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PubMed2006

Prostaglandin production by human osteoclasts in culture.

OBJECTIVE: Prostaglandins (PG) are important mediators of bone metabolism with direct and indirect effects on bone cells. They may have important effects on osteoclasts, but it is not known if these cells can synthesize PG. We used 2 experimental models in order (1) to determine the presence and functionality of cyclooxygenase (COX) and phospholipase A2 (PLA2) enzymes in human osteoclasts and (2) to study their role in cell metabolism. METHODS: Experiments were undertaken on authentic human osteoclasts extracted from human fetuses (fhOC) and on human osteoclast-like (hOCL) cells differentiated from peripheral blood mononuclear cells. The presence of COX proteins was determined by immunohistochemistry. COX and PLA2 enzymatic activity was evaluated at the single-cell level by fluorescence microscopy. An enriched population of hOCL cells was used to evaluate total PG production and the influence of COX activity on bone resorption. RESULTS: COX-1 was expressed in the cytoplasm and COX-2 was distributed mainly near the nuclear membrane of osteoclasts. These cells showed a high basal level of COX activity that could be inhibited by pretreatment with COX inhibitors. Cytosolic PLA2 was present in both models. Human osteoclasts actively produced PG, and the COX-1 pathway was implicated in the control of bone resorption. CONCLUSION: These results indicate that PG may be important autacoids for the control of osteoclast biology and that the COX-1 pathway is implicated in the inhibition of bone resorption.

Animals↗

PubMed2006

[Isolation of bone marrow-derived rat osteoclast-like cells with the digestion of trypsin].

OBJECTIVE: To obtain highly enriched osteoclasts in vitro. METHODS: The bone marrow cells of 1-day, 12-day, 24-day and 36-day old Sprague-Dawley (SD) rats were separately cultured with the presence of 1,25-(OH)2D3 and dexamethasone, and the osteoclast-like cells were identified by Trap staining and scanning electron microscope observation and were purified by trypsin digestion. The count of Trap positive osteoclast-like cells was analyzed statistically. RESULTS: The osteoclast-like cells of each group were Trap positive cells and could form the bone absorption lacunas in vitro. The amounts of osteoclast-like cells were different statistically between the groups (P < 0.05) except between the groups of 1-day old and 12-day old (P > 0.05). Highly enriched osteoclast-like cells were harvested by the digestion of 0.25% trypsin and 0.02% EDTA. CONCLUSION: It's indicated that a large amount of highly enriched osteoclast-like cells could be obtained through the culture of bone marrow cells of 10-day and 12-day old SD rats with 1,25-(OH)2D3 and dexamethasone and the digestion of trypsin/EDTA.

Animals↗

PubMed1989

Immunolocalization of beta 3 subunit of integrins in osteoclast membrane.

Utilizing isolated and cultured osteoclasts it has been possible to establish that they adhere to the substrate through specialized close contact areas, the podosomes, that in fully spread osteoclasts in vitro or in vivo are located within the clear zone. The cytochemical organization of podosomes has further been investigated in order to elucidate their possible involvement in the control of substrate recognition, that precedes bone resorption. An immunofluorescence investigation, performed utilizing human osteoclasts, shows that the beta 2 integrin subunit that in human monocytes is expressed and located in podosomes is absent in human osteoclasts, while the beta 3 subunit of the vitronectin receptor is expressed by osteoclasts, but not by other monocyte-derived cells and colocalizes with vinculin around the actin core of the podosome. The beta 1 subunit of the fibronectin receptors is also found, but with a diffuse pattern, in the osteoclast membrane. These results indicate that podosomes, while present in different cell types, may have in the osteoclast an unique cytochemical organization related to the peculiar function of this cell.

Bone Resorption↗

PubMed1986

Are osteoblastic cells required for the control of osteoclast activity by parathyroid hormone?

Osteoclasts cultured from the endosteal surface of marrow appear to require osteoblasts for PTH to stimulate their resorptive activity, but the contribution of preosteoclasts to these observations has not been ruled out. Other data indicate that an osteoblast-derived factor can influence osteoclast activity, or confer sensitivity of osteoclasts to PTH. Acid phosphatase content of osteoclasts formed in osteoblast-free cultures of marrow cells is stimulated by PTH, but resorption is not. Also, there are conflicting data on the presence of a PTH receptor on osteoclasts. Thus, while PTH may act directly on preosteoclasts, or even on osteoclasts, to enhance resorptive capability, it seems evident that an osteoblastic factor mediates the PTH stimulation of osteoclastic resorptive activity.

Animals↗

PubMed1988

Concurrent activation of granulocytes and osteoclasts in busulfan-suppressed bone marrow in response to transplantation of a mammary carcinoma in mice.

Transplantation of CE mammary adenocarcinoma (CE maca) into normal mice produces both neutrophilia and hypercalcemia due to osteoclastic bone resorption. In order to explore the physiology of osteoclast formation in vivo, the time course of neutrophilia and osteoclast development was examined in mice that had been pretreated with busulfan prior to the CE maca implantation. Busulfan-treated tumor-bearing mice (BUTUM), busulfan-treated control mice (BUCON), tumor-bearing mice with no busulfan (TUM), and normal controls (CON) were sacrificed on days 4, 7, 11, 14, and 17 after tumor implantation. Leukocyte counts, serum calcium levels, marrow cellularity, and marrow colony-forming units (CFU) were determined. Osteoclasts were quantified histologically by the osteoclast: endosteum ratio (OER). BUCON bone marrow was hypoplastic with CFU remaining significantly lower than that of controls over the course of the experiment. In contrast, BUTUM marrow CFU increased dramatically with the growth of the tumor. The most predominant increase was observed in neutrophilic CFU. Development of hypercalcemia closely paralleled neutrophilia in both TUM and BUTUM mice, although these changes were significantly delayed in the BUTUM group. The neutrophil count and serum calcium levels remained within normal control levels for BUCON mice. The OER correlated with serum calcium, and it closely paralleled the neutrophil count in TUM and BUTUM mice. These results clearly indicated the stimulation of bone marrow neutrophilic granulocyte progenitors and osteoclasts by the CE maca, indicating that the bone marrow is the primary target of this tumor. There may be a closely related mechanism in osteoclast and granulocyte stimulation by one or more CE maca factors.

Animals↗

PubMed1987

Reduced life span of the osteoclast in osteopetrotic (mi and midi) mice.

Osteoclasts were enumerated on the parietal bones of mice carrying combinations of alleles (mi, midi and +) at the microphthalmic locus. Homozygous mutants at this locus show varying degrees of osteopetrosis due to defective bone resorption. As the severity of the bone resorption defect increased across the genotypes, there was an increase in the total number of osteoclasts and also an increase in the proportion of uninucleate osteoclasts. In parietal bones incubated for 24 h with parathyroid hormone (PTH) there was no significant difference between osteopetrotic and normal bones in terms of the number of osteoclast nuclei that had taken part in fusion. When bones were incubated for 24 h in the absence of PTH the percentage of osteoclasts remaining was correlated inversely with the severity of the bone resorption defect. Thus, we suggest that the increased proportion of uninucleate osteoclasts in these mutants results from a shortened life span, reducing the time-dependent accumulation of nuclei. This implies a reduced efficiency of uninucleate osteoclasts over multinucleate ones and we speculate on the reason for this.

Alleles↗

PubMed1987

Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption.

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.

Animals↗

PubMed1984

The effect of prostaglandins I2, E1, E2 and dibutyryl cyclic AMP on the cytoplasmic spreading of rat osteoclasts.

We isolated osteoclasts from neonatal rat bone and assessed the effects of prostaglandins (PGs) I2, E1 and E2 on osteoclastic spreading. We found that although the PGs and dibutyryl cyclic AMP (DB-cAMP) acted as direct inhibitors of osteoclastic spreading, if osteoblasts and osteoclasts were co-cultured, the addition of PGs or DB-cAMP caused a considerable increase in spreading. This suggests that the PGs and DB-cAMP induce osteoblasts to stimulate osteoclasts. Osteoblasts are known to produce PGs, and thus possess the capacity to either inhibit (through PGs) or stimulate osteoclasts. Our results suggest that the balance between stimulation and inhibition of osteoclasts by osteoblasts may be determined by osteoblastic cyclic AMP levels.

Alprostadil↗

PubMed1984

Quantitative ultrastructural analyses of rabbit osteoclasts. The effect of in vivo treatment with sodium salicylate.

Rabbit tibial osteoclasts were examined by electron microscopy and quantitative data on their ultrastructural morphology collected by methods described by Holtrop. Osteoclasts from the tibiae of two groups of rabbits were compared: one fed a commercial diet and the other fed the same diet containing 2% sodium salicylate (v/w). No changes in the total number of tibial osteoclasts were detected but average osteoclast size, numbers of nuclei per osteoclast and ruffled border and clear zone areas decreased (p less than 0.05), as did the proportion of osteoclasts directly attached to bone. These results suggest that osteoclast activity is inhibited by in vivo salicylate therapy.

Animals↗

PubMed1981

A quantitative histologic study of avian osteopetrotic bone demonstrating normal osteoclast numbers and increased osteoblastic activity.

Hyperostotic diseases caused by increased osteoblastic activity are poorly understood partially because a suitable animal model is not available for their study. In contrast, a hyperostotic disorder caused by defective osteoclasts (mammalian osteopetrosis) is better understood, largely because of advances made with a murine model of the disease. The avian form of osteopetrosis is caused by RNA tumor viruses, but the role of osteoblasts and osteoclasts is not clear. We have performed a morphometric analysis of osteopetrotic avian bone to provide basic information about changes in the cells of bones from animals developing the disease. Chick embryos were injected at 12 days of incubation with a virus (MAV-2(0)) that induces osteopetrosis at a high frequency. Changes in bone volume were detected 14 days after virus injection. By 3 weeks of age, the bone volumes of osteopetrotic chicks were 4.7-fold larger than controls. The mean caliper diameter of an osteoclast was the same in osteopetrotic and normal bone. The number of osteoclasts per tibia increased in osteopetrotic animals, but the density of osteoclasts decreased. The presence of an increased number of osteoclasts of a normal size rules out the possibility that the virus causes osteopetrosis by selectively killing osteoclasts. The total bone-nonbone interface increased in osteopetrosis, and greater than 90 per cent of interface surface was devoted to bone deposition. These results indicate that avian osteopetrosis is an osteoblastic, proliferative disorder of bone and may serve as an excellent animal model for human diseases in which osteoblasts increase in number.

Animals↗

PubMed1994

Mammalian osteoclasts express a transient potassium channel with properties of Kv1.3.

Previous studies have revealed that expression of K+ channels in osteoclasts correlates with cell morphology and is influenced by interaction with the extracellular matrix. In this study, we investigated the electrophysiological properties of an outwardly rectifying K+ channel in rat and mouse osteoclasts using patch-clamp techniques. Cell-attached patch recordings revealed a channel of approximately 14 pS conductance that opened upon depolarization, and had a reversal potential close to that predicted for a K+ channel. Channel activity was transient; inactivation of ensemble currents, like that of whole-cell currents, occurred as a single exponential process. Both single-channel and macroscopic currents exhibited use-dependent inactivation in response to repetitive depolarizations. Two scorpion toxins, margatoxin and charybdotoxin, blocked this transient K+ channel, with half-maximal inhibition at 200 pM and 5 nM, respectively. In contrast, dendrotoxin (500 nM) had little effect. In summary, the outwardly rectifying K+ channel in osteoclasts resembles the Shaker-related K+ channel, Kv1.3. When membrane potential was recorded in whole-cell configuration, charybdotoxin (50 nM) caused a depolarization of 5 to 10 mV from resting levels of -50 mV or more positive; therefore this K+ channel contributes to the membrane potential of osteoclasts under some conditions. To investigate the molecular nature of osteoclast K+ channels, we performed RT-PCR on osteoclast RNA using primers for Kv1.3 and the inward rectifier, IRK1. mRNA encoded by Kv1.3 and IRK1 was detected and message identity confirmed by restriction enzyme digestion and sequence analysis. We conclude that osteoclasts exhibit, in addition to the previously described inward rectifier, an outwardly rectifying K+ conductance with properties of the Kv1.3. channel.

Animals↗

PubMed1994

The cytoskeletal framework of chick osteoclasts in resin-less sections.

The cytoskeletal framework of chick osteoclasts was examined by the resin-less polyethylene glycol (PEG) method. Resin-less thin sections revealed changes in the 3-dimensional organisation of a microtrabecular lattice during different phases of osteoclast activity. This lattice, composed of interconnecting strands, occupied part of the osteoclast cytoplasm including the ruffled border and clear zone areas. In firmly attached, active osteoclasts, the clear zone exhibited a compact and orderly array with a meshwork appearance. This organelle-free area could be clearly distinguished from other cytoplasmic regions by the compactness of its lattice structure. Well developed membrane infoldings of the ruffled border next to the resorption lacunae were composed of interconnected strands, some of which were in direct contact with the ruffled border membrane. Migrating osteoclasts remote from the bone surface lacked these membrane modifications. Their peripheral cytoplasm exhibited a disorganised meshwork of strands without the clear zone. While migrating, osteoclasts which still adhered to the bone surface, appeared to form loci within the compact lattice structure referred to as podosomes. These results demonstrate the dynamic morphological changes in the organisation of the trabecular lattice that occur within attached and migrating osteoclasts.

Animals↗

PubMed1994

Cytochrome c oxidase activity in osteoclasts appeared in an early stage of parathyroid hormone treatment.

Osteoclasts which appeared in an early stage of parathyroid hormone (PTH) administration were examined using cytochrome c oxidase activity. In the period of 1-2 hr after intraperitoneal administration of PTH, two types of osteoclasts were observed. The first type had a well-developed ruffled border characterized by deeply infolded extracellular channels, and many endocytic vacuoles were observed, as seen in control osteoclasts. Many of the mitochondria contained in the first type were with the reaction products of the enzyme on the crystal membrane and within the intermembrane space (type 1 mitochondria), and the distribution of type 1 mitochondria was similar to that seen in control osteoclasts. The second type had a ruffled border characterized by unclear extracellular channels and cytoplasmic projections. The numerical percentage of type 1 mitochondria in the second type of osteoclasts (45.3%) was significantly smaller than that in the first type (79.9%). Mitochondria which had the reaction products of the enzyme only on the crystal membrane including those with no reaction product (type 2 mitochondria) were predominant in the second type of osteoclasts. These results include two possible ways in which a morphologically regressive change of the ruffled border can occur in osteoclasts during an early stage of PTH treatment, and cytochrome c oxidase activity is closely associated with the change in the ruffled border.

Animals↗

PubMed1993

[Regulation of osteoclastic resorption by prostanoids in vivo].

Prostanoids, and particularly prostaglandins (PGs) E, are potent mediators of bone resorption. However the actual role(s) of these agents in osteoclastic resorption is (are) not yet known. In an attempt to approach the role of prostanoids in the events leading to resorption itself, we used two different animal models. We showed that indomethacin, an inhibitor of prostanoid biosynthesis, drastically reduced osteoclastic resorption at the dose of 7 mg/kg/d in the two models. This effect was dose-dependent as resorption inhibition increased by increasing the dose of indomethacin. However the effect was already significant at the lower dose (2 mg/kg/d). The total number of osteoclasts in the site was according by reduced, showing the involvement of the prostanoids in the events preceding and/or regulating the fusion of the mononucleated preosteoclasts into multinucleated osteoclasts. The calculation of the ratio active/inactive osteoclasts indicated that the activation (i.e. access to the bone surface) of the osteoclast, was prostanoid-dependent; indeed the ratio systematically decreased in the indomethacin-treated animals in the two models. We also observed that the inhibition was not sustained in time as resorption recovered, probably through the starting of PG-independent metabolic pathways. However this starting was rather slow and these pathways apparently were less affective than the PG-dependent ones since the intrinsic activity of the differentiated osteoclasts was significantly reduced.

Animals↗

PubMed1994

Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts.

Osteoclasts are multinucleate giant cells playing key roles in bone resorption. These cells solubilize mineralized bone matrix by means of acid and protease action; however, the precise mechanism of this process is not well known. Recently, we succeeded in the isolation of pure osteoclasts from rabbit bones and constructed a cDNA library. Using a differential screening procedure, two genes expressed predominantly in osteoclasts compared with spleen cells were isolated (Tezuka, K., Sato, T., Kamioka, H., Nijweide, P. J., Tanaka, K., Matsuo, T., Ohta, M., Kurihara, N., Hakeda, Y., and Kumegawa, M. (1992) Biochem. Biophys. Res. Commun. 186, 911-917). One of them, OC-2, was found to encode a possible cysteine proteinase structurally related to cathepsins L and S. By in situ hybridization, OC-2 was confirmed to be expressed in osteoclasts in vivo. By Northern blot analysis, OC-2 was highly and preferentially expressed in osteoclasts compared with other tissues such as kidney, liver, spleen, and lung. The predominant expression of OC-2 in osteoclasts may suggest that OC-2 encodes a protein, possibly a cysteine proteinase, that plays an important role in osteoclastic bone resorption.

Amino Acid Sequence↗

PubMed1996

Stimulation of osteoclast-like cell formation by Pasteurella multocida toxin from hemopoietic progenitor cells in mouse bone marrow cultures.

The effects of purified Pasteurella multocida toxin (PMT) on osteoclast formation from hemopoietic progenitor cells were studied using an in vitro system. Mononuclear adherent mouse bone marrow cells were cultured for 7 or 14 days in the presence of PMT, or 1,25-dihydroxy-vitamin D3, or both. Mononuclear osteoclast-like cells, which are postmitotic osteoclast precursor cells, were identified as tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells possessing calcitonin receptors. Multinucleated osteoclast-like cells were TRAP-positive multinuclear cells with calcitonin receptors. The results demonstrate that, as does 1,25(OH)2D3, Pasteurella multocida toxin stimulates proliferation of adherent bone marrow mononuclear cells (progenitor cells), and their differentiation into postmitotic mononuclear osteoclast precursor cells. It also causes fusion of the latter into multinuclear osteoclasts; however, the number of osteoclasts obtained with PMT is smaller than with 1,25-dihydroxy-vitamin D3.

Acid Phosphatase↗

PubMed1998

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis.

Osteoclasts are bone resorbing cells of hematopoietic origin; however, a progenitor cell population that gives rise to mature osteoclasts remains elusive. We have characterized a unique cell surface phenotype of clonogenic osteoclast progenitors (colony-forming unit-osteoclast [CFU-O]) and obtained a marrow cell population selectively enriched for these progenitors. Whole bone marrow cells were sequentially separated based on physical and cell surface characteristics, and the presence of CFU-O and other hematopoietic progenitors was examined. CFU-O was enriched in a nonadherent, low-density, lineage-marker-negative (Lin-), Thy1.2-negative (Thy1.2-), Sca1-negative (Sca1-), and c-kit-positive (c-kit+) population, as were the progenitors that were responsive to macrophage-colony-stimulating factor(CSF; CFU-M), granulocyte-macrophage-CSF (CFU-GM), and stem cell factor (CFU-SCF). When the Lin-Thy1.2-Sca1- population was divided into c-kithigh and c-kitlow populations based on c-kit fluorescence, over 88% of CFU-M, CFU-GM, and CFU-SCF were found in the c-kithigh population. In relation to the above mentioned hematopoietic progenitors, CFU-O was significantly higher in the c-kitlow population: 80% of progenitors present in the c-kitlow population were CFU-O. The CFU-O in both c-kithigh and c-kitlow populations showed key features of the osteoclast: multinucleated tartrate-resistant acid phosphatase-positive cell formation, expressions of vitronectin receptors, c-src and calcitonin receptors, and bone resorption. We have identified a progenitor cell population in the earliest stage of the osteoclast lineage so far described and developed a method to isolate it from other hematopoietic progenitors. This should help pave the way to understand the molecular mechanisms of osteoclast differentiation.

Animals↗

PubMed1998

Osteoclast-like cells in murine collagen induced arthritis.

OBJECTIVE: To investigate the participation of osteoclast-like bone resorbing cells in the joint destruction of murine collagen induced arthritis (CIA). METHODS: After induction of CIA in DBA/1J mice, a histological time course study was conducted on paw sections stained for tartrate resistant acid phosphatase (TRAP), a marker of osteoclasts. Cells from arthritic paws were cultured in vitro with or without indomethacin (IM) or anti-interleukin 6 neutralizing antibody (anti-IL-6), and stained for TRAP. Levels of prostaglandin E2 (PGE2), IL-1beta, IL-6, and tumor necrosis factor-alpha in the culture supernatants were determined by ELISA. The bone resorbing ability of these cells was examined on dentine slices. In control experiments, cells of normal paws or of arthritic tibiae were cultured in the same manner. RESULTS: TRAP positive osteoclast-like cells were detected late in the development of bone lesions at every eroded front in the pannus-bone and the pannus-subchondral bone junctions of arthritic joints. In vitro, cells of arthritic paws formed bone resorbing osteoclast-like cells spontaneously. However, the control culture failed to form these cells. PGE2 and IL-6 were detected at higher levels in arthritic culture than in control culture. Although both indomethacin and anti-IL-6 reduced osteoclast-like cell formation and indomethacin inhibited PGE2 synthesis, indomethacin failed to reduce IL-6. CONCLUSION: These findings suggest the direct participation of osteoclast-like cells in the joint destruction of CIA, the locally enhanced activity of osteoclast-like cell differentiation in arthritic paws, and the participation of prostaglandins and prostaglandin-independent IL-6 in this differentiation.

Acid Phosphatase↗