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Department of Molecular Pharmacology, Medical Research Institute (Y.A., H.Y., K.T., A.N., M.N.), and Department of Orthopedic Surgery (K.S.), Tokyo Medical and Dental University, Tokyo 101-0062, Japan; and Department of Cell Biology and Neuroscience, Rutgers University (S.R.R., D.T.D.), Piscataway, New Jersey 08854
Address all correspondence and requests for reprints to: Masaki Noda, M.D., Ph.D., Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 2–3-10 Kanda, Surugadai Chiyoda-ku, Tokyo, Japan. E-mail: noda.mph{at}mri.tmd.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteopontin (OPN) is a cytokine and cell attachment protein. It is
produced in many tissues in the body and is abundant in the
extracellular matrix of mineralized tissues (1, 2, 3). The
protein contains an RGDS sequence motif that can promote attachment of
several types of cells, including osteoclasts to plastic or bone
(4, 5, 6). OPN binds to a subset of integrin receptors; most
notably it is a well characterized ligand for the
vß3 integrin that is
expressed at high levels in osteoclasts and endothelial cells (3, 7). Engagement of the
vß3 integrin by OPN
activates intracellular signaling pathways and elevates inositol
polyphosphate and intracellular calcium levels in various cell types
(8, 9). The
vß3 integrin is
required for angiogenesis, and in vitro it is involved in
inhibition of endothelial cell death via an NF-
B-mediated pathway
(10, 11). OPN acts as a cytokine, and its binding to
vß3 activates c-Src
and phosphoinositide-3-hydroxy kinase (12, 13). As one
consequence of the activation of intracellular signaling pathways, OPN
suppresses the induction by lipopolysaccharide and interferon-
of
nitric oxide synthesis induced in kidney proximal tubule epithelial
cells by inhibiting the induction of the inducible nitric oxide
synthase messenger RNA (14). OPN is also produced at high
levels by macrophages in granulomas associated with tuberculosis,
silicosis, or reactions to foreign bodies (15, 16, 17).
In bone, OPN is produced by osteoblasts when they form bone matrix and is subsequently accumulated in the mineralized matrix. OPN binds strongly to hydroxyapatite, possibly explaining its abundance in bone matrix. OPN is also expressed at high levels in osteoclasts (18, 19). These observations suggest that OPN could play a role in the attachment of bone cells to bone and in controlling subsequent bone cell functions such as bone resorption. However, the function of OPN in bone as well as other tissues is not known.
Recently, OPN knockout mouse strains have been established by homologous recombination in mouse embryonic stem cells (20, 21). These mice are fertile and mature normally, with apparently unaltered development of most tissues and organs examined to date. Their skeletal patterning is also normal. Because OPN has been shown to be an extracellular signaling molecule for osteoclasts and endothelial cells in vitro (3), we investigated the consequences of the absence or presence of OPN in vivo on the angiogenesis, accumulation of osteoclasts, and subsequent bone resorption by using an ectopic bone-resorbing model in which wild-type and OPN-deficient bone discs were implanted into wild-type and OPN knockout mice, respectively.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intramuscular implantation of bone discs
A single skin incision was made in the back of the anesthetized
host animal. The bone discs were individually implanted into pockets
made in the paravertebral muscles of either wild-type or OPN-deficient
mice. Wild-type bone discs and OPN-deficient bone discs were implanted
into the muscles of wild-type and OPN-deficient mice, respectively. The
shape and location of the bone discs were monitored by soft x-ray
analysis of the animals under anesthesia. All discs were excised 4 or 8
weeks after implantation, fixed with 2.5% glutaraldehyde/PBS, and then
subjected to soft x-ray examination (25 kV, 50 mA, 2 sec). The levels
of resorption of bone discs were calculated as follows. The resorbed
area of each disc was quantitated using an image analyzer equipped with
the Luzex system (NIRECO, Tokyo, Japan), and the value was normalized
against the initial size of the bone discs (2.5 mm in diameter).
Subcutaneous implantation of bone discs
Subcutaneous pockets were made in the backs of the animals by
opening the tips of the inserted scissors. The bone discs were then
placed into the pockets. The location and shape of the bone discs
in vivo were monitored periodically by soft x-ray analysis
of the animals under anesthesia. All of the sc implanted bone discs
were excised 3 months after implantation. Photographs of the bone discs
were taken at the time of excision, and vessels on the bone discs were
traced to OHP film. Then the lengths of the vessels were quantified
using an image analyzer equipped with the Luzex system. To quantitate
only neovascularization, very thick vessels (>0.04 mm) were eliminated
from consideration. The number of branch points of the vessels was
determined using the photographed records of the excised bones. Some of
the bone discs were subjected to en bloc staining for
tartrate-resistant acid phosphatase (TRAP) activity, and the stained
area was quantitated by the image analyzer.
Histochemical staining for TRAP activity
Bone discs implanted im were excised 4 weeks after implantation,
fixed with 4% paraformaldehyde, and decalcified with 20% EDTA
for 2 weeks at 4 C. After dehydration and paraffin embedding, serial
sections at 5-µm thickness were prepared. Sections were made in a
plane perpendicular to the plane of the bone disc within its central
part along the diameter. To estimate the accumulation of osteoclasts on
the bone discs implanted in muscle, sections were stained for TRAP
activity by immersing the sections in a buffer (pH 5.0) containing
sodium acetate (100 mM), sodium tartrate (50
mM), naphthol phosphate AS-MX (0.1 mg/ml),
N,N-dimethyl formamide (1%), Fast Red Violet
LB salt (0.6 mg/ml), and manganese (II) chloride (0.4
mM) at room temperature for 10 min. The sections
were counterstained with Alcian Blue. Osteoclasts were identified as
TRAP-positive cells attached to bone. These cells were counted in five
sections (for each of the five bones per group), which were 150 µm
apart. The average number of osteoclasts per bone section was obtained
for each of the bone discs.
Immunohistochemistry and terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)
assay
Bone discs implanted im were excised 4 weeks after implantation
and were fixed and decalcified as described above. The serial sections
at 5-µm thickness of the im implanted bone discs obtained as side by
side sections used for TRAP staining were incubated at 4 C overnight
with MEC 13.3 rat monoclonal antimouse PECAM-1 (CD31) antibody
(PharMingen, San Diego, CA). The sections were then
incubated at room temperature for 1 h with biotinylated goat
antirat IgG antibody (Biosource Technologies, Inc.,
Camarillo, CA) and visualized with peroxidase-conjugated avidin using
the Vectastain kit (Vector Laboratories, Inc., Burlingame, CA) and diaminobenzadene. The PECAM-1
(CD31)-positive new vessels around the bone discs within 260 µm at
their edges were counted in five serial sections, 150 µm apart, in
the central part of the bone discs. The average number of vessels per
section was obtained for each of the bone discs. We made sure that the
soft tissues around the implants, which contained the vessels, were
recovered quantitatively for both groups by excising the implants with
relatively large bulks of surrounding muscles. By examination of the
histological sections, it was clear that most of the neovascularization
was within the region 260 µm away from the bone discs, as no major
neovascularization was observed in the outer regions (>260 µm away
from the bone discs), which were still within all histological
sections. The TUNEL assay was conducted using the side by side sections
from these bone discs according to the manufacturer’s instruction
(TaKaRa, Tokyo, Japan).
Restoration of impaired resorption of OPN-deficient bone by
treatment of the bone discs with OPN
Bone discs were prepared as described above from calvarial bones
of wild-type or OPN-deficient mice.
Glutathione-S-transferase (GST)-OPN fusion protein and GST
were prepared as described previously (21). The bone discs
were immersed for 3 h at 4 C in either recombinant GST-OPN fusion
protein solution (0.375 mg/ml in PBS) or GST protein (0.375 mg/ml in
PBS) as controls. These discs were then immersed in 70% ethanol for 1
min, rinsed in PBS, and then immediately implanted into the pockets
made in the right or left paravertebral muscles of host wild-type or
OPN-deficient mice as described above. The GST-immersed bone discs
were implanted into the pocket on the left side, and GST-OPN-immersed
bone discs were implanted in the right side. These experiments were
conducted in a pairwise manner using the bone disc from one side of the
parietal bone for treatment with GST-OPN fusion protein treatment and
that from the other side for GST treatment. Bone discs were monitored
by soft x-ray and were excised 8 weeks after implantation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, soft x-ray
examination indicated that the wild-type bone discs implanted in
wild-type mice were significantly resorbed. In contrast, bone
resorption was severely impaired when bone discs from OPN knockout mice
were implanted im in OPN knockout mice. Quantification of the resorbed
area revealed that about 25% of the total area was lost in wild-type
mice, whereas only 5% was lost in the OPN-deficient group (Fig. 1B). These results indicate that OPN is required for efficient
resorption of im implanted bones.
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). The
resorbed areas in the bone discs in these groups were about 11% and
13%, respectively (Fig. 1B). Overall, these data establish that OPN in
both the host and bone discs is required for efficient resorption of im
implanted bones.
Osteoclast accumulation at the implanted bone disc was impaired in
the absence of OPN
To obtain insight into the underlying mechanism of the effect of
OPN deficiency at the cellular level, we further examined the
osteoclasts attached to the im implanted bone discs using histological
sections of the bone discs stained for TRAP activity. Osteoclasts were
observed at the edge of the bone discs in both wild-type (Fig. 2A) and OPN knockout mice groups (Fig. 2B). The TRAP activity of osteoclasts as well as the morphology of the
lacunae formed by the individual osteoclasts were similar regardless of
the genotype (Fig. 2, A and B). However, quantification of the number
of osteoclasts indicated reduction in the OPN knockout mice group
compared with wild-type group (Fig. 2C). Thus, the reduction in bone
resorption was at least in part due to the reduction in the number of
osteoclasts resorbing the ectopically implanted bone discs in OPN
knockout mice group.
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vß3 integrin-mediated
signals (10, 11), the presence of apoptotic cells around
the bone discs in vivo was examined according to TUNEL assay
using side by side sections of the bone discs used for the
detection of PECAM-1 (CD31). TUNEL signals associated with
PECAM-1 (CD31)-positive cells were very rare in the presence or
absence of OPN in bone discs and host mice (data not shown). Although im implantation experiments were useful for bone resorption assay, they are less useful for analysis of the length or branching of the vasculatures because the surrounding muscle tissues were tightly attached to the bone discs when the bone discs were excised, and this made such analyses impossible. In contrast, sc implantation allows us to examine the OPN effects on the above-mentioned neovascularization due to the characteristics of the loose sc tissues in this environment, although slow rates of vascularization of bone discs result in prolonged times required for observable bone resorption, as we did not see bone resorption within 3 months. Thus, to further analyze the effect of OPN deficiency on the neovascularization, we implanted bone discs sc in the backs of the mice.
When the bone discs were excised under a dissecting microscope 3 months
after implantation, vascularization of the implanted bone discs was
readily observed (Fig. 3AB). To
quantitate the angiogenic response to the wild-type or OPN-deficient
bone discs, the length of the vasculature attached on the surface of
each bone disc was measured. The length of the vessels was about 3-fold
higher in wild-type than in OPN-deficient mice (Fig. 3C). To further
evaluate angiogenic activity, the number of branch points of the
vessels was also counted. As shown in Fig. 3D, the number of branch
points in wild-type animals was 3-fold higher than that in the
OPN-deficient group.
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). In contrast, such accumulation of
osteoclasts was reduced in OPN knockout mice (Fig. 4A). Quantification
of the TRAP-positive area on the bone discs indicated that 35% of the
total area was covered by TRAP staining in wild-type mice, whereas the
TRAP-positive area was reduced to 25% in the absence of OPN (Fig. 4B).
The bone discs were also sectioned after the en bloc
staining for TRAP. These histological examinations indicated the
presence of TRAP-positive multinucleated cells attached to the bone
discs. These observations based on the two types of bone implantation
models establish that OPN facilitates vascularization of ectopically
implanted bone and accumulation of osteoclasts.
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, OPN-deficient bone
discs treated with GST-OPN fusion protein were resorbed more
efficiently than the control GST protein-treated bone discs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone matrix contains type I collagen as the major component, making up
approximately 90% of the organic fraction. Noncollagenous bone matrix
proteins, including osteocalcin, biglycan, osteonectin, and matrix Gla
protein, have been shown to play roles in bone metabolism based on
studies of knockout mice (24, 25, 26, 27). OPN is also one of the
most abundant noncollagenous bone matrix proteins in bone. In
vitro experiments have shown that OPN can promote attachment of
cells and stimulate signaling events in osteoclasts. However, knockout
mice deficient in OPN did not reveal a major bone phenotype, at least
during the early period of their life under typical laboratory
conditions (20). We hypothesized that OPN is required for
the situation where osteoclastic bone resorption is enhanced. As the
data in this paper indicate, the role of OPN in bone resorption is
apparently more pronounced under circumstances of accelerated,
pathological bone loss. Such situations also include the rapid bone
loss that occurs after ovariectomy (1). It may be that
under some circumstances OPN-dependent osteoclastic recruitment can be
compensated, perhaps by the closely related protein BSP-1 and/or
osteoclast-regulating cytokines such as receptor activator of nuclear
factor-B ligand (28, 29). However, under conditions of
rapid bone resorption, there is a strict requirement for OPN function.
Our observation that exogenously supplied OPN increases bone resorption
area in the implanted bone discs confirms the importance of this
protein in this model of bone loss.
On the role of OPN in angiogenesis
As OPN deficiency per se does not suppress osteoclast
development from hemopoietic precursor cells in the cultures of bone
marrow cells and spleen cells (20), we investigated
whether OPN deficiency affects angiogenesis to ectopically implanted
bone discs. The reduced number of PECAM-1-positive vessels on the im
implanted bone discs in the absence of OPN indicated that angiogenesis
was deficient under these conditions, as PECAM-1 is a marker of
endothelial cells. In addition, quantitation of vessels around the bone
discs in sc implantation experiments revealed that angiogenesis was
impaired by the lack of OPN. Thus, OPN is required for efficient
angiogenesis to ectopically implanted bone discs.
Previous in vitro experiments indicated that apoptosis of
endothelial cells is promoted by the absence of the ligands for
vß3 integrin such as
OPN and vitronectin (10), and that endothelial cell
proliferation can be promoted by adhesion to
vß3 ligands in culture
(30). However, our results showed no elevation of TUNEL
signals in the PECAM-1 (CD31)-positive endothelial cells associated
with the bone discs in vivo. This observation indicates that
in vivo, differences in apoptotic rates do not account for
the OPN effects on vascularization. It is likely that vitronectin or
other molecules may have compensated for the absence of OPN in the
prevention of apoptosis in vivo. Whether other mechanisms,
such as a reduction in chemotaxis (31, 32) or
ß3 integrin-dependent phenomena
(33), may be affected by the absence OPN-deficient
resulting in suppression of angiogenesis as well as osteoclast
accumulation and/or function is a subject for further investigation.
Our observation that osteoclasts on the ectopically implanted bone
discs were similar in shape, TRAP activity, and morphology of their
lacunae regardless of the genotype suggests that OPN deficiency did not
significantly affect the differentiation and/or function of individual
osteoclasts in vivo.
On the bone disc implantation
In our experiments, round bone discs were implanted into muscle of
knockout and wild-type mice. Intramuscular implantation revealed the
consequences of OPN deficiency on resorption in 4 weeks. As shown in
the data in Fig. 1, there was a small, but certain, bone resorption in
the OPN-deficient bone discs; therefore, it is reasonable to assume
that they catch up to the wild-type bone at a later time point. This
implantation system using round bone discs has an advantage over the
previously reported bone particle implantation system, as it is
amenable to several kinds of analyses: 1) simple evaluation of the
resorption levels by taking x-ray films of the bone discs before and
after the 4-week implantation in muscle, and 2) sequential monitoring
of the degree of resorption (even a small degree of resorption can be
visualized as a notch in the margin of the bone disc seen in x-ray
analysis).
Although the data obtained in this system support the role for OPN in normal osteoclastic function and bone remodeling, it is not a perfect model of normal and pathological remodeling in bone. These studies could be extended to include studies examining fracture remodeling and PTH-induced remodeling in OPN-deficient mice. In addition, the reduced numbers of osteoclasts and apparent decrease in vascularization could be part of a very complex process involving defective inflammatory responses associated with tissue injury. Therefore, further analyses are required to reveal the mechanisms or sites of action of OPN in these process.
In conclusion, we have demonstrated in vivo that the absence of OPN impairs angiogenesis, subsequent osteoclast accumulation, and eventual bone resorption of ectopically implanted bones. OPN present in bone matrix, therefore, acts as a molecule required for bone resorption in vivo, at least in response to stress as shown in the ectopic bone implantation.
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Footnotes |
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Received August 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 in smooth
muscle cell migration to osteopontin in vitro. J Clin Invest 95:713–724
B mediates
vß3 integrin-induced
endothelial cell survival. J Cell Biol 141:1083–1093Vß3 in the
disruption of the tumor vasculature induced by TNF and IFN-. Nat Med 4:408–414[CrossRef][Medline]
v-subunit.
Endocrinology 137:2432–2440[Abstract]
vß3 integrin,
osteopontin, and thrombin. Am J Pathol 149:293–305[Abstract]
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