Osteoclast Formation In Cultures Research Articles

The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors.

Inflammatory bone diseases, including rheumatoid arthritis, periodontitis and peri-implantitis, are associated not only with the production of inflammatory cytokines but also with local oxidative status, which is defined by intracellular reactive oxygen species (ROS). Osteoclast differentiation has been reported to be related to increased intracellular ROS levels in osteoclast lineage cells. Sudachitin, which is a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions in mammalian cells. However, the effects of sudachitin on inflammatory bone destruction and osteoclastogenesis remain unknown. In calvaria inflamed by a local lipopolysaccharide (LPS) injection, inflammation-induced bone destruction and the accompanying elevated expression of osteoclastogenesis-related genes were reduced by the co-administration of sudachitin and LPS. Moreover, sudachitin inhibited osteoclast formation in cultures of isolated osteoblasts and osteoclast precursors. However, sudachitin rather increased the expression of receptor activator of NF-κB ligand (RANKL), which is an important molecule triggering osteoclast differentiation, and the mRNA ratio of RANKL/osteoprotegerin that is a decoy receptor for RANKL, in the isolated osteoblasts, suggesting the presence of additional target cells. When osteoclast formation was induced from osteoclast precursors derived from bone marrow cells in the presence of soluble RANKL and macrophage colony-stimulating factor, sudachitin inhibited osteoclastogenesis without influencing cell viability. Consistently, the expression of osteoclast differentiation-related molecules including c-fos, NFATc1, cathepsin K and osteoclast fusion proteins such as DC-STAMP and Atp6v0d2 was reduced by sudachitin. In addition, sudachitin decreased activation of MAPKs such as Erk and JNK and the ROS production evoked by RANKL in osteoclast lineage cells. Our findings suggest that sudachitin is a useful agent for the treatment of anti-inflammatory bone destruction.

PPAR agonists stimulate adipogenesis at the expense of osteoblast differentiation while inhibiting osteoclast formation and activity

Drugs used in the treatment of type 2 diabetes and cardiovascular disease, specifically peroxisome proliferator-activated receptor (PPAR) agonists, have been reported to affect bone cell function and fracture risk. In this study, we assessed the direct effects of PPAR-γ agonists (rosiglitazone and troglitazone), used in the treatment of diabetes, and a PPAR-α agonist (fenofibrate), used to treat hyperlipidaemia, on the function of primary osteoblasts and osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by up to 85% in cultures treated with rosiglitazone and by 45% in troglitazone-treated or fenofibrate-treated cultures; at the same time, lipid droplet formation was increased by 40-70%. The expression of key osteogenic markers was similarly downregulated in cultures treated with PPAR agonists, whereas adipogenesis markers were upregulated. Formation of osteoclasts in cultures derived from mouse marrow diminished with fenofibrate treatment, whereas both glitazones reduced resorptive activity without affecting osteoclast number. Metformin, although not a PPAR agonist, is also commonly used in the treatment of type 2 diabetes. Here, metformin was found to have no effect on bone cell function. Taken together, these data suggest that PPAR-γ agonists may enhance bone loss via increased adipogenesis at the expense of osteoblast formation. In contrast, PPAR-α agonists may prevent bone loss. Given that the prevalence of diabetes and cardiovascular disease is expected to rise significantly, greater attention may need to be paid to the effects of PPAR agonists on bone homeostasis.

Nordihydroguaiaretic acid inhibition of NFATc1 suppresses osteoclastogenesis and arthritis bone destruction in rats

Nordihydroguaiaretic acid (NDGA) is known to have prominent anticancer activity against several cancers, and is also known to be an inhibitor of 5-lipoxygenase (5-LO). In this study, we investigated the regulatory function of NDGA on inflammatory bone destruction mediated by osteoclasts. NDGA markedly inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced formation of osteoclasts in cultures of murine osteoclast precursor cell line RAW-D cells and primary bone marrow-derived macrophages culture systems. The inhibitory effect of NDGA on osteoclastogenesis did not arise from the inhibition of 5-LO activity. NDGA did not affect MAPKs, such as p38, JNK, and NF-κB, but significantly inhibited the induction of NFATc1, a key transcription factor for osteoclastogenesis. NDGA also suppressed activation of ERK in osteoclast precursors. RANKL-induced calcium oscillation observed in osteoclast precursors was completely diminished by the addition of NDGA. In mature osteoclasts, RANKL-induced nuclear translocation of NFATc1 was clearly inhibited by NDGA treatment. Finally, in vivo studies demonstrated that administration of NDGA significantly reduced severe bone destruction and osteoclast recruitment in the ankle joint of rats with adjuvant-induced arthritis. These results indicate the potential utility of NDGA as a therapeutic agent for ameliorating inflammatory bone destruction in rheumatoid arthritis.

Influence of melatonin on murine osteoclast formation in culture — Automated detection and quantification by a novel image analysis system

Influence of melatonin on murine osteoclast formation in culture — Automated detection and quantification by a novel image analysis system

Direct cell–cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell-cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell-cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co-culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule-1 (ICAM-1) and of osteoclastogenesis-related genes (RANKL, RANK, TNF-alpha, and IL-1beta) was highly up-regulated in the co-cultures compared to mono-cultures and the 5-10-fold up-regulation reflected a synergistic increase due to direct cell-cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)(2)VitD(3) and dexamethasone. In case of indirect cell-cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast-like cells that were formed in co-culture with periodontal ligament fibroblasts was significantly augmented compared to mono-cultures. Our data indicate that cell-cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts.

Diphenylhydantoin Inhibits Osteoclast Differentiation and Function Through Suppression of NFATc1 Signaling

Diphenylhydantoin (DPH) is widely used as an anticonvulsant drug. We examined the effects of DPH on osteoclast differentiation and function using in vivo and in vitro assay systems. Transgenic mice overexpressing a soluble form of RANKL (RANKL Tg) exhibited increased osteoclastic bone resorption. Injection of DPH into the subcutaneous tissue overlying calvaria of RANKL Tg mice suppressed the enhanced resorption in the calvaria. In co-cultures of mouse osteoblasts and bone marrow cells, DPH inhibited lipopolysaccharide (LPS)-induced osteoclast formation. DPH affected neither the mRNA expression of RANKL and osteoprotegerin nor the growth of mouse osteoblasts in culture. On the other hand, DPH inhibited the RANKL-induced formation of osteoclasts in cultures of mouse bone marrow-derived macrophages (BMMphis) and of human peripheral blood-derived CD14(+) cells. DPH concealed LPS-induced bone resorption in mouse calvarial organ cultures and inhibited the pit-forming activity of mouse osteoclasts cultured on dentine slices. DPH suppressed the RANKL-induced calcium oscillation and expression of nuclear factor of activated T cells c1 (NFATc1) and c-fos in BMMphis. Moreover, DPH inhibited the RANKL-induced nuclear localization and auto-amplification of NFATc1 in mature osteoclasts. Both BMMphis and osteoclasts expressed mRNA of a T-type calcium channel, Cav3.2, a target of DPH. Blocking the expression of Cav3.2 by short hairpin RNAs significantly suppressed RANKL-induced osteoclast differentiation. These results suggest that DPH inhibits osteoclast differentiation and function through suppression of NFATc1 signaling. The topical application of DPH may be a therapeutic treatment to prevent bone loss induced by local inflammation such as periodontitis.

A possible suppressive role of galectin-3 in upregulated osteoclastogenesis accompanying adjuvant-induced arthritis in rats

Galectin-3 is a β-galactoside-binding animal lectin having pleiotropic effects on cell growth, differentiation, and apoptosis. This lectin has been shown to be involved in phagocytosis by macrophages and in inflammation. Here we investigated an involvement of galectin-3 in the regulatory process of inflammatory bone resorption in rats with adjuvant-induced arthritis (AA rats) accompanying severe bone destruction in the ankle joints. The protein level of galectin-3 in the ankle-joint extracts was markedly augmented at week 3 after adjuvant injection, at the time when severe bone destruction was observed. Immunohistochemical analysis revealed an extremely high expression of galectin-3 in macrophages and granulocytes infiltrated in the area of severe bone destruction. To estimate the role of galectin-3 in osteoclastogenesis and osteoclastic bone resorption, recombinant galectin-3 was added to in vitro culture systems. Galectin-3 markedly inhibited the formation of osteoclasts in cultures of murine osteoclast precursor cell line as well as in rat bone marrow culture systems. This inhibition was not observed by heat-inactivated galectin-3 or by galectin-7. Although recombinant galectin-3 did not affect signaling through mitogen-activated protein kinase (MAPK) or nuclear factor-κB (NF-κB), it specifically suppressed the induction of nuclear factor of activated T-cells c1 (NFATc1). Galectin-3 significantly inhibited dentine resorption by mature osteoclasts in vitro. Furthermore, in vivo studies clearly showed a significant suppression of bone destruction and osteoclast recruitment accompanying arthritis, when galectin-3 was injected into the cavity of ankle joint of AA rats. Thus, abundant galectin-3 observed in the area of severe bone destruction may act as a negative regulator for the upregulated osteoclastogenesis accompanying inflammation to prevent excess bone destruction.

Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast‐like cells

Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.

Staphylococcus aureus capsular material promotes osteoclast formation

Osteomyelitis, which is most frequently due to infection by Staphylococcus aureus, commonly causes bone destruction. S. aureus is known to secrete a number of surface-associated proteins that are potent stimulators of bone resorption. The precise cellular and humoral mechanisms that mediate this stimulatory effect are uncertain. In this study, we have determined whether osteoclast formation and resorption is directly promoted by surface-associated proteins. Surface-associated material (SAM) obtained from a 24-hour culture of S. aureus was added to cultures of mouse and human monocytes. Human monocyte cultures were incubated in the presence and absence of a soluble receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). In cultures where M-CSF, RANKL, and SAM were added together, osteoclast formation did not exceed that seen in cultures with M-CSF and RANKL. In keeping with this finding, SAM did not increase osteoclast formation and resorption when mouse monocytes were cocultured with RANKL-expressing osteoblasts. In the absence of RANKL, however, SAM was capable of inducing osteoclast formation in cultures of human monocytes. This finding was evidenced by the generation of vitronectin receptor and tartrate-resistant acid phosphatasepositive multinucleated cells that were capable of lacunar resorption. Inhibitors of RANKL-dependent (RANK:Fc, OPG) and RANKL-independent (anti-TNF-alpha, gp130, IL-8, TGF-beta) osteoclast formation did not inhibit SAM-induced osteoclast formation. SAM did not stimulate mature osteoclast resorption activity. These findings indicate that RANKL, which is present in the circulation as a soluble factor, does not play a role in osteoclast formation in the presence of S. aureus SAM and that S. aureus SAM contains a soluble factor that promotes osteoclast formation by a RANKL-independent mechanism.

Breast Cancer Increases Osteoclastogenesis by Secreting M-CSF and Upregulating RANKL in Stromal Cells

Background. Breast cancer metastasis to bone causes resorption of the mineralized matrix by osteoclasts.Macrophage colony stimulating factor (M-CSF)and receptor activator of the NF-κB ligand (RANKL) are produced by stromal cells and are essential for osteoclast formation. The human breast cancer cell line, MDA-MB-231, reliably forms bone metastases in a murine model and stimulates osteoclast formation in culture. We hypothesized that MDA-MB-231 stimulates osteoclast formation through secretion of M-CSF and/or RANKL.Materials and methods. We cocultured MDA-MB-231 and a bone marrow derived cell line, UAMS-33, and evaluated the expression of M-CSF and RANKL mRNA. Osteoclast formation was assessed using these cells added to hematopoietic cell cultures.Results. MDA-MB-231 exhibited constitutive expression of M-CSF mRNA. As expected, addition of recombinant M-CSF (30 ng/ml) and RANKL (30 ng/ml) to hematopoietic osteoclast precursors supported osteoclast formation, while the addition of soluble RANKL alone or MDA-231 without added RANKL did not. Notably, coculture of MDA-231 with hematopoietic cells and added soluble RANKL stimulated significant osteoclast formation, indicating that MDA-231 served as an effective source for M-CSF. MDA-231 did not express RANKL. However, when cocultured with the murine bone marrow stromal cell line UAMS-33, RANKL expression was significantly increased in the latter cells. MDA-231 also stimulated osteoclast formation in coculture with UAMS-33 and hematopoietic cells.Conclusions. We conclude that MDA-MB-231 increases osteoclast formation by secreting adequate amounts of M-CSF protein and enhancing the expression of RANKL by stromal support cells. The ability to stimulate osteoclasts may explain the ability to metastasize to bone.

IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro.

IL-12, like IL-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that IL-12, like IL-18, was unable to act directly on osteoclastic precursors. IL-12, like IL-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Additionally, IL-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at concentrations 20- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to IL-4, IL-10, or IL-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to IL-12 and IL-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.

Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro.

Nitrogen-containing bisphosphonates were shown to cause macrophage apoptosis by inhibiting enzymes in the biosynthetic pathway leading from mevalonate to cholesterol. This study suggests that, in osteoclasts, geranylgeranyl diphosphate, the substrate for prenylation of most GTP binding proteins, is likely to be the crucial intermediate affected by these bisphosphonates. We report that murine osteoclast formation in culture is inhibited by both lovastatin, an inhibitor of hydroxymethylglutaryl CoA reductase, and alendronate. Lovastatin effects are blocked fully by mevalonate and less effectively by geranylgeraniol whereas alendronate effects are blocked partially by mevalonate and more effectively by geranylgeraniol. Alendronate inhibition of bone resorption in mouse calvaria also is blocked by mevalonate whereas clodronate inhibition is not. Furthermore, rabbit osteoclast formation and activity also are inhibited by lovastatin and alendronate. The lovastatin effects are prevented by mevalonate or geranylgeraniol, and alendronate effects are prevented by geranylgeraniol. Farnesol and squalene are without effect. Signaling studies show that lovastatin and alendronate activate in purified osteoclasts a 34-kDa kinase. Lovastatin-mediated activation is blocked by mevalonate and geranylgeraniol whereas alendronate activation is blocked by geranylgeraniol. Together, these findings support the hypothesis that alendronate, acting directly on osteoclasts, inhibits a rate-limiting step in the cholesterol biosynthesis pathway, essential for osteoclast function. This inhibition is prevented by exogenous geranylgeraniol, probably required for prenylation of GTP binding proteins that control cytoskeletal reorganization, vesicular fusion, and apoptosis, processes involved in osteoclast activation and survival.

プロスタグランディン E_2 存在下での破骨細胞の新生過程における lipopolysaccharide とインターロイキン 1 の作用

Prostaglandin E_2 (PGE_2), interleukin-1 (IL-1), and lipopolysaccharide (LPS) are the candidates for the inducers of osteoclastic bone resorption accompanied with periodontal disease. The purpose of this paper is to show the effect of these substances on osteoclast formation in cell culture. Mouse bone marrow cells were prepared, and the cells were cultured with IL-1 and LPS in the presence of PGE_2. After the culture, tartrate-resistant acid phosphatase positive cells were scored as osteoclasts. The results obtained were as follows : 1. IL-1 increased the multinuclear osteoclast formation in the culture in the presence of 0.1μM PGE_2. 2. The multinuclear osteoclast formation was increased in the culture with IL-1 added in the second half culture period as much as in the culture with the addition throughout the culture period. The multinuclear osteoclast formation decreased in the culture with IL-1 added in the first half culture period. 3. Mononuclear osteoclast formation was increased in the culture with LPS added in the first half period of the culture with 0.1μM PGE_2. Multinuclear osteoclast formation was increased by LPS added in the second half period with 0.1μM PGE_2. 4. Mean nuclear number of multinuclear osteoclasts formed by IL-1 or LPS in the presence of 0.1μM PGE_2 was much higher than that by 0.1μM PGE_2 alone. Mean nuclear number of multinuclear osteoclasts by 1μM PGE_2 alone was much less than that by 0.1μM PGE_2. These results show that osteoclasts are formed by IL-1 and LPS in the presence of low concentration of PGE_2. This suggests the possibility that PGE_2, IL-1 and LPS induce or enhance bone resorption, through osteoclast formation, accompanied with periodontal disease.

Effect of medium pH on osteoclast activity and osteoclast formation in cultures of dispersed rabbit osteoclasts.

We investigated the effect of medium pH on activity of isolated osteoclasts and have also looked at the possibility that medium pH affects osteoclast numbers during culture. Osteoclast-containing cell suspensions prepared from neonatal rabbits were cultured on bovine bone slices at pH 6.5, 7.0, or 7.5. After 24 or 48 h of culture, the cells and bone slices were fixed and stained for tartrate-resistant acid phosphatase (TRAP). After counting the osteoclasts, the cells were removed and the resorption lacunae stained by immunostaining using anticollagen type I antibody and then quantitated. We found that the resorptive activity of isolated rabbit osteoclasts was sharply increased at pH 6.5-7. Osteoclast differentiation and proliferation, on the other hand, were optimal at pH 7.0-7.5 but decreased at pH 6.5. The results thus imply that pH regulation of the bone surface environment can dramatically alter both the number of osteoclasts and their resorptive activity.

Location of osteoclast precursors in fetal rat calvaria cultured on collagen gels

Although osteoclasts are derived from hematopoietic cells, the exact identity of their precursors and the mechanism for their recruitment onto bone surfaces remain unclear. We wished to study their differentiation in the fetal rat calvaria and to locate its source of osteoclast precursor cells. Osteoclasts were detected by neutral red staining or cytochemical reaction for acid phosphatase of intact bone (cell number and area measured by computerized image analysis) or in cryostat sections of bone (enzyme activity measured by quantitative cytochemistry). Histology of semithin sections of fixed bones was also examined. The 19 day calvariae contained few mature osteoclasts. After 48 h culture on gels of type 1 collagen (1.5 mg/ml) supplemented with 5 mM calcium beta-glycerophosphate, 10 mM proline, and 2 micrograms/ml ascorbic acid, numerous large osteoclasts were seen on their endocranial surfaces. In contrast, cell morphology and enzyme activity deteriorated in bones cultured in liquid medium. The cells that formed in vitro rapidly responded to calcitonin by contraction. Stripping of endocranial membranes from the calvariae prevented osteoclast formation in culture, but these cells were seen when "stripped" bones had been cocultured with their membranes for 48 h or with intact 16 day calvariae (well before the onset of osteogenesis). Few osteoclasts were found when an 0.22 micron filter was inserted between the stripped calvaria and the endocranial membranes. We conclude that the endocranial membranes, which contain the meningeal blood vessels, are a major source of osteoclast precursors and that these cells are present in calvarial tissue even before the onset of osteogenesis.