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PubMed2000

Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts.

Osteoclasts are thought to belong to a macrophage lineage. However, the nature of common precursors of osteoclasts and macrophages remains to be investigated. We have characterized the differentiation potential of mouse bone marrow macrophages into mature osteoclasts. Monocyte macrophage-colony-stimulating factor (M-CSF) stimulated the proliferation of bone marrow macrophages in a dose-dependent manner and these M-CSF-dependent bone marrow macrophage (MDBM) cells efficiently differentiated into the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the presence of soluble RANKL (sRANKL) and M-CSF in the in vitro culture. The macrophage-like cell line TMC16 was established from tsA58 (temperature-sensitive SV40 large T-antigen) transgenic mice in the same manner to the preparation of MDBM cells and also differentiated into mature osteoclasts. During this differentiation in vitro, the morphology of the cells changed from spindle to round and smaller (termed pOC) on day 2 and to multinuclear (termed multinucleated cells [MNCs]) on day 4. The surface expression of macrophage marker CD14 was down-regulated and that of CD43 was up-regulated on pOC, analyzed by flow cytometry. RNA analysis revealed that osteoclast marker genes such as calcitonin receptor (CTR), carbonic anhydrase II (CAII), cathepsin K (cath K), MMP9, and TRAP were strongly expressed in MNCs and weakly in pOC whereas MDBM cells did not express these genes. However, the osteopontin (OPN) gene was strongly expressed in MDBM cells and this expression became weakened after differentiation into pOC. The TMC16 cell line weakly expressed cath K, TRAP, and OPN, suggesting that the TMC16 cell line is immortalized at a stage slightly differentiated from MDBM cells. Furthermore, cell sorting analysis revealed that osteoclast early progenitors in bone marrow cells are preferentially present in the Mac-1- F4/80dull population, which differentiated into MDBM cells (the osteoclast progenitor) expressing Mac-1+ F4/80int, suggesting that M-CSF plays roles of a differentiation factor as well as a growth factor for osteoclast early progenitors. These results showed the transition of morphology, surface markers, and gene expression from the early to mature stage in osteoclast differentiation. We propose three differentiation stages in the osteoclast lineage: the pro-osteoclast (spindle-shaped macrophage cells), the pre-osteoclast (small round mononucleated TRAP-positive cells), and the mature osteoclast (multinucleated TRAP-positive cells) stage.

Animals↗
PubMed1993

Long-term culture of disaggregated rat osteoclasts: inhibition of bone resorption and reduction of osteoclast-like cell number by calcitonin and PTHrP[107-139].

The isolated osteoclast bone resorption assay has proved to be a useful means of examining the response of mammalian and avian osteoclasts to a variety of stimuli. The assay has traditionally been performed over a period of 24 hours. By extending the duration of the osteoclast bone resorption assay, we have been able to assess the long-term effects of carboxyl-terminal parathyroid hormone-related protein (hPTHrP[107-139]), salmon calcitonin (sCT) and hPTH[1-34] on bone resorption and TRACP-positive osteoclast-like cell number. We found that, in control cultures over a period of up to 144 hours, the osteoclast-like cells not only remained viable but their numbers also increased. The number of mononucleated and multinucleated osteoclast-like cells doubled in the first 48 hours before stabilizing over the remainder of the incubation period. Osteoblasts also proliferated, resulting in a resorption response to hPTH[1-34] being evident from 48 hours onward. hPTHrP]107-139] persistently inhibited basal and PTH-stimulated bone resorption for at least 96-144 hours, whereas "escape" from the inhibitory effect of sCT was seen after 48-72 hours. Decreased numbers of both mononucleated and multinucleated TRACP-positive osteoclast-like cells were seen by 48 hours in cultures treated with sCT. In contrast, hPTHrP[107-139] reduced the number of mononuclear TRACP-positive cells with only a late effect on multinucleated cells. Furthermore, the increased number of osteoclast-like cells seen in response to hPTH[1-34] was inhibited by carboxyl-terminal PTHrP. In summary, this study indicates that the extended bone resorption assay system is a complex one where both osteoclastic resorption and osteoclast maturation are evident. Using this system, we have shown that hPTHrP[107-139] acts as a potent long-term inhibitor of osteoclastic bone resorption, without evidence of escape from its effect. Its action to reduce the number of mononucleated osteoclast-like cells suggests that it affects several aspects of osteoclast activity.

Acid Phosphatase↗
PubMed2002

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts.

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.

Acid Phosphatase↗
PubMed2006

The dendritic cell-specific transmembrane protein DC-STAMP is essential for osteoclast fusion and osteoclast bone-resorbing activity.

Osteoclasts are bone-resorbing cells that play a critical role for bone destruction in rheumatoid arthritis. It is well known that osteoclasts form multinuclear cells by cell-cell fusion of mononuclear osteoclasts; however, what molecules are required for osteoclast cell-cell fusion, and the role of multinucleation remain uncharacterized. We identified the dendritic cell-specific transmembrane protein DC-STAMP, a putative seven transmembrane protein, and generated DC-STAMP-deficient mice. The cell fusion of osteoclasts was completely abrogated in DC-STAMP-deficient mice, while the transcription factors required for osteoclast differentiation or osteoclast maturation markers were induced as wild type osteoclasts. Interestingly, bone-resorbing activity was reduced in DC-STAMP-deficient osteoclasts compared with wild-type osteoclasts, and DC-STAMP-deficient mice showed osteopetrosis. Thus, we identified DC-STAMP as an essential molecule for osteoclast cell-cell fusion, and found that multinuclear osteoclasts have a higher bone-resorbing activity than mononuclear osteoclastic cells seen in DC-STAMP-deficient mice.

Animals↗
PubMed2000

Sequential requirements for SCL/tal-1, GATA-2, macrophage colony-stimulating factor, and osteoclast differentiation factor/osteoprotegerin ligand in osteoclast development.

OBJECTIVE: Osteoclasts are of hematopoietic origin. The mechanism by which hematopoietic stem cells are specified to the osteoclast lineage is unclear. To understand the process of generation and differentiation of this lineage of cells, we performed in vitro studies on the differentiation of embryonic stem cells. MATERIALS AND METHODS: We examined the potential of mutant embryonic stem cell lines harboring targeted deletions of the GATA-1, FOG, SCL/tal-1, or GATA-2 genes to differentiate into osteoclasts and determined when these molecules function in osteoclast development. RESULTS: The lack of GATA-1 or FOG did not affect osteoclastogenesis. In contrast, SCL/tal-1-null embryonic stem cells generated no osteoclasts. In the case of the loss of GATA-2, a small number of osteoclasts were generated. GATA-2-null osteoclasts were morphologically normal and the terminal maturation was not disturbed, but a defect was observed in the generation of osteoclast progenitors. Experiments using specific inhibitors that block the signaling through macrophage colony-stimulating factor and osteoclast differentiation factor/osteoprotegerin ligand suggested that GATA-2 seems to act earlier in osteoclastogenesis than these cytokines. Interestingly, macrophage colony-forming units were not severely reduced by the loss of GATA-2 compared to osteoclast progenitors. CONCLUSION: These results indicate that osteocalsts need SCL/tal-1 at an early point in development, and that GATA-2 is required for generation of osteoclast progenitors but not for the later stages when macrophage colony-stimulating factor and osteoclast differentiation factor/ osteoprotegerin ligand are needed. We also demonstrated that osteoclast progenitors behave as a different population than macrophage colony-forming units.

Basic Helix-Loop-Helix Proteins↗
PubMed2002

p38 MAPK-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function.

Receptor activator of nuclear factor-kappaB ligand (RANKL)-induced signals play critical roles in osteoclast differentiation and function. SB203580, an inhibitor of p38 MAPK, blocked osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2) in cocultures of mouse osteoblasts and bone marrow cells. Nevertheless, SB203580 showed no inhibitory effect on RANKL expression in osteoblasts treated with 1alpha,25-dihydroxyvitamin D(3) and prostaglandin E(2). RANKL-induced osteoclastogenesis in bone marrow cultures was inhibited by SB203580, suggesting a direct effect of SB203580 on osteoclast precursors, but not on osteoblasts, in osteoclast differentiation. However, SB203580 inhibited neither the survival nor dentine-resorption activity of osteoclasts induced by RANKL. Lipopolysaccharide (LPS), IL-1, and TNFalpha all stimulated the survival of osteoclasts, which was not inhibited by SB203580. Phosphorylation of p38 MAPK was induced by RANKL, IL-1, TNFalpha, and LPS in osteoclast precursors but not in osteoclasts. LPS stimulated phosphorylation of MAPK kinase 3/6 and ATF2, upstream and downstream signals of p38 MAPK, respectively, in osteoclast precursors but not in osteoclasts. Nevertheless, LPS induced degradation of IkappaB and phosphorylation of ERK in osteoclasts as well as in osteoclast precursors. These results suggest that osteoclast function is induced through a mechanism independent of p38 MAPK-mediated signaling.

Animals↗
PubMed1997

Cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity.

Interferon-gamma (IFN-gamma) has been shown to inhibit interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) stimulated bone resorption by strongly stimulating nitric oxide (NO) synthesis. Here we studied the mechanisms underlying this inhibition. Osteoclasts were generated in 10-day cocultures of mouse osteoblasts and bone marrow cells and the effect of cytokine-induced NO on osteoclast formation and activity was determined. Stimulation of the cocultures with IL-1 beta, TNF-alpha and IFN-gamma markedly enhanced NO production by 50- to 70-fold, and this was found to be derived predominantly from the osteoblast cell layer. When high levels of NO were induced by cytokines during early stages of the cocultures, osteoclast formation was virtually abolished and bone resorption markedly inhibited. Cytokine stimulation during the latter stages of coculture also resulted in inhibition of bone resorption, but here the effects were mainly due to an inhibitory effect on osteoclast activity. At all stages, however, the inhibitory effects of cytokines on osteoclast formation and activity were blocked by the NO-synthase inhibitor L-NMMA. Further investigations suggested that the NO-mediated inhibition of osteoclast formation was due in part to apoptosis of osteoclast progenitors. Cytokine stimulation during the early stage of the culture caused a large increase in apoptosis of bone marrow cells, and these effects were blocked by L-NMMA and enhanced by NO donors. We found no evidence of apoptosis in osteoclasts exposed to high levels of cytokine-induced NO at any stage in the culture, however, or of apoptosis affecting mature osteoclasts exposed to high levels of NO, suggesting that immature cells in the bone marrow compartment are most sensitive to NO-induced apoptosis. In summary, these studies identify NO as a potentially important osteoblast-osteoclast coupling factor which has potent inhibitory effects on bone resorption. These actions, in turn, are mediated by inhibition of osteoclast formation probably due to NO-induced apoptosis of osteoclast progenitors and by inhibition of the resorptive activity of mature osteoclasts.

Animals↗
PubMed2003

IL-3 acts directly on osteoclast precursors and irreversibly inhibits receptor activator of NF-kappa B ligand-induced osteoclast differentiation by diverting the cells to macrophage lineage.

Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of the monocyte/macrophage lineage in the presence of M-CSF and receptor activator of NF-kappaB ligand (RANKL). In this study we investigated the role of IL-3 in osteoclast differentiation. We show here that IL-3, a cytokine secreted by activated T lymphocytes, inhibits RANKL-induced osteoclast differentiation by a direct action on early osteoclast precursors. Anti-IL-3 Ab neutralized the inhibitory effect of IL-3 on osteoclast differentiation. In addition, IL-3 inhibits TNF-alpha-induced osteoclast differentiation in bone marrow-derived macrophages. However, IL-3 has no inhibitory effect on mature osteoclasts. In osteoclast precursors, IL-3 prevents RANKL-induced nuclear translocation of NF-kappaB by inhibiting the phosphorylation and degradation of IkappaB. RT-PCR analysis revealed that IL-3 down-regulated c-Fos transcription. Interestingly, the osteoclast precursors in the presence of IL-3 showed strong expression of macrophage markers such as Mac-1, MOMA-2, and F4/80. Furthermore, the inhibitory effect of IL-3 on osteoclast differentiation was irreversible, and the osteoclast precursors preincubated in IL-3 were resistant to RANKL action. Thus, our results reveal for the first time that IL-3 acts directly on early osteoclast precursors and irreversibly blocks RANKL-induced osteoclast differentiation by diverting the cells to macrophage lineage.

Active Transport, Cell Nucleus↗
PubMed1991

Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts.

Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody-coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified populations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.

Animals↗
PubMed1994

Integrins and osteoclastic resorption in three bone organ cultures: differential sensitivity to synthetic Arg-Gly-Asp peptides during osteoclast formation.

We investigated possible inhibitory effects of five synthetic Arg-Gly-Asp (RGD)-containing peptides on osteoclastic resorption in three distinct in vitro resorption assays (17-day-old fetal mouse bone organ cultures) that differ in stages of osteoclast differentiation. RGD peptides, which can bind the adhesion receptors called integrins, inhibited osteoclastic resorption (45Ca release) in fetal mouse bone explants in which osteoclast precursors have yet to adhere to the mineralized matrix and develop into mature osteoclasts (metacarpals and coculture system). Treatment of metacarpals with RGD peptides inhibited the formation of multinucleated TRAP+ osteoclasts in the mineralized matrix because their mononuclear TRAP+ osteoclast precursors remained localized in the periosteum. In particular, echistatin, a viper venom protein with known affinity for alpha v beta 3 integrin, and GdRGDSP inhibited osteoclastic resorption dose dependently in these systems (ED50 10(-9) and 10(-4) M, respectively) but did not alter the activity of mature resorbing osteoclasts in radii. In addition, 45Ca release was significantly inhibited by the cyclic peptide GPenGRGDSPCA, which has a relatively higher affinity for the vitronectin than fibronectin receptor(s). In contrast, GRDGdSP, which has a much higher affinity for the fibronectin receptor (than the vitronectin receptors), had no effect on resorption at similar concentrations in any resorption system used. In summary, the data presented in this paper show that peptides with RGD motifs are capable of inhibiting osteoclastic resorption in bone organ cultures. Our studies not only support the hypothesis concerning the importance of alpha v beta 3 in osteoclastic resorption but also suggest an important role of integrin(s) in events preceding the actual resorption of calcified matrix by osteoclasts.

Amino Acid Sequence↗
PubMed1995

Osteoclast 121F antigen expression during osteoblast conditioned medium induction of osteoclast-like cells in vitro: relationship to calcitonin responsiveness, tartrate resistant acid phosphatase levels, and bone resorptive activity.

Osteoclast differentiation from hematopoietic precursors into multinucleated cells uniquely capable of removing the organic and inorganic components of bone matrix occurs in multistep process, during which osteoclasts acquire the specialized characteristics necessary for bone resorptive activity and physiological regulation. Among those traits is a novel plasma membrane glycoprotein, reactive with the anti-osteoclast monoclonal antibody 121F, which is expressed during the course of osteoclast differentiation, shares structural and functional homologies with Mn2+/Fe2+ superoxide dismutase, and has been hypothesized to protect the osteoclast from the damaging effects of superoxide radicals generated during active bone resorption. We have reported previously that the expression of this membrane antigen is induced on multinucleated giant cells when the prefusion marrow mononuclear cells are cultured in conditioned medium from avian calvaria. The studies reported here were designed to investigate the relationship between expression of the 121F antibody-reactive osteoclast membrane antigen and tartrate resistant acid phosphatase levels, bone resorptive activity, calcitonin responsiveness, and ultrastructural features of avian bone marrow-derived multinucleated giant cells formed either in the presence or absence of diffusible osteoblast secreted factors. Parallel analyses of in vivo formed osteoclasts isolated from the same animals were performed for direct comparisons. In this report we demonstrate: (1) that the 121F monoclonal antibody-reactive osteoclast membrane antigen is stably induced in giant cells by soluble osteoblast-derived factors in a species nonrestricted but concentration- and temporal-dependent manner; (2) that osteoblast-mediated antigen induction is reflected in both increased numbers of cells and elevated expression of individual cells that are reactive with the 121F antibody, as determined by ELISA and histomorphometry; (3) that osteoblast conditioned medium, in addition to inducing this antigen in bone marrow cells, also elevates other defining osteoclast characteristics in these avian giant cells including their TRAP activity, cell retraction from the bone surface in response to calcitonin, bone resorptive function, and expression of a series of additional osteoclast antigenic markers; and (4) that secreted osteoblast products alone do not raise the levels of these traits for in vitro formed marrow giant cells to the extent associated with in vivo formed osteoclasts. Therefore, osteoblast soluble factors alone appear unable to promote the full differentiation of bone marrow cells in vitro into mature bone-resorbing osteoclasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1996

c-fos antisense DNA inhibits proliferation of osteoclast progenitors in osteoclast development but not macrophage differentiation in vitro.

We previously reported that osteoclast formation in vitro, by coculture of mouse bone marrow and primary osteoblastic cells, occurs in two phases: proliferation of osteoclast progenitors followed by terminal differentiation into mature osteoclasts. Using this coculture system, we examined the effects of c-fos antisense and sense phosphorothioate oligonucleotides on osteoclast development and macrophage differentiation. Treatment with c-fos antisense for the first 4 days of coculture inhibited osteoclast formation in a dose-dependent fashion. However, when c-fos antisense was added during the second phase of coculture (4-6 days), osteoclast formation was unaffected. In contrast, c-fos antisense treatment had no effect on the appearance of F4/80 antigen-positive cells of the macrophage lineage in these cultures or on the induction by colony stimulating factor-1 of macrophage colony formation in cultures of mouse bone marrow cells in agar. Neither osteoclast differentiation nor macrophage appearance was inhibited by adding control c-fos sense in the cocultures. When c-fos antisense was added into an assay of bone resorption by mature osteoclasts, pit formation on dentine slices was unaffected. These results indicate that c-fos plays an important role in the proliferative phase of osteoclast progenitors in osteoclast development, but not in the terminal differentiation phase or in the bone resorbing activity of mature osteoclasts. c-fos antisense specifically inhibited osteoclast formation but had no effect on macrophage development.

Analysis of Variance↗
PubMed2005

Proteasome inhibitors abrogate osteoclast differentiation and osteoclast function.

Cancer-induced bone disease results in bone destruction, pathological fractures, and pain. We hypothesized that the inhibition of the proteasome-ubiquitin system in osteoclasts could abolish the receptor activator of NF-kappaB ligand (RANKL) mediated osteoclast differentiation and function, since RANKL-mediated downstream signaling plays a crucial role in osteoclast life cycle. In this study, we examined the effects of the proteasome inhibitors MG-132 and MG-262 on RANKL-induced osteoclast differentiation and function. Osteoclast precursors from peripheral blood mononuclear cells were cultured in the presence of RANKL and M-CSF. Osteoclasts were identified as multi-nucleated TRAP-positive cells. Osteoclast function was quantified with the extent of dentine resorption and TRAP activity in culture supernatants. For the evaluation of the effects of proteasome inhibitors towards osteoclastogenesis, sub-apoptotic concentrations of MG-132 and MG-262 were used. Effects on NF-kappaB were obtained in treated and untreated osteoclasts. MG-132 and MG-262 inhibit both osteoclast differentiation and osteoclast function. 0.01 microM MG-132 induced a 3.2-fold (P = 0.004) and 0.001 microM MG-262 a 3.3-fold (P = 0.004) reduction of osteoclast differentiation, respectively. The resorption capacity was decreased 2.6- and 11.1-fold (P = 0.003) by treatment with 0.01 and 0.1 microM MG-132, and 14.2- and 16.6-fold (P = 0.003) by 0.001 and 0.01 microM MG-262, respectively. This decrease correlated with the extent of NF-kappaB binding capacity. In conclusion, this study shows for the first time that proteasome inhibitors act on osteoclast development and function at low concentrations and should be considered as potential drugs for the treatment of cancer-induced osteolytic bone disease.

Bone Resorption↗
PubMed2002

Bisphosphonates suppress bone resorption by a direct effect on early osteoclast precursors without affecting the osteoclastogenic capacity of osteogenic cells: the role of protein geranylgeranylation in the action of nitrogen-containing bisphosphonates on osteoclast precursors.

Nitrogen-containing bisphosphonates (NBps) are taken up by osteoclasts and inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway. There is evidence, however, that cells other than mature osteoclasts, like osteoclast precursors and osteoblasts, are also involved in the action of Bps on bone resorption in vitro. To examine this issue further, we developed a new in vitro model, which allows the study of the effects of additives on early osteoclast precursors. In this model, osteogenic cells are essential for osteoclastogenesis. The model consists of 15-day-old fetal mouse metatarsals. At time of explantation, these bone rudiments do not yet contain a mineralized matrix or osteoclasts; only early osteoclast precursors are present in the perichondrium. During culture and after the addition of Nabeta-glycerolphosphate, the bones form a mineralized matrix that is consequently resorbed by osteoclasts that develop from their precursors. Short treatment of these explants with Bps, before the formation of a mineralized matrix, resulted in a subsequent dose-dependent inhibition of bone resorption. The relative potencies of eight Bps to suppress resorption were comparable with those observed after the addition of Bps after the formation of a mineralized matrix, the natural target of Bps. In addition, the effects of the NBp olpadronate, but not of clodronate, on osteoclastic resorption, could be partly reversed by geranylgeraniol. Results indicate that Bps can suppress osteoclastic resorption in vitro by a direct action on very early osteoclast precursors at the bone surface, and not by affecting the osteoclastogenic capacity of osteogenic cells. Moreover, the mechanism of action of the NBp olpadronate, but not clodronate, on early tartrate-resistant acid phosphatase-negative osteoclast precursors involves inhibition of protein geranylgeranylation, indicating a molecular mechanism similar to that established for mature osteoclasts.

Animals↗
PubMed1989

Generation of osteoclastic function in mouse bone marrow cultures: multinuclearity and tartrate-resistant acid phosphatase are unreliable markers for osteoclastic differentiation.

The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, and a series of recent experiments has used enumeration of the tartrate-resistant acid phosphatase (TRAP)-positive multinucleate cells that develop in cultures of hemopoietic tissue as a means to analyze the regulation of osteoclast generation. These multinucleate cells have never been definitively characterized as osteoclasts, however, and we elected to assess the relationship among bone resorption (the primary function of the osteoclast), TRAP, and multinuclearity in mouse bone marrow cultures. Mouse bone marrow cells and peritoneal macrophages were incubated on plastic coverslips or bone slices for up to 14 days in the presence or absence of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Osteoclast generation, as judged by bone resorption, occurred in marrow cell cultures only in the presence of 1 alpha,25-(OH)2D3. However, TRAP-positive multinuclear cells developed both with and without the hormone. The multinuclear cells bound F4/80, a marker for macrophages that does not bind to osteoclasts. Peritoneal macrophages became multinucleate and developed TRAP positivity in culture to levels similar to those in freshly isolated osteoclasts, especially with 1 alpha,25-(OH)2D3, but remained nonresorptive. In cultures of marrow cells incubated with 1 alpha,25-(OH)2D3 bone resorption was more extensive than could readily be accounted for by the number of multinucleate cells present, and the size of excavations and extent of resorption suggested a major contribution by mononuclear cells with osteoclastic function. Thus, while TRAP and multinuclearity are reliable markers for osteoclastic phenotype in bone, they are unreliable markers in culture. Experiments designed to evaluate the regulation of osteoclast generation through enumeration of TRAP-positive multinucleate cells formed in bone marrow cultures will not only overstate, to an unknown and probably variable degree, the number of multinucleate osteoclasts that develop, but will also fail to even identify what may be a considerable and more substantial population of mononuclear cells that possess osteoclastic characteristics.

Acid Phosphatase↗
PubMed2001

Transforming growth factor-beta induces osteoclast ruffling and chemotaxis: potential role in osteoclast recruitment.

Transforming growth factor-beta (TGF-beta) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-beta stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-beta (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-beta, indicating chemotaxis. echistatin, an alphavbeta3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-beta. In contrast, a beta1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-beta but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-beta-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-beta, may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-beta-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.

Androstadienes↗
PubMed2002

Nuclear localization of type I parathyroid hormone/parathyroid hormone-related protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-related protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.

Parathyroid hormone-related protein (PTHrP) is not required for osteoclastogenesis during embryonic development; however, after birth it has been shown to regulate osteoclast formation during tooth eruption. Our study explores the hypothesis that PTHrP also may regulate osteoclast differentiation in the regenerating skeletal tissues of deer antlers, bones capable of complete regeneration. Osteoclast-like multinucleated cells (MNCs) formed spontaneously in micromass cultures derived from antler cartilage and these cells had the phenotypic characteristics of osteoclasts. PTHrP and receptor activator of NF-kappaB ligand (RANKL) stimulated antler osteoclast formation although the effect of RANKL was less marked than that of PTHrP. The addition of osteoprotegerin (OPG) only partially decreased (by approximately 65%) the number of osteoclasts in PTHrP-treated cultures. To determine whether PTHrP also potentially could have direct effects on antler osteoclasts, we studied, by confocal microscopy, the expression of the type I PTH/PTHrP receptor (PTH1R) in MNCs cultured on glass and found the receptor protein to have a nuclear localization. In situ hybridization showed that antler MNCs also expressed PTH1R and PTHrP messenger RNAs (mRNAs). PTHrP was immunolocalized in MNCs cultured on glass but was undetectable in cells resorbing a dentine substrate. In tissue sections of antler cartilage, PTHrP and PTH1R were expressed in vitronectin receptor-positive (VNR+) osteoclast-like cells localized in the perivascular stroma. Thus, these data show that PTHrP plays a role in the regulation of osteoclast differentiation in regenerating skeletal tissues and that PTHrP can have effects on osteoclastogenesis that are independent of RANKL synthesis. Ours is the first study to describe the expression of the type I PTH/PTHrP receptor in mammalian osteoclasts at a protein and mRNA level, which indicates that PTHrP also may have a direct effect on osteoclasts. This also is the first study to show a nuclear localization of the PTHIR in cells of the osteoclast lineage, although the functional significance of this observation has yet to be established.

Animals↗
PubMed1989

[Regulation of osteoclastic bone resorption. 1. Limitation of osteoclast motile activity by fibroblast-like cells].

The cell-to-cell interaction between osteoclasts and fibroblast-like cells, which were mechanically disaggregated from long bones of neonatal rabbits, was investigated to clarify the cellular mechanisms responsible for the regulation of osteoclastic bone resorption by using phase-contrast time lapse recording and fluorescent microscopy for visualization of F-actin cytoskeleton. Osteoclasts showed rapid extension and retraction of pseudopodia with intense ruffling activity and active translocation over a glass surface when there was enough open space around them, while fibroblast-like cells seemed much more stationary. Osteoclasts were not able to cross over cell bodies of fibroblast-like cells which interfered with migration of the osteoclasts. As fibroblast-like cells proliferated, they gradually took over the open glass surface and finally tightly surrounded the osteoclasts. Such osteoclasts usually became contracted and pseudopodial and migratory activities decreased. Fluorescent microscopy of well-spread osteoclaststs stained with rhodamine-labeled phalloidin revealed development of a band-like structure composed of a number of brightly fluorescent F-actin dots at the cell periphery and fine F-actin bundles which run through the cell bodies. On the other hand, contracted osteoclasts tightly surrounded by fibroblast-like cells showed conglomerates composed of accumulated F-actin dots instead of the band-like structure. An F-actin pattern of fibroblast-like cells, which was very different from that of osteoclasts, displayed thick and straight bright bundles with in the cell bodies. The present observation suggests that ongoing osteoclastic bone resorption may be regulated by fibroblast-like cells through limitation of osteoclastic motile activity, and alteration of cytoskeleton and cell-substratum interaction which occurred in osteoclasts may be involved in the mechanism.

Actins↗