0.04);thus,trabeculaeinMsx2Tg
Statistics—For statistical testing of dynamic histomor- mice were more plate-like than those of their WT sibs (52).
phometry, micro-CT, and gene expression results, analyses Although Msx2Tg-dependent increases in tibial trabecular
were performed using Student’s t test as described previ- thicknessdidnotreachsignificancebymicro-CT(notshown),
ously (44). All data are presented as the means (cid:6) S.E. of staticmorphometryoffemoraltrabeculaedidestablishasignif-
measurement to facilitate meaningful comparisons (95% icant15%increaseinfemoraltrabecularthickness(Tb.Th)and
confidenceinterval(cid:6)1.96S.E.). confirmedMsx2-dependentincreasesinTb.NandTb.BV/TV
(Fig. 3). Long bone cortical thickness was also significantly
RESULTS increased by 33% in Msx2Tg versus WT siblings (p (cid:7) 0.05).
CMV-Msx2 Transgenic Mice Exhibit Higher Bone Volume Thus, augmenting Msx2 expression in bone significantly
with Greater Numbers of Plate-like Trabeculae as Compared increasestrabecularbonemassaccrual.
withWild-typeLittermates—Wedemonstratedpreviouslythat Msx2Tg Mice Exhibit Greater Osteoblast Numbers and
Msx2 stimulated osteogenic differentiation but inhibited adi- Increased Trabecular Bone Formation—Detailed static and
pogenesisinculturedC3H10T1/2mesenchymalcells(28,29). dynamichistomorphometry(34,53)ofsecondaryspongiosain
Wewishedtobetterunderstandthephysiologicalrelevanceof thedistalfemurrevealedthecellularmechanismsofMsx2long
osteogenic-adipogeniclineageallocationbyMsx2onpostnatal bone anabolism. Significant increases in trabecular bone vol-
bone homeostasis. Therefore, we extended our studies to the ume/total volume (Tb.BV/TV; Fig. 3A), Tb.N (Fig. 3B), and
evaluation of bone and body composition in our CMV-Msx2 Tb.Th(Fig.3C)inMsx2Tgmice(n(cid:8)7)versusnontransgenic
transgenic (Msx2Tg) mice (19), a murine model previously siblings(n(cid:8)11)wereobservedat6monthsofage.Importantly,
demonstrated by Maxson and co-workers (13) to efficiently thetrabecularboneformationrate/tissuevolume(BFR/TV;tis-
recapitulate key features of Msx2 actions in craniosynostosis. suevolumereferent)wasincreased76%(p(cid:8)0.03;Fig.3D).A
JULY18,2008•VOLUME283•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 20509
WntSignalsMediateMsx2BoneAnabolism
FIGURE2.Msx2Tgmiceexhibitgreatertrabecularbonevolumeandnum-
bersofplate-liketrabeculaeascomparedwithnon-TgWTsiblings.Fol-
lowingeuthanasia,lefttibiaefrommaleMsx2Tg(n(cid:8)7)andWTnon-Tgmale
siblings(n(cid:8)11)weredissected,fixedinethanol,andanalyzedbymicro-CT.
A, total bone volume was increased as a percentage of tissue volume in
Msx2Tgversusnon-Tgsibs.Additionally,trabecularnumber(B)andconnec-
tivity density, an index of trabecular strut-like interaction, is increased in
Msx2Tganimals(C).Notethatthestructuralmodelingindex,aparameterthat
decreasesastrabeculaebecomemoresturdyandplate-like(52),isdecreased
in Msx2Tg mice (D). Tb.BV/TV, trabecular bone volume; Tb.N, trabecular
number.
similarchangewasnotedwhenbonesurfacereferentwasused
for BFR (WT (cid:8) 52.5 (cid:6) 9.1 (cid:3)m3/(cid:3)m2/year versus Msx2Tg (cid:8)
73.7 (cid:6) 8.4 (cid:3)m3/(cid:3)m2/year, p (cid:8) 0.065 for the 40% BFR/BS
increase). A 2.7-fold increase in osteoid surface/bone surface
wasseenwithnochangeinosteoidthickness(notshown),indi-
catingnormalandunchangedmatrixmineralization.Because
mineralizingsurface(MS/BS;Fig.3E)wasincreasedwithouta
changeinmatrixappositionrate(MAR;Fig.3F),thisstrongly
suggestedMsx2Tg-inducedincreasesinBFR/TVwerebecause
ofincreasednumbersofosteoblasts.Osteoblastnumberswere
indeed increased; the osteoblast perimeter/bone perimeter
(Ob.Pm/B.Pm)was50%greaterinMsx2Tgversusnon-Tgsibs
(p(cid:8)0.001;Fig.3G).Bycontrast,osteoclastnumberswerenot
increased (Oc.Pm/B.Pm; Fig. 3H). Increased bone formation
without significant changes in osteoclast-mediated bone
resorptionwasconfirmedbyserummarkeranalysis(54);serum
TRAP5c (tartrate-resistant acid phosphatase; WT (cid:8) 1.09 (cid:6)
0.11IU/mlversusMsx2Tg(cid:8)1.01(cid:6)0.17IU/ml;p(cid:8)0.68)and
CTX(collagenC-terminaltelopeptide;WT(cid:8)29.7(cid:6)4.2ng/ml
versus Msx2Tg (cid:8) 23.0 (cid:6) 3.6 ng/ml; p (cid:8) 0.28) did not differ
between Msx2Tg and non-Tg sibs. Thus, Msx2Tg increases
long bone mass and trabecular bone formation by increasing
FIGURE1.SkeletalaccumulationofMsx2proteinisincreasedinMsx2Tg
thetotalnumberoftrabecularbone-formingosteoblasts,with-
mice.MicetransgenicforMsx2expressionenhancedbytheCMVpromoter
weregeneratedasdetailedpreviously(19).Cellularextractspreparedfrom outsignificantchangesinosteoclast-mediatedboneresorption.
primarycellculturesofneonatalcalvarialandlongboneosteoblastswere
analyzedforMsx2proteinaccumulationbyWesternblotanalysis.Expression
levelswerereferencedtothatofactinexpression.A,notethatMsx2protein
accumulationinbothcalvarialandlongbonemesenchymalcellculturesis ossificationcentersofMsx2Tgmice.Nosignalwasobservedintheabsenceof
enhanced2-foldbyMsx2Tg.LevelsofMsx2mRNAaccumulationwereequiv- primaryantibody.C,higherpowermagnificationofchondrocytesinsecond-
alentlyincreased(p(cid:7)0.05,seetext).B,immunoperoxidasestainingforMsx2 aryossificationcenterssuggestedonlynuclearlocalizationofMsx2inboth
expressionwasmoreintenseinlongbonesfromMsx2TgversusWTsiblings, WT(shown)andMsx2Tgmice(notshown).D,inmarrowspongiosa(leftpanel)
localizedtoearlyhypertrophiczone(HZ)chondrocytes,andperiostealand andperiosteal(rightpanel)skeletalcompartments,bothnuclear(whitearrow-
marrow compartments of the metaphysis (MET) and diaphysis (DIA). heads)andnon-nuclear(blackarrowheads)Msx2proteinaccumulationwas
Expanded Msx2 protein accumulation was also observed in secondary observed.Aby,antibody.
20510 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER29•JULY18,2008
WntSignalsMediateMsx2BoneAnabolism
0.37,p(cid:8)0.05)witha10%percentreductionintotalbodyfat
(WT (cid:8) 14.4 (cid:6) 0.53%; Msx2Tg (cid:8) 13.03 (cid:6) 0.42%, p (cid:8) 0.04)
versusnontransgeniclittermates.Nodifferenceinoverallbody
weightwasobserved(WT(cid:8)14.74(cid:6)0.28g;Msx2Tg(cid:8)15.16(cid:6)
0.52 g; p (cid:8) 0.23). The significant differences in BMD and fat
masspersistedafter4monthsofchallengewithhighfatdiets
withcontenttypicalofwesternizedsocieties;wholebodyBMD
remainedsignificantlyelevated(WT(cid:8)51.84(cid:6)0.46mg/cm2
versusMsx2Tg(cid:8)52.92(cid:6)0.42mg/cm2,p(cid:8)0.04),andfatmass
was reduced (WT (cid:8) 45.3 (cid:6) 0.84% versus Msx2Tg (cid:8) 43.1 (cid:6)
0.61%;p(cid:8)0.02)inMsx2Tgmice(Fig.4A).Again,nodifference
inoverallbodyweightwasdetectedbetweenobeseMsx2Tgand
non-Tglittermates(p(cid:8)0.43;Fig.4A).Serumleptin,anadipo-
kine marker of body fat content (55), was also reduced in
Msx2Tg versus non-Tg sibs after dietary challenge (WT (cid:8)
1773(cid:6)384pg/ml;Msx2Tg(cid:8)452(cid:6)174;p(cid:7)0.001;Fig.4B).
Consistentwiththewholebodychangesintissuecomposition,
examination of proximal tibiae revealed reduced marrow fat
accumulation by histology in Msx2Tg mice; Fig. 4C is repre-
sentativeofresultsobservedfromeightanimalsstudied,fourof
eachgenotype(seesupplementalmaterial).Quantitativeanal-
ysisoflongbonemesenchymalcellsisolatedfromfourWTand
seven Msx2Tg mice fed high fat diets for 4 months demon-
stratedareductioninoilredO-positiveadipocyticcolonyfor-
mation(WT(cid:8)76.4(cid:6)0.8%,Msx2Tg(cid:8)35.7(cid:6)6.0%oilred-O
positivecolonies;seeFig.4D).Thus,ascomparedwithnon-Tg
WTlittermates,Msx2Tgmiceexhibitgloballyenhancedbone
mass,withreciprocalreductionsinbodyfatandlongbonemar-
rowfat.
LongBoneMesenchymalCellsDerivedfromMsx2Transgenic
Mice Exhibit Enhanced Osteogenic Differentiation in Vitro in
Addition to Reduced Adipogenic Differentiation—To further
study the mechanisms of increased bone formation and
reducedadiposeinMsx2Tgmice,weisolatedlongbonemes-
enchymalcellsandcalvarialosteoblastsfromWTandMsx2Tg
miceandcomparedtheirosteogenicactivitiesincultures.The
FIGURE3.Boneformationandnumbersofosteoblastsareincreasedin osteogenic potential of Msx2Tg bone mesenchymal cell cul-
Msx2Tgwithoutsignificantchangesinnumbersofosteoclasts.Following tures,whichcontainbothosteoblastandadipocyteprecursor
euthanasia,femursweredissectedandanalyzedbystaticanddynamichisto-
cells, was increased 1.5-fold relative to WT littermate bone
morphometryinthetrabecularspongiosaofthedistalrightfemur(0.5–1.6
mmproximaltothegrowthplate).ForMsx2Tg,n(cid:8)7;fornon-Tgsibs,n(cid:8)11. mesenchymal cell cultures (WT (cid:8) 37.5 (cid:6) 5.6%, Msx2Tg (cid:8)
A, as initially observed by micro-CT, histological assessment confirmed 56.3(cid:6)8.4%mineralizedcolonies;seeFig.5A).Moreover,cal-
increasedbonevolumeaspercentoftotaltissuevolumeinMsx2Tgversus
non-Tgsibs.Trabecularnumber(B)andtrabecularthickness(C)werealso varial osteoblasts or bone mesenchymal cells from Msx2Tg
increased.Dynamichistomorphometryrevealedincreasesinboneformation mice exhibited significantly increased ALP activity (2.08 (cid:6)
(D, tissue volume referent). Mineralizing surface is increased (E) without
0.07-fold;seeFig.5B)andequivalentlyelevatedmRNAaccu-
changesinmineralappositionrate(F),arisingfromincreasesinnumbersof
bone-formingosteoblasts(G)inMsx2Tgmice.Nosignificantchangeinbone- mulation for ALP, osterix (Osx), and bone sialoprotein (Fig.
resorbing osteoclast numbers (H) or activity (see text) was observed in 5C).Intriguingly,nochangewasobservedintherelativeaccu-
Msx2Tgmiceversusnon-Tgsibs.Tb.BV/TV,trabecularbonevolume;Tb.N,tra-
becularnumber;Tb.Th,trabecularthickness;MS,mineralizingsurface;Oc, mulation of Runx2 message (Fig. 5C). Accumulation of the
osteoclast;Ob.Pm,osteoblastperimeter/boneperimeter. nucleartranscriptionalco-adapter,(cid:2)-catenin,hasbeenshown
to be critical to the elaboration of osteoblast development
Msx2TransgenicMiceExhibitGloballyHigherBoneMineral downstreamofosteogenicWntsignaling(56).Therefore,cel-
Density,withLowerBodyandMarrowFatasComparedwith lularextractsofbonemesenchymalcellculturefromWTand
Wild-typeLittermates—Globalanalysisofbodycompositionby Msx2Tgmicewereprepared,and(cid:2)-cateninproteinaccumula-
dual electron x-ray absorptiometry (44) in 1-month-old sibs tionwasquantifiedbyELISA.Totalcellular(cid:2)-cateninprotein
confirmed and extended the detailed histomorphometry of waselevated1.6-foldinMsx2Tgversusnon-Tglongbonecell
hindlimblongbone;wholebodydualelectronx-rayabsorpti- cultures(Fig.5D,p(cid:8)0.02)and2-foldincalvarialosteoblasts
ometryindicatedthattheMsx2Tgskeletongloballyexhibiteda (datanotshown).Thus,theexvivoosteogenicpotentialofmes-
small but statistically significant 3.3% greater bone mineral enchymalprogenitorsisolatedfromMsx2Tgmiceisenhanced
density(BMD;WT(cid:8)30.38(cid:6)0.39mg/cm2;Msx2Tg(cid:8)31.39(cid:6) comparedwithnon-TgWTsibs,consistentwiththeincreased
JULY18,2008•VOLUME283•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 20511
WntSignalsMediateMsx2BoneAnabolism
osteoblast numbers observed in Msx2Tg mice long bone as
quantifiedbyhistomorphometry.
Msx2Tg Activation of Osteogenic Differentiation Is Antago-
nizedbyDkk1,anInhibitorofCanonicalWntSignaling—The
reciprocalcontrolofbonemesenchymalcellosteogenesisand
adipogenesisbytheMsx2transgenewashighlyreminiscentof
the effects of canonical Wnt signaling (22, 23). Wnt10b has
beenshowntopromoteosteogenesisandsuppressadipogenic
differentiationfrommultipotentmarrowosteoprogenitors(22,
23).IfparacrinecanonicalWntsignalscontributetotheeffects
ofMsx2Tgonbonemarrowosteogenesis,thenDkk1,ahighly
specificinhibitorofLRP5/6Wntreceptorsignalingcomplexes
(36),shouldantagonizeMsx2Tgactions.Wethusgenerateda
retroviral expression construct, SFG-Dkk1, to augment Dkk1
production by bone mesenchymal cells. We first tested the
effectsofSGF-Dkk1transductiononC3H10T1/2cellcultures
previously transduced with SFG-Msx2; in this validated cell
culture model, Msx2 actions are antagonized by exogenous
recombinant purified Dkk1 (19). As shown in Fig. 6A, SFG-
Dkk1 completely abrogated the effects of SFG-Msx2 on ALP
inductioninC3H10T1/2cells.Wenextevaluatedtheeffectsof
SFG-Dkk1 transduction on osteogenic differentiation using
calvarialosteoblastsisolatedfromMsx2Tgversusnon-Tgsib-
lings. As compared with control virus (SFG-LacZ), transduc-
tionwithSFG-Dkk1profoundlyinhibitedthemineralizednod-
ule formation up-regulated by the Msx2 transgene (Fig. 6B).
Thus,Msx2Tginductionofosteogenicdevelopmentintrans-
genic osteoblasts is dependent upon endogenous paracrine
canonicalWntsignalingcascades.
TheMsx2TransgeneUp-regulatesWnt7butSuppressesDkk1
ProteinAccumulationinCalvarialOsteoblastsandLongBone
MesenchymalCells—Ourstudiesofarterialcalcificationhave
revealed that Msx2 controls osteogenic lineage allocation of
aorticvascularprogenitors(28)viaparacrineWntsignals(19).
The body composition changes observed in response to
Msx2TgresembledthoseofthecanonicalligandWnt10b(20,
22, 23). Moreover, because SFG-Dkk1 inhibited Msx2Tg
actions, this strongly suggested that modulation of Wnt and
Dkk1proteinbiogenesiscontributestoMsx2boneanabolism
(seeaboveandRef.28).Totestthisnotion,weanalyzedprotein
accumulation of Wnt1, Wnt3a, Wnt7 (antibody recognizes
both Wnt7a and Wnt7b), Wnt10b, and Dkk1-prominent
genomic targets of Msx2 first identified in aortic adventitial
myofibroblaststransducedwithSFG-Msx2(19).Ascompared
with non-Tg sibs, Msx2Tg up-regulated Wnt1 and Wnt7 in
calvarial osteoblasts (cid:3)3-fold, but suppressed Dkk1 protein
weightwereobservedbetweengenotypes;however,wholebodybonemin-
eraldensity(BMD)wasincreasedinMsx2Tgmice(n(cid:8)22)versusnon-TgWT
(n(cid:8)34)siblings.Bycontrast,fatmasspercentagewasdecreasedinMsx2Tg
animals.B,decreasesinfatmassinMsx2Tgmicewereparalleledbyeven
greaterreductionsinserumleptin(WT,n(cid:8)11;Msx2Tg,n(cid:8)7).C,histologic
analysisofWTandMsx2Tgsiblingsdemonstratedthatmarrowfatwasalso
diminishedinMsx2Tganimalsversusnon-Tgsibswhenfedwesternizeddiets.
FIGURE 4. Msx2Tg mice exhibit globally increased bone mass and Thehistologyshownisrepresentativeofresultsobtainedfromeightanimals
reducedfatmassthannon-Tgsiblings.Beginningat1monthofage,male analyzed(WT,n(cid:8)4;Msx2Tg,n(cid:8)4;seesupplementalmaterial).D,adipocytic
(shown)andfemale(notshown)Msx2Tgandnon-Tg(WT)siblingcohorts OilRedO-positivecolonieswerequantifiedinprimarybonemarrowmesen-
werefedhighfatdietcompositiontypicalofwesternsocietiesfor16weeks. chymalcellculturesobtainedfromWT(n(cid:8)4)andMsx2Tg(n(cid:8)7)micethat
Subsequently, animals were weighed, and body composition was deter- hadbeenfedthehighfatdietfor4months(see“ExperimentalProcedures”).
minedbydualelectronx-rayabsorptiometry.A,nodifferencesinoverallbody NotetherelativereductioninadipogenicpotentialbytheMsx2transgene.
20512 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER29•JULY18,2008
WntSignalsMediateMsx2BoneAnabolism
Higher power magnification more
clearly revealed vascular sinusoid
Wnt7 staining pattern in marrow
(seesupplementalmaterial),aswell
as in Haversian bone vasculature
(Fig. 7E, asterisk and stout arrow)
andinosteocytes(Fig.7E,longthin
arrows). Of note, the pattern of
Wnt7 protein accumulation was
completely distinct from that of
Wnt1 immunoperoxidase staining,
which clearly localized to marrow
megakaryocytic cells (data not
shown). Thus, Msx2Tg controls
canonical Wnt ligand protein
expression, consistently augment-
ingtheaccumulationofWnt7while
reducing Dkk1 protein levels in
osteoblasts derived from calvaria
andlongbone.
Msx2TgEnhancesCanonicalWnt
SignalinginLongBoneinVivo—To
confirm that the Msx2Tg enhances
endogenous canonical Wnt signal-
ing in bone, we crossed Msx2Tg
mice with TOPGAL reporter mice
(32) and characterized (cid:2)-galacto-
FIGURE5.PrimarybonemesenchymalcellculturesfromMsx2Tgmiceexhibitgreaterosteogenicdiffer-
entiationascomparedwithnon-Tgsibs.Primaryculturesofhindlimblongbonemesenchymalcellswere sidase(LacZ)staininginlongbones
preparedandculturedunderpro-osteogenicconditions.Numbersofosteogeniccolonieswerescoredby ofneonatalmice.Ascomparedwith
AlizarinRedstainingasdescribedpreviously(28).A,notethatnumbersofosteogeniccolonieswereincreased
by(cid:3)50%inMsx2Tg(n(cid:8)5)versusnon-Tgsibs(n(cid:8)5).BandC,Msx2Tg-enhancedosteogenicdifferentiation TOPGAL siblings, trabecular LacZ
wasconfirmedby2-foldincreasesinbothalkalinephosphataseenzymeactivityofprimarycalvarialosteoblast staining in the trabecular primary
cultures(B)andexpressionoftheosteogenicgenes,ALP,BSP,andOsx(C).ForbothWTandMsx2Tgosteoblast spongiosa and the endosteal bone
cultures,n(cid:8)4.D,totalcellular(cid:2)-cateninlevelsmeasuredbyELISAwerealsoincreasedinculturesderivedfrom
Msx2Tgmice(n(cid:8)3)versusnon-Tgsiblings(n(cid:8)3). envelope was much more promi-
nentinMsx2Tgversusnon-Tgsibs
accumulation(Fig.7A,expressionnormalizedtoeIF2(cid:1)protein (Fig.8A).AnalysisoftotallongboneRNAfromolder2-month-
signalintensity).NochangewasobservedinWnt3aorWnt10b old cohorts confirmed the histochemical results; RT-qPCR
proteinaccumulation.Inlongbonemesenchymalcells,littleif demonstrated higher levels of both lacZ reporter and Wnt7a
anychangeoccurredinWnt1andWnt10bproteinlevels(Fig. gene expression in Msx2Tg(cid:9);TOPGAL versus TOPGAL sibs
7A). However, Wnt7 was once again increased, and Dkk1 (Fig. 8B). By contrast, no significant up-regulation of Wnt1,
decreased,bytheMsx2transgene;accumulationofWnt7pro- Wnt3a, or Wnt10b mRNAs was observed in long bone of
tein was increased (cid:3)10-fold in Msx2Tg animals versus non- Msx2Tgmice(Fig.8B,leftpanel).Wnt7bmRNAalsoaccumu-
transgenic sibs (Fig. 7A). As compared with calvarial osteo- latedto3-foldhigherlevelsinlongbonefromMsx2Tgversus
blasts, Wnt3a protein levels were lower in long bone non-Tganimals(Fig.8B,rightpanel).Moreover,aspredicted
mesenchymal cells but were increased 2-fold by the Msx2 from our Western blot analyses, serum levels of Dkk1 were
transgene. significantly reduced by 43% in Msx2Tg mice versus non-Tg
Because Wnt7 protein accumulation was consistently and siblings(Fig.8C).
dynamically regulated by the Msx2Tg in primary cell culture, WenextstudiedLacZstainingofculturedbonemesenchy-
immunohistochemistry was used to spatially resolve the mal cells from TOPGAL mice (32) to provide an additional
expression of Wnt7 in Msx2Tg long bone. In neonatal mice, indexofcanonicalWnt/(cid:2)-cateninsignaling.Ascomparedwith
Wnt7accumulatedintheendostealcompartmentofthemin- TOPGALsiblings,longbonemesenchymalcellsfromMsx2Tg;
eralizingperiosteumandalongthetrabeculaeofthespongiosa TOPGAL mice exhibit a 5-fold increase in the numbers of
(Fig. 7B). Wnt7 expression was also observed in early hyper- LacZ-stained cells (Fig. 8D; p (cid:7) 0.0001). Similar results were
trophiczonechondrocytes(Fig.7B),thusoverlappingthepat- obtained with primary calvarial osteoblasts (data not shown).
ternofMsx2accumulationinneonatalMsx2Tgmice(Fig.1,B Thus, histochemical, quantitative RT-qPCR, and biochemical
andC).Inadultmouselongbone,Wnt7immunoreactivitywas dataconvergetodemonstratethattheMsx2Tgaugmentsskel-
stillprominent,detectedinbonemarrowvascularstructures, etalcanonicalWntsignalingcascades.
mineralizingspongiosa,andosteocytes(Fig.7C).Nosignalwas Canonical Wnt7-LRP Signaling Contributes to Msx2-de-
observed in the absence of primary Wnt7 antibody (Fig. 7D). pendentInductionofOsteogenesisinC3H10T1/2Mesenchy-
JULY18,2008•VOLUME283•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 20513
WntSignalsMediateMsx2BoneAnabolism
FIGURE6.Msx2Tg-enhancedosteogenicnoduleformationisinhibitedby
Dkk1.Thepan-tropicretroviralvectorSFG(28)wasusedtoexpresseither
Dkk1orlacZcontrolbytransductionofC3H10T1/2cells(A)orprimaryosteo-
blastsfromWTandMsx2Tgmice(B).SFG-Msx2wasusedasastrategyto
up-regulateMsx2-WntsignalinginC3H10T1/2cellsasdetailedpreviously
(19).A,SFG-Dkk1virusabrogatedSFG-Msx2inductionofALPactivity,func-
tionallyvalidatingthebioactivityoftheDkk1expressionvector.Foreach
group,n(cid:8)3.B,ascomparedwiththeSFG-LacZcontrolvirus(upperpanels),
SFG-Dkk1transduction(lowerpanels)markedlydiminishedosteogenicnod-
uleformationinbothWT(leftpanels)andMsx2Tg(rightpanels)bonemesen-
chymalcellcultures.(cid:4)400magnification,scalebar,0.1mm.
mal Cells—Because Wnt7 protein levels were significantly
affectedbytheMsx2Tg,andbothWnt7aandWnt7bareago-
nists for canonical Wnt signaling (21, 57–59), Wnt7 family
members are potential paracrine mediators of Msx2 action.
FIGURE7.TheMsx2TgincreasesWnt7anddecreasesDkk1proteinaccu-
Moreover,asobservedinothercellsystems(21),transientco-
mulation in calvarial osteoblasts and long bone mesenchymal cells.
expression of Wnt7a and Wnt7b eukaryotic expression plas- A,primarycalvarialosteoblastandlongbonemesenchymalcellcultureswere
preparedfromWTandMsx2TgsiblingsandextractsanalyzedforMsx2-re-
midssignificantlyactivatecanonicalTCF/LEF-dependenttran-
sponsivecanonicalWntregulatorsbyWesternblot.Relativeproteinaccumu-
scriptioninC3H10T1/2cells(seesupplementalmaterial).The lation was normalized to that of the housekeeping protein, eIF2(cid:1), and
C3H10T1/2multipotentmesenchymalcelllinefaithfullyreca- expressedasfolddifferenceofthenon-Tgcontrol.Wnt7(antibodyrecog-
nizesbothWnt7aandWnt7b)wasincreasedbytheMsx2Tgincalvarialand
pitulates the osteogenic versus adipogenic differentiation fate
longbonecultures,whereasDkk1proteinwasreciprocallydecreased.Wnt1
choicesofbonemarrowmesenchyme(23,28).Thus,tofurther andWnt3awerealsoregulated,increasedincalvarialandlongbone,respec-
establishtheroleofparacrinecanonicalWnttonetotheactions tively.Althoughexpressed,Wnt10bproteinwasnotinducedbytheMsx2Tg.
B,inneonatallongbone,Wnt7immunolocalizedtoendosteum(largearrow),
of Msx2, we examined the effects of depleting key inhibitors trabecularspongiosa(smallarrow),andearlyhypertrophiczone(HZ)chon-
(Dkk1)andactivators(Wnt1,Wnt7a,andWnt7b)ofthispath- drocytes(brackets).Wnt7proteinaccumulationwasalsodetectedinsecond-
aryossificationcentersoflongbone(notshown).C,strikingWnt7expression
wayontheactionsofMsx2intheC3H10T1/2model.Wefirst
(arrows)wasdetectedinmarrowvascularsinusoids(whitearrows),andthe
establishedthatthereceptorsforDkk1andcanonicalWntsig- mineralizinggrowthplate(blackarrows),aswellasinosteocytes(fineblack
naling,LRP5andLRP6,participatedinMsx2inductionofALP. arrows)ofadultlongbone.D,nosignalwasobservedintheabsenceofWnt7
primaryantibody.E,vascularcellsliningtheHaversiancanal(asteriskand
AsshowninFig.9A,siRNAdirectedtoLRP6,andtoalesser
large arrow) also accumulate Wnt7 protein, as do osteocytes (fine black
extent LRP5, inhibited Msx2 induction of bone ALP in arrows).
20514 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER29•JULY18,2008