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Vol. 95, Issue 7, 3597-3602, March 31, 1998
* Research Institute of Life Science, Snow Brand Milk Products Co.,
Ltd., 519 Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan; and
§ Department of Biochemistry, School of Dentistry, Showa
University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Communicated by Hector F. DeLuca, University of Wisconsin, Madison,
WI, January 22, 1998 (received for review December 15, 1997)
Osteoclasts, the multinucleated cells that resorb bone, develop
from hematopoietic cells of monocyte/macrophage lineage.
Osteoclast-like cells (OCLs) are formed by coculturing spleen cells
with osteoblasts or bone marrow stromal cells in the presence of
bone-resorbing factors. The cell-to-cell interaction between
osteoblasts/stromal cells and osteoclast progenitors is essential for
OCL formation. Recently, we purified and molecularly cloned
osteoclastogenesis-inhibitory factor (OCIF), which was identical to
osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor
necrosis factor receptor family and inhibits osteoclastogenesis by
interrupting the cell-to-cell interaction. Here we report the
expression cloning of a ligand for OPG/OCIF from a complementary DNA
library of mouse stromal cells. The protein was found to be a member of
the membrane-associated tumor necrosis factor ligand family and induced
OCL formation from osteoclast progenitors. A genetically engineered
soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein
is osteoclast differentiation factor (ODF), a long-sought ligand
mediating an essential signal to osteoclast progenitors for their
differentiation into osteoclasts. ODF was found to be identical to
TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only
osteoclastogenesis but also immune system.
Hemopoietic precursor cells differentiate into osteoclasts at
bone-resorbing sites under the control of osteotropic hormones and
local factors produced in the microenvironment (1-4). We previously
established a cell culture system to produce osteoclast-like cells
(OCLs) using cocultures of spleen cells and osteoblasts or bone marrow
stromal cells (5, 6). In the cocultures, OCLs are formed from spleen
cells in the presence of such stimulators of bone resorption as
interleukin 6 (IL-6), IL-11, parathyroid hormone (PTH), prostaglandin
E2 (PGE2), and
1,25-dihydroxyvitamin D3
[1,25(OH)2D3] (1-3, 5,
6). These stimulators are classified into three categories in terms of
signal transduction pathways: vitamin D receptor-mediated signals
[1,25(OH)2D3]; protein
kinase A-mediated signals (PTH and PGE2); and
gp130-mediated signals (IL-6 and IL-11). They appear to transduce the
signals that induce osteoclastogenesis to osteoblasts/stromal cells
(1-3). No OCLs are formed, even in the presence of
1,25(OH)2D3, when the
cocultures of osteoblasts/stromal cells and spleen cells are
separated with a membrane filter (5-7). These observations indicated
that osteoblasts/stromal cells are essential for in vitro
osteoclastogenesis through a cell-to-cell interaction (1-3). Thus, we
proposed a hypothetical membrane-bound factor(s) that is expressed on
osteoblasts/stromal cells in response to the stimulators of bone
resorption and induces osteoclastogenesis by signaling to osteoclast
progenitors. We named the factor "osteoclast differentiation factor
(ODF)" (1-3).
Recently, we reported the purification of osteoclastogenesis-inhibitory
factor (OCIF) from conditioned medium of human fibroblasts (8). OCIF
specifically inhibits in vitro OCL formation elicited through the three distinct signaling pathways stimulated by
1,25(OH)2D3, PTH, or IL-11
(8). The nucleotide sequence analysis of OCIF cDNA (9) has demonstrated
that OCIF is identical to osteoprotegerin (OPG) (10), which was
initially identified as a novel secreted member of the tumor-necrosis
factor (TNF) receptor family in an expressed sequence tag cDNA project
(10). The analyses of transgenic mice overexpressing OPG/OCIF and
animals injected with OPG/OCIF have demonstrated that OPG/OCIF
suppresses bone resorption associated with osteoclast development (9,
10). OPG/OCIF lacks apparent transmembrane domain (9, 10) and seems
to act as a soluble inhibitory receptor in
osteoclastogenesis.
A mouse bone marrow-derived stromal cell line, ST2, is known to support
OCL formation from mouse spleen cells in the presence of
1,25(OH)2D3 and
dexamethasone (Dex) (6). We have recently reported that OPG/OCIF
specifically binds to ST2 cells treated with
1,25(OH)2D3 and Dex, but
not to untreated ST2 cells (8, 9). When the binding sites on the
treated ST2 cells were occupied by OPG/OCIF, the cells failed to
support OCL formation from spleen cells (9). Cross-linking study using
[125I]OPG/OCIF revealed that a 40-kDa protein
on the treated ST2 cells binds to OPG/OCIF (9). These results raised
the possibility that the binding protein of 40 kDa is a ligand for
OPG/OCIF and identical to ODF. Thus, we screened a cDNA expression
library of ST2 cells treated with
1,25(OH)2D3 and Dex to
isolate the cDNA encoding the OPG/OCIF binding protein. Here we
report that the protein is a member of the membrane-associated TNF
ligand family and has characteristics that satisfy the major criteria
of ODF.
Cell Culture.
ST2 cells (Riken Cell Bank, Tsukuba) were
cultured in cDNA Cloning.
A cDNA expression library in pcDL-SR Preparation of a Soluble ODF (sODF).
sODF was prepared by
fusing the extracellular domain of ODF
(Asp76-Asp316) to the
C-terminal end of thioredoxin using ThioFusion Expression System
(Invitrogen). A DNA fragment encoding the extracellular domain of ODF
was prepared by digesting pOBM291 with BamHI and EcoRI. PCR was performed using ODF cDNA as a template to
amplify the 3' portion of the cDNA with the following two primers:
5'-ATCAGAAGACAGCACTCACT-3' and
5'-GGGGTCGACCTAGGACATCCATGCTAATGTTCC-3'. The PCR product
was then digested with SalI and EcoRI and a
160-bp fragment was isolated. The BamHI-EcoRI
and SalI-EcoRI fragments and pTrxFus vector
digested with BamHI and SalI were ligated to
yield pTrx-ODF. An Escherichia coli strain GI724 was
transformed with pTrx-ODF. sODF was purified from the soluble
cytoplamic fraction of the transformants by affinity chromatography on
an OPG/OCIF-immobilized column and by gel filtration chromatography.
Expression and Molecular Characterization of Full-Length ODF.
COS-7 cells transfected with the ODF expression vector or the empty
vector were cultured for 2 days. The cells were pulse-labeled with 0.2 mCi/ml (1 Ci = 37 Gbq) Express Protein Labeling Mix (NEN) for
4 h in cysteine-methionine-free medium, washed twice with PBS, and
lysed in 10 mM of Tris·HCl, pH 8.0, containing 1% Triton X-100,
0.14 M of NaCl, 0.1% bovine hemoglobin, and protease inhibitors (12).
After removal of cell debris by centrifugation, the lysates (100 µl)
were incubated with or without 1 µg of OPG/OCIF for 1 h at
4°C. Five micrograms of anti-OPG/OCIF or mouse immunogloblin (IgG)
was added to the mixture and the complexes formed were bound to protein
A-Sepharose gel. OPG/OCIF-associated immunoprecipitates were
analyzed on a 12.5% SDS/polyacrylamide gel under reducing conditions.
OCL Formation Assay.
COS-7 cells transfected with the ODF
expression vector or the empty vector were cultured for 2 days on
coverslips in a 24-well plate, fixed in PBS containing 1%
paraformaldehyde for 8 min at room temperature, and washed four times
with PBS. Mouse spleen cells (7 × 105
cells) obtained from 6- to 15-week-old male ddY mice (6) were cultured
for 6 days either on the fixed cells in Dentine Resorption Assay.
Spleen cells (2 × 107 cells) were cultured in Binding Analysis of [125I]sODF to C7 Cells.
Radioiodination of sODF with
[125I]Bolton-Hunter Reagent (NEN) was
performed as described (14). C7 cells (1 × 105 cells) were cultured in a 24-well plate.
Binding analysis was performed by incubating C7 cells with 1.0 × 106 cpm of [125I]sODF for
1.5 h at 37°C in the presence or absence of 400-fold excess
unlabeled sODF as described previously (9).
Northern Blot Analysis.
Isolation of total RNA and
hybridization were done as described (15).
Poly(A)+ RNA was isolated from total RNA using an
oligo(dT) cellulose spin column. A blot contained 2 µg per lane of
poly(A)+ RNA from trabecular bone and bone marrow
of mouse femur (see Fig. 4C). Mouse multitissue Northern
blots (OriGene Technologies, Rockville, MD) contained 2 µg per lane
of poly(A)+ RNA (Fig. 4C). Other blots
contained 20 µg per lane of total RNA (Fig. 4 A and
B). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
was used as an internal control.
Preparation of Primary Osteoblasts.
Primary osteoblasts were
prepared from calvaria of newborn ddY mice as described (16). The
osteoblasts were cultured in Cloning of Mouse ODF cDNA.
By screening a cDNA expression
library of ST2 cells treated with
1,25(OH)2D3 and Dex using
[125I]OPG/OCIF, a cDNA clone, designated
pOBM291, was isolated from
Cell Biology
Osteoclast differentiation factor is a ligand for
osteoprotegerin/osteoclastogenesis-inhibitory factor and is
identical to TRANCE/RANKL
,,,
,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-minimal essential medium (MEM) (GIBCO/BRL) containing
10% fetal calf serum (FCS). COS-7 cells (Riken Cell Bank) were
cultured in Dulbecco's modified eagle's medium (DMEM; GIBCO/BRL)
containing 10% FCS. C7 cells, which were a generous gift from S.-I.
Hayashi (Tottori University, Yonago), were cultured in -MEM
containing 10% FCS and 1 ng/ml of human macrophage
colony-stimulating factor (M-CSF) (Green Cross, Osaka).
296 (11)
was constructed using Great Lengths cDNA synthesis kit (CLONTECH) from
poly(A)+ RNA of ST2 cells cultured in the
presence of 10
8 M of
1,25(OH)2D3 and
107 M of Dex for 5 days. The resulting ST2
expression library was plated onto 24-well plates at a density of
100 bacterial colonies per well. Subpools of plasmid DNA were
prepared from each bacterial plate using a Qiawell plasmid purification
system (Qiagen, Chatwsworth, CA) and were transfected into COS-7 cells
grown in 24-well plates using Lipofectamine (GIBCO/BRL).
Transfectants were cultured for 2 days and incubated with 3 × 105 cpm of
[125I]OPG/OCIF at 37°C for 1 h as
described (9). A positive pool obtained was partitioned into smaller
subpools until a single cDNA clone, designated pOBM291, was isolated.
The cDNA was sequenced using AmpliTaq DyeDeoxy Terminator
Cycle Sequencing on an ABI 377 sequencer (Perkin-Elmer). For the
binding analysis using [125I]OPG/OCIF, COS-7
cells transfected with pOBM291 (the ODF expression vector) or
pcDL-SR296 (the empty vector) were cultured for 2 days, and then
incubated with 1 nM of [125I]OPG/OCIF (1 × 106 cpm) at 37°C for 1 h in the
presence or absence of 400 nM of unlabeled OPG/OCIF as described (9).
-MEM containing 10% FCS and
10 ng/ml of human M-CSF or in the same medium in the presence or
absence of various concentrations of sODF in a 24-well plate. After
treatment, the cells were subjected to tartrate-resistant acid
phosphatase (TRAP) staining, calcitonin binding, and autoradiography. For TRAP staining, the cells were fixed and stained for acid
phosphatase in the presence of 50 mM of sodium tartrate (6). For
calcitonin binding, the cells were incubated with 0.25 nM of
[125I]salmon calcitonin (Amersham) for 1 h
at 37°C in the presence or absence of 100 nM of unlabeled calcitonin.
For autoradiography, the cells cultured in LaboTech chamber slides
(Nunc) were incubated with [125I]calcitonin,
stained for TRAP, dipped in NTB-2 emulsion (Kodak), and subjected to
autoradiography. For OCL formation from C7 cells, the cells (5 × 104 cells) were cultured for a week in the
presence of 10 or 20 ng/ml of M-CSF and various concentrations of
sODF, and stained for TRAP.
-MEM containing
10% FCS, 10 ng/ml of M-CSF, and 30 ng/ml of sODF for 6 days in a
10-cm dish. The cultured cells were recovered from the culture with a
scraper and were collected by centrifugation. The cells (1 × 105 cells) were cultured for additional 3 days on
dentine slices in the same medium. After removal of the cells, the
slices were stained with Mayer's hematoxylin to visualize resorption
pits (13).
-MEM containing 10% FCS in the
presence of 108 M of
1,25(OH)2D3,
108 M of IL-11, 106 M
of PGE2, or 5 × 108
M of PTH for 6 days and subjected to the isolation of RNA.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
5 × 104
colonies. pOBM291 contained a 1.65-kb insert with an ORF encoding 316 amino acids (Mr 36K) (Fig.
1A). Hydropathy
analysis showed the absence of a signal sequence and the presence of an
internal 24-residue hydrophobic domain, which presumably represents a
transmembrane domain. This structure is typical of a type II
transmembrane protein with an extracellular C-terminal region. A
homology search of the GenBank sequence database revealed that the
C-terminal 165 residues of the protein had a significant homology to
the extracellular domains of the TNF ligand family members (17-19)
(data not shown). The protein satisfied the major criteria for ODF, the
predicted mediator of osteoclastogenesis (1, 2), in the light of its biological activity and its gene expression regulated by bone resorbing
factors (see below). We renamed the protein as ODF. Almost
simultaneously, a new member of the TNF ligand family designated as
TRANCE (20) or RANKL (21) was independently cloned. Comparison of the
predicted amino-acid sequences of ODF and TRANCE/RANKL revealed
identity between the two proteins.
View larger version (35K):
[in a new window]
Fig. 1.
ODF is an OPG/OCIF-binding protein.
(A) Schematic structure of ODF. Domains: C, cytoplasmic; TM,
transmembrane; E, extracellular. Arrowhead represents the N terminus
(Asp76) of ODF, which is fused to the C-terminal end of
thioredoxin. (B) Binding analysis of
[125I]OPG/OCIF to COS-7 cells transfected with the ODF
expression vector (COSODF) or COS-7 cells transfected with
the empty vector (COSVec). Data are expressed as mean ± SD of three cultures. (C) Coimmunoprecipitation of
ODF-OPG/OCIF complexes with anti-OPG/OCIF antibody (Ab).
Specific Binding of OPG/OCIF to ODF. [125I]OPG/OCIF was incubated with COS-7 cells transfected with the ODF expression vector or an empty vector. As shown in Fig. 1B, OPG/OCIF specifically bound to COS-7 cells expressing ODF (COSODF), but not to control COS-7 cells transfected with the empty vector (COSVec). To determine molecular weight of ODF, we pulse-chase labeled COSODF cells and co-immunoprecipitated the cell lysates with anti-OPG/OCIF antibody in the presence of OPG/OCIF. The results demonstrated that ODF is a 40-kDa protein (Fig. 1C).
Biological Function of ODF. To examine whether ODF mediates a cell-to-cell signal responsible for osteoclastogenesis, we carried out an in vitro OCL formation assay by evaluating TRAP activity and calcitonin binding, a combination of which is unique to osteoclasts (1, 22). When COSODF or COSVec cells were fixed with paraformaldehyde and then mouse spleen cells were cultured on the fixed cells for 6 days in the presence of M-CSF, TRAP-positive mononuclear and multinucleated cells appeared on the COSODF cells but not on the COSVec cells (Table 1). In addition, [125I]calcitonin specifically bound to the cells cultured on the COSODF cells. Concurrent addition of OPG/OCIF to the cultures inhibited both the TRAP-positive cell formation and the calcitonin binding in a dose-dependent manner (Table 1). These results suggest that ODF mediates the cell-to-cell signaling essential for osteoclastogenesis.
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sODF Induces OCL Formation From Spleen Cells in the Absence of Osteoblasts/Stromal Cells. The results from the experiments using COSODF cells did not exclude the possibility that other proteins co-expressed on COSODF cells might be involved in the signaling for osteoclastogenesis. Therefore, we next examined the effect of sODF, which is produced by fusing the extracellular domain of ODF to thioredoxin, on in vitro OCL formation. When mouse spleen cells were cultured in the presence of 10 ng/ml of M-CSF and various concentrations of sODF, TRAP-positive multinucleated cells were formed in a dose-dependent manner (Fig. 2 A and B). In the cocultures, giant TRAP-positive cells were formed in the presence of 30 ng/ml of sODF and the cells with numerous nuclei ranged in size up to 500 µm in diameter (Fig. 2A). Strong TRAP stain was observed in the periphery of the nuclei. The morphological features of the TRAP-positive multinucleated cells were quite similar to those formed in cocultures of spleen cells and stromal cells. Autoradiography using [125I]calcitonin confirmed the presence of calcitonin receptors on the induced TRAP-positive cells (Fig. 2C). OPG/OCIF negated the effect of sODF in a dose-dependent manner (data not shown). Furthermore, when these OCLs were cultured on dentine slices for 3 days in the presence of M-CSF and sODF, numerous resorption pits were formed on the slices (Fig. 2D).
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sODF Acts Directly on Osteoclast Progenitors. Mature monocytes and alveolar macrophages as well as several cell lines of the macrophage lineage can differentiate into OCLs, when cocultured with stromal cells in the presence of 1,25(OH)2D3 (23-25). A macrophage cell line, C7, is also capable of differentiating into OCLs in such a coculture system (26). Because spleen cells are a mixture of hematopoietic cell and stromal cell populations, we used C7 cells to examine whether ODF acts directly on osteoclast progenitors. sODF induced the formation of TRAP-positive multinucleated cells from C7 cells in a dose-dependent manner in the presence of M-CSF (Fig. 3A). The TRAP-positive multinucleated cells formed from C7 cells were morphologically similar to those formed from spleen cells in the presence of M-CSF and sODF (Fig. 3B; see also Fig. 2A) and produced numerous resorption pits on dentine slices (data not shown). Binding analysis showed that [125I]sODF specifically bound to C7 cells [7663 ± 125 cpm (minus unlabeled sODF) vs. 2735 ± 158 cpm (plus unlabeled sODF), mean ± SD].
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Expression of ODF Gene in Stromal Cells, Osteoblasts, and
Tissues.
Northern blot analysis using a 1.0-kb ODF cDNA probe
showed that a single mRNA transcript of approximately 2.4 kb was
present in ST2 cells treated with
1,25(OH)2D3 and Dex, but
not in the untreated cells (Fig.
4A). The results are
consistent with the observation that OPG/OCIF binds to ST2 cells
treated with 1,25(OH)2D3 and Dex (9). Expression of the ODF gene was found to be strikingly up-regulated by 1,25(OH)2D3
in a dose-dependent manner in a range of 1010
to 107 M (Fig. 4A). Dex alone
did not show any effects on the expression of the ODF gene (data not
shown). The combination of 108 M of
1,25(OH)2D3 and
107 M of Dex, which is known to show a maximal
effect on OCL formation in the cocultures of spleen cells and bone
marrow stromal cells (6), gave the highest up-regulation of the ODF
gene expression (Fig. 4A). Up-regulation of the ODF
gene expression was also observed in mouse primary osteoblasts cultured
in the presence of
1,25(OH)2D3, IL-11,
PGE2, or PTH (Fig. 4B). The
up-regulation of ODF expression was consistent with the observation
that the binding of [125I]OPG/OCIF to
osteoblasts increased when the cells were treated with each stimulator
(data not shown). OPG/OCIF inhibited OCL formation elicited by each
stimulator in the cocultures of spleen cells and osteoblasts (data not
shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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During the past decade, we demonstrated that a cell-to-cell contact between osteoblasts/stromal cells and osteoclast progenitors is important for osteoclastogenesis (1-3, 5, 6). In the present study we molecularly cloned a mouse OPG/OCIF-binding protein and identified it as ODF (1-3), the long-sought-after factor essential for osteoclastogenesis. The following evidence demonstrates that ODF is expressed on the membrane of osteoblasts/stromal cells in response to three distinct bone-resorbing signals and mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts: (i) the fixed COSODF cells induced the OCL formation from spleen cells; (ii) sODF induced the OCL formation from spleen cells or C7 cells in the absence of osteoblasts/stromal cells, and these OCLs were capable of forming numerous pits on dentine slices; (iii) the three distinct signals, through vitamin D receptor, protein kinase A, and gp130, independently up-regulated the expression of ODF gene in osteoblasts and induced ODF on the membrane; and (iv) the specific binding of [125I]sODF to C7 cells suggested the direct action of ODF on osteoclast progenitors of the monocyte/macrophage lineage.
Because ODF is identified as an OPG/OCIF binding protein, it is important to understand the relationship between ODF and OPG/OCIF. In the present study, we demonstrated that OPG/OCIF specifically abolishes the effect of both membrane-bound- and soluble-form ODF. OPG/OCIF also inhibited the OCL formation elicited through the three distinct signaling pathways in cocultures of spleen cells and osteoblasts, as it did in the culture of unfractionated bone cells or cocultures of spleen cells and stromal cell lines (8). These results strongly suggest that OPG/OCIF inhibits in vitro osteoclastogenesis by directly binding to ODF on osteoblasts/stromal cells and eventually interrupting ODF-mediated signaling from osteoblasts/stromal cells to osteoclast progenitors. On the analogy of the TNF ligand family that interacts with a parallel TNF receptor family (17-19), we suspect that OPG/OCIF acts as a soluble form competitor of ODF receptor presumably expressed on osteoclast progenitors (see below).
M-CSF produced by osteoblasts/stromal cells appears to be essential
for the proliferation and differentiation of osteoclast progenitors
(27-31). This is consistent with our observation that M-CSF was
indispensable for ODF-mediated OCL formation. The concentration of
M-CSF seems to be crucial for ODF-mediated OCL formation. A high
concentration of M-CSF (
40 ng/ml) suppressed OCL formation from
sODF-stimulated spleen cells (N.S., unpublished observation). Perkins
and Kling (32) also reported that addition of exogenous M-CSF to the
cocultures of ST2 cells and spleen cells causes a dose-dependent
decrease in the number of OCLs accompanied by an increase in the number
of macrophage. These results suggest that not only ODF expression but
also local concentrations of M-CSF influence osteoclast
differentiation. We previously reported that the expression of
OPG/OCIF gene in ST2 cells is down-regulated by
1,25(OH)2D3 and
up-regulated by calcium ions (9). In addition, the level of OPG/OCIF
expression in trabecular bones of ovariectomized rats decreased to 60%
of that of sham-operated rats (H.Y., unpublished observation). These
results imply a possible role of OPG/OCIF as a local regulator for
osteoclastogenesis. Northern blot analysis revealed that ODF gene was
highly expressed in not only osseous tissues but also nonosseous
tissues such as thymus and lung. At present, we cannot explain why
osteoclasts are not seen in nonosseous tissues expressing a high level
of ODF mRNA. The microenvironment suitable for osteoclastogenesis seems
to require a balance in the local concentrations of M-CSF and
OPG/OCIF, presence of osteoclast progenitors, and ODF expression on
osteoblast/stromal cells.
During the preparation of this manuscript, a new member of the TNF
ligand family designated TRANCE (20) or RANK ligand (RANKL; ref. 21)
was independently cloned. Analysis of the predicted amino-acid sequence
revealed that ODF is identical to TRANCE/RANKL. TRANCE was identified
as an activator of c-Jun N-terminal kinase in T-cells. TRANCE is
abundantly expressed in T cells but not in B cells. RANKL was
identified as a ligand for RANK, a new member of the TNF receptor
family derived from dendritic cells. RANKL activates NF-
B and
enhances T cell growth and dendritic cell function. Together,
TRANCE/RANKL/ODF seems to be an important regulator of T cells,
dendritic cells, and osteoclasts. It should be noted that both
dendritic cells and osteoclasts are generated from hematopoietic cells
of the monocyte-macrophage lineage (33). Recently, Iotsova et
al. (34) reported that NF-B1 and NF-B2 double-knockout mice
develop osteopetrosis because of a defect of osteoclast
differentiation. This report suggests that the activation of NF-B is
important for osteoclastogenesis. Taken all together, these
observations imply the identity of RANK with ODF receptor. The
mechanism by which ODF mediates signals to osteoclast progenitors through a possible ODF receptor, RANK, should be elucidated. We found
that the expression of the TRANCE/RANKL/ODF gene is up-regulated by
osteotropic factors, including
1,25(OH)2D3. Vitamin D and
other osteotropic factors may also be involved in the
TRANCE/RANKL/ODF-mediated immune responses. Our findings appear to
give an insight into the understanding of the potential immunological
roles of vitamin D (35).
The present study indicates that osteoblasts/stromal cells play an essential role in osteoclastogenesis through the expression of ODF on the membrane (Fig. 5). Identification of ODF as TRANCE/RANKL and as a ligand for OPG/OCIF raises the possibility that ODF and OPG/OCIF are involved in the regulation of not only osteoclastogenesis but also immune system. Further characterization of TRANCE/RANKL/ODF, OPG/OCIF, and the possible ODF receptor, RANK, will shed light on osteoclast biology and immunology.
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ACKNOWLEDGEMENTS |
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We thank F. Kobayashi and C. Mashiyama for technical assistance, and M. Hosono, T. Kawaguchi, T. Nakakarumai, H. Kawahara, and S. Ishida for the preparation of recombinant human OPG/OCIF.
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FOOTNOTES |
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These authors contributed equally to this work.
To whom reprint requests should be addressed. e-mail:
fvbd7042{at}mb.infoweb.or.jp.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AB008426).
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ABBREVIATIONS |
|---|
1, 25(OH)2D3,
1,25-dihydroxyvitamin D3;
Dex, dexamethasone;
OCIF, osteoclastogenesis-inhibitory factor;
OCL, osteoclast-like cell;
ODF, osteoclast differentiation factor;
OPG, osteoprotegerin;
PGE2, prostaglandin E2;
PTH, parathyroid
hormone;
sODF, soluble osteoclast differentiation factor;
TRAP, tartrate-resistant acid phosphatase;
TNF, tumor necrosis factor;
M-CSF, macrophage colony-stimulating factor;
MEM, -minimal essential
medium.
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REFERENCES |
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Abstract
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Materials & Methods
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Discussion
References
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 M.-H. Gannage-Yared, C. Yaghi, B. Habre, S. Khalife, R. Noun, M. Germanos-Haddad, and V. Trak-Smayra Osteoprotegerin in relation to body weight, lipid parameters insulin sensitivity, adipocytokines, and C-reactive protein in obese and non-obese young individuals: results from both cross-sectional and interventional study Eur. J. Endocrinol., March 1, 2008; 158(3): 353 - 359. [Abstract] [Full Text] [PDF]
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 J. A. Fretz, N. K. Shevde, S. Singh, B. G. Darnay, and J. W. Pike Receptor Activator of Nuclear Factor-{kappa}B Ligand-Induced Nuclear Factor of Activated T Cells (C1) Autoregulates Its Own Expression in Osteoclasts and Mediates the Up-Regulation of Tartrate-Resistant Acid Phosphatase Mol. Endocrinol., March 1, 2008; 22(3): 737 - 750. [Abstract] [Full Text] [PDF]
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 I. A. Nakchbandi, R. Lang, B. Kinder, and K. L. Insogna The Role of the Receptor Activator of Nuclear Factor-{kappa}B Ligand/Osteoprotegerin Cytokine System in Primary Hyperparathyroidism J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 967 - 973. [Abstract] [Full Text] [PDF]
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 A. Sultan, A. Avignon, F. Galtier, C. Piot, D. Mariano-Goulart, A. M. Dupuy, and J. P. Cristol Osteoprotegerin, Thiazolidinediones Treatment, and Silent Myocardial Ischemia in Type 2 Diabetic Patients Diabetes Care, March 1, 2008; 31(3): 593 - 595. [Full Text] [PDF]
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 H. Ha, J.-H. Lee, H.-N. Kim, H. B. Kwak, H.-M. Kim, S. E. Lee, J. H. Rhee, H.-H. Kim, and Z. H. Lee Stimulation by TLR5 Modulates Osteoclast Differentiation through STAT1/IFN-{beta} J. Immunol., February 1, 2008; 180(3): 1382 - 1389. [Abstract] [Full Text] [PDF]
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 M. K. Shea, S. L. Booth, J. M. Massaro, P. F. Jacques, R. B. D'Agostino Sr, B. Dawson-Hughes, J. M. Ordovas, C. J. O'Donnell, S. Kathiresan, J. F. Keaney Jr, et al. Vitamin K and Vitamin D Status: Associations with Inflammatory Markers in the Framingham Offspring Study Am. J. Epidemiol., February 1, 2008; 167(3): 313 - 320. [Abstract] [Full Text] [PDF]
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K. Kim, S.-H. Lee, J. Ha Kim, Y. Choi, and N. Kim NFATc1 Induces Osteoclast Fusion Via Up-Regulation of Atp6v0d2 and the Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Mol. Endocrinol., January 1, 2008; 22(1): 176 - 185. [Abstract] [Full Text] [PDF]
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