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The Journal of Immunology, 1999, 163: 434-442.
Copyright © 1999 by The American Association of Immunologists

Osteoclast Differentiation Factor Acts as a Multifunctional Regulator in Murine Osteoclast Differentiation and Function1

Eijiro Jimi*, Shuichi Akiyama*, Taro Tsurukai*, Nobuo Okahashi{dagger}, Kanichiro Kobayashi*, Nobuyuki Udagawa*, Tatsuji Nishihara{dagger}, Naoyuki Takahashi* and Tatsuo Suda2,*

* Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan; and {dagger} Department of Oral Science, National Institute of Infectious Diseases, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclast differentiation factor (ODF), a novel member of the TNF ligand family, is expressed as a membrane-associated protein by osteoblasts/stromal cells. The soluble form of ODF (sODF) induces the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here, the effects of sODF on the survival, multinucleation, and pit-forming activity of murine osteoclasts were examined in comparison with those of M-CSF and IL-1. Osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts and bone marrow cells expressed mRNA of RANK (receptor activator of NF-{kappa}B), a receptor of ODF. The survival of OCLs was enhanced by the addition of each of sODF, M-CSF, and IL-1. sODF, as well as IL-1, activated NF-{kappa}B and c-Jun N-terminal protein kinase (JNK) in OCLs. Like M-CSF and IL-1, sODF stimulated the survival and multinucleation of prefusion osteoclasts (pOCs) isolated from the coculture. When pOCs were cultured on dentine slices, resorption pits were formed on the slices in the presence of either sODF or IL-1 but not in that of M-CSF. A soluble form of RANK as well as osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF, blocked OCL formation and prevented the survival, multinucleation, and pit-forming activity of pOCs induced by sODF. These results suggest that ODF regulates not only osteoclast differentiation but also osteoclast function in mice through the receptor RANK.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Osteoclastic bone resorption consists of multiple steps such as the differentiation of osteoclast precursors into mononuclear prefusion osteoclasts (pOC),3 the fusion of pOCs to form multinucleated osteoclasts, and the activation of osteoclasts to resorb bone (1, 2, 3). Multinucleated cells (MNC) with osteoclast characteristics, including tartrate-resistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit-forming activity on dentine slices are formed in a coculture of murine hemopoietic cells and osteoblasts/stromal cells in the presence of osteotropic factors such as 1{alpha},25-dihydroxyvitamin D3, parathyroid hormone, and IL-11 (3). The target cells of those osteotropic factors are osteoblasts/stromal cells but not osteoclast progenitors in the induction of osteoclasts in vitro (3). From these results, we proposed a hypothesis that osteoblasts/stromal cells express a common factor, osteoclast differentiation factor (ODF), in response to those osteotropic factors. ODF appeared a membrane-associated factor, since cell-to-cell contact between osteoclast precursors and osteoblasts/stromal cells was prerequisite for osteoclast formation. Thus, osteoclast precursors of the monocyte-macrophage lineage recognize ODF through cell-to-cell interaction with osteoblasts/stromal cells, then differentiate into osteoclasts (3).

Osteoblasts/stromal cells also play an essential role in inducing osteoclast function (osteoclast activation) (4). When purified osteoclast-like cells (OCLs) formed in vitro were cultured on dentine slices, they failed to form resorption pits (4). Osteoblasts/stromal cells, simultaneously added, greatly enhanced the pit-forming activity of OCLs through a mechanism involving cell-to-cell contact with OCLs (4). OCLs rapidly died via spontaneously occurring apoptosis in the absence of osteoblasts/stromal cells. The cytokines IL-1 and M-CSF were shown to prolong the survival of purified OCLs (5). The activation of NF-{kappa}B was involved in the IL-1-induced survival of OCLs (6). Using pOCs obtained from the echistatin-treated coculture of murine osteoblastic cells and bone marrow cells, we showed that both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs, but only IL-1 induced the pit-forming activity of pOCs, even in the absence of osteoblasts/stromal cells (7). These results indicate that some factors can be replaced with osteoblasts/stromal cells in the induction of the survival, multinucleation, and activation of osteoclasts.

The identical proteins osteoprotegerin (OPG) and osteoclastogenesis inhibitory factor (OCIF), which inhibit osteoclast development in vitro and in vivo, have recently been cloned independently (8, 9). OPG/OCIF is a member of the TNF receptor family, but it does not have a transmembrane domain, suggesting that OPG/OCIF functions as a circulating factor. Subsequently, the cDNA encoding the binding molecule of OPG/OCIF was isolated from an expression library of the murine stromal cell line ST2, which supports OCL formation in coculture with hemopoietic cells (10). The binding molecule of OPG/OCIF was a membrane-associated protein of the TNF ligand family. This molecule satisfied all of the criteria of ODF and was thus renamed ODF. ODF was also found to be identical to TNF-related activation-induced cytokine (TRANCE) and receptor activator of NF-{kappa}B ligand (RANKL), which were independently cloned from murine T cell hybridomas and murine dendritic cells, respectively (11, 12). Lacey et al. (13) also succeeded in the molecular cloning of a ligand for OPG from an expression library of the murine myelomonocytic cell line 32D. The OPG ligand (OPGL) was identical to ODF (TRANCE/RANKL). The administration of OPGL (ODF) to mice caused reduced bone volume and extreme hypercalcemia without a significant increase in the number of osteoclasts, suggesting that ODF is involved in the activation of osteoclasts as well (13). Fuller at al. (14) also recently reported that TRANCE (ODF) is involved in the osteoclast activation induced by osteoblastic cells treated with parathyroid hormone. A soluble form of TRANCE induced a striking change in the motility and spreading of isolated rat osteoclasts, and inhibited their apoptosis (14). These results suggest that ODF is necessary for the osteoblast-mediated activation of mature osteoclasts.

The utilization of a soluble form of ODF (sODF) has allowed us to elucidate the role of ODF in osteoclast function in more detail. In the present study, we examined the effects of ODF on the survival, multinucleation, and activation of osteoclasts in comparison with those of M-CSF and IL-1. sODF, M-CSF, and IL-1 promoted the survival and multinucleation of pOCs through their respective receptors. sODF and IL-1, but not M-CSF, stimulated the pit-forming activity of OCLs. sODF, as well as IL-1, activated NF-{kappa}B and c-Jun N-terminal protein kinase (JNK) in OCLs. Not only OCIF but also a soluble form of RANK inhibited all of the events induced by sODF. These results indicate that ODF expressed by osteoblasts/stromal cells as a membrane-associated protein is responsible for inducing not only osteoclast differentiation but also osteoclast function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and chemicals

A recombinant murine sODF purified by affinity chromatography on an OPG/OCIF-immobilized column and by gel filtration chromatography was kindly provided by Snow Brand Milk Products (Tochigi, Japan). The purity of sODF in this preparation was >95% in SDS polyacrylamide gel electrophoresis. Recombinant human IL-1{alpha}, recombinant human M-CSF, and murine IL-1 receptor antagonist (IL-1ra) were obtained from R&D Systems (Minneapolis, MN). Echistatin was purchased from Sigma (St. Louis, MO). Anti-human I{kappa}B{alpha} rabbit polyclonal Abs were purchased from New England BioLabs (Lake Placid, NY). Anti-human Bcl-2, Bcl-xL, and RelA (p65) rabbit polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-c-Fms mAb was kindly provided by Dr. S-I. Nishikawa (Kyoto University, Kyoto, Japan). Anti-human ß-actin mAb was obtained from Boehringer Mannheim Biochemica (Mannheim, Germany).

Coculture system and enrichment of osteoclast-like cells

Osteoblasts obtained from the calvariae of newborn mice and bone marrow cells obtained from the tibiae of male mice were cocultured in {alpha}MEM (Life Technologies, Grand Island, NY) containing 10% FBS, 1{alpha},25-dihydroxyvitamin D3 (10-8 M) (Wako Pure Chemical, Osaka, Japan) and PGE2 (10-6 M) (Sigma) in 100-mm-diameter dishes coated with collagen gels (Nitta Gelatin, Osaka). OCLs were formed within 6 days in culture and were removed from the dishes by treating with 0.2% collagenase (Wako). The purity of OCLs in this fraction (crude OCL preparation) was about 5%. To further purify the OCLs, the crude OCL preparation was replated on culture dishes. After culture for 8 h, osteoblasts were removed with PBS containing 0.001% pronase E (Calbiochem, La Jolla, CA) and 0.02% EDTA according to the method described previously (15).

Mononuclear and binuclear pOCs were prepared as described previously (16) with a slight modification. Cocultures of bone marrow cells and a murine calvaria-derived osteoblastic cell line, KS4 (17), were maintained in 100-mm-diameter dishes for 6 days as described above. The KS4 cells were removed first from the coculture, using a mixture of collagenase-dispase (Boehringer Mannheim), followed by washing three times with 0.1% BSA in {alpha}MEM. pOCs were then released from the dish with 30 nM echistatin. More than 90% of the cells in the pOC preparation used in the present study were positive for TRAP.

Northern blot analysis

Total RNA was extracted from murine spleen cells, bone marrow cells, primary osteoblasts, purified OCLs, murine myoblastic C2C12 cells, and murine bone marrow-derived stromal ST2 cells using Trizol solution (Life Technologies). The total RNA (10 µg) was electrophoresed in 1.0% agarose-formaldehyde gels, and the RNA was transferred on nylon membrane filters (Hybond-N, Amersham International, Little Chalfont, U.K.). The membranes were hybridized for 15 h at 42°C with radioactive cDNA probes for murine RANK and murine TNF type I and type II receptors, which were cloned by RT-PCR and labeled using a multirandom primer oligonucleotide labeling kit (Takara Shuzo, Osaka, Japan). As an internal control, the membrane was rehybridized with a radioactive cDNA probe for mouse GAPDH. Each membrane was then exposed to an x-ray film.

EMSA and JNK assay

For the EMSA, nuclear extracts were prepared according to the method described by Dignam et al. (18). The sequence of the NF-{kappa}B-binding oligonucleotide used as a radioactive DNA probe was 5'-AGCTTGGGGACTTTCCGAG-3'. The DNA binding reaction was performed at room temperature in a volume of 20 µl, which contained the binding buffer (10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 4% glycerol, 100 mM NaCl, 5 mM DTT, 100 mg/ml BSA), 3 µg of poly(dI-dC), 1 x 105 cpm of a 32P-labeled probe, and 8 µg of nuclear proteins. After incubation for 15 min, the samples were electrophoresed on native 5% acrylamide/0.25x TBE gels. The gels were dried and exposed to an x-ray film. For the determination of JNK activity, purified OCLs treated with sODF or IL-1 were washed twice with ice-cold PBS, then lysed in a lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF). The JNK activity of the cell lysates was determined using a stress-activated protein kinase (SAPK)/JNK kinase assay kit (New England BioLabs).

Survival and cell fusion assays

The survival rate of OCLs was measured as reported previously (5, 19). After OCLs were purified, some of the cultures were subjected to TRAP staining. TRAP-positive MNCs containing more than 3 nuclei were counted as living OCLs. Other cultures were further incubated in the presence or absence of sODF. After incubation for indicated periods, the remaining OCLs were counted. To examine the effect of sODF on the fusion of osteoclasts, we replated pOCs (15,000 cells/well) on 96-well culture plates with or without various increasing concentrations of sODF. After culture for 18 h, the cells were fixed and stained for TRAP. Some cultures were also treated with IL-1ra (1 µg/ml), anti-c-Fms Ab (10 µg/ml), or OCIF (100 ng/ml). The number of TRAP-positive MNCs with more than 10 nuclei was counted as pOC-derived OCLs. Actin rings in pOC-derived OCLs were also visualized by rhodamine-conjugated phalloidin staining, as previously described (15). Results are expressed as the means ± SEM of three cultures.

Immunoblotting analysis and immunofluorescence microscopy

After purified OCL preparations were cultured for various periods in the presence of ODF, the cells were washed twice with ice-cold PBS and then lysed in a lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 10 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 mM PMSF). The cell lysates (20 µg of protein) were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). After blocking with 2% BSA in TBST, the I{kappa}B{alpha}, Bcl-2 and Bcl-xL Abs (1/1000 dilution) were added in TBST containing 2% BSA and visualized by an enhanced chemiluminescence assay using reagents from Amersham and exposed to an x-ray film. The immunofluorescence analysis was done as described previously (6). Anti-RelA (p65) Abs (1 µg/ml) was used for the immunostaining.

Pit formation assay

pOC preparations (15,000 cells/0.1 ml/well) were seeded on dentine slices (4-mm diameter) which had been placed in 96-well plates. After incubation for 2 h, dentine slices were transferred to 48-well plates (one slice/well) in the presence or absence of ODF. Pit formation by pOCs was determined after culture for 24 h. For the pit formation assay, cells were removed from dentine slices, and the resorbed area was stained with Mayer’s hematoxylin (15). The numbers of pits on the slices were counted.

Expression and purification of soluble RANK

A FLAG-tagged soluble form of RANK (sRANK) was generated by cloning a PCR product encoding the RANK ectodomain (amino acids 1–213) into the EcoRI site of an expression vector that carries the promoter region of human EF1{alpha} gene (pEF-BOS) (20). COS 7 cells were transfected with the expression vectors (16.6 µg/100-mm-diameter dish) by cationic liposomes (DMRIE-C, Life Technologies) according to the manufacture’s recommendation. The supernatant was harvested 48 h later, passed through a 0.45-µm filter, incubated with anti-FLAG M2 affinity gel (Kodak, New Haven, CT), and eluted with FLAG peptide (250 µg/ml, Kodak) as outlined in the manufacturer’s protocol. The eluant was dialyzed against PBS, and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RANK, a novel TNF receptor family member, has been shown to be a signal-transducing receptor of ODF (12, 21). Total RNA was obtained from spleen cells, bone marrow cells, purified OCLs, primary osteoblasts, myoblastic C2C12 cells, and bone marrow-derived stromal ST2 cells, and the expression of RANK mRNA was examined in the RNA preparations (Fig. 1Go). Few contaminating osteoblasts (alkaline phosphatase-positive cells) or monocyte-macrophages (F4/80 Ag-positive cells) were detected in the purified OCL preparations in this study (data not shown). The expression of other TNF receptor family members, such as TNFRI and TNFRII, was also examined in the same membrane. TNFRI mRNA was similarly expressed in most of the cells examined in this study. TNFRII mRNA was highly expressed by OCLs, and the expression level by the other cells was much lower (Fig. 1Go). OCLs also highly expressed RANK mRNA (Fig. 1Go).



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  		FIGURE 1. Expression of RANK mRNA by OCLs. Total RNA (10 mg) extracted from spleen cells (lane 1), bone marrow cells (lane 2), purified OCLs (lane 3), C2C12 cells (lane 4), ST2 cells (lane 5), and primary osteoblasts (lane 6) was separated in a 1.0% formaldehyde-agarose gel and hybridized with the cDNA probes for RANK, TNF type I receptor, TNF type II receptor, and GAPDH as described under Materials and Methods.

 
The overexpression of RANK was sufficient to activate NF-{kappa}B (12). The ligand-dependent NF-{kappa}B activation was also demonstrated by the cotransfection of RANKL with RANK in human 293 cells and T cells (12). We then examined whether sODF activates NF-{kappa}B in OCLs. sODF activated NF-{kappa}B in OCLs in a dose-dependent manner (Fig. 2GoA). Fig. 2GoB shows the time course of change in the activation of NF-{kappa}B and the levels of I{kappa}B{alpha} in OCLs after stimulation with sODF. sODF transiently activated NF-{kappa}B in OCLs, and the maximal activation occurred at 30 min. The degradation of I{kappa}B{alpha} coincided with the activation of NF-{kappa}B (Fig. 2GoB). OCIF, a decoy receptor of ODF, prevented the sODF-induced NF-{kappa}B activation, but it did not affect at all the IL-1-induced NF-{kappa}B activation in the OCLs (Fig. 2GoC). The immunocytochemical analysis revealed that RelA (p65) was translocated from the cytoplasm into almost all the nuclei of the OCLs within 30 min after the sODF stimulation (Fig. 2GoD).



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  		FIGURE 2. sODF activates NF-{kappa}B in purified OCLs. A, Purified OCLs were treated with increasing concentrations of sODF for 30 min. B, Time course of changes in the activation of NF-{kappa}B and levels of I{kappa}B{alpha} in OCLs after stimulation with sODF. Purified OCLs were treated with sODF (100 ng/ml) for the indicated periods. C, Effects of OCIF on the activation of NF-{kappa}B in OCLs treated with sODF. sODF (100 ng/ml) (lanes 2 and 3) or IL-1 (10 ng/ml) (lanes 4 and 5) was incubated with OCIF (100 ng/ml) for 1 h at 37°C. Purified OCLs were treated with sODF or IL-1 in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of OCIF for 30 min. NF-{kappa}B activity in the nuclear extracts was determined by an EMSA, and the amount of I{kappa}B{alpha} was determined by immunoblotting, as described under Materials and Methods. D, Translocation of RelA (p65) into the nuclei of OCLs in response to sODF. Purified OCLs were treated with sODF (100 ng/ml) for 30 min. Cells were then fixed and incubated with Abs against RelA (p65), followed by FITC-conjugated anti-rabbit immunoglobulins. The subcellular localization of FITC-labeled RelA (P65) was observed by fluorescence microscopy.

 
JNK is a signal transducer commonly activated by TNF-related ligands (22). TRANCE (ODF) has been shown to activate JNK in T cells and in bone marrow-derived dendritic cells (11). We examined whether sODF activates JNK in mature OCLs. JNK was rapidly activated in OCLs after the stimulation with sODF, in a dose-dependent manner (Fig. 3Go, A and B). The activation of JNK was also induced in OCLs treated with 10 ng/ml of IL-1 (Fig. 3GoC). OCIF blocked the JNK activation induced by sODF but not that induced by IL-1 (Fig. 3Go, B and C).



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  		FIGURE 3. sODF activates JNK in purified OCLs. A, Purified OCLs were treated with increasing concentrations of sODF for 30 min. B, Purified OCLs were treated with sODF (100 ng/ml) for the indicated periods in the absence (lanes 1–4) or presence (lanes 5–8) of OCIF (100 ng/ml). C, Purified OCLs were treated with IL-1 in the presence (lane 4) or absence (lane 3) of OCIF for 30 min. JNK activities were measured using a SAPK/JNK assay kit as described under Materials and Methods.

 
We reported that the activation of NF-{kappa}B is involved in the survival of OCLs promoted by IL-1 (6). Like IL-1, sODF prolonged the survival of OCLs in a dose-dependent manner (Fig. 4GoA). Addition of OCIF with sODF prevented the sODF-induced survival of OCLs. To the contrary, OCIF could not prevent the IL-1- or M-CSF-induced survival of OCLs. OCIF did not accelerate spontaneous cell death of OCLs (Fig. 4GoA). TRANCE (ODF) has been shown to induce the survival of dendritic cells, which was mediated by up-regulation of Bcl-xL (23). To determine whether sODF induces Bcl-2 or Bcl-xL, we measured their expression in OCLs treated with sODF. A Western blot analysis showed that the expression of Bcl-2 and Bcl-xL in OCLs was not affected by sODF (Fig. 4GoB).



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  		FIGURE 4. sODF prolongs the survival of OCLs. A, Purified OCLs were incubated for 24 h with sODF (100 ng/ml, 1 µg/ml), IL-1 (10 ng/ml), or M-CSF (100 ng/ml) in the presence (hatched columns) or absence (open columns) of OCIF (100 ng/ml). TRAP-positive OCLs containing more than three nuclei were then counted after TRAP staining. Similar results were obtained from three independent experiments. *, Significantly different from cultures treated with ODF alone; p < 0.01. B, Western blot analysis of Bcl-2 and Bcl-xL in purified OCLs. Purified OCLs were cultured in the absence (lanes 2 and 4) or presence (lanes 3 and5) of sODF (100 ng) for 6 h (lanes 2 and 3) or 18 h (lanes 4 and 5). The murine leukemia cell line M1 (lane 6) was used as a positive control.

 
Wesolowski et al. (16) developed a method for obtaining highly purified (>90%) TRAP-positive mononuclear or binuclear pOCs released by the "disintegrin" echistatin from cocultures of murine osteoblastic cells (MB1.8 cells) with murine bone marrow cells. We reported that IL-1 and M-CSF both prolonged the survival and induced the multinucleation of pOCs in the absence of osteoblasts/stromal cells (7). A small number of TRAP-positive MNCs (pOC-derived OCLs) were formed within 2–5 h in the control cultures (7), but they disappeared by 18 h of the incubation period (Fig. 5GoA). To determine whether the effects of sODF, IL-1, and M-CSF on the survival and multinucleation of pOCs are mediated through their respective receptors, OCIF, IL-1ra, and Ab against c-Fms (M-CSF receptor) were added to pOC cultures (Fig. 5GoB). IL-1ra inhibited only the IL-1-induced multinucleation of pOCs, and anti-c-Fms Ab inhibited only M-CSF-induced multinucleation of pOCs (Fig. 5GoB). The survival and multinucleation of pOCs induced by sODF were inhibited by adding OCIF but not by adding IL-1ra or anti-c-Fms Ab (Fig. 5GoB). The formation of ringed structures of F-actin dots (actin rings) in osteoclasts is closely related to the osteoclast function (24). Actin rings were distributed at the periphery of the sODF-induced OCLs with smooth contours (Fig. 5GoC). This suggests that the smooth contour of pOC-derived OCLs is somehow related to the sODF-induced pit-forming activity.



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  		FIGURE 5. sODF induces the multinucleation of pOCs. A, Purified pOCs were incubated with or without sODF (100 ng/ml) for 18 h. The cells were then stained for TRAP. B, Effects of IL-1ra, anti-c-Fms Ab, and OCIF on the IL-1-, M-CSF-, and sODF-induced multinucleation of pOCs. pOCs were pretreated for 30 min with or without IL-1ra (1 µg/ml), anti-c-Fms Ab (10 µg/ml), or OCIF (100 ng/ml) and further incubated for 18 h with IL-1 (10 ng/ml) (open columns), M-CSF (100 ng/ml) (hatched columns), or sODF (100 ng/ml) (closed columns). TRAP-positive MNCs with more than 10 nuclei were counted as pOC-derived OCLs. The results shown are the means ± SEM of three independent experiments. *, Significantly different from the control cultures; p < 0.001. C, Actin ring formation in pOC-derived OCLs induced by sODF. pOCs were incubated with sODF (100 ng/ml) for 18 h, and then cells were fixed and stained for TRAP. F-actin was detected with rhodamine-conjugated phalloidin. Bar = 100 µm.

 
pOCs were then cultured for 24 h on dentine slices in the presence or absence of sODF (Fig. 6Go). The number of pOC-derived OCLs remaining on the slices in the cultures treated with sODF was much greater than that in the control cultures (data not shown). Resorption pits on dentine slices were observed in the culture treated with sODF (Fig. 6GoA). The pit-forming activity of pOCs induced by sODF was inhibited by adding OCIF (Fig. 6GoB). M-CSF also prolonged the survival and induced the multinucleation of pOCs, but no resorption pits were formed in the cultures treated with M-CSF (7).



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  		FIGURE 6. sODF induces the pit-forming activity of pOCs. pOCs were plated on dentine slices for 2 h. The dentine slices were then transferred to 48-well plates and incubated for 24 h in the absence or presence of sODF (100 ng/ml) with or without OCIF (100 ng/ml). A, Resorption pits formed on dentine slices were stained with Mayer’s hematoxylin. B, The number of resorption pits was determined. The results shown are the means ± SEM of three independent experiments. *, Significantly different from the control cultures; p < 0.001. Bar = 100 µm.

 
We then examined the effect of interactions between ODF and RANK on the activation of NF-{kappa}B and JNK by adding sRANK to purified OCLs. Addition of sRANK significantly reduced the number of OCLs in a dose-dependent manner (Fig. 7Go). Proliferation of stromal cells in bone marrow cultures appeared to not be affected by the addition of sRANK or OCIF. Addition of sRANK as well as OCIF reduced the activity of NF-{kappa}B and JNK (Fig. 7GoB). Moreover, the sODF-mediated stimulation of the survival, multinucleation, and pit-forming activity of pOCs was also abrogated by the addition of sRANK (Table IGo).



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  		FIGURE 7. sRANK prevents both the sODF-induced OCL formation and activation of NF-{kappa}B and JNK in OCLs. A, Murine bone marrow cells were cultured with M-CSF (50 ng/ml) for 4 days in the absence or presence of OCIF (50 ng/ml) sRANK (50 ng/ml, 500 ng/ml) or sODF (50 ng/ml) in 96-well plates. The cells were then fixed and stained for TRAP, and TRAP-positive MNCs containing more than three nuclei were counted as OCLs. The results shown are the means ± SEM of three cultures. Significantly different from the culture treated with ODF and M-CSF: *, p < 0.001; **, p < 0.005. B, Effects of sRANK on the activation of NF-{kappa}B and JNK in OCLs treated with sODF. sODF (100 ng/ml) was preincubated with or without OCIF (50 ng/ml, 100 ng/ml) or sRANK (100 ng/ml, 1 µg/ml) for 1 h at 37°C. Purified OCLs were treated for 30 min with sODF in the absence (lanes 1, 4,and 5) or presence of OCIF (lanes 2, 6,and 7), or sRANK (lanes 3, 8, and 9). NF-{kappa}B activity in the nuclear extracts was determined by an EMSA, and JNK assay was performed using a SAPK/JNK assay kit as described under Materials and Methods.

 

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  		Table I. Effects of sRANK on the survival, multinucleation, and pit-forming activity of osteoclasts promoted by sODF1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously reported that both IL-1 and M-CSF induced the survival and multinucleation of OCLs/pOCs, but only IL-1 stimulated their pit-forming activity (7). The present study clearly showed that, like IL-1, sODF stimulated not only the survival and multinucleation of OCLs/pOCs but also their pit-forming activity. Our results are consistent with the findings by Fuller et al. (14), who demonstrated that TRANCE (ODF) is involved in the osteoclast activation induced by osteoblastic UMR 106 cells treated with parathyroid hormone. It was also shown that OPGL (ODF) administered into mice induced osteoclastic bone resorption without a significant increase in the number of osteoclasts (13). These results suggest that ODF is a factor essentially involved in not only osteoclast differentiation but also osteoclast activation.

The survival and multinucleation of pOCs induced by M-CSF, IL-1, and sODF were inhibited by anti-c-Fms (M-CSF receptor) Ab, IL-1ra (antagonist of IL-1 type 1 receptor), and OPG/OCIF (a decoy receptor of ODF), respectively (Fig. 5Go). OPG/OCIF specifically inhibited activation of NF-{kappa}B and JNK in OCLs induced by sODF but not that induced by IL-1 (Figs. 2Go and 3Go). These results suggest that M-CSF, IL-1, and ODF have similar effects on the survival and multinucleation of osteoclasts via respective receptors. A soluble form of TNFR has been shown to inhibit the activity of TNF-{alpha} and TNF-ß (25, 26, 27, 28). Here, sRANK inhibited the OCL formation in bone marrow cultures treated with sODF together with M-CSF and blocked the sODF-induced activation of NF-{kappa}B and JNK in OCLs (Fig. 7Go). Nakagawa et al. (21) recently reported that polyclonal Abs against the extracellular domains of RANK induced OCL formation in spleen cell cultures in the presence of M-CSF. This indicates that the clustering of RANK is required for the RANK-mediated signal transduction for osteoclastogenesis. In contrast, the Fab fragment of anti-RANK Abs completely inhibited ODF-mediated osteoclastogenesis (21). These results suggest that RANK is the sole receptor of ODF responsible for inducing differentiation and activation of osteoclasts.

We have reported that M-CSF is indispensable for both the proliferative phase and the differentiation phase of osteoclast development (29). ODF and M-CSF cannot be replaced by other cytokines in inducing osteoclast differentiation. The present study shows that M-CSF and IL-1, as well as ODF, prolonged the survival of OCLs and induced their fusion. ODF and IL-1 but not M-CSF induced the pit-forming activity of purified OCLs/pOCs in culture. These results suggest that ODF is the sole factor for inducing osteoclast differentiation, but factors other than ODF are also able to support the survival, fusion, and activation of mature osteoclasts. Fig. 8Go summarizes the role of cytokines examined in this study in the regulation of osteoclast differentiation and function.



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  		FIGURE 8. The differentiation pathway of osteoclast progenitors into functionally active osteoclasts and the cytokines required for each step of the pathway.

 
sODF activated NF-{kappa}B in OCLs, which coincided with the degradation of I{kappa}B{alpha} (Fig. 2GoB). Our immunocytochemical analysis revealed that RelA (p65) was translocated from the cytoplasm into almost all of the nuclei of OCLs within 30 min after sODF stimulation (Fig. 2GoD). This action of ODF was quite similar to that of IL-1 previously reported (6, 7). Wong et al. (11) reported that a soluble form of TRANCE (ODF) induced JNK activation in T cells but not in splenic B cells and bone marrow-derived dendritic cells. sODF as well as IL-1-activated JNK in OCLs (Fig. 3Go). In contrast, M-CSF activated JNK but not NF-{kappa}B in OCLs, though it supported the survival and fusion of OCLs/pOCs (our unpublished observation). These results suggest that the survival and fusion of osteoclasts are not sufficient for inducing osteoclast function.

TRANCE (ODF) has been shown to induce the survival of dendritic cells through the up-regulation of Bcl-xL (23). It was also reported that targeting of both Bcl-xL and SV40 large T Ag to cells of the osteoclast lineage immortalized osteoclast precursors (30). These results suggest that antiapoptotic proteins such as Bcl-xL and Bcl-2 are involved in the survival of osteoclasts. However, neither Bcl-2 nor Bcl-xL in OCLs was up-regulated by sODF in our culture condition. IL-1 failed to induce the expression of Bcl-2 and Bcl-xL in OCLs (31). This suggests that ODF supports the survival of osteoclasts through a mechanism different from the up-regulation of Bcl-2 and Bcl-xL.

The activation of NF-{kappa}B has been shown to increase cellular resistance to apoptosis (32, 33, 34, 35, 36). Using antisense oligodeoxynucleotides to NF-{kappa}B (RelA/p65 and p50) and proteasome inhibitors that inhibit the degradation of I{kappa}B, we have shown that the activation of NF-{kappa}B is involved in the survival of OCLs promoted by IL-1 (6). The activation of NF-{kappa}B appears to be also involved in the ODF-induced survival of OCLs/pOCs. The precise role of JNK in apoptosis is controversial. Strong activation of JNK was induced by apoptosis-inducing stresses such as UV and hydrogen peroxide (37, 38, 39). Using the knockout mice of the SEK1 gene, which encodes a direct upstream kinase of JNK, SEK1-induced signals were shown to play a protective role against various cytotoxic stimuli (40). TNF receptor-associated factor (TRAF) 2 is a signal-transducing protein of the TNF receptor family (39). The activation of JNK was impaired, but the activation of NF-{kappa}B was induced in thymocytes obtained from dominant negative (DN) TRAF2-transgenic mice and in embryonic fibroblasts obtained from TRAF2-deficient mice (41, 42). These thymocytes and fibroblasts were rather apoptotic in the presence of TNF-{alpha}. Furthermore, thymocytes from I{kappa}B{alpha} DN and TRAF2 DN double-transgenic mice were more sensitive to TNF-induced apoptosis than those from normal mice and I{kappa}B{alpha} DN- or TRAF2 DN-transgenic mice (43). Therefore, JNK-meditated signals appear to collaborate with NF-{kappa}B in inducing the antiapoptotic action induced by sODF.

We previously showed that OCLs expressed IL-1 type 1 receptors (6). Xu et al. (44) reported that intense signals for IL-1 type I receptor mRNA were detected in active osteoclasts in an adjuvant arthritis model in rats, whereas mRNA of IL-1 type II receptor, which serves as a decoy receptor, was expressed preferentially in resting osteoclasts. Bone histological studies of OPG/OCIF knockout mice revealed that physiological bone resorption was regulated mainly by ODF and OPG/OCIF (45). IL-1 appears to be involved in pathological bone resorption, such as that observed in rheumatoid arthritis and periodontal bone diseases.

Recent studies indicate that the cytoplasmic tail of RANK interacts with TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 (46, 47, 48, 49). Mapping of the structural requirements for TRAF/RANK interaction revealed that selective TRAF-binding sites clustered in two distinct domains of the RANK cytoplasmic tail. In particular, TRAF6 interacted with the membrane-proximal domain of the cytoplasmic tail distinct from binding sites for TRAF1, -2, -3, and -5. When the TRAF6 interaction domain was deleted, RANK-mediated NF-{kappa}B activation was completely inhibited, and JNK activation was partially inhibited (48). N-terminal truncation of TRAF6 (TRAF6 DN) also inhibited RANKL-induced NF-{kappa}B activation (48, 49). These results suggest that TRAF6 transduces a signal involved in RANK-mediated activation of osteoclast function.

Double knockout mice of p50 (NF-{kappa}B1) and p52 (NF-{kappa}B2), subunits of NF-{kappa}B, showed severe osteopetrosis because of the impaired osteoclast differentiation (50, 51). The osteopetrotic disorder was cured by normal bone marrow transplantation. These results indicate that osteoclast progenitors are impaired in the deficient mice. sODF activated NF-{kappa}B in the target cells including osteoclasts. This suggests that the ODF-induced activation of NF-{kappa}B in osteoclast progenitors also plays a crucial role in their differentiation into osteoclasts. Further studies are necessary to elucidate the molecular mechanism of the action of ODF in osteoclast differentiation and function.


   Acknowledgments
 
We thank Dr. Takeshi Yanai (Showa University School of Dentistry) for his technical assistance, and Dr. Hisataka Yasuda (Snow Bland Milk Products) for helpful discussion.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid 09771546 and 08557101 from the Ministry of Education, Science and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Tatsuo Suda, Department of Biochemistry, School of Dentistry, Showa University 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. E-mail address: Back

3 Abbreviations used in this paper: pOC, prefusion osteoclast-like cell; TRAP, tartrate-resistant acid phosphatase; ODF, osteoclast differentiation factor; OCL, osteoclast-like cell; OPG, osteoprotegerin; OCIF, osteoclastogenesis inhibitory factor; TRANCE, TNF-related activation-induced cytokine; RANK, receptor activator of NF-{kappa}B; L, ligand; sODF, soluble form of ODF; JNK, c-Jun N-terminal protein kinase; sRANK, soluble form of RANK; TRAF, TNF receptor-associated factor; DN, dominant negative; MNC, multinucleated cell; IL-1ra, IL-1 receptor antagonist; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; SEK1, SAPK-ERK kinase 1. Back

Received for publication January 20, 1999. Accepted for publication April 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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