IFT80 negatively regulates osteoclast differentiation via association with Cbl-b to disrupt TRAF6 stabilization and activation.

Excess bone loss due to increased osteoclastogenesis is a significant clinical problem. Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation. The role of IFT80, an IFT complex B protein, in osteoclasts (OCs) is completely unknown. Here, we demonstrate that deletion of IFT80 in the myeloid lineage led to increased OC formation and activity accompanied by severe bone loss in mice. IFT80 regulated OC formation by associating with Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) to promote protein stabilization and proteasomal degradation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). IFT80 knockdown resulted in increased ubiquitination of Cbl-b and higher TRAF6 levels, thereby hyperactivating the receptor activator of nuclear factor-κß (NF-κß) ligand (RANKL) signaling axis and increased OC formation. Ectopic overexpression of IFT80 rescued osteolysis in a calvarial model of bone loss. We have thus identified a negative function of IFT80 in OCs.

Multifaceted MRGPRX2: New insight into the role of mast cells in health and disease.

Cutaneous mast cells (MCs) express Mas-related G protein-coupled receptor-X2 (MRGPRX2; mouse ortholog MrgprB2), which is activated by an ever-increasing number of cationic ligands. Antimicrobial host defense peptides (HDPs) generated by keratinocytes contribute to host defense likely by 2 mechanisms, one involving direct killing of microbes and the other via MC activation through MRGPRX2. However, its inappropriate activation may cause pseudoallergy and likely contribute to the pathogenesis of rosacea, atopic dermatitis, allergic contact dermatitis, urticaria, and mastocytosis. Gain- and loss-of-function missense single nucleotide polymorphisms in MRGPRX2 have been identified. The ability of certain ligands to serve as balanced or G protein-biased agonists has been defined. Small-molecule HDP mimetics that display both direct antimicrobial activity and activate MCs via MRGPRX2 have been developed. In addition, antibodies and reagents that modulate MRGPRX2 expression and signaling have been generated. In this article, we provide a comprehensive update on MrgprB2 and MRGPRX2 biology. We propose that harnessing MRGPRX2's host defense function by small-molecule HDP mimetics may provide a novel approach for the treatment of antibiotic-resistant cutaneous infections. In contrast, MRGPRX2-specific antibodies and inhibitors could be used for the modulation of allergic and inflammatory diseases that are mediated via this receptor.

Estrogen deficiency attenuates fluid flow-induced [Ca2+]i oscillations and mechanoresponsiveness of MLO-Y4 osteocytes.

It has been proposed that estrogen regulates the mechanosensitivity of osteocytes; however, the effects of estrogen deficiency that arises during postmenopausal osteoporosis on mechanical stimulation-induced calcium signaling in osteocytes remain elusive. Here, we pretreated MLO-Y4 osteocytes with 10 nM E2 for 2, 3 and 5 d, then simulated postmenopausal conditions either by estrogen withdrawal (EW) from culture medium, or by inhibiting the estrogen receptor by using fulvestrant and estrogen (FE; ICI 182,780) in vitro We investigated [Ca2+]i oscillations and mechanobiologic responses of osteocytes (EW and FE) that were exposed to oscillatory fluid flow (OFF; 1 Pa, 0.5 Hz). We demonstrated that estrogen treatment enhanced OFF-induced [Ca2+]i oscillations and that this effect was abrogated both by FE and EW. Moreover, osteocytes in both estrogen-depleted groups (EW and FE) had reduced levels of NO and prostaglandin E2 release, down-regulated dentin matrix protein-1, sclerostin, osteopontin, osteocalcin, and alkaline phosphatase mRNA expression, and reduced F-actin fiber formation after OFF stimulation compared with estrogen-treated cells. We propose a link between estrogen deficiency and alterations in [Ca2+]i-mediated mechanosensitivity of osteocytes, which ultimately alter osteocyte function and differentiation.-Deepak, V., Kayastha, P., McNamara, L. M. Estrogen deficiency attenuates fluid flow-induced [Ca2+]i oscillations and mechanoresponsiveness of MLO-Y4 osteocytes.

Carvacrol Inhibits Osteoclastogenesis and Negatively Regulates the Survival of Mature Osteoclasts.

Bone is a dynamic tissue that undergoes continuous remodeling coupled with the action of osteoblasts and osteoclasts. Osteoclast activity is elevated during osteoporosis and periodontitis resulting in excessive loss of trabecular and alveolar bone. Osteoclasts are formed in an inflammatory response to cytokine production receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL) and bacterial challenge lipopolysaccharide (LPS). Carvacrol, a monoterpenic phenol present in Origanum vulgare and Thymus vulgaris, is a natural compound with known medicinal properties. We investigated the effects of carvacrol on osteoclast formation induced by RANKL and LPS. Carvacrol suppressed RANKL-induced formation of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells in RAW264.7 macrophages and human CD14(+) monocytes. Furthermore, carvacrol inhibited LPS-induced osteoclast formation in RAW264.7 macrophages. Investigation of the underlying molecular mechanisms revealed that carvacrol downregulated RANKL-induced NF-κB activation in a dose-dependent manner. Furthermore, the suppression of NF-κB activation correlated with inhibition of inhibitor of kappaB (IκB) kinase (IKK) activation and attenuation of inhibitor of NF-κB (IκBa) degradation. Carvacrol potentiated apoptosis in mature osteoclasts by caspase-3 activation and DNA fragmentation. Moreover, carvacrol did not affect the viability of proliferating MC3T3-E1 osteoblast-like cells. Collectively, these results demonstrate that carvacrol mitigates osteoclastogenesis by impairing the NF-κB pathway and induction of apoptosis in mature osteoclasts.

Constitutive L-Sox5 overexpression delays differentiation of ATDC5 cells into chondrocytes and correlates with reduced expression of differentiation markers.

L-Sox5 is a member of sex-determining region Y-type high mobility group box (SOX) family of transcription factors. We assessed the effects of retroviral overexpression of L-Sox5 on chondrocyte differentiation using the clonal murine cell line ATDC5. We observed a temporal-restricted expression pattern of L-Sox5 in insulin-induced ATDC5 cells differentiating toward chondrocyte lineage. The protein expression levels of L-Sox5 showed a drastic decrease in contrast to unaltered mRNA levels during differentiation. L-Sox5 delayed the differentiation of ATDC5 cells as evidenced by Alcian blue staining for proteoglycan synthesis. The mRNA levels of chondrocyte and hypertrophic/osteoarthritic markers were markedly decreased or delayed in L-Sox5 overexpressing cells. L-Sox5 abrogated the promoter activity of Runx2. These results suggest that L-Sox5 protein expression may diminish along with the progress of chondrogenic differentiation. L-Sox5 may act as a negative regulator if expressed aberrantly at least in part by regulating the critical fate of chondrogenesis.

Inhibitory effects of eugenol on RANKL-induced osteoclast formation via attenuation of NF-κB and MAPK pathways.

Bone loss diseases are often associated with increased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. Compounds that can attenuate RANKL-mediated osteoclast formation are of great biomedical interest. Eugenol, a phenolic constituent of clove oil possesses medicinal properties; however, its anti-osteoclastogenic potential is unexplored hitherto. Here, we found that eugenol dose-dependently inhibited the RANKL-induced multinucleated osteoclast formation and TRAP activity in RAW264.7 macrophages. The underlying molecular mechanisms included the attenuation of RANKL-mediated degradation of IκBα and subsequent activation of NF-κB pathway. Furthermore, increase in phosphorylation and activation of RANKL-induced mitogen-activated protein kinase pathways (MAPK) was perturbed by eugenol. RANKL-induced expression of osteoclast-specific marker genes such as TRAP, cathepsin K (CtsK) and matrix metalloproteinase-9 (MMP-9) was remarkably downregulated by eugenol. These findings provide the first line of evidence that eugenol mediated attenuation of RANKL-induced NF-κB and MAPK pathways could synergistically contribute to the inhibition of osteoclast formation. Eugenol could be developed as therapeutic agent against diseases with excessive osteoclast activity.

Ratjadone C-mediated nuclear accumulation of HDAC4: implications on Runx2-induced osteoblast differentiation of C3H10T1/2 mesenchymal stem cells.

Histone deacetylases (HDACs) are a group of enzymes that deacetylate ε-N-acetyl lysine residues of histone and non-histone proteins and play an important role in gene regulation. HDAC4, a class-IIa HDAC, has been reported to shuttle between nucleus and cytoplasm in response to various cellular stimuli. The nucleo-cytoplasmic shuttling of HDAC4 is critical, and an anomalous nuclear localization might affect the cellular differentiation program. While the subcellular localization of HDAC4 has been reported to be vital for myoblast differentiation and chondrocyte hypertrophy, nuclear accumulation of HDAC4 during Runx2-induced osteoblast differentiation of stem cells has not been characterized. Ratjadone C is a natural compound that inhibits the nuclear export of proteins. Here, we show that Runx2 is a more potent transcription factor than Osterix in inducing osteoblast differentiation. Under the influence of ratjadone C, HDAC4 is retained in the nucleus and co-localizes with Runx2. However, forced nuclear accumulation of HDAC4 by ratjadone C or overexpression of the nuclear resident form of HDAC4 does not inhibit osteoblast differentiation, suggesting that the Runx2- induced osteogenic program of C3H10T1/2 cells is not affected by HDAC4. Even though phosphorylation of HDAC4 affects its compartmentalization and the stemness of progenitor cells, we found that total HDAC4 and phosphorylated HDAC4 remain cytoplasmic under both osteogenic and nonosteogenic conditions. Collectively, this work demonstrates that, regardless of the nucleo-cytoplasmic presence of HDAC4, the Runx2-induced osteoblast differentiation program of C3H10T1/2 cells remains unaffected. Additionally, the ratjadone C-mediated nuclear retention assay can potentially be used as a screening tool to identify novel regulatory mechanisms of HDAC4 and its functional partners in various pathophysiological conditions.

Thrombospondin-1 is a putative target gene of Runx2 and Runx3.

Thrombospondin-1 (TSP-1), a matricellular protein widely acclaimed to be involved in the inhibition of angiogenesis and tumorigenesis, is synthesized and secreted by many cell types, including osteoblast and cancer cells. TSP-1 is highly upregulated during early stage of osteogenesis, whereas it inhibits terminal osteoblast differentiation. Expression of TSP-1 is downregulated in cancer cells, and its ectopic expression has been shown to restrain tumor growth. Transcriptional regulation of TSP-1 in osteogenesis and cancer is poorly understood; this prompted us to study its regulation by the two key regulators of the aforementioned processes: Runx2 and Runx3. Through a PCR-based cDNA subtraction technique, we identified and cloned a cDNA fragment for mouse TSP-1, whose expression was dramatically upregulated in response to Runx2 expression in mesenchymal stem cells. Moreover, TSP-1 expression was considerably reduced in the lung of Runx2 knockout mouse. On the other hand, TSP-1 gene expression drastically increased at both the transcriptional and translational levels in response to Runx3 expression in B16-F10 melanoma cells. In line with this, Runx2 and Runx3 bound to the TSP-1 promoter and stimulated its activity. Hence, these results provide first line of evidence that TSP-1 is a transcriptional target gene of Runx2 and Runx3.

Analysis of HDAC1-mediated regulation of Runx2-induced osteopontin gene expression in C3h10t1/2 cells.

Histone deacetylases (HDACs) deacetylate lysine residues of histone and non-histone proteins and thereby regulate the cell-cycle, gene expression, and several other processes. We have analyzed the effects of HDAC1 on Runx2-mediated regulation of osteopontin (OPN) promoter activation and gene expression in mesenchymal progenitor C3h10t1/2 cells and show that co-expression of HDAC1 along with Runx2 results in down-regulation of Runx2-induced OPN mRNA expression during both the proliferation and differentiation stages of C3h10t1/2 cells. Luciferase assay results revealed that HDAC1 efficiently down-regulated Runx2-stimulated OPN promoter activity in a dose-dependent manner whereas TSA relieved the HDAC1-mediated repression and up-regulated the Runx2-induced OPN promoter activity and mRNA expression. In vivo HDAC1 co-localized and physically interacted with Runx2 and associated with the OPN promoter. Thus, HDAC1 not only plays a critical role in regulation of Runx2-stimulated expression of osteogenic genes, like OPN, but also regulate the proliferation and differentiation stages of mesenchymal progenitor cells, such as C3h10t1/2.

Reduced activity and cytoplasmic localization of Runx2 is observed in C3h10t1/2 cells overexpressing Tbx3.

Tbox3, a T-box containing transcription factor, has been reported to negatively regulate osteoblastogenesis. Here, we studied the effects mediated by Tbx3 on the master osteogenic transcription factor Runx2 in C3h10t1/2 cells. Dual-luciferase assay results showed that Tbx3 interferes in the Runx2-mediated activation of osteopontin promoter by abrogating Runx2 activity. Immunofluorescence study demonstrated that Runx2 was simultaneously localized in the nucleus and the cytoplasm of Tbx3-overexpressing cells. The results obtained in this study indicate that Tbx3 abrogates Runx2 activity and elevated expression levels of Tbx3 in the cells can result in mislocalization of Runx2.

Identification of Cilia in Different Mouse Tissues.

Cilia are microtubule-based hair-like organelles that extend from the cell surface. However, the existence and distribution of cilia in each organ and tissue at the postnatal stage in vivo remain largely unknown. In this study, we defined cilia distribution and arrangement and measured the ciliary lengths and the percentage of ciliated cells in different organs and tissues in vivo by using cilium dual reporter-expressing transgenic mice. Cilia were identified by the presence of ARL13B with an mCherry+ signal, and the cilium basal body was identified by the presence of Centrin2 with a GFP+ signal. Here, we provide in vivo evidence that chondrocytes and cells throughout bones have cilia. Most importantly, we reveal that: 1. primary cilia are present in hepatocytes; 2. no cilia but many centrioles are distributed on the apical cell surface in the gallbladder, intestine, and thyroid epithelia; 3. cilia on the cerebral cortex are well oriented, pointing to the center of the brain; 4. ARL13B+ inclusion is evident in the thyroid and islets of Langerhans; and 5. approximately 2% of cilia show irregular movement in nucleus pulposus extracellular fluid. This study reveals the existence and distribution of cilia and centrioles in different tissues and organs, and provides new insights for further comprehensive study of ciliary function in these organs and tissues.

PI3K is involved in L-selectin- and PSGL-1-mediated neutrophil rolling on E-selectin via F-actin redistribution and assembly.

L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) are adhesion molecules that play critical roles in neutrophil rolling during inflammation and lymphocyte homing. On the other hand they also function as signaling receptors to induce cytoskeleton changes. The present study is to investigate the signaling kinases responsible for the F-actin changes mediated by L-selectin and PSGL-1 during neutrophil rolling on E-selectin. Western blot analysis demonstrated that PI3K activation, peaking within 5 min, was induced by ligation of L-selectin and PSGL-1 with E-selectin, and that Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) was recruited to the membrane and tyrosine-phosphorylated, depending on PI3K. Furthermore, the F-actin redistribution and assembly mediated by ligation with E-selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E-selectin. However, Syk/Zap70, the well-known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F-actin-based cytoskeleton changes during neutrophil rolling on E-selectin, which may consequently regulate the rolling event.

Rooibos tea extracts inhibit osteoclast formation and activity through the attenuation of NF-κB activity in RAW264.7 murine macrophages.

Rooibos tea is a naturally sweet and aromatic tea that is native to the Western Cape province of South Africa. Rooibos is usually fermented to produce the traditional reddish brown colour and has been found to have numerous health benefits. These include beneficial effects on osteoblasts; however, its effects on osteoclast formation and activity are unknown. Osteoclasts are large, multinucleated cells responsible for bone resorption. Binding of RANKL to its receptor on osteoclast precursors triggers the NF-κB signalling pathway leading to the formation of osteoclasts. Certain bone destructive diseases, such as osteoporosis, are characterised by overactive osteoclasts. The inhibition of osteoclasts may offer a potential mode to prevent these diseases. The polyphenol contents of both fermented and unfermented tea extracts were similar although the radical scavenging activity of fermented rooibos tea was lower. Both tea extracts were not cytotoxic and inhibited osteoclast formation. Fermented rooibos tea extract caused a greater reduction in osteoclast resorption and the associated gene expression when compared with unfermented rooibos tea. Both tea extracts were shown to attenuate NF-κB activity. Fermented rooibos was found to have a more potent inhibitory effect on osteoclasts than unfermented rooibos extract and therefore may have a beneficial effect on bone health.

Ellagic acid inhibits RANKL-induced osteoclast differentiation by suppressing the p38 MAP kinase pathway.

Bone undergoes continuous remodeling by a coupled action between osteoblasts and osteoclasts. During osteoporosis, osteoclast activity is often elevated leading to increased bone destruction. Hence, osteoclasts are deemed as potential therapeutic targets to alleviate bone loss. Ellagic acid (EA) is a polyphenol reported to possess anticancer, antioxidant and anti-inflammatory properties. However, its effects on osteoclast formation and function have not yet been examined. Here, we explored the effects of EA on RANKL-induced osteoclast differentiation in RAW264.7 murine macrophages (in vitro) and human CD14+monocytes (ex vivo). EA dose-dependently attenuated RANKL-induced TRAP+ osteoclast formation in osteoclast progenitors with maximal inhibition seen at 1 µM concentration without cytotoxicity. Moreover, owing to perturbed osteoclastogenesis, EA disrupted actin ring formation and bone resorptive function of osteoclasts. Analysis of the underlying molecular mechanisms revealed that EA suppressed the phosphorylation and activation of the p38 MAP kinase pathway which subsequently impaired the RANKL-induced differentiation of osteoclast progenitors. Taken together, these novel results indicate that EA alleviates osteoclastogenesis by suppressing the p38 signaling pathway downstream of RANKL and exerts inhibitory effects on bone resorption and actin ring formation.

In silico design and bioevaluation of selective benzotriazepine BRD4 inhibitors with potent antiosteoclastogenic activity.

The bromodomain (BRD) and extra-terminal domain (BET) protein family bind to acetylated histones on lysine residues and act as epigenetic readers. Recently, the role of this protein family in bone loss has been gaining attention. Earlier studies have reported that benzotriazepine (Bzt) derivatives could be effective inhibitors of BET proteins. In this study, using in silico tools we designed three Bzt analogs (W49, W51, and W52). By docking, molecular simulations, and chemiluminescent Alpha Screen binding assay, we show that the studied analogs were selective at inhibiting BRD4 when compared to BRD2. Furthermore, we tested the effectiveness of these analogs on osteoclast formation and function. Among the examined analogs, Bzt-W49 and Bzt-W52 were found to be the most potent inhibitors of osteoclastogenesis without cytotoxicity in murine RAW264.7 osteoclast progenitors. Both the compounds also inhibited osteoclast formation without affecting cell viability in human CD14+ monocytes. Moreover, owing to attenuated osteoclastogenesis, actin ring formation and bone resorptive function of osteoclasts were severely perturbed. In conclusion, these results suggest that the novel BRD4-selective Bzt analogs designed in this study could be explored further for developing therapeutics against bone loss diseases characterized by excessive osteoclast activity.

Antibacterial activity of diisocyanate-modified chitosan for biomedical applications.

A diisocyanate-modified chitosan (DIMC) was synthesized via a cross-linking reaction with chitosan and diphenyl methane diisocyanate. The structural and thermal properties of the DIMC were systematically characterized by FTIR, UV-vis, TGA, DSC, XRD and SEM. In addition, the optical properties were evaluated by photoluminescence. Finally, the antibacterial activities of the synthesized DIMC were examined against Escherichia coli and Staphylococcus pyogenes bacteria by agar plate diffusion method. The DIMC showed better degree of bacterial growth inhibition against E. coli as compared with unaltered chitosan. These results suggest that the synthesized chitosan xerogel could be used as a novel biodegradable material with improved antibacterial properties for biomedical applications.

Ferulic acid impairs osteoclast fusion and exacerbates survival of mature osteoclasts.

Elevated bone loss induced by osteoclasts is a critical and most commonly observed pathological complication during osteolytic diseases such as osteoporosis. Hence, attenuation of osteoclast formation or function is a classical therapeutic approach to regulate bone loss. In this study, we found that ferulic acid (FA), a natural compound potently inhibited osteoclast formation in human CD14+ peripheral blood monocytes ex vivo with an IC50 of 39 µM. Moreover, due to impaired differentiation of osteoclast progenitors, actin ring formation and bone resorption activity were also perturbed. Investigation of underlying molecular mechanisms revealed that FA inhibited the RANKL-induced expression of dendritic cell-specific transmembrane protein (DC-STAMP), a critical regulator of osteoclast fusion. In addition, expression of matrix metalloproteinase-9 (MMP-9) and cathepsin K, the key osteoclast specific lysosomal proteases involved in bone matrix resorption were severely aggravated by FA. A significant reduction in mature osteoclast numbers was detected in the presence of FA accompanied by increased caspase-3 activity and DNA-fragmentation, a characteristic hallmark of apoptosis. Collectively, these results suggested that FA inhibited osteoclast fusion by suppressing the expression of DC-STAMP and induced apoptosis in mature osteoclasts by the caspase-3 pathway.

Piperine alleviates osteoclast formation through the p38/c-Fos/NFATc1 signaling axis.

Increased bone fracture is one of the health risk factors in patients with bone loss related disorders such as osteoporosis and breast cancer metastasis to bone. Over activity of osteoclasts leads to uncoupling of bone remodeling favoring bone loss over bone formation. Receptor activator of nuclear factor-κß ligand (RANKL) triggers the differentiation pathway leading to multinucleated osteoclast formation. Modulation of RANKL or its downstream signaling pathways involved in osteoclast formation is of significant interest in the development of anti-resorptive agents. In this study, the effects of piperine, an alkaloid present in Piper nigrum L. on osteoclast formation was investigated. Piperine inhibited tartrate-resistant acid phosphatase-positive multinucleated osteoclast formation in murine RAW264.7 macrophages and human CD14+ monocytes induced by RANKL and breast cancer cells. Piperine attenuated the p38-mitogen activated protein kinase pathway activation, while the extracellular-signal-regulated kinase, c-Jun N-terminal kinase, or NF-κß pathways downstream of RANKL remained unaffected. Concomitantly, expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), the key transcription factors involved in osteoclastogenesis were remarkably inhibited by piperine. Furthermore, piperine disrupted the actin ring structure and bone resorption, a characteristic hallmark of osteoclasts. Collectively, these results suggested that piperine inhibited osteoclast differentiation by suppressing the p38/NFATc1/c-Fos signaling axis..

Arachidonic acid and docosahexaenoic acid suppress osteoclast formation and activity in human CD14+ monocytes, in vitro.

An unbalanced diet can have adverse effects on health. Long chain polyunsaturated fatty acids (LCPUFAs) have been the focus of research owing to their necessity of inclusion in a healthy diet. However, the effects of LCPUFAs on human osteoclast formation and function have not been explored before. A human CD14+ monocyte differentiation model was used to elucidate the effects of an ω-3 LCPUFA, docosahexaenoic acid (DHA), and an ω-6 LCPUFA, arachidonic acid (AA), on osteoclast formation and activity. CD14+ monocytes were isolated from peripheral blood of healthy donors and stimulated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand to generate osteoclasts. Data from this study revealed that both the LCPUFAs decreased osteoclast formation potential of CD14+ monocytes in a dose-dependent manner when treated at an early stage of differentiation. Moreover, when exposed at a late stage of osteoclast differentiation AA and DHA impaired the bone resorptive potential of mature osteoclasts without affecting osteoclast numbers. AA and DHA abrogated vitronectin receptor expression in differentiating as well as mature osteoclasts. In contrast, the degree of inhibition for calcitonin receptor expression varied between the LCPUFAs with only AA causing inhibition during osteoclast differentiation. Furthermore, AA and DHA down regulated the expression of key osteoclast-specific genes in differentiating as well as mature osteoclasts. This study demonstrates for the first time that LCPUFAs can modulate osteoclast formation and function in a human primary osteoclast cell line.