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Background: Increased bone resorption is driven by augmented osteoclast activity in pathological states of the bone, including osteoporosis, fracture and metastatic bone cancer. Pain is a frequent co-morbidity in bone pathologies and adequate pain management is necessary for symptomatic relief. Bone cancer is associated with severe skeletal pain and dysregulated bone remodelling, while increased osteoclast activity and bone pain are also observed in osteoporosis and during fracture repair. However, the effects of altered osteoclast activity and bone resorption on nociceptive processing of bone afferents remain unclear., Methods: This study investigates whether physiologic osteoclasts and resulting changes in bone resorption can induce skeletal pain. We first assessed correlation between changes in bone microarchitecture (through µCT) and skeletal pain using standardized behavioural phenotyping assays in a mouse model of metastatic bone cancer. We then investigated whether increased activity of physiologic osteoclasts, and the associated bone resorption, is sufficient to induce skeletal pain using mouse models of localized and widespread bone resorption following administration of exogenous receptor activator of nuclear factor kappa-B ligand (RANKL)., Results: Our data demonstrates that mice with bone cancer exhibit progressive pain behaviours that correlate with increased bone resorption at the tumour site. Systemic RANKL injections enhance osteoclast activity and associated bone resorption, without producing any changes in motor function or pain behaviours at both early and late timepoints., Conclusion: These findings suggest that activation of homeostatic osteoclasts alone is not sufficient to induce skeletal pain in mice., Significance Statement: The role of osteoclasts in peripheral sensitization of sensory neurones is not fully understood. This study reports on the direct link between oestrogen-independent osteoclast activation and skeletal pain. Administration of exogenous receptor activator of nuclear factor kappa-B ligand (RANKL) increases bone resorption, but does not produce pro-nociceptive changes in behavioural pain thresholds. Our data demonstrates that physiologic osteoclasts are not essential for skeletal pain behaviours., (© 2020 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFIC®.)

Objective: To explore the effect of advanced glycation end products (AGEs) on osteoclasts at different stages of differentiation., Methods: Raw264.7 cells cultured in vitro were induced for osteoclastogenesis using RANKL, and the stages of differentiation of the osteoclasts were determined with TRAP staining. The cells were then randomly divided into control group, early-stage AGEs intervention group and late-stage AGEs intervention group. The viability of the cells after AGEs treatment was assessed using CCK-8 method. The cells were examined after the induction for osteoclastogenesis using TRAP staining, and the expression levels of RANK, NFATC-1, TRAF-6, TRAP and CTSK mRNAs were tested with RT-PCR; the expressions of CTSK and RANK proteins were detected using Western boltting., Results: We defined the initial 3 days of induction as the early stage of differentiation and the time beyond 3 days as the late stage of differentiation of Raw264.7 cells. Intervention with AGEs at 100 mg/L produced no significant effects on the viability of the cells, but AGEs suppressed the cell proliferation at a concentration exceeding 100 mg/L. The number of osteolasts in the early- and late-stage intervention groups was greater than that in the control group, but the cell count differed significantly only between the early-stage intervention group and control group ( P < 0.05). The gene expressions of RANK, NFATC-1, TRAF-6, TRAP and CTSK all increased after the application of AGEs in both the early and late stages of differentiation, but the changes were significant only in the early-stage intervention group ( P < 0.05). The changes in CTSK and RANK protein expressions were consistent with their mRNA expressions., Conclusions: AGEs can affect the differentiation of osteoclasts differently when applied at different stages, and intervention with AGEs at the early stage produces stronger effect to promote osteoclast differentiation than its application at a late stage.

Neutrophils are one of the most abundant leukocytes in the sites of lesion of inflammatory diseases such as periodontitis and rheumatoid arthritis. These diseases are accompanied by bone loss, which worsens the quality of life of the patients. However, the role of neutrophils in the inflammatory bone loss has not been fully investigated. In the present study, we found that human neutrophils enhanced osteoclast differentiation from mouse bone marrow cells co-cultured with mouse osteoblasts in the presence of active vitamin D 3 . The enhanced osteoclast differentiation was significantly suppressed by elastatinal, a synthetic inhibitor of neutrophil elastase. Also, we found that human neutrophils degraded human recombinant osteoprotegerin (OPG), a decoy receptor for nuclear factor κB (RANK) ligand (RANKL), the essential osteoclast differentiation-inducing factor, expressed by osteoblasts. Degradation of OPG by neutrophils was suppressed by human α 1 -protease inhibitor, the major endogenous inhibitor of neutrophil elastase. Recombinant human neutrophil elastase degraded human OPG in its death domain-like region. These results indicated that the degradation of OPG by elastase contributed at least in part to the enhanced osteoclast differentiation by neutrophils. There is a possibility that neutrophils play an important role in inflammatory bone loss., Competing Interests: Declaration of competing interest All authors declare that the research was conducted in the absence of financial interest and they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Osteoclasts are multinucleated, giant cells originating from myeloid hematopoetic stem cells. These are the only cells in nature which can resorb bone. Differentiation of mononucleated osteoclast progenitor cells requires stimulation with M-CSF (macrophage colony-stimulating factor) for the cells to proliferate and survive and with RANKL (receptor activator of nuclear factor kappa B ligand) for differentiation along the osteoclastic lineage to cells which eventually fuse to the mature, multinucleated osteoclasts. Therefore, most hormones and cytokines stimulating osteoclastogenesis do so indirectly by regulating the expression in osteoblasts of RANKL and its inhibitory decoy receptor OPG. Antibodies neutralizing RANKL is a common therapy to inhibit excessive osteoclast formation in diseases such as osteoporosis and malignant tumors with skeletal metastasis. Mature osteoclasts resorb bone by stimulating acid release into the resorption lacunae, followed by proteolytic degradation of bone matrix proteins. Loss-of-function mutations of proteins involved in acidification and proteolysis cause osteopetrosis, a disease with sclerotic bone due to non-functional osteoclasts. Osteoclasts are important for a healthy skeleton by removing damaged bone during remodeling of the skeleton, but are also important for modeling of bone, calcium homeostasis and tooth eruption, and possibly also for glucose and fat metabolism. Loss of bone in inflammatory disease, metastasizing tumors and osteoporosis is due to increased RANKL expression and enhanced osteoclast formation. The present overview aims to summarize how osteoclasts are formed and resorb bone in health and disease., (Copyright© of YS Medical Media ltd.)

Background: Radix Paeoniae Rubra (RPR), a traditional Chinese herb, has anti-inflammatory and immuno-regulatory properties. This study explored the effects of RPR on stimulation of osteoclast differentiation in RAW264.7 cells and peripheral blood mononuclear cells (PBMC)s., Methods: The mature osteoclasts were measured by bone resorption assays and TRAP staining. JNK, ERK, p38 and NF-κB inhibitors were used applied in order to verify their contribution in RPR-induced osteoclast differentiation. The NF-κB and MAPK pathways were evaluated by western blotting, RT-PCR and luciferase assay., Results: RPR induced osteoclast differentiation in a dose-dependent manner and induced the resorption activity of osteoclasts differentiation of RAW264.7 cells and PBMCs. Western blotting showed that RPR treatment induced phosphorylation of JNK, ERK, and p38 in RAW 264.7 cells. Treatment of JNK, ERK, and p38 MAP kinase inhibitors verified the contribution of JNK, ERK and p38. RPR treatment induced c-Fos and NFATc1 protein expression; NF-κB inhibitor treatment and luciferase assay verified the contribution of the NF-κB pathway., Conclusions: This study demonstrated the interesting effect, in which RPR stimulated osteoclast differentiation in murine RAW264.7 cells and human monocytes.

Objective: This work aims to examine the effects of paeonol treatment on the ability of bone marrow-derived macrophage (BMM) to excrete inflammatory factors and to differentiate into osteoclasts upon induction with Porphyromonas gingivalis (P. gingivalis). This work also aims to investigate the underlying mechanisms of these abilities., Methods: BMM culture was treated with different paeonol concentrations at for 1 h and then stimulated with P. gingivalis for 24 h before programmed death-ligand 1 (PD-L1) was quantified with flow cytometry. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The BMM culture was treated with the receptor activator for nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and then with paeonol for 1 h prior to induction with P. gingivalis. Then, osteoclast formation was assessed using tartrate resistant acid phosphatase (TRAP) staining. The osteoclast-related proteins TRAP and receptor activator of nuclear factor-κB (RANK) were quantified by Western blotting., Results: Paeonol was nontoxic to BMM within a range of 10-50 μmol·L⁻¹. Flow cytometry showed that paeonol inhibited PD-L1 expression in P. gingivalis-induced BMM in a dose-dependent manner. ELISA indicated that paeonol dose-dependently inhibited the excretion of TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM (P<0.01). TRAP staining revealed that paenol treatment inhibited the differentiation of P. gingivalis-induced BMM into osteoclasts. Western blot results suggested that paeonol decreased the expression of TRAP and RANK in BMM., Conclusions: Paeonol dose-dependently inhibited the excretion of the inflammatory factors TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM in a dose-dependent manner. Moreover, paenol treatment prevented the differentiation of P. gingivalis-induced BMM differentiation into osteoclasts.
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Objective: To investigate the effect of zoledronate on protein interaction between Ca(2+)/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation. Methods: Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis, and group B was treated with 1×10(-6) zoledronate for two days from day 2. Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMKⅡ and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc1 and TRAP in both groups. Osteoclast formation was also analyzed. Results: In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3±1.5, 8.7±2.1 and (5 034.4±775.4) μm(2) respevtively, which were significantly lower than those (37.7±5.7, 23.0±4.0 and [15 042.7±1 906.0] μm(2)) in group A ( P< 0.01). Co-IP and reverse Co-IP examination indicated that protein-binding between CaMKⅡ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A ( P< 0.01). The protein level of calmodulin and CaMKⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A ( P< 0.01). NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A ( P< 0.01), respectively. Conclusions: Zoledronate could significantly inhibit protein-binding between CaMKⅡ and calmodulin and down-regulate protein level of NFATc1 and TRAP.

Objective: Human gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts., Design: Two hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at -80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay., Results: The hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture., Conclusion: We concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factors (MCSF) in vitro., Methods: The bone marrow stem cells (BMSCs) of Rosa(-notch1) mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein (GFP) virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Deltal, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase (TRAP) were detected at the defined stage by reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was analyzed by TRAP assay., Results: The number of TRAP-positive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The mRNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hesl of the experimental group were significantly higher than those of the control group, whereas the TRAP mRNA expression of the experimental group was significantly lower than that of the control group (P<0.05)., Conclusion: Up-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.

Background and Objective: The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal tissues and may contribute to periodontitis progression. TNF-α stimulates formation and activity of osteoclasts, the cells that are recruited in periodontitis, that cause alveolar bone degradation and subsequent tooth loss. We previously showed that TNF-α is elevated in co-cultures of periodontal ligament fibroblast (PDLF) and peripheral blood mononuclear cells (PBMC). Hence, TNF-α could be a determining factor in osteoclast formation in these cultures, as osteoclasts are formed despite the fact that prototypical osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand is outnumbered at least 100-fold by its inhibitor osteoprotegerin in these cultures., Material and Methods: To assess the role of TNF-α in periodontitis-associated osteoclast formation in vitro, osteoclast formation was analyzed in the presence of the anti-TNF-α therapeutic agent infliximab in two culture systems: (i) PBMC in co-culture with PDLFs from controls and patients with periodontitis, or (ii) with PBMC only. PDLFs from control and patients with periodontitis were exposed to infliximab, PBMCs were added and the formation of osteoclast-like cells was assessed., Results: TNF-α was highest levels in supernatants at 7 d in co-cultures and declined at 14 and 21 d. TNF-α was undetectable in cultures that received infliximab. The formation and activity of osteoclasts in co-cultures was not affected by infliximab. In contrast, infliximab in cultures of only PBMC significantly reduced the formation of osteoclasts. This reduction was accompanied by a decreased number and size of cell clusters, a step that precedes the formation of osteoclasts. TNF-α was again undetectable in the supernatant of infliximab-treated cultures, but was detectable at similar levels in cell lysates of control and infliximab-treated PBMC cultures., Conclusion: Our study shows that the contribution of TNF-α to osteoclast formation is cell system dependent. It contributes to PBMC-induced osteoclast formation, possibly by establishing stronger cell-cell interactions that precede osteoclast formation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil's claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

Objective: To observe the osteoclast differentiation of Raw264.7 strain stably expressing enhanced green fluorescent protein (EGFP)., Methods: Raw264.7 cells were transfected with EGFP-Lifeact gene via retrovirus. The G3 cell clone was obtained by limited dilution technique which stably expressed EGFP under the fluorescence microscope. The morphology of G3 cells were observed. The effects of transfection on receptor activator of nuclear factor kappaB ligand (RANKL)--induced osteoclastogenesis and bone resorbing function of G3 cells were examined by tartrate resistant acid phosphatase (TRAP) staining, immunoblotting detection of cathepsin K and bone pit resorption assay. The real-time images of podosome dynamics were taken by laser confocal microscopy during osteoclast differentiation of G3 cells., Results: The Raw264.7 cells were successfully transfected with EGFP-Lifeact gene. The G3-EGFP cloned strain which could stably express EGFP even after 20 passages was constructed. There was no significant difference in morphology between G3-EGFP and wild Raw264.7 cells. The fusion rates of the transfection group and of the wild control group were (35±5)% and (39±5)%, respectively, which were not significantly different (P>0.05). The semi-quantitative ratio of cathepsin K/β-actin in the wild control group and in the transfection group was 0.83±0.07 and 1.02±0.08 (P>0.05), respectively. Bone pit results showed that the total area of the bone resorption was respectively 272,252±36,193 and 262,408±23,243 (P>0.05) and the number of the bone pits was respectively 320±51 and 339±55 (P>0.05). The photos of laser confocal microscopy showed the constant cell-cell fusion during osteoclast differentiation of G3-EGFP cells. In addition, the dynamic self-organized podosome initially assembled podosome clusts, then dynamic rings, finally formed the characteristic podosome belt pattern in mature osteoclasts., Conclusions: Enhanced green fluorescent protein high effectively expressed in Raw264.7. Biological character does not change after transfection.

Objective: To review the recent research progress of the regulatory mechanisms of osteoclast (OC) formation and functions., Methods: The related literature on OC formation, activation, and functions was reviewed, analyzed, and summarized., Results: Macrophage colony stimulating factor and receptor activator of nuclear factor-κB ligand are essential cytokines which regulate all aspects of OC formation and functions. In additional to mediating bone resorption, OCs also participate in regulation of osteoblast formation and immune responses., Conclusion: The researches on the regulation of OC formation and functions have further revealed the destruction mechanisms of various kinds of bone diseases, which will facilitate to find more suitable biological targets for clinical therapy.

The receptor activator of nuclear factor-κB ligand (RANKL) is a key factor in regulating osteoclastogenesis and in maintaining the survival of mature osteoclasts. We screened differentially expressed genes in RAW264.7 cells in response to RANKL and found osteoclast stimulatory transmembrane protein (OC-STAMP) as one of the RANKL-induced genes of interest. Recently, OC-STAMP has been identified as the RANKL-induced protein that promotes osteoclast differentiation, but the mechanism that regulates its expression is not understood. Therefore, the tissue distribution of OC-STAMP and the signaling pathways that regulate its expression were studied here. Similar to osteoclasts, OC-STAMP was expressed in most tissues, suggesting its involvement in the function of other tissues. Interestingly, OC-STAMP was downregulated by 17β-estradiol at high concentrations, suggesting the potential relationship between OC-STAMP and estrogen. Importantly, the knockdown of OC-STAMP at the transcript level resulted in the inhibition of multinucleated osteoclast formation and the decreased expression of genes including transcription factor (such as c-Jun), receptors (such as RANK and c-Fms), a signaling molecule (such as TRAF6), and a cell fusion-related molecule (such as meltrin-α), suggesting that the osteoclast differentiation needs the coordinated expression of OC-STAMP with several molecules required for transcription, signaling transduction, and cell fusion. Additionally, the treatment of its specific antibody inhibited the formation and bone resorptive activity of mature osteoclasts, suggesting its involvement in the function of mature osteoclasts. Furthermore, studies with pharmacological inhibitors suggested PKCβ or Akt might be the major signaling molecules to regulate the expression of OC-STAMP during osteoclastogenesis.

IL-1 is a potent cytokine that can induce bone erosion in inflammatory sites such as rheumatoid joint regions via activation of osteoclasts. Not only is IL-1 capable of activating osteoclasts, but it is also a key cytokine involved in the differentiation, multinucleation, and survival of osteoclasts. Herein, we show that IL-1 has the potential to drive osteoclast differentiation via a receptor activator of NF-kappaB ligand (RANKL)/RANK-independent mechanism. Although IL-1 has a synergistic effect on RANKL-induced osteoclast formation, IL-1 alone cannot induce osteoclast differentiation from osteoclast precursors (bone marrow-derived macrophages (BMMs)) due to a lack of IL-1 signaling potential in these cells. However, we demonstrate that overexpression of the IL-1RI receptor in BMMs or induction of IL-1RI by c-Fos overexpression enables IL-1 alone to induce the formation of authentic osteoclasts by a RANKL/RANK-independent mechanism. The expression of IL-1RI is up-regulated by RANKL via c-Fos and NFATc1. Furthermore, the addition of IL-1 to IL-1RI overexpressing BMMs (IL-1/IL-1RI) strongly activates NF-kappaB, JNK, p38, and ERK which is a hallmark gene activation profile of osteoclastogenesis. Interestingly, IL-1/IL-1RI does not induce expression of c-Fos or NFATc1 during osteoclast differentiation, although basal levels of c-Fos and NFATc1 seem to be required. Rather, IL-1/IL-1RI strongly activates MITF, which subsequently induces osteoclast-specific genes such as osteoclast-associated receptor and tartrate-resistant acid phosphatase. Together, these results reveal that IL-1 has the potential to induce osteoclast differentiation via activation of microphthalmia transcription factor under specific microenvironmental conditions.

Objective: To establish a culture method for a large amount of highly purified osteoclast-like cells in vitro. To investigate the gene expression of some osteoclast marker enzymes. To lay the foundation for the further study of the signal path on the differentiation and formation of osteoclast-like cells., Methods: The bone marrow mononuclear cells of rat were treated with 30 ng/mL macrophagecolony-stimulating factor (M-CSF) and 50 ng/mL receptor activator of NF-kappaB ligand (RANKL) and cultured for 6 days. After culturing, cells were evaluated by morphology observation, tartrate-resistant acid phosphatase (TRAP) staining, Giemsa staining, pit staining, and the gene expression of some osteoclast marker enzymes., Results: The TRAP-positive mononuclear cells were more frequently observed than the multinucleated cells and pit staining could be seen on the dentine slice. The transcription expression of TRAP, matrix metalloproteinase-9 (MMP-9), membrane-type1-matrix metalloproteinase (MT1-MMP) and cathepsin K were detected by RT-PCR., Conclusion: The cooperation of M-CSF and RANKL could induce a large amount of highly purified osteoclast-like cells formation in rat bone marrow culture. The typical characteristics of osteoclast-like cells were demonstrated and the enriched osteoclast-like cells expressed TRAP, MMP-9, MT1-MMP and cathepsin K.

Objective: CD14(+) monocyte cell lines can differentiate into an osteoclast (OC)-like lineage. However, the identification of human cell lines with stem cell characteristics, capable of differentiating into OCs, would provide a tool for the study of the molecular mechanisms regulating their commitment, differentiation, and function. Since the human acute myeloid leukemia cell line MUTZ-3 contains both CD34(+) stem cell and CD14(+) cell populations, we investigated the capacity of the stem/progenitor CD34(+) population to differentiate into functional OCs., Materials and Methods: Sorted MUTZ-3-CD34(+) and MUTZ-3-CD14(+) cells were cultured in presence of M-CSF, RANK-L, and TNF-alpha to generate OCs. Differentiation was evaluated by TRAP staining and RT-PCR, which assessed the expression of c-fms, RANK, MMP-9, CATK, TRAP, and CTR in -CD34(+)OC and -CD14(+)OC cells. Resorption pit formation was also evaluated. CD34, CD14, M-CSF-R, RANK, and CTR expression was assessed by FACS analysis., Results: MUTZ-3-CD34(+) differentiated into OCs, displaying the full range of differentiation markers; MMP-9, CATK, TRAP, and RANK mRNA were detected from day 3 of culture, whereas CTR from day 12. Stimulated MUTZ-3-CD34(+) generated functional osteoclasts that formed extensive resorption lacunae on both mineralized surface and bone slices. Surprisingly, in both sorted populations we identified a population M-CSF-R(+)/RANK(+) that at the same time co-expressed CD14 and CD34., Conclusions: These findings demonstrate that MUTZ-3 cells constitute an invaluable model to study the expression pattern in different developmental stages of commitment and differentiation. Importantly, the data indicate that the CD14(+)CD34(+)M-CSF-R(+)RANK(+) population represents an intermediate stage of differentiation from CD34 precursors and monocytes to osteoclast.

Osteoclasts are cells of monocyte/macrophage origin that degrade bone matrix. Receptor activator of NF-kappaB ligand (RANKL) induces osteoclast differentiation in the presence of macrophage colony-stimulating factor. RANKL activates the tumor necrosis factor receptor-associated factor 6, c-Fos, and calcium signaling pathways, all of which are indispensable for the induction and activation of nuclear factor of activated T cells (NFAT) c1. NFATc1 is the master transcription factor for osteoclast differentiation, which regulates many osteoclast-specific genes. Multiple immunoglobulin-like receptors associated with immunoreceptor tyrosine-based activation motif (ITAM)-harboring adapters, Fc receptor common chi subunit (FcRgamma), and DNAX-activating protein (DAP) 12 mediate costimulatory signals for RANK, which activate calcium signaling through phospholipase Cgamma (PLCgamma). In addition to calcineurin-NFATc1, calcium signaling activates the CaMK-CREB (calcium/calmodulin activated kinase-cyclic AMP-response element binding protein) pathway, which also plays a critical role in osteoclastogenesis. This review summarizes recent advances in the study of signaling mechanisms of osteoclast differentiation.

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved., (Copyright 2006 Wiley-Liss, Inc.)

Wear debris-induced vascularized granulomatous periprosthetic tissue may augment the progress of prosthetic loosening, a major clinical problem after total joint replacement. The purpose of this study is to investigate the association of ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced inflammatory osteoclastogenesis and expression of RANK/RANKL and VEGF/VEGF receptors (Flt-1 and Flk-1) using a mouse osteolysis model. UHMWPE particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. Mice were injected with either recombinant VEGF or VEGF inhibitor (VEGF R2/F(c) Chimera). Mice without drug treatment, as well as mice injected with saline alone were included. Each group contains 10 mice. Pouch tissues were harvested 2 weeks after bone implantation for histological and molecular analysis. UHMWPE stimulation significantly increased VEGF gene expression, and exerted a lower enhancement effect on the gene expression of Flt-1 and Flk-1. UHMWPE-stimulated VEGF production was markedly reduced by VEGF inhibitor treatment. Immunofluorescent staining indicated that pouch tissue macrophages were the main source of both VEGF and Flt-1 production. A positive association was observed between tissue inflammation and the levels of VEGF and Flt-1 gene transcripts. Both RANK and RANKL gene transcripts were significantly increased by UHMWPE stimulation, which was subsequently reduced by VEGF inhibitor treatment (p<0.05). VEGF treatment increased TRAP(+) cells in pouches either with or without UHMWPE particle stimulation, and VEGF inhibitor treatment caused a significant reduction in the number of TRAP(+) cells in UHMWPE-containing pouches. This study suggests that VEGF has a role in the regulation of RANK/RANKL-mediated osteoclastogenesis, and warrant future investigations to elucidate the role of VEGF signaling in the pathogenesis of prosthetic loosening.

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-kappaB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-gamma, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-kappaB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-alpha in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.

Multicentric reticulohistiocytosis (MR) is a systemic disease of unknown cause characterized by the presence of a heavy macrophage infiltrate in skin and synovial tissues and the development of an erosive polyarthritis. The synovial fluid in MR is known to contain numerous mononuclear cells. In this study, we have determined whether these cells contribute to joint destruction in MR by differentiating them into osteoclasts. Synovial fluid mononuclear cells were isolated from the knee joint of a 44-year-old male with MR. These cells were cultured with various combinations of macrophage-colony stimulating factor, receptor activator for nuclear factor kappaB ligand (RANKL), tumor necrosis factor alpha, interleukin-1alpha, osteoprotegerin, and zoledronate. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase, vitronectin receptor, and the extent of lacunar resorption. Most MR synovial fluid mononuclear cells expressed a macrophage phenotype (CD14(+), CD68(+), HLA-DR(+), CD11b(+)). Extensive osteoclast formation and lacunar resorption were noted in macrophage-colony stimulating factor/RANKL-treated cell cultures. MR synovial fluid contained increased tumor necrosis factor alpha and decreased osteoprotegerin compared with osteoarthritis synovial fluid. Lacunar resorption was inhibited in cultures containing zoledronate. Pamidronate treatment of the patient also reduced the number of synovial fluid macrophages and resulted in less osteoclast formation and lacunar resorption. MR synovial fluid contains numerous macrophages that are capable of differentiating into osteoclasts by the RANKL signaling pathway. Inhibitors of osteoclast formation and resorption activity may be of use in preventing the severe joint destruction that commonly occurs in MR.

Dendritic cells (DC) are innate immune effectors and are critically involved in regulating T cell immunity. Osteoclasts (OC) are bone-resorbing cells derived from the monocyte/macrophage lineage in response to receptor activator of NF-kappaB ligand (RANKL). DC and T cells form aggregates in the inflammatory infiltrates at active disease sites in human and in experimental rheumatoid arthritis and periodontitis. We investigated whether DC interactions with T cells in the bone environment can support the development of functional OC. In the present study, we demonstrate that upon proper activation by microbial or protein Ags (namely Actinobacillus actinomycetemcomitans, bovine insulin, and outer membrane protein-1) and during immune interactions with CD4+ T cells in vitro, murine BM-derived and splenic CD11c+ DC (CD11b- F4/80- Ly-6C- CD31-) develop into TRAP+ CT-R+ cathepsin-k+ functional OC in a RANKL/RANK-dependent manner. Rescue and blocking experiments using CD11c+ DC derived from Csf-1(-/-) op/op mice show that M-CSF is required "before" developing such osteoclastogenic potential upstream of RANKL/RANK signaling, suggesting that immature CD11c+ DC can indeed act like OC precursors. In addition, these CD11c+ DC-derived OC are capable of inducing bone loss after adoptive transfer in vivo. These data suggest a direct contribution of DC during immune interactions with CD4+ T cells to inflammation-induced osteoclastogenesis. Therefore, our findings not only provide further evidence for DC plasticity, but also extend the current paradigm of osteoimmunology.

Unlabelled: RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool., Introduction: The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs., Materials and Methods: Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions., Results: RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells., Conclusions: These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.

Unlabelled: We showed that IGF-I deficiency impaired osteoclastogenesis directly and/or indirectly by altering the interaction between stromal/osteoblastic cells and osteoclast precursors, reducing RANKL and M-CSF production. These changes lead to impaired bone resorption, resulting in high BV/TV in IGF-I null mice., Introduction: Although IGF-I has been clearly identified as an important growth factor in regulating osteoblast function, information regarding its role in osteoclastogenesis is limited. Our study was designed to analyze the role of IGF-I in modulating osteoclastogenesis using IGF-I knockout mice (IGF-I(-/-))., Materials and Methods: Trabecular bone volume (BV/TV), osteoclast number, and morphology of IGF-I(-/-) or wildtype mice (IGF-I(+/+)) were evaluated in vivo by histological analysis. Osteoclast precursors from these mice were cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) or co-cultured with stromal/osteoblastic cells from either genotype. Osteoclast formation was assessed by measuring the number of multinucleated TRACP+ cells and pit formation. The mRNA levels of osteoclast regulation markers were determined by quantitative RT-PCR., Results: In vivo, IGF-I(-/-) mice have higher BV/TV and fewer (76% of IGF-I(+/+)) and smaller osteoclasts with fewer nuclei. In vitro, in the presence of RANKL and M-CSF, osteoclast number (55% of IGF-I(+/+)) and resorptive area (30% of IGF-I(+/+)) in osteoclast precursor cultures from IGF-I(-/-) mice were significantly fewer and smaller than that from the IGF-I(+/+) mice. IGF-I (10 ng/ml) increased the size, number (2.6-fold), and function (resorptive area, 2.7-fold) of osteoclasts in cultures from IGF-I(+/+) mice, with weaker stimulation in cultures from IGF-I(-/-) mice. In co-cultures of IGF-I(-/-) osteoblasts with IGF-I(+/+) osteoclast precursors, or IGF-I(+/+) osteoblasts with IGF-I(-/-) osteoclast precursors, the number of osteoclasts formed was only 11% and 48%, respectively, of that from co-cultures of IGF-I(+/+) osteoblasts and IGF-I(+/+) osteoclast precursors. In the long bones from IGF-I(-/-) mice, mRNA levels of RANKL, RANK, M-CSF, and c-fms were 55%, 33%, 60%, and 35% of that from IGF-I(+/+) mice, respectively., Conclusions: Our results indicate that IGF-I regulates osteoclastogenesis by promoting their differentiation. IGF-I is required for maintaining the normal interaction between the osteoblast and osteoclast to support osteoclastogenesis through its regulation of RANKL and RANK expression.

Unlabelled: RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology., Introduction: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis., Materials and Methods: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest., Results: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts., Conclusions: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis, periodontitis, and osteoporosis. Inflammation-induced bone loss of this sort results from increased numbers of bone-resorbing osteoclasts. Numerous studies have indicated that conjugated linoleic acid (CLA) positively influences calcium and bone metabolism. Gene-deletion studies have shown that receptor activator of nuclear factor-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. In this report, we examine the ability of CLA to suppress RANKL signaling and osteoclastogenesis in RAW264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated nuclear factor-kappaB (NF-kappaB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-kappaB activation, including phosphorylation of I-kappaBalpha, degradation of I-kappaBalpha, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-alpha production and osteoclast differentiation, including osteoclast-specific genes such as tartrate-resistant acid phosphatase, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9 expression and osteoclast-specific transcription factors such as c-Fos, nuclear factor of activated T-cells expression, and bone resorption pit formation. CLA also inhibited RANKL-induced activation of mitogen-activated protein kinase p38 but had little effect on c-Jun N-terminal kinase activation. Collectively, these data demonstrate for the first time that CLA inhibits osteoclastogenesis by modulating RANKL signaling. Thus, CLA may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption by excessive osteoclastogenesis.

The effect of beta-cryptoxanthin, a kind of carotenoid, on osteoclastic cells in mouse marrow culture system in vitro was investigated. The macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages were cultured in the presence of M-CSF (10 ng/ml) and receptor activator of NF-kappaB ligand (RANKL; 25 ng/ml) for 4 days. The osteoclastic cells formed were further cultured in medium containing either vehicle or beta-cryptoxanthin (10(-8)-10(-6) M) with or without M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 24-72 h. Osteoclastic cells were significantly decreased with culture of beta-cryptoxanthin (10(-7) or 10(-6) M) with or without M-CSF and RANKL for 24, 48, or 72 h. beta-Cryptoxanthin (10(-8) M)-induced decrease in osteoclastic cells were significantly inhibited in the presence of caspase-3 inhibitor (10(-8) or 10(-7) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent cells cultured with beta-cryptoxanthin (10(-7) or 10(-6) M) for 24 or 48 h, indicating that the carotenoid induces apoptotic cell death. Apoptosis-related gene expression was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with beta-cryptoxanthin (10(-7) or 10(-6) M) for 24 or 48 h caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL, while Bcl-2 and Apaf-2 mRNA expressions were significantly increased with culture of beta-cryptoxanthin (10(-7) or 10(-6) M) without M-CSF and RANKL for 24 or 48 h. Akt-1 mRNA expression was not significantly changed with culture of the carotenoid (10(-7) or 10(-6) M) for 24 or 48 h. Moreover, tartrate-resistant acid phosphatase (TRACP) activity, or TRACP and cathepsin K mRNA expressions were significantly decreased with culture of beta-cryptoxanthin (10(-6) M) in the presence or absence of M-CSF and RANKL for 48 h. This study demonstrates that beta-cryptoxanthin has stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function., ((c) 2006 Wiley-Liss, Inc.)

Background and Objective: Lipopolysaccharide is thought to be a major virulence factor of pathogens associated with periodontal diseases and is believed to stimulate bone resorption in vivo. Although Prevotella nigrescens has been implicated in periodontitis, its role in osteoclastogenesis has not been reported. In this study, we investigated the effects of lipopolysaccharide from P. nigrescens on the formation of osteoclasts and the production of cytokines related to osteoclast differentiation., Material and Methods: Mouse bone marrow mononuclear cells were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL), with or without lipopolysaccharide. Bone marrow mononuclear cells were also cocultured with calvarial osteoblastic cells in the presence or absence of lipopolysaccharide. Osteoclast formation was determined by tartrate-resistant acid phosphatase cytochemistry. The production of osteoprotegerin (OPG), M-CSF, tumor necrosis factor alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and prostaglandin E2 (PGE2) was determined by enzyme-linked immunosorbent assay (ELISA)., Results: P. nigrescens lipopolysaccharide inhibited osteoclast differentiation from bone marrow mononuclear cells cultured in the presence of M-CSF and RANKL. However, in the coculture system, P. nigrescens lipopolysaccharide stimulated osteoclastogenesis. Notably, P. nigrescens lipopolysaccharide decreased OPG production but increased TGF-beta secretion. In addition, treatment with P. nigrescens lipopolysaccharide increased PGE2 production during the late stage of the culture period. There was no difference in M-CSF and TNF-alpha production., Conclusion: These results demonstrate that P. nigrescens lipopolysaccharide stimulates osteoclastogenesis in the coculture system by decreasing the production of OPG and increasing the production of TGF-beta and PGE2. Through the mechanisms involving these factors, P. nigrescens lipopolysaccharide may cause alveolar bone resorption in periodontal diseases.

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Background and Objective: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7., Material and Methods: Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD., Results: Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL., Conclusion: Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.

Lipopolysaccharide (LPS) in the outer layers of Gram-negative bacteria plays an important role in initiating and sustaining periapical lesions. To understand the mechanisms of osteoclastic bone resorption in periapical lesions induced by LPS, we stimulated osteoclast precursors, RAW 264.7 cells with LPS. LPS stimulated osteoclastogenesis when osteoclast precursors were primed with activator for NF-kappaB ligand (RANKL) as little as 24 h. By employing real-time PCR analysis, we have confirmed that osteoclast-like cells stimulated by LPS express high level of osteoclast-specific gene markers such as TRAP, cathepsin K, and calcitonin receptor. These results suggest that bone-resportive action by LPS is partially independent of RANKL.

Paget's disease of bone (PDB) is a debilitating bone disorder characterized by giant osteoclasts, enhanced bone destruction, and irregular bone formation. Recently, mutations in SQSTM1 (also known as p62) have been detected in PDB sufferers, with all mutations resulting in either loss of function or truncation/deletion of the ubiquitin binding-associated (UBA) domain. We hypothesized that mutation in the p62 gene resulting in either deletion or premature termination of the UBA domain accounts for the elevated osteoclastic formation and bone resorption associated with PDB. Remarkably, overexpression of the p62 UBA domain deletion mutant (p62DeltaUBA) significantly enhanced osteoclastogenesis in vitro compared to cells expressing either wild-type p62 (p62WT) or a control vector in a RAW264.7 osteoclastogenic system. Overexpression of p62DeltaUBA potentiated the formation of abnormally large multinucleated osteoclasts and resorption of bone, reminiscent of PDB. Consistent with the enhancement of osteoclastogenesis, overexpression of p62DeltaUBA potentiated receptor activator of nuclear factor-kappaB ligand-induced activation of nuclear factor-kappaB, NFAT, and ERK phosphorylation. Furthermore, as determined by confocal microscopy, deletion of the p62 UBA domain impaired the association of p62 with TRAF6 in the proteasomal compartment. These results suggest that the UBA domain encodes essential regulatory elements required for receptor activator of nuclear factor-kappaB ligand-induced osteoclast formation and bone resorption that may be directly associated with the progression of PDB.

Malignant infantile osteopetrosis is a rare and lethal disease characterized by the absence of bone resorption due to inactive osteoclasts (OCLs). Among the murine models of osteopetrosis, the Tcirg1oc/oc mouse is the most resembling to the human pathology. In the majority of patients as in Tcirg1oc/oc mouse, the gene involved is the Tcirg1 gene, encoding the a3 subunit of the vacuolar proton pump. However, to date, no osteoclastic cell lines from osteopetrotic mice are available to facilitate the study of either OCL differentiation in osteopetrosis or the factors involved in the control of Tcirg1 gene expression. Heterozygotes Tcirg1+/oc mice were crossed with p53+/- mice to obtain homozygotes p53-/-Tcirg1oc/oc and p53-/-Tcirg1+/+ animals. The p53-/-Tcirg1oc/oc mice display the same bone and hematological phenotype as the original Tcirg1oc/oc mice. From the bone marrow of these mice, we have derived cell lines named POC-MGoc/oc and POC-MG+/+. These cell lines express standard osteoclastogenic markers and differentiate into OCLs in the presence of RANK-L and M-CSF. Furthermore, both cell lines can be transduced by a lentiviral vector with a high efficiency and without alteration of their OCL differentiation potential. Therefore, these cell lines provide valuable new tools to study the differentiation and function of osteoclasts in normal and resorption defective conditions.

Objectives: The objective of this study was to investigate how the expression of the RANK-RANKL-OPG system mediates the formation and differentiation of osteoclasts and causes bone resorption in cholesteatoma., Methods: An immunohistochemical analysis was carried out in 22 cholesteatoma tissues obtained during middle ear surgery and 15 normal postauricular skin tissues to examine the expression of RANKL and OPG., Results: All 22 cases of cholesteatoma and the 15 cases of normal postauricular skin expressed RANKL and OPG. The count and rate of RANKL-positive cells in cholesteatoma was significantly higher than in normal postauricular skin. The count and rate of OPG-positive cells in normal postauricular skin was significantly higher than in cholesteatoma. The ratio of the positive expression rates of RANKL and OPG in cholesteatoma was statistically higher than in normal postauricular skin., Conclusions: We provide evidence suggesting that RANKL, which activates osteoclasts, plays a significant role in the mechanism of bone destruction in cholesteatoma, and that the ratio of RANKL to OPG may be a reliable indicator of bone destruction in cholesteatoma.

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.

Unlabelled: In this study, overexpression of noggin, a BMP antagonist, in developing bone caused significantly decreased osteoclast number as well as bone formation rate, resulting in increased bone mass with immature bone quality. BMP signaling plays important roles in normal bone development and regulation of bone resorption., Introduction: Bone morphogenetic proteins (BMPs) act on various types of cells. Although involvement of BMP signals in osteoblast differentiation has been studied extensively, the effects of BMPs on osteoclasts have not been widely researched. Consequently, the net effects of BMPs on bone remain unclear. The purpose of this study was to delineate more fully the role of BMPs in skeletal biology., Materials and Methods: We generated transgenic mice that express BMP4 or noggin in bone under the control of the 2.3-kb alpha1(I) collagen chain gene (Col1a1) promoter, and analyzed their bone phenotype. We also analyzed bone of transgenic mice expressing BMP4 specifically in cartilage., Results: Mice overexpressing BMP4 in bone developed severe osteopenia with increased osteoclast number. Mice overexpressing noggin, a BMP antagonist, in bone showed increased bone volume associated with decreased bone formation rate and decreased osteoclast number. The noggin-transgenic tibias exhibited reduced periosteal bone formation and reduced resorption of immature bone in marrow spaces, associated with frequent fractures at the diaphysis. Co-culture of primary osteoblasts prepared from noggin-transgenic calvariae and wildtype spleen cells resulted in poor osteoclast formation, which was rescued by addition of recombinant BMP2, suggesting that noggin inhibits osteoclast formation by attenuating BMP activities in noggin-transgenic mice. The expression levels of Rankl were not decreased in primary osteoblasts from noggin transgenic mice. Immunoblot analysis showed increased phosphorylation of Smad1/5/8 in osteoclast precursor cells after 20-minute treatment with BMPs, suggesting that these cells are stimulated by BMPs. Mice overexpressing BMP4 in cartilage had enlarged bones containing thick trabeculae, possibly because of expansion of cartilage anlagen., Conclusions: Overexpression of noggin in bone revealed that BMP signals regulate bone development through stimulation of osteoblasts and osteoclasts.

Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-kappaB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed beta3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-kappaB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.

Unlabelled: Asymptomatic gene carriers and clinically affected ADO2 subjects have the same ClCN7 mutation. We examined osteoclastic bone resorption in vitro as well as osteoclast formation, several markers, acid secretion, and cytoskeletal structure. We found that ADO2 expression results from osteoclast specific properties., Introduction: Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder that results from heterozygous mutations in the ClCN7 gene. However, of those individuals with a ClCN7 mutation, one third are asymptomatic gene carriers who have no clinical, biochemical, or radiological manifestations. Disease severity in the remaining two thirds is highly variable., Materials and Methods: Human peripheral blood mononuclear cells were isolated and differentiated into osteoclasts by stimulation with hRANKL and human macrophage-colony stimulating factor (hM-CSF). Study subjects were clinically affected subjects, unaffected gene carriers, and normal controls (n = 6 in each group). Pit formation, TRACP staining, RANKL dose response, osteoclast markers, acid secretion, F-actin ring, and integrin alpha(v)beta3 expression and co-localization were studied., Results: Osteoclasts from clinically affected subjects had severely attenuated bone resorption compared with those from normal controls. However, osteoclasts from unaffected gene carriers displayed similar bone resorption to those from normal controls. In addition, the resorption lacunae from both unaffected gene carriers and normal controls appeared much earlier and spread much more rapidly than those from clinically affected subjects. As time progressed, the distinction between clinically affected subjects and the other two groups increased. No significant difference was found in acidic secretion or osteoclast formation between the three groups. Osteoclast cytoskeletal organization showed no difference between the three groups but there was low cellular motility in clinically affected subjects., Conclusions: Osteoclasts from the unaffected gene carriers, in contrast to those from the clinically affected subjects, functioned normally in cell culture. This finding supports the hypothesis that intrinsic osteoclast factors determine disease expression in ADO2. Further understanding of this mechanism is likely to lead to the development of new approaches to the treatment of clinically affected patients.

In the present study, dexamethasone treatment of neonatal mouse calvarial bones increased mRNA expression of tartrate-resistant acid phosphatase, calcitonin receptor (CTR), cathepsin K, carbonic anhydrase II, osteoprotegerin (OPG), and receptor activator of nuclear factor-kappaB (RANK) as well as mRNA and protein expression of RANK ligand (RANKL). The increase in OPG mRNA noted with dexamethasone was in contrast to 1,25(OH)(2)-vitamin D3 (D3) treatment, which decreased OPG expression. Stimulation of (45)Ca release by dexamethasone and hydrocortisone in calvariae was blocked by OPG. Stimulation of RANKL, RANK, OPG, and CTR mRNA expression by dexamethasone in calvariae was blocked by the glucocorticoid receptor antagonist RU 38,486. Greater than additive potentiations of CTR mRNA and RANKL mRNA and protein were observed when D3 and dexamethasone were combined. Vitamin D receptor mRNA was increased by dexamethasone and D3, whereas glucocorticoid receptor (GR) mRNA was decreased by dexamethasone and unaffected by D3. No synergistic interaction between dexamethasone and D3 on either vitamin D receptor or GR mRNA expression was noted. The data demonstrate that dexamethasone-induced bone resorption in calvarial bones is associated with increased differentiation of osteoclasts and regulation of the RANKL-RANK-OPG system. The increase in OPG expression and the decrease of GR expression noted with dexamethasone offer an explanation for why bone breakdown in mouse calvariae treated with glucocorticoids is less than that caused by resorptive agents like D3. The synergistic stimulation of RANKL by dexamethasone and D3 offers an explanation of how glucocorticoids and D3 interact to potentiate bone resorption.

Bone is a dynamic tissue that is regulated by the activity of bone-resorbing osteoclasts and bone-forming osteoblasts. Excessive osteoclast formation causes diseases such as osteoporosis and rheumatoid arthritis. Natural substances may be useful as therapeutic drugs to prevent many diseases in humans because they avoid the many side effects of treatment with chemical compounds. Here we show that tanshinone IIA isolated from Salvia miltiorrhiza Bunge inhibits the receptor activator of NF-kappaB ligand (RANKL)-mediated osteoclast differentiation of osteoclast precursors. Tanshinone IIA suppressed the expression levels of c-Fos and NFATc1 induced by RANKL. However, retrovirus-mediated overexpression of c-Fos induced the expression of NFATc1 despite the presence of tanshinone IIA and reversed the inhibitory effect of tanshinone IIA on osteoclast differentiation. Also, the introduction of osteoclast precursors with the NFATc1 retrovirus led to osteoclast differentiation in the presence of tanshinone IIA. Our results suggest that tanshinone IIA may have a role as a therapeutic drug in the treatment of bone disease such as osteoporosis.

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.

Osteoclast differentiation is tightly regulated by osteoblasts. Osteoblasts express two cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL), essential for osteoclast differentiation. Osteoclast precursors firstly differentiate into mononuclear osteoclasts in response to M-CSF and RANKL. Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor, which is involved in the determination of osteoclast differentiation. The mononuclear osteoclast then fuses each other to form the multinucleated cell. Dendritic cell-specific transmembrane protein (DC-STAMP) plays an important role in the fusion of mononuclear osteoclasts. Osteoclasts recognize bone and form ruffled borders and clear zones on the bone surface (polarization). Polarizing osteoclasts construct active vascular transportation systems to resorb bone efficiently. RANK-TRAF6 (TNF receptor-associated factor 6) -mediated signals key roles in both osteoclast differentiation and function. In this article, I will review the recent findings on osteoclast differentiation and function.

Objective: To determine whether CD40 ligation of rheumatoid arthritis synovial fibroblasts (RASFs) is able to induce RANKL expression and osteoclastogenesis in RASFs, and to identify its mechanism of action in patients with RA., Methods: CD40 of RASFs was ligated with CD40 ligand (CD40L)-transfected L cells or activated T cells. The formation of osteoclasts in cocultures of CD40-ligated RASFs and T lymphocyte-depleted peripheral blood mononuclear cells was evaluated by tartrate-resistant acid phosphatase staining, detection of calcitonin receptor, and resorption pit formation assay. The expression of NF-kappaB, IkappaB alpha, ERK-1/2, phospho-ERK-1/2, p38, phospho-p38, and RANKL was examined by immunoblotting and/or semiquantitative reverse transcription-polymerase chain reaction., Results: CD40 ligation of RASFs by CD40L-transfected L cells or activated T cells induced RANKL expression and enhanced osteoclastogenesis. CD40 ligation of RASFs also induced activation of ERK-1/2, p38 MAPK, and NF-kappaB and up-regulation of CD40 ligation-induced RANKL expression, whereas osteoclastogenesis was reduced in RASFs transfected with a dominant-negative mutant of IkappaB alpha or by an NF-kappaB inhibitor. However, specific inhibitors of MAPK/ERK-1/2 and p38 MAPK partially blocked the induction of RANKL expression and osteoclastogenesis. Monoclonal antibodies against interleukin-1 and tumor necrosis factor alpha partially inhibited CD40 ligation-mediated osteoclastogenesis., Conclusion: These results indicate that CD40 ligation of RASFs induces RANKL expression mainly via NF-kappaB activation and also results in enhanced osteoclast formation, both of which might play important roles in bone and cartilage destruction in RA. Inhibition of the CD40-CD40L interaction is a potential strategy for the prevention of bone damage in RA.

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTPepsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

Mitogen-activated protein kinase (MAPK) pathways are implicated in joint destruction in rheumatoid arthritis (RA) by modulating the production and functions of inflammatory cytokines. Although p38 MAPK (p38) participates in signaling cascades leading to osteolysis in arthritis, the mechanisms of its action in this process remain incompletely understood. Here, we found that the osteoclast (Ocl) precursors expressed p38alpha, but not p38beta, p38delta, and p38gamma isoforms. Treatment of these cells with receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) resulted in p38 activation. Importantly, Ocl development induced by RANKL or RANKL and tumor necrosis factor (TNF)-alpha was blocked with the novel p38 inhibitor 4-(3-(4-chlorophenyl)-5-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)pyrimidine (SC-409). To validate in vitro data, p38 role was further investigated in streptococcal cell wall (SCW)-induced arthritis in rats. We found that SCW-induced joint swelling and bone destruction were attenuated by SC-409. Mechanistically, the data show that SCW-stimulated DNA binding activity of the transcription factor myocyte-enhancing factor 2 C, which is downstream of p38, was inhibited by SC-409. In addition, SC-409 inhibited SCW-stimulated expression of numerous factors, including TNF-alpha, interleukin-1beta, and RANKL. Although c-Jun NH2-terminal kinase and NF-kappaB pathways were activated in vitro by RANKL and in vivo by SCW, SC-409 had no significant effect on these pathways. In conclusion, our data show that p38 modulates the production and signaling of cytokines, thus providing a mechanism of the bone-sparing effect of SC-409 in rat arthritis. These data present SC-409 as a novel potent p38 inhibitor and suggest that p38-based therapies may be beneficial in preventing bone loss associated with RA.

Unlabelled: We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called "lipid raft," and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system., Introduction: Osteoclasts are bone-resorbing multinuclear polykaryons that are essential for bone remodeling and are formed through cell fusion of mononuclear macrophage/monocyte lineage precursors. Although osteoclastogenesis has been shown to be critically regulated by the RANK/RANKL system, the mechanism how precursor cells fuse with each other remains unclear. We examined the function of CD9, a member of tetraspanin superfamily, which has previously been shown to form macromolecular membrane microdomains and to regulate cell-cell fusion in various cell types., Materials and Methods: We used RAW264.7, a macrophage/monocyte lineage cell line, which can differentiate into osteoclast-like polykaryons on the application of RANKL. Expression and distribution of CD9 was assessed by Western blotting, fluorescence-assorted cell sorting (FACS) and immunohistochemistry with light and electron microscopy. A specific neutralizing antibody and RNA interference were used to inhibit the function of CD9, and green fluorescent protein (GFP)-CD9 was exogenously expressed to enhance the effect of CD9. The distribution of CD9 in lipid microdomain was examined by biochemical (sucrose density gradient) isolation and imaging technique., Results: CD9 is expressed on cell surfaces of RAW264.7, which is enhanced by RANKL. Targeted inhibition of CD9 decreases the number of osteoclast-like cells. On the other hand, overexpression of CD9 promotes spontaneous cell fusion even in the absence of RANKL. CD9 is localized in detergent-insoluble "lipid raft" microdomain in RANKL stimulation, and disruption of lipid rafts markedly reduces the formation of osteoclast-like polykaryons. Immunohistochemical studies of bone tissues revealed the expression of CD9 in osteoclasts in vivo., Conclusions: These data suggest that function of tetraspanin CD9 and its expression in lipid rafts are crucial for cell fusion during osteoclastogenesis.

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.