Macrophage colony stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors

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Macrophage colony-stimulating factor is

indispensable for both proliferation and

differentiation of osteoclast progenitors.

S Tanaka, … , T Kurokawa, T Suda

J Clin Invest.





The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast

development was examined in a co-culture system of mouse osteoblastic cells and spleen

cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d

in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture.

Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of

cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation.

Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors

primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred

predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When

anti-M-CSF antibody or anti-anti-M-CSF receptor antibody was added either for the first 4 d or for the

final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells

and osteoblastic cells obtained from op/op mice, which cannot produce functionally active

M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in

response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that

M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation

into mature osteoclasts.

Research Article



Colony-stimulating Factor Is Indispensable for

both Proliferation and Differentiation of Osteoclast


SakaeTanaka,**NaoyukiTakahashi,* Nobuyuki Udagawa,*Tatsuya Tamura,*TakuhikoAkatsu, *

E.Richard Stanley,' Takahide Kurokawa,* and Tatsuo Suda*

*Department of Biochemistry, Showa University,School ofDentistry, Tokyo 142, Japan;tDepartmentof Orthopedics, University of Tokyo, School of Medicine, Tokyo112, Japan;and


of DevelopmentalBiology and Cancer,

AlbertEinsteinCollege ofMedicine,Bronx, NY10461


The mechanism of action ofmacrophage colony-stimulating factor(M-CSF)inosteoclastdevelopmentwasexaminedin a co-culture systemofmouseosteoblasticcells and spleen cells. Inthisco-culture, osteoclast-likemultinucleatedcells(MNCs) were formed within 6 d inresponse to 10 nM la,25(0H)2D3 added onlyforthefinal2 d ofculture.Simultaneously adding hydroxyurea for thefinal2 dcompletely inhibited proliferation ofcultured cells withoutaffecting la,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using

13H1-thymidinerevealed that osteoclastprogenitors primarily prolif-erated duringthe first4 d, whereas their differentiation into MNCsoccurredpredominantlyduringthefinal2dofculture in response to 1a,25(0H)2D3. When anti-M-CSF antibody or anti-M-CSFreceptorantibodywasadded either forthefirst4 d orforthefinal2d, the MNC formationwassimilarly inhibited. Inco-cultures ofnormal spleen cells andosteoblasticcells ob-tainedfromop/op mice, whichcannot producefunctionally ac-tiveM-CSF,thelackof M-CSFeitherforthefirst4 d orforthe final 2dfailed to form MNCs in response to1a,25(0H)2D3 added for the last 2 d. These results clearly indicatethat M-CSF isindispensable forbothproliferation ofosteoclast pro-genitors and theirdifferentiation into mature osteoclasts.(J. Clin.Invest.1993.


Keywords:osteopetrotic mice *la,25-dihydroxyvitaminD3 *anti-macrophage colony-stimu-lating factor antibody*




Itiswellestablishedthatosteoclasts, multinucleatedgiant cells responsible for boneresorption,arederived from hemopoietic progenitors(1).Over the pastfewyears, many attempts have been made to reveal the mechanism of osteoclast formation, and severalin vitrosystemsfor examiningosteoclastformation have beendeveloped(2-5). Wepreviouslyreported that

osteo-AddresscorrespondencetoDr.TatsuoSuda, Department of Biochem-istry, School of DentBiochem-istry, ShowaUniversity, 1-5-8 Hatanodai, Shina-gawa-ku, Tokyo 142, Japan.

Receivedfor publication26May 1992 andinrevisedform21

Au-gust 1992.

clast-like multinucleated cells (MNCs)' were formed when mousespleen cellswereco-cultured with primaryosteoblastic cells, obtained from mouse calvaria, in the presence ofbone-re-sorbing agents such as la,25(OH)2D3, PTH, and PGs (6). Like authentic osteoclasts, MNCsformed in our co-culture sys-tem were positive for tartrate-resistant acid phosphatase (TRAP),possessedanumber of calcitoninreceptors, had ruf-fledborders and clear zones, and formed resorption pits on dentine slices(6).

Inapreviousreport(7),we showed that notonlymouse

spleen cells but also blood monocytes and alveolar macro-phages formedsingle cell-derived colonieson the marrow-de-rived stromal ST2 cell layers. All of the colonies consisted of mainly nonspecific esterase (a macrophage marker en-zyme)-positive cells, and TRAP-positivecells alsoappearedin the colonies in response to 1a,25 (OH )2D3and dexamethasone (7). Theseresultssuggestthat osteoclastsarederived from cells of the monocyte-macrophage


Thishypothesisis sup-ported bythe recentfindingthat osteoclastdeficiencyin osteo-petrotic (op/op) mice isdueto an




func-tionally active macrophage


factor(M-CSF, also known ascolony-stimulatingfactor-1)(8, 9).Yoshidaet

al.clearly demonstratedan extrathymidineinsertionatbpNo. 262 in thecoding


of the M-CSF gene inop/op mice, whichgenerated aTGAstopcodon, 21 bpdownstream(10). Almostsimultaneously, it wasshown that administration of recombinanthumanM-CSFtoop/op mice cured their osteo-petrotic bone disorders (11). This in vivo observation was

quickly confirmed



( 12)


Wiktor-Jedrzejc-zak et al.(13).Wealso


thatosteoblastic cellsobtained from op/op mice couldnotsupport osteoclastdifferentiation in co-cultures with normal


cells ( 14).


M-CSF together with 1a,25 (OH )2D3 induced osteoclast-likeMNCs in co-cultures with op/op osteoblasticcells

( 14).

Thesefindings strongly indicate that M-CSF produced by osteoblastic cells playsacriticalrolein osteoclast development.

In1986,MacDonald et al. reportedthat M-CSFstimulated osteoclast-likecellformationinhumanbone marrow cultures ( 15). Morerecently,wedemonstratedthatM-CSFeffectively stimulates


of osteoclast progenitors,butthatthis effect can beduplicatedby other hemopoietic growth factors such asGM-CSF and IL-3 (16). These findings support the hypothesisthatM-CSF playsanimportantrole not only in the

1.Abbreviations used in thispaper:M-CSF,macrophage

colony-stimu-lating factor; MNCs, multinucleated cells; TRAP, tartrate-resistant acidphosphatase.


©TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/93/01/0257/07 $2.00


growthof osteoclast progenitorsbut alsointheir terminal dif-ferentiation into mature osteoclasts.

In the present study, we have attempted to separate the process of osteoclast development into two phases, prolifera-tive phase and differentiation phase, to elucidate the mecha-nism of action of M-CSF in osteoclast development. We report here that M-CSF is critical not only for proliferation of osteo-clast progenitors but also for their terminaldifferentiationinto mature osteoclasts.


Antibodies and chemicals.Goatanti-mouseM-CSF antiserum

(anti-M-CSFantibody) ( 17) and mouse c-fms/M-CSF receptor

anti-serum(anti-c-fmsantibody) ( 18) were prepared as described. These antibodies added at 0.1%weresufficient to inhibit colony formation of mousemarrow cellsinduced by murine M-CSF. Anti-mouse GM-CSF polyclonalantibody(anti-GM-CSF antibody) was kindly provided by SumitomoPharmaceutical Co. (Osaka, Japan). This antibody added

at0.4% completelyinhibited theDNAsynthesis in mouse marrow cells induced by 2 ng/ml of murine GM-CSF. la,25(0H)2D3 was pur-chased fromPhilips-Duphar(Amsterdam, The Netherlands). Salmon calcitonin waskindlysuppliedbyChugai Pharmaceutical Co. (Tokyo, Japan).


salmoncalcitonin was prepared as previously described (19). Thespecific activity of the product was 18.5 TBq/mmol. Hy-droxyureawasobtained from Sigma Chemical Co. (St. Louis, MO).

[3H]Thymidine (specific activity, 3.11 TBq/mmol) waspurchased from Amersham International plc. (Amersham, UK). FCSwas ob-tainedfrom Gibco Laboratories (Grand Island, NY). a-MEM was

purchased from FlowLaboratories,Inc.(McLean,VA).Culturedishes andplates were obtainedfrom ComingGlassInc. (Corning, NY).

Recombinant human M-CSFwaskindly providedbyMorinagaMilk Co.(Tokyo,Japan).

Cell cultures. Male mice (7- to9-wk-old)andnewbornmice, ddy strain,wereobtained from Shizuoka Laboratories Animal Center

(Shi-zuoka,Japan).Primaryosteoblastic cellswerepreparedfrom newborn mouse calvariaaspreviouslyreported(6).Male andfemale

heterozy-gotes(+ /op) of B6C3 micewereobtained from The Jackson Labora-tory(BarHarbor,ME).Aquarter of their littermatesareexpectedto

beosteopetrotic(op/op).Theop/op homozygoteswereradiologically distinguishedatbirth fromphenotypically normal, +/?siblings. Osteo-blastic cells ofop/op mice were obtained accordingto the method

previouslyreported(14). In short, calvaria ofop/opmicewere cul-tured in typeIcollagengels, andoutgrowingcellswereusedasop/op

osteoblastic cells.Spleencellswereobtained fromsplenictissuesof 7-to9-wk-oldddymice. Osteoblastic cells(1xI04cells/well)andspleen

cells (7.5xI05 cells/well)wereco-cultured for 6 d in a-MEM contain-ing 10% FCS in the presenceorabsence of 10nM Ia,25(OH)2D3in 24-well plates (0.5 ml/well).Some culturesweretreatedwith 10 nM

Ia,25(OH)2D3only for the final2dof the 6-d co-cultureperiod.To inhibit DNAsynthesis, hydroxyurea(0.2-1.0mM)wasaddedtothe co-cultures together with lIa,25(OH)2D3 (10 nM)for the final2dof culture.In someexperiments,co-culturesweretreatedondays0-4or

days4-6 with either anti-M-CSFantibody, anti-c-fmsantibody, or

anti-GM-CSFantibody. In otherexperiments, osteoblastic cells ob-tainedfromop/opcalvariawereco-cultured with normalspleencells from ddy mice in the presence ofla,25(OH)2D3added onlyfor the last

2d. RecombinanthumanM-CSFwasaddedat100 ng/mltothe

cul-tureseither for the first4d,for the final2d,orthroughouttheentire 6-d co-cultureperiod.In somecultures,1mMhydroxyureawasadded

totheco-cultureforthefinal2d.All cultures weremaintainedat37°C

in ahumidified atmosphere of5%CO2inair.

Identification ofthe osteoclast-like MNCs. Afterbeingculturedfor

6d, cellswerefixedandstained forTRAPin accordancewitha

previ-ouslyreported method (20). TRAP-positive cells with three ormore

nucleiwerecountedasMNCs. Expression of calcitonin receptorswas

also assessed by autoradiography using [125I]salmon calcitonin as de-scribed previously (21 ). More than 95% of the TRAP-positive MNCs formed in theco-cultures treated withIa,25(OH)2D3 for the final 2 d of culture showed specific binding of labeled calcitonin (data not in-cluded). Therefore, we referred TRAP-positive MNCs formed inour

co-culture system to osteoclast-like MNCs in this article.

Assessmentofcell proliferation. We evaluated cell proliferation us-ing two experimental procedures; one involved determinus-ing [3H

]-thymidine incorporation into acid- insoluble fractions ofcultured cells, andthe other was autoradiographic observation using [ 3H ] thymidine. In theexperiment assessing [ 3H ] thymidine incorporation, the radioiso-tope(3.7 x 10'Bq/0.5 ml) was added to co-cultures on day 6. After incubation for 12 h, cells were washed twice with ice-cold PBS. The radioactivity incorporated into TCA-insoluble fractions was counted inscintillation fluid (ACS II; Amersham Corp., Arlington Heights, IL).For autoradiographic studies, co-cultures were performed on cov-erslips (13.5 mm; Sumitomo Bakelite Co., Tokyo, Japan) placed in 24-well plates.[3H


Thymidine (3.7 x104Bq/0. 5 ml) was added to the co-cultures for 12 h either on day 3 or day 5. In some experiments, co-cultures were treated with 1 mM hydroxyurea together with Ia,25(OH)2D3 for the final 2 d of culture to inhibit cell proliferation. 1 mMhydroxyurea was sufficient to inhibit completely DNA synthesis in our co-cultures. After being cultured for 6 d, cells were fixed, stained for TRAP, and processed for autoradiography as described previously (21 ).Nuclei which contained > 50 grains were counted as labeled nu-clei.

Statisticalanalysis. Each series of experiments was repeated at least threetimes. The results obtained from a typical experiment were ex-pressed as the means±SEM (standard error of the mean) of




Whenmousespleen cellswereco-cultured for 6 d withmouse osteoblastic cells in the presenceof 10 nM la,25(OH)2D3, a

number ofosteoclast-like MNCs were formed in response to the vitamin(Fig. 1). Adding1a,25 (OH)2D3 only for the final 2 d of the 6-d co-culture period also induced osteoclast-like MNC formation, though thenumberofosteoclast-likeMNCs formedwasslightly smaller than that observed when the

vita-minwasadded throughout the co-culture period (Fig. 1). A

small number of osteoclast-like MNCs were also formed in

control co-cultures after the cells had been cultured for 6 d

without 1a,25(OH)2D3, but no osteoclast-like MNCs ap-peared on day 4 even in co-cultures treated with 1a,25(OH)2D3 (Fig. 1). We repeated these experiments 18

times,and obtained similar resultsin 15 of them. In the other

three experiments, only a few osteoclast-like MNCs were

formed on day 6 in response to 1a,25(OH)2D3 even when

Ca,25(OH)2D3wasaddedthroughoutthe entire 6-d co-culture period. Theseresultsindicate thatosteoclastprecursors differ-entiate into osteoclasts duringthe final 2 d of culture(days 4-6)in responseto Ca,25(OH)2D3.

To determine whether cellgrowthmust occurduring

termi-naldifferentiation ofosteoclast



os-teoclast-like MNC formationinthe presenceofhydroxyurea. Adding hydroxyurea togetherwith 10nM I



the final 2 d of culture


inhibited [3H]-thymidine incorporation into acid-insoluble fractions of


in-0 0





._ _

I 0

B0 3: .9

on E 0 =







O - 4 days 14-6



1,25D3 I 1,25D3 |


_ jI 1,25D3 |


Days ofCultures

Figure1.Time courseof changes in osteoclast-like MNC formation in co-cultures ofosteoblastic cells and spleen cells in the presenceor

absenceofla,25(OH)2D3. Mouseprimaryosteoblastic cells and spleen cellswereco-culturedfor 6 d without (o) or with

la,25(OH)2D3(1,25D3). Ia,25(OH)2D3wasaddedat10nMeither throughout the6-dco-cultureperiod(o)oronly for the final 2 dof

culture(.).Theresultsareexpressedasthemeans±SEMof four


hibit completely DNAsynthesis. Nevertheless, osteoclast-like MNCformation induced by


was notaffected by

simultaneously adding hydroxyurea (Table I).


osteo-clast-like MNCswereformed when hydroxyureawasaddedat 1 mMfor the first4d(datanotincluded).


studies also showed that when [3H]


wasadded for 12 h onday5 toco-cultures, which had been treated with hydroxy-ureatogether with 1a,25(OH)2D3 for the final2dof culture, none ofthe nuclei in the osteoclast-like MNCswerelabeled with


onday 6 (Fig. 2 C). This


con-firmed that theinduction of differentiation by


(OH)2D3 ofosteoclast precursorsintoosteoclast-like MNCsoccurs dur-ing days 4-6, and that thisprocessis absolutely


of cell


We next comparedtherelative proliferation activity ofthe osteoclast




3 and 5 of co-culture. The

co-cultured cellswerelabeledwith


onday 3, and then were treated with



with 1a,25 (OH )2D3for the final2 d toinhibit cellproliferation after


Aboutahalf(5 1%) of the nuclei in the osteoclast-like MNCsformed in cultures given


thymidineonday3were densely labeled (TableIIand


2A). Many osteoblastic cells also contained labeled nuclei. When the labeling was per-formedonday5in theabsenceof hydroxyurea, only 7.3% of the nuclei in osteoclast-like MNCs formed in response to 1a,25 (OH)2D3werelabeled with


(Fig. 2Band TableII). The numberof osteoclast-likeMNCsformedin co-cultureslabeledwith


]thymidine onday 3 was much less,

ascompared withthe numberobtained inco-cultures labeled onday5(TableII). Even when hydroxyurea was not added to theco-cultureslabeled with


on day 3, the num-ber of osteoclast-like MNCs formed in response to 1a,25


wassimilarlydecreased(datanot shown).This suggests that in the





sensitive toradioactive thymidine. From these

re-sults, weconcludedthatosteoclastprogenitorsmostly prolifer-ateduring the first 4 days (proliferative phase) and that their differentiation into osteoclast-like MNCs occurs preferentially during the final 2 d of culture in the absence of appreciable DNAsynthesis (differentiation phase).

Toexamine how M-CSF is involved in osteoclast develop-ment, co-cultures were treated with anti-M-CSF antibody,


antibody, and anti-GM-CSF antibody during the proliferativephase (thefirst4 d) orduringthedifferentiation phase (thefinal 2 d). Fig. 3 shows the dose-response effect of theseantibodies added either during the first4d(Fig. 3 A)or during the final 2 d (Fig. 3 B)onosteoclast-like MNC forma-tion induced by 1a,25(OH)2D3. In either case, anti-M-CSF antibody and anti-c-fms antibody inhibited osteoclast-like MNC formationdose-dependently


3,AandB). The ap-pearance of TRAP-positive mononuclearcells(possibly pre-cursorsof osteoclast-like MNCs)wasalsosuppressed by treat-ment with the respective antibodies (data not included). In contrast, neither osteoclast-like MNC formation nor TRAP-positive mononuclear cell formation induced by lIa,25(0H)2D3 was inhibited by adding GM-CSF anti-body bothintheproliferative phaseand differentiationphase (Fig. 3, A andB). Ineither case,preimmune serum had no inhibitory effect onosteoclast-like MNC formation (data not shown).

To furtherinvestigate the importance of M-CSFin osteo-clastdevelopment,weperformed co-cultures of normal spleen cells obtained fromddymice and osteoblastic cells fromop/op mice, which donotproducefunctionally active M-CSF.When recombinant human M-CSF was present at 100 ng/ml throughout the




osteoclast-like MNCs

TableI. Effects ofHydroxyureaon[3H]ThymidineIncorporation

intoAcid-insolubleFractionsandon Osteoclast-likeMNC

Formation inCo-culturesofMouseOsteoblastic Cells andSpleenCells


[3H]Thymidine TRAP-positive

Treatment incorporation MNCsformed

cpm/well number/well

Vehicle 120,320±15,677 28±5*

1a,25(OH)2D3 (10 nM) 91,030±9,176 144±2

la,25(OH)2D3 (10nM)

+hydroxyurea (0.2mM) 14,364±2,640* 137±9

la,25(OH)2D3 (10 nM)

+hydroxyurea(1.0 mM) 2,133±374* 132±17

Mouseprimaryosteoblastic cells and spleen cellswereco-cultured in the absenceofla,25(OH)2D3for the first 4 d then withoutorwith 10nM Ia,25(OH)2D3 foranadditional 2d.Hydroxyureawasadded

tothe co-culturesat0.2mM or1.0mMfor the final 2 dof culture together with 10nM la,25(OH)2D3.


(3.7x 104Bq/ 0.5ml)wasaddedtosomeof the culturesonday 6.Afterincubation for 12 h, theradioactivity incorporatedinto TCA-insoluble fractions

wascounted.Intheremaining cultures, cells were fixed and stained forTRAP onday 6, andTRAP-positiveMNCswerecounted. * Sig-nificantly different from the co-cultures treated withIa,25(OH)2D3



% 0








p ''>Ss 3t i ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Ww~~~~~~~~~o


¢r= _R

t .r


Figure 2.Autoradiography of[3H ] thymidine incorporation into co-cultures of osteoblastic cells and spleen cells. Mouse primary osteoblastic cells andspleencells were co-cultured in the absenceofIa,25(OH)2D3 for thefirst 4 d then with10nM Ia,25(OH )2D3 for an additional 2 d.[

3H]-thymidine (3.7x 104Bq/0.5 ml)wasaddedfor12 honday 3 (A) or day 5 (B and C). In experiments (A) and (C), hydroxyurea was added at 1 mM for the final 2 d of culture together with Ia,25(OH)2D3. After being cultured for 6 d, cells were fixed, stained for TRAP, and processed for autoradiography. Arrows and arrow heads indicate TRAP-positive MNCs and their[3H]thymidine-labelednuclei, respectively.

wereformed inresponse to 1a,25(OH)2D3, whichwasadded only forthefinal2d(Fig. 4). Osteoclast-like MNCswerealso formedevenin the presenceof hydroxyurea, whichwasadded

at1 mMforthefinal2 dtogether with 1a,25(OH)2D3(Fig. 4). However, lackofM-CSFeitherforthefirst4d(proliferative phase) orforthefinal2d(differentiationphase)failedtoform osteoclast-likeMNCs almostcompletely(Fig. 4).Theseresults demonstratethat M-CSFisrequired bothin theproliferative phase and inthe differentiation phase ofosteoclast develop-ment.

TableII. LabelingofNuclei with[3H]Thymidine in

TRAP-positive MNCs Formedin Co-culturesofMouseOsteoblastic Cells andSpleenCells

Day of Number Number Percentage Numberof

[3H]thymidine of nuclei ofnuclei of labeled TRAP-positive labeling scored* labeledt nuclei MNCs/coverslip

Day3 141 72 51 43±4.8

Day5 804 59 7.3 233±15

Mouseprimary osteoblastic cells and spleen cellswereco-culturedon

coverslipsin the absenceofIa,25(OH)2D3for thefirst4d thenwith 10nMlIa,25(OH)2D3foranadditional2 d.


(3.7x 104 Bq/0.5 ml)wasaddedfor 12 honday 3orday5. Theco-cultures labeledonday 3weretreatedwith 1mMhydroxyureaforthefinal 2 dof culturetoinhibit cellproliferationafterlabeling.Afterbeing

culturedfor6d, cellswerefixed,stained forTRAP,andprocessed

forautoradiography. *Onlynuclei ofTRAP-positiveMNCswhich could bedistinguishedfrom those ofTRAP-negativeosteoblastic

cells werescored. *Nuclei containing>50grainswerecountedas labelednuclei.


The present studyclearlyshows that the 6-d cultureperiodof

ourco-culture system can be separated intotwo phases: the first 4 d, in which theproliferation of osteoclast progenitors primarilyoccurs,and thefinal 2 d,inwhich their differentia-tion intomatureosteoclasts ispredominant. First,no TRAP-positiveosteoclast-like MNCs appeared onday 4even in the presence of 1 a,25 (OH )2D3(Fig. 1). Second, TRAP-positive MNCswereformed duringthefinal2dof culturein response

to1a,25(OH )2D3evenunderconditions in whichDNA synthe-siswasalmostcompletely inhibited bytheaddition of1 mM hydroxyurea (Table I). Third, the labeling index of

[3H]-thymidine in the nuclei of


MNCs was 51% when[3H


thymidinewasadded onday 3andonly7%onday5 (Fig. 2 and Table II). Itshould be noted that the number of osteoclast-like MNCs countedonday6 in co-cultures labeled with[3H


thymidineonday 3 was markedly decreased as com-paredwith the numberobtainedin co-cultures labeledonday 5.Thissuggests thatosteoclastprogenitorsarehighly sensitive

to [3H]thymidine duringtheproliferative phase. Adding hy-droxyurea at 1 mM forthefirst 4 d inhibited osteoclast-like MNCformation completely (data notshown).Inagreement withour findings, Schevenetal. (22) reported that prolifera-tion ofosteoclastprogenitorsinembryonicmousemetatarsal boneswassensitivetoionizingirradiation,but theformation of multinucleatedosteoclastswasrelatively resistant.

Usingamodifiedco-culture system,weexaminedtherole ofM-CSF in osteoclastdevelopment.M-CSF isa


factor responsibleforproliferation, differentiationandsurvivalof he-mopoietic cells of the monocyte-macrophage


(23). Treatment ofco-cultures with anti-M-CSF


for the first4 ddose-dependentlyinhibited osteoclast-like MNC




,qw 7'

V .71









Concentrationofantibodies (%)

0 0.1 0.33


tem(16). However, in the presentstudy, anti-GM-CSF






proliferative phase

failed to suppress

osteoclast formation. We also confirmed that the proliferation ofmacrophagesmaintained on the op/oposteoblasticcellswas very poor (unpublished observation), though GM-CSF was normally produced byop/op osteoblasticcells (8). These re-sultsindicatethat inthis modified co-culture system, osteoclast progenitors proliferated preferentiallyin response to M-CSF. Thisnotion isin accordancewiththatof Corbozetal. (24), who reported that irradiation inhibited the M-CSF-induced


boneresorption ina mousemetatarsal boneculture system. It


is therefore concluded that M-CSF is


as a

prolifera-tion factor of osteoclastprogenitors.

Additionofeither anti-M-CSFantibodyoranti-c-fms anti-bodytoco-culturesduring the differentiation


also sup-pressed the appearanceof


MNCs dose-depen-dently(Fig. 3 B). As was shown in the experiments with hy-droxyurea, differentiation ofprecursorcells intoosteoclast-like anti-GM-CSF MNCs occurredby amechanism independentof cellgrowth duringthe final 2 d of culture(Fig. 1 and TableI). Anti-GM-CSF antibody added during the differentiation phase did not affect osteoclast-like MNC induction by





Experiments using osteoblasticcellsobtained from op/op mouse calvaria made ourconclusion more


The op/op osteoblastic cells donotproducefunctionallyactive M-CSF.Noosteoclast-likeMNCswereformed in co-cultures of



osteoblastic cells and normal spleen cells even in the

anti-M-CSF presence of 1 a,25 (OH)2D3. Adding recombinant human M-CSF (100 ng/ml) throughout the 6-d culture


and la,25(OH)2D3 during the final 2 d induced osteoclast-like MNC formation.Anumberofosteoclast-like MNCswere

simi-Figure3. Dose-responseeffects ofanti-M-CSF, anti-c-fmsand anti-GM-CSF antibodiesonosteoclast-like MNC formationinducedby

a,25(OH)2D3 inco-culturesof osteoblastic cells andspleencells.

Mouseprimaryosteoblastic cells andspleencellswereco-cultured

in theabsenceofla,25(OH)2D3for the first4dthen with 10 nM

a,25(OH)2D3 foranadditional2d.Increasingconcentrations of

anti-M-CSFantibody (.), anti-c-fms antibody (o)oranti-GM-CSF

antibody (m)wereadded eitherfor the first 4 d(A)orfor the final

2 d(B)of culture. After 6 d ofculture, TRAP-positiveMNCswere

counted. The resultsareexpressedasthepercentages(the

means±SEM of fourcultures)ofTRAP-positiveMNCsformed in

experimentalculturestothosein control cultures treated with

la,25(OH)2D3alone for thefinal 2d of culture. Numbers of

TRAP-positive MNCs formed in the control cultures for the

respec-tiveantibody experimentswere121±9.6 foranti-M-CSF, 172±12 for

anti-c-fms,and 164±15 for anti-GM-CSFexperiments (the means±SEM/wellof fourcultures). *Significantlydifferent from the cultures treatedwith la,25(OH)2D3 alone,P<0.01.



0 10


zX a

-Y c 0






1u.,25(OH)2D3 (10 nM)4-6days _

rhM-CSF(100 ng/ml) (0-4days

4-6days _

Hydroxyurea (1 mM) 4-6days _


* *


+ + +






+ + +

_ _ _ _ +


mation inducedby 1a,25(OH)2D3, whichwasadded for the

final 2 d of culture (Fig. 3 A). This indicates that M-CSF is

necessaryforpromoting proliferation of osteoclast progenitors.

Similar resultswere obtainedinexperiments with anti-c-fms

antibody, but anti-GM-CSF antibody hadnoinhibitory effect

(Fig. 3 A). We previously reported that M-CSFwasthemost

efficient stimulator of osteoclast progenitor proliferation, but that GM-CSF and IL-3 could be substituted for M-CSF to

someextentinproducing this effect intwo-stepco-culture

sys-Figure4.Osteoclast-likeMNC formationinco-cultures of osteoblas-tic cells obtained fromop/opmiceandnormalmousespleencells obtained fromddymice. Cellswereculturedinthe absence of

la,25(OH)2D3for the first 4 d then with 10 nM la,25(OH)2D3 for anadditional2d. Recombinant human M-CSF(rhM-CSF)was addedat 100ng/mltothe cultureseither for the first4d,forthefinal

2d,orthroughouttheentire cultureperiod. Hydroxyureawasadded

totheculturesonlyfor the final 2 dtogetherwith a,25(OH)2D3in thepresenceof rhM-CSFthroughoutthe 6-d cultureperiod. *Signifi-cantlydifferent from the cultures treated with rhM-CSF whichwas

presentthroughoutthe cultureperiod,P<0.01. 150



0 0


c ,Lo







0° do





4E _


z 100





larly formed in response to Ia,25(OH)2D3 even in the pres-enceof1 mMhydroxyurea whichwasaddedduringthe last 2d (Fig. 4). In contrast, lackofM-CSF either in the proliferative phase orinthedifferentiation phasefailedtoform osteoclast-likeMNCs(Fig.4). Theseresults clearly demonstrate that M-CSF playsacritical rolein inducing not onlyproliferation of osteoclastprogenitorsbut also their differentiation into mature osteoclasts.This is consistentwith the finding of Hattersley et al., who reported that GM-CSF increased the appearance of F4/80-positive macrophages but not osteoclasts in co-cultures of op/op osteoblasticcells and normal spleen cells (25).

Theactionof M-CSF has been reportedtobemediated bya specific receptorencodedbythe


protooncogene, which exhibitstyrosine kinase


(26). Kodamaetal.reported that M-CSF receptors existed in osteoclast-like MNCs and theirprecursorsbutnotinosteoblastic cells

(27). Therefore,

in ourco-cultures of osteoblasticcells andspleen cells, thetarget cells of M-CSF are probably osteoclast

progenitors. Signal

transduction pathways through M-CSF receptors have been investigated in bone marrow-derived macrophages and several established cell lines of the monocyte-macrophage


In the M-CSF(CSF-1)-dependent mouse macrophage cell line BAC1.2F5, M-CSFstimulated the rapid tyrosine phosphoryla-tion of several,predominantly cytoplasmic, proteins (18, 28, 29)that wasfollowed by serine phosphorylation ofthe cyto-plasmicprotooncogeneproduct,RAF- 1, andactivation of the RAF-


seine kinase (30). Usingamurine macro-phage cellline, P388D1,Varticovskiet al.reportedthatM-CSF receptorsactivated

phosphatidylinositol-3 kinase,

which may play animportantroleinthesignal transduction pathway of M-CSF (31 ). It was alsoshown thatM-CSFwasrequired by bone marrow-derived macrophages


the G1



ensurebothcellsurvivaland entryinto the S phase, butwas no longerrequired after cells had entered the S phase (32).More recently, Matsushimeetal.(33) reported that regulators


the G 1/S transition could be

cyclin-like proteins



wasregulated by M-CSF. Itisnot knownwhethersuchM-CSF


transduction pathwaysarealso


inthe develop-mentofosteoclasts. Mostof thework sofar reportedpointsto an


rolefor M-CSFasa


of monocyte-macro-phage


cells,but verylittle isknown about the mecha-nism of action of M-CSF incelldifferentiation.


the in vitrosystem for osteoclast developmentused inthis study will beauseful model for investigating the role of M-CSF in cell differentiation. Further studies are needed to elucidate the mechanismunderlyingthe


transduction induced by M-CSF-activated




hormones in




This workwassupported bygrants-in-aid(02454429, 03454437) from theMinistryofScience,Education, and Culture of Japan, andagrant CA26509 (E.R.Stanley) from theNational Institutes of Health.


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