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PubMed1991

Production and characterization of osteoclast-selective monoclonal antibodies that distinguish between multinucleated cells derived from different human tissues.

Osteoclastoma-derived giant cells were used to produce 11 mouse monoclonal antibodies (MAb) reactive against human osteoclasts on undecalcified sections of adult human bone. All exhibited unique reactivities across a wide range of human tissues. Three in particular demonstrated distinctive reactivities; C35 was highly selective for bone osteoclasts, C27 showed selective reactivity for osteoclasts, tissue macrophages and blood-borne monocytes, and C22 showed selective membrane staining of osteoclasts. Consequently, C22 was used to coat Dynabeads to affinity-purify viable human osteoclasts from osteoclastoma-derived cell suspensions. Immunocytochemical staining of inflammatory osteoarthritic synovium/granulation tissue demonstrated positivity in the majority of giant cells with MAb C22 and C27. In contrast, C35 reacted with only very occasional giant cells. Furthermore, multinucleated cells formed in long-term human bone marrow cultures demonstrated similar selective staining. C27 stained all giant cells and the majority of mononuclear cells. C22 detected only a small proportion of giant cells. In contrast to its staining on bone osteoclasts, C22 demonstrated granular cytoplasmic staining in cultured giant cells. C35 stained no cells at all in these cultures. These MAb can therefore distinguish between giant cells of various origins and authentic mature osteoclasts. Alternatively, they can recognize antigens expressed at different stages of osteoclast differentiation and therefore provide an excellent tool for the study of the human osteoclast lineage.

Antibodies, Monoclonal↗
PubMed2003

Osteoclast apoptosis: the role of Fas in vivo and in vitro.

Both the number and the activity of osteoclasts are critical for maintaining normal bone turnover. The number is determined by rates of cell differentiation and death. Fas-mediated apoptosis is a dominant mechanism for apoptosis. Here, we show the presence of the Fas receptor on mouse, human, avian, and cultured RAW264.7 (murine) derived osteoclasts and the up-regulation of its expression during mouse osteoclast differentiation. Additionally, Fas is a fully functional death receptor in osteoclasts, and its signaling pathway is consistent with classical Fas signaling in other cell systems, involving mitochondrial release of cytochrome c and activation of caspases 3 and 9. This demonstration of Fas-mediated apoptosis in mature osteoclasts provides a new and potent mechanism for the regulation of osteoclast life span. The in vivo significance of Fas-mediated apoptosis in bone (osteoclasts) was demonstrated in aged Lpr and Gld mice, which have a dysfunctional immune system. Lpr mice, which have a defect in the Fas gene, have decreased bone mineral density, bone volume, trabecular thickness, and increased osteoclast number. Gld mice, which have a Fas ligand mutation, have a slight yet insignificant decrease in bone mineral density, but a highly significant increase in osteoclast number. Taken together, these data demonstrate that the Fas/Fas ligand system is important in the regulation of bone turnover and may represent a critical link between the immune system and bone remodeling in development and in various diseases.

Animals↗
PubMed2006

Colony-stimulating factor-1 increases osteoclast intracellular pH and promotes survival via the electroneutral Na/HCO3 cotransporter NBCn1.

Colony-stimulating factor-1 (CSF-1) promotes the survival of osteoclasts, short-lived cells that resorb bone. Although a rise in intracellular pH (pH(i)) has been linked to inhibition of apoptosis, the effect of CSF-1 on pH(i) in osteoclasts has not been reported. The present study shows that, in the absence of CO(2)/HCO(3)(-), CSF-1 causes little change in osteoclast pH(i). In contrast, exposing these cells to CSF-1 in the presence of CO(2)/HCO(3)(-) causes a rapid and sustained cellular alkalinization. The CSF-1-induced rise in pH(i) is not blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-) transporters but is abolished by removing extracellular sodium. This inhibition profile is similar to that of the electroneutral Na/HCO(3) cotransporter NBCn1. By RT-PCR, NBCn1 transcripts are present in both osteoclasts and osteoclast-like cells (OCLs), and by immunoblotting, the protein is present in OCLs. Moreover, CSF-1 promotes osteoclast survival in the presence of CO(2)/HCO(3)(-) buffer but not in its absence. Preventing the activation of NBCn1 markedly attenuates the ability of CSF-1 to 1) block activation of caspase-8 and 2) prolong osteoclast survival. Inhibiting caspase-3 or caspase-8 in OCLs prolongs osteoclast survival to the same extent as does CSF-1. This study provides the first evidence that osteoclasts express a CSF-1-regulated Na/HCO(3) cotransporter, which may play a role in cell survival.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones.

It has previously only been possible to assess osteoclastic bone resorption in intact bone, where other cell types may modify or mediate osteoclastic responses to environmental agents. We have recently developed techniques which enable us to measure bone resorption by osteoclasts extracted from bone and have used these techniques to assess the effects of prostaglandins (PGs) and calcium-regulating hormones on bone resorption by these cells. Osteoclasts were mechanically disaggregated from neonatal rabbit long bones and cultured on slices of devitalized cortical bone for 8 or 24 h. After this time, osteoclasts were associated with the appearance in the scanning electron microscope of characteristic resorption pits, the volume of which was calculated by computer-assisted morphometric and stereophotogrammetric techniques after removal of cells. Salmon calcitonin inhibited osteoclastic bone resorption at concentrations of 1 pg/ml and above, while PTH and 1,25-dihydroxyvitamin D3 were without significant effect. This suggests that the latter hormones do not increase bone resorption in intact bone through a direct effect on osteoclasts. PGI2, PGE1, and PGE2, all of which are known to stimulate resorption when added to intact bone, paradoxically reduced resorption in our cultures. It appears likely that PGs act as direct inhibitors of osteoclastic bone resorption but have an additional effect on other cells in bone, which are induced by PGs to cause osteoclastic stimulation.

Alprostadil↗
PubMed1986

Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone.

Indirect evidence suggests that cells of the osteoblastic lineage may mediate augmented osteoclastic bone resorption induced by PTH. To test this suggestion, osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Resorption was measured by computer-assisted morphometric and stereophotogram-metric quantification of osteoclastic excavations, identified in the scanning electron microscope after culture. We compared the effect of PTH on bone resorption by osteoclasts incubated alone with the effect of the hormone on resorption by osteoclasts cocultured with osteoblastic cells. PTH had no effect on bone resorption by osteoclasts alone, but in the presence of any of three osteoblast-containing cell populations, or in the presence of cloned, hormone-responsive osteosarcoma cells, PTH caused a 2- to 4-fold increase in osteoclastic resorption. Significant stimulation was observed at 10(-4) IU/ml PTH. None of the osteoblastic cell populations caused morphologically detectable bone resorption in the absence of osteoclasts. These results indicate that PTH acts primarily on osteoblasts, which are induced by the presence of the hormone to stimulate osteoclastic bone resorption.

Animals↗
PubMed1992

Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin.

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function.

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.

Animals↗
PubMed2001

Cytokine-activated endothelium recruits osteoclast precursors.

Osteoclast precursors reach sites of osteoclast formation and remodelling via the vasculature and are therefore destined to encounter endothelium before migrating to the bone surface. Here we investigated the hypothesis that endothelium may be involved in the regulation of osteoclast precursor recruitment to sites of bone resorption. Osteoclast precursors in human peripheral blood were identified by their ability to form mature osteoclasts in 21-day cultures supplemented with RANKLigand, M-CSF, 1,25(OH)(2)-vitamin D(3), dexamethasone and prostaglandin E(2). Under control conditions few osteoclast precursors adhered to endothelial cells (the human bone marrow-derived endothelial cell line BMEC-1). However, BMEC-1 cells treated with the resorption stimulating cytokines IL-1beta and TNFalpha depleted the PBMC population of all osteoclast precursors. These results provide the first evidence that osteoclast precursors can adhere to endothelium and suggest that endothelium could play an important role in the recruitment of osteoclast precursors to sites of bone resorption.

Bone Marrow Cells↗
PubMed2002

Transcriptional activity of nuclei in multinucleated osteoclasts and its modulation by calcitonin.

The function of osteoclasts is to digest the calcified bone matrix. Osteoclasts, together with myotubes, are among the rare examples of multinucleated cells found in higher vertebrates, resulting from the fusion of mononucleated progenitors belonging to the monocyte/macrophage lineage. So far, no information is available about function and transcriptional activity of multiple nuclei in osteoclasts. We have used a run-on technique to visualize RNA synthesis in individual nucleus. We provide the first evidence that nuclei of resorbing osteoclasts, isolated from chick embryo long bones, or differentiated in vitro from murine spleen cells in presence of RANKL and macrophage-colony stimulating factor, are all transcriptionally active. Nevertheless, if transcriptional activity is the same for all the nuclei within a cell, its level varies between osteoclasts: osteoclasts with highly active nuclei are always associated with resorption pits. We found that global transcription activity of resorbing osteoclasts seeded on calcified matrix is down-regulated after 5-h treatment with calcitonin, which transiently blocks resorption. This effect is reversible because calcitonin removal led to nuclear transcription activation. These results indicate a strong correlation between transcription and resorption. Finally, our data indicate that the resorption pit surface is linearly related to the nuclei number per osteoclast, strongly suggesting that functional advantage of osteoclast multinucleation is to improve resorption efficiency.

Animals↗
PubMed1995

Localisation of mRNA for collagenase in osteocytic, bone surface and chondrocytic cells but not osteoclasts.

Osteoclasts resorb the extracellular matrix of bone by secreting protons and enzymes into a circumpherentially sealed compartment between the osteoclast and the bone surface. Although the lysosomal cysteine proteinases play a major role in matrix degradation by osteoclasts, collagenase (matrix metalloproteinase-1, EC 3.4.24.7) is also required for osteoclastic bone resorption, and may be directly involved in collagen degradation in the hemivacuole. We assessed the effects of inhibitors of cysteine proteinases and collagenase on bone resorption by osteoclasts isolated from rodent bone. We found that while inhibition of cysteine proteinases strongly suppressed osteoclastic resorption, inhibitors of collagenase were without effect on the number, size, or demineralised fringe of excavations. We could find no evidence of expression of mRNA for collagenase in rat osteoclasts by in situ hybridisation, but found that it was expressed by chondrocytes, bone surface cells and osteocytes adjacent to osteoclasts. The distribution of these cells, and the correlation between increased collagenase production and increased stimulation of osteoclastic resorption in vitro by bone cells, suggests that these cells might be involved in the regulation of bone resorption in situ, and that collagenase production might play a role in this process.

Animals↗
PubMed1998

Adhesive and bone resorptive activities of isolated osteoclasts from hen medullary bone.

In the present study, osteoclasts were isolated from hen medullary bones at the formative and resorptive phases. The cells were cultured on glass culture dishes and bone slices. After culturing, the adhesion activity of the isolated osteoclasts with the substrates was estimated with a light microscope, and the surfaces of the bone slices were observed with a scanning electron microscope. The results showed that the adhesion activity of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts is higher at the bone resorptive phase than at the bone formative phase, and this tendency in isolated osteoclasts was observed more frequently on the bone slices than on the glass culture dishes. Furthermore, scanning electron microscopy showed that the isolated osteoclasts in the bone resorptive phase adhered to the bone surface with developed-cytoplasmic projections and formed broad pits where collagen fibrils were exposed. On the other hand, isolated osteoclasts in the bone formative phase adhered to the bone slice with board-shaped cytoplasmic projections and did not form any pits. These results suggest that isolated osteoclasts in the bone resorptive phase have a high level of adhesion activity and actively resorb the bone, whereas isolated osteoclasts in the bone formative phase have a low level of adhesion activity and cease bone resorption. The procedure reported here is useful for studying the bone-resorptive mechanism of authentic osteoclasts.

Acid Phosphatase↗
PubMed2005

Regulation of osteoclast apoptosis and motility by small GTPase binding protein Rac1.

UNLABELLED: The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts. INTRODUCTION: Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated. MATERIALS AND METHODS: We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1(DN)) or constitutively active Rac1 (Rac1(CA)) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assays, survival assays, and Western blotting, including an activated-Rac1 pull-down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1(DN) were recorded with a time-lapse video microscope. RESULTS: Adenovirus vector-mediated dominant negative Rac1 (Rac1(DN)) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival effect of M-CSF for OCLs was abrogated by Rac1(DN) overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3'-kinase (PI3K) inhibitors, whereas a Mek inhibitor had only partial effect. Rac1(DN) also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1(DN) expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF. CONCLUSIONS: Small guanosine triphosphatase (GTPase) Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.

Actins↗
PubMed2006

Effect of monoterpenes on the formation and activation of osteoclasts in vitro.

UNLABELLED: Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. INTRODUCTION: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. MATERIALS AND METHODS: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. RESULTS: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. CONCLUSIONS: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.

Animals↗
PubMed1998

The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors.

The most obvious proteolytic event controlled by the osteoclast is bone matrix removal in the resorption compartment. Here, however, we investigated whether matrix metalloproteinase (MMP) activity of the osteoclast might be involved in its migration to its future bone resorption site. We seeded either nonpurified or purified osteoclasts onto either uncoated or collagen-coated dentine slices and cultured them in the presence or absence of specific MMP inhibitors. When nonpurified osteoclasts were cultured on uncoated dentine, MMP inhibitors did not prevent pit formation, as previously reported. However, when collagen-coated dentine was used, pit formation was strongly inhibited by MMP inhibitors. The same results were obtained when performing these experiments with purified osteoclasts, thus demonstrating the ability of osteoclasts by themselves to migrate through collagen via an MMP-dependent pathway. This demonstration was confirmed by using collagen-coated invasion chambers. In addition, the invasions were not, or only slightly, inhibited by inhibitors of serine proteinases, cysteine proteinases, and carbonic anhydrase, though the latter two are well established bone resorption inhibitors that strongly inhibited pit formation. It is concluded that osteoclasts can migrate through collagen in the absence of other cells and that this migration relies on MMP activity, whereas other enzymes typically required for bone removal in the resorption compartment are not essential for migration. Some of the osteoclast MMPs might thus be relevant to the migratory/invasive activity of the osteoclast, rather than to its bone resorptive activity itself.

Amino Acids, Branched-Chain↗
PubMed2000

Force-induced osteoclast apoptosis in vivo is accompanied by elevation in transforming growth factor beta and osteoprotegerin expression.

The mechanism controlling the disappearance of osteoclasts from bone surfaces after bone resorption in vivo is largely unknown. This is because there is no suitable experimental system to trace the final fate of osteoclasts. Here, we used an experimental model of tooth movement in rats to show that preexisting osteoclasts disappeared from the bone surface through apoptosis during a force-induced rapid shift from bone resorption to formation. On the distal alveolar bone surface of the maxillary molar in growing rats, many mature osteoclasts were present. When light tensional force was applied to the bone surface through an orthodontic appliance, these preexisting osteoclasts gradually disappeared. One day after the application of force, about 24% of the osteoclasts exhibited apoptotic morphology and the proportion of apoptotic cells was increased to 41% by day 2, then decreased afterward. These changes were undetectable on the control distal alveolar bone surface, which is free from tensional force. As shown by in situ hybridization, a marked increase in transforming growth factor beta1 (TGF-beta1) and osteoprotegerin (OPG) messenger RNA (mRNA) was observed in the stretched cells on the tensioned distal bone surface, simultaneously with the loss of osteoclasts. Both of these factors are known to have a negative effect on osteoclast recruitment and survival. As early as 2 days after force application, some of these stretched cells were identified as cuboidal osteoblasts showing intense signals for both factors. Our data suggest there may be a sequential link in tensional force applied on the bone lining cells, up-regulation of TGF-beta1/OPG, and disappearance of osteoclasts.

Acid Phosphatase↗
PubMed2001

Generation of bone-resorbing osteoclasts from B220+ cells: its role in accelerated osteoclastogenesis due to estrogen deficiency.

Estrogen deficiency stimulates both osteoclastic bone resorption and pre-B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220+ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220+ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220+ and B220- cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross-contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate-resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220+ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220+ cells are capable of generating osteoclasts and that the increase in the number of B220+ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.

Acid Phosphatase↗
PubMed2001

Mechanical strain effect on bone-resorbing activity and messenger RNA expressions of marker enzymes in isolated osteoclast culture.

Adaptive modeling and remodeling are controlled by the activities of osteoblasts and osteoclasts, which are capable of sensing their mechanical environments and regulating deposition or resorption of bone matrix. The effects of mechanical stimuli on isolated osteoclasts have been scarcely examined because it has proven to be difficult to prepare a number of pure osteoclasts and to cultivate them on mineralized substratum during mechanical stimulation. Recently, we developed an apparatus for applying mechanical stretching to the ivory slice/plastic plate component on which cells could be cultured. The loading frequency, strain rate, and generated strain over an ivory surface could be controlled by a personal computer. Using this apparatus, we examined the role of mechanical stretching on the bone-resorbing activity of the osteoclasts. Mature and highly enriched osteoclasts were cultured for 2, 12, and 24 h on the ivory/plate component while being subjected to intermittent tensile strain. The stretched osteoclasts showed enhanced messenger RNA (mRNA) expression levels of osteoclast marker enzymes, tartrate-resistant acid phosphatase (TRAP), and cathepsin K and increases of resorbed-pit formation, suggesting that the mechanical stretching up-regulated the bone-resorbing activity of the osteoclasts. A stretch-activated cation (SA-cat) channel blocker significantly inhibited the increases of the mRNA level and pit formation after 24 h of stretching. This study suggested the possibility that the mature osteoclasts responded to mechanical stretching through a mechanism involving a SA-cat channel in the absence of mesenchymal cells and, as a result, up-regulated their bone-resorbing activity.

Acid Phosphatase↗
PubMed2002

NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.

Expression of RANKL by stromal cells and of RANK and both NF-kappaB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF-KB signaling in this process, we used NF-kappaB p50-/- ;p52-/- double knockout (dKO) and wild-type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony-stimulating factor (M-CSF). NF-kappaB dKO splenocytes formed more colony-forming unit granulocyte macrophage (CFU-GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU-GM colonies. RANKL increased the number of CFU-GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from NF-kappaB dKO mice revealed a two-to threefold increase in the percentage of CD11b (Mac-1) and RANK double-positive cells compared with WT controls. Treatment of NF-kappaB dKO splenocytes with interleukin (IL)-1, TNF-alpha, M-CSF, GM-CSF, and IL-6 plus soluble IL-6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF-kappaB dKO or p50-/-;p52+/- (3/4KO) mice. Thus, NF-kappaB p50 and p52 expression is not required for formation of RANK-expressing osteoclast progenitors but is essential for RANK-expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.

Animals↗