BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 253, 395– 400 (1998)
ARTICLE NO. RC989788
RANK Is the Essential Signaling Receptor for Osteoclast
Differentiation Factor in Osteoclastogenesis
Nobuaki Nakagawa,1 Masahiko Kinosaki,1 Kyoji Yamaguchi, Nobuyuki Shima,
Hisataka Yasuda, Kazuki Yano, Tomonori Morinaga, and Kanji Higashio2
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., 519 Ishibashi-machi,
Shimotsuga-gun, Tochigi 329-0512, Japan
Received October 31, 1998
Osteoclast differentiation factor (ODF) is a ligand
for osteoclastogenesis-inhibitory factor/osteoprotegerin
(OCIF/OPG), and mediates an essential signal for os-
teoclastogenesis. Soluble-form ODF binds directly to
osteoclast progenitors, suggesting the presence of a
membrane-bound receptor for ODF (ODFR) on the
cells. To understand the ODF-mediated signal trans-
duction mechanism in osteoclastogenesis, we molecu-
larly cloned ODFR from a mouse macrophage-like os-
teoclast progenitor cell line, C7. Nucleotide sequence
analysis revealed that ODFR is identical to RANK, a
recently identified member of the tumor necrosis fac-
tor receptor (TNFR) family, which is involved in the
regulation of dendritic cell function. A polyclonal an-
tibody against the extracellular domain of RANK in-
duced osteoclastogenesis in the presence of macro-
phage colony-stimulating factor (M-CSF). In contrast,
both a genetically engineered soluble RANK and Fab
fragment of the antibody blocked the binding of ODF
to RANK and ODF-mediated osteoclastogenesis. These
results indicate that RANK is the signaling receptor
essential for ODF-mediated osteoclastogenesis. © 1998
Academic Press
Bone remodeling involves bone resorption by osteo-
clasts and bone synthesis by osteoblasts (1–3). Osteo-
clasts are multinucleated cells that derive from hema-
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed. Fax: 81-285-53-
1314. E-mail: fvbd7042@mb.infoweb.ne.jp.
Abbreviations used: ODF, osteoclast differentiation factor; OCIF,
osteoclastogenesis-inhibitory factor; OPG, osteoprotegerin; TNF, tu-
mor necrosis factor; M-CSF, macrophage colony-stimulating factor;
OCL, osteoclast-like cell; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3;
PTH, parathyroid hormone; PGE2, prostaglandin E2; IL, interleukin;
sODF, soluble ODF; sRANK, soluble RANK; RANK-Ab, anti-RANK
polyclonal antibody; Fab-RANK, Fab fragment of RANK-Ab; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; TRAP, tartrate-resistant
acid phosphatase.
395
topoietic cells of the monocyte-macrophage lineage. A
co-culture system of spleen cells and osteoblasts or
bone marrow stromal cells is established to produce
osteoclast-like cells (OCLs) (4, 5). A variety of osteo-
tropic factors, such as 1,25-dihydroxyvitamin D3
[1,25(OH)2D3], parathyroid hormone (PTH), prosta-
glandin E2 (PGE2), and interleukin 11 (IL-11), are es-
sential for inducing OCL formation in the co-cultures
(1, 2, 6). These osteotropic factors seem to transduce
their signals to osteoblasts/stromal cells. Suda and co-
workers hypothesized that a membrane-bound factor,
designated “osteoclast differentiation factor (ODF)” is
expressed on osteoblasts/stromal cells in response to
osteotropic factors, and that it transduces a signal for
osteoclastogenesis to osteoclast progenitors through
cell-to-cell interaction (1, 2, 6) (Fig. 1).
We recently purified and molecularly cloned
osteoclastogenesis-inhibitory factor (OCIF) (7, 8) (also
called osteoprotegerin [OPG] (9)). OCIF/OPG is a se-
creted member of the tumor necrosis factor receptor
(TNFR) family, and inhibits osteoclastogenesis stimu-
lated by 1,25(OH)2D3, PTH ,or IL-11 (7, 8) (Fig. 1).
Analyses of rats injected with OCIF/OPG (8, 9), trans-
genic mice overexpressing OCIF/OPG (9), and OCIF/
OPG knockout mice (10, 11) demonstrated that OCIF/
OPG plays an important role as an inhibitor of
osteoclastogenesis in vivo. Subsequently, we succeeded
in molecular cloning of ODF as a ligand for OCIF/OPG
(12). ODF (also called OPG ligand [OPGL] (13)) is a
member of the membrane-associated tumor necrosis
factor (TNF) ligand family, and induces osteoclast dif-
ferentiation from mouse spleen cells, mouse bone mar-
row cells, C7 mouse macrophage-like cells, or human
peripheral blood mononuclear cells, which cells are
co-treated with macrophage colony-stimulating factor
(M-CSF) in the absence of osteoblasts/stromal cells and
osteotropic factors (12–14) (Fig. 1). In a fetal mouse
organ culture system, a genetically engineered soluble
form ODF (sODF) induces bone resorption. Both anti-
ODF antibody and OCIF/OPG negated bone resorption
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Vol. 253, No. 2, 1998
FIG. 1. A model illustrating a molecular mechanism by which osteoblasts/stromal cell regulate osteoclastogenesis (1, 6, 12).
elicited by osteotropic factors, such as 1,25(OH)2D3,
PTH, and PGE2 (15). The results established that ODF
is a long sought ligand expressed on osteoblasts/
stromal cells in response to osteotropic factors and that
it mediates an essential signal to osteoclast progenitors
for their differentiation into active osteoclasts. Binding
analysis demonstrated that sODF specifically binds to
C7 cells (12), suggesting the presence of a membrane-
bound ODF receptor (ODFR) on osteoclast progenitors
(Fig. 1). ODF was found to be identical to TRANCE (16,
17)/RANK ligand (RANKL) (18), a factor regulating
dendritic-cell function. This raised the possibility that
ODF mediates osteoclastogenesis through RANK (12,
13, 19) (Fig. 1). Alternatively, it is possible that an-
other receptor transduces the signaling for osteoclas-
togenesis since several members of the TNF ligand
family, such as TNFa, LTa, LTb, and TRAIL, utilize
multiple receptors (20 –24).
In the present study, we screened a C7 cDNA expres-
sion library by panning to isolate a functional ODFR
for osteoclastogenesis. Sequencing analysis of the pu-
tative ODFR cDNAs showed that they encoded RANK.
To examine whether RANK is the only signaling recep-
tor for ODF, we prepared a genetically engineered sol-
uble RANK (sRANK), anti-RANK polyclonal antibody
(RANK-Ab), and Fab fragment of the antibody (Fab-
RANK). RANK-Ab induced OCLs from mouse spleen
cells. Both sRANK and Fab-RANK suppressed the
binding of ODF to RANK and the ODF-mediated OCL
formation. This report provides the first direct evi-
dence that RANK is a receptor for ODF and is essential
for osteoclastogenesis.
MATERIALS AND METHODS
Reagents. Recombinant OCIF was prepared as described previ-
ously (25). sODF was prepared as described previously (12, 15). C7
cells were a generous gift from Dr. S.-I. Hayashi (Tottori University).
Screening of cDNA expression library. An expression vector,
pSRaEX was constructed by inserting the annealed product of two
oligonucleotides: 59-GAATTCTAGAGCTAGCGGCCGCGG-39 (sense)
and 59-GTACCCGCGGCCGCTAGCTCTAGAATTCTGCA-39 (anti-
sense) into the PstI-KpnI site of pcDL-SRa296 (26). A cDNA expres-
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sion library in pSRaEX was constructed using the Great length
cDNA synthesis kit (Clontech) from poly(A)1RNA of C7 cells. Screen-
ing of the C7 cDNA expression library was performed by several
rounds of transfection and selection by panning (27). After four cycles
of panning, plasmid DNA was prepared from randomly-selected
transformants and subjected to a binding assay as described previ-
ously (12). The insert fragments in these cDNA clones obtained were
sequenced using AmpliTaq DyeDeoxy Terminator Cycle Sequencing
on an ABI 377 sequencer (Perkin-Elmer).
Binding analysis of 125I-labeled sODF to COS7 cells expressing
RANK. Radioiodination of sODF was performed as described (12).
COS7 cells transfected with pRANK (a RANK expression vector) or
pcDL-SRa296 (an empty vector) were cultured for two days, and then
they were incubated with 1.5 nM of 125I-labeled sODF (2 3 105 cpm)
at 37¡C for 1 hr in the presence or absence of 600 nM of unlabeled
sODF as described previously (12). Cells were washed with
phosphate-buffered saline (PBS) three times, and were lysed in 500
ml of 0.1 M NaOH. Radioactivity of the lysate was measured as
described previously (12).
Northern blot analysis. Isolation of total and poly(A)1RNAs and
hybridization, were done as described previously (12, 28).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was
used as an internal control.
Preparation of sRANK. The vector to express sRANK was con-
structed as follows. A DNA fragment encoding myc-His tags was
amplified using pSecTag A (Invitrogen) as a template and TagF
(59-GCTGGCTAGCCACCATGGAGACAGACACACTC-39) and BgR
(59-CCAGATCTTGATCAGCGGGTTTAAACTCAATGATG-39) as prim-
ers by polymerase chain reaction (PCR). The amplified fragment was
FIG. 2. sODF specifically binds to RANK. Binding analysis of
125
I-labeled sODF to COS7 cells transfected with a RANK expression
vector (COSRANK) or with the empty vector (COSVec). Data are ex-
pressed as means6standard errors (SE) of three cultures.
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Vol. 253, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M-CSF in the presence or absence of 100 mg/ml of RANK-Ab. The

cells were subjected to TRAP staining as described previously (5).
Effect of Fab-RANK, sRANK, or OCIF on the binding of sODF to
sRANK. Each well in a 96-well plate was coated overnight at 4°C with
sRANK in PBS (312 ng/ml for the assay of Fab-RANK and 1.25 mg/ml
for the assay of sRANK and OCIF). The wells were blocked with 300 ml
of 50% BlockAce (Snow Brand, Tokyo Japan) in PBS for 1 hr at room
temperature and washed three times with PBS containing 0.05%
Tween 20 (PBS-T). For the assay of Fab-RANK, serially diluted Fab-
RANK or Fab-control was added to the wells of the sRANK-immobilized
plate. The plate was incubated at room temperature for 2 hr and then
washed three times with PBS-T. Then, 100 ng/ml of sODF was added to
the wells, and the plate was further incubated at room temperature for
2 hr. The wells were washed three times with PBS-T, and sODF in the
wells was detected using horseradish peroxidase (HRP)-labeled anti-
ODF antibody by enzyme-linked immunosorbent assay (ELISA). For
the assay of sRANK and OCIF, 25 ng/ml of sODF was premixed with
FIG. 3. Expression of the RANK gene. Two blots loaded with 20
serially-diluted sRANK or OCIF, incubated at room temperature for 1
mg of total RNA from C7 cells, or OCLs induced from spleen cells
hr, and then the mixture was added to the sRANK-immobilized plate.
with sODF and M-CSF, and a blot of mouse tissue poly(A)1RNA (2
The plate was incubated at room temperature for 2 hr. The wells were
mg) were probed with RANK cDNA. The blot of tissue poly(A)1RNA
washed three times with PBS-T, and sODF in the wells was detected by
was reprobed with GAPDH cDNA. RNA size markers (Invitrogen)
ELISA as described above.
are shown on the left of each panel.

RESULTS
digested with restriction endonucleases NheI and BglII and inserted Cloning of ODF Receptor
between NheI and BamHI sites of pCEP4 (Invitrogen) to yield
pCEP4-myc-His. A DNA fragment encoding Igk chain leader se- By screening a cDNA expression library of C7 cells
quence was amplified by PCR using pSecTag A as a template and using sODF, eleven positive clones were isolated. Se-
TagF and mRANKR (59-AGGAGTGACCTGGTCACCAGTGGAACC-
TGGAACCCA-39) as primers. A DNA fragment encoding the extra-
cellular domain of RANK (Gln30-Pro213) was amplified by PCR
using pRANK as a template and mRANKF (59-TCCACTGGTGACC-
AGGTCACTCCTCCATGCACCCAG-39) and YLPXhR (59-CCCTCG-
AGGGGCAGGTAAGCCTGGGCCTCC-39) as primers. The two frag-
ments were fused by the secondary PCR using primers TagF and
YLPXhR. The chimeric DNA fragment encoding Igk chain leader
sequence and the extracellular domain of RANK was digested with
restriction endonucleases NheI and XhoI and the NheI/XhoI frag-
ment obtained was inserted between NheI and XhoI sites of pCEP4-
myc-His to yield pCEP4-sRANK. pCEP4-sRANK was transfected
into 293-EBNA cells (Invitrogen) using FuGene 6 (Boehringer Mann-
heim) according to the manufacturer’s protocol. sRANK was purified
from the conditioned medium of the transfectants by Ni-NTA column
chromatography (QIAGEN), followed by gel filtration.
Preparation of anti-RANK polyclonal antibody and its Fab frag-
ment. Male JW rabbits were subcutaneously immunized, four
times at two weeks intervals, with 1 ml of emulsion prepared by
mixing sRANK (200 mg/ml) with equal volume of complete Freund’s
adjuvant (Difco). Anti-RANK polyclonal antibody (RANK-Ab) was
purified from serum obtained from the rabbits 10 days after the final
injection using a HiTrap Protein A column (Amersham Pharmacia
Biotech) according to the manufacturer’s protocol. Fab fragment
prepared from either RANK-Ab (Fab-RANK) or non-immunized rab-
bit immunoglobulin (IgG) (Fab-control) was prepared using the Im-
munoPure Fab preparation kit (PIERCE).
OCL formation assay. For the examination of the effect of
sRANK, Fab-RANK or OCIF on the ODF-mediated OCL formation,
mouse spleen cells (7 3 105 cells) obtained from 6- to 15-week-old FIG. 4. sRANK inhibited ODF-mediated osteoclastogenesis. (A)
male ddY mice were cultured for 3 or 6 days in a 24-well plate in Effect of sRANK and OCIF on ODF-mediated OCL formation. Mouse
a-MEM containing 10% fetal calf serum (FCS), 20 ng/ml of human spleen cells were cultured in a-MEM containing 10% FCS and 20 ng/ml
M-CSF (Green Cross, Osaka, Japan), 50 or 100 ng/ml of sODF, and of M-CSF for a week in the presence or absence of the indicated con-
serially-diluted sRANK, Fab-RANK or OCIF. Activity of tartrate- centrations of sODF, sRANK, and OCIF. OCL formation was evaluated
resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) by measuring TRAP activity. Data are expressed as means6SE of three
in the cells was measured as described (7). For the examination of cultures. (B) Effect of sRANK (open circle) and OCIF (closed circle) on
the effect of RANK-Ab on OCL formation, mouse spleen cells were the binding of sODF to the immobilized sRANK was assessed by a
cultured for 6 days in a-MEM containing 10% FCS and 20 ng/ml of solid-phase ELISA as described in Materials and Methods.

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Vol. 253, No. 2, 1998
FIG. 5. Morphology of OCLs induced from spleen cells by RANK-Ab. Mouse spleen cells were cultured in a-MEM containing 10% FCS
and 20 ng/ml of M-CSF for a week, in the presence (right panel) or absence (left panel) of 100 mg/ml of RANK-Ab. The cells were stained for
TRAP. TRAP-positive cells appeared as red cells. Bar 5 100 mm.
quence analysis revealed that all these clones encoded
RANK. 125I-labeled sODF specifically bound to COS-7
cells transfected with a RANK expression vector
(COSRANK), but not to COS-7 cells transfected with the
empty vector (COSVec) (Fig. 2). When a 400-fold excess
of unlabeled sODF was simultaneously added, the
binding was completely abolished (Fig. 2).
Expression of the RANK Gene in Osteoclast
Progenitors, OCLs, and Trabecular Bone
Northern blot analysis using a 1.0 kb RANK probe
detected transcripts of approximately 5.5, 4.0, 3.5 and
3.0 kb in C7 cells and OCLs induced from spleen cells
(Fig. 3). The 5.5 kb mRNA was expressed strongly in
trabecular bone and weakly in spleen and bone marrow
(Fig. 3). Strong signal of 5.5 kb was also seen in lung,
brain, kidney, small intestine, and thymus, although
the gene was found to be expressed ubiquitously (data
not shown).
Soluble RANK (sRANK) Abolishes ODF-Mediated
Osteoclastogenesis
sODF induced OCL formation from spleen cells in
the presence of M-CSF (Fig. 4A). Addition of sRANK to
the culture inhibited the OCL formation in a dose-
dependent manner (Fig. 4A). Addition of OCIF also
inhibited the OCL formation, and the effective dose of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OCIF was much lower than that of sRANK (Fig. 4A). A
solid-phase ELISA using immobilized sRANK revealed
that OCIF is much more effective than sRANK as a
competitor for the binding of sODF to sRANK (Fig. 4B).
Anti-RANK Polyclonal Antibody (RANK-Ab) Mimics
ODF-Mediated Osteoclastogenesis
To understand the role of RANK in osteoclastogen-
esis, we examined the effect of RANK-Ab on the OCL
formation. RANK-Ab induced OCL formation from
spleen cells in the absence of sODF (Fig. 5). The OCLs
formed were morphologically similar to those induced
by sODF. M-CSF was essential for the RANK-Ab-
mediated osteoclastogenesis. While simultaneous addi-
tion of sRANK with the RANK-Ab completely inhibited
the OCL formation, OCIF did not affect the RANK-Ab-
mediated osteoclastogenesis (data not shown).
Fab Fragment of Anti-RANK Polyclonal Antibody
(Fab-RANK) Abolishes the ODF-Mediated
Osteoclastogenesis
To elucidate whether ODF mediates osteoclastogen-
esis through RANK, we examined the effect of Fab-
RANK on the OCL formation induced by sODF. As
shown in Fig. 6A, Fab-RANK strongly inhibited the
ODF-mediated OCL formation in a dose-dependent
manner. In contrast, Fab-control had no effect on the
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Vol. 253, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 6. Fab-RANK inhibited ODF-mediated OCL formation. (A)
Effect of Fab-RANK on the ODF-mediated OCL formation. Mouse
spleen cells were precultured for 1 hr in a-MEM containing 10% FCS
and 20 ng/ml of M-CSF in the presence or absence of the indicated
concentrations of Fab-RANK, Fab-control or OCIF. Then sODF was
added to some cultures, and the cells were cultured for 3 days. OCL
formation was evaluated by measuring TRAP activity. Data are ex-
pressed as mean6SE of three cultures. (B) Fab-RANK blocked the
binding of sODF to sRANK. The effect of Fab-RANK (open circle) and
Fab-control (closed circle) on the binding of sODF to sRANK was as-
sessed by a solid-phase ELISA as described in Materials and Methods.
OCL formation. A solid-phase ELISA employing immo-
bilized sRANK confirmed that Fab-RANK blocked the
binding of sODF to sRANK in a dose-dependent man-
ner (Fig. 6B). Fab-control had no effect on the binding.
DISCUSSION
In this report, we described the identification of the
receptor for ODF in osteoclastogenesis. From the fol-
lowing evidence, we conclude that RANK transduces
an essential signal for osteoclastogenesis into osteo-
clast progenitors by binding to ODF. 1) All putative
ODFR cDNA clones isolated from a C7 cDNA library
encoded RANK. 2) sRANK and Fab-RANK completely
inhibited ODF-mediated osteoclastogenesis by binding
to ODF and RANK, respectively. 3) RANK-Ab mim-
icked the ODF function.
We prepared two types of antibody against RANK: a
polyclonal divalent antibody, RANK-Ab, and a mono-
valent form of RANK-Ab, Fab-RANK. RANK-Ab was
399
expected to transduce RANK-mediated signal because
of its ability to cross-link and cluster antigens as is the
case with agonist antibodies against TNFR (29 –33). In
contrast, Fab-RANK was thought to be incapable of
clustering the surface receptors and act as an antago-
nist by blocking the ligand-receptor binding. Use of the
two antibodies enabled us to further characterize the
role of RANK in osteoclastogenesis. The complete inhi-
bition of ODF-mediated osteoclastogenesis by Fab-
RANK indicates that RANK is the only signaling re-
ceptor for ODF. The mimicking of the ODF function by
RANK-Ab suggests that the clustering of RANK is
required for the RANK-mediated signaling.
OCIF/OPG was thought to be a naturally occurring
decoy receptor for ODF (12–15, 19). In this report, we
demonstrated that both OCIF/OPG and sRANK inhib-
ited ODF-mediated osteoclastogenesis by interfering
the binding of ODF to RANK. We also showed that
OCIF/OPG had no effect on RANK-Ab-mediated osteo-
clastogenesis, while sRANK completely inhibited it.
These results provide the first direct evidence that
OCIF/OPG acts as a decoy receptor for ODF to compete
against RANK.
Northern blot analysis shows that expression of the
RANK gene is strong in bone, the only tissue where
osteoclasts are present. Both an osteoclast progenitor
cell line C7 and OCLs expressed abundant RANK
mRNA. Other tissues with osteoclast progenitors such
as spleen and bone marrow also expressed the mRNA.
These results suggest that RANK expression is neces-
sary but not sufficient for osteoclastogenesis. On the
other hand, the involvement of RANK and ODF/
TRANCE/RANKL in T-cell activation has recently
been reported (16 –18). The ubiquitous expression of
the RANK gene may suggest its new role(s) in other
organs besides osseous and immune tissues.
In conclusion, ODF mediates a signal through RANK
expressed on the osteoclast progenitors for their differ-
entiation into osteoclasts. The discovery of RANK, as
well as ODF and OCIF/OPG, opens new avenues of
research into the mechanisms regulating osteoclast
differentiation and activation. Further characteriza-
tion of the signaling pathway downstream of RANK is
urgently needed.
ACKNOWLEDGMENTS
We thank K. Aoki and K. Yoshiba for preparing anti-RANK poly-
clonal antibody.
REFERENCES
1. Suda, T., Takahashi, N., and Martin, T. J. (1992) Endocr. Rev.
13, 66 – 80.
2. Suda, T., Udagawa, N., and Takahashi, N. (1996) in Principles of
Bone Biology (Bilezikian, J. P., Raisz, L. G., and Rodan, G. A.,
Eds.), pp. 87–102, Academic Press, San Diego.
3. Roodman, G. D. (1996) Endocr. Rev. 17, 308 –332.
Vol. 253, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4. Takahashi, N., Akatsu, T., Udagawa, N., Sasaki, T., Yamaguchi,
A., Moseley, J. M., Martin, T. J., and Suda, T. (1988) Endocri-
nology 123, 2600 –2602.
5. Udagawa, N., Takahashi, N., Akatsu, T., Sasaki, T., Yamaguchi,
A., Kodama, H., Martin, T. J., and Suda, T. (1989) Endocrinology
125, 1805–1813.
6. Suda, T., Udagawa, N., Nakamura, I., Miyaura, C., and Taka-
hashi, N. (1995) Bone 17, 87S–91S.
7. Tsuda, E., Goto, M., Mochizuki, S., Yano, K., Kobayashi, F.,
Morinaga, T., and Higashio, K. (1997) Biochem. Biophys. Res.
Commun. 234, 137–142.
8. Yasuda, H., Shima, N., Nakagawa, N., Mochizuki, S., Yano, K.,
Fujise, N., Sato, Y., Goto, M., Yamaguchi, K., Kuriyama, M.,
Kanno, T., Murakami, A., Tsuda, E., Morinaga, T., and Higashio,
K. (1998) Endocrinology 139, 1329 –1337.
9. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang,
M.-S., Luthy, R., Nguyen, H. Q., Wooden, S., Bennett, L., Boone,
T., Shimamoto, G., DeRose, M., Eliott, R., Colombero, A., Tan,
H.-L., Trail, G., Sullivan, J., Davy, E., Bucay, N., Renshaw-Gegg,
L., Hughes, T. M., Hill, D., Pattison, W., Campbell, P., Sander,
S., Van, G., Tarpley, J., Derby, P., Lee, R., Amgen EST Program,
and Boyle, W. J. (1997) Cell 89, 309 –319.
10. Mizuno, A., Amizuka, N., Irie, K., Murakami, A., Fujise, N.,
Kanno, T., Sato, Y., Nakagawa, N., Yasuda, H., Mochizuki, S.,
Gomibuchi, T., Yano, K., Shima, N., Washida, N., Tsuda, E.,
Morinaga, T., Higashio, K., and Ozawa, H. (1998) Biochem.
Biophys. Res. Commun. 247, 610 – 615.
11. Bucay, N., Sarosi, I., Dunstan, C., Morony, S., Tarpley, J., Cap-
parelli, C., Scully, S., Tan, H., Xu, W., Lacey, D., Boyle, W., and
Simonet, W. (1998) Genes Dev 12, 1260 –1268.
12. Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki,
M., Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M., Murakami,
A., Tsuda, E., Morinaga, T., Higashio, K., Udagawa, N., Taka-
hashi, N., and Suda, T. (1998) Proc. Natl. Acad. Sci. USA 95,
3597–3602.
13. Lacey, D., Timms, E., Tan, H., Kelley, M., Dunstan, C., Burgess,
T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H.,
Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian,
Y., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J.,
Delaney, J., and Boyle, W. (1998) Cell 93, 165–176.
14. Matsuzaki, K., Udagawa, N., Takahashi, N., Yamaguchi, K.,
Yasuda, H., Shima, N., Morinaga, T., Toyama, Y., Yabe, Y.,
Higashio, K., and Suda, T. (1998) Biochem. Biophys. Res. Com-
mun. 246, 199 –204.
15. Tsukii, K., Shima, N., Mochizuki, S., Yamaguchi, K., Kinosaki,
400
M., Yano, K., Shibata, O., Udagawa, N., Yasuda, H., Suda, T.,
and Higashio, K. (1998) Biochem. Biophys. Res. Commun. 246,
337–341.
16. Wong, B. R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao,
M., Kalachikov, S., Cayani, E., Bartlett III, F. S., Frankel, W. N.,
Lee, S. Y., and Choi, Y. (1997) J. Biol. Chem. 272, 25190 –25194.
17. Wong, B. R., Josien, R., Lee, S. Y., Sauter, B., Li, H.-L., Stein-
man, R. M., and Choi, Y. (1997) J. Exp. Med. 186, 2075–2080.
18. Anderson, D. A., Maraskovsky, E., Billingsley, W. L., Dougall,
W. C., Tometsko, M. E., Roux, E. R., Teepe, M. C., DuBose, R. F.,
Cosman, D., and Galibert, L. (1997) Nature 390, 175–179.
19. Fuller, K., Wong, B., Fox, S., Choi, Y., and Chambers, T. J. (1998)
J. Exp. Med. 188, 997–1001.
20. Beutler, B., and van Huffel, C. (1994) Science 264, 667– 668.
21. Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76,
959 –962.
22. Cosman, D. (1994) Stem Cells 12, 440 – 455.
23. Gruss, H.-J., and Dower, S. K. (1995) Blood 85, 3378 –3404.
24. Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305–1308.
25. Tomoyasu, A., Goto, M., Fujise, N., Mochizuki, S., Yasuda, H.,
Morinaga, T., Tsuda, E., and Higashio, K. (1998) Biochem. Bio-
phys. Res. Commun. 245, 382–387.
26. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K.,
Yoshida, M., and Arai, N. (1988) Mol. Cell Biol. 8, 466 – 472.
27. Aruffo, A., and Seed, B. (1987) Proc. Natl. Acad. Sci. USA 84,
8573– 8577.
28. Yasuda, H., Imai, E., Shiota, A., Fujise, N., Morinaga, T., and
Higashio, K. (1996) Hepatology 24, 636 – 642.
29. Engelmann, H., Holtmann, H., Brakebush, C., Avni, Y. S., Sarov,
I., Nophar, Y., Hadas, E., Leitner, O., and Wallach, D. (1990)
J. Biol. Chem. 265, 14497–14504.
30. Espevik, T., Brockhaus, M., Loetscher, H., Nonstad, U., and
Shalaby, R. (1990) J. Exp. Med. 171, 415– 426.
31. Tartaglia, L. A., Weber, R. F., Figari, I. S., Reynolds, C., Palla-
dino, M. A., and Goeddel, D. V. (1991) Proc. Natl. Acad. Sci. USA
88, 9292–9296.
32. Tartaglia, L. A., Goeddel, D. V., Reynolds, C., Figari, I. S., We-
ber, R. F., Fendly, B. M., and Palladino, M. A. (1993) J. Immunol.
151, 4637– 4641.
33. Degli-Esposti, M. A., Davis-Smith, T., Din, W. S., Smolak, P. J.,
Goodwin, R. G., and Smith, C. A. (1997) J. Immunol. 158, 1756 –
1762.