of September 1, 2018.This information is current as Factor

StimulatingLigand and Macrophage-Colony- BκCombination of Receptor Activator of NF-

for Osteoclastogenesis Induced by a Is EssentialβEndogenous Production of TGF-

Masayoshi Kumegawa and Yoshiyuki HakedaOgasawara, Hironori Kaneko, Takuya Sato, Hiroshi Mano, Toshio Kaneda, Takaki Nojima, Mari Nakagawa, Aichi

http://www.jimmunol.org/content/165/8/4254doi: 10.4049/jimmunol.165.8.4254

2000; 165:4254-4263; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/8/4254.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Endogenous Production of TGF-b Is Essential forOsteoclastogenesis Induced by a Combination of ReceptorActivator of NF- kB Ligand and Macrophage-Colony-Stimulating Factor1

Toshio Kaneda,* Takaki Nojima,* Mari Nakagawa,* Aichi Ogasawara,* Hironori Kaneko,*Takuya Sato,* Hiroshi Mano,† Masayoshi Kumegawa,* and Yoshiyuki Hakeda2*

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormonesand cytokines. Of these factors, receptor activator of NF-kB (RANK) ligand (RANKL) has been recently cloned as an essentialinducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage(M/M f)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclastsby treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting therequirement for additional factors. The isolated M/Mf-like hemopoietic cells expressed TGF-b and type I and II receptors ofTGF-b. Therefore, we examined the effect of TGF-b on osteoclastogenesis. TGF-b with a combination of sRANKL and M-CSFpromoted the differentiation of nearly all M/M f-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-bAb abrogated the osteoclast generation. These TGF-b effects were also observed in cultures of unfractionated bone cells, andanti-TGF-b blocked the stimulatory effect of 1,25-dihydroxyvitamin D3. Translocation of NF-kB into nuclei induced by sRANKLin TGF-b-pretreated M/M f-like hemopoietic cells was greater than that in untreated cells, whereas TGF-b did not up-regulatethe expression of RANK, the receptor of RANKL. Our findings suggest that TGF-b is an essential autocrine factor forosteoclastogenesis.The Journal of Immunology,2000, 165: 4254–4263.

Osteoclasts are the cells primarily responsible for boneresorption, and are of hemopoietic stem cell origin. Pre-cursors of osteoclasts have been demonstrated to share

common properties with those of a monocyte/macrophage (M/Mf)3 cell lineage (1, 2). Recent extensive studies have increasedour understanding of osteoclast biology, particularly osteoclasto-genesis. Many systemic hormones and local cytokines pathophysi-ologically participate in regulating osteoclast differentiation, in-cluding M-CSF, IL-1, IL-6, IL-11, TNF-a, 1,25-dihydroxyvitaminD3 (1,25(OH)2D3), parathyroid hormone, and PGs (3, 4). Oste-oclast differentiation factor/osteoprotegerin ligand/TNF-related ac-tivation-induced cytokine/receptor activator of NF-kB (RANK) li-gand (RANKL) has recently been identified as the most importantand critical molecule for osteoclast development (5, 6). Bone mar-row stroma/osteoblasts produce this molecule on the plasma mem-brane in response to several osteotropic factors, and osteoclast pre-

cursors express a receptor of RANKL (RANK). Most recently,mice with a disrupted RANKL gene were found to have severeosteopetrosis (7). Therefore, the RANKL/RANK system is con-sidered to be an essential signal for osteoclast differentiation in theinteraction between stromal cells and cells of the osteoclast lin-eage. Like the interaction of osteoclast precursors/stromal cells,RANKL is expressed on activated T cells and activates maturedendritic cells that express RANK on their plasma membrane, im-plying a role for T cell-dendritic cell interaction during an immuneresponse (7–9).

Soluble RANKL (sRANKL) lacking transmembrane and intra-cellular regions is now available and has allowed us to elucidatethe role of RANKL in osteoclast development and function inmore detail. Osteoclasts have been shown to be formed fromspleen cells, nonadherent bone marrow cells, or peripheral blood-derived monocytes in the presence of M-CSF and sRANKL in theabsence of stromal cells (5, 6, 10, 11). In addition, a macrophage-like cell line has been demonstrated to potentially differentiate intoosteoclasts when treated with M-CSF and sRANKL (12). How-ever, the efficiency for osteoclast formation was low in the abovecultures, suggesting the requirement for other factors for osteoclas-togenesis. Furthermore, due to the lack of a population of oste-oclast progenitors that synchronously differentiate into osteoclasts,the molecular mechanisms regulating the process of osteoclasto-genesis have remained uncertain.

In this study we developed a new isolation method for obtainingosteoclast progenitors. By this procedure, we isolated M/Mf-likehemopoietic cells from mouse unfractionated bone cells; these iso-lated cells are potentially capable of differentiating into osteoclastsin response to M-CSF and sRANKL. Surprisingly, neutralizing Abagainst TGF-b completely blocked osteoclast formation from the

*Department of Oral Anatomy, Meikai University School of Dentistry, Sakado,Saitama, Japan; and†Department of Bioscience, Faculty of Applied Bioscience, To-kyo University of Agriculture, Setagaya, Tokyo, Japan

Received for publication February 28, 2000. Accepted for publication July 24, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by a grant-in aid for scientific research from theMinistry of Education, Science, and Culture of Japan.2 Address correspondence and reprint requests to Dr. Yoshiyuki Hakeda, Departmentof Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283,Japan. E-mail address: y-hakeda@dent.meikai.ac.jp3 Abbreviations used in this paper: M/Mf, monocyte/macrophage; RANK, receptoractivator of NF-kB; RANKL, receptor activator of NF-kB ligand; sRANKL, solubleRANKL; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; TGFR, TGF-b receptor; TRAP,tartrate-resistant acid phosphatase; MNCs, multinucleate cells; JNK, c-Jun N-terminalprotein kinase; TRAF, TNF receptor-associated factor.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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precursors induced by sRANKL/M-CSF signaling. In addition, ex-ogenous TGF-b induced the further commitment and maturationof osteoclast progenitors into mature osteoclasts in the absence ofstromal cells. In contrast, many studies using in vitro culture sys-tems containing stromal cells have demonstrated that TGF-b in-hibited the differentiation and function of osteoclasts (13, 14).Thus, TGF-b possesses multifunctional biological activities. Tar-get cells of TGF-b are heterogeneous in bone, including bone-forming and -resorbing cells, hemopoietic cells, and bone marrowstromal cells (15–17). Therefore, it has been difficult to elucidatethe precise and direct action of TGF-b on osteoclast development.Here we report that endogenous production of TGF-b by M/Mf-like hemopoietic cells and the derived osteoclast precursors is es-sential for osteoclastogenesis induced by a combination ofRANKL and M-CSF. Our findings expand the established roles ofTGF-b in osteoclastogenesis and provide a novel insight into bonemetabolism.

Materials and MethodsAntibodies

Neutralizing mAb (clone, 1D11) against TGF-b1, -2, and -3 and isotypecontrol mouse IgG1 were obtained from R&D Systems (Minneapolis,MN). Polyclonal rabbit anti-RANK Ab was provided by Snow Brand MilkProducts Co. Ltd. (Tochigi, Japan). Unlabeled anti-CD16/32 (clone 2.4G2)Ab, biotinylated anti-CD11b (Mac-1a-chain; clone M1/70), anti-CD11a(LFA-1 a-chain; clone 2D7), anti-CD44 (clone IM-7) and anti-CD61 (in-tegrinb3; clone C9.G2) Abs and PE-labeled anti-CD14 (clone rmC5-3) Abwere purchased from PharMingen International (San Diego, CA). Biotinylatedanti-F4/80 (clone A3-1), FITC-labeled anti-integrinav, and unlabeled anti-DEC-205 (clone NLDC-145) were obtained from Serotec (Kidlington, U.K.),Sumitomo Electronic (Osaka, Japan), and BMA Biomedicals (Augst, Swit-zerland), respectively. Anti-p50 (sc-114X) and anti-p65 (sc-109X) Abs werepurchased from Santa Cruz Biotechnology (San Diego, CA).

Isolation of M/Mf-like hemopoietic cells from mouseunfractionated bone cells

Mouse unfractionated bone cells were prepared from femora and tibiae of4- to 5-wk-old ICR mice (Shizuoka Laboratories Animal Center, Shizuoka,Japan). After removal of connective soft tissues, the bones were mincedinto small pieces ina-MEM (ICN Biomedicals, Aurora, OH) supple-mented with 10% FBS (Intergen, Purchase, NY) and 100 U/ml of penicil-lin. The cells were dissociated from the bone fragments by vortexing andwere filtered through a nylon mesh with a 70-mm pore size. The cellsobtained in suspension were used as mouse unfractionated bone cells. Theunfractionated bone cells (108 cells) were seeded and cultured for 6 days ina-MEM containing 10% FBS and PGE2 (1028 M; Sigma, St. Louis, MO)in 100-mm tissue culture dishes in a humidified atmosphere of 5% CO2.The medium was exchanged on day 4 of culture. During the 6 days inculture, the stromal cells derived from the unfractionated bone cells pro-liferated to become overconfluent, forming a stromal cell layer sheet. Pok-ing at the end of the stromal cell layer caused the layer to spontaneouslyroll up and detach from the dish. When the unfractionated cells were pre-cultured in the presence of high concentration of PGE2 (1026 M), numer-ous tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cellsand TRAP-positive multinucleate cells (MNCs) were generated in the cul-ture, consistent with the previous studies (18). However, in the preculturepretreated with the lower dose of PGE2 (1028 M), the cells remaining onthe bottom of the dishes consisted of a large population of M/Mf-like cells,a small population of nonadherent cells and stromal cells, and few TRAP-positive cells. After removal of nonadherent cells and stromal cells bywashing with PBS and incubating in 0.25% trypsin/0.05% EDTA, M/Mf-like hemopoietic cells were harvested in PBS by vigorously pipetting. Inthe population of isolated hemopoietic cells, contaminating stromal cellsand TRAP-positive cells represented,0.01% of the total cells.

Estimation of osteoclastogenesis from M/Mf-likehemopoietic cells

Isolated M/Mf-like hemopoietic cells were seeded at an initial density of1 or 2.5 3104 cells/cm2 and cultured ina-MEM/10% FBS with or withoutseveral cytokines and/or other agents. The culture medium was exchangedevery 3 days. After a culture period of the desired length, the cells werefixed in 10% formalin and stained for TRAP activity with a leukocyte acid

phosphatase kit (Sigma). The numbers of total cells, TRAP-positive mono-nuclear cells, and TRAP-positive MNCs were counted under a microscope.TRAP-positive mononuclear cells and MNCs were considered to be preos-teoclastic and osteoclastic cells, respectively. Thereafter, nuclei of thesecells were again stained with propidium iodide (50mg/ml) in 0.1% sodiumcitrate, and the numbers of nuclei of total cells, TRAP-positive mononu-clear cells, and TRAP-positive MNCs in the culture were counted under afluorescence microscope. The total nuclei number represents the rate of celldivision. The number of nuclei in TRAP-positive cells (mononuclear cellsplus MNCs) was used as an indicator of the commitment to the osteoclastlineage. A fusion index was calculated as the percentage of nuclei inTRAP-positive MNCs per those in total cells, and the value was consideredthe percentage of cells that participated in the cell fusion, indicating oste-oclast maturation.

Pit formation assay

Osteoclastic cells were generated ina-MEM/10% FBS containing M-CSF(10 ng/ml; Chemicon International, Temecula, CA) with sRANKL (40 ng/ml; PeproTech EC, London, U.K.) and/or various concentrations ofTGF-b1 (Austral Biologicals, San Ramon, CA) for 5 days. Then, aftertreatment with trypsin/EDTA, the cells in the culture were pipetted off andharvested. The cells obtained (800 cells) were seeded on each dentine sliceand incubated for 1 day. At the end of the incubation, the cells on thedentine slices were stained for TRAP activity to confirm their survival.Then, the cells were scraped off the dentine slices, and the slices werestained with acid hematoxylin (Sigma). The number of the stained pits wascounted under a microscope.

Flow cytometry

After isolation of M/Mf-like hemopoietic cells, the cells were suspendedin ice-cold PBS containing 0.5% BSA, 0.1% sodium azide, and 1 mMglucose. Before being stained for cell surface Ags, the progenitors werepreincubated with anti-CD16/32 Ab or an excess of mouse IgG (Sigma) toreduce nonspecific binding of Abs. The pretreated cells were stained for 30min with biotinylated anti-CD11b, anti-CD11a, anti-CD44, anti-F4/80, oranti-integrinb3 Abs; with FITC-labeled anti-integrinav; with PE-labeledanti-CD14; or with unlabeled anti-DEC-205 Abs. For staining with thebiotinylated and the unlabeled Abs, the stained cells were secondarily in-cubated with avidin-FITC (PharMingen) and FITC-conjugated anti-rat IgG(PharMingen), respectively, for 30 min in PBS containing anti-CD16/32Ab or an excess of mouse IgG. Then, the cells were analyzed withoutgating on a FACStar (Becton Dickinson, San Jose, CA).

RT-PCR

Total RNA (1mg) extracted from cells in the culture was used as a templatefor cDNA synthesis. cDNA was prepared by use of a Superscript II pre-amplification system (Life Technologies, Gaithersburg, MD). Primers weresynthesized on the basis of the reported mouse cDNA sequences for TRAP,integrin av, integrin b3, calcitonin receptor, cathepsin K, RANK, CD14,TGF-b1, TGF-b2, TGF-b3, TGF-b receptor I (TGFR-I), and TGFR-II.Sequences of the primers used for PCR were as follows: TRAP forward,59-CACGATGCCAGCGACAAGAG-39; TRAP reverse, 59-TGACCCCGTATGTGGCTAAC-39; integrinav forward, 59-GCCAGCCCATTGAGTTTGATT-39; integrin av reverse, 59-GCTACCAGGACCACCGAGAAG-39; integrinb3 forward, 59-TTACCCCGTGGACATCTACTA-39; integrinb3 reverse, 59-AGTCTTCCATCCAGGGCAATA-39; cathepsin K forward,59-GGAAGAAGACTCACCAGAAGC-39; cathepsin K reverse, 59-GTCATATAGCCGCCTCCACAG-39; calcitonin receptor forward, 59-ACCGACGAGCAACGCCTACGC-39; calcitonin receptor reverse, 59-GCCTTCACAGCCTTCAGGTAC-39. CD14 forward, 59-AAGTTCCCGACCCTCCAAGTT-39; CD14 reverse, 59-CTGCCTTTCTTTCCTTACATC-39; RANK for-ward, 59-CTCTGCGTGCTGCTCGTTCC-39; RANK reverse, 59-TTGTCCCCTGGTGTGCTTCT-39; TGF-b1 forward, 59-GGACCGCAACAACGCCATCTA-39; TGF-b1 reverse, 59-CGCACACAGCAGTTCTTCTCT-39;TGF-b2 forward, 59-CATCCCGAATAAAAGCGAAGA-39; TGF-b2 re-verse, 59-AAAACTCCCTCCCTCCTGTCA-39. TGF-b3 forward, 59-TTTTCCTCCCCCTTTCTACTG-39; TGF-b3 reverse, 59-GGTTCCATTTTTCTCCACTGA-39; TGFR-I forward, 59-GAAGGGCTCATCACCACCAAT-39;TGFR-I reverse, 59-AGGCAGCTAACCGTATCCAGA-39; TGFR-II for-ward, 59-GGCATCGCTCATCTCCACAGT-39; TGFR-II reverse, 59GCCCTCGGTCTCTCAGCACAC-39; b-actin forward, 59-TCACCCACACTGTGCCCATCTAC-39; and b-actin reverse, 59-GAGTACTTGCGCTCAGGAGGAGC-39. Amplification was conducted for 22–32 cycles, each of94°C for 30 s, 58°C (TGF-b2 and TGF-b3, 56°C) for 30 s, and 72°C for 1 minin a 25-ml reaction mixture containing 0.5ml of each cDNA, 25 pmol of eachprimer, 0.2 mM dNTP, and 1 U of Tap DNA polymerase (Qiagen, Valencia,CA). After amplification, 15ml of each reaction mixture was analyzed by 1.5%

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agarose gel electrophoresis, and the bands were then visualized by ethidiumbromide staining.

Western blot analysis

After the isolated M/Mf-like hemopoietic cells had been treated with M-CSF and/or TGF-b for 2 days, the cells were washed with PBS; scrapedinto a solution consisting of 10 mM sodium phosphate (pH 7.5), 150 mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mMEDTA, 1 mM aminoethylbenzenesulfonyl fluoride, 10mg/ml leupeptin,and 10mg/ml aprotinin; and sonicated for 15 s. The protein concentrationin the cell lysate was measured with a bicinchoninic acid protein assay kit(Pierce, Rockford, IL). Each sample containing equal amounts of proteinwas subjected to 10% SDS-PAGE, and the proteins separated in the gelwere subsequently electrotransferred onto a polyvinylidene difluoridemembrane. After having been blocked with 5% skim milk, the membranewas incubated with anti-RANK Abs or nonimmune rabbit IgG and subse-quently with peroxidase-conjugated anti-rabbit IgG Ab. Immunoreactiveproteins were visualized with Western blot chemiluminescence reagents(DuPont-New England Nuclear Products, Boston, MA) following the man-ufacturer’s instructions.

EMSA

Nuclear extracts were prepared from M/Mf-like hemopoietic cells pre-treated for 2 days with M-CSF alone or with M-CSF and TGF-b as pre-viously described (19). Double-stranded oligonucleotides containing anNF-kB binding site (59-AGTTGAGGGGACTTTCCCAGGC-39) were ra-diolabeled with [g-32P]ATP and combined with 1mg of nuclear extracts for20 min at room temperature using a gel shift assay system (Promega, Mad-ison, WI). The specificity of the reaction was confirmed by competitionwith a 50-fold molar excess of nonlabeled oligonucleotides. The protein-DNA complexes were resolved by 7.2% PAGE in 0.53 TBE buffer andvisualized by autoradiography. In the supershift experiment, the nuclearextracts were incubated with anti-p50 or anti-p65 Ab for 30 min on iceafter binding to the oligonucleotides, and then were subjected to PAGE.

Statistical analysis

Significant differences between means of group were analyzed by one-wayANOVA and Dunnett’s test.

ResultsCharacterization of M/Mf-like hemopoietic cells isolated from aculture of mouse unfractionated bone cells

When unfractionated bone cells prepared from 4- to 5-wk-old micewere cultured in the presence of a low dose of PGE2 (1028 M) for6 days, the stromal cells proliferated to overconfluence in the cul-ture and formed a sheet of cells. Morphologically appearing mac-rophage-like cells adhered to the substratum under the stromal celllayer. After the stromal cell sheet was detached, the remainingcells were isolated. As shown in Fig. 1,A and B, these cells re-vealed a mononuclear macrophage-like shape with a relativelylarge cytoplasm, and all the cells were capable of phagocytosinglatex beads. These cells required M-CSF for their survival (datanot shown). In addition, they were TRAP negative. Cell surfacemolecules expressed on the isolated cells were analyzed by flowcytometry using various Abs (Fig. 1C). The cells were positive forCD11b (Mac-1a-chain), CD44, F4/80, and CD11a (LFA-1) andweakly positive for CD14 and integrinav, but negative for integrinb3, which is expressed on mature osteoclasts, and DEC205, whichis expressed on dendritic cells. Taken together, these findings in-dicate that these cells belonged to the M/Mf lineage.

In vivo and in vitro studies have demonstrated that RANKL incooperation with M-CSF is essential for generation of osteoclastsfrom hemopoietic cells (5, 6). When the cells isolated by the aboveprocedure were incubated for 6 days in the presence of M-CSF andsRANKL, TRAP-positive MNCs were formed in the culture in adose-dependent manner, as shown in Fig. 2. The total number ofnuclei in the cultures was increased by the addition of M-CSF,indicating that the proliferation of the cells was dependent on M-CSF. M-CSF also increased the percentage of nuclei in TRAP-positive cells as an indicator of commitment to the osteoclast lin-

eage and the fusion index of TRAP-positive MNCs, defined as thepercentage of cells participating in the fusion. These values at 20ng/ml of M-CSF were 28 and 17%, respectively, producing a max-imal effect; and no further stimulation was observed at higher con-centrations. On the other hand, although the total nuclear numberwas not changed by the addition of sRANKL, the percentage ofnuclei in TRAP-positive cells and the fusion index of TRAP-pos-itive MNCs were increased in a concentration-dependent fashion,with a maximal stimulation at 40 ng/ml. When TRAP-positiveMNCs generated in the presence of M-CSF and sRANKL werereplated on dentine slices, these cells resorbed dentine and formedpits on the surface (Fig. 2D). In addition, semiquantitative PCRanalysis revealed that treatment with M-CSF and sRANKL for 6days induced an increase in the levels of mRNA for TRAP, ca-thepsin K, calcitonin receptor, and integrinb3, all of which areabundantly expressed in osteoclasts (Fig. 3). On the other hand, theaddition of sRANKL decreased the level of CD14 mRNA. Theseeffects of M-CSF and sRANKL indicate that the isolated hemo-poietic cells were potentially capable of differentiating into matureosteoclasts. In addition, these results demonstrate that M-CSF andRANKL play crucial roles in survival, proliferation, and differen-tiation of osteoclast progenitors, consistent with the conclusion ofprevious studies (5, 6). However, the percentages of nuclei inTRAP-positive cells and the fusion index were not very high, in-dicating that all the cells did not differentiate into the osteoclastlineage.

Exogenous TGF-b in combination with RANKL/M-CSF inducesosteoclast formation by all isolated hemopoietic cells

Cells of the M/Mf lineage are known to produce several cytokinesand growth factors, and their proliferation and differentiation areregulated in an autocrine and paracrine manner (20, 21). Of thesefactors, TGF-b is expressed not only by monocyte/macrophagesbut also by MNCs (22). The expression of two types (TGFR-I andTGFR-II) of TGF-b receptor on isolated hemopoietic cells wasconfirmed by RT-PCR analysis (Fig. 4). These results indicate thatthe isolated hemopoietic cells are potentially responsive to TGF-b.Simultaneous addition of TGF-b with sRANKL (40 ng/ml) andM-CSF (10 ng/ml) dose dependently increased the number ofTRAP-positive MNCs among the cells cultured for 6 days, with amaximal effect of 12-fold at 1.25–20 ng/ml. At 20 ng/ml ofTGF-b, the fusion index was 60%, i.e., 12-fold greater than that inthe absence of TGF-b. Besides the TRAP-positive MNCs, almostall the mononuclear cells were TRAP positive (Fig. 5,A andB).However, since the cells died in the presence of TGF-b andsRANKL without M-CSF, TGF-b could not replace M-CSF forthe survival of the osteoclast progenitors (data not shown). In ad-dition, the combination of TGF-b and M-CSF without sRANKLsupported the survival of the cells, but did not induce the formationof osteoclastic TRAP-positive MNCs. Associated with the en-hancement of differentiation into osteoclasts, TGF-b further in-creased the mRNA levels of TRAP, cathepsin K, calcitonin recep-tor, and integrinsav and b3 and further decreased the CD14mRNA level (Fig. 3). The number of pits excavated by the cellscultured with TGF-b, M-CSF, and sRANKL was much greaterthan that with M-CSF and sRANKL (Fig. 5C). As shown in Fig.6, the stimulatory effect of TGF-b was dose dependent, consistentwith the increase in osteoclastic cell formation. Taken together, thedata show exogenous TGF-b to be a potent, but additive, inducerof osteoclastogenesis.

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Endogenous TGF-b as well as M-CSF and RANKL participatein osteoclastogenesis

As shown in Fig. 4, the isolated hemopoietic cells expressedTGF-b1 and -b2 as well as their receptors. Therefore, we nextexamined whether endogenous TGF-b is involved in osteoclastgeneration in an autocrine fashion. Addition of neutralizing Abagainst TGF-b abrogated the stimulation of osteoclast-like cell for-mation induced by M-CSF and sRANKL, whereas the nonimmuneIgG had no effect (Fig. 7). This result shows that osteoclastogen-esis induced by M-CSF and RANKL requires the endogenous pro-

duction of TGF-b by the osteoclast progenitors. Next, to ascertainthe action point of endogenous TGF-b in osteoclast development,we examined the effects of different treatment periods with anti-TGF-b on the osteoclast-like cell formation from the isolated he-mopoietic cells pretreated with M-CSF and/or TGF-b. When theisolated hemopoietic cells were cultured for 6 days in the presenceof M-CSF and sRANKL without TGF-b after pretreatment withTGF-b and M-CSF for the first 2 days, the fusion index of TRAP-positive osteoclastic MNCs formed in the cultures was equivalentto that in the cultures treated for the last 6 days with a combination

FIGURE 1. Phase-contrast micrographs of iso-lated M/Mf-like hemopoietic cells. After removalof stromal cell layer, the remaining cells in the cul-ture dishes were isolated, replated, and cultured ina-MEM containing 10% FBS for 3 h (A). The iso-lated cells morphologically appeared to be M/Mf.The isolated cells were incubated with 0.2% latexbeads for 3 h and were washed with PBS to removeunphagocytosed beads. Most cells showed phagocy-totic activity (B). The bar in each photograph indi-cates 100mm. The isolated cells were analyzed fortheir cell surface Ags by flow cytometry (C). AfterFc block or excessive mouse IgG pretreatment, thecells were stained with various Abs as shown in theunshaded area under the curve. The shaded area un-der the curve shows control staining with FITC-avi-din or FITC-anti-rat IgG.

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of TGF-b, sRANKL, and M-CSF (Fig. 8B; lanes 2and 3 fromleft). The cultures pretreated only with M-CSF required the con-tinuous presence of TGF-b for the high efficiency of osteoclasticcell formation (Fig. 8A;lanes 2and3 from left). These data sug-gest that 2-day pretreatment with TGF-b allows osteoclast progen-itors to prime to commit to an osteoclast lineage. However, theexpression of the RANK receptor (RANK) in the isolated hemo-poietic cells was not up-regulated by 2-day pretreatment withTGF-b at mRNA and protein levels, whereas the expression wasenhanced by M-CSF (Fig. 9,A and B). Instead, the TGF-b pre-treatment synergistically stimulated activation of NF-kB evokedby sRANKL as determined by direct EMSA (Fig. 9C). Followingthe TGF-b pretreatment, treatment for 6 days with anti-TGF-bgreatly reduced the osteoclast generation induced by the combi-nation of sRANKL and M-CSF. Likewise, the inhibition was seenin the cultures treated with anti-TGF-b for the last 5 and 3 days,implying that endogenous production of TGF-b is involved in theprocesses of osteoclast differentiation, including priming and mat-uration (Fig. 8,A andB; lanes 4–6 from left). Finally, as in the

FIGURE 4. Expression of TGF-bs and TGF-b receptors. Total RNA ofthe M/Mf-like hemopoietic cells was immediately extracted after isolationof the cells (lane 1), or the isolated M/Mf-like hemopoietic cells weretreated with M-CSF (10 ng/ml) alone (lane 2), M-CSF plus sRANKL (40ng/ml; lane 3), or M-CSF, sRANKL, and TGF-b1 (10 ng/ml;lane 4) for6 days, and then total RNA was extracted from each culture. The primersused were designed for mouse genes of TGF-b1, TGF-b2, TGF-b3, andtypes I and II of TGFR. The numbers in parentheses indicate the numbersof PCR cycles.

FIGURE 2. Isolated M/Mf-like hemopoietic cells potentially differen-tiate into mature osteoclasts. The isolated cells (23 104 cells) were cul-tured with various concentrations of M-CSF (A) in the presence ofsRANKL (40 ng/ml), or the cells (53 104 cells) were cultured with variousdoses of sRANKL (B) in the presence of M-CSF (10 ng/ml) for 6 days. Atthe end of the culture period, the cells were stained for nuclei and TRAPactivity. The numbers of total nuclei and of TRAP-positive mononuclearcells and MNCs were counted. The total number of nuclei (M) representsthe proliferation of the cells in the culture. The percentages (F) of nucleiin TRAP-positive mononuclear cells and of MNCs per total nuclear num-ber show the rate of commitment of the M/Mf-like hemopoietic cells intocells of the osteoclast lineage. Fusion index (E) indicates the rate of TRAP-positive osteoclast precursors that participate in cell fusion resulting inmature osteoclasts. Values are the mean6 SE for three cultures in a rep-resentative experiment.C, Photomicrograph of the TRAP-stained cells cul-tured for 6 days with sRANKL (40 ng/ml) and M-CSF (10 ng/ml).D, The cellstreated for 5 days with sRANKL and M-CSF were replated on dentine slicesand incubated for 1 day. Resorption pits formed by the cells were stained withacid hematoxylin. The bar in each photograph indicates 100mm.

FIGURE 3. Semiquantitative RT-PCR analysis for the expression ofvarious mRNAs. Total RNA of the M/Mf-like hemopoietic cells was im-mediately extracted after isolation of the cells (lanes 1,5, and9), or theisolated M/Mf-like hemopoietic cells were treated with M-CSF (10 ng/ml)alone (lanes 2,6, and10), M-CSF plus sRANKL (40 ng/ml;lanes 3,7, and11), or M-CSF, sRANKL, and TGF-b1 (10 ng/ml;lanes 4,8, and12) for6 days, and then total RNA was extracted from each culture. The primersused were designed for mouse genes of TRAP, cathepsin K, CD14, inte-grins av andb3, calcitonin receptor, andb-actin. Each cDNA was ampli-fied for the indicated number of PCR cycles.

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cultures of osteoclast progenitors, anti-TGF-b Ab abolished theformation of TRAP-positive osteoclastic MNCs induced by M-CSF plus sRANKL or 1,25(OH)2D3 in cultures of unfractionatedbone cells (Fig. 10), indicating that the requirement of endogenousTGF-b for osteoclastogenesis is not restricted to cultures of iso-lated hemopoietic cells.

DiscussionIn this study we succeeded in developing a new isolation methodfor obtaining a homogenous population of osteoclast progenitors.The progenitors possessed common phenotypes of monocyte/mac-rophages, representing M/Mf-like hemopoietic cells. Using thestroma-free culture system of isolated M/Mf-like hemopoietic

FIGURE 5. Effect of exogenous addition of TGF-b on osteoclastogen-esis from isolated M/Mf-like hemopoietic cells. The isolated M/Mf-likehemopoietic cells were cultured for 6 days with various concentrations ofTGF-b1 in the presence of M-CSF (10 ng/ml) and sRANKL (40 ng/ml).A,At the end of the culture, the cells were stained for nuclei and TRAPactivity. Total nuclear number, percentage of that number in TRAP-posi-tive cells, and fusion index were calculated. Values are the mean6 SE forthree cultures in a representative experiment.B, Photomicrograph ofTRAP-positive cells cultured for 6 days with TGF-b1 (10 ng/ml),sRANKL (40 ng/ml), and M-CSF (10 ng/ml).C, The cells treated for 5days with TGF-b, sRANKL, and M-CSF were replated on dentine slicesand incubated for 1 day. Resorption pits formed by the cells were stainedwith acid hematoxylin. The bar in each photograph indicates 100mm.

FIGURE 6. Pits excavated by osteoclastic cells generated by treatmentwith M-CSF, sRANKL, and TGF-b1. Isolated M/Mf-like hemopoieticcells were cultured for 5 days without or with various concentrations ofTGF-b1 in the presence of M-CSF (10 ng/ml) and sRANKL (40 ng/ml).Then, after treatment with trypsin/EDTA, the cells obtained (800 cells)were seeded on each dentine slice and incubated for 1 day. At the end ofthe incubation, the cells were scraped off the dentine slices, the slices werestained with acid hematoxylin, and the number of resorption pits on thedentine slice was counted under a microscope. Values are the mean6 SEfor four cultures in a representative experiment.

FIGURE 7. Neutralizing anti-TGF-b Ab abolishes generation of oste-oclasts induced by M-CSF and sRANKL. The isolated M/Mf-like hemo-poietic cells were treated for 6 days with neutralizing anti-TGF-b Ab (20mg/ml) or isotype control IgG1 (20mg/ml) in the presence of variouscombinations of M-CSF (10 ng/ml), TGF-b1 (10 ng/ml), and/or sRANKL(40 ng/ml). Then, the fusion index in each culture was calculated. Valuesare the mean6 SE for three cultures in a representative experiment.p, p ,0.01 vs cultures treated with M-CSF and sRANKL, or cultures treated withM-CSF, TGF-b1, and sRANKL, by Dunnett’s analysis.

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cells, we demonstrated that TGF-b directly acts on the hemopoi-etic cells to enhance the osteoclast formation elicited by a combi-nation of sRANKL and M-CSF. The isolated hemopoietic cellsexpressed mRNAs of TGF-b1 and -b2 and TGFR-I and -IIthroughout the culture period, suggesting that osteoclast progeni-tors are both the TGF-b-producing cells and cells responsive toTGF-b. Because various hemopoietic cells express TGF-b andTGF-b receptors and their proliferation and differentiation are

widely regulated by TGF-b (23, 24), and because osteoclasts are ofhemopoietic origin (25), these expressions in the isolated cells arenot surprising. In fact, the production of TGF-b and the expressionof TGF-b receptors have been previously reported in chick oste-oclasts (22) and in osteoclastic MNCs derived from human giantcell tumors of bone (26). In addition, since anti-TGF-b Ab greatlysuppressed the osteoclast formation from isolated cells, endoge-nous production of autocrine-acting TGF-b by hemopoietic cellsappears to be required for osteoclastic differentiation. Furthermore,TGF-b induced both the priming of hemopoietic cells to differen-tiate into the cells of osteoclast lineage and the maturation of thesecells.

We isolated M/Mf-like hemopoietic cells from cultures of un-fractionated bone cells treated with PGE2. These cells requiredM-CSF for their survival and growth. In addition, the isolated cellsexpressed various monocyte/macrophage-phenotypic surface Ags,and showed phagocytotic activity. GM-CSF also supported thesurvival, but neither stimulated the proliferation nor induced oste-oclast generation even in the presence of sRANKL (data notshown). It has been recently demonstrated that human cells sharingmonocyte/macrophage phenotypes were capable of differentiatinginto dendritic cells and osteoclasts dependent on GM-CSF andM-CSF, respectively (27). In addition, when the isolated M/Mf-like hemopoietic cells were cultured with LPS (28), these cells didnot differentiate into an osteoclastic cell lineage even withsRANKL, M-CSF, and TGF-b (data not shown). Taken together,the available data indicate that the isolated cells represent bipo-tential immature monocytes/macrophages.

TGF-bs are multifunctional cytokines that widely regulate theproliferation and differentiation of a variety of cell types, includingepithelial and mesenchymal cells (29, 30). Numerous studies onbone cells have indicated that TGF-b stimulates the growth anddifferentiation of osteoprogenitors to become bone matrix-produc-ing cells (31, 32). Thus, TGF-bs are positive regulators of boneformation. However, the effect of TGF-b on bone resorption iscontroversial. A stimulatory effect of TGF-b on bone resorptionwas observed in organ cultures of mouse calvariae (33). In con-trast, TGF-b inhibited the osteoclastic bone resorption in fetal ratlong bones (34). Furthermore, Hughes et al. (35) showed thatTGF-b promoted the apoptosis of osteoclasts in culture of bone

FIGURE 8. Effect of neutralizing anti-TGF-b Ab on priming and maturation of osteoclast differentiation. The isolated M/Mf-like hemopoietic cellswere pretreated for 2 days with M-CSF (10 ng/ml) alone (A) or with TGF-b1 (10 ng/ml) plus M-CSF (B). Then, after the factors had been removed bywashings, the pretreated cells were further treated for 6 days with various combinations of M-CSF, TGF-b, and/or sRANKL (40 ng/ml). Neutralizinganti-TGF-b Ab (20 mg/ml) or isotype control IgG1 (20mg/ml) was added to the cultures at day 0 (D-0), day 1 (D-1), or day 3 (D-3) after the start oftreatment. Values are the mean6 SE for three cultures in a representative experiment.p, p , 0.01 vs culture treated with M-CSF and sRANKL, byDunnett’s analysis.

FIGURE 9. Expression of RANKL receptor (RANK) and activation ofNF-kB in M/Mf-like hemopoietic cells.A andB, Total RNA and mem-brane proteins were immediately extracted from the isolated untreatedM/Mf-like cells (A,lanes 1and4; B, lanes 1,4, and7) and from the cellstreated for 2 days with M-CSF at 10 ng/ml (A,lanes 2and5; B, lanes 2,5, and8), or with M-CSF and TGF-b1 at 10 ng/ml (A, lanes 3 and 6; B,lanes 3, 6 and 9). Western blotting (A) and RT-PCR (B) analyses forRANK expression were performed. Each cDNA was amplified for the in-dicated number of PCR cycles.C, EMSA. The isolated M/Mf-like cellswere precultured in the absence (lanes 1–5) or presence (lanes 6–12) ofTGF-b1 (10 ng/ml) with M-CSF (10 ng/ml) for 2 days. After preculture,the cells were treated with sRANKL (40 ng/ml) for 0 h (lanes 1and6),0.5 h (lanes 2and7), 1 h (lanes 3and8), 2 h (lanes 4and9), and 4 h (lanes5 and10). Then, nuclear proteins in the cells were extracted and subjectedto EMSA. Lanes 11and12, EMSAs using a sample from the cells treatedwith sRANKL for 1 h after TGF-b pretreatment (lane 8) were performedin the presence of anti-p65 and anti-p50 Abs, respectively.

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marrow cells consisting of a heterogeneous population. Therefore,the inhibition of bone resorption by TGF-b may in part be attrib-uted to the induced osteoclast apoptosis, although we did not ob-serve such a stimulatory effect of TGF-b on osteoclast apoptosis inour culture system. Regarding osteoclastogenesis, the inhibition byTGF-b was demonstrated in cultures of bone marrow cells, whichcontained stromal cells, and in cocultures of bone marrow cells orspleen cells and stromal cells (13). On the other hand, TGF-b wasreported to stimulate the formation of osteoclast-like cells in cul-tures of a human leukemia cell line, FLG 29.1, in an autocrinemanner (36). Transgenic mice overexpressing TGF-b2 exhibitedan osteoporosis-like phenotype due to the increased osteoclasticfunction (37, 38), and transgenic mice expressing dominant neg-ative type II TGF-b receptor decreased osteoclastic bone resorp-tion (39), suggesting a locally positive participation of TGF-b inosteoclast development. Sells et al. (40) recently demonstrated thatTGF-b in combination with RANKL and M-CSF enhanced oste-oclast-like formation in cultures of bone marrow cells and spleencells containing few osteoblastic/stromal cells. In addition, TGF-bwas demonstrated to stimulate osteoclast formation in coculturesof spleen cells and T lymphocytes expressing RANKL (41). Ourfindings are consistent with those results, although target cells ofTGF-b were not defined due to the heterogeneity of hemopoieticcells in those culture systems (40, 41). The isolated cells examinedin this study consisted of a homogenous population with mono-cyte/macrophage phenotypes, and all of them differentiated intocells of osteoclast lineage by TGF-b treatment in the presence ofsRANKL and M-CSF. Therefore, TGF-b directly acts on oste-oclast progenitors to stimulate their differentiation into osteoclasts.Taken together, the overall effects of TGF-b on osteoclastogenesisare dependent on the cell population.

We also indicated that TGF-b in combination with sRANKLand M-CSF stimulated osteoclast formation in the cultures of un-fractionated bone cells. These cultures contained stromal cells, butthe stromal cells somehow did not expansively proliferate in thecultures. The inhibitory effect of TGF-b seems to be observedwhen a large number of osteoblastic/stromal cells are present. Itwas recently demonstrated that TGF-b increased the expression ofosteoprotegerin (identical with osteoclastogenesis-inhibitory fac-tor), which strongly inhibits osteoclastogenesis as a decoy receptorof RANKL (42). Therefore, osteoprotegerin may be at least in part

a mediator of the TGF-b inhibitory effect via stromal cells. How-ever, endogenous TGF-b is intrinsically essential for osteoclastdevelopment, and the stimulatory effect of exogenous TGF-b isseen under the condition of a minimal number of stromal cells.

PGE2 has recently been reported to cooperate with RANKL andM-CSF in the promotion of osteoclast formation from hemopoieticcells (43). In a variety of cell types, TGF-b induces the productionof PGs mediated by up-regulation of prostaglandin G/H synthase-2(44, 45). Those studies suggest that the enhancement of osteoclastformation by TGF-b presented in this study is mediated by en-dogenous synthesis of PGs. However, since NS-398, a selectiveinhibitor of PGG/H synthase-2, did not affect the stimulation ofosteoclast generation by TGF-b (data not shown), the stimulatoryeffect of TGF-b is not endogenous PG dependent.

RANKL has been demonstrated to activate NF-kB and c-JunN-terminal protein kinase (JNK) through RANK in osteoclasticcells as well as in dendritic cells (12, 46, 47). Recent studies in-dicate that binding of RANKL to RANK caused association of thereceptor with several TNF receptor-associated factors (TRAFs),resulting in the activation of NF-kB (46–48). Knockout mice ofboth NF-kB1 and NF-kB2, and of TRAF6, exhibited severe os-teopetrosis due to impaired osteoclast differentiation (49, 50).Therefore, TRAF6 and NF-kB seem to be involved in osteoclas-togenesis. In this study we demonstrated that TGF-b synergisti-cally increased the translocation of NF-kB into nuclei induced byRANKL in M/M f-like osteoclast progenitors, although TGF-bdid not affect the expression of RANK, suggesting an intracellularcross-talk in signalings of TGF-b and RANKL. At present, thedetailed molecular signalings of TGF-b that strongly promote os-teoclast formation are not known. It was recently reported thatTGF-b-activated kinase 1 functionally interacted with IkB kinaseto stimulate NF-kB (51). Such an interaction of TGF-b receptordownstream signaling molecules with RANK-associated mole-cules may at least in part account for the synergistic induction ofosteoclastogenesis by TGF-b and RANKL.

Involvement of TGF-b in the pathogenesis of osteopenic disor-ders has been suggested. It was demonstrated in rheumatoid ar-thritis that the synovium contained a large number of macrophage-like cells that have a strong ability to produce TGF-b as well asother inflammatory cytokines, such as IL-1 and TNF-a (52, 53),and a high level of endogenous TGF-b was also detectable in other

FIGURE 10. Effects of exogenous TGF-b and neutralizing anti-TGF-b Ab on osteoclast generation in cultures of mouse unfractionated bone cells.A,Unfractionated bone cells (13 105) were cultured in each well of 96-well plates for 5 days in the presence of M-CSF (10 ng/ml) with various combinationsof TGF-b1 (10 ng/ml), sRANKL (40 ng/ml), anti-TGF-b Ab (20 mg/ml), and/or nonimmune IgG1 (20mg/ml). B, Unfractionated bone cells (53 105) werecultured for 7 days with or without 1,25(OH)2D3 (10 nM) in the absence or the presence of neutralizing anti-TGF-b. Then, the cells in the culture werestained for TRAP activity, and the TRAP-positive MNCs were counted. Values are the mean6 SE for three cultures in a representative experiment.A:p, p , 0.05;pp, p , 0.01 (vs cultures treated with M-CSF and sRANKL, by Dunnett’s analysis).B: pp, p , 0.01 (vs cultures treated with 1,25(OH)2D3,by Dunnett’s analysis).

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types of arthritides, including osteoarthritis (54). In addition, theTGF-b concentration in serum was shown to be elevated in osteo-porotic women, with good correlation with the bone loss (55).Taken together with our findings, TGF-b may contribute to de-struction of bone as well as bone formation in vivo.

In conclusion, TGF-b is intrinsically required for osteoclasto-genesis in combination with RANKL and M-CSF. The results pre-sented here expand our knowledge about the multifunctional rolesof TGF-b in bone metabolism.

AcknowledgmentsWe thank Drs. L. G. Raisz (University of Connecticut Health Center), K.Higashio (Snow Brand Milk Products Co. Ltd.), and T. Kuriki (AventisPharma Ltd.) for suggestions and critical review, for the generous gift ofanti-RANK Ab, and for operating the FACStar, respectively.

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