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Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Giant cell tumour of bone (GCTB) is a histologically benign, locally aggressive skeletal lesion with an unpredictable propensity to relapse after surgery and a rare metastatic potential. The microscopic picture of GCTB shows different cell types, including multinucleated giant cells, mononuclear cells of the macrophage-monocyte lineage, and spindle cells. The histogenesis of GCTB is still debated, and morphologic, radiographic or molecular features are not predictive of the clinical course. Characterization of the unexplored cell metabolism of GCTB offers significant clues for the understanding of this elusive pathologic entity. In this study we aimed to characterize GCTB energetic metabolism, with a particular focus on lactate release and the expression of monocarboxylate transporters, to lie down a novel path for understanding the pathophysiology of this tumour. We measured the expression of glycolytic markers (GAPDH, PKM2, MCT4, GLUT1, HK1, LDHA, lactate release) in 25 tissue samples of GCTB by immunostaining and by mRNA and ELISA analyses. We also evaluated MCT1 and MCT4 expression and oxidative markers (JC1 staining and Bec index) in tumour-derived spindle cell cultures and CD14+ monocytic cells. Finally, we quantified the intratumoural and circulating levels of lactate in a series of 17 subjects with GCTB. In sharp contrast to the benign histological features of GCTB, we found a high expression of glycolytic markers, with particular reference to MCT4. Unexpectedly, this was mainly confined to the giant cell, not proliferating cell component. Accordingly, GCTB patients showed higher levels of blood lactate as compared to healthy subjects. In conclusion, taken together, our data indicate that GCTB is characterized by a highly glycolytic metabolism of its giant cell component, opening new perspectives on the pathogenesis, the natural history, and the treatment of this lesion.

The major organic component of bone is collagen type I. Osteoclasts are terminally differentiated multinucleated cells of hematopoietic origin that are essential for physiological development of bone and teeth. We examined if osteoclast differentiation from murine bone marrow precursors is affected by collagen type I, or by its degradation products produced by human recombinant cathepsin K. Osteoclasts formation was dose-dependently inhibited in the presence of full length collagen type I or its 30–75 kDa degradation products added to the osteoclast differentiation media for the duration of an experiment. Collagen degradation fragments signaled through SH-2 phosphatases, inhibiting calcium signaling and NFATc1 translocation in osteoclast precursors. Osteoclasts and their precursors expressed a collagen receptor of leukocyte receptor complex family, LAIR-1. Importantly, collagen fragments failed to inhibit osteoclast formation from LAIR-1 deficient murine osteoclast precursors. This study demonstrates that collagen degradation fragments inhibit osteoclast formation acting through LAIR-1, providing a novel mechanism for the physiologically-relevant negative control of osteoclastogenesis.

Osteoclasts are defined as cells capable of excavating 3-dimensional resorption pits in bone and other mineralised tissues. They are derived from the differentiation/fusion of promonocytic precursors, and are usually large, multinucleated cells. In common with other cells from this myeloid lineage such as macrophages and dendritic cells, they are adapted to function in hypoxic, acidic environments. The process of bone resorption is rapid and is presumably highly energy-intensive, since osteoclasts must actively extrude protons to dissolve hydroxyapatite mineral, whilst secreting cathepsin K to degrade collagen, as well as maintaining a high degree of motility. Osteoclasts are well known to contain abundant mitochondria but they are also able to rely on glycolytic (anaerobic) metabolism to generate the ATP needed to power their activity. Their primary extracellular energy source appears to be glucose. Excessive accumulation of mitochondrial reactive oxygen species in osteoclasts during extended periods of high activity in oxygen-poor environments may promote apoptosis and help to limit bone resorption - a trajectory that could be termed "live fast, die young". In general, however, the metabolism of osteoclasts remains a poorly-investigated area, not least because of the technical challenges of studying actively resorbing cells in appropriate conditions.

Fluoroquinolones (FQs) are a class of antibiotics with a broad spectrum of activity, known to disturb bone metabolism. The aim of this work was to characterize the cellular and molecular effects of five FQs (ofloxacin, norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in unstimulated and stimulated human osteoclast precursors. Peripheral blood mononuclear cells (PBMC) were cultured in the absence (unstimulated) or in the presence of osteoclastogenic factors (M-CSF and RANKL, stimulated), and were treated with FQs (0.3 × 10− 9–10− 3 M), for 21 days. In unstimulated PBMC cultures, FQs (excepting moxifloxacin) exhibited a high osteoclastogenic potential, as shown by a significant increase in the expression of osteoclastic genes, TRAP activity and, specially, number of TRAP-positive multinucleated cells and calcium phosphate resorbing ability, suggesting the presence of mature and functional osteoclasts. Norfloxacin and levofloxacin induced the higher effect, followed by ciprofloxacin and ofloxacin. A decrease on apoptosis and an increase on M-CSF expression might have a possible contribution in the observed cellular behavior. In stimulated PBMC cultures, FQs further increase the osteoclastogenic response induced by M-CSF and RANKL (except ofloxacin). However, the osteoclastogenic response was much lower than that observed in unstimulated PBMC cultures. Both in unstimulated and stimulated PBMC cultures, for most of the FQs, the osteoclastogenic effects were observed in a wide range of concentrations, representative of plasmatic and tissue levels attained in several clinical settings. The various FQs differed on the stimulatory concentration range, the extent of the induced osteoclastogenic response and, also, on the dose- and time-dependent profile. Nevertheless, at high concentrations all the FQs seemed to elicit an increase on apoptosis. Additionally, some differences were noted in the intracellular signaling pathways tested, namely NFkB, MEK and PGE2 production. Results suggest that, considering the inter-individual variability of the FQs pharmacokinetics, the detailed biological profile of each FQ on bone cells is of utmost importance to clarify the effects of these compounds on bone metabolism.

Osteoclasts are multinucleated, bone-resorbing cells formed via fusion of monocyte progenitors, a process triggered by prolonged stimulation with RANKL, the osteoclast master regulator cytokine. Monocyte fusion into osteoclasts has been shown to play a key role in bone remodeling and homeostasis; therefore, aberrant fusion may be involved in a variety of bone diseases. Indeed, research in the last decade has led to the discovery of genes regulating osteoclast fusion; yet the basic cellular regulatory mechanism underlying the fusion process is poorly understood. Here, we applied a novel approach for tracking the fusion processes, using live-cell imaging of RANKL-stimulated and non-stimulated progenitor monocytes differentially expressing dsRED or GFP, respectively. We show that osteoclast fusion is initiated by a small (~ 2.4%) subset of precursors, termed “fusion founders”, capable of fusing either with other founders or with non-stimulated progenitors (fusion followers), which alone, are unable to initiate fusion. Careful examination indicates that the fusion between a founder and a follower cell consists of two distinct phases: an initial pairing of the two cells, typically lasting 5–35 min, during which the cells nevertheless maintain their initial morphology; and the fusion event itself. Interestingly, during the initial pre-fusion phase, a transfer of the fluorescent reporter proteins from nucleus to nucleus was noticed, suggesting crosstalk between the founder and follower progenitors via the cytoplasm that might directly affect the fusion process, as well as overall transcriptional regulation in the developing heterokaryon.

Bone-resorbing osteoclasts exhibit polarized morphological structures such as actin rings, clear zones, and ruffled borders. To gain insight into the mechanism of bone-resorbing activity of osteoclast and to discover new types of anti-resorptive agents, we have screened for natural compounds that inhibit the bone-resorbing activity of osteoclast-like multinucleated cells (OCLs). Destruxin B (DestB) and E (DestE), cyclodepsipeptides, were found to inhibit pit formation without affecting osteoclast differentiation and survival. Destruxins reversibly induced morphological changes in OCLs in a dose-dependent manner (DestB, 0.2-1 microM; DestE, 0.01-0.05 microM) and inhibited pit formation. Destruxin-induced morphological changes were accompanied by disruption of the actin rings in OCLs. The formation of actin rings in OCLs after adhesion was also inhibited by destruxins. Electron microscopical analysis revealed that destruxin-treated OCLs on dentine slices have no prominent clear zones and ruffled borders. The effective concentrations of destruxins on the morphological changes were almost the same as those that inhibited bone resorption in organ culture system. These results suggest that the anti-resorptive effects of destruxins result from induction of a disorder of the morphological structures in polarized OCLs.

The osteopetrotic grey-lethal (gl) mouse mutant displays many similarities to the human malignant autosomal-recessive form of osteopetrosis. In this study, we show that the gl osteopetrotic bone phenotype is characterized by the presence of numerous differentiated multinucleated osteoclasts. A significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was detected in vivo, suggesting induction of differentiation in the osteoclast lineage as a compensatory mechanism. These gl osteoclast cells demonstrated a defective cytoskeletal reorganization and an underdeveloped ruffled border, a membrane structure essential for active bone resorption. Accordingly, resorption activity of these cells is markedly impaired by four- to tenfold as evaluated with the pit formation assay. This low bone resorption in gl osteoclasts is highly reminiscent of the loss in key enzymes, V-ATPase or cathepsin-K, and in signaling factors, Src or TRAF-6, which were shown not to be significantly altered in gl osteoclasts. Thus, independently of a deficiency in V-ATPase, Src, cathepsin-K, and TRAF-6, the gl mutation results in increased number of osteoclasts, characterized by a disrupted cytoskeleton and an underdeveloped ruffled border.

In mouse bone marrow cultures, the formation of osteoclast-like, that is, tartrate-resistant acid phosphatase-positive (TRAP+) and calcitonin (CT) receptor-positive multinucleated cells (MNCs), induced by 10(-10) to 10(-8) mol/L 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], could be augmented by triiodothyronine (T3), which alone had no effect on osteoclast-like cell formation. The permissive effect of T3 increased the response to 1alpha,25(OH)2D3 by approximately one order of magnitude. Linear concentration dependence was observed between 10(-11) and 10(-8) mol/L T3. Importantly, inhibition of prostaglandin synthesis by indomethacin significantly impeded osteoclast-like cell formation by 1alpha,25(OH)2D3 and abrogated the effect of T3 thereon. Basal interleukin-6 (IL-6) production by cultured marrow cells was significantly stimulated by 1alpha,25(OH)2D3. However, even at an exceedingly high concentration of 20 ng/mL, IL-6 was ineffective in inducing osteoclast-like cell formation. Therefore, any hormonally induced rise in IL-6 release from bone marrow cells could not account for the observed changes in TRAP+ MNC numbers. Nevertheless, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis was partially dependent on IL-6 because it could be significantly blocked by a neutralizing monoclonal anti-IL-6 antibody, and to the same extent by a monoclonal anti-IL-6 receptor antibody. Unimpaired signaling through the IL-6/IL-6R system is also a prerequisite for the auxiliary effect of T3 on induction of osteoclast-like cells by 1alpha,25(OH)2D3. Our data provide evidence that 1alpha,25(OH)2D3 induces osteoclast-like cell formation, at least in part, in an IL-6-dependent mode of action, which is also subject to modulation by T3. The mechanism of interaction of the two hormones apparently involves joint stimulation of prostaglandin synthesis.

The study of osteoclast differentiation, function, and fate has been hampered by the lack of nonavian, nonrodent models in which biochemical and molecular studies can be conducted. The present study was undertaken to determine if osteoclasts could be generated from porcine bone marrow cells. Bone marrow from the long bones of neonatal female pigs was enriched for mononuclear cells and cultured in the presence or absence of 1,25-(OH)2D3, rhIL-11, or PGE2. A confluent layer of stromal cells was observed after 4–8 days in culture and multinucleated giant cells formed after 6–10 days of culture. The multinucleated cells stained positively for tartrate-resistant acid phosphatase and formed resorption lacunae when exposed to bovine cortical bone slices. When examined by transmission electron microscopy, abundant mitochondria, perinuclear Golgi complexes, numerous variably sized vacuoles, prominent rough endoplasmic reticulum, and free polysomes were observed in the mutlinucleated cells. Stimulation of the in vitro generated osteoclasts with 10−8 mol/L salmon calcitonin resulted in a three to fivefold increase in cAMP production and in cell retraction. Although the osteoclasts formed in the presence or absence of 1,25-(OH)2D3 10–50-fold more osteoclasts were observed in the cultures treated with 1,25-(OH)2D3 in comparison to cultures without 1,25-(OH)2D3. Osteoclast differentiation was also stimulated by rhIL-11 and PGE2; although, the number of cells generated in 6–7 days was significantly less than the number obtained with 1,25-(OH)2D3 treatment. In addition, these multinucleated cells expressed high levels of Src kinase activity and responded to bafilomycin Al, an inhibitor of the vacuolar type H+-ATPase, treatment with a decrease in osteoclastic bone resorption. In summary, the porcine cells possess the major distinguishing characteristics of osteoclasts and provide an alternative mammalian model to study osteoclast differentiation and resorptive activity.

Cells exhibiting osteoclast characteristics of calcitonin receptors (CTRs) and tartrate-resistant acid phosphatase (TRAP) histochemistry are formed in murine bone marrow cultures treated with lα,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ]. We have previously demonstrated that CTR mRNA is highly expressed in these cultures. The aim of this study was to investigate homologous regulation of the CTR, and regulation of TRAP expression in osteoclast-like cells after brief treatment with salmon CT (sCT). Murine bone marrow cells were cultured in 9 cm dishes in the presence of 10 nmol/L 1,25 (OH) 2 D 3 . On day 6 of culture, when multinucleated cells were abundant, the cells were treated with 1 nmol/L sCT for 1 h. Both control and treated cells were then harvested at intervals up to 72 h posttreatment, and both CTR and TRAP mRNA levels assessed by reverse-transcription PCR (RTPCR). In parallel cultures, cells with CTR expression detectable by autoradiography, and TRAP positivity by histochemistry, were counted. The effects of brief sCT treatment could be seen 6 h after treatment when the CTR RT-PCR product was markedly reduced. Total recovery of CTR mRNA levels had not occurred even after 72 h. Calcitonin treatment had little effect on TRAP mRNA levels. There was no difference in the numbers of multinucleated TRAP(+) osteoclast-like cells between treated and control cells. These results indicate that brief sCT treatment, while not influencing multinucleated osteoclast-like cell number, causes specific, acute reduction of CTR mRNA in bone marrow culture-derived osteoclasts. The prolonged decrease in CTR mRNA levels suggests that recovery may require new osteoclast formation, and indicates that regulation of the CTR in cells of the osteoclast lineage is different from that in nonosteoclastic cells and tissues.

Tartrate-resistant acid phosphatase (TRAP) histochemistry has been used to follow chondro/osteoclasts and their precursors during ontogenesis of the mandibular condyle of the mouse (from day 16 of gestation until day 15 of neonatal life). TRAP+ mono-, bi-, and multinuclear cells were counted separately in the perichondrium, along the resorption front and in the subchondral spongiosa. Index of cellular density was calculated by dividing the absolute numbers of cells by the length of resorption front or the area of spongiosa, respectively. The study revealed that TRAP+ cells are present in the perichondrium of the mandibular condylar cartilage from the first day of its existence as an organ, namely day 17 of gestation. These cells are more numerous in the posterior part of the condyle, and reach their maximal number on the third day of neonatal life. Along the resorption front, mono-, bi-, and multinucleated TRAP+ cells were counted. Their total number and their respective indexes of cellular density changed variably during the observation period: (a) the number of mononuclear cells increased gradually and, by the end of the observation period, was 13 times greater than it was at the beginning; (b) the number of binuclear cells increased threefold; and (c) the number of multinuclear cells stayed constant, except for a small peak around the time of birth. The changes in the spongiosa showed an opposite trend. In the spongiosa, the index of cellular density of the mononuclear TRAP+ cells increased slightly during ontogenesis. The number of binuclear cells increased twofold, while the number of multinuclear cells increased 17-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of action of tiludronate [(4-chlorophenyl)-thiomethylene bisphosphonate] on osteoclastic bone resorption was examined in mouse culture systems. Tiludronate did not inhibit the formation of osteoclast-like multinucleated cells (OCLs) induced by 1α,25-dihydroxyvitamin D 3 in co-cultures of mouse osteoblastic cells and bone marrow cells. OCLs obtained from cocultures on collagen gel-coated dishes, treated with tiludronate, formed as many resorption pits on dentine slices as those obtained from the control co-cultures. However, pit formation by OCLs was dose-dependently inhibited when tiludronate was added directly to the pit formation assay. Other bisphosphonates such as alendronate and endronate dose-dependently inhibited pit formation according to the in vivo potencies of the respective bisphosphonates to inhibit bone resorption. However, they had no inhibitory effect on the recruitment of OCLs induced by 1α,25-dihydroxyvitamin D 3 in the cocultures. When OCLs were placed on dentine slices, they formed the ringed structure of F-actin-containing podosomes and ruffled borders (polarized OCLs) even in the presence of tiludronate. However, the actin rings in OCLs were disrupted by the addition of tiludronate soon after they began to resorb dentine. In contrast, OCLs placed on collagen gel formed neither actin rings nor ruffled borders (nonpolarized OCLs), and showed no response to tiludronate. OCLs formed from the spleen cells of osteosclerotic ( oc/oc ) mice developed the ringed structure of podosomes, but not ruffled borders, on dentine slices. The actin ring in the oc/oc spleen cell-derived OCLs placed on dentine slices was not disrupted by the addition of tiludronate. However, the actin rings formed in normal OCLs and in oc/oc OCLs placed on plastic dishes were similarly and rapidly disrupted by the microinjection of tiludronate into their cytoplasm. These results indicate that tiludronate is selectively incorporated into polarized OCLs that have a ruffled border, suggesting that the bisphosphonate is a specific inhibitor of functioning osteoclasts.

Unfractionated mouse long-bone cells prestimulated with rat parathyroid hormone (PTH) for 7 days (PTH-prestimulated long-bone cells; PTH-psLB cells) contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells and about 0.2% TRAP-positive multinucleate cells. When PTH-psLB cells (1.0 x 10(5)/slice) were cultured on dentine slices, about 100 resorption lacunae (pits) per slice were formed by 6 h after the start of culture, while the number of TRAP-positive multinucleate cells on the dentine slices did not increase. When the number of inoculated cells was plotted versus the number of pits formed during the 6-h cultivation, a sigmoid curve was generated. When PTH-psLB cells (1.0 x 10(3)/slice) were cocultured with various mouse stromal cell lines, the MC3T3-G2/PA6 line was found to increase the number of pits. Since this activity was manifested within a short period of time (4 h), and as there was no change in the number of TRAP-positive multinucleate cells, we postulate that the activity of MC3T3-G2/PA6 is exerted on mature osteoclastic cells capable of resorbing bone rather than on some differentiation step of the osteoclast cell lineage. With respect to the mechanism of expression of this activity, it is unlikely that MC3T3-G2/PA6 produces a soluble factor, because no activity was seen when MC3T3-G2/PA6 and PTH-psLB cells were cultured separately across a membrane in a Transwell. These results suggest that cell-to-cell contact and communication between PTH-psLB cells and MC3T3-G2/PA6 may be a crucial event for the stimulation of osteoclastic bone resorption by MC3T3-G2/PA6.

Enzymatically released cells from neonatal mouse calvarial bones are frequently used as primary mouse osteoblasts for in vitro studies. We, here, report that although these cells lack mRNA expression of the osteoclastic genes Calcr, Acp5 and Mmp-9 at early time points after their isolation, these transcripts are gradually upregulated when the calvarial osteoblast cultures are differentiated to more mature osteoblasts in long term cultures. Similarly, Calcr, Acp5, Mmp-9, as well as Rank and Nfatc1 mRNA expressions are robustly increased when the osteoblast cultures were induced to differentiate by treatment with bone morphogenetic protein (BMP-2). The increased Calcr mRNA resulted in functionally active calcitonin receptors. Enhanced osteoblastic differentiation was associated with increased Rankl mRNA expression and decreased Opg and Cfs1 mRNA expression. Treatment of the osteoblastic cells with BMP-2 or RANKL, either on plastic dishes or bone slices, resulted in the formation of multinucleated tartrate-resistant acid phosphatase positive cells, which were able to excavate resorption pits and release CTX from the bones. In contrast, increased osteoblastic differentiation induced by BMP-2 in the mouse calvarial osteoblastic cell line MC3T3-E1 was not associated with increased mRNA expression of Calcr, Acp5, Rank, c-Fms or Oscar. Interestingly, Ctsk mRNA was increased during osteoblastic differentiation in both mouse calvarial osteoblast cultures and in MC3T3-E1 cultures. Also osteoblasts isolated from mouse long bones by outgrowth from explant cultures were contaminated with osteoclast progenitors able to differentiate into bone resorbing osteoclasts. These data indicate that mouse calvarial osteoblast cultures contain osteoclast progenitor cells, which will be differentiated along the osteoclastic lineage during osteoblastic differentiation. Moreover, the data show that BMP-2 not only stimulates osteoblastic differentiation but can also induce osteoclastogenesis through increased RANKL.

Paget's disease of bone is characterized by large numbers of osteoclasts that have viral-like nuclear and/or cytoplasmic inclusions. Pagetic osteoclasts express respiratory syncytial viral (RSV) and measles viral (MV) nucleocapsid antigens. The data suggest a possible viral etiology for Paget's disease. However, studies to characterize further the putative viral inclusions in Paget's osteoclasts have been severely hampered by the extreme difficulty in isolating large numbers of osteoclasts from pagetic bone. The recent demonstration that osteoclast-like multinucleated cells (MNC), that had certain characteristics of pagetic osteoclasts formed in marrow cultures from Paget's patients, may permit studies to describe this virus further. Therefore, we have cultured marrow samples from involved and uninvolved bones from Paget's patients and from normal subjects to determine if the MNC formed in these cultures express viral antigens. RSV and/or MV antigens were expressed in the mononuclear cells and/or the MNC formed in 12 of 12 marrow cultures from active lesions of patients with Paget's disease, with 40–50% of the cells expressing viral antigens. In contrast, less than 5% of cells isolated from cultures from normal subjects expressed RSV and/or MV. These results suggest that MNC formed in long-term marrow cultures from patients with Paget's disease frequently express paramyxoviral antigens and are very similar to pagetic osteoclasts. Thus, these marrow cultures may be useful for further characterizing the virus in Paget's disease.

We established a useful assay system for evaluating osteoclast-mediated bone resorption based on the use of unfractionated bone cells obtained from 10- to 11-day-old mice. When cells from 10 to 11 mice were treated for 7 days with rat parathyroid hormone (rPTH, 10(-8) M), a total of 4 to 5 x 10(7) cells could be obtained from the culture by treatment with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells. When the harvested cells were cultured on dentine slices without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone resorption. Eel calcitonin (eCT) decreased the number of pits in a dose-dependent manner, and its half maximal inhibition dose (ID50) was 1.08 x 10(-11) M. Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable for forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclastic cell line for evaluating bone resorption is available at present, this system can provide a useful, practical means for assaying osteoclastic bone-resorbing activity.

Rat bone marrow cultures containing 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] formed multinucleated cells (MNCs) that had many characteristics of osteoclasts. These MNCs, which have a tartrate-resistant acid phosphatase (TRAP) activity, could be classified into two morphological types: one type had smooth cellular margins (smooth-margined MNCs) and the other type had irregular spike-like margins (stellate MNCs). When bone marrow cells depleted of authentic osteoclasts were seeded and cultured on dentine slices, only low numbers of resorption lacunae could be detected. However, when preformed MNCs were detached by trypsinization and replated on dentine slices, numerous resorption lacunae were observed by scanning electron microscopy on these slices. Formation of lacunae occurred reproducibly during the five to ten days of culture. We also examined the effect of retinoic acid on TRAP-positive MNC formation in this bone marrow culture system. Although RA inhibited total TRAP-positive MNC formation, it increased the ratio of stellate MNCs to smooth-margined MNC, suggesting that RA may have the ability to regulate the formation of active osteoclasts.

Osteoclasts are multinucleated bone-resorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna. Carbonic anhydrase II and vacuolar H(+)-ATPases produce and transport protons, while chloride channels provide a Cl(-) flux into the resorption site. These activities are required for inorganic matrix dissolution that precedes enzymatic removal of organic bone matrix. In other cell types it has become evident that carbonic anhydrase isoenzymes interact with AE proteins to form transport metabolons that regulate intracellular pH. Membrane-bound carbonic anhydrase isoenzymes may also compensate for the lack of cytoplasmic carbonic anhydrase II. Therefore, our goal was to explore the expression of membrane-bound carbonic anhydrase (CA) isoenzymes CA IV, CA IX, CA XII and CA XIV in bone-resorbing osteoclasts. Immunohistochemistry and confocal microscopy showed expression of CA IV, CA XII and CA XIV in cultured rat and human osteoclasts. To confirm these results, RT-PCR was used. Immunohistochemistry revealed distinct staining patterns for CA IV, CA XII and CA XIV in rat trabecular bone specimens. A plasma membrane staining was observed in bone lining cells with the CA XII antibody while osteoclast plasma membranes were stained with CA IV and CA XIV antibodies. Confocal microscopy of cultured human osteoclasts showed a punctated intracellular CA IV staining and a perinuclear CA XIV staining while no CA IX or CA XII staining was observed. To evaluate the physiological role of membrane-bound CAs in osteoclasts, we used PCS, a novel membrane-impermeable CA inhibitor. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of PCS while higher concentrations affected cell survival. PCS treatment also disturbed intracellular acidification in osteoclasts, as determined by live cell microscopy. In conclusion, our data shows that membrane-bound carbonic anhydrase isoenzymes CA IV and CA XIV are expressed both at mRNA and protein levels in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to support the hypothesis that intracellular pH regulation in osteoclasts may indeed involve transport metabolons.

Putative sites of bone resorption in the acellular bony skeleton of the medaka fish (Oryzias latipes) were investigated primarily by RNA in situ hybridization and histological analysis. Numerous cells that displayed intense enzymatic activity of tartrate-resistant acid phosphatase (TRAP), the main marker of osteoclasts, were distributed in the pharyngeal region of this fish. Moreover, these cells expressed cathepsin K, an osteoclast-specific gene, as well as the genes for TRAP and vacuolar-type proton ATPase (V-ATPase). Some of the TRAP-positive cells displayed all of the morphological characteristics equivalent to those of mammalian- and bird-type osteoclasts. These cells were associated primarily with the shedding teeth and their supporting bones (pedicles), where alkaline phosphatase (ALPase)-positive osteoblasts were also located, implying progressive bone remodeling associated with tooth replacement in these regions. In contrast, the inner aspects of the neural and hemal arches of the vertebral column, which were the only sites of bone resorption other than the tooth-bearing bones, showed sporadically aligned flat mononuclear TRAP-positive cells without a ruffled border, indicating a different mode of bone remodeling in these regions. These results suggest the feasibility of medaka as a model animal for the investigation of bone-related abnormalities and their genetic backgrounds.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.

Studies were carried out to characterize the effects of cyclosporines and FK506 on the formation and survival of osteoclasts deriving from mouse bone marrow cultures. Cyclosporin A (CsA), cyclosporin B (CsB), cyclosporin H (CsH), and FK506 all inhibited receptor activator of NFkappaB ligand (RANKL)-stimulated tartrate-resistant acid phosphatase (TRAP) activity and generation of TRAP+ multinucleated cells in the cultures. CsA and CsG were approximately equipotent, CsH was approximately one order of magnitude less potent than the other cyclosporines, and FK506 was approximately two orders of magnitude more potent than CsA and CsG. All of the inhibitors demonstrated greater potency and efficacy on decreasing the number of TRAP+ multinucleated cells than on decreasing total TRAP activity. Further evidence that late stages were more sensitive to inhibition was obtained in experiments in which CsA was present for different segments of the RANKL-stimulated culture period. CsA was as efficacious when added for the final 2 days of a 4-day culture as when added for the entire culture period, whereas it was less effective if added for only the first 2 days of the culture. When CsA or FK506 were added for 1 day to cultures in which osteoclasts had already formed, the numbers of TRAP+ osteoclasts decreased. Treatment with CsA or FK506 produced nuclear fragmentation and disruption of the multinucleated osteoclasts and an increase in caspase-3 activity. The apoptosis inhibitor z-VAD partially prevented the inhibitory effects of CsA and FK506 on the survival of TRAP+ multinucleated cells in the cultures and also preserved the normal osteoclast morphology. The data indicate that an important component of the inhibitory effects of CsA and FK506 on marrow-derived osteoclasts is the induction of apoptosis.

Interleukin-6 (IL-6) has been postulated as a possible mediator of bone loss after estrogen deficiency, and its signal is transduced via glycoprotein 130 (gp130) after binding IL-6 receptor (IL-6R) in the membrane of target cells. In this study, the expression of IL-6R and gp130 in bone marrow cells during osteoclast differentiation was investigated. Mouse bone marrow cells were isolated and cultured with or without 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. During the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), IL-6R and gp130 expression in the mononuclear cells, stromal cells, and TRAP-positive MNCs were quantitated, using a laser cytometer with a fluorescence confocal microscopy. With 1,25(OH)2D3 stimulus, the level of gp130 significantly increased, but that of IL-6R did not in the stromal cells. In contrast, the levels of both gp130 and IL-6R significantly increased in the mononuclear cells by the treatment with 1,25(OH)2D3. The high expression of both gp130 and IL-6R was observed in the TRAP-positive mononuclear cells. Moreover, both IL-6R and gp130 were expressed in the TRAP-positive MNCs and isolated murine osteoclasts. The treatment of TRAP-positive MNCs with IL-6 caused enhancement of the resorbing activity in a dose-dependent manner, and the effect was prevented by a neutralizing antibody against IL-6R. These data suggest that gp130 and IL-6R, as well as IL-6, are involved in the formation and activation of osteoclasts.

We examined the mode of action of bisphosphonates on osteoclastic cell recruitment using mouse marrow cultures with or without osteoblastic cells. Tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+) MNC] formed in cultures were determined to be osteoclastic cells. In marrow cultures, TRAP(+) MNC formation in the presence of 10 −8 mol/L 1,25(OH) 2 D 3 was not affected by the addition of 10 −6 mol/L dihydrogen (cycloheptylamino)-methylenebisphosphonate monohydrate (YM175). However, it was inhibited in cocultures of marrow cells with osteoblastic cells. The inhibitory effect was evident throughout the entire culture period. YM175 dose dependently inhibited TRAP(+) MNC formation, and other bisphosphonates—pamidronate and alendronate—also inhibited TRAP(+) MNC formation in the coculture. Similar observations were also made in the coculture of spleen cells with osteoblastic cells. The conditioned media of osteoblastic cells treated with 10 −6 mol/L YM175 inhibited TRAP(+) MNC formation in marrow cultures. The presence of YM175 in methylcellulose cultures affected neither the colony formation of monocyte-macrophage lineage, nor TRAP(+) MNC formation in the succeeding cocultures of recovered cells with osteoblastic cells. These results indicate that YM175 and probably other bisphosphonates as well preferentially inhibit the later stage of osteoclastogenesis through its action on osteoblastic cells. Our findings suggest that part of the inhibitory action by osteoblastic cells in the presence of bisphosphonates is mediated through soluble factor(s).

We have previously described a model system, using 15-day fetal mouse metatarsals cultured in serumless medium, in which osteoclasts and their precursors develop from in situ progenitors in a manner which is similar, both temporally and spatially, to that which occurs in vivo. In this report we evaluate the role of the osteotropic hormone 1,25-dihydroxyvitamin D3 (1,25-D3) on osteoclast formation in this model system by characterizing its effects on proliferation, differentiation, and fusion of cells of the osteoclast lineage. Morphologic evaluation was used to enumerate osteoclast precursors, mono- and multinucleate osteoclasts, and osteoclast nuclei in serial paraffin sections. Dose response data reveal a significant stimulation of osteoclast formation by 1,25-D3 in the range of 0.6 nM to 40 nM, and kinetic analyses suggest that these effects are on proliferation of osteoclast progenitors as well as on differentiation of precursors to form osteoclasts. A single 48 h exposure between day 4 and 6 of culture is necessary and sufficient to induce maximal osteoclast formation, while continuous exposure beyond this "critical period" inhibits multinucleate osteoclast formation. Simultaneous treatment with indomethacin inhibits the effects of 1,25-D3, while treatment with PGE2 stimulates osteoclast formation without significantly increasing precursor numbers, or inhibiting multinucleate osteoclast formation. These results suggest that the effect of 1,25-D3 to induce differentiation of precursors to form mono- and multinucleate osteoclasts is mediated by endogenous prostaglandin synthesis. On the other hand, the inhibition of polykaryon formation observed with continuous 1,25-D3 treatment, does not appear to be a prostaglandin mediated phenomena.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence was sought for the presence of osteoclasts or preosteoclasts in cells prepared from neonatal murine calvaria by sequential enzymatic digestion. Freshly isolated cells released late in the digestion process resorbed both living and devitalized calvarial bone matrix in response to PTH, accompanied by the development of osteoclasts. Light and scanning electron microscopy of these cells after 1 to 2 days in culture revealed the presence of round cells (10 to 15 μ in diameter) with minimal surface microvilli in addition to the larger osteoblastic cells. Few cells contained tartrate-resistant acid phosphatase (TRAPase). If initially seeded at confluent density, more cells positive for TRA-Pase developed during subsequent culture, and these cells retained the ability to resorb calvarial bone matrix upon treatment with parathyroid hormone (PTH). These cultured cells responded to PTH with increased secretion of TRA-Pase into the medium and increased numbers of TRAPase positive cells. These were 20 to 50 μ in diameter, sometimes multinucleated, and some were spread 100 to 200 μ in length. Observations of living cells that took up neutral red showed that, upon treatment with calcitonin (CT), surface filopodia of some but not all of the labeled cells retracted within 30 minutes. Loss of resorptive response to PTH, as well as PTH-stimulated development of TRAPasepositive cells, occurred if the cells were initially seeded at low density and grown to confluence before exposure to hormone. This correlated with the loss of most of the 10 to 15 μ diameter round cells. These observations suggest that preosteoclasts are present among these small cells that can give rise to osteoclasts upon treatment with PTH

We studied the influence of skeletal tissue on expression of the osteoclastic phenotype in the mouse, in vivo and in vitro . In various soft and hard tissues of adult and fetal mice the distribution of mono- and multinucleate cells showing tartrate-resistant acid phosphatase was studied, using enzyme histochemistry on undecalcified plastic sections. Cells with strong TRAcP activity were only observed in mineralized tissues. Multinucleate TRAcP cells were exclusively found in close correlation with mineral resorption. In fetal bones mononuclear TRAcP cells appeared in the surrounding soft tissue prior to osteoclast formation. In addition, conditioned media of fetal long bone rudiments (FBCM) increased the number of mononuclear cells showing strong TRAcP activity in 7 d cultures of adult bone marrow. FBCM stimulated DNA synthesis in TRAcP cells and induced their multi-nuclearity. Pretreatment with FBCM increased the capacity of bone marrow cultures to form osteoclasts in coculture with fetal bone rudiments. However, FBCM did not change the number of cells with tartrate-sensitive acid phosphatase activity (TSAcP cells). The activity of FBCM was heat labile and was not detectable in CM of killed bones. CM of embryonic mouse fibroblasts which contains M-CSF activity, strongly increased the number of TSAcP cells but reduced the number of TRAcP cells. These data suggest that fetal mouse bone tissue induces the differentiation of osteoclast precursors. In addition, fetal bone rudiments but not embryonic fibroblasts seem to produce a factor(s) which stimulate(s) the formation of cells showing characteristics of osteoclast precursor cells. Osteoclasts and macrophages seem to have different growth requirements, indicating that they represent separate cell lines which may nevertheless derive from a common progenitor.