Legends to Supplementary Information Figures
Figure 1 Analysis of the rescue effects by osteoblast and the involvement of
FcR. a, Coculture of osteoclast precursor cells derived from WT, DAP12,
FcRand DAP12FcR (DKO) mice with osteoblasts derived from the
mutant mice. Osteoblasts derived from WT, DAP12, FcRand
DAP12FcRmice have the same ability to support osteoclastogenesis,
although the in vitro rescue effect by osteoblasts was only partial. The normal
in vivo osteoclastogenesis in FcRmice may be explained as follows: the
level of in vitro osteoclast differentiation depends on both FcR- and DAP12mediated signals and FcR-mediated signals alone cannot induce the full
activation in vitro. On the other hand, DAP12 deficiency may be fully
compensated in vivo through complex mechanisms such as the elevation of
RANKL/M-CSF expression and upregulated FcR-mediated signalling. A recent
report on the partial rescue of osteoclastogenesis from DAP12 BMMs by high
dose M-CSF is consistent with this idea (J. Cell. Biochem. 90, 871, 2003). In
addition, we speculate that the defective bone resorbing activity may upregulate
the osteoclastogenesis through a feedback mechanism. b, The effect of the
culture medium of osteoblasts on RANKL/M-CSF-induced osteoclastogenesis in
DAP12BMMs. Osteoblasts were cultured in the presence of 1, 25 (OH)2
vitamin D3 and dexamethasone under the same conditions as those for
coculture experiments. Culture supernatant of osteoblasts (OB sup) was
recovered after incubation for 1 d () or 2 d (). DAP12BMMs were cultured
in these culture mediums with 10% FBS, 100 ng ml1 soluble RANKL and 10 ng
ml1 M-CSF. In contrast to the positive effect by coculture, we observed no
rescue effect by the culture medium of osteoblasts. These results suggest that
the osteoblast culture medium does not contain soluble factors involved in this
rescue effect, although additional experimentation is required to confirm the
necessity of cell-cell contact. c, Association of FcR or DAP12 with chimaeric
receptors overexpressed in 293T cells. FcR associated with RIIB-OSCAR and
RIIB-FcRIII (left). Association of DAP12 with RIIB-TREM-1, but not with RIIBOSCAR in 293T cells (right). d, Bone morphometric analysis of FcR mice.
There was no significant difference in trabecular bone volume or the number of
osteoclasts. n.s.: not significant. e, Osteoclastogenesis in FcR BMMs
stimulated with RANKL/M-CSF. There was no significant difference in
osteoclast differentiation between WT and FcR BMMs.
Figure 2 Bone morphometric and histological analysis of DAP12FcR
(DKO) mice. a, Osteoblastic parameters in the bone morphometric analysis of
the tibia of WT and DAP12FcR mice (12 weeks of age). For other
parameters, see Fig. 2d in the main text. These results reveal that the
osteoblastic bone formation is also decreased, excluding the possibility that the
increase in bone formation contributes to the increased bone mass. b, Lower
magnification view of the histology of the tibia of WT and DAP12FcR mice.
Arrowheads indicate a small number of osteoclasts observed below epiphyseal
plates in DAP12FcR mice. The appearance of a few number of osteoclasts
below epiphyseal plates and the normal tooth eruption contrast with severe
osteopetrosis in the bone marrow. The detailed mechanism remains to be
elucidated, but it is possible that FcR and DAP12 are required only in the bone
marrow, but not in the periosteal/periodontal environments. In terms of the
osteoclast differentiation in the bone marrow, DAP12 and FcR compensate for
each other by activating the ITAM-mediated common downstream signalling
pathways, but mild osteopetrosis in DAP12 mice is attributed to a specific
function of DAP12 in the regulation of bone-resorbing activity of osteoclasts,
which cannot be compensated by FcR.
Figure 3 ITAM-harbouring adaptor-associated immunoreceptors in osteoclast
lineage cells. a, mRNA expression of ITAM-harbouring adaptors, associated
molecules and immunoreceptors (Affymetrix GeneChip). Multiple
immunoreceptors including inhibitory receptors (indicated by the asterisks) were
expressed by osteoclast precursor cells (pOC) and mature osteoclasts (mOC).
b, FcR-associating proteins on the surface of the osteoclast lineage cells. In
pOC glycosylated proteins with predicted core size of 74 and 33 kDa seemed to
be major whereas in mOC the 33-kDa species became major. The 33-kDa
species disappeared in both OCs from FcRIII mice, indicating that this band
represents FcRIII (relative molecular mass (Mr) 40–60 kDa; core size 33 kDa [J.
Cell. Sci. Suppl. 9, 45, 1988]). Moreover, in the absence of the 33-kDa species
the 74-kDa protein species became the major protein. This glycoprotein may
be PIR-A (Mr 85 kDa; core size 73 kDa [Proc. Natl. Acad. Sci. USA 94, 5261,
1997]). Other minor protein species might include FcRI (Mr 70 kDa; core size
49 kDa [Immunology 78, 358, 1993]), OSCAR (Mr is not known; core size 27
kDa [J. Exp. Med. 195, 201, 2002]) and DCAR (Mr is not known; core size 24
kDa [J. Biol. Chem. 278, 32645, 2003]). Thus, the FcR-associating receptors
on the OC surface may include FcRIII, PIR-A, FcRI, OSCAR and DCAR.
Despite the association of FcR with FcRIII, we observed that the antibody
against FcRIIB/III (2.4G2) had no stimulatory effect even on FcRIIB BMMs
(data not shown). In addition, unlike DAP12FcR mice, the bone phenotype
of DAP12FcRIII mice is almost the same as that of DAP12 mice (M. I.
and T. Takai, unpublished observation), suggesting that FcRIII is not critically
involved in osteoclastogenesis. c, DAP12-associating proteins on the surface
of the osteoclast lineage cells. DAP12-associating proteins with a core size of
16–45 kDa are significant on the cell surface. Among these, in pOC
glycosylated proteins with 45, 39, 29, 25 kDa backbones seemed to be major,
whereas in mOC those with 45, 39, 29 kDa backbones and non-glycosylated
16-kDa protein were eminent. Referring to the data of known core size for each
candidate receptor (TREM-2b and -3, 23 kDa and 18 kDa, respectively [Eur. J.
Immunol. 31, 783, 2001]; MDL-1, 22 kDa [Proc. Natl. Acad. Sci. USA 96,
9792,1999] SIRP1, 42 kDa [H. O. and T. M. unpublished observation]), the
45-kDa protein species might be SIRP1, and 25–28-kDa species might include
TREM-2b and MDL-1. A weak 18-kDa signal might represent TREM-3. These
results suggest that the receptor repertoires associating with DAP12 may
include TREM-2, -3, MDL-1, and SIRP1. d, Effect of plate-bound antibodies
against immunoreceptors on the rate of apoptotic cells in RANKL-stimulated
BMMs. BMMs were stimulated with plate-bound antibodies in the presence of
RANKL (100 ng ml1) and M-CSF (10 ng ml1) for 3 d, and were stained for
TUNEL. There was no significant difference between control IgG and any
antibody stimulation.
Figure 4 Intracellular signalling events downstream of ITAM-harbouring
adaptors. a, Expression of NFATc1, c-Fos and TRAF6 in WT and
DAP12FcR (DKO) cells stimulated with RANKL/M-CSF. Osteoclast
precursor cells were cultured in the presence of RANKL (100 ng ml1) and MCSF (10 ng ml1) for 72 h and were stained with anti-NFATc1 and anti-cFos/anti-TRAF6 antibodies (Santa Cruz) as described (Dev. Cell 3, 889, 2002).
NFATc1 expression in DAP12FcR cells is barely detectable, but the
expression of c-Fos or TRAF6 is still observed. Ph: phase contrast. b,
Induction of NFATc1 in DAP12cellsby PIR stimulation. DAP12 BMMs
were stimulated with plate-bound anti-PIR antibody in the presence of RANKL
(100 ng ml1) and M-CSF (10 ng ml1) for 3 d, and were immunostained with
anti-NFATc1 antibody as described above. Stimulation with anti-PIR antibody
rescued the NFATc1 expression in DAP12 cells. c, Phosphorylation of p38,
JNK and IB by RANKL in WT and DAP12FcR cells. Osteoclast precursor
cells were stimulated by RANKL (100 ng ml1) for indicated periods and cell
lysates were separated by SDS–PAGE followed by immunoblot with
phosphorylation-specific antibodies (Cell Signaling) or other antibodies (p38,
JNK; Cell Signaling, -actin; Sigma). In contrast, the phosphorylation of PLC,
which may be dependent on the phosphorylation of ITAM, is impaired in
DAP12FcR cell (Fig. 4e). RANK is not a tyrosine kinase receptor but
associates with TRAF6 linked to various kinases such as c-Src, TAK1 and MAP
kinases, among which c-Src may be involved in the phosphorylation of ITAM by
RANKL. d, Effect of piceatannol, an inhibitor of the Syk family kinases on
RANKL/M-CSF-induced osteoclastogenesis. The effect of piceatannol was
examined as described (Nature 416, 744, 2002). The proliferation of BMMs
was not affected at these concentrations (data not shown).