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

Macrophage colony-stimulating factor is

indispensable for both proliferation and

differentiation of osteoclast progenitors.

S Tanaka, … , T Kurokawa, T Suda

J Clin Invest.

1993;

91(1)

:257-263.

https://doi.org/10.1172/JCI116179

.

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

Macrophage

Colony-stimulating Factor Is Indispensable for

both Proliferation and Differentiation of Osteoclast

Progenitors

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

ODepartment

of DevelopmentalBiology and Cancer,

AlbertEinsteinCollege ofMedicine,Bronx, NY10461

Abstract

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.

91:257-263.)

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

anti-c-fms

antibody

Introduction

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

lineage.

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

inability

to

produce

func-tionally active macrophage

colony-stimulating

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

al.clearly demonstratedan extrathymidineinsertionatbpNo. 262 in thecoding

region

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

by

Kodamaetal.

( 12)

and

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

reported

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

spleen

cells ( 14).

Adding

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

proliferation

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.

J.Clin.Invest.

©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.

Methods

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).

_['25I]

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

I

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

quadrupli-catecultures.SignificantdifferencesweredeterminedusingStudent'st

test.

Results

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

progenitors,

weexamined

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

a,25(OH)2D3

for

the final 2 d of culture

dose-dependently

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

in-0 0

(A

z

2

C

._ _

I ₀

B0 3: .9

on E 0 =

E

z

-

0--

-0--0

I

O - 4 days 14-6

days]

I

1,25D3 I 1,25D3 |

I

_ jI 1,25D3 |

2

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

cultures.

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

Ia,25(OH)2D3

was notaffected by

simultaneously adding hydroxyurea (Table I).

No

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

Autoradiographic

studies also showed that when [3H]

thymidine

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

[3H]thymidine

onday 6 (Fig. 2 C). This

further

con-firmed that theinduction of differentiation by

Ila,25

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

independent

of cell

proliferation.

We next comparedtherelative proliferation activity ofthe osteoclast

progenitors

on

days

3 and 5 of co-culture. The

co-cultured cellswerelabeledwith

[3H]thymidine

onday 3, and then were treated with

hydroxyurea

together

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

labeling.

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

_[3HI

thymidineonday3were densely labeled (TableIIand

Fig.

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

[3H]thymidine

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

[3H

]thymidine onday 3 was much less,

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

[3H]thymidine

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

(OH)2D3

wassimilarlydecreased(datanot shown).This suggests that in the

proliferative

phase,osteoclastprogenitors

are

highly

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,

anti-c-fms

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

(Fig.

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

6-d

co-culture

period,

osteoclast-like MNCs

TableI. Effects ofHydroxyureaon[3H]ThymidineIncorporation

intoAcid-insolubleFractionsandon Osteoclast-likeMNC

Formation inCo-culturesofMouseOsteoblastic Cells andSpleenCells

Numberof

[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.

[3H]Thymidine

(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

p~~~I

% 0

-1:

;

0

.::,

...I

p.

9

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

wo.jl'

¢r= _R

t .r

.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[3_H]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.

[3H]Thymidine

(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.

Discussion

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

TRAP-positive

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

growth

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

lineage

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

antibody

for the first4 ddose-dependentlyinhibited osteoclast-like MNC

for-X,401

;w'..

V

,qw 7'

V .71

7

..t

.0

.",

I

100

50

Concentrationofantibodies (%)

0 0.1 0.33

Concentrationofantibodies(%)

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

anti-iti-GM-CSF

body

added

during

the

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

ti-c-tms

boneresorption ina mousemetatarsal boneculture system. It

iM-CsF

is therefore concluded that M-CSF is

important

as a

prolifera-tion factor of osteoclastprogenitors.

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

phase

also sup-pressed the appearanceof

TRAP-positive

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

la,25(OH)2D3

(Fig.

3

_B).

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

convincing.

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

anti-c-fms

_op/op

_{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

period

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

6

0 10

_4

zX a

-Y c 0

_;

-0.0

02!

E

z

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

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

4-6days _

Hydroxyurea (1 mM) 4-6days _

**

* *

+1

+ + +

_

+

+

_

+

+ + +

_ _ _ _ +

Treatment

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

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0 0

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B

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

c-fms

protooncogene, which exhibitstyrosine kinase

activity

(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

lineage.

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-

1-associated

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

during

the G1

phase

to

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

of

the G 1/S transition could be

cyclin-like proteins

whose

expression

wasregulated by M-CSF. Itisnot knownwhethersuchM-CSF

signal

transduction pathwaysarealso

important

inthe develop-mentofosteoclasts. Mostof thework sofar reportedpointsto an

important

rolefor M-CSFasa

mitogen

of monocyte-macro-phage

lineage

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

Therefore,

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

signal

transduction induced by M-CSF-activated

tyrosine

kinaseand

osteotropic

hormones in

os-teoclast

development.

Acknowledgments

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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