[Frontiers in Bioscience 16, 21-30, January 1, 2011]
Regulatory Mechanism of Osteoclastogenesis by RANKL and Wnt Signals
Naoyuki Takahashi1, Kazuhiro Maeda1,4, Akihiro Ishihara1,2, Shunsuke Uehara3, Yasuhiro Kobayashi1
1
TABLE OF CONTENTS
1. ABSTRACT
Osteoclasts develop from monocyte-macrophage lineage cells under the regulation of osteoblasts. Osteoblasts express two cytokines essential for osteoclastogenesis, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-B ligand (RANKL). Osteoblasts also produce osteoprotegerin (OPG), a decoy receptor for RANKL, which inhibits the interaction between RANKL and RANK, a receptor of RANKL. Bone resorption-stimulating factors act on osteoblasts to regulate RANKL and OPG expression. Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) is a master transcription factor for osteoclast differentiation. The immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal was discovered as a co-stimulatory signal in RANKL-induced osteoclastogenesis. Wnt proteins activate two pathways: -catenin-dependent canonical and -catenin-independent noncanonical pathways. Wnt proteins promote differentiation of osteoblasts through the canonical pathway. The canonical pathway in osteoblasts also suppresses osteoclastogenesis through up-regulation of OPG expression and down-regulation of RANKL expression. In contrast, activation of the noncanonical pathway in osteoclast precursors enhances RANKL-induced osteoclastic differentiation. Thus, Wnt signals in osteoblasts and osteoclast precursors play important roles in osteoclastogenesis. This review summarizes the regulatory mechanism of osteoclastogenesis by RANKL and Wnt signals.
2. INTRODUCTION
Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption, respectively. Osteoblasts produce bone matrix proteins and lead mineralization of the tissue. Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte-macrophage lineage (1-3). We developed a mouse coculture system of osteoblasts/stromal cells and hematopoietic cells in which osteoclasts are formed in response to bone resorption-stimulating factors such as 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH), prostaglandin E2 (PGE2), and interleukin 11 (IL-11) (1,4). A series of experiments using this coculture system established the concept that osteoblasts/stromal cells are closely involved in osteoclast development (5).
Advances in bone cell biology have elucidated the precise mechanism by which osteoblasts regulate osteoclast differentiation and function. Osteoblasts express two cytokines, macrophage colony-stimulating factor (M-CSF, also called CSF-1) and receptor activator of nuclear factor B ligand (RANKL), essential for osteoclast differentiation (1,6). Bone resorption-stimulating factors all stimulate RANKL expression in osteoblasts/stromal cells. The identification of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis, provided major insights into the molecular mechanism of osteoclast differentiation (7). The discovery of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signals as a co-stimulatory signal in RANKL-induced osteoclastogenesis has confirmed that osteoblasts play another important role in osteoclastogenesis (7).
Wnt proteins (Wnts) are a family of 19 glycoproteins (8, 9). Wnt binds to two distinct receptor complexes: a complex of Frizzled and low density lipoprotein receptor-related protein 5/6 (LRP5/6) and another complex of Frizzled and receptor tyrosine kinase orphan receptors (Rors). The binding of Wnts to the receptors activates two classes of signalling pathways: a -catenin-dependent canonical pathway and a - catenin-independent noncanonical pathway. The importance of the canonical pathway in bone biology has been emphasized by the identification of a link between bone mass and mutations in the LRP5 gene (10). Loss-of-function mutations in LRP5 reduce the number of osteoblasts and cause osteoporosis.
Recent findings have shown that the -catenin-dependent canonical pathway in osteoblasts/stromal cells suppresses osteoclastogenesis through the up-regulation of osteoprotegerin (OPG) expression and the down-regulation of RANKL expression (11,12). In addition, the activation of the noncanonical pathway in osteoclast precursors enhances RANKL-induced osteoclastic differentiation, in a cell-autonomous manner (13). These results suggest that Wnts play important roles not only in bone formation, but also in bone resorption. In this review, we summarize the regulatory mechanism of osteoclastogenesis by RANKL and Wnt signals. The word "osteoblasts" is used as a generic name for osteoblast lineage cells, including bone marrow stromal cells, in this article.
3. REGULATION OF OSTEOCLAST DIFFERENTIATION AND FUNCTION
3.1. M-CSF
Experiments with an osteopetrotic op/op mouse model have established that an osteoblast product, M-CSF, is crucial for osteoclast formation (14) (Figure 1). The M-CSF gene of op/op mice cannot functionally code active M-CSF protein due to an extra thymidine insertion in the coding region of the M-CSF gene. Administration of recombinant human M-CSF restored impaired bone resorption in op/op mice (15). Calvarial osteoblasts obtained from op/op mice failed to support osteoclast development in cocultures with normal spleen cells, but the addition of M-CSF to cocultures induced osteoclast formation in response to 1,25(OH)2D3 (16). These findings indicate that M-CSF produced by osteoblasts plays an essential role in osteoclast development. M-CSF is involved in not only proliferation of osteoclast progenitors of the monocyte/macrophage lineage, but also their differentiation into osteoclasts. Osteoblasts constitutively express M-CSF (17) (Figure 1).
3.2. RANKL
RANKL is a member of the tumor necrosis factor (TNF) family (TNF superfamily member 11, TNFSF11), which is also identified as "osteoclast differentiation factor" (18-21) (Figure 1). In contrast to M-CSF, the expression of RANKL by osteoblasts is inducible. Osteoblasts express RANKL as a membrane-associated form in response to stimuli of bone resorption-stimulating hormones and factors such as 1,25(OH)2D3, PTH, PGE2, and IL-11 (1,6,18). Osteoclast precursors express c-Fms (M-CSF receptors) and RANK (TNF receptor superfamily member 11A, TNFRSF11A). Osteoclast precursors recognize RANKL through cell-to-cell interactions with osteoblasts and differentiate into osteoclasts in the presence of M-CSF. RANKL expressed by osteoblasts also stimulates the survival and bone-resorbing activity of osteoclasts. Thus, RANKL is a cytokine responsible for the whole life cycle of osteoclasts. Osteoblasts also produce OPG (TNFRSF11B), a soluble decoy receptor for RANKL, which inhibits osteoclastogenesis by blocking RANKL-RANK interaction (1, 6, 22, 23). OPG production by osteoblasts is also regulated by osteotropic factors. Most bone resorption-stimulating hormones and cytokines down-regulate the expression of OPG in osteoblasts (1, 6). Both RANKL-deficient (RANKL-/-) mice (24) and RANK-/- mice (25) developed severe osteopetrosis with no osteoclasts in bone tissues. In contrast, OPG -/- mice exhibited severe trabecular and cortical bone porosity with enhanced osteoclastic bone resorption (26, 27). Administration of OPG or blocking antibodies against RANKL to normal mice strongly suppressed bone-resorbing activity of osteoclasts, as well as osteoclast differentiation (22, 28) (Figure 1). Taken together, these findings confirm the notion that RANKL expressed by osteoblasts also plays an essential role in osteoclast differentiation and function in humans.
3.3. DC-STAMP
Multinucleated osteoclasts are formed by cell-cell fusion of mononuclear preosteoclasts. Recent studies have shown that the dendritic cell-specific transmembrane protein (DC-STAMP), a seven transmembrane protein, is responsible for the cell-cell fusion of osteoclasts (29, 30). DC-STAMP expression in osteoclast precursors was up-regulated during differentiation into osteoclasts. No multinucleated osteoclasts were observed in DC-STAMP knockout (DC-STAMP -/-) mice, but many mononuclear preosteoclasts expressing osteoclast-specific markers were detected in those bone tissues. Bone-resorbing activity was considerably lower in DC-STAMP-/- osteoclasts than in wild-type osteoclasts (30). DC-STAMP-/- mice developed mild osteopetrosis. These results suggest that DC-STAMP is an essential molecule for osteoclast fusion, and multinucleated osteoclasts have higher bone-resorbing activity than mononuclear preosteoclasts. Thus, osteoclasts are specifically differentiated multinucleated cells specialized for bone resorption.
4. SIGNAL TRANSDUCTION IN osteoclastogenesis
4.1. NFATc1
The cytoplasmic tail of RANK interacts with TNF receptor-associated factor 1 (TRAF1), TRAF2, TRAF3, TRAF5 and TRAF6 (31, 32). Of these, TRAF6-mediated signals appear to be important for osteoclast differentiation and function because TRAF6 -/- mice develop osteopetrosis (33, 34). TRAF6-mediated signals in osteoclast precursors enhance activation of nuclear factor-B (NF-B) and c-Jun N-terminal kinase (JNK), and induction of c-Fos. Mice deficient in both the p50 and p52 subunits of NF-B (35, 36) and those lacking c-Fos (37) develop osteopetrosis due to an early block of differentiation in the osteoclast lineage. Using mice lacking JNK1 or JNK2, David et al. (38) showed that JNK1 activation essentially modulate osteoclastogenesis. Ikeda et al. (39) also reported that RANK-mediated signaling on osteoclasts was inhibited by overexpression of dominant-negative JNK1. These results suggest that JNK1 plays a role in osteoclastogenesis.
RAW264.7, a transformed mouse myeloid cell line, differentiates into osteoclasts in response to RANKL. Ishida et al. (40) reported that another transcription factor, NFATc1 (also called NFAT2), was specifically induced in RAW264.7 cells by treatment with RANKL. The translocation of NFATc1 into the nucleus is an essential step in autoamplification, followed by exerting its function. Calcineurin is a Ca2+-dependent phosphatase which is activated by calcium-calmodulin signalling. The calcineurin inhibitor cyclosporine A inhibited NFATc1 activity and suppressed osteoclast differentiation. Antisense oligonucleotides to NFATc1 also suppressed osteoclast differentiation. Using a cDNA microarray, Takayanagi et al. (41) independently identified NFATc1 as a master transcription factor for osteoclast differentiation. RANKL evoked Ca2+ oscillations that led them to calcineurin-mediated activation of NFATc1 (7, 41).
4.2. Co-stimulatory ITAM signals
ITAM is an important signalling component for a number of receptors in the immune system, including T-cell, B-cell, NK-cell, and Fc receptors (42, 43). Kim et al. (44) succeeded in the molecular cloning of a new member of the leukocyte receptor complex (LRC) family specifically expressed by osteoclasts, and named it "the osteoclast-associated receptor (OSCAR)" (Figure 2). Genes in the LRC family produce immunoglobulin-like surface receptors and play critical roles in the regulation of both innate and adaptive immune responses (45). OSCAR is detected specifically in murine mononuclear preosteoclasts or multinucleated osteoclasts (44). The putative ligand of OSCAR was shown to be expressed by osteoblasts. Addition of a soluble form of OSCAR in cocultures with osteoblasts inhibited the formation of osteoclasts from bone marrow precursor cells in the presence of bone-resorbing factors (44). These results suggest that OSCAR may be an important bone-specific regulator of osteoclast differentiation. However, the physiological role of OSCAR in osteoclastogenesis remains to be elucidated.
Intracellular calcium oscillation is an important stimulus for NFATc1 activation. Koga et al. (46) showed that molecules containing ITAM, such as DNAX-activating protein 12 (DAP12) and Fc receptor common chain (FcR), facilitate the calcium-mobilizing mechanism during osteoclastogenesis (Figure 2). FcR and DAP12 are adaptor molecules that associate with immunoglobulin-like receptors such as OSCAR, triggering receptors expressed on myeloid cells 2 (TREM2), signal-regulatory protein 1 (SIRP1) and paired immunoglobulin-like receptor A (PIR-A). OSCAR and PIR-A use FcR, while TREM2 and SIRP1 associate with DAP12. Deficiency in both FcR and DAP12 caused osteopetrotic phenotypes in mice (46). ITAM-mediated signals cooperate with RANK to stimulate calcium oscillation through ITAM phosphorylation and the resulting activation of spleen tyrosine kinase (SYK) and phospholipase C (PLC). Recently, it was shown that RANK and ITAM signalling induced the formation of Tec family non-receptor tyrosine kinases (Tec/Bruton's tyrosine kinase (Btk)) and B cell linker protein (BLNK), and resulted in PLC-mediated activation, an essential calcium signal for osteoclastogenesis (47). Because this pathway is crucial for the robust induction of NFATc1 that leads to osteoclastogenesis, these signals are known as co-stimulatory signals for RANK in osteoclastogenesis. Although the ligands for of these receptors will have to be identified in the future, osteoblasts have been proposed to express molecules which stimulate ITAM signals. Recent studies have shown that the system of semaphoplin 6D and its receptor plexin A1 has important roles in osteoclast differentiation through activation of DAP12-mediated ITAM signals (48). This series of experiments has established a new research area, "Osteoimmunology" (7).
5. Wnt signalling pathways
5.1. Canonical and noncanonical Wnt signals
In the absence of Wnt signalling, glycogen synthase kinase-3 (GSK-3) phosphorylates -catenin in the target cells (8,9) (Figure 3). Phosphorylated -catenin is degraded through the ubiquitin-proteasome pathway. Wnt1 class ligands such as Wnt1 and Wnt3a activate the canonical pathway through the formation of a complex of Wnt, Frizzled, and LRP5 or LRP6 (49). This complex in turn promotes the phosphorylation of GSK-3, which inhibits the kinase activity of GSK-3. Inactivation of GSK-3 induces the accumulation of -catenin in the target cells, followed by translocation of accumulated -catenin into the nucleus. Accumulation of -catenin leads to its translocation into the nucleus, where it interacts with T-cell factor/lymphoid enhancer factor (TCF/LEF) family members to initiate transcription of the target genes. LRP5 and LRP6 act as Wnt coreceptors for the canonical pathway (8,9,49). The importance of the canonical pathway in bone biology has been emphasized by the identification of a link between bone mass and mutations in the LRP5 gene (10). Loss-of-function mutations in LRP5 reduce the number of osteoblasts and cause osteoporosis (10, 50). A recent report has suggested that LRP5 signals control bone formation by inhibiting serotonin synthesis in the duodenum (51).
Noncanonical Wnt signalling is classified into two subpathways: the Wnt/Ca2+ and Wnt/planer cell polarity pathways (49). Both pathways are activated by Wnt5a class ligands such as Wnt5a and Wnt11. The Ca2+ pathway activates Ca2+-sensitive enzymes, such as Ca2+-calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC) (52-55). The planer cell polarity pathway is mediated through JNK-dependent signals, and this pathway plays a critical role in morphogenetic processes, including convergent-extension movements during gastrulation and the alignment of the sensory hair cells of the inner ears (56-58). The mammalian Ror family consists of two structurally related proteins, Ror1 and Ror2. Ror1 and Ror2 act as alternative receptors or coreceptors for Wnt5a (59). Wnt5a binds to the cysteine-rich domain of Ror2, which then associates with Frizzled2 and activates JNK in cultured cells (60). Recently, Sato et al. (61) showed that Frizzled2 was internalized through a clathrin-mediated route in response to Wnt5a, and Ror1 or Ror2 were required for this action. These results suggest that both Ror2 and Ror1 mediate Wnt5a signaling by activating the non-canonical Wnt pathway.
5.2. Role of canonical Wnt signals in bone resorption
Osteoclasts are formed in cocultures of osteoblasts and bone marrow cells in the presence of bone resorption-stimulating factors such as 1,25(OH)2D3 and PTH. Wnt3a strongly inhibits 1,25(OH)2D3-induced osteoclast formation in cocultures of stromal ST2 cells and bone marrow cells (62). However, Wnt3a fails to inhibit RANKL-induced osteoclast formation in bone marrow macrophage cultures. These results suggest that the inhibitory effect of Wnt3a on osteoclast formation is mediated by osteoblasts. Glass et al. (11) developed mice expressing a stabilized form of -catenin in their osteoblasts (-catenin mutant mice), and reported that bone mass was increased, but the number of osteoblasts and other parameters of osteoblast function remained unchanged in those mice (Figure 4). Interestingly, the -catenin mutant mice developed osteopetrosis with tooth eruption defects and a decreased number of osteoclasts. Urinary deoxypyridinoline, a marker of osteoclastic bone resorption, was also decreased in the -catenin mutant mice. Micro-array analysis comparing gene expression changes in LRP5-/- mice and -catenin mutant mice showed that OPG was up-regulated in osteoblasts in those mice (11). When -catenin was inactivated selectively in mature osteoblasts using 1(I) collagen Cre mice, the bone mass was decreased due to enhancement of bone resorption. Activation of the canonical Wnt pathway stimulated OPG expression in osteoblasts. In addition, the canonical Wnt pathway suppressed the expression of RANKL in MC3T3E1 cells and MG-63 cells (12). These results suggest that the activation of the canonical Wnt pathway in osteoblasts suppresses bone resorption through up-regulation of OPG expression and down-regulation of RANKL expression (Figure 4).
LRP5 and LRP6 are expressed in bone marrow macrophages, suggesting that Wnt3a can stimulate canonical Wnt pathways in osteoclast precursors (13). Wnt3a stimulated cytosolic -catenin accumulation in bone marrow macrophages, but showed no effect on osteoclast formation in bone marrow macrophage cultures treated with RANKL and M-CSF (13). When -catenin in bone marrow macrophages was depleted by short hairpin RNAs, those macrophages normally differentiated into osteoclasts in response to RANKL and M-CSF. These results suggest that the canonical Wnt pathway plays important roles in osteoblasts, but not in osteoclast precursors, to regulate bone resorption.
5.3. Role of noncanonical Wnt signals in bone resorption
Wnt5a stimulates the noncanonical Wnt pathway in target cells. A recent study showed that Wnt5a enhanced RANKL-induced osteoclast formation in mouse bone marrow macrophage cultures (13) (Figure 4). Wnt3a showed no effect on osteoclast formation in the same culture system. Mouse bone marrow macrophages expressed Frizzleds 2 and 5 and Ror2, the receptor components of Wnt5a. Knock-down of Ror2 by short hairpin RNA abolished the synergistic effect of Wnt5a on osteoclast formation, suggesting that the synergistic effect of Wnt5a on osteoclast formation is mediated by the Wnt5a-Ror2 axis. Wnt5a stimulated phosphorylation of PKC and enhanced RANKL-induced phosphorylation of JNK in bone marrow macrophages. Thus, JNK appeared to be involved in the crosstalk between RANK- and Ror2-medated signals. Accumulation of -catenin was not induced by Wnt5a in osteoclast precursors. RT-PCR analysis also revealed that osteoblasts expressed higher amounts of Wnt5a than bone marrow macrophages. These results suggest that Wnt5a produced by osteoblasts enhances osteoclast differentiation through the noncanonical Wnt pathway in osteoclast precursors. It was also reported that synovial tissues from rheumatoid arthritis patients produce large amounts of Wnt5a (63). This suggests that Wnt5a secreted from the synovial tissue is involved in bone destruction in rheumatoid arthritis. These results suggest that Wnt5a promotes RANKL-induced osteoclast formation through Ror2 expressed by osteoclast precursors in physiological and pathological situations (Figure 4).
Ror1 and Ror2 exhibit different expression patterns in the developing limbs, where the expression of Ror2 is broad compared with that of Ror1 (64). Ror2-/- mice exhibit a perinatal lethality and profound skeletal abnormalities with shortened limbs (65). Ror2 is expressed in chondrocytes of developing cartilage anlagen, but not in osteogenic cells during intramembranous ossification. Wnt5a-/- mice also die during the perinatal period and this is associated with severe reductions in length of the individual skeletal elements (66). Reduced proliferation of progenitor cells was observed in the progress zone of forelimbs in Wnt5a-/- mice, and Wnt5a stimulated proliferation and differentiation of chondrocytes. These results suggest that Wnt5a-Ror2 signaling regulates chondrogenesis. However, it is still not known how this signaling pathway regulates bone remodeling. Our findings suggest that defects of Wnt5a-Ror2 signals in osteoclast precursors may be involved in the abnormality of developing limbs in these deficient mice.
6. Conclusions
Osteoblasts closely regulate osteoclast differentiation and function through the expression of two cytokines, M-CSF and RANKL. Osteoblasts also express OPG, a soluble decoy receptor of RANKL, as an inhibitor of osteoclastogenesis. Most bone resorbing factors enhance RANKL expression and suppress OPG expression in osteoblasts. NFATc1 has been identified as a master transcription factor for osteoclast differentiation. Recent studies have also shown that immunoglobulin-like receptor-mediated ITAM signals are involved in the robust induction of NFATc1 in osteoclast precursors. Osteoblasts have been proposed to express molecules which stimulate ITAM signals. ITAM signals are known as co-stimulatory signals for RANK in osteoclastogenesis.
Wnt signalling regulates physiological development of organs, whereas deregulated signals induce diseases including cancer, arthritis and osteoporosis. Wnts mediate biological processes via two signalling pathways: the -catenin-dependent canonical and -catenin-independent noncanonical pathways. Wnts act on osteoblast precursors and promote their differentiation into mature osteoblasts through the canonical pathway. The canonical pathway in osteoblasts suppresses osteoclastogenesis through down-regulation of the RANKL/OPG ratio. In contrast, the activation of the noncanonical pathway in osteoclast precursors enhances RANKL-induced osteoclastic differentiation in a cell-autonomous manner. Osteoclast precursors express Ror2, a coreceptor for noncanonical signalling, while osteoblasts express Wnt5a, a Ror2 ligand. Synovial tissues from rheumatoid arthritis patients produce large amounts of Wnt5a. Based on these findings, we propose that the noncanonical Wnt pathway is a newly discovered co-stimulatory signal in osteoclastogenesis. This co-stimulatory signal plays an important role under not only physiological, but also pathological, conditions such as rheumatoid arthritis. These results also suggest that the Wnt5a-Ror2 pathway could be a therapeutic target for bone diseases with abnormal bone resorption.
7. References
1. T. Suda, N. Takahashi, N. Udagawa, E. Jimi, M.T. Gillespie, and T.J. Martin: Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20, 345-357 (1999)
doi:10.1210/er.20.3.345
2. S.L. Teitelbaum and F.P. Ross: Genetic regulation of osteoclast development and function. Nat Rev Genet 4, 638-649 (2003)
doi:10.1038/nrg1122
3. G.D. Roodman: Regulation of osteoclast differentiation. Ann N Y Acad Sci 1068, 100-109 (2006)
doi:10.1196/annals.1346.013
4. N. Takahashi, T. Akatsu, N. Udagawa, T. Sasaki, A. Yamaguchi, J.M. Moseley, T.J. Martin, and T. Suda: Osteoblastic cells are involved in osteoclast formation. Endocrinology 123, 2600-2602 (1988)
doi:10.1210/endo-123-5-2600
5. T. Suda, N. Takahashi, and T.J. Martin: Modulation of osteoclast differentiation. Endocr Rev 13, 66-80 (1992)
doi:10.1210/edrv-13-1-66
6. W.J. Boyle, W.S. Simonet, and D.L. Lacey: Osteoclast differentiation and activation. Nature 423, 337-342 (2003)
doi:10.1038/nature01658
7. H. Takayanagi: Osteoimmunology and the effects of the immune system on bone. Nat Rev Rheumatol 5, 667-676 (2009)
doi:10.1038/nrrheum.2009.217
8. J.R. Miller: The Wnts. Genome Biol 3, 3001.1-3001.15 (2002)
No DOI found
9. C.Y. Logan and R. Nusse: The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20, 781-810 (2004)
doi:10.1146/annurev.cellbio.20.010403.113126
10. Y. Gong, R.B. Slee, N. Fukai, G. Rawadi, S. Roman-Roman, A.M. Reginato, H. Wang, T. Cundy, F.H. Glorieux, D. Lev, M. Zacharin, K. Oexle, J. Marcelino, W. Suwairi, S. Heeger, G. Sabatakos, S. Apte, W.N. Adkins, J. Allgrove, M. Arslan-Kirchner, J.A. Batch, P. Beighton, G.C. Black, R.G. Boles, L.M. Boon, C. Borrone, H.G. Brunner, G.F. Carle, B. Dallapiccola, A. De Paepe, B. Floege, M.L. Halfhide, B. Hall, R.C. Hennekam, T. Hirose, A. Jans, H. Juppner, C.A. Kim, K. Keppler-Noreuil, A. Kohlschuetter, D. LaCombe, M. Lambert, E. Lemyre, T. Letteboer, L. Peltonen, R.S. Ramesar, M. Romanengo, H. Somer, E. Steichen-Gersdorf, B. Steinmann, B. Sullivan, A. Superti-Furga, W. Swoboda, M.J. van den Boogaard, W. Van Hul, M. Vikkula, M. Votruba, B. Zabel, T. Garcia, R. Baron, B.R. Olsen, and M.L. Warman: LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107, 513-523 (2001)
doi:10.1016/S0092-8674(01)00571-2
11. D.A. Glass, 2nd, P. Bialek, J.D. Ahn, M. Starbuck, M.S. Patel, H. Clevers, M.M. Taketo, F. Long, A.P. McMahon, R.A. Lang, and G. Karsenty: Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev Cell 8, 751-764 (2005)
doi:10.1016/j.devcel.2005.02.017
12. G.J. Spencer, J.C. Utting, S.L. Etheridge, T.R. Arnett, and P.G. Genever: Wnt signalling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro. J Cell Sci 119, 1283-1296 (2006)
doi:10.1242/jcs.02883
13. K. Maeda, Y. Kobayashi, T. Mizoguchi, Y. Nakamich, T. Yamashita, S. Kinugawa, N. Udagawa, K. Marumo, N. Takahashi: Wnt5a enhances RANKL-induced osteoclastogenesis. J Bone Miner Res 22 (Suppl. 1), S43 (2007)
No DOI found
14. H. Yoshida, S. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, T. Sudo, and L.D. Shultz: The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442-444 (1990)
doi:10.1038/345442a0
15. R. Felix, M.G. Cecchini, and H. Fleisch: Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse. Endocrinology 127, 2592-2594 (1990)
doi:10.1210/endo-127-5-2592
16. N. Takahashi, N. Udagawa, T. Akatsu, H. Tanaka, Y. Isogai, and T. Suda: Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128:1792-1796 (1991)
doi:10.1210/endo-128-4-1792
17. S. Tanaka, N. Takahashi, N. Udagawa, T. Tamura, T. Akatsu, E.R. Stanley, T. Kurokawa, and T. Suda: Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. J Clin Invest 91, 257-263 (1993)
doi:10.1172/JCI116179
18. B.R. Wong, J. Rho, J. Arron, E. Robinson, J. Orlinick, M. Chao, S. Kalachikov, E. Cayani, F.S. Bartlett, 3rd, W.N. Frankel, S.Y. Lee, and Y. Choi: TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 272, 25190-25194 (1997)
doi:10.1074/jbc.272.40.25190
19. D.M. Anderson, E. Maraskovsky, W.L. Billingsley, W.C. Dougall, M.E. Tometsko, E.R. Roux, M.C. Teepe, R.F. DuBose, D. Cosman, and L. Galibert: A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390, 175-179 (1997)
doi:10.1038/36593
20. H. Yasuda, N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, and T. Suda: Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 95, 3597-3602 (1998)
doi:10.1073/pnas.95.7.3597
21. D.L. Lacey, E. Timms, H.L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, H. Hsu, J. Sullivan, N. Hawkins, E. Davy, C. Capparelli, A. Eli, Y.X. Qian, S. Kaufman, I. Sarosi, V. Shalhoub, G. Senaldi, J. Guo, J. Delaney, and W.J. Boyle: Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165-176 (1998)
doi:10.1016/S0092-8674(00)81569-X
22. W.S. Simonet, D.L. Lacey, C.R. Dunstan, M. Kelley, M.S. Chang, R. Luthy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, G. Shimamoto, M. DeRose, R. Elliott, A. Colombero, H.L. Tan, G. Trail, J. Sullivan, E. Davy, N. Bucay, L. Renshaw-Gegg, T.M. Hughes, D. Hill, W. Pattison, P. Campbell, S. Sander, G. Van, J. Tarpley, P. Derby, R. Lee, and W.J. Boyle: Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89, 309-319 (1997)
doi:10.1016/S0092-8674(00)80209-3
23. E. Tsuda, M. Goto, S. Mochizuki, K. Yano, F. Kobayashi, T. Morinaga, and K. Higashio: Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem Biophys Res Commun 234, 137-142 (1997)
doi:10.1006/bbrc.1997.6603
24. Y.Y. Kong, U. Feige, I. Sarosi, B. Bolon, A. Tafuri, S. Morony, C. Capparelli, J. Li, R. Elliott, S. McCabe, T. Wong, G. Campagnuolo, E. Moran, E.R. Bogoch, G. Van, L.T. Nguyen, P.S. Ohashi, D.L. Lacey, E. Fish, W.J. Boyle, and J.M. Penninger: Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402, 304-309 (1999)
doi:10.1038/46303
25. W.C. Dougall, M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt, E. Daro, J. Smith, M.E. Tometsko, C.R. Maliszewski, A. Armstrong, V. Shen, S. Bain, D. Cosman, D. Anderson, P.J. Morrissey, J.J. Peschon, and J. Schuh: RANK is essential for osteoclast and lymph node development. Genes Dev 13, 2412-2424 (1999)
doi:10.1101/gad.13.18.2412
26. N. Bucay, I. Sarosi, C.R. Dunstan, S. Morony, J. Tarpley, C. Capparelli, S. Scully, H.L. Tan, W. Xu, D.L. Lacey, W.J. Boyle, and W.S. Simonet: Osteoprotegerin－deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12, 1260-1268 (1998)
doi:10.1101/gad.12.9.1260
27. A Mizuno, N. Amizuka, K. Irie, A. Murakami, N. Fujise, T. Kanno, Y. Sato, N. Nakagawa, H. Yasuda, S. Mochizuki, T. Gomibuchi, K. Yano, N. Shima, N. Washida, E. Tsuda, T. Morinaga, K. Higashio, and H. Ozawa: Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 247, 610-615 (1998)
doi:10.1006/bbrc.1998.8697
28. P.I. Croucher, C.M. Shipman, J. Lippitt, M. Perry, K. Asosingh, A. Hijzen, A.C. Brabbs, E.J. van Beek, I. Holen, T.M. Skerry, C.R. Dunstan, G.R. Russell, B. Van Camp, and K. Vanderkerken: Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma. Blood 98:3534-3540 (2001)
doi:10.1182/blood.V98.13.3534
29. T. Kukita, Wada, A. Kukita, T. Kakimoto, F. Sandra, K. Toh, K. Nagata, T. Iijima, M. Horiuchi, H. Matsusaki, K. Hieshima, O. Yoshie, and H. Nomiyama: RANKL-induced DC-STAMP is essential for osteoclastogenesis. J Exp Med 200, 941-946 (2004)
doi:10.1084/jem.20040518
30. M. Yagi, T. Miyamoto, Y. Sawatani, K. Iwamoto, N. Hosogane, N. Fujita, K. Morita, K. Ninomiya, T. Suzuki, K. Miyamoto, Y. Oike, M. Takeya, Y. Toyama, and T. Suda: DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 202, 345-351 (2005)
doi:10.1084/jem.20050645
31. B.G. Darnay, V. Haridas, J. Ni, P.A. Moore, and B.B. Aggarwal: Characterization of the intracellular domain of receptor activator of NF-kappaB (RANK). Interaction with tumor necrosis factor receptor-associated factors and activation of NF-kappab and c-Jun N-terminal kinase. J Biol Chem 273, 20551-20555 (1998)
doi:10.1074/jbc.273.32.20551
32. J. Inoue, J. Gohda, and T. Akiyama: Characteristics and biological functions of TRAF6. Adv Exp Med Biol 597, 72-79 (2007)
doi:10.1007/978-0-387-70630-6_6
33. M.A. Lomaga, W.C. Yeh, I. Sarosi, G.S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, A. van der Heiden, A. Itie, A. Wakeham, W. Khoo, T. Sasaki, Z. Cao, J.M. Penninger, C.J. Paige, D.L. Lacey, C.R. Dunstan, W.J. Boyle, D.V. Goeddel, and T.W. Mak: TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev 13, 1015-1024 (1999)
doi:10.1101/gad.13.8.1015
34. A Naito, S. Azuma, S. Tanaka, T. Miyazaki, S. Takaki, K. Takatsu, K. Nakao, K. Nakamura, M. Katsuki, T. Yamamoto, and J. Inoue: Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4, 353-362 (1999)
doi:10.1046/j.1365-2443.1999.00265.x
35. V. Iotsova, J. Caamano, J. Loy, Y. Yang, A. Lewin, and R. Bravo: Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat Med 3, 1285-1289 (1997)
doi:10.1038/nm1197-1285
36. G. Franzoso, L. Carlson, L. Xing, L. Poljak, E.W. Shores, K.D. Brown, A. Leonardi, T. Tran, B.F. Boyce, and U. Siebenlist: Requirement for NF-kappaB in osteoclast and B-cell development. Genes Dev 11, 3482-3496 (1997)
doi:10.1101/gad.11.24.3482
37. Z.Q. Wang, C. Ovitt, A.E. Grigoriadis, U. Mohle-Steinlein, U. Ruther, and E.F. Wagner: Bone and haematopoietic defects in mice lacking c-fos. Nature 360, 741-745 (1992)
doi:10.1038/360741a0
38. J.P. David, K. Sabapathy, O. Hoffmann, M.H. Idarraga, and F.F. Wagner EF: JNK1 modulates osteoclastogenesis through both c-Jun. J Cell Sci 115:4317-4325 (2002)
doi:10.1242/jcs.00082
39. F. Ikeda, T. Matsubara, T. Tsurukai, K. Hata, R. Nishimura, and T. Yoneda: JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts. J Bone Minr Res 23, 907-914 (2008)
doi:10.1359/jbmr.080211
40. N. Ishida, K. Hayashi, M. Hoshijima, T. Ogawa, S. Koga, Y. Miyatake, M. Kumegawa, T. Kimura, and T. Takeya: Large scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator. J Biol Chem 277, 41147-41156 (2002)
doi:10.1074/jbc.M205063200
41. H. Takayanagi, S. Kim, T. Koga, H. Nishina, M. Isshiki, H. Yoshida, A. Saiura, M. Isobe, T. Yokochi, J. Inoue, E.F. Wagner, T.W. Mak, T. Kodama, and T. Taniguchi: Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 3, 889-901 (2002)
doi:10.1016/S1534-5807(02)00369-6
42. J.G. Monroe, J.G: ITAM-mediated tonic signalling through pre-BCR and BCR complexes. Nat Rev Immunol 6, 283-294 (2006)
doi:10.1038/nri1808
43. A.S. Tessarz, and A. Cerwenka: The TREM-1/DAP12 pathway. Immunol Lett 116, 111-116 (2008)
doi:10.1016/j.imlet.2007.11.021
44. N. Kim, M. Takami, J. Rho, R. Josien, and Y. Choi: A novel member of the leukocyte receptor complex regulates osteoclast differentiation. J Exp Med 195, 201-209 (2002)
No DOI found
45. J. Trowsdale, R. Barten, A. Haude, C.A. Stewart, S. Beck, and M.J. Wilson: The genomic context of natural killer receptor extended gene families. Immunol Rev 181:20-38, (2001)
doi:10.1034/j.1600-065X.2001.1810102.x
46. T. Koga, M. Inui, K. Inoue, S. Kim, A. Suematsu, E. Kobayashi, T. Iwata, H. Ohnishi, T. Matozaki, T. Kodama, T. Taniguchi, H. Takayanagi, and T. Takai: Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 428, 758-763 (2004)
doi:10.1038/nature02444
47. M. Shinohara, T. Koga, K. Okamoto, S. Sakaguchi, K. Arai, H. Yasuda, T. Takai, T. Kodama, T. Morio, R.S. Geha, D. Kitamura, T. Kurosaki, W. Ellmeier, and H. Takayanagi: Tyrosine kinases Btk and Tec regulate osteoclast differentiation by linking RANK and ITAM signals. Cell 132:794-806.(2008)
doi:10.1016/j.cell.2007.12.037
48. N. Takegahara, H. Takamatsu, T. Toyofuku, T. Tsujimura, T. Okuno, K. Yukawa, M. Mizui, M. Yamamoto, D.V. Prasad, K. Suzuki, M. Ishii, K. Terai, M. Moriya, Y. Nakatsuji, S. Sakoda, S. Sato, S. Akira, K. Takeda, M. Inui, T. Takai, M. Ikawa, M. Okabe, A. Kumanogoh, and H. Kikutani: Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis. Nat Cell Biol 8, 615-622 (2006)
doi:10.1038/ncb1416
49. M.D. Gordon, and R. Nusse: Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 281, 22429-22433 (2006)
doi:10.1074/jbc.R600015200
50. M. Kato, M.S. Patel, R. Levasseur, I. Lobov, B.H. Chang, D.A. Glass, 2nd, C. Hartmann, L. Li, T.H. Hwang, C.F. Brayton, R.A. Lang, G. Karsenty, and L. Chan: Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 157, 303-314 (2002)
doi:10.1083/jcb.200201089
51. V.K. Yadav, J.H. Ryu, N. Suda, K.F. Tanaka, J.A. Gingrich, G. Schutz, F.H. Glorieux, C.Y. Chiang, J.D. Zajac, K.L. Insogna, J.J. Mann, R. Hen, P. Ducy, and G. Karsenty: Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 135, 825-837 (2008)
doi:10.1016/j.cell.2008.09.059
52. A.T. Weeraratna, Y. Jiang, G. Hostetter, K. Rosenblatt, P. Duray, M. Bittner, and J.M. Trent: Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell 1, 279-288 (2002)
doi:10.1016/S1535-6108(02)00045-4
53. J. Dejmek, A. Safholm, C. Kamp Nielsen, T. Andersson, and K. Leandersson: Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells. Mol Cell Biol 26, 6024-6036 (2006)
doi:10.1128/MCB.02354-05
54. T. Ishitani, S. Kishida, J. Hyodo-Miura, N. Ueno, J. Yasuda, M. Waterman, H. Shibuya, R.T. Moon, J. Ninomiya-Tsuji, and K. Matsumoto: The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca2+ pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol, 23, 131-139 (2003)
doi:10.1128/MCB.23.1.131-139.2003
55. L.C. Sheldahl, D.C. Slusarski, P. Pandur, J.R. Miller, M. Kuhl, and R.T. Moon: Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos. J Cell Biol 161, 769-777 (2003)
doi:10.1083/jcb.200211094
56. A. Schambony and D. Wedlich: Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway. Dev Cell 12, 779-792 (2007)
doi:10.1016/j.devcel.2007.02.016
57. D. Qian, C. Jones, A. Rzadzinska, S. Mark, X. Zhang, K.P. Steel, X. Dai, and P. Chen: Wnt5a functions in planar cell polarity regulation in mice. Dev Biol 306, 121-133 (2007)
doi:10.1016/j.ydbio.2007.03.011
58. S. Yamamoto, O. Nishimura, K. Misaki, M. Nishita, Y. Minami, S. Yonemura, H. Tarui, and H. Sasaki: Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell 15, 23-36 (2008)
doi:10.1016/j.devcel.2008.05.007
59. A. Mikels, Y. Minami, and R. Nusse: Ror2 receptor requires tyrosine kinase activity to mediate Wnt5A signaling. J Biol Chem 284, 30167-30176 (2009)
doi:10.1074/jbc.M109.041715
60. I. Oishi, H. Suzuki, N. Onishi, R.Takada, S. Kani, B. Ohkawara, I. Koshida, K. Suzuki, G. Yamada, G.C. Schwabe, S Mundlos, H Shibuya, S Takada, and Y Minami: The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway Genes Cells 8, 645-654 (2003) doi:10.1046/j.1365-2443.2003.00662.x
61. A. Sato, H. Yamamoto, H. Sakane, H. Koyama, A. Kikuchi: Wnt5a regulates distinct signalling pathways by binding to Frizzled2. EMBO J 29:41-54 (2010)
doi:10.1038/emboj.2009.322
62. T. Yamane, T. Kunisada, H. Tsukamoto, H. Yamazaki, H. Niwa, S. Takada, and S.I. Hayashi: Wnt signaling regulates hemopoiesis through stromal cells. J Immunol 167, 765-772 (2001)
No DOI found
63. M. Sen, K. Lauterbach, H. El-Gabalawy, G.S. Firestein, M. Corr, and D.A. Carson: Expression and function of wingless and frizzled homologs in rheumatoid arthritis. Proc Natl Acad Sci U S A 97, 2791-2796 (2000)
doi:10.1073/pnas.050574297
64. T. Matsuda, M. Nomi, M. Ikeya, S. Kani, I. Ohishi, T. Terashima, S. Takada, and Y. Minami: Expression of the receptor tyrosin kinase genes, Ror1 and Ror2, during mouse development. Mech Dev 105, 153-156 (2001)
doi:10.1016/S0925-4773(01)00383-5
65. T.M. DeChiara, R.B. Kimble, W.T. Poueymirou, J. Rojas, P. Masiakowski, D.M. Valenzuela, G.D. Yancopoulos: Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development. Nat Genet 24:271-274 (2003)
doi:10.1038/73488
66. T.P. Yamaguchi, A. Bradley, A.P. McMahon, S. Jones: A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126:1211-1223 (1999)
No DOI found
Abbreviations: M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor-B ligand; OPG, osteoprotegerin; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; ITAM, immunoreceptor tyrosine-based activation motif; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PTH, parathyroid hormone; PGE2, prostaglandin E2; IL-11, interleukin 11; LRP, low density lipoprotein receptor-related protein; Ror, receptor tyrosine kinase orphan receptor; TNF, tumor necrosis factor; DC-STAMP, dendritic cell-specific transmembrane protein; TRAF, TNF receptor-associated factor; NF-B, nuclear factor-B; JNK, c-Jun N-terminal kinase; LRC, leukocyte receptor complex; OSCAR, osteoclast-associated receptor; DAP12, DNAX-activating protein 12; FcR, Fc receptor common chain; TREM2, triggering receptors expressed on myeloid cells 2; SIRP1, signal-regulatory protein 1; PIR-A, paired immunoglobulin-like receptor A; SYK, spleen tyrosine kinase; PLC, phospholipase C; Btk, Bruton's tyrosine kinase; BLNK, B cell linker protein; GSK-3, glycogen synthase kinase-3; TCF/LEF, T-cell factor/lymphoid enhancer factor, CAMKII, Ca2+-calmodulin-dependent protein kinase II; PKC, protein kinase C
Key words: Osteoclasts, RANKL, RANK, Wnt, Ror, Frizzled, LRP
Send correspondence to: Naoyuki Takahashi, Institute for Oral Science, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781, Japan, Tel: 81-263-51-2135, Fax: 81-263-51-2223, E-mail:takahashinao@po.mdu.ac.jp