/00/$03.00/0 Vol. 141, No. 9. Osteoprotegerin Produced by Osteoblasts Is an Important Regulator in Osteoclast Development and Function*

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1 /00/$03.00/0 Vol. 141, No. 9 Endocrinology Printed in U.S.A. Copyright 2000 by The Endocrine Society Osteoprotegerin Produced by Osteoblasts Is an Important Regulator in Osteoclast Development and Function* NOBUYUKI UDAGAWA, NAOYUKI TAKAHASHI, HISATAKA YASUDA, ATSUKO MIZUNO, KANAMI ITOH, YUTAKA UENO, TOSHIMASA SHINKI, MATTHEW T. GILLESPIE, T. JOHN MARTIN, KANJI HIGASHIO, AND TATSUO SUDA Department of Biochemistry (N.U., N.T., K.I., Y.U., T.S., T.S.), School of Dentistry, Showa University, Tokyo, ; Snow Brand Milk Products Company (H.Y., A.M., K.H.), Tochigi, , Japan; and St. Vincent s Institute of Medical Research (M.T.G., T.J.M.), Fitzroy, Victoria 3065, Australia WE SUCCEEDED in molecular cloning of osteoclast differentiation factor as a ligand for osteoprotegerin (OPG) (1). Osteoclast differentiation factor, a member of the tumor necrosis factor (TNF) ligand family, was identical to receptor activator of nuclear factor (NF)- B (RANK) ligand (RANKL) (2), TNF-related activation-induced cytokine (TRANCE) (3), and OPG ligand (4), which were independently identified by other investigators. RANKL induced osteoclast differentiation from mouse hemopoietic cells and human peripheral blood mononuclear cells in the presence of macrophage colony-stimulating factor (M-CSF) (5, 6). Neither osteoblasts/stromal cells nor osteotropic factors were necessary for osteoclast formation from spleen cells induced by RANKL and M-CSF (5, 6). In 1997, we purified osteoclastogenesis inhibitory factor Received February 22, Address all correspondence and requests for reprints to: Dr. Tatsuo Suda, Department of Biochemistry, School of Dentistry, Showa University, Hatanodai, Shinagawa-ku, Tokyo , Japan. suda@showa-u.ac.jp. * This work was supported, in part, by a grant-in-aid ( ) and the High-Technology Research Center Project from the Ministry of Education, Science, Sport and Culture of Japan. ABSTRACT Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor- B ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type ( / ) or heterozygous ( / ) mouse-derived osteoblasts with bone marrow cells prepared from homozygous ( / ) mice. In contrast, homozygous ( / ) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous ( / ) bone marrow cells, even in the absence of boneresorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous ( / ) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] to these cocultures significantly enhanced osteoclast differentiation. In addition, boneresorbing activity in organ cultures of fetal long bones derived from homozygous ( / ) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type ( / ) mice. Osteoblasts prepared from homozygous ( / ), heterozygous ( / ), and wild-type ( / ) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1,25(OH) 2 D 3. When homozygous ( / ) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1,25(OH) 2 D 3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form. (Endocrinology 141: , 2000) (OCIF) from conditioned medium of human embryonic fibroblasts, IMR-90, which had a capability to inhibit osteoclast formation in mouse cocultures of hemopoietic cells and osteoblasts/stromal cells (7). OCIF is a heparin-binding basic glycoprotein, which has been isolated as a monomer with an apparent M r of 60K and a disulfide-linked homodimer with an M r of 120K (7). Cloning of its complementary DNA revealed that OCIF is a TNF receptor family member (8, 9), identical to OPG (10) and TNF receptor-like molecule 1 (11, 12). Hepatic expression of OPG in transgenic mice resulted in osteopetrosis (10). OPG inhibited osteoclast formation by directly binding to a ligand for OPG expressed on osteoblasts/stromal cells (8). We previously reported that not only osteoclast progenitors but also mature osteoclasts strongly expressed messenger RNA (mrna) of RANK, a receptor for RANKL (13, 14). Like OPG, a soluble form of RANK inhibited osteoclast differentiation and function of mature osteoclasts (13, 14). In addition, polyclonal antibodies against the extracellular domain of RANK induced osteoclast formation in spleen cell cultures, in the presence of M-CSF, because of clustering of the surface receptors (14, 15). These findings indicate that RANK is the signaling receptor essential for RANKL-medi- 3478

2 OSTEOCLAST FORMATION IN OPG-DEFICIENT MICE 3479 ated osteoclastogenesis and that OPG acts as a decoy receptor for RANKL to compete against RANK. Physiological roles of OPG have been studied in OPGdeficient mice produced by targeted disruption of the gene (16, 17). OPG-deficient mice were viable and fertile, but they exhibited severe osteoporosis caused by enhanced osteoclast formation and function (16, 17). Destruction of growth plates and lack of trabecular bone, with an increase in the number of osteoclasts, were detected in long bones of adult OPGdeficient mice. The strength and mineral density of their bones were markedly reduced. These findings demonstrate that OPG is a key factor acting as a negative regulator against osteoclastogenesis and bone resorption in vivo. To elucidate the potential roles of OPG produced by osteoblasts in osteoclast differentiation and function, we examined osteoclast formation in a mouse coculture system and bone-resorbing activity in a long-bone organ culture system using the wildtype and OPG-deficient mice. The supporting activity of osteoblasts, to induce osteoclast formation in cocultures, was significantly increased when OPG-deficient mouse-derived osteoblasts were used. In addition, bone-resorption in organ cultures of fetal long bones obtained from OPG-deficient mice was markedly increased. Because RANKL is a type II transmembrane protein, this cytokine seems to support osteoclast formation as a membrane-associated factor. However, recent reports indicate another possibility: that RANKL also acts as a soluble form to induce osteoclast formation. Lum et al. (18) reported that RANKL was released form cells by shedding with a TNF converting enzyme (TACE). Kong et al. (19) also showed that activated murine T lymphocytes secreted an active form of RANKL into culture medium. These findings indicate the possibility that RANKL produced by osteoblasts/stromal cells may be neutralized by OPG simultaneously produced by osteoblasts/stromal cells. Using OPG-deficient mice, we show here that RANKL expressed by osteoblasts functions as a membrane-associated form. Materials and Methods Preparation of OPG-deficient mice OPG-deficient mice with the genetic background of a mixture of CD-1 and C57BL/6 were generated by targeted disruption of the gene, as described previously (16). After heterozygous ( / ) matings, homozygous ( / ), heterozygous ( / ), and wild-type ( / ) mice were identified by PCR analysis of tail DNA from each mouse, as previously described (16). All procedures for animal care were approved by the Showa University Animal Management Committee. Drugs Bacterial collagenase and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Recombinant human M-CSF (Leukoprol) was obtained from Yoshitomi Pharmaceutical Company (Osaka, Japan). Human PTH (1 34) was obtained from Peptide Institute (Osaka, Japan). Recombinant human OPG and soluble form of mouse RANKL (srankl) were prepared as described previously (1, 8). Type I collagen gel solution (Cell matrix Type-IA) was obtained from Nitta Gelatin, Inc. (Osaka, Japan). 125 I-labeled human calcitonin and 45 CaCl2 were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Other chemicals and reagents used were of analytical grade. Mouse coculture system for osteoclastogenesis To isolate primary osteoblasts from either OPG-deficient or wild-type mice, calvaria from 2-day-old mice were cut into small pieces and cultured for 5 days in collagen-gel prepared in -MEM containing 10% FBS (JRH Biosciences, Lenexa, KS) (20 22). Osteoblasts grown from the calvarium were collected by treatment of collagen gel cultures with collagenase, and they were stored at 80 C until use. Osteoblasts (10 4 cells/well) prepared from each mouse were coculture, for 7 days, with bone marrow cells ( cells/well) obtained from OPG-deficient mice, in a 48-well plate with 0.3 ml of -MEM containing 10% FBS, in the presence or absence of 10 8 m 1,25(OH) 2 D 3. In some experiments, bone marrow cells ( cells) prepared from homozygous ( / ) and wild-type ( / ) adult mice (11-week-old males) were cultured in the presence of srankl (100 ng/ml) and M-CSF (50 ng/ml) for 5 days (1). The culture medium was changed every 3 days. Adherent cells were then fixed with 10% formaldehyde in PBS, treated with ethanol-acetone (50:50), and stained for tartrate-resistant acid phosphatase (TRAP), as described previously (23). TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts. Expression of calcitonin receptors was assessed by autoradiography using 125 I-labeled human calcitonin (23). Fetal long-bone organ culture system Bone-resorbing activity was measured using a modification of Raisz s organ culture method (24). In short, pregnant OPG heterozygous ( / ) mice were injected sc with 25 Ci of 45 Ca on day 16 of gestation. Twenty-four hours after injection, shafts of radii and ulnae were dissected from fetuses, cleaned free of surrounding muscle and fibrous tissues, and precultured in serum- or BSA-free BGJb medium (Life Technologies, Grand Island, NY). After preincubation for 48 h, the bones were transferred into 0.5 ml BGJb medium containing 0.2% BSA and incubated for 72 h in the presence or absence of test materials. Boneresorbing activity was expressed as the percent release of 45 Ca from prelabeled bones, as described previously (24). PCR amplification of reverse-transcribed mrna For semiquantitative RT-PCR analysis, total cellular RNA was extracted from calvarial osteoblasts treated, with or without 10 8 m 1,25(OH) 2 D 3, for 48 h using Trizol solution (Life Technologies). Firststrand complementary DNA was synthesized from total RNA with random primers and was subjected to PCR amplification with EX Taq polymerase (Takara Biochemicals, Shiga, Japan) using specific PCR primers: mouse RANKL, 5 -CGCTCTGTTCCTGTACTTTCGAGCG-3 (forward, nucleotides ) and 5 -TCGTGCTCCCTCCTTTCAT- CAGGTT-3 (reverse, nucleotides ); mouse glyceraldehyde-3- phosphate dehydrogenase (GAPDH), 5 -ACCACAGTCCATGCCAT- CAC-3 (forward, nucleotides ) and 5 -TCCACCACC-CTGT- TGCTGTA-3 (reverse, nucleotides ). Preliminary experiments were performed to ensure that the number of PCR cycles was within the exponential phase of the amplification curve. PCR products were resolved on a 2% agarose gel, and the reaction was confirmed by Southern blot transfer onto nylon membranes (Hybond-N; Amersham Pharmacia Biotech) and hybridization with 32 P-labeled internal sense oligonucleotide probes; mouse RANKL, 5 -GAGCCTCAGGCTTGCCCCGC- CGGGCCACATCGA-GCCACGAACCTTCCATCATAGCT GGAA-3 (forward, nucleotides ); mouse GAPDH, 5 -GGGGCAGCCCA- GAACATCATCCCTGCATCCACTGGTGCTGCCAAGGC-TGTGGGC- AAGGTC-3 (forward, nucleotides ). Statistical analysis The data were analyzed by one-factor ANOVA and Student s t test (Stat View; Abacus Concepts Inc., Berkeley, CA). The Student s t test was performed when the ANOVA test indicated significance at the P level. All values were represented as the means sem of quadruplicate cultures. Results Osteoblasts were prepared from each calvarium of OPGdeficient homozygous ( / ) (n 10), heterozygous ( / )

3 3480 OSTEOCLAST FORMATION IN OPG-DEFICIENT MICE Endo 2000 Vol 141 No 9 (n 21), and wild-type ( / ) (n 9) mice. To explore the precise roles of OPG in osteoclast formation, these calvarial osteoblasts were cocultured with bone marrow cells derived from OPG-deficient mice. No TRAP-positive osteoclasts were formed in cocultures of bone marrow cells from OPGdeficient homozygous ( / ) mouse and osteoblasts prepared from wild-type ( / ) or heterozygous ( / ) mouse, when no bone-resorbing factors were added (Fig. 1; A-a, A-b, and B). In contrast, homozygous ( / ) mouse-derived osteoblasts strongly supported formation of TRAP-positive mononuclear and multinucleated cells, which appeared as dark red cells in the coculture, even in the absence of boneresorbing factors (Fig. 1, A-c and B). An autoradiographic study, using labeled calcitonin, revealed that TRAP-positive cells formed in these cocultures possessed calcitonin receptors (data not shown). Addition of OPG (10 ng/ml) to the cocultures of osteoblasts and bone marrow cells derived from homozygous ( / ) mouse completely inhibited the spontaneously occurring differentiation of TRAP-positive osteoclasts (Fig. 1A-d). Treatment of these cocultures with 1,25(OH) 2 D 3 (10 8 m) significantly enhanced osteoclast formation in all cocultures (Fig. 1B). We next examined whether bone-resorbing activity was also spontaneously enhanced in OPG-deficient bone organ cultures. Fetal long bones were prepared from OPG-deficient homozygous ( / ), heterozygous ( / ), and wild-type ( / ) mice; and bone-resorbing activity was assessed in the presence and absence of PTH (10 6 m). Spontaneous release of 45 Ca from homozygous ( / ) bone was significantly increased, in comparison with that from wild-type ( / ) bone (Fig. 2). In addition, PTH (10 6 m) markedly stimulated 45 Ca release from homozygous ( / ) bone, which was much higher than that from wild-type ( / ) bone treated with PTH (Fig. 2). Similarly, bone-resorbing activity induced by 1,25(OH) 2 D 3 (10 8 m) in homozygous ( / ) (n 2) mice was significantly higher than that in wild-type ( / ) mice (n 4) ( % vs %, P 0.01). OPG (1000 ng/ml) almost completely inhibited 45 Ca release induced by PTH (10 6 m) in organ cultures of OPG-deficient bone, as well as in heterozygous ( / ) and wild-type ( / ) bone (Fig. 2). Expression of RANKL mrna in osteoblasts of OPGdeficient ( / ) mice was compared with that of heterozygous ( / ) and wild-type ( / ) mice. Osteoblasts prepared from homozygous ( / ), heterozygous ( / ), and wild-type ( / ) mice constitutively expressed similar levels of RANKL mrna; and RANKL mrna expression in each mouse genotype was equally elevated by the treatment with 1,25(OH) 2 D 3 for 3 days (Fig. 3). To examine whether stimulation of osteoclast formation in OPG-deficient mice was attributable to the increment of osteoclast progenitors, bone marrow cells were prepared from homozygous ( / ) and wild-type ( / ) mice and were cultured for 7 days in the presence of srankl (100 ng/ml) and M-CSF (50 ng/ml). Bone marrow cells obtained from homozygous ( / ) or wild-type ( / ) mice differentiated into osteoclasts similarly, in response to srankl and M-CSF [homozygous ( / ), ; wild-type ( / ), , n 3]. This indicates that there are similar numbers of FIG. 1. TRAP-positive osteoclast formation in cocultures of OPG-deficient mouse-derived osteoblasts and bone marrow cells. A, Calvarial osteoblasts (10 4 cells/well) prepared from wild-type ( / ) (a), heterozygous ( / ) (b), and OPG-deficient homozygous ( / ) (c and d) mice were cocultured with bone marrow cells ( cells/well) obtained from homozygous ( / ) mice in a 48-well plate with 0.3 ml/well of -MEM containing 10% FBS. Cocultures were performed in the presence (d) or absence (a, b, and c) of OPG (10 ng/ml) for 7 days. After culture for 7 days, cells were fixed and stained for TRAP. TRAP-positive cells appeared as dark red cells. Arrows in the cocultures of homozygous ( / ) mice indicate TRAP-positive multinucleated cells formed. Bars, 100 m. B, Calvarial osteoblasts (10 4 cells/well) prepared from wild-type ( / )(n 9), heterozygous ( ) (n 21) and OPG-deficient homozygous ( / ) (n 10) mice were cocultured with bone marrow cells ( cells/well) obtained from homozygous ( / ) mice in a 48-well plate in the presence or absence of 10 8 M 1,25(OH) 2 D 3. After culturing for 7 days, cells were fixed and stained for TRAP. The number of TRAP-positive osteoclasts was scored. Values were expressed as the means SEM of quadruplicate cultures. Experiments were repeated five times, with similar results. *, Significantly different from the cultures with wild-type ( / ) mice (P 0.01).

4 OSTEOCLAST FORMATION IN OPG-DEFICIENT MICE 3481 TRAP-positive osteoclasts were formed exclusively inside the colony of osteoblasts (Fig. 4, A and B). Addition of srankl (100 ng/ml) and M-CSF (50 ng/ml) to the spot coculture stimulated osteoclast formation both inside and outside the colony (Fig. 4C). These findings suggest that membrane- or matrix-associated RANKL is important for osteoclast differentiation induced by osteoblasts. FIG. 2. Bone-resorbing activity in organ cultures of mouse fetal long bone. Pregnant OPG heterozygous ( / ) mice were injected sc with 25 Ci of 45 Ca on day 16 of gestation. The following day, shafts of the radii and ulnae were dissected from fetuses, cleaned free of surrounding muscle and fibrous tissue. After preincubation for 48 h, long bones prepared from wild-type ( / ) (n 5), heterozygous ( / ) (n 8), and OPG-deficient homozygous ( / ) (n 5) mice were cultured in the absence or presence of PTH (10 6 M) or OPG (1000 ng/ml) for 72 h. Bone-resorbing activity was expressed as the percent release of 45 Ca from prelabeled bones. Values are expressed as means SEM of five cultures. *, Significantly different from the cultures with wild-type ( / ) mice (P 0.01). FIG. 3. Expression of RANKL mrna by calvarial osteoblasts. Calvarial osteoblasts were prepared from wild-type ( / ), heterozygous ( / ), and OPG-deficient homozygous ( / ) mice. Total cellular RNA was extracted from calvarial osteoblasts treated with or without 10 8 M 1,25(OH) 2 D 3 for 3 days. Total cellular RNA was reverse transcribed and amplified by 28 cycles of PCR for mouse RANKL mrna and 20 cycles for mouse GAPDH mrna using the specific primers described in Materials and Methods. PCR products were transferred to a nylon membrane and hybridized with 32 P-labeled internal sense oligonucleotides specific for mouse RANKL or GAPDH sequences. osteoclast progenitors in bone marrow cavities of homozygous ( / ) and wild-type ( / ) mice. Recent studies have shown that RANKL may act as a soluble factor for inducing osteoclast formation in vitro. To address this issue, we examined whether osteoblasts produce a soluble form of RANKL, using OPG-deficient mice because the inhibitory effects elicited by OPG are eliminated. Osteoblasts obtained from homozygous ( / ) mice were spot-cultured for 2 h in the left side of a single culture dish, then mouse bone marrow cells also obtained from homozygous ( / ) mice were uniformly plated over the culture dish. When the spot coculture was treated with 1,25(OH) 2 D 3 (10 8 m) and M-CSF (50 ng/ml) for 7 days, Discussion RANKL and M-CSF are two essential factors responsible for inducing osteoclast differentiation (5, 6). This is supported by the finding that the targeted disruption of the gene encoding either RANKL (25) or RANK (26, 27) similarly leads to severe osteopetrosis with a complete absence of TRAPpositive cells in the bone tissue in mice. Another osteoclastdeficient osteopetrotic model, op/op mice, cannot produce functionally active M-CSF because of a frame shift-type mutation in the coding region of the M-CSF gene (28). OPG is a soluble receptor for RANKL that inhibits osteoclast differentiation in vitro as a decoy receptor (13 15). Recombinant OPG acts on bone tissues and increases bone mineral density and bone volume associated with a decease in active osteoclast numbers in normal rats (8). Injection of OPG into hypercalcemic tumor-bearing nude mice reduced serum Ca concentration (29). Simonet et al. (10) reported that hepatic expression of OPG in transgenic mice resulted in osteopetrosis caused by the failure of osteoclast formation. Two independent research groups, including ours (16, 17), showed that OPG knockout mice developed severe osteoporosis caused by enhanced osteoclast formation and function with age. These experimental findings suggest that OPG is a key factor acting as a negative regulator against osteoclastic bone resorption in vivo. OPG has been shown to be ubiquitously expressed in many tissues (8, 10, 12). It was also reported that the serum concentration of OPG is increased in postmenopausal women who have a high rate of bone turnover (30). In the present study, the lack of OPG production by osteoblasts stimulated osteoclast formation in cocultures of osteoblasts and bone marrow cells. Bone-resorbing activity was also elevated in OPG-deficient long bones in the presence and absence of bone-resorbing factors. The enhanced osteoclastogenesis and bone resorption in vitro in OPG-deficient mice were completely inhibited by adding OPG. The OPG deficiency did not affect the levels of RANKL mrna, which was constitutively expressed by osteoblasts from wild-type ( / ), heterozygous ( / ), and OPG-deficient ( / ) mice at an equivalent level. Furthermore, RANKL mrna levels in osteoblasts were similarly up-regulated by the treatment with 1,25(OH) 2 D 3 in each of the three OPG genetic backgrounds. Consistent with the present findings, anti-opg antibody enhanced osteoclast formation induced by 1,25(OH) 2 D 3 in mouse bone marrow cultures (12). These findings indicate that, besides RANKL and M-CSF, OPG produced by osteoblasts is also a physiologically important regulator of osteoclast formation and function of mature osteoclasts. OPG gene expression in osteoblasts/stromal cells is downregulated by 1,25(OH) 2 D 3 and dexamethasone (8, 31), and

5 3482 OSTEOCLAST FORMATION IN OPG-DEFICIENT MICE Endo 2000 Vol 141 No 9 FIG. 4. Cell-to-cell contact between osteoclast progenitors and osteoblasts is essential for osteoclast formation. OPGdeficient homozygous ( / ) mousederived calvarial osteoblasts (10 4 cells/ 0.05 ml) were spot-cultured for 2 h in -MEM containing 10% FBS, in the left side of a single culture well of 12-well plates. Mouse bone marrow cells ( cells) obtained from OPG-deficient homozygous ( / ) mice were then uniformly plated over the culture well. The spot coculture was treated for 6 days in the presence of 1,25(OH) 2 D 3 (10 8 M) (A, B) or srankl (100 ng/ml) and M- CSF (50 ng/ml) (C). B, Left and right panels show the portions inside and outside the colony of calvarial osteoblasts (A), respectively. Cells were fixed and stained for TRAP and Carrazi s Hematoxylin. Bar, 100 m.

6 OSTEOCLAST FORMATION IN OPG-DEFICIENT MICE 3483 the combination of these agents is known to support osteoclastogenesis in the coculture system. In contrast, transforming growth factor- inhibits osteoclast formation in mouse cocultures, and this factor induces OPG production by osteoblasts or bone marrow stromal cells (ST2) (32, 33). These observations raise the possibility that osteoclast differentiation is critically regulated by OPG, which is produced as a local factor by osteoblasts/stromal cells in response to osteotropic factors or cytokines. Additionally, we reported that the concentration of OPG in synovial fluids was significantly lower in patients with rheumatoid arthritis than patients with gout (34), suggesting a potential role of OPG in the protection from accelerated osteoclastic bone resorption. Recently, Kong et al. (19) and our group (35) independently reported that activated T cells supported osteoclast differentiation via RANKL-RANK interaction. Kong et al. (19) also reported that, unlike osteoblasts/stromal cells, activated T lymphocytes seemed to secrete a soluble form of RANKL into culture medium, which induced osteoclastogenesis in vitro. This suggests that there is a difference between osteoblasts/ stromal cells and T lymphocytes in the mode of RANKL production. Osteoblasts/stromal cells express RANKL as a membrane-bound or matrix-associated form to promote differentiation of osteoclast progenitors into osteoclasts through a mechanism involving cell-to-cell contact. Elaboration of a soluble form of RANKL may be the result of processing the mature protein by TACE or a TACE-like enzyme (18). Furthermore, it has been reported that osteoclasts are formed when hemopoietic cells are cocultured with activated T cells fixed with paraformaldehyde (19). This indicates that membrane-bound RANKL is also important for the activated T- cell-mediated bone destruction. Under physiological conditions, osteoclast formation requires cell-to-cell contact with osteoblasts/stromal cells, which generate RANKL as a membrane-bound factor in response to several bone resorbing factors. In normal bone remodeling, osteoblastic bone formation always occurs as a programmed manner accurately and quantitatively just after osteoclastic bone resorption. In contrast, in pathological bone resorption, as in rheumatoid arthritis, T cells seem to secrete a soluble form of RANKL, which acts directly on osteoclast progenitors without cell-to-cell contact. Thus, it is attractive to consider that cell-to-cell contact between osteoclast progenitors and osteoblasts/stromal cells may leave some memory for bone formation in osteoblasts/stromal cells. This possibility is currently being explored in our laboratory. Acknowledgments We thank Dr. S. Kotake (The Institute of Rheumatology, Tokyo Women s Medical University) and Dr. N. 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