From the Department Molecular Pharmacology, Medical
Research Institute, and the ** Department Oral Surgery,
Tokyo Medical and Dental University, Tokyo 101-0062, Japan, the
§ Department of Cell Biology and Neuroscience, Rutgers
University, Piscataway, New Jersey 08854, and the ¶ Department of
Internal Medicine, Washington University, St. Louis, Missouri
63110, and
Snow Milk Brand Co., Tochigi 329-05,
Japan
Received for publication, December 4, 2000, and in revised form, January 19, 2000
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ABSTRACT |
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Osteopontin is an RGDS-containing
protein that acts as a ligand for the
Osteoclastic bone resorption is a key event in the pathophysiology
of osteopenic diseases such as osteoporosis and hyperparathyroidism. Osteoclastic bone resorption involves a number of sequential events, including differentiation and activation of osteoclasts. These processes are regulated both by key cytokines and hormones interacting with various cell surface receptors and by interactions of osteoclast precursors with osteoblasts/stromal cells (1-3). The
Osteopontin (14), is a mineralized matrix protein and a cytokine that
has been suggested to act in several types of organs and systems,
including bone (15), the immune system (16), the vascular system (17),
and kidney (18). In addition, osteopontin is expressed at sites of
inflammation. Osteopontin modifies cell behavior (19, 20) and alters
gene expression. In mineralized tissues, osteopontin is produced by
osteoblasts and osteoclasts and has been proposed to play a key role in
both types of cells (21-25). In bone, osteopontin is particularly
concentrated in cement lines and the lamina limitans (26). However, the
role of osteopontin in bone has not yet been fully elucidated.
Recently, osteopontin-deficient mice and osteopontin/vitronectin
double-null mutant mice have been created (27, 28). Although these
animals show normal development, we recently found that the lack of
osteopontin makes osteopontin-deficient mice resistant to
ovariectomy-induced bone resorption in vivo, in an
established animal model of postmenopausal osteoporosis (29). However,
the mechanism through which osteopontin deficiency protects bone is not
understood. Namely, whether osteopontin deficiency directly causes a
reduction in bone resorption or not has yet been known. To elucidate
the mechanism of osteopontin action, we investigated bone resorption in
organ cultures in which osteoclastic activity was stimulated by
PTH.1
Reagents--
Recombinant soluble RANKL was provided by Snow
Brand Milk Products Co. (30). Recombinant murine M-CSF was purchased
from R&D Systems (Minneapolis, MN). Human PTH was obtained from Bachem (Torrance, CA) and was dissolved in 1 mM acetic acid
containing 0.1% bovine serum albumin (Sigma Chemical Co.-Aldrich, St.
Louis, MO). The osteopontin-deficient mice were produced as described by Rittling et al. (28).
Organ Cultures--
Either wild type or osteopontin-deficient
mice at the ages of 3-10 months in a background of 129/S3 × C57BL/6 F2 were mated, and 17- or 18-day pregnant (dated from time of
sperm-positive vaginal smears) mice were injected with 50 µCi of
45Ca (Amersham Pharmacia Biotech, Buckinghamshire, United
Kingdom) as calcium chloride as described previously (31). 24 h
later, the 18- or 19-day post coitum embryos were removed, and the
radii and ulnae were dissected out under a binocular microscope. The cartilaginous ends of the long bones were cut off, and the shafts were
rinsed in PBS with antibiotics. The bone shafts were cultivated on
pieces of filter membrane (Millipore) placed in a 96-well plate (Corning Glass, Corning, NY) containing 50 µl of Bone Resorption Assay--
When the culture terminated, the
amount of 45Ca released from the bones into the medium was
measured by counting the radioactivity in the medium. The radioactivity
in the media was quantified using a scintillation counter. Although
fetal bovine serum (FBS) has been used in the organ culture system, we
replaced FBS with mouse serum obtained from the mothers at the time of
sacrifice; this was to exclude the influence by the possible presence
of osteopontin in FBS. Therefore, the radioactivities in the mother's
serum used for the cultures were also examined to estimate the
background levels. The bone-resorbing activity was estimated using the
culture media taken on day 6 of the organ cultures according to the
following formula. Typically, the amount of radioactivity in the mother mouse's serum was less than 10% of the amount released from the bone
as follows: 45Ca released into 50 µl of medium = (45Ca activity in 20 µl of sample medium × 2.5
Values in calcium 45 counts in dpm at the 6-day period of contralateral
side limb bones, which were treated with vehicle, were defined as
100%. The equation is as follows. The calcium 45 counts in dpm at the
6-day period from bone cultures treated with PTH or RANKL were divided
by the calcium 45 counts released into the cultures of vehicle-treated
bone specimens.
Radiographic Analysis--
The bones cultured in the presence or
absence of RANKL and M-CSF for 6 days were fixed in 4%
paraformaldehyde, followed by soft x-ray examination using FUJI
industrial FR film (Minami-Asigara City, Kanagawa, Japan). The distance
between the anode to film was set to be 540 mm, and the exposure time,
current, and voltage were 120 s, 3 mA, and 15 kV, respectively.
The radiographs of the bones were subjected to densitometry, which was
based on the digitalization of the image pixels whose intensity was
coded on the gray scale with 256 levels (corresponding to 8 bits) using a digital image processor. The threshold was set at level 50 of the 256 gray scale. The number obtained by integration of the counts of the
levels of the density scales (over 50) was divided by the area of bone
to yield bone density.
To make sure that the comparisons of relative bone density by digital
morphometric analysis was not influenced by the differences in bone
density at different anatomical sites, we have compared the entire bone
of the forelimbs of the mice used for the organ cultures for this
quantification. Thus, in doing this type of analysis, the x-ray picture
of the whole cultured bone was taken, and the density of the whole
cultured bone was measured. The values for that density were normalized
against the size (area) of the bones.
Osteoclast Formation in Spleen Cell Culture--
Spleens from
12-month-old osteopontin-deficient mice or wild type mice were used as
described (32) to make a suspension of spleen cells in Immunofluorescence Examinations--
For immunofluorescence
examinations, spleen cells were prepared as described (32) and were
cultured on glass coverslips. On the next day of the plating, the cells
on the coverslips were rinsed with PBS twice and fixed for 10 min at
room temperature with 4% paraformaldehyde. The cells were rinsed with
PBS twice and were subjected to membrane permeabilization for 2 min
with 0.1% Triton X-100. After rinsing with PBS twice, the cells were incubated for 30 min with 1% bovine serum albumin to block nonspecific binding of antibodies. Then the cells were incubated for 2 h at room temperature with 1:200 dilution of anti-CAS or Src antibodies followed by the treatment with second antibodies conjugated with Alexa
to visualize the fluorescein isothiocyanate fluorescence (33). The
cells were subsequently stained for actin-fibers by using
rhodamin-phalloidin (Molecular Probe Inc.) and were also stained for
TRAP.
Osteoclast Number in Forelimb Bone--
At the end of the
culture period of PTH experiments, bones were rinsed in PBS, fixed for
7 days in 4% paraformaldehyde acid, and decalcified by 10% EDTA, pH
7.4, for 3 days. Serial sections were prepared from paraffin blocks at
7-µm thickness and were stained for TRAP (tartrate resistant acid
phosphatase) activity, with Alcian blue for counter-staining. The
TRAP-positive cells with more than three nuclei were counted using
fifteen 7-µm-thick serial sections from each bone. Each of the
fifteen sections was taken at five-section intervals from a total of 75 sections per bone. The total number of osteoclasts per bone was
obtained by summing the number in all fifteen sections. For RANKL
experiments, both mononuclear and multinucleated TRAP(+) cells were counted.
RT-PCR--
RT-PCR reactions were carried out according to
the standard method using 10× PCR buffer (Takara, Japan), 1.25 mM dNTP, primer 3, primer 4, cDNA, and Taq
polymerase. Based on preliminary experiments, the cycle number was
optimized to be 35 cycles. PCR primers for osteopontin and GAPDH
were as follows. OPNEX6, 5'-TCCAATGAAAGCCATGACC-3'; OPNUNT6,
5'-GAAGAGTGAGTGAATCTGC-3'. The bands of osteopontin were 331 bp. The
GAPDH primer was purchased from system Science Co. (Hokkaido, Japan).
GAPDH Sense. 5'-ACCACAGTCCATGCCATCAC-3'; GAPDH antisense,
5'-TCCACCACCCTGTTGCTGTA-3'. The bands were examined after fractionation
in 1% agarose gel stained with ethidium bromide.
Statistical Analysis--
The statistical significance of the
data was evaluated by using Fisher's protected least significant
difference or Mann-Whitney's U test.
We examined whether organ cultures of bones derived from
osteopontin-deficient fetal mice differed from those from wild type mice with respect to their response to PTH, which is known to activate
bone resorption by osteoclasts (31, 34). The basal levels of
45Ca release from the organ cultures of the wild type and
osteopontin-deficient bones were similar. As reported previously, PTH
treatment increased resorption of the wild type fetal bones by about
40% as judged by the amount of 45Ca release (Fig.
1). In contrast, 45Ca release
from the osteopontin-deficient bones in organ culture was not enhanced
by the treatment with PTH.
v
3 integrin, which is abundantly
expressed in osteoclasts, cells responsible for bone resorption in
osteopenic diseases such as osteoporosis and hyperparathyroidism.
However, the role of osteopontin in the process of bone resorption has
not yet been fully understood. Therefore, we investigated the direct
function of osteopontin in bone resorption using an organ culture
system. The amount of 45Ca released from the
osteopontin-deficient bones was not significantly different from the
basal release from wild type bones. However, in contrast to the
parathyroid hormone (PTH) enhancement of the 45Ca release
from wild type bones, PTH had no effect on 45Ca release
from organ cultures of osteopontin-deficient bones. Because PTH is
located upstream of receptor activator of NF-
B ligand (RANKL), that
directly promotes bone resorption, we also examined the effect of
RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced
45Ca release from the bones of wild type fetal mice but not
from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in
osteopontin-deficient bone, we investigated the number of
tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones
subjected to PTH treatment in cultures. The number of TRAP-positive
cells was increased significantly by PTH in wild type bone; however, no
such PTH-induced increase in TRAP-positive cells was observed in
osteopontin-deficient bones. These results indicate that the absence of
osteopontin suppressed PTH-induced increase in bone resorption via
preventing the increase in the number of osteoclasts in the local
milieu of bone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
v
3 integrin is a major receptor on the
osteoclast that can interact with RGD sequence-containing
ligands such as osteopontin and vitronectin (4). It possibly
promotes osteoclast attachment to bone and, when engaged by either
immobilized or soluble ligands, stimulates signaling pathways that
regulates osteoclast migration and function. Bone matrix consists of
about 90% type I collagen and about 5% noncollagenous
proteins. Among these noncollagenous proteins, at least five proteins,
osteopontin, bone sialoprotein, thrombospondin, fibronectin, and
vitronectin, contain RGD sequences that can be recognized by some
integrins (5-8). These noncollagenous bone proteins are candidate
ligands for the
v
3 integrin expressed on
the osteoclasts. In vitro data suggest that the
3 integrin subunit is involved in the attachment of
osteoclasts to osteopontin and bone sialoprotein, whereas the
1 integrin subunit is responsible for the attachment of
these cells to fibronectin (9). With regard to osteoclastic bone
resorption in vitro, interactions between osteopontin and/or
bone sialoprotein and the
v
3 integrin (10, 11) have been proposed to play a crucial role. The signaling pathways stimulated by the integrin lead to modulation of cytoskeletal reorganization via regulatory molecules such as gelsolin (12). A recent
study also indicates that the
3 integrin is
important in osteoclastic bone resorption in vivo. (13)
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-modified minimum essential medium (
-MEM, Sigma-Aldrich, Steinheim, Germany)
supplemented with 5% serum prepared from the mother mice of the
corresponding genotype. The bones were preincubated for 1day, and then
they were subsequently cultured in fresh media for 6 days under an atmosphere of 5% CO2, at 37 °C with one change of the
half of medium on day 3. In each experiment, bones from the right and left side of the embryos were collected. The bone from one side was
used as an experimental group and treated with either PTH (10
7 M) or soluble RANKL (100 ng/ml) plus
M-CSF (10 ng/ml), and the other was used as untreated control.
45Ca activity in 2.5 µl (5%) serum sample).
-MEM
containing 10% FBS (Life Technologies, Gaithersburg, MD). Briefly,
spleen cells were squeezed out through the opening of the capsule made
at the distal end of the spleen, were subjected to pipetting, and were
spun down to form a pellet. The cells were resuspended in 1 ml of
ice-cold double-distilled water for less than 10 s to remove
reticulocytes, immediately resuspended in 10 ml of medium supplemented
with 10% FBS, and spun down. The cells were resuspended again in
FBS-supplemented medium and filtered through a cell strainer before
estimating the viable cell number based on trypan blue exclusion method
using one volume of an aliquot of the cell suspension mixed with 3 volumes of the dye solution. Spleen cells at 3.5 × 105 cells/cm2 were plated into 6-well plates
(Corning Costar Co., Cambridge, MA) and were cultured in the presence
of 30 ng/ml soluble RANKL and 10 ng/ml M-CSF. Cells were maintained
(under an atmosphere of 5% CO2, at 36 °C) in 4.0 ml of
-MEM/FBS for up to 11days. Following incubation for the appropriate
period, the cultures were fixed and stained histochemically for the
osteoclast-associated enzyme TRAP (30). TRAP-positive multinucleated
(cells containing more than three nuclei) cells were counted at 100×
magnification in 40 rectangular fields of 41.6 mm2 in each
of the wells. In separate cultures, spleen cells were cultured in the
presence of 100 ng/ml soluble RANKL and 30 ng/ml M-CSF on dentin slices
placed in a 96-well plate in the media supplemented with 5% wild type
serum or osteopontin-deficient mice serum. Some cells were subjected to
RNA isolation and PCR analyses.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PTH-induced 45Ca release is
impaired in osteopontin-deficient bones. 45Ca-labeled
bones were cultured in the presence of 10 7 M
PTH or vehicle for 6 days, and the levels of 45Ca in the
medium were measured. Data are expressed as ratios against that in wild
type control, which was treated with vehicle alone (acetic acid 0.15%
and bovine serum albumin 0.1%). Columns and bars
are mean ± S.E., respectively. Data represent the values obtained
from a total of 28 bones (WT, PTH (
), 7 bones;
WT, PTH (+), 7 bones; OPN-KO,
PTH (
), 7 bones; OPN-KO, PTH (+), 7 bones). These experiments were performed three times with similar
results. The significance of the data was evaluated using Wilcoxon's
paired signed-rank U test. The asterisk indicates
a significant difference from the control, p < 0.05.
PTH binds to receptors expressed on osteoblasts and/or stromal cells
and enhances expression of membrane-bound RANKL, which in turn binds to
its receptor, receptor activator of NFB, on osteoclasts or
their precursors, to enhance osteoclast development and/or osteoclast
activity (35-37). It is also known that PTH suppresses osteopontin
expression in osteoblasts (38). Therefore, we examined whether absence
of bone resorption resulting from osteopontin deficiency was between
PTH and RANKL expression or downstream of RANKL. Soluble RANKL in
combination with M-CSF promoted development of TRAP(+) cells in
osteopontin-deficient spleen cells at levels comparable to those in
wild type cells (Fig. 2). Therefore,
osteopontin deficiency does not affect osteoclastogenesis induced by
RANKL. To examine the effect of osteopontin deficiency on RANKL-induced bone resorption, we tested whether RANKL stimulated bone resorption by
measuring 45Ca release in the organ culture assay.
Treatment for 6 days with soluble RANKL in combination with M-CSF
enhanced 45Ca release by about 40% in wild type bones. In
contrast, treatment with soluble RANKL and M-CSF did not increase
45Ca release from the osteopontin-deficient bones (Fig.
3).
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To evaluate morphologically the effect of soluble RANKL and M-CSF on
bones in organ culture, soft x-ray pictures of the bones were taken and
bone density was quantitatively determined. The bone density of
untreated group was similar between control wild type and the control
osteopontin-deficient bones. The density of wild type bones was
decreased by the treatment with soluble RANKL and M-CSF. However, the
density of osteopontin-deficient bones was not affected by the
treatment with soluble RANKL and M-CSF (Fig.
4, A and B). These
observations substantiated those obtained in 45Ca release
assays.
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To obtain insight into the mechanism underlying the defect in
45Ca release resulting from osteopontin-deficiency at
cellular levels, we conducted a histological examination of the bones
cultured for 6 days in the presence or the absence of PTH (Fig.
5A). We counted TRAP-positive
cells that were observed in the forelimb bones. The number of
TRAP-positive cells containing three or more nuclei was similar between
wild type and osteopontin-deficient bones when cultured in the absence
of PTH. In wild type bones, the number of TRAP-positive cells was
higher in the bones treated with PTH than that in the control (Fig.
5B). However, no such increase in the number of
TRAP-positive cells was observed in osteopontin-deficient bones even
after culture in the presence of PTH (Fig. 5B). Similarly,
RANKL treatment increased TRAP-positive cells in wild type but not in
osteopontin-deficient bones in organ culture (data not shown).
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To estimate the effect of wild type serum, which would contain
soluble osteopontin on TRAP(+) MNC formation, we cultured wild type or osteopontin-deficient spleen cells on dentin slice in the
presence of soluble RANKL and M-CSF in medium dentin slices contain osteopontin (26). In this setting, the presence of
osteopontin in wild type spleen cells cultured in the medium
supplemented with wild type serum allowed RANKL-induced development of
TRAP-positive multinucleated cells more efficiently than
osteopontin-deficient spleen cells cultured in the medium supplemented
with osteopontin-deficient mice serum (Fig.
6). These observations suggest that
osteoclastic precursors exist in osteopontin-deficient bone tissue but
require osteopontin either from themselves and/or from the serum to
mature into TRAP-positive multinucleated cells.
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We further tested whether RANKL regulates the expression of osteopontin
gene in spleen cells. As indicated in Fig.
7, RANKL and M-CSF treatment suppressed
osteopontin gene expression. This is a specific effect, because GAPDH
levels were not altered by the treatment with RANKL and M-CSF. This
observation suggests the presence of a negative feedback system
possibly to keep the balance of bone resorption.
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Finally, we investigated the expression and localization of signaling
molecules known to be involved in regulation of osteoclastic function.
The expression of Src and CAS and their intracellular localization as
well as actin cytoskeleton pattern were similar between the wild type
and osteopontin-deficient osteoclasts developed from the spleen cells
treated with RANKL and M-CSF (Fig.
8).
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DISCUSSION |
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Our experiments indicate that PTH-induced bone resorption does not occur in osteopontin-deficient bones. Although previous ovariectomy experiments indicated that osteopontin-deficiency protected bone against bone resorption, the mechanism of this protection was not clear. Our organ culture data clearly indicate that bone resorption induced by PTH in the local milieu of bone does not occur in the absence of osteopontin, indicating that the protection of bone against bone resorption was via a direct action of osteopontin in bone but not an indirect systemic effect.
PTH exerts its regulatory effects on calcium homeostasis in part by stimulating the release of calcium from bone (31, 34). This PTH-stimulated bone resorption involves at least two cell types: osteoblasts and osteoclasts (34, 40). The binding of PTH to its receptors on osteoblasts initiates several events that activate osteoclasts and stimulate bone resorption (34, 40). These include enhancement of RANKL expressions, which promote both the proliferation and differentiation of osteoclast precursors and the activation of mature osteoclasts (41, 42). We therefore investigated which PTH-induced events were deficient in the absence of osteopontin. We observed that soluble RANKL with M-CSF did not increase 45Ca release in osteopontin-deficient bones, indicating that the critical point where osteopontin is required lies downstream of RANKL action.
We further investigated the cellular basis for the mechanism of protection against bone resorption by osteopontin deficiency and found that, in parallel to the requirement for osteopontin in bone resorption induced by PTH in a bone organ culture system, the increase in osteoclast number in the bones in organ cultures in response to PTH treatment also required osteopontin. We also observed that osteopontin-deficient spleen cells or bone marrow cells could give rise to osteoclasts in vitro in the presence of vitamin D (27) or RANKL and M-CSF (Fig. 2). The osteoclasts developed from either wild type or osteopontin-deficient spleen cells by the treatment with RANKL revealed similar expression and localization of signaling and attachment molecules including Src, CAS, vinculin, and actin. These results indicate that the development of osteoclasts per se is not impaired in osteopontin-deficient bones; rather the specific signaling pathways activated by PTH and RANKL, resulting in an increase in the number of osteoclasts in bone, are inhibited in the absence of osteopontin in these organ cultures.
This notion was also supported by the reduction in the RANKL-induced TRAP(+)-MNC formation on the dentin slice in the osteopontin-deficient spleen cells cultured in the serum lacking osteopontin. supplemented with either wild type mouse serum or osteopontin-deficient mouse serum. Crossing combinations of the genotypes of spleen cells and those of serum may yield further information on the roles of osteopontin from the different sources. Human serum osteopontin levels range from 16 to 64 ng/ml according to Harris (39) and our semi-quantitative measurements of mouse serum have yielded similar results (data not shown).
It appears that, either as a solid phase (osteopontin coating the slide) or as a soluble protein, exogenous osteopontin can assist the formation of TRAP+ cells from osteopontin-deficient spleen cells via RANKL treatment. Therefore, endogenous production of osteopontin may not be necessary to develop TRAP+ cells at least in such in vitro situation, where osteoclastogenesis is fully activated by the presence of relatively high concentrations of RANKL and M-CSF. As mentioned above, analysis on the mechanism that requires osteopontin to convey PTH signal to induce final bone resorption revealed that osteopontin is not required in the pathway between PTH and RANKL but rather downstream of RANKL, because RANKL activation of bone resorption in the organ cultures was also impaired. Thus our current hypothesis is that PTH-induced RANKL signaling resulting in either an increase in osteoclast number and/or activation is disrupted by the absence osteopontin.
To estimate the activity of osteopontin-deficient TRAP-positive cells, we examined pit formation on hydroxyapatite-coated slide glass after stimulation with 1,25(OH)2-vitamin D3 and dexamethasone. Significant reduction (p < 0.05) was observed in the number of pits in the case of osteopontin-deficient TRAP-positive cells compared with wild type (Y. Muguruma and M. Noda, data not shown) in two out of four independent experiments. These observations suggest that osteopontin is required for the activity of osteoclasts as well. Obviously, it is possible that the effect on resorption needs a more detailed analysis on bone or dentin to better determine whether the observed change in hydroxyapatite removal represents true changes in resorption or another change in cell dynamics such as motility and phagocytic activity.
In the bone organ culture environment, which was used in our experiments, bone matrix does not contain osteopontin. Therefore, it is still possible that the absence of osteopontin in bone matrix may prevent rudimentary osteoclastic precursors derived from blood from homing and residing in bone tissue (while the bones were in the body).
Because osteopontin-deficient mice have been reported to grow without any retardation, it is possible that chondroclasts, which are responsible for the resorption of calcified cartilage matrix, may be functioning normally. Based on our observation on the defects in PTH-induced bone resorption in osteopontin-deficient bones in organ culture, it is possible that osteoclasts, which resorb bones, may be more susceptible to the osteopontin deficiency in the case of situations with accelerated bone resorption. If the growth plate chondroclasts may not be affected by the absence of osteopontin, it may provide differential properties to bone and cartilage with regard to its effects on the two resorbing cells, i.e. chondroclasts and osteoclasts, respectively.
It is intriguing to compare in vivo and in vitro situations in osteopontin-deficient mice. Bone resorption appears to be normal in osteopontin-deficient mice at birth. In the organ cultures of osteopontin-deficient mice bones, PTH and RANKL signaling were not able to develop TRAP-positive multinucleated cells. It appears that certain molecules supplied from the tissues outside bones could compensate for the lack of osteopontin in in vivo situation. However, in organ cultures, such supply of compensatory molecules from tissues outside bones may not be available. Thus, as shown in the present data, osteopontin-deficient bones cannot respond to PTH and RANKL with regard to the development of TRAP-positive multinucleated cells as well as calcium 45 release. Our data also suggest that RANKL treatment suppressed osteopontin expression in spleen cell cultures, suggesting the presence of a certain negative feedback system.
In conclusion, our observations indicate that osteopontin is directly
required for bone resorption activated by PTH-RANKL axis via increasing
the number of osteoclasts in the microenvironment of bone.
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FOOTNOTES |
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* This research was supported by grants-in-aid from the Japanese Ministry of Education (12557123, 12026212, 12215060, 0930734), by grants from Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation, by a grant from the Research for the Future Program of the Japan Society for the Promotion of Science (96100205), by the Tokyo Biochemical Research Foundation, and by National Space Development Agency. Research at Rutgers was supported by National Institutes of Health Grants AR44434 and ES06897 (to D. T. D.) and CA72740 (to S. R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Pharmacology Medical Research Institute, Tokyo Medical and Dental
University, 3-10 Kanda Surugadai 2-Chome, Chiyoda-ku, Tokyo, Japan.
Tel.: 011-81-3-5280-8066; Fax: 011-81-3-5280-8066; E-mail: noda.mph@mri.tmd.ac.jp.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010938200
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ABBREVIATIONS |
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The abbreviations used are:
PTH, parathyroid hormone;
RANKL, receptor activator of NF-B ligand;
M-CSF, macrophage-colony stimulating factor;
PBS, phosphate-buffered
saline;
-MEM,
-modified minimum essential medium;
FBS, fetal
bovine serum;
RT, reverse transcription;
PCR, polymerase chain
reaction;
CAS, CRK-associated substrate;
TRAP, tartrate-resistant acid
phosphatase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
bp, base pair(s).
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