[Show abstract][Hide abstract] ABSTRACT: Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.
The Journal of Cell Biology 05/2012; 197(4):553-68. · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fos plays essential roles in the osteoclastic differentiation of precursor cells generated by colony-stimulating factor 1 (CSF-1) and receptor activator of NF-κB ligand (RANKL; also known as tumor necrosis factor ligand superfamily member 11, Tnsf11). RANKL-deficient (RANKL(-/-)) mice and Fos(-/-) mice exhibit osteopetrosis due to an osteoclast deficiency. We previously reported that RANK-positive osteoclast precursors are present in bone of RANKL(-/-) mice but not Fos(-/-) mice. Here we report the role of Fos in RANK expression in osteoclast precursors. Medullary thymic epithelial cells and intestinal antigen-sampling microfold cells have been shown to express RANK. High expression of RANK was observed in some epithelial cells in the thymic medulla and intestine but not in osteoclast precursors in Fos(-/-) mice. RANK mRNA and protein levels in bone were lower in Fos(-/-) mice than RANKL(-/-) mice, suggesting that Fos-regulated RANK expression is tissue specific. When wild-type bone marrow cells were inoculated into Fos(-/-) mice, RANK-positive cells appeared along bones. RANK expression in wild-type macrophages was upregulated by coculturing with RANKL(-/-) osteoblasts as well as wild-type osteoblasts, suggesting that cytokines other than RANKL expressed by osteoblasts upregulate RANK expression in osteoclast precursors. CSF-1 receptor-positive cells were detected near CSF-1-expressing osteoblastic cells in bone in Fos(-/-) mice. CSF-1 upregulated RANK expression in wild-type macrophages but not Fos(-/-) macrophages. Overexpression of Fos in Fos(-/-) macrophages resulted in the upregulation of RANK expression. Overexpression of RANK in Fos(-/-) macrophages caused RANKL-induced signals, but failed to recover the RANKL-induced osteoclastogenesis. These results suggest that Fos plays essential roles in the upregulation of RANK expression in osteoclast precursors within the bone environment.
[Show abstract][Hide abstract] ABSTRACT: Multi-walled carbon nanotubes (MWCNTs) promote calcification during hydroxyapatite (HA) formation by osteoblasts. Primary cultured osteoblasts are incubated with MWCNTs or carbon black. After culture for 3 weeks, the degree of calcification is very high in the 50 μg mL(-1) MWCNT group. Transmission electron microscopy shows needle-like crystals around the MWCNTs, and diffraction patterns reveal that the peak of the crystals almost coincides with the known peak of HA.
[Show abstract][Hide abstract] ABSTRACT: The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.
Nature medicine 02/2012; 18(3):405-12. · 28.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetracyclines, such as doxycycline and minocycline, are used to suppress the growth of bacteria in patients with inflammatory diseases. Tetracyclines have been shown to prevent bone loss, but the mechanism involved is unknown. Osteoclasts and dendritic cells (DCs) are derived from common progenitors, such as bone marrow-derived macrophages (BMMs). In this article, we show that tetracyclines convert the differentiation pathway, resulting in DC-like cells not osteoclasts. Doxycycline and minocycline inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis of BMMs, but they had no effects on cell growth and phagocytic activity. They influenced neither the proliferation nor the differentiation of bone-forming osteoblasts. Surprisingly, doxycycline and minocycline induced the expression of DC markers, CD11c and CD86, in BMMs in the presence of RANKL. STAT5 is involved in DC differentiation induced by GM-CSF. Midostaurin, a STAT5-signaling inhibitor, and an anti-GM-CSF-neutralizing Ab suppressed the differentiation induced by GM-CSF but not by tetracyclines. In vivo, the injection of tetracyclines into RANKL-injected mice and RANKL-transgenic mice suppressed RANKL-induced osteoclastogenesis and promoted the concomitant appearance of CD11c(+) cells. These results suggested that tetracyclines prevent bone loss induced by local inflammation, including rheumatoid arthritis and periodontitis, through osteoclast-DC-like cell conversion.
The Journal of Immunology 02/2012; 188(4):1772-81. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts.
[Show abstract][Hide abstract] ABSTRACT: Eldecalcitol (ED-71) is a new vitamin D₃ derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED-71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED-71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED-71 (50 ng/kg body weight) to 8-week-old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED-71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle-arrested receptor activator of NF-κB (RANK)-positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED-71 affected neither the number of RANK-positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED-71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL-positive cell surface around the trabecular bone was significantly reduced in ED-71-treated mice than in the control mice. ED-71 administration also increased BMD in 12-week-old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED-71 increases BMD by suppressing RANKL expression in trabecular bone in vivo.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 11/2011; 27(2):461-73. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are terminally differentiated multinucleated cells that are the principal resorptive cells of bone, playing a central role in the formation of the skeleton and regulation of its mass. The molecular events involved in the differentiation and function of osteoclasts had not been clarified for a long time. Over the past two decades, several novel approaches have been developed and adopted to investigate osteoclast biology. In the present review, we would like to update recent progress in the elucidation of the molecular mechanism of osteoclast activation and function.
Modern Rheumatology 09/2011; 22(2):167-77. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.
Journal of Biological Chemistry 08/2011; 286(42):37023-31. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. We previously showed that cell cycle-arrested quiescent osteoclast precursors (QOPs) were detected along bone surfaces as direct osteoclast precursors. Here we show that receptor activator of NF-κB (RANK)-positive cells isolated from bone marrow and peripheral blood possess characteristics of QOPs in mice. RANK-positive cells expressed c-Fms (receptors of macrophage colony-stimulating factor) at various levels, but scarcely expressed other monocyte/granulocyte markers. RANK-positive cells failed to exert phagocytic and proliferating activities, and differentiated into osteoclasts but not into dendritic cells. To identify circulating QOPs, collagen disks containing bone morphogenetic protein-2 (BMP disks) were implanted into mice, which were administered bromodeoxyuridine daily. Most nuclei of osteoclasts detected in BMP-2-induced ectopic bone were bromodeoxyuridine-negative. RANK-positive cells in peripheral blood proliferated more slowly and had a much longer lifespan than F4/80 (a macrophage marker)-positive macrophages. When BMP disks and control disks were implanted in RANK ligand-deficient mice, RANK-positive cells were observed in the BMP disks but not in the controls. F4/80-positive cells were distributed in both disks. Administration of FYT720, a sphingosine 1-phosphate agonist, promoted the egress of RANK-positive cells from hematopoietic tissues into bloodstream. These results suggest that lineage-determined QOPs circulate in the blood and settle in the bone.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 08/2011; 26(12):2978-90. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the
effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL neutralizing monoclonal antibody (Mab, clone OYC1)
and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of
5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density (BMD) in the distal femoral metaphysis from day
2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRACP-5b, a marker
for osteoclasts) on day 1 and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (ALP,
a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts
in femurs of the treated mice on day 4 and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection
of parathyroid hormone (PTH) for 2 weeks increased the BMD in trabecular and cortical bone by stimulating bone formation in
the OYC1-treated mice. These results suggest that PTH exerted its bone anabolic activity in mice with few osteoclasts. The
mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.
Journal of Biological Chemistry 08/2011; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoporosis is caused by imbalance between osteoclastic bone resorption and osteoblastic bone formation. From the recent results of several kinds of knockout mice, osteoclast differentiation factor (RANKL) and its soluble decoy receptor for RANKL (OPG) are essentially involved in pathogenesis of osteoporosis. Deficiency of RANKL in human has been shown to result in osteopetrosis. Furthermore, it has been reported that anti-RANKL neutralizing antibody (denosumab) will be effective new drug for osteoporosis.
[Show abstract][Hide abstract] ABSTRACT: The effects of gravity on bone metabolism are unclear, and little has been reported about the effects of hypergravity on the mature skeleton. Since low gravity has been shown to decrease bone volume, we hypothesized that hypergravity increases bone volume. To clarify this hypothesis, adult female rats were ovariectomized and exposed to hypergravity (2.9G) using a centrifugation system. The rats were killed 28 days after the start of loading, and the distal femoral metaphysis of the rats was studied. Bone architecture was assessed by micro-computed tomography (micro-CT) and bone mineral density was measured using peripheral quantitative CT (pQCT). Hypergravity increased the trabecular bone volume of ovariectomized rats. Histomorphometric analyses revealed that hypergravity suppressed both bone formation and resorption and increased bone volume in ovariectomized rats.Further, the cell morphology, activity, proliferation, and differentiation of osteoclasts and osteoblasts exposed to hypergravity were evaluated in vitro. Hypergravity inhibited actin ring formation in mature osteoclasts, which suggested that the osteoclast activity was suppressed. However, hypergravity had no effect on osteoblasts. These results suggest that hypergravity can stimulate an increase in bone volume by suppressing bone resorption in ovariectomized rats.
Advances in Space Research 04/2011; · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the middle ear, a chain of three tiny bones (ie, malleus, incus, and stapes) vibrates to transmit sound from the tympanic membrane to the inner ear. Little is known about whether and how bone-resorbing osteoclasts play a role in the vibration of auditory ossicles. We analyzed hearing function and morphological features of auditory ossicles in osteopetrotic mice, which lack osteoclasts because of the deficiency of either cytokine RANKL or transcription factor c-Fos. The auditory brainstem response showed that mice of both genotypes experienced hearing loss, and laser Doppler vibrometry revealed that the malleus behind the tympanic membrane failed to vibrate. Histological analysis and X-ray tomographic microscopy using synchrotron radiation showed that auditory ossicles in osteopetrotic mice were thicker and more cartilaginous than those in control mice. Most interestingly, the malleal processus brevis touched the medial wall of the tympanic cavity in osteopetrotic mice, which was also the case for c-Src kinase-deficient mice (with normal numbers of nonresorbing osteoclasts). Osteopetrotic mice showed a smaller volume of the tympanic cavity but had larger auditory ossicles compared with controls. These data suggest that osteoclastic bone resorption is required for thinning of auditory ossicles and enlargement of the tympanic cavity so that auditory ossicles vibrate freely.
American Journal Of Pathology 03/2011; 178(3):1270-8. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity. The aim of this study was to investigate whether MTA may prevent osteoclast differentiation in vitro. MTA solution, but not other commonly used retrofilling materials, such as Dycal, Super-EBA, or intermediate restorative material (IRM) solution, dose-dependently inhibited osteoclastogenesis in cocultures of mouse bone marrow cells (BMCs) with primary osteoblast cells (POBs) induced by 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2) D(3) ]. Exogenous CaCl(2) medium supplementation did not inhibit osteoclastogenesis in cocultures. Furthermore, MTA solution did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, suggesting that POBs are targets of MTA. MTA solution suppressed the 1α,25(OH)(2) D(3) -induced reduction of osteoprotegerin (OPG) mRNA and protein production without changing RANKL expression in POBs. Consistent with this result, MTA solution did not inhibit osteoclastogenesis in cocultures of BMCs and POBs from OPG-deficient mice. Therefore, the maintenance of OPG expression in POBs appears to be critical for the inhibitory effect of MTA solution on osteoclast differentiation.
Journal of Biomedical Materials Research Part A 02/2011; 96(2):358-64. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy.
Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers.
The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium.
The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA.
[Show abstract][Hide abstract] ABSTRACT: Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte-macrophage lineage. Bone-forming osteoblasts play a role in the formation of osteoclasts. Osteoblasts/stromal cells express two cytokines essential for osteoclastogenesis: receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Using RANKL and M-CSF, osteoclasts can be induced from monocyte-macrophage lineage cells even in the absence of osteoblasts. We describe here methods for the identification of osteoclasts formed in vitro.
[Show abstract][Hide abstract] ABSTRACT: To present a vertical ridge augmentation with a composite synthetic bone graft by the mixed bone-filling materials and autogenous bone chips retrieved from the implant osteotomy site for a case for esthetic implant prosthesis on the atrophied alveolar bone.
Nonresorbable and resorbable hydroxyapatite and demineralized freeze-dried bone allograft particles were used at ratio of 1:2:2 (volume ratio). Titanium micromesh was used for alveolar ridge configuration and retention. After 8 months, implant placement and interimplant bone augmentation were performed.
15-mm vertical and 10-mm horizontal bone augmentation has been achieved. Marginal bone heights and periimplant mucosa remained stable in cases of loading after >4 years of follow-up.
These results suggest that this method has the potential for use in esthetic implant rehabilitation on the highly atrophied alveolar bone.
[Show abstract][Hide abstract] ABSTRACT: The quantity of the receptor activator of NF-κB ligand (RANKL) expressed at the cell surface of osteoblastic cells is an important factor regulating osteoclast activation. Previously, RANKL was found to be localized to secretory lysosomes in osteoblastic cells and to translocate to the cell surface in response to stimulation with RANK-Fc-conjugated beads. However, the in vivo significance of stimulation-dependent RANKL release has not been elucidated. In this study we show that small GTPases Rab27a and Rab27b are involved in the stimulation-dependent RANKL release pathway in osteoblastic cells. Suppression of either Rab27a or Rab27b resulted in a marked reduction in RANKL release after stimulation. Slp4-a, Slp5, and Munc13-4 acted as effector molecules that coordinated Rab27a/b activity in this pathway. Suppression of Rab27a/b or these effector molecules did not inhibit accumulation of RANKL in lysosomal vesicles around the stimulated sites but did inhibit the fusion of these vesicles to the plasma membrane. In osteoblastic cells, suppression of the effector molecules resulted in reduced osteoclastogenic ability. Furthermore, Jinx mice, which lack a functional Munc13-4 gene, exhibited a phenotype characterized by increased bone volume near the tibial metaphysis caused by low bone resorptive activity. In conclusion, stimulation-dependent RANKL release is mediated by Rab27a/b and their effector molecules, and this mechanism may be important for osteoclast activation in vivo.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 10/2010; 26(4):689-703. · 6.04 Impact Factor