Parathyroid Hormone-Smad3 Axis Exerts Anti-apoptotic Action and Augments Anabolic Action of ...

14 Pages·2003·0.79 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY Vol.278,No.52,IssueofDecember26,pp.52240–52252,2003
©2003byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedinU.S.A.
Parathyroid Hormone-Smad3 Axis Exerts Anti-apoptotic
Action and Augments Anabolic Action of Transforming
Growth Factor (cid:1)in Osteoblasts*
Receivedforpublication,March13,2003,andinrevisedform,July22,2003
Published,JBCPapersinPress,September29,2003,DOI10.1074/jbc.M302566200
HideakiSowa,HiroshiKaji,MeiFwayIu,TatsuoTsukamoto,ToshitsuguSugimoto‡,
andKazuoChihara
FromtheDivisionofEndocrinology/Metabolism,NeurologyandHematology/Oncology,DepartmentofClinicalMolecular
Medicine,KobeUniversityGraduateSchoolofMedicine,7-5-2Kusunoki-cho,Chuo-ku,Kobe650-0017,Japan
Although several studies indicated that parathyroid consists of two different events, resorption by osteoclasts and
hormone(PTH)exertedanabolicactiononbone,itspre- formationbyosteoblasts.Foranincreaseinbonemineralden-
cise mechanisms have been unknown. On the other sity,apositivebalance,inwhichtheformationispriortothe
hand, transforming growth factor (cid:1) (TGF-(cid:1)), abun- resorption, should be gained. The ability to gain the positive
dantlystoredinbonematrix,stimulatesboneformation balancemaybeatherapeuticstrategyforosteoporosis.
withalocalinjectioninrodents.Althoughourprevious Although several agents are capable of decreasing bone re-
study suggested that Smad3 is an important molecule sorptionandhaltingfurtherbonelossinosteopenicstates,the
for the stimulation of bone formation, no reports have idealdrugwouldbeananabolicagentthatincreasesbonemass
been available about the effects of PTH on Smad3. In
by stimulating bone formation. It has been well established
this present study, we examined the effects of PTH on
that daily injections of low dose of parathyroid hormone
Smad3 and the physiological significance in mouse os-
(PTH),1 a main regulatory hormone in calcium and bone me-
teoblasticcells.PTHpromotedtheexpressionofSmad3
tabolism, increase bone mass in animals and humans (1–8).
mRNAwithin10minandtheproteinlevelinadose-de-
However,themechanismsbywhichPTHpossessesboneana-
pendentmannerinMC3T3-E1andratosteoblasticUMR-
bolicactioninvivoarenotfullyknown.
106 cells. Protein kinase A (PKA) activator as well as
Ontheotherhand,transforminggrowthfactor(cid:1)(TGF-(cid:1))is
proteinkinaseC(PKC)activatorsincreasedSmad3pro-
mostabundantinbonematrix,comparedwithothertissues(9).
tein level, and both PKA and PKC inhibitors antago-
nized PTH-induced Smad3, indicating that PTH pro- TGF-(cid:1)is stored in an inactive form, released from the bone
motes the production of Smad3 through both PKA and matrix,andactivatedinthebonemicroenvironment(10).Itis
PKC pathways. Next, we examined anti-apoptotic ef- producedbyosteoblastsandappearstoregulatebonemetabo-
fectsofPTHandSmad3inthesecells,employingtrypan lisminvariousways,includingskeletaldevelopmentandbone
blue, transferase-mediated nick end labeling, and Ho- remodeling (11). TGF-(cid:1)modulates the proliferation, differen-
echst staining. Pretreatment with PTH or overexpres- tiation, and production of bone matrix proteins of osteoblasts
sion of Smad3 decreased the number of apoptotic cells (10). Several reports demonstrated that TGF-(cid:1)induced bone
induced by dexamethasone and etoposide. Moreover, a formationwhenitwaslocallyadministeredintobonetissuesin
dominant negative mutant, Smad3(cid:2)C, abrogated PTH- rat(12–15).TheSmadfamilyproteinsarecriticalcomponents
inducedanti-apoptoticeffects.Ontheotherhand,PTH oftheTGF-(cid:1)signalingpathways(16,17),andTGF-(cid:1)regulates
augmentedTGF-(cid:1)-inducedtranscriptionalactivity.Fur- the transcriptional response of the target genes through the
thermore, PTH enhanced TGF-(cid:1)-induced production of two receptor-regulated Smads, Smad2 and Smad3 (16, 17).
typeIcollagen,whereasitdidnotaffectTGF-(cid:1)-reduced
Receptor-mediated phosphorylation of Smad2 or Smad3 in-
proliferation in MC3T3-E1 cells. These observations in-
duces their association with the common partner Smad4, fol-
dicatedthatPTHamplifiedtheanaboliceffectsofTGF-(cid:1)
lowedbytranslocationintothenucleuswherethesecomplexes
byacceleratingthetranscriptionalactivityofSmad3.In
activate transcription of specific genes (16, 17). We recently
conclusion,wefirstdemonstratedthatPTH-Smad3axis
reported that Smad3 promotes the production of type I colla-
exerts anti-apoptotic effects in osteoblasts and rein-
forces the anabolic action by TGF-(cid:1) in osteoblasts. gen, alkaline phosphatase activity, and mineralization in
mouseosteoblasticMC3T3-E1cells(18,19).Moreover,themice
Hence, PTH-Smad3 axis might be involved in the bone
with the target disruption of Smad3 exhibited the osteopenia
anabolicactionofPTH.
caused by the decreased bone formation (20). Based on these
data,wehaveproposedthatSmad3isamoleculeofpromoting
The bone is a highly specialized and dynamic organ with boneformation.
continuous regeneration, called remodeling. Bone remodeling
1The abbreviations used are: PTH, parathyroid hormone; TGF-(cid:1),
*ThisworkwassupportedinpartbyagrantfromKanzawaMedical transforming growth factor (cid:1); PKA, protein kinase A; PKC, protein
ResearchFoundation(toH.K.);Grants-in-aid15590977(toH.K.)and kinase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS,
14571064(toT.S.)fromtheMinistryofScience,Education,andCul- phosphate-buffered saline; (cid:2)-MEM, (cid:2)-minimal essential medium;
tureofJapan;andagrant-in-aidfromtheHormoneReceptorAbnor- DMEM,Dulbecco’smodifiedEagle’smedium;db-cAMPS,N6,O2(cid:1)-dibu-
malityResearchCommitteeMinistryofHealthandWelfareofJapan tyryl adenosine 3(cid:1),5(cid:1)-cyclic monophosphate; (S )-cAMPS, S diaste-
(toT.S.).Thecostsofpublicationofthisarticleweredefrayedinpartby reomer of adenosine cyclic 3(cid:1),5(cid:1)-phosphorothioapte; FBS, fetapl bovine
the payment of page charges. This article must therefore be hereby serum;PMSF,phenylmethylsulfonylfluoride;DTT,dithiothreitol;RT,
marked “advertisement” in accordance with 18 U.S.C. Section 1734 reverse transcriptase; TUNEL, transferase-mediated dUTP nick end
solelytoindicatethisfact. labeling; IGF, insulin-like growth factor; Runx2, Runt-related tran-
‡To whom correspondence should be addressed. Tel.: [phone]- scriptionalfactor2;AP-1,activatorprotein-1;CREB,cAMP-responsive
5885;Fax:[phone];E-mail:[email protected]. element-bindingprotein.
52240 Thispaperisavailableonlineathttp://www.jbc.org
This is an Open Access article under the CC BY license.
Bone Anabolic Action of PTH-Smad3 Axis 52241
Although several studies indicated that PTH increases SubcellularFractionation—Culturesweretrypsinized,andthecells
TGF-(cid:1)expression and secretion in osteoblasts (21, 22), there werewashedwithPBSandcollectedbycentrifugation(24).Cellswere
havebeennopapersthatreportedtheeffectsofPTHonSmad3 gentlyresuspendedin2mlofbuffercontaining5mMKCl,1mMMgCl2,
20mMHEPES,10mMEDTA,0.5mMPMSF,0.5%aprotinin,and0.5%
in osteoblasts. Hence, in our present study, we examined the
leupeptin; allowed to swell for 10 min; processed by 20 strokes in a
effectsofPTHontheexpressionandthetranscriptionalactiv- Dounce tissue homogenizer; and centrifuged at 2000 (cid:4) g for 10 min.
ity of Smad3, and also its physiological significance in Afterdecantingthesupernatant,thepelletwasresuspendedin1mlof
osteoblasts. radioimmunoprecipitationbuffer(150mMNaCl,10mMTris-HCl(pH
7.4),1%deoxycholate,0.1%SDS,0.5%aprotinin,and0.5mMPMSF)
EXPERIMENTALPROCEDURES andbrieflysonicated.Nuclearpelletswereobtainedbycentrifugation
Materials—MC3T3-E1andUMR-106cellswerekindlyprovidedby at15,000(cid:4)gfor20minat4°C;resuspendedin20mMHEPES(pH7.9),
Dr.H.Kodama(OhuDentalCollege,Ohu,Japan)andDr.T.J.Martin 25%glycerol,0.42MNaCl,1.5mMMgCl2,0.2mMEDTA,0.5mMDTT;
(St. Vincent’s Institute of Medical Research, Melbourne, Australia), andagainprocessedinaDouncehomogenizer.Aftera20-mincentrif-
respectively. Human recombinant TGF-(cid:1), human PTH-(1–34), cyclo- ugationat15,000(cid:4)g,supernatantsweredialyzedfor5hagainst20
heximide, actinomycin D, phorbol 12-myristate 13-acetate, forskolin, mMHEPES(pH7.9),20%glycerol,0.1MKCl,0.2mMEDTA,0.5mM
N6,O2(cid:1)-dibutyryl adenosine 3(cid:1),5(cid:1)-cyclic monophosphate (db-cAMPS), PMSF,and0.5mMDTT.ProteinquantitationwasperformedwithBCA
PTH-(3–34)aminopeptide,staurosporine,H7,andH89werepurchased proteinassayreagent(Pierce).Westernblotanalysiswasperformedas
fromSigma,andS diastereomerofadenosinecyclic3(cid:1),5(cid:1)-phosphoro- describedabove.
p
thioate ((S )-cAMPS) from Biolog Life Science Institute (Bremen, RNA Extraction and Northern Analysis—Total RNA was prepared
p
Germany). Anti-Smad3, Smad2, Smad4, and anti-phosphorylated from cells using the acid guanidinium-thiocyanate-phenol-chloroform
Smad3antibodieswerepurchasedfromSantaCruzBiotechnology,Inc. extractionmethod.Twenty(cid:3)goftotalRNAwasdenatured,runona1%
(SantaCruz,CA).Allotherchemicalsusedwereofanalyticalgrade. agarosegelcontaining2%formaldehyde,transferredtoanitrocellulose
Cell Culture—MC3T3-E1 and UMR-106 cells were cultured in membrane,andfixedwithultravioletlight(Funa-UV-Linker,Funako-
(cid:2)-MEM(containing50mg/mlascorbicacid)andDMEMsupplemented shi, Tokyo, Japan). The membrane was hybridized to a DNA probe
with 10% FBS and 1% penicillin-streptomycin (Invitrogen), respec- labeledwith32P(AmershamBiosciences)overnightat42°C.Thehy-
tively.Themediumwaschangedtwiceaweek. bridization probe was the 2.8-kb fragment of the (cid:2)1 gene of type I
Construct and Transient Transfection—Myc-tagged Smad2 and procollagen(agiftfromDr.T.Kimura,OsakaUniversity,Osaka,Ja-
Smad3andflag-taggedSmad4wereprepared,aspreviouslydescribed pan).Afterhybridization,thefilterwaswashedtwicewith2(cid:4)standard
(23).Smad3DNAwasderivedfromrat.AmutantformofMyc-tagged saline citrate (SSC) containing 0.5% SDS and subsequently washed
Smad3(Smad3(cid:2)C),inwhichtheMH2domaincorrespondingtoamino twicewith0.1(cid:4)SSCcontaining0.5%SDSat58°Cfor1h.Thefilter
acid residues 278–425 was removed, was kindly provided by Dr. Y. was exposed to x-ray film, using intensifying screen at (cid:5)80°C. All
Chen. Myc-Smad3, Myc-Smad3(cid:2)C, and empty vector (pcDNA3.1(cid:3)) valueswerenormalizedforRNAloadingbyprobingblotswithhuman
(each 3 (cid:3)g) were transfected to MC3T3-E1 and UMR-106 cells with (cid:1)-actincDNA(WakoPureChemicalIndustries,Ltd.,Osaka,Japan).
LipofectAMINE(Invitrogen).Sixhlater,thecellswerefedwithfresh SemiquantitativeReverseTranscription-PolymeraseChainReaction
mediumcontaining10%FBS.Forty-eighthlater,thetransfectedcells (RT-PCR)—Reversetranscriptionof5(cid:3)gofculturedcelltotalRNAwas
were used for the experiments. To rule out the possibility of clonal carriedoutfor50minat42°Candthen15minat70°C,usingSuper
variation,wecharacterizedatleastthreeindependentclonesforeach ScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen),
transfection.Emptyvector-transfectedcellswereusedasthecontrol. whichcontainedRTbuffer,oligo(dT) ,5(cid:4)First-StrandSolution,10
12–18
LuciferaseAssay—Cellswereseededatadensityof2(cid:4)105cells/6- mM dNTP, 0.1 M DTT, SuperScript II (RT-enzyme), and RNase H
wellplate.Twenty-fourhlater,cellsweretransfectedwith3(cid:3)gofthe (RNase inhibitor). PCR using primers to unique sequences in each
reporterplasmid(p3TP-Lux)andthepCH110plasmidexpressing(cid:1)-ga- cDNAwascarriedoutinavolumeof10(cid:3)lofreactionmixtureforPCR
lactosidase(1(cid:3)g),usingLipofectAMINE(Invitrogen).Fifteenhlater, (assuppliedbyTaKaRa,Otsu,Japan),supplementedwith2.5unitsof
themediumwaschangedtoafreshonecontaining4%FBS,andthe TaKaRaTaqTM,1.5mMamountofeachdNTP(TaKaRa),and10(cid:4)PCR
cellswereincubatedforanadditional9h.Thereafter,cellswerecul- buffer,whichcontained100mMTris-HCl(pH8.3),500mMKCl,and15
turedfor24hinthepresenceorabsenceof5.0ng/mlTGF-(cid:1)inmedium mMMgCl2.Twenty-fivengofeachprimerand1(cid:3)loftemplate(froma
containing0.2%FBS.Cellswerelysed,andtheluciferaseactivitywas 50-(cid:3)lRTreaction)wereused.Thermalcyclingconditionsandprimer
measured and normalized to the relative (cid:1)-galactosidase activity as sequences are described below: 1) initial denaturation at 96°C for 2
described(18). min; 2) cycling for cDNA-specific number of cycles; 96°C for 1 min,
ProteinExtractionandWesternAnalysis—Cellswerelysedwithra- cDNA-specificannealingtemperaturefor2min,and72°Cfor2min;
dioimmunoprecipitationbuffercontaining0.5mMPMSF,completepro- and3)finalextensionat72°Cfor5min.Primersequences,annealing
teaseinhibitormixture,1%TritonX-100,and1mMsodiumorthovana- temperature, and cycle numbers were as follows. Smad3 sequences
date.Celllysateswerecentrifugedat12,000(cid:4)gfor20minat4°C,and were5(cid:1)-GAGTAGAGACGCCAGTTCTACC-3(cid:1)and5(cid:1)-GGTTTGGAGAA-
the supernatants were stored at (cid:5)80°C. Protein quantitation was CCTGCGTCCAT-3(cid:1) (62°C; 25 cycles) (25), and glyceraldehyde-3-
performed with BCA protein assay reagent (Pierce). Twenty (cid:3)g of phosphatedehydrogenase(GAPDH)sequenceswere5(cid:1)-ATCCCATCA-
proteinsweredenaturedinSDSsamplebufferandseparatedon10% CCATCTTCCAGGAG-3(cid:1) and 5(cid:1)-CCTGCTTCACCACCTTCTTGATG-3(cid:1)
polyacrylamide-SDSgels.Proteinsweretransferredin25mMTris,192 (58°C;22cycles).ForsemiquantitativeRT-PCR,thenumberofcycles
mMglycine,and20%methanoltopolyvinylidenedifluoride.Blotswere waschosensothatamplificationremainedwellwithinthelinearrange,
blockedwithTris-bufferedsaline(20mMTris-HCl(pH7.5)and137mM asassessedbydensitometry(NIHImageJ,version1.08i,publicdomain
NaCl) plus 0.1% Tween 20 containing 3% dried milk powder. The program).AnequalvolumefromeachPCRwasanalyzedby6%non-
antigen-antibodycomplexeswerevisualizedusingtheappropriatesec- denaturingpolyacrylamidegelelectrophoresis,andethidiumbromide-
ondary antibodies (Sigma), and the enhanced chemiluminescence de- stained PCR products were evaluated. Marker gene expression was
tectionsystem,asrecommendedbythemanufacturer(AmershamBio- normalizedtoGAPDHexpressionineachsample.
sciences,Buckinghamshire,UnitedKingdom). Determination of Osteoblast Apoptosis—Trypan blue staining (In-
For co-immunoprecipitation experiments, cells were lysed with a vitrogen;0.1%finalconcentration)wasusedforroutinequantification
buffercontaining1%TritonX-100,1%deoxycholate,50mMTris-HCl ofapoptosis(26).Inbrief,adropofthecellsuspensionwasmixedwith
(pH7.4),100mMNaCl,25mMsodiumfluoride,10mMsodiumpyro- adropofthetrypanbluesolution.Theratioofeachvolumewas1:1.The
phosphate,2mMsodiumorthovanadate,1.5mMMgCl2,2mMEGTA, totalnumbersofviableandnonviablecellswerecalculatedunderthe
plus a protease inhibitor mixture for 30 min at 4°C, and insoluble light microscopy (27). Apoptosis cleaves cellular DNA into histone-
materials were separated by centrifugation at 4°C for 30 min at associatedfragments.Asthemorespecificstainingtodetectapoptosis,
14,000(cid:4)g.Thesupernatantcontaining1mgofproteinwasclarified transferase-mediated dUTP nick end labeling (TUNEL) staining and
andincubatedwithanti-flagantibody(Sigma)onarockingplatformat Hoechststainingwereemployed.Inbrief,cellswereculturedonround
4°Covernight.TheimmunecomplexeswerecollectedwithProteinG coverglasses(FisherScientific,Pittsburgh,PA)setin6-wellplatesand
Plus/Protein A-agarose beads (Calbiochem-Novabiochem Corp., San theglassesattachedbycellswererinsedwithice-coldPBStwotimes,
Diego,CA)for30minat4°C.Thebeadswerewashedthreetimeswith followedbyfixationwith4%neutralformaldehyde.Then,thesefixed
thelysisbuffer,resuspendedin2(cid:4)samplebuffer,andboiledfor5min. celllayersontheglasseswerestained.TUNELreactionwasperformed
Immunoprecipitated proteins were then analyzed by SDS-PAGE and usinganApoptosisinSituDetectionkit(Wako),followingthestandard
subjected to Western blot analysis employing anti-Myc antibody protocol,asdescribed(28).Hoechststainingwasperformedtoviewthe
(Sigma)asdescribedabove. pyknotic fragmented nuclei typical of apoptotic cells, using Hoechst
52242 Bone Anabolic Action of PTH-Smad3 Axis
FIG.1. PTH stimulates the expres-
sion of Smad3 in a dose-dependent
manner. A, after confluent MC3T3-E1
cellswereculturedinserum-free(cid:2)-MEM
for12h,cellsweretreatedwiththeindi-
cated concentrations of 10(cid:5)8 M PTH-(1–
34) for 1 h. Then, RNA extraction and
semiquantitative RT-PCR assay were
performed, as described under “Experi-
mental Procedures.” B and C, confluent
MC3T3-E1cells(B)orUMR-106cells(C)
wereculturedinserum-free(cid:2)-MEMand
DMEM, respectively, for 12 h, and then
cellsweretreatedwiththeindicatedcon-
centrationsofPTH-(1–34)for6h.Then,
proteinextractionandWesternblotanal-
ysis were performed, as described under
“ExperimentalProcedures.”
33258 (Sigma). In each experiment, the percentages of numbers of within1hinMC3T3-E1andUMR-106cells(Fig.2,BandC).
apoptoticcellspertotalonesinrandomlyselectedfieldswerecalculated These effects of PTH-(1–34) were sustained at least for 24 h.
usinghemocytometer.Eachexperimentwasperformedatleastthree
Moreover, PTH-(1–34) increased the level of phosphorylated
times.
[3H]ThymidineIncorporationAssay—MC3T3-E1cellswereseededat Smad3 within 6 h (Fig. 2D). Because the up-regulation of
2 (cid:4) 104 cells/well in 24-well plates. These cells were maintained in Smad3byPTH-(1–34)wasinitiatedinearlytimes,PTHmight
(cid:2)-MEMwith10%FBS.After48hofculture,cellswerelabeledwith0.5 stimulate the expression of Smad3 without protein synthesis
(cid:3)Ci/ml[3H]thymidine(AmershamBiosciences)for4h.Theincubation denovo.Wethereforeemployedcycloheximide,aproteinsyn-
wasterminatedbyremovalofthemedium,washedwithPBStwice,and
thesis inhibitor, and actinomycin D, a transcription inhibitor.
followedbytheadditionof5%trichloroaceticacidonicein10min.After
removalofthetrichloroaceticacid,theresiduewasdissolvedin20mM Although10(cid:3)McycloheximidedidnotaffectPTH-inducedex-
NaOHat37°C,andscintillationmixturewasadded.Eachsamplewas pression of Smad3 mRNA, PTH-induced Smad3 protein was
countedinaliquidscintillationcounter. reduced with 10 (cid:3)M actinomycin D (Fig. 3). These findings
Statistics—Datawereexpressedasmeans(cid:6)S.E.Statisticalanalysis suggestedthatPTHpromotedtheexpressionofSmad3atthe
wasperformedusinganunpairedttestoranalysisofvariance.
transcriptionallevelindependentlyofproteinsynthesisdenovo
RESULTS inosteoblasts.
PTH Stimulated the Expression of Smad3 mRNA and Pro- PTH Promoted Smad3 Expression via PKA and PKC Path-
tein—First,weexaminedtheeffectsofPTHontheexpressions ways—PTHbindstoPTH/PTH-relatedproteinreceptor,which
ofSmad3mRNAandprotein.InMC3T3-E1cells,PTH-(1–34) is a G-protein-coupled seventh transmembrane-type receptor.
stimulated the expression of Smad3 mRNA in a dose-depend- ThePTHsignalsareknowntobetransducedthroughPKAand
entmanner(Fig.1A).PTH-(1–34)alsostimulatedtheexpres- PKC pathways (1). We therefore investigated whether PTH
sion of Smad3 protein in both MC3T3-E1 and UMR-106 cells wouldup-regulatetheSmad3expressionthroughPKAand/or
(Fig. 1, B and C). Next, we performed the time-course experi- PKCpathway(s).AsshowninFig.4,activatorsforPKApath-
ments.AsshowninFig.2A,PTH-(1–34)promotedtheexpres- ways, db-cAMP and (S )-cAMPS as well as forskolin, and an
p
sion of Smad3 mRNA within 10 min in MC3T3-E1 cells. The activator for PKC pathway, phorbol 12-myristate 13-acetate,
expression of Smad3 protein was enhanced by PTH-(1–34) promoted the expression of Smad3 mRNA and protein in
Bone Anabolic Action of PTH-Smad3 Axis 52243
FIG.2.PTHstimulatestheexpressionofSmad3intheearlytimes.A,afterconfluentMC3T3-E1cellswereculturedinserum-free(cid:2)-MEM
for 12 h, cells were treated with 10(cid:5)8 M PTH-(1–34) for the indicated time. Then, RNA extraction and semiquantitative RT-PCR assay were
performed,asdescribedunder“ExperimentalProcedures.”BandC,afterconfluentMC3T3-E1cells(B)orUMR-106cells(C)wereculturedin
serum-free(cid:2)-MEMorDMEM,respectively,for12h,cellsweretreatedwith10(cid:5)8MPTH-(1–34)fortheindicatedtime.Then,proteinextraction
andWesternblotanalysiswereperformed,asdescribedunder“ExperimentalProcedures.”D,ThemembraneusedinBwasre-hybridizedby
anti-phosphorylatedSmad3antibody(P-Smad3).
MC3T3-E1cells(Fig.4,AandB)andUMR-106cells(Fig.4C). expressionofSmad3mRNAandproteinthroughbothPKAand
Inaddition,aspecificinhibitorofPKApathway,H89,aswell PKCpathwaysinosteoblasts.
as the inhibitors of PKC pathways, staurosporine and H7, Smad3 Is an Essential Molecule for PTH-induced Anti-apo-
antagonized PTH-induced expression of Smad3 mRNA and ptoticEffectsinOsteoblasts—PTHexertsanti-apoptoticsignals
proteininMC3T3-E1cells(Fig.5,AandB)andUMR-106cells in osteoblasts (29). Recent study revealed that mice with the
(Fig. 5C). These findings indicated that PTH stimulates the targeted disruption of Smad3 exhibited osteopenia caused by
52244 Bone Anabolic Action of PTH-Smad3 Axis
soneincreasedthenumbersofTUNEL-positivecells(Fig.7,A
and C). PTH-(1–34) did not affect dexamethasone-induced
TUNEL-positive cell number in Smad3(cid:2)C-transfected cells,
althoughPTHantagonizeditinemptyvector-transfectedcells
(Fig.7,AandC).Hoechststainingmethodisgenerallyrecom-
mendedtodetectapoptoticnucleusmorphologically.Asshown
in Fig. 7B, the nucleus of the empty vector-transfected cells
treated with dexamethasone exhibited the dot-spot appear-
ance,indicatingthatthecellsfellinapoptosis.AsseeninFig.
7D, dexamethasone increased the number of apoptotic cells
transfected with empty vector and Smad3(cid:2)C. However, PTH
did not affect the number of apoptotic cells transfected with
Smad3(cid:2)C,althoughPTHantagonizeditinemptyvector-trans-
fectedcells(Fig.7D).ThesefindingsindicatedthatSmad3isan
essential molecule for PTH-induced anti-apoptotic action in
osteoblasts.
PTH Augmented Smad3-induced Transcriptional Activity—
OurpreviousstudyrevealedthatSmad3promotedtheexpres-
sionoftypeIprocollagen,resultinginaccelerationofmineral-
ization in MC3T3-E1 cells (19). We therefore examined the
effectsofPTHonSmad3-inducedtranscriptionalactivitywith
luciferase assay using 3TP-Lux containing the promoter of
plasminogeninhibitor1withaSmad3-specificresponsiveele-
ment. TGF-(cid:1)increased the transcriptional activity in the ab-
senceofPTH-(1–34)inMC3T3-E1cells(Fig.8A).PTH-(1–34)
significantly augmented TGF-(cid:1)-induced transcriptional activ-
ity, although PTH-(1–34) alone did not affect them (Fig. 8A).
FIG.3. PTH stimulates the expression of Smad3 at the tran- These results suggested that PTH-(1–34) augmented TGF-(cid:1)-
scriptionallevel.A,afterconfluentMC3T3-E1cellswereculturedin induced transcriptional activity of Smad3. Although PTH in-
serum-free(cid:2)-MEMfor12h,cellswerepretreatedwith10(cid:3)Mcyclohex- creased the expression of TGF-(cid:1)in MC3T3-E1 and UMR-106
imide(CHX)for6handtreatedwith10(cid:5)8MPTH-(1–34)for1h.Then, cells (Fig. 8B), PTH alone did not affect the transcriptional
RNA extraction and semiquantitative RT-PCR analysis were per-
formed,asdescribedunder“ExperimentalProcedures.”B,afterconflu- activityinMC3T3-E1cells,andTGF-(cid:1)pretreatmentaswellas
entMC3T3-E1cellswereculturedinserum-free(cid:2)-MEMfor12h,cells PTH pretreatment alone did not affect the transcriptional ac-
werepretreatedwith10(cid:3)MactinomycinD(Act.D)for6handtreated tivity (data not shown). Moreover, PTH pretreatment did not
with10(cid:5)8MPTH-(1–34)for6h.Then,proteinextractionandWestern affect TGF-(cid:1)-induced phosphorylation of Smad3 (data not
blot analysis were performed, as described under “Experimental
Procedures.” shown). These findings suggest that PTH augmented TGF-(cid:1)-
induced transcriptional activity in a manner independent of
PTH-induced expression of TGF-(cid:1)and Smad3. In TGF-(cid:1)sig-
decreasedboneformation,includingthepromotedapoptosisof
naling,receptor-mediatedphosphorylationofSmad2orSmad3
osteoblastsandosteocytes(20).Theseevidencesuggestedthat
induces their association with the common partner Smad4,
Smad3, as well as PTH, possesses anti-apoptotic effects in
followed by translocation into the nucleus where these com-
osteoblasts. We therefore examined the effects of PTH and
plexes activate transcription of specific genes (16, 17). We ex-
Smad3 on dexamethasone- or etoposide-induced apoptosis in
amined the effects of PTH on the association of Smad2 and
MC3T3-E1 and UMR-106 cells. We employed trypan blue
Smad3 with Smad4. As shown in Fig. 8 (D and E), when
staining, TUNEL staining, and Hoechst staining methods to
detect the apoptotic cells. The trypan blue stain method is Myc-Smad2orMyc-Smad3wasco-transfectedwithflag-Smad4
generally used to distinguish the viable cells from nonviable in MC3T3-E1 and UMR-106 cells, Smad2 or Smad3 was co-
ones.AsshowninFig.6,dexamethasoneandetoposideinduced immunoprecipitatedwithSmad4withTGF-(cid:1)treatment.How-
the number of nonviable cells in MC3T3-E1 (A and B) and ever, PTH-(1–34) did not affect the association of Smad2 or
UMR-106cells(CandD).TreatmentwithPTH-(1–34)aswell Smad3withSmad4inTGF-(cid:1)signaling(Fig.8,D(forSmad2)
as Smad3 overexpression antagonized dexamethasone- and and E (for Smad3)). Moreover, PTH did not affect the expres-
etoposide-induced cell death. The truncated Smad3 mutant, sion of Smad2 and Smad4 in MC3T3-E1 and UMR-106 cells
Smad3(cid:2)C, lacks its MH2 region in C terminus and possesses (Fig. 8C). These findings suggest that PTH specifically in-
dominant negative effects on endogenous Smad3 activity, as creased the expression of Smad3, and that PTH-augmented
previously described (18). Smad3 inactivation with Smad3(cid:2)C transcriptional activity by TGF-(cid:1)are independent of Smad2
antagonizedthePTH-inducedanti-apoptoticeffectsonthecells and Smad4. Next, we examined the nuclear translocation of
treated with dexamethasone and etoposide, although Smad3byusingSmad3-transfectedMC3T3-E1cells.Asshown
Smad3(cid:2)Cexpressionitselfdidnotaffectthem(Fig.6,AandC). inFig.8F,PTH-(1–34)didnotaffectTGF-(cid:1)-inducedtransloca-
ThesefindingssuggestedthatPTHandtheoverexpressionof tionofSmad3intonucleus.TheseresultssuggestedthatPTH
Smad3antagonizeddexamethasone-andetoposide-inducedap- stimulatedthetranscriptionalactivityofSmad3,afterSmad3
optosis,andthatSmad3wasindispensableinthePTH-induced wastranslocatedintonucleus.
anti-apoptotic signals in osteoblasts. Cell death detected by PTHAugmentedAnabolicbutNotCatabolicEffectsofTGF-(cid:1)
trypan blue assay may include death by mechanisms other in Osteoblasts—TGF-(cid:1)stimulates type I collagen expression
than apoptosis. TUNEL stain method is more specific for the and inhibits proliferation of MC3T3-E1 cells, as previously
detectionofapoptoticcells,becausecellswithTUNEL-positive described (30). As shown in Fig. 9 (A and B), PTH-(1–34)
nucleus mean ones with DNA fragmentations. In both empty accelerated TGF-(cid:1)-induced expression of type I procollagen
vector-andSmad3(cid:2)C-transfectedUMR-106cells,dexametha- mRNA and type I collagen protein in MC3T3-E1 cells. These
Bone Anabolic Action of PTH-Smad3 Axis 52245
FIG.4.PKAandPKCagonistsstimulatetheexpressionofSmad3.A,afterconfluentMC3T3-E1cellswereculturedinserum-free(cid:2)-MEM
for 12 h, cells were treated with 10(cid:5)5 M phorbol 12-myristate 13-acetate (PMA), 10(cid:5)5 M forskolin (fors), 10(cid:5)4 M N6,O2(cid:1)-dibutyryl adenosine
3(cid:1),5(cid:1)-cyclicmonophosphate(db-cAMPs),and10(cid:5)5MSpdiastereomerofadenosinecyclic3(cid:1),5(cid:1)-phosphorothioate(Sp-cAMPs)for1h.Cont.,control.
Then,RNAextractionandsemiquantitativeRT-PCRassaywereperformed,asdescribedunder“ExperimentalProcedures.”BandC,confluent
MC3T3-E1cells(B)orUMR-106cells(C)wereculturedinserum-free(cid:2)-MEMorDMEM,respectively,for12h,andthencellsweretreatedwith
thePKAorPKCagonistsfor6h(BandC).Then,proteinextractionandWesternblotanalysiswereperformed,asdescribedunder“Experimental
Procedures.”
findings suggested that PTH-(1–34) augmented TGF-(cid:1)-in- volvedintheanti-apoptoticeffectsofPTH.Finally,PTHaug-
ducedexpressionandsynthesisoftypeIcollagen.Ontheother mentedboneanabolicactionsofTGF-(cid:1).
hand,PTH-(1–34)didnotaffectTGF-(cid:1)-reduced[3H]thymidine In this study, PTH promoted the expression of Smad3 in
incorporation in MC3T3-E1 cells (Fig. 9C). These results sug- osteoblasts.Smad3isacriticalcomponentoftheTGF-(cid:1)signal-
gested that PTH-(1–34) augmented anabolic but not catabolic ing pathways, and receptor-mediated phosphorylation of
effectsofTGF-(cid:1)inosteoblasts. Smad2 or Smad3 induces their association with the common
partner Smad4, followed by translocation into the nucleus
DISCUSSION wherethesecomplexesactivatetranscriptionofspecificgenes
The present study indicated three important new points in (16, 17). Thus, Smad3 is considered to play as a signal trans-
osteoblasts. First, PTH promoted the expression of Smad3, a ductionmoleculeandatranscriptionalregulatorincytoplasm
crucial mediator in TGF-(cid:1)signaling. Second, Smad3 was in- andnucleus,respectively.PreviousstudiesindicatedthatPTH
52246 Bone Anabolic Action of PTH-Smad3 Axis
FIG.5.BothPKAandPKCinhibitorsantagonizePTH-inducedexpressionofSmad3.A,afterconfluentMC3T3-E1cellswerecultured
inserum-free(cid:2)-MEMfor12h,cellswerepretreatedwithaPKAinhibitor(10(cid:5)8MH89)orPKCinhibitors(10nMstaurosporineor50(cid:3)MH7)for
6handtreatedwith10(cid:5)8MPTH-(1–34)for1h.Then,RNAextractionandsemiquantitativeRT-PCRanalysiswereperformed,asdescribedunder
“ExperimentalProcedures.”BandC,afterconfluentMC3T3-E1cells(B)andUMR-106cells(C)wereculturedinserum-free(cid:2)-MEMorDMEM,
respectively,for12h,cellswerepretreatedwithPKAorPKCinhibitorsfor6handtreatedwith10(cid:5)8MPTH-(1–34)for6h.Then,proteinextraction
andWesternblotanalysiswereperformed,asdescribedunder“ExperimentalProcedures.”
stimulatesTGF-(cid:1)expressioninosteoblasts(22).Inthepresent scriptionalactivator(40–42),anditinducedpromoteractivity
study,PTHstimulatedSmad3expressionwithinonly1hand in the osteocalcin, osteopontin, type I collagen, and collagen-
its effects were independent of the protein synthesis de novo. ase-3 promoters (40, 43–48). In UMR-106–01 cells, a rat os-
These findings indicated that Smad3 expression induced by teoblasticcellline,PTHledtoactivationofRunx2(49)andthe
PTH was not through TGF-(cid:1). Smad3 might be a transcrip- physicalinteractionbetweenAP-1andRunx2wasrequiredfor
tional regulator for bone formation partly in a manner inde- the PTH-stimulated transcriptional activity of the collagen-
pendent of TGF-(cid:1)in osteoblasts, as previously reported (18, ase-3 promoter in MC3T3-E1 cells (50). These data suggest
19).IthasbeenreportedthatPTHaffectsthetranscriptional that those transcriptional regulators are important in PTH
factors through activator protein-1 (AP-1), cAMP-responsive actionsinbone.OurpreviousstudysuggestedthatSmad3isa
element-binding protein (CREB), and Runt-related transcrip- crucial molecule in bone formation (18, 19). The present find-
tional factor 2 (Runx2) (31). As for the AP-1 family, c-Fos, ings,therefore,suggestedthatSmad3functions,asatranscrip-
Fra-1,and(cid:2)FosBplayimportantrolesinboneformationinthe tionalregulatordownstreamofPTHsignaling.
previousstudies(32–34),andPTHstimulatedtheexpressionof Itwasreportedthatseveralnuclearreceptorsofthesteroid
c-Fos, c-Jun, Fra-1, Fra-2, and FosB (35–39). Moreover, the hormones interacted with Smad3; Smad3 potentiated ligand-
phosphorylation of CREB in response to PTH treatment was inducedtransactivationofvitaminDreceptorasacoactivator
required for PTH-stimulated expression of c-Fos (35, 39). ofthisreceptor(51),andglucocorticoidreceptorinhibitedtran-
Runx2 has been characterized as an osteoblast-specific tran- scriptionalactivationofSmad3(52).Smad3wasanandrogen
Bone Anabolic Action of PTH-Smad3 Axis 52247
FIG.6.TheeffectsofPTHandSmad3onthecellviabilityinosteoblasts.Emptyvector-,Smad3(cid:2)C-,orSmad3-transfectedMC3T3-E1
cells(AandB)andUMR-106cells(CandD)weretreatedwith10(cid:5)7Mdexamethasone(Dex)or5(cid:4) 10(cid:5)5Metoposide(Etop)for6hwithorwithout
pretreatment with 10(cid:5)8 M PTH-(1–34) for 1 h inserum-free (cid:2)-MEM or DMEM. Apoptotic cells were enumerated by trypan blue staining, as
described under “Experimental Procedures.” Bars represent mean (cid:6) S.E. of ratio of trypan blue-positive cell numbers in untreated empty
vector-transfectedcells(controlgroup).*,p(cid:7)0.01fromPTH-untreatedgroup.**,p(cid:7)0.01fromcorrespondingvector-transfectedgroup.
receptorco-regulatorinprostatecancercells,whereasthean- Smad3(55).AlthoughthesefindingssuggestedthatSmad3is
drogenreceptorrepressedTGF-(cid:1)signalingthroughinteraction relatedtotheactionofsteroidhormones,noreportshavebeen
with Smad3 (53, 54). Estrogen receptor-mediated transcrip- availableaboutthesignificanceoftheseinteractionsinosteo-
tional activation enhanced by TGF-(cid:1)signaling was through blasts.Moreover,whetherthepeptidehormonewouldregulate
52248 Bone Anabolic Action of PTH-Smad3 Axis
FIG.7.Smad3isinvolvedinPTH-inducedanti-apoptoticactioninosteoblasts.Emptyvector-orSmad3(cid:2)C-transfectedUMR-106cells
weretreatedwith10(cid:5)7Mdexamethasone(Dex)for6hwithorwithoutpretreatmentwith10(cid:5)8MPTH-(1–34)for1hinserum-freeDMEM. Aand
B,apoptoticcellsweredetectedwithTUNELstaining(A)orHoechststain(B).CandD,thenumbersofapoptoticcells,theTUNEL-positivecells
(C)andcellswhosenucleishowdot-spotappearance(D),werecalculated.Barsrepresentmean(cid:6)S.E.ofratioofthevaluesincontrolgroup.*,
p(cid:7)0.01fromPTH-untreatedgroup.
the expression and the transcriptional activity of Smad3 was PTHresultedinincreasedbonemineraldensity,whereascon-
not known. Our present report was the first that calciotropic tinuous exposure to high concentration of PTH in vivo led to
hormoneaffectedtheexpressionandtranscriptionalactivityof progressivebonelossandosteopenia(1–8).Ontheotherhand,
Smad3. PTHpossessesbidirectionalactionsinvitro,boneanabolic,and
SeveralstudiesindicatedthatPTHexertedskeletalanabolic catabolic ones. PTH increased the expression of insulin-like
effects. In animals and humans, intermittent treatment with growthfactorI(IGF-I)(56,57)andosteocalcin(58),whereasit
Bone Anabolic Action of PTH-Smad3 Axis 52249
FIG.8. PTH stimulates the transcriptional activity of Smad3. MC3T3-E1 cells were transfected with 3 (cid:3)g of the reporter plasmid
(p3TP-Lux),thepCH110plasmidexpressing(cid:1)-galactosidase(1(cid:3)g)perwellin6-wellplate.Forty-eighthlater,cellsweretreatedwith10(cid:5)8MPTH
for12h,andfedwithfresh(cid:2)-MEMwithorwithout5ng/mlTGF-(cid:1).Then,24hlater,cellswereharvestedandrelativeluciferaseactivitywas
measured. Values of relative luciferase activity represent the mean (cid:6) S.E. *, p (cid:7) 0.01 from TGF-(cid:1)-untreated group. **, p (cid:7) 0.01 from
PTH-untreatedgroup.BandC,afterconfluentMC3T3-E1cellswereculturedinserum-freemediumfor12h,cellsweretreatedwith10(cid:5)8M
PTH-(1–34) for the indicated times. Then, protein extraction and Western blot analysis were performed, as described under “Experimental
Procedures.”DandE,Myc-taggedSmad2(D)orSmad3(E)wasco-transfectedintoMC3T3-E1cellswithflag-taggedSmad4.Cellsweretreated
with5ng/mlTGF-(cid:1)for1hafterpretreatmentwith10(cid:5)8PTH-(1–34)for12h.Cellextractswereimmunoprecipitated(IP)withanti-flag,followed
byimmunoblotting(IB)withanti-Myc,asdescribedunder“ExperimentalProcedures.”F,Smad3-transfectedMC3T3-E1cellsweretreatedwith
10(cid:5)8MPTHfor12h,andfedwithfresh(cid:2)-MEMwithorwithout5ng/mlTGF-(cid:1).Onehlater,subcellularfractionationsandWesternblotanalysis
wereperformed,asdescribedunder“ExperimentalProcedures.”
decreased the expression of type I collagen (59, 60), alkaline inosteoblasts(21,70,71).IGF-Iisknowntostimulatebonecell
phosphatase (61, 62), osteonectin (63), osteopontin (64), and replication and matrix synthesis (72, 73), and mice with a
DNA synthesis (65). The different effects of PTH were also bone-specific mutation in IGF-I receptor exhibited a striking
reportedamongcellsfromdifferentspecies.Forexample,PTH decrease in bone volume (74). Moreover, a neutralizing anti-
stimulated proliferation of primary osteoblastic cells isolated body against IGF-I blocked the stimulatory effect on alkaline
fromhumantrabeculae(66)andchickcalvariae(67),although phosphataseactivityandtheexpressionofosteocalcininduced
PTH inhibited proliferation of UMR-106 cells (68, 69). Thus, bytheintermittentexposuretoPTHinosteoblastcellsfromrat
theactionsofPTHonosteoblastsarestillcontroversial. calvariae(75).Theselinesofevidencesuggestedthattheana-
Theanabolicmechanisminvivomaybepartiallyexplained bolicactionofPTHinboneispartlymediatedbyIGF-I(21,70,
by the growth factor genes expressed in activated osteoblasts 76, 77). Furthermore, IGF-I-binding proteins also might be
following exposure to PTH. PTH is known to stimulate the related to anabolic action of PTH-IGF-I signals (78). On the
expression of IGF-I, IGF-II, IGF-binding proteins, and TGF-(cid:1) other hand, the anabolic action of PTH has been partly ex-