1. Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
2. Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, and COE Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549, Japan and PRESTO, Japan Science and Technology Agency (JST), Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan
3. Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo 4-1, Aoba-ku, Sendai 980-8575, Japan and CREST, JST, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan
4. Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Showa-machi 3-39-15, Maebashi 371-8512, Japan
5. Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan
6. These authors contributed equally to this work

Correspondence to: Hiroshi Takayanagi^1,2Toshiyuki Takai³ Email: taka.cell@tmd.ac.jp
Email: tostakai@idac.tohoku.ac.jp

Osteoclast differentiation induced by RANKL and M-CSF is severely blocked in the bone marrow monocyte/macrophage lineage cells (BMMs) derived from mice lacking DAP12 (ref. 16), also known as KARAP or TYROBP^{9, 10}. DAP12 is a membrane adaptor molecule that contains an ITAM motif, which activates calcium signalling in immune cells. Despite this in vitro blockage, DAP12-deficient (DAP12^-/-) mice exhibit only mild osteopetrosis and contain a normal number of osteoclasts. This suggests that the DAP12-mediated signal plays a crucial role in the RANKL/M-CSF-induced osteoclast formation system but that another molecule(s) can rescue the DAP12 deficiency in vivo¹⁶.

To investigate the molecular basis of this discrepancy, we stimulated BMMs with RANKL and M-CSF after strictly depleting the stromal/osteoblastic cells¹⁷. In this osteoblast-free system, osteoclast differentiation is completely abrogated in DAP12^-/- BMMs (Fig. 1a); we observed no formation of multinucleated cells (MNCs) positive for the marker enzyme of osteoclasts, tartrate-resistant acid phosphatase (TRAP). This differentiation block is efficiently rescued by retroviral expression of DAP12, but not by DAP12Y65F, a DAP12 mutant that does not transmit the ITAM signal¹⁸ (Fig. 1b), indicating that the DAP12-mediated ITAM signal is required for osteoclastogenesis in this system. Interestingly, DAP12^-/- BMMs undergo osteoclast differentiation in co-culture with osteoblasts¹⁹ (Fig. 1c), showing that osteoblasts can stimulate the signal that compensates for the loss of the DAP12-mediated ITAM signal. This compensatory mechanism may explain the normal osteoclast differentiation in DAP12^-/- mice in vivo (see Supplementary Fig. 1a, b).

Figure 1: Impaired osteoclastogenesis in the absence of DAP12 and the compensatory mechanism by osteoblasts.

a, Complete lack of osteoclastogenesis in DAP12^-/- BMMs stimulated with RANKL/M-CSF. b, Rescue of osteoclastogenesis by retrovirus-mediated expression of DAP12, but not DAP12Y65F, in DAP12^-/- BMMs. These multinucleated cells have a bone-resorbing activity (not shown). c, Osteoclast formation from DAP12^-/- BMMs in co-culture with osteoblasts. Bone-resorbing activity was examined on dentine slices (not shown). d, Flow-cytometric analysis of cell-surface expression of FcRIIB after overexpression of the chimaeric receptor of RIIB–OSCAR or FcRIIB (left). Weak expression of FcRIIB is detected by transfection of RIIB–OSCAR alone (pink line, left and right panels), but cotransfection with FcR exclusively increased this expression (violet line, right). e, Histology of tibia (upper, toluidine blue staining) and microradiographic analysis of femur (lower, microcomputed tomography, left; microradiograph, right) of FcR^-/- mice. There was no significant abnormality.

High resolution image and legend (162K)

The osteoclast-associated receptor (OSCAR) is an activating-type immunoglobulin-like receptor induced in RANKL-stimulated BMMs¹¹. Expression of a putative OSCAR ligand in osteoblasts¹¹ led us to investigate OSCAR as a candidate receptor that compensates for the loss of DAP12-mediated signalling. To explore the adaptor molecules with which OSCAR associates, we constructed a chimaeric receptor composed of the extracellular portion of type IIB FcR for IgG (FcRIIB) and the transmembrane and cytoplasmic portion of OSCAR (RIIB–OSCAR). Flow-cytometric analysis revealed that RIIB–OSCAR expression was significantly enhanced only when it was cotransfected with FcR (Fig. 1d). Thus, RIIB–OSCAR preferentially associates with FcR, another adaptor protein containing an ITAM motif, but not with DAP12 or DAP10. Immunoprecipitation analysis also verified the exclusive association of RIIB–OSCAR with FcR (Supplementary Fig. 1c).

The results prompted us to investigate the bone phenotype of mice deficient in FcR (FcR^-/- mice)²⁰, but there was no significant difference in the osteoclast number and the trabecular bone volume between wild-type and FcR^-/- mice (Fig. 1e and Supplementary Fig. 1d). In addition, there was little, if any, difference between wild-type and FcR^-/- mice in the differentiation of osteoclasts in the RANKL/M-CSF system (Supplementary Fig. 1e) and the co-culture system (data not shown). Although OSCAR–Fc, a fusion protein of OSCAR ectodomain and Fc portion of IgG, suppresses osteoclastogenesis in vitro¹¹, our results suggest that FcR-mediated signals can be compensated for by other signals in vivo.

Considering the possibility that DAP12 and FcR functionally compensate for each other²¹, we generated mice lacking both molecules (DAP12^-/- FcR^-/- mice). These mice exhibit severe osteopetrosis, with bone marrow filled with unresorbed bone (Fig. 2a). DAP12^-/- mice show mild osteopetrosis due to impaired osteoclast activity¹⁶, but the osteopetrosis in DAP12^-/- FcR^-/- mice is much more severe, as seen in microradiographs (Fig. 2a) and trabecular bone volume (Fig. 2b). Importantly, we observed few osteoclasts in DAP12^-/- FcR^-/- mice, indicating that the osteopetrosis is caused by defective differentiation rather than by defective activity of osteoclasts (Fig. 2c, e). Bone-morphometric analysis revealed that osteoblastic bone formation also decreases (Fig. 2d and Supplementary Fig. 2a). Thus, the ITAM-harbouring adaptors FcR and DAP12 are essential for osteoclast differentiation in vivo. Despite the severe osteopetrosis in DAP12^-/- FcR^-/- mice, these mice have no defect in tooth eruption (data not shown). In addition, we observed a very small number of TRAP⁺ MNCs in limited areas just below the epiphyseal plate (Fig. 2e and Supplementary Fig. 2b), suggesting that another adaptor molecule(s) may compensate for the function under specific conditions. In the culture system, DAP12^-/- FcR^-/- osteoclast precursor cells cannot undergo osteoclast differentiation in response to RANKL and M-CSF (Fig. 3a). Retroviral transfer of DAP12, but not DAP12Y65F or FcR, into DAP12^-/- FcR^-/- cells efficiently rescued osteoclast differentiation induced by RANKL and M-CSF. In addition, DAP12^-/- FcR^-/- precursor cells barely differentiate into osteoclasts even when co-cultured with osteoblasts (Fig. 3b). In the co-culture system, retroviral transfer of FcR, but not ITAM-deficient FcRY65F, also rescued osteoclast differentiation, albeit partially. These results suggest that the ITAM signal mediated through FcR and DAP12 is indispensable for RANKL-induced osteoclastogenesis and that each adaptor-mediated signal is differentially regulated.

Figure 2: Severe osteopetrosis in DAP12^-/-FcR^-/- (DKO) mice due to impaired osteoclast differentiation.

a, Histology of tibia and microradiographic analysis of femur of DAP12^-/- and DAP12^-/- FcR^-/- mice (12 weeks of age). Bone-marrow cavity is absent in DAP12^-/- FcR^-/- mice. Higher radiopacity shows that DAP12^-/- FcR^-/- mice have a much more severe osteopetrotic phenotype than DAP12^-/- mice. b, Increased trabecular bone volume in DAP12^-/- FcR^-/- mice. c, The number of osteoclasts is not altered in DAP12^-/- mice, but is markedly decreased in DAP12^-/- FcR^-/- mice. d, Osteoblastic parameters in the bone-morphometric analysis of the tibia of wild-type and DAP12^-/- FcR^-/- mice (12 weeks of age). e, Histology of the tibia of DAP12^-/- and DAP12^-/- FcR^-/- mice (TRAP and toluidine blue staining). Bone-marrow cavity is filled with unresorbed bone in the diaphysis of DAP12^-/- FcR^-/- mice. No osteoclasts are observed in the diaphysis of DAP12^-/- FcR^-/- mice, whereas the osteoclast number is normal in DAP12^-/- mice. Typical sites of cartilage remnant are indicated by asterisks. In the metaphyseal area, a small number of osteoclasts are observed below epiphyseal plates in DAP12^-/- FcR^-/- mice (arrowheads).

High resolution image and legend (191K)

Figure 3: Distinct roles of FcR- and DAP12-associating immunoreceptors in the regulation of osteoclastogenesis.

a, Complete block of in vitro osteoclastogenesis in DAP12^-/- FcR^-/- (DKO) cells stimulated with RANKL and M-CSF. This was rescued by pMX-DAP12, but not pMX-DAP12Y65F or pMX-FcR. b, Severe impairment of osteoclastogenesis in DAP12^-/- FcR^-/- cells even in co-culture with osteoblasts. Osteoclastogenesis was rescued by pMX-DAP12 or pMX- FcR in an ITAM-dependent manner. c, Association of FcR with PIR-A, FcRIII and OSCAR (upper). pOC, osteoclast precursor cells stimulated with RANKL. For the comments on the association of FcR with FcRIII, see Supplementary Fig. 3b, c. Association of DAP12 with TREM-2 and SIRP1, but not with NKG2D, in pOC (lower). For the positive control, DAP12 associates with SIRP1 and NKG2D in dendritic cells (DC) and NK cells (NK), respectively. d, Treatment of BMMs with plate-bound monoclonal antibodies against OSCAR, PIR, TREM-2 or SIRP1 promotes osteoclastogenesis stimulated by RANKL and M-CSF. The stimulatory effect by antibody-mediated crosslinking is more obvious at a low concentration of RANKL (5 ng ml^-1) than at a high concentration (100 ng ml^-1). e, Effect of plate-bound antibodies on osteoclastogenesis from FcR^-/- BMMs. Anti-OSCAR and anti-PIR antibodies have no effect on FcR^-/- BMMs. f, Effect of plate-bound antibodies on osteoclastogenesis from DAP12^-/- BMMs or DAP12^-/- FcR^-/- cells stimulated with RANKL/M-CSF. Anti-OSCAR and anti-PIR antibodies rescued osteoclastogenesis from DAP12^-/-BMMs, but such an effect was not observed in anti-TREM-2 or anti-SIRP1 antibodies. No stimulatory effect was observed on osteoclastogenesis from DAP12^-/- FcR^-/- cells.

High resolution image and legend (49K)

FcR or DAP12 associates with several specific immunoreceptors for cell activation in myeloid lineage cells^{13, 21, 22}. To identify receptors that associate with FcR and DAP12 in osteoclast lineage cells, we screened the expression profiles of messenger RNAs for known candidate receptors using GeneChip analysis. We found that a series of receptors and ITAM-associated molecules, as well as FcR and DAP12, are expressed in the osteoclast lineage (Supplementary Fig. 3a). These putative FcR- and DAP12-associating receptors^{11, 12, 13, 14, 15} were detected on the surface of the osteoclast lineage using cell-surface labelling with biotin (Supplementary Fig. 3b, c). Among these, immunoprecipitation experiments confirmed that paired immunoglobulin-like receptor (PIR)-A, FcRIII and OSCAR each pair with FcR and that triggering receptor expressed by myeloid cells (TREM)-2 and signal-regulatory protein (SIRP)1, but not NKG2D, pair with DAP12 in the osteoclast lineage (Fig. 3c).

To test whether these receptor-mediated signals promote osteoclast differentiation by associating with FcR or DAP12, we stimulated BMMs with plate-bound monoclonal antibodies against OSCAR, PIR, TREM-2 and SIRP1. Triggering of either receptor by crosslinking with an antibody accelerated RANKL-induced osteoclast differentiation, indicating that these receptors activate osteoclastogenesis, although the stimulatory effect is more obvious at a low concentration of RANKL (Fig. 3d and Supplementary Fig. 3d). In the absence of RANKL, the stimulation of these receptors alone could not induce osteoclast differentiation (data not shown), suggesting that these receptor-mediated signals act cooperatively with RANKL but cannot substitute for the signal.

A stimulating effect by anti-OSCAR and anti-PIR antibodies was not observed in FcR^-/- BMMs, whereas anti-TREM-2 and anti-SIRP1 antibodies stimulated FcR^-/- BMMs, the same as they did with wild-type cells (Fig. 3e). This indicates that FcR is required for the stimulatory function of OSCAR and PIR-A. Consistent with the observation that osteoblasts rescue DAP12 deficiency through FcR-associated receptors, anti-OSCAR and anti-PIR antibodies could rescue the osteoclastogenesis from DAP12^-/- BMMs but not from DAP12^-/- FcR^-/- cells (Fig. 3f). Stimulation of DAP12-associating receptors such as TREM-2 and SIRP1 did not rescue DAP12^-/- BMMs. This indicates that DAP12 is required for the stimulatory effect through TREM-2 and SIRP1 (Fig. 3f).

How does the ITAM signal contribute to RANKL-induced signalling events? To address this question, we performed a genome-wide screening of mRNA expression in RANKL-stimulated osteoclast precursor cells derived from wild-type and DAP12^-/- FcR^-/- mice. Among the transcription factors and effector molecules involved in RANKL signalling⁶, the induction of nuclear factor of activated T cells c1 (NFATc1) is most strongly suppressed in DAP12^-/- FcR^-/- cells (Fig. 4a). We analysed the protein expression of essential molecules for osteoclastogenesis^{2, 4, 6} including NFATc1, c-Fos and TRAF6, and revealed that NFATc1 expression in DAP12^-/- FcR^-/- cells stimulated with RANKL is barely detectable, but the expression of c-Fos or TRAF6 is still observed under the same conditions (Supplementary Fig. 4a). To confirm the crucial role of NFATc1 as a downstream target, we examined whether ectopic expression of NFATc1, c-Fos or TRAF6 by retrovirus-mediated gene transfer rescues the differentiation block of osteoclasts in DAP12^-/- FcR^-/- cells. Ectopic expression of NFATc1, but not c-Fos or TRAF6, resulted in efficient osteoclast formation even in DAP12^-/- FcR^-/- cells (Fig. 4b).

Figure 4: ITAM-harbouring adaptors are essential for RANKL induction of calcium signalling and NFATc1.

a, GeneChip analysis of mRNA expression of transcription factors and effector molecules involved in RANKL signalling. NFATc1 induction is most strongly impaired in DAP12^-/- FcR^-/- (DKO) cells. b, Introduction of NFATc1 by pMX retrovirus vector (pMX-NFATc1) into the DAP12^-/- FcR^-/- precursor cells efficiently rescued the osteoclastogenesis (with bone-resorbing activity; not shown) in the presence of RANKL and M-CSF. c, Calcium signalling in osteoclast precursor cells stimulated with RANKL and M-CSF for 24 h. Calcium oscillation was not observed in DAP12^-/- FcR^-/- or DAP12^-/- cells. Stimulation with plate-bound anti-PIR antibody rescued the calcium signalling in DAP12^-/- cells, but not in DAP12^-/- FcR^-/- cells. d, Phosphorylation of FcR and DAP12 by RANKL. Phosphorylation of FcR and DAP12 induced by RANKL in BMMs as well as in RAW 264.7 cells. PY, phosphotyrosine. e, Impaired phosphorylation of PLC1 by RANKL in DAP12^-/- FcR^-/- cells. f, A schematic model of ITAM-mediated costimulatory signal in RANKL-stimulated induction of osteoclast differentiation. Phosphorylation of ITAM stimulated by immunoreceptors and RANKL–RANK interaction results in the recruitment of Syk family kinases, leading to the activation of PLC and calcium signalling, which is critical for NFATc1 induction. NFATc1 induction is also dependent on c-Fos and TRAF6, both of which are activated by RANKL. RANKL may also contribute to efficient ITAM signalling through the induction of immunoreceptors or their putative ligands.

High resolution image and legend (70K)

RANKL-induced calcium signalling is essential for autoamplification of the NFATc1 gene during osteoclastogenesis⁶, whereas immunoglobulin-like receptors activate phospholipase C (PLC) and calcium signalling through ITAM in immune cells^{13, 21}. We therefore examined calcium signalling in RANKL-stimulated osteoclast precursor cells derived from DAP12^-/- FcR^-/- mice. As shown in Fig. 4c, calcium oscillation induced by RANKL is not significantly observed in DAP12^-/- FcR^-/- cells, which suggests that the inhibition of this calcium signalling explains the impaired induction of NFATc1. Furthermore, stimulation with the anti-PIR antibody rescued the defect in calcium signalling and NFATc1 expression in DAP12^-/- cells, suggesting that the stimulation of an immunoreceptor is required for RANKL-induced activation of the calcium signal leading to NFATc1 induction and osteoclastogenesis (Fig. 4c and Supplementary Fig. 4b). These findings suggest that FcR and DAP12 are required for the induction of NFATc1, the crucial step in the RANKL-induced osteoclast differentiation programme, through activation of calcium signalling.

To gain further insight into the mechanism linking RANKL and ITAM-mediated calcium signalling, we analysed the phosphorylation events induced by RANKL. Our analysis revealed that RANKL induces the phosphorylation of both FcR and DAP12 in BMMs as well as in RAW 264.7 cells (Fig. 4d). We further demonstrated that RANKL-induced phosphorylation of PLC is impaired in DAP12^-/- FcR^-/- cells (Fig. 4e), but RANKL-induced phosphorylation of p38, JNK and IB is not affected (Supplementary Fig. 4c). This suggests that FcR and DAP12 are specifically involved in the PLC–calcium pathway. In addition, piceatannol, the inhibitor of Syk kinase, which is recruited to ITAM in immune cells, has an inhibitory effect on osteoclastogenesis (Supplementary Fig. 4d).

Our results show that the ITAM-harbouring adaptors FcR and DAP12 deliver essential signals, in concert with RANK-induced signalling cascades, for terminal differentiation of osteoclasts. Our study establishes the importance of the ITAM-mediated costimulatory signal in RANKL-induced osteoclast differentiation (Fig. 4f), which is analogous to the role of costimulatory signals in the immune system^{13, 21}. Although most of the ligands for the immunoreceptors remain to be identified, putative ligands are provided by distinct cell types: osteoclast precursor cells themselves stimulate exclusively DAP12-associated receptors, but osteoblasts also stimulate FcR-associated receptors. The defective osteoblast function in DAP12^-/- FcR^-/- mice suggests that the osteoclast–osteoblast communication through these receptors may couple bone resorption to formation. It remains to be elucidated whether inhibitory receptors such as FcRIIB and PIR-B (Supplementary Fig. 3a) contribute to counterbalancing the ITAM signal in osteoclast differentiation. It is notable that osteoclastogenesis is enhanced in mice lacking phosphatases such as SHP-1 or SHIP-1, which counterbalance the ITAM signal in the immune system^{23, 24}. The mutations of the ITAM-harbouring adaptors and their associating receptors are related to pathological conditions in the skeletal system^{25, 26, 27}, suggesting that the regulation of costimulatory signals may provide a novel strategy for the treatment of skeletal disorders.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank J. V. Ravetch, H. Kubagawa and M. D. Cooper for providing materials, and M. Kaji, A. Sugahara, Y. Ito, K. Maya, A. Sato, A. Nakamura, M. Isobe, T. Yokochi, A. Izumi, T. Kohro, Y. Matsui, H. Murayama, K. Sato, M. Asagiri and I. Kawai for technical assistance and discussion. This work was supported in part by a grant for Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the CREST and PRESTO programs of Japan Science and Technology Agency (JST), grants for the 21st century COE program 'Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone' and 'Center for Innovative Therapeutic Development Towards the Conquest of Signal Transduction Diseases', Grants-in-Aid for Scientific Research from JSPS and MEXT, Health Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan, grants of the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim, Mochida Medical and Pharmaceutical Research Foundation and a grant from Japan Orthopaedics and Traumatology Foundation.

Competing interests statement

The authors declare no competing financial interests.

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