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Sample records for rankl-induced osteoclast differentiation

  1. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

    PubMed

    Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

    2015-10-01

    To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p < 0.01) in a dose dependent manner. These results suggest that vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

  2. Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

    SciTech Connect

    Lee, Sang-Hyun; Jang, Hae-Dong

    2015-02-15

    Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

  3. Suppression of RANKL-induced osteoclast differentiation by cilostazol via SIRT1-induced RANK inhibition.

    PubMed

    Park, So Youn; Lee, Sung Won; Kim, Hye Young; Lee, Sang Yeob; Lee, Won Suk; Hong, Ki Whan; Kim, Chi Dae

    2015-10-01

    Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage hematopoietic lineages and degrade bone matrix by secretion of lytic enzymes. The regulation of osteoclast differentiation provides a potential strategy for treatment of bone-lytic damage. In this study, cilostazol, an inhibitor of type III phosphodiesterase, inhibited RANKL [receptor activator of nuclear factor kappa B (RANK) ligand]-induced RANK expression in bone marrow-derived monocyte/macrophage precursors (BMMs) and Raw 264.7 cells by inhibiting PU.1 via SIRT1 activation. RANKL-induced RANK expression was attenuated by cilostazol and rSIRT1 in Raw 264.7 cells, and these were blocked by sirtinol. In line with these, cilostazol elevated SIRT1 mRNA and protein levels in 12-24h and increased SIRT1 activity, and these effects were inhibited by sirtinol. Furthermore, the RANKL-induced nuclear expression of PU.1, a transcription factor required for macrophage differentiation, was suppressed by cilostazol. Additionally, marked RANKL-induced RANK immunofluorescence staining in Raw 264.7 cells was attenuated by cilostazol and rSIRT1, and both attenuations were prevented by sirtinol. Extensive RANK staining of knee synovial tissues in a mouse model of collagen-induced arthritis (CIA) was markedly reduced by cilostazol (30mg/kg/day). In line with these results, both RANKL- and M-CSF-induced differentiation of BMMs to multinucleated TRAP(+) giant cells and resorption pit formation were inhibited by cilostazol associated with a decrease in TRAP (a marker enzyme of osteoclasts) activity. In conclusion, cilostazol activates SIRT1, which suppresses the nuclear translocation of PU.1, and thus, inhibits RANKL-stimulated RANK expression and causes anti-osteoclast formation in BMMs in vitro and in their murine model of CIA.

  4. Berberine Sulfate Attenuates Osteoclast Differentiation through RANKL Induced NF-κB and NFAT Pathways.

    PubMed

    Zhou, Lin; Song, Fangming; Liu, Qian; Yang, Mingli; Zhao, Jinmin; Tan, Renxiang; Xu, Jun; Zhang, Ge; Quinn, Julian M W; Tickner, Jennifer; Xu, Jiake

    2015-11-13

    Osteoporosis, a metabolic bone disease, is characterized by an excessive formation and activation of osteoclasts. Anti-catabolic treatment using natural compounds has been proposed as a potential therapeutic strategy against the osteoclast related osteolytic diseases. In this study, the activity of berberine sulfate (an orally available form of berberine) on osteoclast differentiation and its underlying molecular mechanisms of action were investigated. Using bone marrow macrophages (BMMs) derived osteoclast culture system, we showed that berberine sulfate at the dose of 0.25, 0.5 and 1 μM significantly inhibited the formation of osteoclasts. Notably, berberine sulfate at these doses did not affect the BMM viability. In addition, we observed that berberine sulfate inhibited the expression of osteoclast marker genes, including cathepsin K (Ctsk), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP, Acp5) and Vacuolar-type H+-ATPase V0 subunit D2 (V-ATPase d2). Luciferase reporter gene assay and Western blot analysis further revealed that berberine sulfate inhibits receptor for activation of nuclear factor ligand (RANKL)-induced NF-κB and NFAT activity. Taken together, our results suggest that berberine sulfate is a natural compound potentially useful for the treatment of osteoporosis.

  5. Effect of amorphous silica nanoparticles on in vitro RANKL-induced osteoclast differentiation in murine macrophages

    NASA Astrophysics Data System (ADS)

    Nabeshi, Hiromi; Yoshikawa, Tomoaki; Akase, Takanori; Yoshida, Tokuyuki; Tochigi, Saeko; Hirai, Toshiro; Uji, Miyuki; Ichihashi, Ko-Ichi; Yamashita, Takuya; Higashisaka, Kazuma; Morishita, Yuki; Nagano, Kazuya; Abe, Yasuhiro; Kamada, Haruhiko; Tsunoda, Shin-Ichi; Itoh, Norio; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2011-07-01

    Amorphous silica nanoparticles (nSP) have been used as a polishing agent and/or as a remineralization promoter for teeth in the oral care field. The present study investigates the effects of nSP on osteoclast differentiation and the relationship between particle size and these effects. Our results revealed that nSP exerted higher cytotoxicity in macrophage cells compared with submicron-sized silica particles. However, tartrate-resistant acid phosphatase (TRAP) activity and the number of osteoclast cells (TRAP-positive multinucleated cells) were not changed by nSP treatment in the presence of receptor activator of nuclear factor κB ligand (RANKL) at doses that did not induce cytotoxicity by silica particles. These results indicated that nSP did not cause differentiation of osteoclasts. Collectively, the results suggested that nanosilica exerts no effect on RANKL-induced osteoclast differentiation of RAW264.7 cells, although a detailed mechanistic examination of the nSP70-mediated cytotoxic effect is needed.

  6. Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis.

    PubMed

    Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi; Shin, Dong Min

    2012-02-01

    The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca(2+) signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP(3) and evaluated IP(3)-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca(2+) signaling proteins such as IP(3) receptors (IP(3)Rs), plasma membrane Ca(2+) ATPase, and sarco/endoplasmic reticulum Ca(2+) ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP(3) was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP(3) levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP(3)Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP(3)Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP(3) levels and the IP(3)-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

  7. Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

    PubMed Central

    Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi

    2012-01-01

    The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis. PMID:22416217

  8. Relationship between fluoride exposure and osteoclast markers during RANKL-induced osteoclast differentiation.

    PubMed

    Junrui, Pei; Bingyun, Li; Yanhui, Gao; Xu, Jiaxun; Darko, Gottfried M; Dianjun, Sun

    2016-09-01

    Skeletal fluorosis is a metabolic bone disease caused by excessive accumulation of fluoride. Although the cause of this disease is known, the mechanism by which fluoride accumulates on the bone has not been clearly defined, thus there are no markers that can be used for screening skeletal fluorosis in epidemiology. In this study, osteoclasts were formed from bone marrow cells of C57BL/6 mice-treated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand. The mRNA expression of tartrate-resistant acid phosphatase 5b (TRAP5b), osteoclast-associated receptor (OSCAR), calcitonin receptor (CTR), matrix metalloproteinase 9 (MMP9) and cathepsin K (CK) were detected using real-time PCR (RT-PCR). Results showed that fluoride between 0.5 and 8mg/l had no effect on osteoclast formation. However fluoride at 0.5mg/l level significantly decreased the activity of osteoclast bone resorption. Fluoride concentration was negatively correlated with the activity of osteoclast bone resorption. On day 5 of osteoclast differentiation maturity, MMP9 and CK mRNA expression were not only negatively correlated with fluoride concentration, but directly correlated with the activity of osteoclast bone resorption. TRAP5b, CTR and OSCAR mRNA expression were positively correlated with the number of osteoclast and they had no correlation with the activity of osteoclast bone resorption. Thus, it can be seen that MMP9 and CK may reflect the change of activity of bone resorption as well the degree of fluoride exposure. TRAP5b, CTR and OSCAR can represent the change of number of osteoclast formed. PMID:27500448

  9. The effects of Lycii Radicis Cortex on RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells

    PubMed Central

    KIM, JAE-HYUN; KIM, EUN-YOUNG; LEE, BINA; MIN, JU-HEE; SONG, DEA-UK; LIM, JEONG-MIN; EOM, JI WHAN; YEOM, MIJUNG; JUNG, HYUK-SANG; SOHN, YOUNGJOO

    2016-01-01

    Post-menopausal osteoporosis is a serious age-related disease. After the menopause, estrogen deficiency is common, and excessive osteoclast activity causes osteoporosis. Osteoclasts are multinucleated cells generated from the differentiation of monocyte/macrophage precursor cells such as RAW 264.7 cells. The water extract of Lycii Radicis Cortex (LRC) is made from the dried root bark of Lycium chinense Mill. and is termed 'Jigolpi' in Korea. Its effects on osteoclastogenesis and post-menopausal osteoporosis had not previously been tested. In the present study, the effect of LRC on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation was demonstrated using a tartrate-resistant acid phosphatase (TRAP) assay and pit formation assay. Moreover, in order to analyze molecular mechanisms, we studied osteoclastogenesis-related markers such as nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, receptor activator of NF-κB (RANK), TRAP, cathepsin K (CTK), matrix metallopeptidase-9 (MMP-9), calcitonin receptor (CTR) and carbonic anhydrase II (CAII) using RT-qPCR and western blot analysis. Additionally, we also determined the effect of LRC on an ovariectomized (OVX) rat model. We noted that LRC inhibited RANKL-induced osteoclast differentiation via suppressing osteoclastogenesis-related markers. It also inhibited osteoporosis in the OVX rat model by decreasing loss of bone density and trabecular area. These results suggest that LRC exerts a positive effect on menopausal osteoporosis. PMID:26848104

  10. Role of CrkII Signaling in RANKL-Induced Osteoclast Differentiation and Function.

    PubMed

    Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Seong, Semun; Nam, Kwang-Il; Lee, Seoung Hoon; Kim, Kyung Keun; Kim, Nacksung

    2016-02-01

    Rac1, a member of small GTPases, is a key regulator of osteoclast differentiation and function. The Crk family adaptor proteins, consisting of Src homology (SH) 2 and SH3 protein-binding domains, regulate cell proliferation, migration, and invasion through Rac1 activation. In this study, we examined the role of CrkII in osteoclast differentiation and function. Retroviral overexpression of CrkII in osteoclast precursors enhanced osteoclast differentiation and resorptive function through Rac1 activation. The knockdown of CrkII in osteoclast precursors using small interfering RNA inhibited osteoclast differentiation and its resorption activity. Unlike wild-type CrkII, overexpression of the three SH domains in mutant forms of CrkII did not enhance either osteoclast differentiation or function. Phosphorylation of p130 Crk-associated substrate (p130Cas) by osteoclastogenic cytokines in preosteoclasts increased the interaction between p130Cas and CrkII, which is known to be involved in Rac1 activation. Furthermore, transgenic mice overexpressing CrkII under control of a tartrate-resistant acid phosphatase promoter exhibited a low bone mass phenotype, associated with increased resorptive function of osteoclasts in vivo. Taken together, our data suggest that the p130Cas/CrkII/Rac1 signaling pathway plays an important role in osteoclast differentiation and function, both in vitro and in vivo.

  11. A novel PPAR{gamma} agonist, KR62776, suppresses RANKL-induced osteoclast differentiation and activity by inhibiting MAP kinase pathways

    SciTech Connect

    Park, Ju-Young; Bae, Myung-Ae; Cheon, Hyae Gyeong; Kim, Sung Soo; Hong, Jung-Min; Kim, Tae-Ho; Choi, Je-Yong; Kim, Sang-Hyun; Lim, Jiwon; Choi, Chang-Hyuk; Shin, Hong-In; Kim, Shin-Yoon Park, Eui Kyun

    2009-01-16

    We investigated the effects of a novel peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, KR62776, on osteoclast differentiation and function, and on the underlying signaling pathways. KR62776 markedly suppressed differentiation into osteoclasts in various osteoclast model systems, including bone marrow mononuclear (BMM) cells and a co-culture of calvarial osteoblasts and BMM cells. KR62776 suppressed the activation of tartrate-resistant acid phosphatase (TRAP) and the expression of genes associated with osteoclast differentiation, such as TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated receptor (OSCAR). Furthermore, KR62776 reduced resorption pit formation in osteoclasts, and down-regulated genes essential for osteoclast activity, such as Src and {alpha}v{beta}3 integrin. An analysis of a signaling pathway showed that KR62776 inhibited the receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and nuclear factor-{kappa}B (NF-{kappa}B). Together, these results demonstrate that KR62776 negatively affects osteoclast differentiation and activity by inhibiting the RANKL-induced activation of MAP kinases and NF-{kappa}B.

  12. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression

    PubMed Central

    Zeng, Xiang-zhou; He, Long-gang; Wang, Song; Wang, Keng; Zhang, Yue-yang; Tao, Lei; Li, Xiao-juan; Liu, Shu-wen

    2016-01-01

    Aim: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. Methods: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. Results: Aconine (0.125, 0.25 μmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. Conclusion: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP. PMID:26592521

  13. Celastrol attenuates bone erosion in collagen-Induced arthritis mice and inhibits osteoclast differentiation and function in RANKL-induced RAW264.7.

    PubMed

    Gan, Ke; Xu, Lingxiao; Feng, Xiaoke; Zhang, Qiande; Wang, Fang; Zhang, Miaojia; Tan, Wenfeng

    2015-02-01

    Recently, the traditional Chinese medicine Tripterygium wilfordii Hook f (TwHF) of the Celastraceae family has attracted increasing attention for its potential therapeutic application in patients with rheumatoid arthritis (RA). It is well accepted that TwHF exerts the antirheumatic activity and mainly depends on its potent anti-inflammatory property. To further explore the therapeutic potential of the well-defined TwHF-derived single compound - celastrol in RA, we study the therapeutic efficacy of celastrol on bone erosion in collagen-induced arthritis (CIA) mice and delineate its effects on osteoclast differentiation and functions in RANKL-induced osteoclast precursors RAW264.7 cell line. In CIA mice, daily injection of celastrol (beginning on day 28 after arthritis induction) markedly suppressed arthritis, and reduced bone damage in the joints as demonstrated by histology and bone micro-computed tomography (CT). The effects were accompanied by reductions of osteoclast cells in joints, serum tartrate-resistant acid phosphatase (TRAP) 5b, and expression of osteoclastic genes (Trap, Ctsk, Ctr, Mmp-9) and transcriptional factors (c-Fos, c-Jun and NFATc1). When RAW264.7 cells were treated with RANKL, celastrol inhibited the formation of TRAP+ multinucleated cells and the bone-resorbing activity in dose-dependent manners. Furthermore, celastrol reduced the RANKL-induced expression of osteoclastic genes and transcriptional factors, as well as phosphorylation of NF-kB and mitogen-activated protein kinases (MAPK). These findings show that celastrol could directly inhibit osteoclast formation and function, suggesting a novel therapeutic strategy of celastrol for managing RA, especially in preventing bone destruction.

  14. Curcumol suppresses RANKL-induced osteoclast formation by attenuating the JNK signaling pathway

    SciTech Connect

    Yu, Mingxiang; Chen, Xianying; Lv, Chaoyang; Yi, Xilu; Zhang, Yao; Xue, Mengjuan; He, Shunmei; Zhu, Guoying; Wang, Hongfu

    2014-05-02

    Highlights: • Curcumol suppresses osteoclasts differentiation in vitro. • Curcumol impairs JNK/AP-1 signaling pathway. • Curcumol may be used for treating osteoclast related diseases. - Abstract: Osteoclasts, derived from hemopoietic progenitors of the monocyte/macrophage lineage, have a unique role in bone resorption, and are considered a potential therapeutic target in the treatment of such pathologic bone diseases as osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we demonstrate that curcumol, one of the major components of the essential oil of Rhizoma Curcumae, exhibits an inhibitory effect on receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast differentiation with both bone marrow-derived macrophages and RAW264.7 cells in a dose-dependent manner. In addition, RANKL-induced mRNA expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K, is prominently reduced in the presence of curcumol. Furthermore, the molecular mechanism of action was investigated, and curcumol inhibited osteoclastogenesis by specifically impairing RANKL-induced c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling, which was further identified in rescue studies by means of anisomycin, a JNK signaling-specific activator. Taken together, these findings suggest that curcumol suppresses RANKL-induced osteoclast differentiation through the JNK/AP-1 signaling pathway, and may be useful as a therapeutic treatment for bone resorption-associated diseases.

  15. A medium-chain fatty acid, capric acid, inhibits RANKL-induced osteoclast differentiation via the suppression of NF-κB signaling and blocks cytoskeletal organization and survival in mature osteoclasts.

    PubMed

    Kim, Hyun-Ju; Yoon, Hye-Jin; Kim, Shin-Yoon; Yoon, Young-Ran

    2014-08-01

    Fatty acids, important components of a normal diet, have been reported to play a role in bone metabolism. Osteoclasts are bone-resorbing cells that are responsible for many bone-destructive diseases such as osteoporosis. In this study, we investigated the impact of a medium-chain fatty acid, capric acid, on the osteoclast differentiation, function, and survival induced by receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). Capric acid inhibited RANKL-mediated osteoclastogenesis in bone marrow-derived macrophages and suppressed RANKL-induced IκBα phosphorylation, p65 nuclear translocation, and NF-κB transcriptional activity. Capric acid further blocked the RANKL-stimulated activation of ERK without affecting JNK or p38. The induction of NFATc1 in response to RANKL was also attenuated by capric acid. In addition, capric acid abrogated M-CSF and RANKL-mediated cytoskeleton reorganization, which is crucial for the efficient bone resorption of osteoclasts. Capric acid also increased apoptosis in mature osteoclasts through the induction of Bim expression and the suppression of ERK activation by M-CSF. Together, our results reveal that capric acid has inhibitory effects on osteoclast development. We therefore suggest that capric acid may have potential therapeutic implications for the treatment of bone resorption-associated disorders.

  16. 1'-Acetoxychavicol acetate inhibits RANKL-induced osteoclastic differentiation of RAW 264.7 monocytic cells by suppressing nuclear factor-kappaB activation.

    PubMed

    Ichikawa, Haruyo; Murakami, Akira; Aggarwal, Bharat B

    2006-04-01

    Osteoclastogenesis is commonly associated with various age-related diseases, including cancer. A member of the tumor necrosis factor superfamily, receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL), has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this report, we investigated the effect of 1'-acetoxychavicol acetate (ACA), a component of Alpina galanga, on RANKL signaling and consequent osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and coexposure of the cells to ACA completely suppressed RANKL-induced NF-kappaB activation in a time- and concentration-dependent manner. The suppression of NF-kappaB by ACA was mediated through suppression of RANKL-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Furthermore, incubation of monocytic cells with RANKL induced osteoclastogenesis, and ACA suppressed it. Inhibition of osteoclastogenesis was maximal when cells were simultaneously exposed to ACA and RANKL and minimum when ACA was added 2 days after RANKL. ACA also inhibited the osteoclastogenesis induced by human breast cancer MCF-7 cells, multiple myeloma MM1 cells, and head and neck squamous cell carcinoma LICR-LON-HN5 cells. These results indicate that ACA is an effective blocker of RANKL-induced NF-kappaB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss.

  17. 5-Lipoxygenase inhibitors suppress RANKL-induced osteoclast formation via NFATc1 expression.

    PubMed

    Kang, Ju-Hee; Ting, Zheng; Moon, Mi-ran; Sim, Jung-Seon; Lee, Jung-Min; Doh, Kyung-Eun; Hong, Sunhye; Cui, Minghua; Choi, Sun; Chang, Hyeun Wook; Park Choo, Hea-Young; Yim, Mijung

    2015-11-01

    5-Lipoxygenase synthesizes leukotrienes from arachidonic acid. We developed three novel 5-LO inhibitors having a benzoxazole scaffold as a potential anti-osteoclastogenics. They significantly suppressed RANKL-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, one compound, K7, inhibited the bone resorptive activity of osteoclasts. The anti-osteoclastogenic effect of K7 was mainly attributable to reduction in the expression of NFATc1, an essential transcription factor for osteoclast differentiation. K7 inhibited osteoclast formation via ERK and p38 MAPK, as well as NF-κB signaling pathways. K7 reduced lipopolysaccharide (LPS)-induced osteoclast formation in vivo, corroborating the in vitro data. Thus, K7 exerted an inhibitory effect on osteoclast formation in vitro and in vivo, properties that make it a potential candidate for the treatment of bone diseases associated with excessive bone resorption.

  18. FOXO1 mediates RANKL-induced osteoclast formation and activity.

    PubMed

    Wang, Yu; Dong, Guangyu; Jeon, Hyeran Helen; Elazizi, Mohamad; La, Lan B; Hameedaldeen, Alhassan; Xiao, E; Tian, Chen; Alsadun, Sarah; Choi, Yongwon; Graves, Dana T

    2015-03-15

    We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in osteoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-κB ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow macrophages and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre(+)FOXO1(L/L)) compared with matched controls (LyzM.Cre(-)FOXO1(L/L)). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream factors, including dendritic cell-specific transmembrane protein, ATP6vod2, cathepsin K, and integrin αv. Lastly, FOXO1 deletion reduced M-CSF-induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions.

  19. Decursin from Angelica gigas suppresses RANKL-induced osteoclast formation and bone loss.

    PubMed

    Wang, Xin; Zheng, Ting; Kang, Ju-Hee; Li, Hua; Cho, Hyewon; Jeon, Raok; Ryu, Jae-Ha; Yim, Mijung

    2016-03-01

    Osteoclasts are the only cells capable of breaking down bone matrix, and excessive activation of osteoclasts is responsible for bone-destructive diseases. In this study, we investigated the effects of decursin from extract of Angelica gigas root on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast formation using mouse bone marrow-derived macrophages (BMMs). Decursin inhibited RANKL-induced osteoclast formation without cytotoxicity. In particular, decursin maintains the characteristics of macrophages by blocking osteoclast differentiation by RANKL. Furthermore, the RANKL-stimulated bone resorption was diminished by decursin. Mechanistically, decursin blocked the RANKL-triggered ERK mitogen-activated protein kinases (MAPK) phosphorylation, which results in suppression of c-Fos and the nuclear factor of activated T cells (NFATc1) expression. In accordance with the in vitro study, decursin reduced lipopolysaccharide (LPS)- or ovariectomy (OVX)-induced bone loss in vivo. Therefore, decursin exerted an inhibitory effect on osteoclast formation and bone loss in vitro and in vivo. Decursin could be useful for the treatment of bone diseases associated with excessive bone resorption.

  20. IL-33 inhibits RANKL-induced osteoclast formation through the regulation of Blimp-1 and IRF-8 expression

    SciTech Connect

    Kiyomiya, Hiroyasu; Ariyoshi, Wataru; Okinaga, Toshinori; Kaneuji, Takeshi; Mitsugi, Sho; Sakurai, Takuma; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; and others

    2015-05-01

    Interleukin (IL)-33 is a recently discovered proinflammatory cytokine that belongs to the IL-1 family. Several studies have reported that IL-33 inhibits osteoclast differentiation. However, the mechanism of IL-33 regulation of osteoclastogenesis remains unclear. In the present study, we examined the effect of IL-33 on osteoclast formation in vitro. IL-33 suppressed osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL) and/or macrophage stimulating factor (M-CSF). IL-33 also inhibited the expression of RANKL-induced nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), thereby decreasing the expression of osteoclastogenesis-related marker genes, including Cathepsin K, Osteoclast stimulatory transmembrane protein (Oc-stamp) and Tartrate-resistant acid phosphatase (Trap). Blockage of IL-33-ST2 binding suppressed the IL-33-mediated inhibition of NFATc1. RANKL-induced B-lymphocyte-induced maturation protein-1 (Blimp-1) expression was also suppressed by IL-33, which was followed by the stimulation of anti-osteoclastic genes such as interferon regulatory factor-8 (IRF-8). These results suggest that IL-33-ST2 interactions down-regulate both RANKL-induced NFATc1 activation and osteoclast differentiation via the regulation of Blimp-1 and IRF-8 expression. - Highlights: • IL-33 inhibits RANKL-induced osteoclast formation. • IL-33 has inhibitory effect on the RANKL-induced NFATc1 expression. • IL-33-induced NFATc1 suppression depends on the regulation of Blimp-1 and IRF-8.

  1. Inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ).

    PubMed

    Odkhuu, Erdenezaya; Koide, Naoki; Haque, Abedul; Tsolmongyn, Bilegtsaikhan; Naiki, Yoshikazu; Hashimoto, Shoji; Komatsu, Takayuki; Yoshida, Tomoaki; Yokochi, Takashi

    2012-02-29

    The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL-stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-κB and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN-β-mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN-β signaling due to augmented IFNAR expression.

  2. Diarylheptanoid from Curcuma comosa Roxb. suppresses RANKL-induced osteoclast differentiation by decreasing NFATc1 and c-Fos expression via MAPK pathway.

    PubMed

    Chawalitpong, Supatta; Sornkaew, Nilubon; Suksamrarn, Apichart; Palaga, Tanapat

    2016-10-01

    Osteoporosis is caused by a functional imbalance between osteoblasts and osteoclasts. The increased activation of osteoclasts that is a hallmark of osteoporosis results in the progressive loss of bone mass and therefore in an increased susceptibility to bone fractures. Diarylheptanoids are a group of phytoestrogens that have been isolated from a number of plant species, including the rhizomes of Curcuma comosa Roxb. In this study, the effect of one of diarylheptanoids, (3S)-1-(3,4-dihydroxyphenyl)-3-hydroxy-7-phenyl-(6E)-6-heptene (DHPH), was investigated for anti-inflammatory and anti-osteoclastogenic activity. DHPH significantly inhibited nitric oxide production in RAW264.7 cell line following their activation by lipopolysaccharide and interferon-γ, with no cytotoxicity. In primary mouse bone-marrow-derived macrophage precursors, DHPH suppressed osteoclastogenesis induced by receptor activator of nuclear factor-κB (RANK) ligand at an inhibitory concentration 50 of 325±1.37nM. DHPH treatment delayed and reduced the expression of master regulators of osteoclast differentiation, NFATc1 and c-Fos. Consistent with this result, the mRNA level of cathepsin K, associated with osteoclast differentiation, was decreased whereas the reduction in the mRNA of irf8, a negative regulator of osteoclast differentiation, was similar to that measured in the vehicle-treated control cells. DHPH reduced the phosphorylation of p38 MAPK, ERK (p44/42). Furthermore, DHPH suppressed the bone absorption activity of osteoclasts and enhanced osteoblast differentiation. Taken together, DHPH interrupts the immediate downstream signaling cascade of RANK and interferes with osteoclast differentiation and its function while enhances osteoblast differentiation. These results demonstrate the potential of this diarylheptanoid as a new therapeutic agent in osteoporosis. PMID:27523282

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

    PubMed

    Deepak, Vishwa; Kasonga, Abe; Kruger, Marlena C; Coetzee, Magdalena

    2015-06-01

    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.

  4. Amyloid β Peptide Enhances RANKL-Induced Osteoclast Activation through NF-κB, ERK, and Calcium Oscillation Signaling

    PubMed Central

    Li, Shangfu; Yang, Bu; Teguh, Dian; Zhou, Lin; Xu, Jiake; Rong, Limin

    2016-01-01

    Osteoporosis and Alzheimer’s disease (AD) are common chronic degenerative disorders which are strongly associated with advanced age. We have previously demonstrated that amyloid beta peptide (Aβ), one of the pathological hallmarks of AD, accumulated abnormally in osteoporotic bone specimens in addition to having an activation effect on osteoclast (Bone 2014,61:164-75). However, the underlying molecular mechanisms remain unclear. Activation of NF-κB, extracellular signal-regulated kinase (ERK) phosphorylates, and calcium oscillation signaling pathways by receptor activator NF-κB ligand (RANKL) plays a pivotal role in osteoclast activation. Targeting this signaling to modulate osteoclast function has been a promising strategy for osteoclast-related diseases. In this study, we investigated the effects of Aβ on RANKL-induced osteoclast signaling pathways in vitro. In mouse bone marrow monocytes (BMMs), Aβ exerted no effect on RANKL-induced osteoclastogenesis but promoted osteoclastic bone resorption. In molecular levels, Aβ enhanced NF-κB activity and IκB-α degradation, activated ERK phosphorylation and stimulated calcium oscillation, thus leading to upregulation of NFAT-c1 expression during osteoclast activation. Taken together, our data demonstrate that Aβ enhances RANKL-induced osteoclast activation through IκB-α degradation, ERK phosphorylation, and calcium oscillation signaling pathways and that Aβ may be a promising agent in the treatment of osteoclast-related disease such as osteoporosis. PMID:27735865

  5. Methotrexate inhibits osteoclastogenesis by decreasing RANKL-induced calcium influx into osteoclast progenitors.

    PubMed

    Kanagawa, Hiroya; Masuyama, Ritsuko; Morita, Mayu; Sato, Yuiko; Niki, Yasuo; Kobayashi, Tami; Katsuyama, Eri; Fujie, Atsuhiro; Hao, Wu; Tando, Toshimi; Watanabe, Ryuichi; Miyamoto, Kana; Morioka, Hideo; Matsumoto, Morio; Toyama, Yoshiaki; Saya, Hideyuki; Miyamoto, Takeshi

    2016-09-01

    The increasing number of osteoporosis patients is a pressing issue worldwide. Osteoporosis frequently causes fragility fractures, limiting activities of daily life and increasing mortality. Many osteoporosis patients take numerous medicines due to other health issues; thus, it would be preferable if a single medicine could ameliorate osteoporosis and other conditions. Here, we screened 96 randomly selected drugs targeting various diseases for their ability to inhibit differentiation of osteoclasts, which play a pivotal role in development of osteoporosis, and identified methotrexate (MTX), as a potential inhibitor. MTX is currently used to treat sarcomas or leukemic malignancies or auto-inflammatory diseases such as rheumatoid arthritis (RA) through its anti-proliferative and immunosuppressive activities; however, a direct effect on osteoclast differentiation has not been shown. Here, we report that osteoclast formation and expression of osteoclastic genes such as NFATc1 and DC-STAMP, which are induced by the cytokine RANKL, are significantly inhibited by MTX. We found that RANKL-dependent calcium (Ca) influx into osteoclast progenitors was significantly inhibited by MTX. RA patients often develop osteoporosis, and osteoclasts are reportedly required for joint destruction; thus, MTX treatment could have a beneficial effect on RA patients exhibiting high osteoclast activity by preventing both osteoporosis and joint destruction.

  6. Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

    PubMed

    Takegahara, Noriko; Kim, Hyunsoo; Mizuno, Hiroki; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Tomura, Michio; Kanagawa, Osami; Ishii, Masaru; Choi, Yongwon

    2016-02-12

    Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation.

  7. Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

    SciTech Connect

    Kim, Hyun-Ju; Yoon, Hye-Jin; Yoon, Kyung-Ae; Gwon, Mi-Ri; Jin Seong, Sook; Suk, Kyoungho; Kim, Shin-Yoon; Yoon, Young-Ran

    2015-06-10

    Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells. - Highlights: • LCN2 expression is regulated during osteoclast development. • LCN2 suppresses M-CSF-mediated osteoclast precursor proliferation. • LCN2 inhibits RANKL-induced osteoclast differentiation.

  8. Water extract of Acer tegmentosum reduces bone destruction by inhibiting osteoclast differentiation and function.

    PubMed

    Ha, Hyunil; Shim, Ki-Shuk; Kim, Taesoo; An, Hyosun; Lee, Chung-Jo; Lee, Kwang Jin; Ma, Jin Yeul

    2014-04-01

    The stem of Acer tegmentosum has been widely used in Korea for the treatment of hepatic disorders. In this study, we investigated the bone protective effect of water extract of the stem of Acer tegmentosum (WEAT). We found that WEAT inhibits osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL), an essential cytokine for osteoclast differentiation. In osteoclast precursor cells, WEAT inhibited RANKL-induced activation of JNK, NF-κB, and cAMP response element-binding protein, leading to suppression of the induction of c-Fos and nuclear factor of activated T cells cytoplasmic 1, key transcription factors for osteoclast differentiation. In addition, WEAT inhibited bone resorbing activity of mature osteoclasts. Furthermore, the oral administration of WEAT reduced RANKL-induced bone resorption and trabecular bone loss in mice. Taken together, our study demonstrates that WEAT possesses a protective effect on bone destruction by inhibiting osteoclast differentiation and function.

  9. NRROS Negatively Regulates Osteoclast Differentiation by Inhibiting RANKL-Mediated NF-N:B and Reactive Oxygen Species Pathways.

    PubMed

    Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Seong, Semun; Kim, Nacksung

    2015-10-01

    Negative regulator of reactive oxygen species (NRROS) is known to repress ROS generation in phagocytes. In this study, we examined the roles of NRROS in both osteoclasts and osteoblasts. Our results demonstrate that NRROS negatively regulates the differentiation of osteoclasts, but not osteoblasts. Further, overexpression of NRROS in osteoclast precursor cells attenuates RANKL-induced osteoclast differentiation. Conversely, osteoclast differentiation is enhanced upon siRNA-mediated knockdown of NRROS. Additionally, NRROS attenuates RANKL-induced NF-N:B activation, as well as degradation of the NOX1 and NOX2 proteins, which are required for ROS generation. Based on our observations, we present NRROS as a novel negative regulator of RANKL-induced osteoclastogenesis.

  10. NRROS Negatively Regulates Osteoclast Differentiation by Inhibiting RANKL-Mediated NF-κB and Reactive Oxygen Species Pathways

    PubMed Central

    Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Seong, Semun; Kim, Nacksung

    2015-01-01

    Negative regulator of reactive oxygen species (NRROS) is known to repress ROS generation in phagocytes. In this study, we examined the roles of NRROS in both osteoclasts and osteoblasts. Our results demonstrate that NRROS negatively regulates the differentiation of osteoclasts, but not osteoblasts. Further, overexpression of NRROS in osteoclast precursor cells attenuates RANKL-induced osteoclast differentiation. Conversely, osteoclast differentiation is enhanced upon siRNA-mediated knockdown of NRROS. Additionally, NRROS attenuates RANKL-induced NF-κB activation, as well as degradation of the NOX1 and NOX2 proteins, which are required for ROS generation. Based on our observations, we present NRROS as a novel negative regulator of RANKL-induced osteoclastogenesis. PMID:26442864

  11. STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis

    PubMed Central

    Lee, Jongwon; Seong, Semun; Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-chul; Nam, Kwang-Il; Kim, Kyung Keun; Hennighausen, Lothar; Kim, Nacksung

    2016-01-01

    Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways. PMID:27485735

  12. STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis.

    PubMed

    Lee, Jongwon; Seong, Semun; Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-Chul; Nam, Kwang-Il; Kim, Kyung Keun; Hennighausen, Lothar; Kim, Nacksung

    2016-01-01

    Among the diverse cytokines involved in osteoclast differentiation, interleukin (IL)-3 inhibits RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. Here we demonstrate that the activation of signal transducers and activators of transcription 5 (STAT5) by IL-3 inhibits RANKL-induced osteoclastogenesis through the induction of the expression of Id genes. We found that STAT5 overexpression inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate the expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting a key role of STAT5 in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated the expression of Id1 and Id2, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Importantly, microcomputed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in the STAT5 conditional knockout mice than in the wild-type mice after RANKL injection. Taken together, our findings indicate that STAT5 contributes to the remarkable IL-3-mediated inhibition of RANKL-induced osteoclastogenesis by activating Id genes and their associated pathways. PMID:27485735

  13. Sodium-Dependent Phosphate Transporters in Osteoclast Differentiation and Function

    PubMed Central

    Dolder, Silvia; Siegrist, Mark; Wagner, Carsten A.; Biber, Jürg; Hernando, Nati; Hofstetter, Willy

    2015-01-01

    Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies. PMID:25910236

  14. Sodium-dependent phosphate transporters in osteoclast differentiation and function.

    PubMed

    Albano, Giuseppe; Moor, Matthias; Dolder, Silvia; Siegrist, Mark; Wagner, Carsten A; Biber, Jürg; Hernando, Nati; Hofstetter, Willy; Bonny, Olivier; Fuster, Daniel G

    2015-01-01

    Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

  15. Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways.

    PubMed

    Ma, Xiaojun; Liu, Yupeng; Zhang, Yao; Yu, Xiaobing; Wang, Weiming; Zhao, Dewei

    2014-03-01

    Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.

  16. Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells.

    PubMed

    Kim, Hyun-Ju; Yoon, Hye-Jin; Yoon, Kyung-Ae; Gwon, Mi-Ri; Jin Seong, Sook; Suk, Kyoungho; Kim, Shin-Yoon; Yoon, Young-Ran

    2015-06-10

    Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells.

  17. Specificity of RGS10A as a key component in the RANKL signaling mechanism for osteoclast differentiation

    PubMed Central

    Yang, Shuying; Chen, Wei; Stashenko, Philip; Li, Yi-Ping

    2013-01-01

    Summary Significant progress has been made in studies of the mechanisms by which RANKL induces terminal osteoclast differentiation. However, many crucial details in the RANKL-evoked signaling pathway for osteoclast differentiation remain to be defined. We characterized genes specifically expressed in osteoclasts by differential screening of a human osteoclastoma cDNA library, and found that the regulator of G-protein signaling 10A (RGS10A), but not the RGS10B isoform, was specifically expressed in human osteoclasts. The expression of RGS10A is also induced by RANKL in osteoclast precursors and is prominently expressed in mouse osteoclast-like cells. RGS10A silencing by RNA interference blocked intracellular [Ca2+]i oscillations, the expression of NFAT2, and osteoclast terminal differentiation in both bone marrow cells and osteoclast precursor cell lines. Reintroduction of RGS10A rescued the impaired osteoclast differentiation. RGS10A silencing also resulted in premature osteoclast apoptosis. RGS10A silencing affected the RANKL-[Ca2+]i oscillation-NFAT2 signaling pathway but not other RANKL-induced responses. Our data demonstrate that target components of RGS10A are distinct from those of RGS12 in the RANKL signaling mechanism. Our results thus show the specificity of RGS10A as a key component in the RANKL-evoked signaling pathway for osteoclast differentiation, which may present a promising target for therapeutic intervention. PMID:17881498

  18. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades

    SciTech Connect

    Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei; Ouyang, Zhengxiao; Wu, Chuanlong; Liu, Guangwang; Fan, Qiming; Tang, Tingting; Qin, An; Dai, Kerong

    2014-01-10

    Highlights: •A natural-derived compound, dioscin, suppresses osteoclast formation and bone resorption. •Dioscin inhibits osteolytic bone loss in vivo. •Dioscin impairs the Akt signaling cascades pathways during osteoclastogenesis. •Dioscin have therapeutic value in treating osteoclast-related diseases. -- Abstract: Bone resorption is the unique function of osteoclasts (OCs) and is critical for both bone homeostasis and pathologic bone diseases including osteoporosis, rheumatoid arthritis and tumor bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. In this study, we for the first time demonstrated that dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. Moreover, in vivo studies further verified the bone protection activity of dioscin in osteolytic animal model. Together our data demonstrate that dioscin suppressed RANKL-induced osteoclast formation and function through Akt signaling cascades. Therefore, dioscin is a potential natural agent for the treatment of osteoclast-related diseases.

  19. Fucoidan, a Sulfated Polysaccharide, Inhibits Osteoclast Differentiation and Function by Modulating RANKL Signaling

    PubMed Central

    Kim, Young Woo; Baek, Seung-Hoon; Lee, Sang-Han; Kim, Tae-Ho; Kim, Shin-Yoon

    2014-01-01

    Multinucleated osteoclasts differentiate from hematopoietic progenitors of the monocyte/macrophage lineage. Because of its pivotal role in bone resorption, regulation of osteoclast differentiation is a potential therapeutic approach to the treatment of erosive bone disease. In this study, we have found that fucoidan, a sulfated polysaccharide extracted from brown seaweed, inhibited osteoclast differentiation. In particular, addition of fucoidan into the early stage osteoclast cultures significantly inhibited receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast formation, thus suggesting that fucoidan affects osteoclast progenitors. Furthermore, fucoidan significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases (MAPKs) such as JNK, ERK, and p38, and also c-Fos and NFATc1, which are crucial transcription factors for osteoclastogenesis. In addition, the activation of NF-κB, which is an upstream transcription factor modulating NFATc1 expression, was alleviated in the fucoidan-treated cells. These results collectively suggest that fucoidan inhibits osteoclastogenesis from bone marrow macrophages by inhibiting RANKL-induced p38, JNK, ERK and NF-κB activation, and by downregulating the expression of genes that partake in both osteoclast differentiation and resorption. PMID:25334060

  20. Placotylene A, an Inhibitor of the Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclast Differentiation, from a Korean Sponge Placospongia sp.

    PubMed Central

    Kim, Hiyoung; Kim, Kwang-Jin; Yeon, Jeong-Tae; Kim, Seong Hwan; Won, Dong Hwan; Choi, Hyukjae; Nam, Sang-Jip; Son, Young-Jin; Kang, Heonjoong

    2014-01-01

    A new inhibitor, placotylene A (1), of the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, and a regioisomer of placotylene A, placotylene B (2), were isolated from a Korean marine sponge Placospongia sp. The chemical structures of placotylenes A and B were elucidated on the basis of 1D and 2D NMR, along with MS spectral analysis and revealed as an iodinated polyacetylene class of natural products. Placotylene A (1) displayed inhibitory activity against RANKL-induced osteoclast differentiation at 10 μM while placotylene B (2) did not show any significant activity up to 100 μM, respectively. PMID:24705502

  1. RGS12 Is Essential for RANKL-Evoked Signaling for Terminal Differentiation of Osteoclasts In Vitro

    PubMed Central

    Yang, Shuying; Li, Yi-Ping

    2013-01-01

    How RANKL evokes [Ca2+]i oscillations and leads to osteoclast differentiation is unclear. We identified a new signaling protein, RGS12, and found that RGS12 is essential for [Ca2+]i oscillations and osteoclast differentiation induced by RANKL. RGS12 may play a critical role in the RANKL-evoked PLCγ– calcium channels–[Ca2+]i oscillation–NFAT2 pathway. Introduction RANKL-induced [Ca2+]i oscillations play a switch-on role in NFAT2 expression and osteoclast differentiation. However, RANKL evokes [Ca2+]i oscillations and leads to osteoclast differentiation by an unknown mechanism. In this study, we identified a new RANKL-induced signaling protein, regulator of G signaling protein 12 (RGS12), and investigated its effect on osteoclast differentiation in vitro. Materials and Methods We used a genome-wide screening approach to identify genes that are specifically or prominently expressed in osteoclasts. To study the role of the RGS12 in osteoclast differentiation, we used vector and lentivirus-based RNAi gene silencing technology to silence the RGS12 gene in the monocyte progenitor cell lines and primary bone marrow–derived monocytes (BMMs). The interaction between RGS12 and N-type calcium channels was elucidated using co-immunoprecipitation and immunoblotting. Results We found that RGS12 was prominently expressed in osteoclast-like cells (OLCs) induced by RANKL. This result was further confirmed at both the mRNA and protein level in human osteoclasts and mouse OLCs. Silence of RGS12 expression using vector and lentivirus based RNA interference (RNAi) impaired phosphorylation of phospholipase C (PLC)γ and blocked [Ca2+]i oscillations, NFAT2 expression, and osteoclast differentiation in RANKL-induced RAW264.7 cells and BMMs. We further found that N-type calcium channels were expressed in OLCs after RANKL stimulation and that RGS12 directly interacted with the N-type calcium channels. Conclusions These results reveal that RGS12 is essential for the terminal

  2. Methylsulfonylmethane Inhibits RANKL-Induced Osteoclastogenesis in BMMs by Suppressing NF-κB and STAT3 Activities

    PubMed Central

    Joung, Youn Hee; Darvin, Pramod; Kang, Dong Young; SP, Nipin; Byun, Hyo Joo; Lee, Chi-Ho; Lee, Hak Kyo; Yang, Young Mok

    2016-01-01

    Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3’s involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate

  3. Methylsulfonylmethane Inhibits RANKL-Induced Osteoclastogenesis in BMMs by Suppressing NF-κB and STAT3 Activities.

    PubMed

    Joung, Youn Hee; Darvin, Pramod; Kang, Dong Young; Sp, Nipin; Byun, Hyo Joo; Lee, Chi-Ho; Lee, Hak Kyo; Yang, Young Mok

    2016-01-01

    Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3's involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that

  4. AG490 inhibits NFATc1 expression and STAT3 activation during RANKL induced osteoclastogenesis

    SciTech Connect

    Li, Chang-hong; Zhao, Jin-xia; Sun, Lin; Yao, Zhong-qiang; Deng, Xiao-li; Liu, Rui; Liu, Xiang-yuan

    2013-06-14

    Highlights: •AG490 inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells. •AG490 affects cell proliferation and cell cycle distribution. •AG490 reduces NFATc1 expression during RANKL-induced osteoclastogenesis. •AG490 disrupts the activation of RANKL-mediated JAK2/STAT3 signaling pathway. •STAT3 depletion partly mimics the effect of AG490 on RANKL-induced osteoclastogenesis. -- Abstract: Commonly, JAK/STAT relays cytokine signals for cell activation and proliferation, and recent studies have shown that the elevated expression of JAK/STAT is associated with the immune rejection of allografts and the inflammatory processes of autoimmune disease. However, the role which JAK2/STAT3 signaling plays in the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis is unknown. In this study, we investigated the effects of AG490, specific JAK2 inhibitor, on osteoclast differentiation in vitro. AG490 significantly inhibited osteoclastogenesis in murine osteoclast precursor cell line RAW264.7 induced by RANKL. AG490 suppressed cell proliferation and delayed the G1 to S cell cycle transition. Furthermore, AG490 also suppressed the expression of nuclear factor of activated T cells (NFAT) c1 but not c-Fos in RAW264.7. Subsequently, we investigated various intracellular signaling components associated with osteoclastogenesis. AG490 had no effects on RANKL-induced activation of Akt, ERK1/2. Interestingly, AG490 partly inhibited RANKL-induced phosphorylation of Ser{sup 727} in STAT3. Additionally, down-regulation of STAT3 using siRNA resulted in suppression of TRAP, RANK and NFATc1 expression. In conclusion, we demonstrated that AG490 inhibited RANKL-induced osteoclastogenesis by suppressing NFATc1 production and cell proliferation via the STAT3 pathway. These results suggest that inhibition of JAK2 may be useful for the treatment of bone diseases characterized by excessive osteoclastogenesis.

  5. Eriodicyol inhibits osteoclast differentiation and ovariectomy-induced bone loss in vivo.

    PubMed

    Lee, Juhyun; Noh, A Long Sae Mi; Zheng, Ting; Kang, Ju-hee; Yim, Mijung

    2015-12-10

    Osteoclasts are responsible for bone erosion in diseases such as osteoporosis and rheumatoid arthritis. In the present study, we investigate the effects of eriodictyol, a flavonoid found naturally in citrus fruits, on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation using mouse bone marrow macrophages (BMMs). Eriodictyol inhibited RANKL-induced osteoclast formation in a dose-dependent manner without cytotoxicity. In addition, eriodictyol suppressed bone resorption activity of differentiated osteoclasts. The inhibitory effect of eriodictyol was associated with impaired activation of multiple signaling events downstream of RANK, including extracellular signal-regulated kinase, p38, and c-Jun terminal kinase phosphorylation, followed by decreased nuclear factor of activated T cells (NFAT)c1 expression. Ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of eriodictyol, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Consistent with the in vitro anti-osteoclastogenic effect, eriodictyol suppressed lipopolysaccharide-induced osteoclast formation in the calvarial model and ovariectomy-induced bone loss in vivo. Taken together, our data demonstrate that eriodictyol is a new therapeutic agent with the potential to prevent bone destructive diseases by reducing both osteoclast differentiation and function.

  6. Inhibition of differentiation and function of osteoclasts by dimethyl sulfoxide (DMSO).

    PubMed

    Yang, Chunxi; Madhu, Vedavathi; Thomas, Candace; Yang, Xinlin; Du, Xeujun; Dighe, Abhijit S; Cui, Quanjun

    2015-12-01

    Dimethyl sulfoxide (DMSO) is an FDA-approved organosulfur solvent that is reported to have therapeutic value in osteoarthritis and osteopenia. DMSO is used as a cryoprotectant for the cryopreservation of bone grafts and mesenchymal stem cells which are later used for bone repair. It is also used as a solvent in the preparation of various scaffolds used for bone tissue engineering purposes. DMSO has been reported to inhibit osteoclast formation in vitro but the mechanism involved has remained elusive. We investigated the effect of DMSO on osteoclast differentiation and function using a conventional model system of RAW 264.7 cells. The differentiation of RAW 264.7 cells was induced by adding 50 ng/ml RANKL and the effect of DMSO (0.01 and 1% v/v) on RANKL-induced osteoclastogenesis was investigated. Addition of 1% DMSO significantly inhibited RANKL-induced formation of TRAP+, multinucleated, mature osteoclasts and osteoclast late-stage precursors (c-Kit(-) c-Fms(+) Mac-1(+) RANK(+)). While DMSO did not inhibit proliferation per se, it did inhibit the effect of RANKL on proliferation of RAW 264.7 cells. Key genes related to osteoclast function (TRAP, Integrin αVβ3, Cathepsin K and MMP9) were significantly down-regulated by DMSO. RANKL-induced expression of RANK gene was significantly reduced in the presence of DMSO. Our data, and reports from other investigators, that DMSO enhances osteoblastic differentiation of mesenchymal stem cells and also prevents bone loss in ovarietcomized rats, suggest that DMSO has tremendous potential in the treatment of osteoporosis and bone diseases arising from uncontrolled activities of the osteoclasts.

  7. ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation

    PubMed Central

    Kim, Min Jae; Kim, Hyunsoo; Lee, Seoung Hoon; Gu, Dong Ryun; Lee, Soo Young; Lee, Kyunghee; Jeong, Daewon

    2015-01-01

    Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation PMID:26690137

  8. ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation.

    PubMed

    Kim, Min Jae; Kim, Hyunsoo; Lee, Seoung Hoon; Gu, Dong Ryun; Lee, Soo Young; Lee, Kyunghee; Jeong, Daewon

    2015-12-09

    Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation.

  9. The Protein Kinase 2 Inhibitor CX-4945 Regulates Osteoclast and Osteoblast Differentiation In Vitro

    PubMed Central

    Son, You Hwa; Moon, Seong Hee; Kim, Jiyeon

    2013-01-01

    Drug repositioning can identify new therapeutic applications for existing drugs, thus mitigating high R&D costs. The Protein kinase 2 (CK2) inhibitor CX-4945 regulates human cancer cell survival and angiogenesis. Here we found that CX-4945 significantly inhibited the RANKL-induced osteoclast differentiation, but enhanced the BMP2-induced osteoblast differentiation in a cell culture model. CX-4945 inhibited the RANKL-induced activation of TRAP and NFATc1 expression accompanied with suppression of Akt phosphorylation, but, in contrast, it enhanced the BMP2-mediated ALP induction and MAPK ERK1/2 phosphorylation. CX-4945 is thus a novel drug candidate for bone-related disorders such as osteoporosis. PMID:24293011

  10. Cytoplasmic hnRNPK interacts with GSK3β and is essential for the osteoclast differentiation

    PubMed Central

    Fan, Xiaoqin; Xiong, Haiting; Wei, Jinmei; Gao, Xuejuan; Feng, Yuan; Liu, Xiaohui; Zhang, Gong; He, Qing-Yu; Xu, Jiake; Liu, Langxia

    2015-01-01

    Osteoclast differentiation is a complex and finely regulated physiological process that involves a variety of signaling pathways and factors. Recent studies suggested that the Ser9 phosphorylation of Glycogen synthase kinase-3β (GSK3β) is required for the osteoclast differentiation. However, the precise underlying mechanism remains unclear. We have previously identified the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a putative GSK3β interactor. In the present study, we demonstrate that, during the RANKL-induced osteoclast differentiation, the PI3K/Akt-mediated Ser9 phosphorylation of GSK3β provokes the nuclear-cytoplasmic translocation of hnRNPK in an ERK-dependent manner, enhancing the cytoplasmic co-localization and interaction of GSK3β and hnRNPK. We show that hnRNPK is essential for the osteoclast differentiation, and is involved in several reported functions of GSK3β, including the activation of NF-κB, the expression of NFATc1, and the acetylation of tubulin, all known to be critical for osteoclast differentiation and functions. We find that hnRNPK is localized in the actin belt, and is important for the mature osteoclast formation. Taken together, we demonstrate here the critical role of hnRNPK in osteoclast differentiation, and depict a model in which the cytoplasmic hnRNPK interacts with GSK3β and regulates its function. PMID:26638989

  11. Geraniin suppresses RANKL-induced osteoclastogenesis in vitro and ameliorates wear particle-induced osteolysis in mouse model

    SciTech Connect

    Xiao, Fei; Zhai, Zanjing; Jiang, Chuan; Liu, Xuqiang; Li, Haowei; Qu, Xinhua; Ouyang, Zhengxiao; Fan, Qiming; Tang, Tingting; Qin, An; Gu, Dongyun

    2015-01-01

    Wear particle-induced osteolysis and subsequent aseptic loosening remains the most common complication that limits the longevity of prostheses. Wear particle-induced osteoclastogenesis is known to be responsible for extensive bone erosion that leads to prosthesis failure. Thus, inhibition of osteoclastic bone resorption may serve as a therapeutic strategy for the treatment of wear particle induced osteolysis. In this study, we demonstrated for the first time that geraniin, an active natural compound derived from Geranium thunbergii, ameliorated particle-induced osteolysis in a Ti particle-induced mouse calvaria model in vivo. We also investigated the mechanism by which geraniin exerts inhibitory effects on osteoclasts. Geraniin inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, evidenced by reduced osteoclast formation and suppressed osteoclast specific gene expression. Specially, geraniin inhibited actin ring formation and bone resorption in vitro. Further molecular investigation demonstrated geraniin impaired osteoclast differentiation via the inhibition of the RANKL-induced NF-κB and ERK signaling pathways, as well as suppressed the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Collectively, our data suggested that geraniin exerts inhibitory effects on osteoclast differentiation in vitro and suppresses Ti particle-induced osteolysis in vivo. Geraniin is therefore a potential natural compound for the treatment of wear particle induced osteolysis in prostheses failure. - Highlights: • Geraniin suppresses osteoclasts formation and function in vitro. • Geraniin impairs RANKL-induced nuclear factor-κB and ERK signaling pathway. • Geraniin suppresses osteolysis in vivo. • Geraniin may be used for treating osteoclast related diseases.

  12. Salicortin inhibits osteoclast differentiation and bone resorption by down-regulating JNK and NF-κB/NFATc1 signaling pathways.

    PubMed

    Nie, Shaobo; Xu, Jiawei; Zhang, Chenghua; Xu, Chen; Liu, Ming; Yu, Degang

    2016-01-29

    Receptor activator of nuclear factor (NF)-κB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation, and survival. Salicortin is a phenolic glycoside that has been isolated from many plants such as Populus and Salix species, and has been shown to have anti-amnesic and anti-adipogenic effects. In this study, we investigated the effect of salicortin on RANKL-induced osteoclasts formation, bone resorption, and activation of osteoclast-related signaling pathways. Salicortin suppressed RANKL-induced osteoclastogenesis in bone marrow macrophage cultures in a dose-dependent manner, and inhibited osteoclastic bone resorption activity without any cytotoxicity. Salicortin inhibited RANKL-induced c-Jun N-terminal kinase and NF-κB activation, concomitant with retarded IκBα phosphorylation and inhibition of p65 nuclear translocation, leading to impaired transcription of nuclear factor of activated T cells c1 (NFATc1) and expression of osteoclastic-specific genes. Taken together, our findings demonstrate that salicortin inhibits NF-κB and NFATc1 activation, leading to attenuation of osteoclastogenesis and bone resorption. Thus, salicortin may be of interest in developments of treatment for osteoclast related diseases.

  13. Mst2 Controls Bone Homeostasis by Regulating Osteoclast and Osteoblast Differentiation.

    PubMed

    Lee, Jongwon; Youn, Bang Ung; Kim, Kabsun; Kim, Jung Ha; Lee, Da-Hye; Seong, Semun; Kim, Inyoung; Han, Seung-Hee; Che, Xiangguo; Choi, Je-Yong; Park, Yong-Wook; Kook, Hyun; Kim, Kyung Keun; Lim, Dae-Sik; Kim, Nacksung

    2015-09-01

    Mammalian sterile 20-like kinase 2 (Mst2) plays a central role in the Hippo pathway, controlling cell proliferation, differentiation, and apoptosis during development. However, the roles of Mst2 in osteoclast and osteoblast development are largely unknown. Here, we demonstrate that mice deficient in Mst2 exhibit osteoporotic phenotypes with increased numbers of osteoclasts and decreased numbers of osteoblasts as shown by micro-computed tomography (µCT) and histomorphometric analyses. Osteoclast precursors lacking Mst2 exhibit increased osteoclastogenesis and Nfatc1, Acp5, and Oscar expression in response to receptor activator of NF-κB ligand (RANKL) exposure. Conversely, Mst2 overexpression in osteoclast precursors leads to the inhibition of RANKL-induced osteoclast differentiation. Osteoblast precursors deficient in Mst2 exhibit attenuated osteoblast differentiation and function by downregulating the expression of Runx2, Alpl, Ibsp, and Bglap. Conversely, ectopic expression of Mst2 in osteoblast precursors increases osteoblastogenesis. Finally, we demonstrate that the NF-κB pathway is activated by Mst2 deficiency during osteoclast and osteoblast development. Our findings suggest that Mst2 is involved in bone homeostasis, functioning as a reciprocal regulator of osteoclast and osteoblast differentiation through the NF-κB pathway.

  14. (-)-Epigallocatechin gallate inhibition of osteoclastic differentiation via NF-{kappa}B

    SciTech Connect

    Lin, R.-W.; Chen, C.-H.; Wang, Y.-H.; Ho, M.-L.; Hung, S.-H.; Chen, I.-S. Wang, G.-J.

    2009-02-20

    People who regularly drink tea have been found to have a higher bone mineral density (BMD) and to be at less risk of hip fractures than those who do not drink it. Green tea catechins such as (-)-epigallocatechin gallate (EGCG) have been reported to increase osteogenic functioning in mesenchymal stem cells. However, its effect on osteoclastogenesis remains unclear. In this study, we investigated the effect of EGCG on RANKL-activation osteoclastogenesis and NF-{kappa}B in RAW 264.7, a murine preosteoclast cell line. EGCG (10-100 {mu}M) significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in murine RAW 264.7 cells and bone marrow macrophages (BMMs). EGCG appeared to target osteoclastic differentiation at an early stage but had no cytotoxic effect on osteoclast precursors. In addition, it significantly inhibited RANKL-induced NF-{kappa}B transcriptional activity and nuclear translocation. We conclude that EGCG inhibits osteoclastogenesis through its activation of NF-{kappa}B.

  15. Lanthanum Chloride Attenuates Osteoclast Formation and Function Via the Downregulation of Rankl-Induced Nf-κb and Nfatc1 Activities.

    PubMed

    Jiang, Chuan; Shang, Jiangyinzi; Li, Zhe; Qin, An; Ouyang, Zhengxiao; Qu, Xinhua; Li, Haowei; Tian, Bo; Wang, Wengang; Wu, Chuanlong; Wang, Jinwu; Dai, Min

    2016-01-01

    The biological activities of lanthanum chloride (LaCl3 ) and the molecular mechanisms of action underlying its anti-inflammatory, anti-hyperphosphatemic, and osteoblast-enhancing effects have been studied previously, but less is known about the effects of LaCl3 on osteoclasts. The present study used in vivo and in vitro approaches to explore the effects of LaCl3 on osteoclasts and osteolysis. The results indicated that LaCl3 concentrations that were non-cytotoxic to mouse bone marrow-derived monocytes attenuated receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis, bone resorption, mRNA expression of osteoclastogenic genes in these cells, including cathepsin K, calcitonin receptor, and tartrate-resistant acid phosphatase (TRAP). Further, LaCl3 inhibited RANKL-mediated activation of the nuclear factor-κB (NF-κB) signaling pathway, and downregulated mRNA and protein levels of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), and c-fos. In vivo, LaCl3 attenuated titanium (Ti) particle-induced bone loss in a murine calvarial osteolysis model. Histological analyses revealed that LaCl3 ameliorated bone destruction and decreased the number of TRAP-positive osteoclasts in this model. These results demonstrated that LaCl3 inhibited osteoclast formation, function, and osteoclast-specific gene expression in vitro, and attenuated Ti particle-induced mouse calvarial osteolysis in vivo, where the inhibition of NF-κB signaling and downregulation of NFATc1 and c-fos played an important role.

  16. Inactivation of Glycogen Synthase Kinase-3β Is Required for Osteoclast Differentiation*

    PubMed Central

    Jang, Hyun Duk; Shin, Ji Hye; Park, Doo Ri; Hong, Jin Hee; Yoon, Kwiyeom; Ko, Ryeojin; Ko, Chang-Yong; Kim, Han-Sung; Jeong, Daewon; Kim, Nacksung; Lee, Soo Young

    2011-01-01

    Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3β in osteoblasts is well characterized as a negative regulator of β-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3β inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3β activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3β (GSK3β-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3β (GSK3β-K85R) or small interfering RNA (siRNA)-mediated GSK-3β silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3β further confirmed the negative role of GSK-3β in osteoclast formation. We also show that overexpression of the GSK3β-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca2+ oscillations. Remarkably, transgenic mice expressing the GSK3β-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3β in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling. PMID:21949120

  17. Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

    SciTech Connect

    Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae; E-mail: jwoo@isc.chubu.ac.jp

    2007-03-30

    Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-{kappa}B ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway.

  18. Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells

    PubMed Central

    Kim, Bong Gyu; Kwak, Han Bok; Choi, Eun-Yong; Kim, Hun Soo; Kim, Myung Hee; Kim, Seong Hwan; Choi, Min-Kyu; Chun, Churl Hong; Oh, Jaemin

    2010-01-01

    Among the several rotenoids, amorphigenin is isolated from the leaves of Amopha Fruticosa and it is known that has anti-proliferative effects and anti-cnacer effects in many cell types. The main aim of this study was to investigate the effects of amorphigenin on osteoclast differentiation in vitro and on LPS treated inflammatory bone loss model in vivo. We show here that amorphigenin inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages in a dose dependent manner without cellular toxicity. Anti-osteoclastogenic properties of amorphigenin were based on a down-regulation of c-fos and NFATc1. Amorphigenin markedly inhibited RANKL-induced p38 and NF-κB pathways, but other pathways were not affected. Micro-CT analysis of the femurs showed that amorphigenin protected the LPS-induced bone loss. We concluded that amorphigenin can prevent inflammation-induced bone loss. Thus we expect that amorphigenin could be a treatment option for bone erosion caused by inflammation. PMID:21267405

  19. The 1,2,3-triazole derivative KP-A021 suppresses osteoclast differentiation and function by inhibiting RANKL-mediated MEK-ERK signaling pathway.

    PubMed

    Ihn, Hye Jung; Lee, Doohyun; Lee, Taeho; Shin, Hong-In; Bae, Yong Chul; Kim, Sang-Hyun; Park, Eui Kyun

    2015-12-01

    The triazole family of compounds has been implicated in modulating various biological processes such as inflammation, tumorigenesis, and infection. To our knowledge, this is the first study to demonstrate the effects of 1,2,3-triazole substituted biarylacrylonitrile compounds, including KP-A021, on the differentiation and function of osteoclasts. KP-A021 and its triazole derivatives, at a concentration that does not cause a cytotoxic response in bone marrow macrophages (BMMs), significantly inhibited osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as assessed by tartrate-resistant acid phosphatase (TRAP) staining. KP-A021 also dramatically inhibited the expression of marker genes associated with osteoclast differentiation, such as TRAP, cathepsin K (Cat K), dendritic cell-specific transmembrane protein (DC-STAMP), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). Furthermore, KP-A021 inhibited actin ring formation in osteoclasts as well as resorption pit formation induced by osteoclasts. Analysis of the signaling pathway for KP-A021 indicated that this triazole compound inhibited the RANKL-induced activation of extracellular signal-regulated kinase (ERK) and its upstream signaling molecule, mitogen-activated protein kinase kinase1/2 (MEK1/2). Taken together, these results demonstrate that KP-A021 has an inhibitory effect on the differentiation and function of osteoclasts via modulation of the RANKL-induced activation of the MEK-ERK pathway.

  20. Diamagnetic levitation promotes osteoclast differentiation from RAW264.7 cells.

    PubMed

    Sun, Yu-Long; Chen, Zhi-Hao; Chen, Xiao-Hu; Yin, Chong; Li, Di-Jie; Ma, Xiao-Li; Zhao, Fan; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

    2015-03-01

    The superconducting magnet with a high magnetic force field can levitate diamagnetic materials. In this study, a specially designed superconducting magnet with large gradient high magnetic field (LGHMF), which provides three apparent gravity levels (μg, 1 g, and 2 g), was used to study its influence on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation from preosteoclast cell line RAW264.7. The effects of LGHMF on the viability, nitric oxide (NO) production, morphology in RAW264.7 cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the Griess method, and the immunofluorescence staining, respectively. The changes induced by LGHMF in osteoclast formation, mRNA expression, and bone resorption were determined by tartrate-resistant acid phosphatase staining, semiquantity PCR, and bone resorption test, respectively. The results showed that: 1) LGHMF had no lethal effect on osteoclast precursors but attenuated NO release in RAW264.7 cells. 2) Diamagnetic levitation (μg) enhanced both the formation and bone resorption capacity of osteoclast. Moreover, diamagnetic levitation up-regulated mRNA expression of RANK, Cathepsin K, MMP-9, and NFATc1, while down-regulated RunX2 in comparison with controls. Furthermore, diamagnetic levitation induced obvious morphological alterations in osteoclast, including active cytoplasmic peripheral pseudopodial expansion, formation of pedosome belt, and aggregation of actin ring. 3) Magnetic field produced by LGHMF attenuated osteoclast resorption activity. Collectively, LGHMF with combined effects has multiple effects on osteoclast, which attenuated osteoclast resorption with magnetic field, whereas promoted osteoclast differentiation with diamagnetic levitation. Therefore, these findings indicate that diamagnetic levitation could be used as a novel ground-based microgravity simulator, which facilitates bone cell research of weightlessness condition

  1. Diamagnetic levitation promotes osteoclast differentiation from RAW264.7 cells.

    PubMed

    Sun, Yu-Long; Chen, Zhi-Hao; Chen, Xiao-Hu; Yin, Chong; Li, Di-Jie; Ma, Xiao-Li; Zhao, Fan; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

    2015-03-01

    The superconducting magnet with a high magnetic force field can levitate diamagnetic materials. In this study, a specially designed superconducting magnet with large gradient high magnetic field (LGHMF), which provides three apparent gravity levels (μg, 1 g, and 2 g), was used to study its influence on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation from preosteoclast cell line RAW264.7. The effects of LGHMF on the viability, nitric oxide (NO) production, morphology in RAW264.7 cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the Griess method, and the immunofluorescence staining, respectively. The changes induced by LGHMF in osteoclast formation, mRNA expression, and bone resorption were determined by tartrate-resistant acid phosphatase staining, semiquantity PCR, and bone resorption test, respectively. The results showed that: 1) LGHMF had no lethal effect on osteoclast precursors but attenuated NO release in RAW264.7 cells. 2) Diamagnetic levitation (μg) enhanced both the formation and bone resorption capacity of osteoclast. Moreover, diamagnetic levitation up-regulated mRNA expression of RANK, Cathepsin K, MMP-9, and NFATc1, while down-regulated RunX2 in comparison with controls. Furthermore, diamagnetic levitation induced obvious morphological alterations in osteoclast, including active cytoplasmic peripheral pseudopodial expansion, formation of pedosome belt, and aggregation of actin ring. 3) Magnetic field produced by LGHMF attenuated osteoclast resorption activity. Collectively, LGHMF with combined effects has multiple effects on osteoclast, which attenuated osteoclast resorption with magnetic field, whereas promoted osteoclast differentiation with diamagnetic levitation. Therefore, these findings indicate that diamagnetic levitation could be used as a novel ground-based microgravity simulator, which facilitates bone cell research of weightlessness condition.

  2. RGS10-null mutation impairs osteoclast differentiation resulting from the loss of [Ca2+]i oscillation regulation

    PubMed Central

    Yang, Shuying; Li, Yi-Ping

    2007-01-01

    Increased osteoclastic resorption leads to many bone diseases, including osteoporosis and rheumatoid arthritis. While rapid progress has been made in characterizing osteoclast differentiation signaling pathways, how receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) evokes essential [Ca2+]i oscillation signaling remains unknown. Here, we characterized RANKL-induced signaling proteins and found regulator of G-protein signaling 10 (RGS10) is predominantly expressed in osteoclasts. We generated RGS10-deficient (RGS10−/−) mice that exhibited severe osteopetrosis and impaired osteoclast differentiation. Our data demonstrated that ectopic expression of RGS10 dramatically increased the sensitivity of osteoclast differentiation to RANKL signaling; the deficiency of RGS10 resulted in the absence of [Ca2+]i oscillations and loss of NFATc1; ectopic NFATc1 expression rescues impaired osteoclast differentiation from deletion of RGS10; phosphatidylinositol 3,4,5-trisphosphate (PIP3) is essential to PLCγ activation; and RGS10 competitively interacts with Ca2+/calmodulin and PIP3 in a [Ca2+]i-dependent manner to mediate PLCγ activation and [Ca2+]i oscillations. Our results revealed a mechanism through which RGS10 specifically regulates the RANKL-evoked RGS10/calmodulin–[Ca2+]i oscillation–calcineurin–NFATc1 signaling pathway in osteoclast differentiation using an in vivo model. RGS10 provides a potential therapeutic target for the treatment of bone diseases. PMID:17626792

  3. Irreversible inhibition of RANK expression as a possible mechanism for IL-3 inhibition of RANKL-induced osteoclastogenesis

    SciTech Connect

    Khapli, Shruti M.; Tomar, Geetanjali B.; Barhanpurkar, Amruta P.; Gupta, Navita; Yogesha, S.D.; Pote, Satish T.; Wani, Mohan R.

    2010-09-03

    Research highlights: {yields} IL-3 inhibits receptor activator of NF-{kappa}B ligand (RANKL)-induced osteoclastogenesis. {yields} IL-3 inhibits RANKL-induced JNK activation. {yields} IL-3 down-regulates expression of c-Fos and NFATc1 transcription factors. {yields} IL-3 down-regulates RANK expression posttranscriptionally and irreversibly. {yields} IL-3 inhibits in vivo RANK expression. -- Abstract: IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation and survival of pluripotent hematopoietic stem cells. In this study, we investigated the mechanism of inhibitory action of IL-3 on osteoclast differentiation. We show here that IL-3 significantly inhibits receptor activator of NF-{kappa}B (RANK) ligand (RANKL)-induced activation of c-Jun N-terminal kinase (JNK). IL-3 down-regulates expression of c-Fos and nuclear factor of activated T cells (NFATc1) transcription factors. In addition, IL-3 down-regulates RANK expression posttranscriptionally in both purified osteoclast precursors and whole bone marrow cells. Furthermore, the inhibitory effect of IL-3 on RANK expression was irreversible. Interestingly, IL-3 inhibits in vivo RANK expression in mice. Thus, we provide the first evidence that IL-3 irreversibly inhibits RANK expression that results in inhibition of important signaling molecules induced by RANKL.

  4. Aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells

    PubMed Central

    Zeng, Yan-Ping; Yang, Chao; Li, Yuan; Fan, Yong; Yang, Hong-Jun; Liu, Bin; Sang, Hong-Xun

    2016-01-01

    Aspirin is a commonly used medicine as an effective antipyretic, analgesic and anti-inflammatory drug. Previous studies have demonstrated its potential effects of anti-postmenopausal osteoporosis, while the molecular mechanisms remain unclear. The effects of aspirin on receptor-activator of nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclasts were investigated in RAW264.7 cells in the current study. Using tartrate-resistant acid phosphatase (TRAP) staining, it was observed that aspirin inhibited the differentiation of RANKL-induced RAW264.7 cells. The mRNA expression of osteoclastic marker genes, including cathepsin K, TRAP, matrix metalloproteinase 9 and calcitonin receptor, were suppressed by aspirin as identified using reverse transcription-quantitative polymerase chain reaction analysis. The immunofluorescence assay indicated that aspirin markedly inhibited NF-κB p65 translocation to the nucleus in RANKL-induced RAW264.7 cells. In addition, aspirin also suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), observed by western blot analysis. Taken together, these data identified that aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells, implying that aspirin may possess therapeutic potential for use in the prevention and treatment of osteoporosis. PMID:27430169

  5. Synchronized Cell Cycle Arrest Promotes Osteoclast Differentiation

    PubMed Central

    Kwon, Minsuk; Kim, Jin-Man; Lee, Kyunghee; Park, So-Young; Lim, Hyun-Sook; Kim, Taesoo; Jeong, Daewon

    2016-01-01

    Osteoclast progenitors undergo cell cycle arrest before differentiation into osteoclasts, induced by exposure to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The role of such cell cycle arrest in osteoclast differentiation has remained unclear, however. We here examined the effect of synchronized cell cycle arrest on osteoclast formation. Osteoclast progenitors deprived of M-CSF in culture adopted a uniform morphology and exhibited cell cycle arrest at the G0–G1 phase in association with both down-regulation of cyclins A and D1 as well as up-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Such M-CSF deprivation also promoted the differentiation of osteoclast progenitors into multinucleated osteoclasts expressing high levels of osteoclast marker proteins such as NFATc1, c-Fos, Atp6v0d2, cathepsin K, and integrin β3 on subsequent exposure to M-CSF and RANKL. Our results suggest that synchronized arrest and reprogramming of osteoclast progenitors renders them poised to respond to inducers of osteoclast formation. Further characterization of such effects may facilitate induction of the differentiation of heterogeneous and multipotent cells into desired cell lineages. PMID:27517906

  6. Synchronized Cell Cycle Arrest Promotes Osteoclast Differentiation.

    PubMed

    Kwon, Minsuk; Kim, Jin-Man; Lee, Kyunghee; Park, So-Young; Lim, Hyun-Sook; Kim, Taesoo; Jeong, Daewon

    2016-01-01

    Osteoclast progenitors undergo cell cycle arrest before differentiation into osteoclasts, induced by exposure to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The role of such cell cycle arrest in osteoclast differentiation has remained unclear, however. We here examined the effect of synchronized cell cycle arrest on osteoclast formation. Osteoclast progenitors deprived of M-CSF in culture adopted a uniform morphology and exhibited cell cycle arrest at the G₀-G₁ phase in association with both down-regulation of cyclins A and D1 as well as up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Such M-CSF deprivation also promoted the differentiation of osteoclast progenitors into multinucleated osteoclasts expressing high levels of osteoclast marker proteins such as NFATc1, c-Fos, Atp6v0d2, cathepsin K, and integrin β3 on subsequent exposure to M-CSF and RANKL. Our results suggest that synchronized arrest and reprogramming of osteoclast progenitors renders them poised to respond to inducers of osteoclast formation. Further characterization of such effects may facilitate induction of the differentiation of heterogeneous and multipotent cells into desired cell lineages. PMID:27517906

  7. Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation

    PubMed Central

    Yu, Chunxiao; Zhang, Xu; Zhang, Haiqing; Guan, Qingbo; Zhao, Jiajun; Xu, Jin

    2015-01-01

    Objective The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH) and bone mineral density (BMD) in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells. Methods We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA) was used to detect serum FSH, luteinizing hormone (LH), and estradiol (E2). Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E2. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL), and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP) staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank), Trap, matrix metalloproteinase-9 (Mmp-9) and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction). Results 1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner. Conclusions The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro. PMID:26241313

  8. Fisetin Inhibits Osteoclast Differentiation via Downregulation of p38 and c-Fos-NFATc1 Signaling Pathways

    PubMed Central

    Choi, Sik-Won; Son, Young-Jin; Yun, Jung-Mi; Kim, Seong Hwan

    2012-01-01

    The prevention or therapeutic treatment of loss of bone mass is an important means of improving the quality of life for patients with disorders related to osteoclast-mediated bone loss. Fisetin, a flavonoid dietary ingredient found in the smoke tree (Continus coggygria), exhibits various biological activities, but its effect on osteoclast differentiation is unknown. In this study, fisetin dose-dependently inhibited the RANKL-induced osteoclast differentiation with downregulation of the activity or expression of p38, c-Fos, and NFATc1 signaling molecules. The p38/c-Fos/NFATc1-regulated expression of genes required for cell fusion and bone resorption, such as DC-STAMP and cathepsin K, was also inhibited by fisetin. Considering the rescue of fisetin's inhibitory action by NFATc1 over-expression, the cascade of p38-c-Fos-NFATc1 could be strongly involved in the inhibitory effect of fisetin on osteoclast differentiation. Furthermore, fisetin inhibited the bone-resorbing activity of mature osteoclasts. In conclusion, fisetin may be of use in the treatment of osteoclast-related disorders, including osteoporosis. PMID:23008743

  9. Induction of c-Fos and NFATc1 during RANKL-stimulated osteoclast differentiation is mediated by the p38 signaling pathway

    SciTech Connect

    Huang, Hao; Chang, Eun-Ju; Ryu, Jiyoon; Lee, Zang Hee; Lee, Youngkyun . E-mail: yklee@snu.ac.kr; Kim, Hong-Hee . E-mail: hhbkim@snu.ac.kr

    2006-12-08

    The crucial role of p38 mitogen-activated protein kinase for osteoclast differentiation has been suggested from studies with specific pharmacological inhibitors and dominant-negative forms of p38. However, the targets through which p38 regulates osteoclast differentiation have not been clearly revealed. Here, we show that inhibition of p38 activity with SB203580 reduced osteoclastogenesis from primary precursor cells, with concomitant suppression in the induction of both c-Fos and nuclear factor of activated T cells (NFAT) c1 by receptor activator of nuclear factor {kappa}B ligand (RANKL), the key osteoclast differentiation factor. Overexpression of dominant-negative forms of p38 upstream kinases MKK3 and MKK6 elicited similar reduction in RANKL-stimulated elevation of c-Fos and NFATc1. Interestingly, overexpression of c-Fos restored RANKL-induced osteoclast differentiation from and NFATc1 expression in SB203580-treated precursor cells. Our results demonstrate a previously unknown function of the p38 pathway in up-regulating c-Fos and NFATc1 expression during RANKL-induced osteoclastogenesis.

  10. Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

    SciTech Connect

    Hiruma, Yoshiharu; Hirai, Takehiro; Tsuda, Eisuke

    2011-06-10

    Highlights: {yields} Siglec-15 was identified as a gene overexpressed in giant cell tumor. {yields} Siglec-15 mRNA expression increased in association with osteoclast differentiation. {yields} Polyclonal antibody to Siglec-15 inhibited osteoclast differentiation in vitro. -- Abstract: Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor {kappa}B ligand (RANKL) or tumor necrosis factor (TNF)-{alpha}. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D{sub 3} and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.

  11. Ly49Q, an ITIM-bearing NK receptor, positively regulates osteoclast differentiation

    SciTech Connect

    Hayashi, Mikihito; Nakashima, Tomoki; Kodama, Tatsuhiko; Makrigiannis, Andrew P.; Toyama-Sorimachi, Noriko; Takayanagi, Hiroshi

    2010-03-12

    Osteoclasts, multinucleated cells that resorb bone, play a key role in bone remodeling. Although immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling is critical for osteoclast differentiation, the significance of immunoreceptor tyrosine-based inhibitory motif (ITIM) has not been well understood. Here we report the function of Ly49Q, an Ly49 family member possessing an ITIM motif, in osteoclastogenesis. Ly49Q is selectively induced by receptor activator of nuclear factor-{kappa}B (NF-{kappa}B) ligand (RANKL) stimulation in bone marrow-derived monocyte/macrophage precursor cells (BMMs) among the Ly49 family of NK receptors. The knockdown of Ly49Q resulted in a significant reduction in the RANKL-induced formation of tartrate-resistance acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a decreased expression of osteoclast-specific genes such as Nfatc1, Tm7sf4, Oscar, Ctsk, and Acp5. Osteoclastogenesis was also significantly impaired in Ly49Q-deficient cells in vitro. The inhibitory effect of Ly49Q-deficiency may be explained by the finding that Ly49Q competed for the association of Src-homology domain-2 phosphatase-1 (SHP-1) with paired immunoglobulin-like receptor-B (PIR-B), an ITIM-bearing receptor which negatively regulates osteoclast differentiation. Unexpectedly, Ly49Q deficiency did not lead to impaired osteoclast formation in vivo, suggesting the existence of a compensatory mechanism. This study provides an example in which an ITIM-bearing receptor functions as a positive regulator of osteoclast differentiation.

  12. Niclosamide suppresses RANKL-induced osteoclastogenesis and prevents LPS-induced bone loss.

    PubMed

    Cheon, Yoon-Hee; Kim, Ju-Young; Baek, Jong Min; Ahn, Sung-Jun; So, Hong-Seob; Oh, Jaemin

    2016-02-01

    Niclosamide (5-chloro-salicyl-(2-chloro-4-nitro) anilide) is an oral anthelmintic drug used for treating intestinal infection of most tapeworms. Recently, niclosamide was shown to have considerable efficacy against some tumor cell lines, including colorectal, prostate, and breast cancers, and acute myelogenous leukemia. Specifically, the drug was identified as a potent inhibitor of signal transducer and activator of transcription 3 (STAT3), which is associated with osteoclast differentiation and function. In this study, we assessed the effect of niclosamide on osteoclastogenesis in vitro and in vivo. Our in vitro study showed that receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation was inhibited by niclosamide, due to inhibition of serine-threonine protein kinase (Akt) phosphorylation, inhibitor of nuclear factor-kappaB (IκB), and STAT3 serine(727). Niclosamide decreased the expression of the major transcription factors c-Fos and NFATc1, and thereafter abrogated the mRNA expression of osteoclast-specific genes, including TRAP, OSCAR, αv/β3 integrin (integrin αv, integrin β3), and cathepsin K (CtsK). In an in vivo model, niclosamide prevented lipopolysaccharide-induced bone loss by diminishing osteoclast activity. Taken together, our results show that niclosamide is effective in suppressing osteoclastogenesis and may be considered as a new and safe therapeutic candidate for the clinical treatment of osteoclast-related diseases such as osteoporosis. PMID:26792726

  13. Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1

    PubMed Central

    Chen, Yueqi; Sun, Jingjing; Dou, Ce; Li, Nan; Kang, Fei; Wang, Yuan; Cao, Zhen; Yang, Xiaochao; Dong, Shiwu

    2016-01-01

    The healthy skeleton requires a perfect coordination of the formation and degradation of bone. Metabolic bone disease like osteoporosis is resulted from the imbalance of bone formation and/or bone resorption. Osteoporosis also reflects lower level of bone matrix, which is contributed by up-regulated osteoclast-mediated bone resorption. It is reported that monocytes/macrophage progenitor cells or either hematopoietic stem cells (HSCs) gave rise to multinucleated osteoclasts. Thus, inhibition of osteoclastic bone resorption generally seems to be a predominant therapy for treating osteoporosis. Recently, more and more natural compounds have been discovered, which have the ability of inhibiting osteoclast differentiation and fusion. Alliin (S-allyl-l-cysteine sulfoxides, SACSO) is the major component of aged garlic extract (AGE), bearing broad-spectrum natural antioxidant properties. However, its effects on bone health have not yet been explored. Hence, we designed the current study to explore its effects and role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast fusion and differentiation. It was revealed that alliin had an inhibitory effect in osteoclasteogenesis with a dose-dependent manner via blocking the c-Fos-NFATc1 signaling pathway. In addition, alliin decreased the generation of reactive oxygen species (ROS) and down-regulated the expression of NADPH oxidase 1 (Nox1). The overall results revealed that alliin could be a potential therapeutic agent in the treatment of osteoporosis. PMID:27657047

  14. Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

    PubMed

    Xie, Wenjie; Lorenz, Sebastian; Dolder, Silvia; Hofstetter, Willy

    2016-03-01

    Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

  15. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

    NASA Astrophysics Data System (ADS)

    Kuo, Chun-Liang; Kao, Chia-Tze; Fang, Hsin-Yuan; Huang, Tsui-Hsien; Chen, Yi-Wen; Shie, Ming-You

    2015-03-01

    Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment.

  16. Malignant Melanoma With Osteoclast-Like Differentiation.

    PubMed

    Wasserman, Jason K; Sekhon, Harmanjatinder S; Ayroud, Yasmine

    2015-09-01

    Osteoclast-like giant cells are frequently encountered in nonskeletal malignancies; however, the evidence to date suggests that they represent a tissue response to the lesion rather than neoplastic differentiation. We describe a case of metastatic melanoma demonstrating osteoclast-like differentiation in the lung. The lung nodule was diagnosed as a metastatic melanoma by histological features and confirmed by immunohistochemistry. Resection specimen showed numerous multinucleated giant cells exhibiting osteoclast-like morphology dispersed throughout the lesion. Both the neoplastic melanocytes and giant cells were reactive for HMB-45, Melan-A, and S100. In addition, the multinucleated neoplastic giant cells were also reactive for the monocyte/macrophage lineage markers CD68 and CD163, and alkaline phosphatase, an enzyme present in normal osteoclasts. The neoplastic melanocytes and the multinucleated neoplastic giant cells were also reactive for microphthalmia-associated transcription factor, a protein required for the development of both melanocytes and osteoclasts. Collectively, a co-expression of monocyte/macrophage markers along with melanocytic markers and alkaline phosphatase in the multinucleated neoplastic giant cells in metastatic melanoma suggest that malignant melanocytes are capable of differentiating into osteoclast-like cells and consequently aid invasion into various structures and eliciting the aggressive behavior.

  17. The 1,2,3-triazole derivative KP-A021 suppresses osteoclast differentiation and function by inhibiting RANKL-mediated MEK-ERK signaling pathway

    PubMed Central

    Ihn, Hye Jung; Lee, Doohyun; Lee, Taeho; Shin, Hong-In; Bae, Yong Chul; Kim, Sang-Hyun

    2015-01-01

    The triazole family of compounds has been implicated in modulating various biological processes such as inflammation, tumorigenesis, and infection. To our knowledge, this is the first study to demonstrate the effects of 1,2,3-triazole substituted biarylacrylonitrile compounds, including KP-A021, on the differentiation and function of osteoclasts. KP-A021 and its triazole derivatives, at a concentration that does not cause a cytotoxic response in bone marrow macrophages (BMMs), significantly inhibited osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as assessed by tartrate-resistant acid phosphatase (TRAP) staining. KP-A021 also dramatically inhibited the expression of marker genes associated with osteoclast differentiation, such as TRAP, cathepsin K (Cat K), dendritic cell-specific transmembrane protein (DC-STAMP), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). Furthermore, KP-A021 inhibited actin ring formation in osteoclasts as well as resorption pit formation induced by osteoclasts. Analysis of the signaling pathway for KP-A021 indicated that this triazole compound inhibited the RANKL-induced activation of extracellular signal-regulated kinase (ERK) and its upstream signaling molecule, mitogen-activated protein kinase kinase1/2 (MEK1/2). Taken together, these results demonstrate that KP-A021 has an inhibitory effect on the differentiation and function of osteoclasts via modulation of the RANKL-induced activation of the MEK-ERK pathway. PMID:25769316

  18. Wnt16 regulates osteoclast differentiation in conjunction with Wnt5a.

    PubMed

    Kobayashi, Yasuhiro; Thirukonda, Gnanasagar J; Nakamura, Yukio; Koide, Masanori; Yamashita, Teruhito; Uehara, Shunsuke; Kato, Hiroyuki; Udagawa, Nobuyuki; Takahashi, Naoyuki

    2015-08-01

    The canonical Wnt/β-catenin signaling pathway in osteoblast-lineage cells inhibits osteoclastogenesis through the expression of osteoprotegerin (Opg), a decoy receptor of receptor activator of Nf-κb (Rank) ligands. Wnt5a, a typical non-canonical Wnt ligand, enhances the expression of Rank in osteoclast precursors, which, in turn, promotes the Rank ligand (Rankl)-induced formation of osteoclasts. In contrast, Wnt16 and Wnt4 have been shown to inhibit the Rankl-induced formation of osteoclasts through non-canonical Wnt signals. However, the relationships among these Wnt ligands in osteoclastogenesis remained to be elucidated. We herein showed that Wnt16, but not Wnt4, inhibited the Rankl-induced osteoclastogenesis in bone marrow-derived macrophage (BMM) cultures. Wnt3a and Wnt4 inhibited the 1α,25-dihydroxy vitamin D3 (1,25D3)-induced osteoclastogenesis in co-cultures prepared from wild-type mice, but not in those from Opg(-/-) nice. Wnt16 inhibited the 1,25D3-induced formation of osteoclasts in both wild-type and Opg(-/-) co-cultures. Wnt16, Wnt4, and Wnt3a failed to inhibit the pit-forming activity of osteoclasts. Wnt16 failed to inhibit the Wnt5a-induced expression of Rank in osteoclast precursors. In contrast, Wnt5a abrogated the inhibitory effects of Wnt16 on Rankl-induced osteoclastogenesis. These results suggested that Wnt16 inhibited osteoclastogenesis, but not the function of osteoclasts and that Wnt16, an inhibitory Wnt ligand for osteoclastogenesis, regulates bone resorption in conjunction with Wnt5a.

  19. Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

    SciTech Connect

    Franco, Gilson C.N.; Nakanishi, Tadashi; Ohta, Kouji; Rosalen, Pedro L.; Groppo, Francisco C.; Bartlett, John D.; Stashenko, Philip; Taubman, Martin A.; Kawai, Toshihisa

    2011-06-10

    Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

  20. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway.

    PubMed

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun; Lee, Myeung Su; Oh, Jaemin; Kim, Ju-Young

    2015-05-29

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis.

  1. Cyclolinopeptides, cyclic peptides from flaxseed with osteoclast differentiation inhibitory activity.

    PubMed

    Kaneda, Toshio; Yoshida, Haruka; Nakajima, Yuki; Toishi, Minako; Nugroho, Alfarius Eko; Morita, Hiroshi

    2016-04-01

    Flaxseed (Linum usitatissimum seed) is widely used in food and natural health products. In our search for osteoclast differentiation inhibitors, some cyclic peptides isolated from flaxseed, known as the cyclolinopeptides, were discovered to have osteoclast differentiation inhibition activity. The osteoclast differentiation inhibition activity of cyclolinopeptides A-I (1-9) and their related derivatives (10-14) are described herein. Cyclolinopeptides F, H and I (6, 8 and 9), in particular, showed potent osteoclast differentiation inhibition activity. PMID:26923696

  2. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression

    PubMed Central

    Shevde, Nirupama K.; Bendixen, Amy C.; Dienger, Krista M.; Pike, J. Wesley

    2000-01-01

    Loss of ovarian function following menopause results in a substantial increase in bone turnover and a critical imbalance between bone formation and resorption. This imbalance leads to a progressive loss of trabecular bone mass and eventually osteoporosis, in part the result of increased osteoclastogenesis. Enhanced formation of functional osteoclasts appears to be the result of increased elaboration by support cells of osteoclastogenic cytokines such as IL-1, tumor necrosis factor, and IL-6, all of which are negatively regulated by estrogens. We show here that estrogen can suppress receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced differentiation of myelomonocytic precursors into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts through an estrogen receptor-dependent mechanism that does not require mediation by stromal cells. This suppression is dose-dependent, isomer-specific, and reversed by ICI 182780. Furthermore, the bone-sparing analogues tamoxifen and raloxifene mimic estrogen's effects. Estrogen blocks RANKL/M-CSF-induced activator protein-1-dependent transcription, likely through direct regulation of c-Jun activity. This effect is the result of a classical nuclear activity by estrogen receptor to regulate both c-Jun expression and its phosphorylation by c-Jun N-terminal kinase. Our results suggest that estrogen modulates osteoclast formation both by down-regulating the expression of osteoclastogenic cytokines from supportive cells and by directly suppressing RANKL-induced osteoclast differentiation. PMID:10869427

  3. Tenuigenin inhibits RANKL-induced osteoclastogenesis by down-regulating NF-κB activation and suppresses bone loss in vivo.

    PubMed

    Yang, Shuo; Li, Xianan; Cheng, Liang; Wu, Hongwei; Zhang, Can; Li, Kanghua

    2015-10-30

    Tenuigenin, a major active component of polygala tenuifolia root, has been used to treat patients with insomnia, dementia, and neurosis. In this study, we aimed to investigate the effects of tenuigenin on osteoclastogenesis and clarify the possible mechanism. We showed that tenuigenin inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption without cytotoxicity, which was further demonstrated by reduced osteoclast specific gene expression such as TRAP, c-Src, ATP6v0d2, etc. Moreover, the inhibitory effect of tenuigenin was associated with impaired NF-κB activity owing to delayed degradation/regeneration of IkBa and inhibition of p65 nuclear translocation. Consistent with the in vitro results, micro-ct scanning and analysis data showed that tenuigenin suppressed RANKL-induced bone loss in an animal model. Taken together, our data demonstrate that tenuigenin inhibit osteoclast formation and bone resorption both in vitro and in vivo, and comprise a potential therapeutic alternative for osteoclast-related disorders such as osteoporosis and cancer-induced bone destruction. PMID:26392312

  4. Inhibitory effects of the leaves of loquat (Eriobotrya japonica) on bone mineral density loss in ovariectomized mice and osteoclast differentiation.

    PubMed

    Tan, Hui; Furuta, Syoko; Nagata, Toshiro; Ohnuki, Koichiro; Akasaka, Taiki; Shirouchi, Bungo; Sato, Masao; Kondo, Ryuichiro; Shimizu, Kuniyoshi

    2014-01-29

    The loquat, Eriobotrya japonica Lindl. (Rosaceae), is a small tree native to Japan and China that is widely cultivated for its succulent fruit. Its leaves are used as an ingredient of a tasty tea called "Biwa cha" in Japanese. The anti-osteoporosis effects of the leaves of loquat in vitro and in vivo have been investigated. After 15 days of feeding normal diet or diet supplemented with 5% loquat leaves, the body weight, viscera weights, and bone mineral density (BMD) of both groups of eight ovariectomized (OVX) mice were compared. The result showed that the loss of BMD in loquat-fed mice was significantly prevented in three parts of the body, especially in the trabecular bone of the head (P < 0.05), abdomen (P < 0.01), and lumbar (P < 0.05) compared to the control group. No hypertrophy in the uterus by the loquat leaves diet was observed. The effect of the extract (447.25 g) prepared from the dried leaves of loquat (2.36 kg) was further studied on RANKL-induced osteoclast differentiation and cell viability. The extract suppressed the differentiation of osteoclasts under 50, 125, 250, and 500 μg/mL. Through bioactivity-guided fractionation, ursolic acid (1) was isolated and inhibited osteoclast differentiation under 4 and 10 μg/mL. It was concluded that loquat leaves possess the potential to suppress ovariectomy-induced bone mineral density deterioration.

  5. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    PubMed

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  6. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    PubMed

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit

  7. RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

    SciTech Connect

    Sundaram, Kumaran; Nishimura, Riko; Senn, Joseph; Youssef, Rimon F.; London, Steven D.; Reddy, Sakamuri V. . E-mail: reddysv@musc.edu

    2007-01-01

    Osteoclast differentiation is tightly regulated by receptor activator of NF-{kappa}B ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+ 1 to - 1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to + 1 bp to - 446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from - 446 bp to - 1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (- 1123 bp to - 1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity

  8. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

    SciTech Connect

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun; Lee, Myeung Su; Oh, Jaemin; Kim, Ju-Young

    2015-05-29

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.

  9. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.

    PubMed

    Glass, Donald A; Bialek, Peter; Ahn, Jong Deok; Starbuck, Michael; Patel, Millan S; Clevers, Hans; Taketo, Mark M; Long, Fanxin; McMahon, Andrew P; Lang, Richard A; Karsenty, Gerard

    2005-05-01

    Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis. PMID:15866165

  10. ERK5 activation is essential for osteoclast differentiation.

    PubMed

    Amano, Shigeru; Chang, Yu-Tzu; Fukui, Yasuhisa

    2015-01-01

    The MEK/ERK pathways are critical for controlling cell proliferation and differentiation. In this study, we show that the MEK5/ERK5 pathway participates in osteoclast differentiation. ERK5 was activated by M-CSF, which is one of the essential factors in osteoclast differentiation. Inhibition of MEK5 by BIX02189 or inhibition of ERK5 by XMD 8-92 blocked osteoclast differentiation. MEK5 knockdown inhibited osteoclast differentiation. RAW264.7D clone cells, which are monocytic cells, differentiate into osteoclasts after stimulation with sRANKL. ERK5 was activated without any stimulation in these cells. Inhibition of the MEK5/ERK5 pathway by the inhibitors also blocked the differentiation of RAW264.7D cells into osteoclasts. Moreover, expression of the transcription factor c-Fos, which is indispensable for osteoclast differentiation, was inhibited by treatment with MEK5 or ERK5 inhibitors. Therefore, activation of ERK5 is required for the induction of c-Fos. These events were confirmed in experiments using M-CSF-dependent bone marrow macrophages. Taken together, the present results show that activation of the MEK5/ERK5 pathway with M-CSF is required for osteoclast differentiation, which may induce differentiation through the induction of c-Fos.

  11. Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation

    PubMed Central

    Araujo, John C.; Poblenz, Ann; Corn, Paul G.; Parikh, Nila U.; Starbuck, Michael W.; Thompson, Jerry T.; Lee, Francis; Logothetis, Christopher J.; Darnay, Bryant G.

    2013-01-01

    Purpose Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Results Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. Experimental design We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Conclusion Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases. PMID:19855158

  12. Tea polyphenols inhibit rat osteoclast formation and differentiation.

    PubMed

    Oka, Yoshiomi; Iwai, Shinichi; Amano, Hitoshi; Irie, Yuko; Yatomi, Kentaro; Ryu, Kakei; Yamada, Shoji; Inagaki, Katsunori; Oguchi, Katsuji

    2012-01-01

    Matrix metalloproteinases (MMPs) play an important role in degeneration of the matrix associated with bone and cartilage. Regulation of osteoclast activity is essential in the treatment of bone disease, including osteoporosis and rheumatoid arthritis. Polyphenols in green tea, particularly epigallocatechin-3-gallate (EGCG), inhibit MMPs expression and activity. However, the effects of the black tea polyphenol, theaflavin-3,3'-digallate (TFDG), on osteoclast and MMP activity are unknown. Therefore, we examined whether TFDG and EGCG affect MMP activity and osteoclast formation and differentiation in vitro. TFDG or EGCG (10 and 100 µM) was added to cultures of rat osteoclast precursors cells and mature osteoclasts. Numbers of multinucleated osteoclasts and actin rings decreased in polyphenol-treated cultures relative to control cultures. MMP-2 and MMP-9 activities were lower in TFDG- and EGCG-treated rat osteoclast precursor cells than in control cultures. MMP-9 mRNA levels declined significantly in TFDG-treated osteoclasts in comparison to control osteoclasts. TFDG and EGCG inhibited the formation and differentiation of osteoclasts via inhibition of MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Thus, TFDG and EGCG may be suitable agents or lead compounds for the treatment of bone resorption diseases.

  13. Serum amyloid A inhibits osteoclast differentiation to maintain macrophage function.

    PubMed

    Kim, Jiseon; Yang, Jihyun; Park, Ok-Jin; Kang, Seok-Seong; Yun, Cheol-Heui; Han, Seung Hyun

    2016-04-01

    Serum amyloid A is an acute phase protein that is elevated under inflammatory conditions. Additionally, the serum levels of serum amyloid A are associated with the progression of inflammatory arthritis; thus, serum amyloid A might be involved in the regulation of osteoclast differentiation. In the present study, we examined the effects of serum amyloid A on osteoclast differentiation and function. When bone marrow-derived macrophages, as osteoclast precursors, were stimulated with serum amyloid A in the presence of M-CSF and receptor activator of nuclear factor-κB ligand, osteoclast differentiation and its bone-resorption activity were substantially inhibited. TLR2 was important in the inhibitory effect of serum amyloid A on osteoclast differentiation, because serum amyloid A stimulated TLR2. The inhibitory effect was absent in bone marrow-derived macrophages obtained from TLR2-deficient mice. Furthermore, serum amyloid A inhibited the expression of c-Fos and nuclear factor of activated T cells c1, which are crucial transcription factors for osteoclast differentiation, but prevented downregulation of IFN regulatory factor-8, a negative regulator of osteoclast differentiation. In contrast, serum amyloid A sustained the endocytic capacity of bone marrow-derived macrophages and their ability to induce the proinflammatory cytokines, IL-6, IL-1β, and TNF-α. Taken together, these results suggest that serum amyloid A, when increased by inflammatory conditions, inhibits differentiation of macrophages to osteoclasts, likely to maintain macrophage function for host defense.

  14. Benzo[a]pyrene inhibits osteoclastogenesis by affecting RANKL-induced activation of NF-kappaB.

    PubMed

    Voronov, I; Li, K; Tenenbaum, H C; Manolson, M F

    2008-05-15

    Exposure to polycyclic aryl hydrocarbons is linked to cancer, immunosuppression and other numerous health problems. We previously demonstrated that exposure to benzo[a]pyrene (BaP), an environmental pollutant present in high concentrations in urban smog and cigarette smoke, inhibits osteoclast differentiation and bone resorption. We hypothesized that this inhibition could be due to crosstalk between the receptor activator of NF-kappaB ligand (RANKL) and AhR signaling cascades competing for NF-kappaB, a common transcription factor for both pathways. RAW264.7 cells (a mouse macrophage cell line capable of differentiating into osteoclasts in the presence of RANKL) were exposed to different concentrations of RANKL and BaP and the effect on NF-kappaB activation, nuclear translocation, as well as the effect of NF-kappaB inhibitors on BaP-mediated CYP1B1 gene expression was measured. The results demonstrated that BaP inhibited both RANKL-induced NF-kappaB activation and nuclear translocation. At the same time, BaP-induced CYP1B1 gene expression was inhibited by two NF-kappaB inhibitors in a dose-dependent manner, demonstrating that NF-kappaB is involved in a BaP-mediated signaling pathway. A reporter gene assay showed that both BaP and RANKL-induced luciferase reporter gene transcription under the control of NF-kappaB response elements. Co-immunoprecipitation results demonstrated that AhR interacted with NF-kappaB p65 in RAW cells and BaP appeared to enhance this interaction. However, in the presence of RANKL, we did not observe any interaction between AhR and p65. These results support our hypothesis that BaP-mediated inhibition of osteoclastogenesis is a consequence of crosstalk between AhR and RANKL signaling pathways competing for the common transcription factor NF-kappaB. PMID:18396263

  15. Osteoclast differentiation and function in aquaglyceroporin AQP9 null mice

    PubMed Central

    Liu, Yangjian; Song, Linhua; Wang, Yiding; Rojek, Aleksandra; Nielsen, Søren; Agre, Peter; Carbrey, Jennifer M.

    2008-01-01

    Background Information Osteoclasts are cells specialized for bone resorption and play important roles in bone growth and calcium homeostasis. Differentiation of osteoclasts involves fusion of bone marrow macrophage mononuclear precursors in response to extracellular signals. A dramatic increase in osteoclast cell volume occurs during osteoclast biogenesis and is believed to be mediated by Aquaporin 9 (AQP9), a membrane protein that can rapidly transport water and other small neutral solutes across cell membranes. Results Here we report an increase in expression of AQP9 during differentiation of a mouse macrophage cell line into osteoclasts. Bone marrow macrophages from wild type and AQP9 null mice differentiate into osteoclasts that have similar morphology, contain comparable numbers of nuclei, and digest synthetic bone to the same extent. Bones from wild type and AQP9 null mice contain similar numbers of osteoclasts and have comparable density and structure as measured by X-ray absorptiometry and micro-computed tomography. Conclusions Our data confirm that AQP9 expression rises during osteoclast biogenesis but indicate that AQP9 is not essential for osteoclast function or differentiation under normal physiological conditions. PMID:18666888

  16. Retinoid X receptors orchestrate osteoclast differentiation and postnatal bone remodeling

    PubMed Central

    Menéndez-Gutiérrez, María P.; Rőszer, Tamás; Fuentes, Lucía; Núñez, Vanessa; Escolano, Amelia; Redondo, Juan Miguel; De Clerck, Nora; Metzger, Daniel; Valledor, Annabel F.; Ricote, Mercedes

    2015-01-01

    Osteoclasts are bone-resorbing cells that are important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the Mafb promoter, and this interaction is required for proper osteoclast proliferation, differentiation, and activity. Pharmacological activation of RXRs inhibited osteoclast differentiation due to the formation of RXR/liver X receptor (LXR) heterodimers, which induced expression of sterol regulatory element binding protein-1c (SREBP-1c), resulting in indirect MAFB upregulation. Our study reveals that RXR signaling mediates bone homeostasis and suggests that RXRs have potential as targets for the treatment of bone pathologies such as osteoporosis. PMID:25574839

  17. Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro

    SciTech Connect

    Teramachi, Junpei; Morimoto, Hiroyuki; Baba, Ryoko; Doi, Yoshiaki; Hirashima, Kanji; Haneji, Tatsuji

    2010-11-15

    Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and {alpha}-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic-polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-{kappa}B protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important

  18. Bropirimine inhibits osteoclast differentiation through production of interferon-β.

    PubMed

    Suzuki, Hiroaki; Mochizuki, Ayako; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Inoue, Tomio; Chikazu, Daichi; Takami, Masamichi; Kamijo, Ryutaro

    2015-11-01

    Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D3. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D3. Finally, the anti-interferon-β (IFN-β) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-β.

  19. The study of mechanisms of protective effect of Rg1 against arthritis by inhibiting osteoclast differentiation and maturation in CIA mice.

    PubMed

    Gu, Yanqing; Fan, Weimin; Yin, Guoyong

    2014-01-01

    Ginsenoside Rg1 is a natural product extracted from Panax ginseng C.A. Although Rg1 protects tissue structure and functions by inhibiting local inflammatory reaction, the mechanism remains poorly understood. In vitro, Rg1 dose-dependently inhibited TRAP activity in receptor activator of nuclear factor-κB ligand- (RANKL-) induced osteoclasts and decreased the number of osteoclasts and osteoclast resorption area. Rg1 also significantly inhibited the RANK signaling pathway, including suppressing the expression of Trap, cathepsin K, matrix metalloproteinase 9 (MMP9), and calcitonin receptor (CTR). In vivo, Rg1 dramatically decreased arthritis scores in CIA mice and effectively controlled symptoms of inflammatory arthritis. Pathologic analysis demonstrated that Rg1 significantly attenuated pathological changes in CIA mice. Pronounced reduction in synovial hyperplasia and inflammatory cell invasion were observed in CIA mice after Rg1 therapy. Alcian blue staining results illustrated that mice treated with Rg1 had significantly reduced destruction in the articular cartilage. TRAP and cathepsin K staining results demonstrated a significant reduction of numbers of OCs in the articular cartilage in proximal interphalangeal joints and ankle joints in Rg1-treated mice. In summary, this study revealed that Rg1 reduced the inflammatory destruction of periarticular bone by inhibiting differentiation and maturation of osteoclasts in CIA mice.

  20. Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts.

    PubMed

    Baron, R; Neff, L; Tran Van, P; Nefussi, J R; Vignery, A

    1986-02-01

    Positive identification of osteoclast percursors has not yet been possible. The authors have, in the present report, used a model system in the rat in which it is possible to induce the formation of multinucleated osteoclasts at a predictable and reproducible site and time (Tran Van P, Vignery A, Baron R. Anat Rec 1982, 202:445-451; Cell Tissue Res 1982, 225:283-292). This system allowed the investigation of the cellular events occurring locally during the recruitment and differentiation of osteoclast precursors. Prior to the formation of multinucleated osteoclasts, mononuclear cells positive for fluoride-inhibitable nonspecific esterase and cells positive for tartrate-resistant acid phosphatase increase in number locally. Double staining procedures demonstrated the presence of both enzymes in a number of cells, thereby suggesting that they are steps in the differentiation of a single cell population. Ultrastructural studies show that lysosomal enzymes are present in every compartment of the biosynthetic pathway, in small primary lysosomes and various forms of storage granules. As these precursors arrive at the bone surface, the storage granule lysosomes are markedly depleted. It is concluded that mononuclear precursors of the osteoclast are members of the mononuclear-phagocyte lineage and differentiate early to synthesize, store, and later secrete large quantities of lysosomal enzymes. The mature osteoclast, which, as its precursor, is positive for the mononuclear-phagocyte marker enzyme nonspecific esterase, results from the fusion of these mononuclear precursors, which occurs only after their attachment to the bone surface to be resorbed.

  1. Exposure to receptor-activator of NFkappaB ligand renders pre-osteoclasts resistant to IFN-gamma by inducing terminal differentiation.

    PubMed

    Huang, Willis; O'Keefe, Regis J; Schwarz, Edward M

    2003-01-01

    While it has been established that IFN-gamma is a strong activator of macrophages and a potent inhibitor of osteoclastogenesis in vitro, it is also known that this cytokine is produced in particular settings of inflammatory bone loss, such as infection and psoriatic arthritis. Because of the different kinetics between rapid IFN-gamma macrophage activation (<24 hours) and the slower receptor-activator of NFkappaB ligand (RANKL) osteoclast differentiation (7 days), we postulated that IFN-gamma would have different effects on early-stage and late-stage osteoclast precursors. In RAW264.7 cells and primary splenocyte cultures, pretreatment with RANKL rendered these cells resistant to maximally anti-osteoclastogenic doses of IFN-gamma. These cells were also resistant to IFN-gamma-induced nitric oxide production, morphological change, and surface upregulation of CD11b and receptor-activator of NFkappaB, suggesting that early exposure of osteoclast precursors to RANKL induces a broad resistance to the cellular effects of IFN-gamma. Changes in STAT1 activation did not correlate with this resistance, as IFN-gamma activated STAT1 equally in both early-stage and late-stage pre-osteoclasts. Furthermore, we failed to observe changes in TRAF6 expression following IFN-gamma treatment in pre-osteoclasts. Together these data support a model of inflammatory bone loss in which early exposure to RANKL can prime osteoclast precursors to form in the presence of high levels of IFN-gamma using mechanisms independent of the signal molecules STAT1 and TRAF6.

  2. Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice.

    PubMed

    Jhun, JooYeon; Lee, Seung Hoon; Byun, Jae-Kyeong; Jeong, Jeong-Hee; Kim, Eun-Kyung; Lee, Jennifer; Jung, Young-Ok; Shin, Dongyun; Park, Sung Hwan; Cho, Mi-La

    2015-08-01

    Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant synthesized in human body. This enzyme promotes immune system function and can be used as a dietary supplement. Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. RA results in severe destruction of cartilage and disability. This study aimed to investigate the effect of CoQ10 on inflammation and Th17 cell proliferation on an experimental rheumatoid arthritis (RA) mice model. CoQ10 or cotton seed oil as control was orally administrated once a day for seven weeks to mice with zymosan-induced arthritis (ZIA). Histological analysis of the joints was conducted using immunohistochemistry. Germinal center (GC) B cells, Th17 cells and Treg cells of the spleen tissue were examined by confocal microscopy staining. mRNA expression was measured by real-time PCR and protein levels were estimated by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis (FACS) was used to evaluate Th17 cells and Treg cells. CoQ10 mitigated the severity of ZIA and decreased serum immunoglobulin concentrations. CoQ10 also reduced RANKL-induced osteoclastogenesis, inflammatory mediators and oxidant factors. Th17/Treg axis was reciprocally controlled by CoQ10 treatment. Moreover, CoQ10 treatment on normal mouse and human cells cultured in Th17 conditions decreased the number of Th17 cells and enhanced the number of Treg cells. CoQ10 alleviates arthritis in mice with ZIA declining inflammation, Th17 cells and osteoclast differentiation. These findings suggest that CoQ10 can be a potential therapeutic substance for RA.

  3. Hypoxic regulation of osteoclast differentiation and bone resorption activity

    PubMed Central

    Knowles, Helen J

    2015-01-01

    Bone integrity is maintained throughout life via the homeostatic actions of bone cells, namely, osteoclasts, which resorb bone, and osteoblasts, which produce bone. Disruption of this balance in favor of osteoclast activation results in pathological bone loss, which occurs in conditions including osteoporosis, rheumatoid arthritis, primary bone cancer, and cancer metastasis to bone. Hypoxia also plays a major role in these conditions, where it is associated with disease progression and poor prognosis. In recent years, considerable interest has arisen in the mechanisms whereby hypoxia and the hypoxia-inducible transcription factors, HIF-1α and HIF-2α, affect bone remodeling and bone pathologies. This review summarizes the current evidence for hypoxia-mediated regulation of osteoclast differentiation and bone resorption activity. Role(s) of HIF and HIF target genes in the formation of multinucleated osteoclasts from cells of the monocyte–macrophage lineage and in the activation of bone resorption by mature osteoclasts will be discussed. Specific attention will be paid to hypoxic metabolism and generation of ATP by osteoclasts. Hypoxia-driven increases in both glycolytic flux and mitochondrial metabolic activity, along with consequent generation of mitochondrial reactive oxygen species, have been found to be essential for osteoclast formation and resorption activity. Finally, evidence for the use of HIF inhibitors as potential therapeutic agents targeting bone resorption in osteolytic disease will be discussed. PMID:27774484

  4. CD147 promotes the formation of functional osteoclasts through NFATc1 signalling.

    PubMed

    Nishioku, Tsuyoshi; Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi

    2016-04-29

    CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis.

  5. The Inhibitory Effect of Angelica tenuissima Water Extract on Receptor Activator of Nuclear Factor-Kappa-B Ligand-Induced Osteoclast Differentiation and Bone Resorbing Activity of Mature Osteoclasts.

    PubMed

    Ahn, Sung-Jun; Baek, Jong Min; Cheon, Yoon-Hee; Park, Sun-Hyang; Lee, Myeung Su; Oh, Jaemin; Kim, Ju-Young

    2015-01-01

    Angelica tenuissima has been traditionally used in oriental medicine for its therapeutic effects in headache, toothache, and flu symptoms. It also exerts anti-inflammatory activity via the inhibition of the expression of cyclooxygenase-2 (COX-2). However, the effect of Angelica tenuissima on osteoclast differentiation has not been identified until recently. In this study, we first confirmed that Angelica tenuissima water extract (ATWE) significantly interrupted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in a dose-dependent manner without any cytotoxicity. Next, we clarified the underlying mechanisms linking the suppression effects of ATWE on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. At the molecular level, ATWE induced the dephosphorylation of c-Jun N-terminal kinase (JNK) and Akt and decreased the degradation of IκB in RANKL-dependent early signaling pathways. Subsequently, ATWE caused impaired activation of the protein and mRNA levels of c-Fos and nuclear factor of activated T cell c1 (NFATc1). Moreover, the disassembly of filamentous actin (F-actin) ring and anti-resorptive activity of mature osteoclasts were triggered by ATWE treatment. Although ATWE did not enhance osteogenesis in primary osteoblasts, our results showed that ATWE is a potential candidate for anti-resorptive agent in osteoporosis, a common metabolic bone disorder.

  6. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival

    PubMed Central

    Fujiwara, T; Zhou, J; Ye, S; Zhao, H

    2016-01-01

    The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21WAF-1 protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation. PMID:27441652

  7. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival.

    PubMed

    Fujiwara, T; Zhou, J; Ye, S; Zhao, H

    2016-01-01

    The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21(WAF-1) protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation. PMID:27441652

  8. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival.

    PubMed

    Fujiwara, T; Zhou, J; Ye, S; Zhao, H

    2016-01-01

    The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21(WAF-1) protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation.

  9. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-κB and MAPK pathways.

    PubMed

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

    2011-11-18

    Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions. PMID:22037580

  10. Osteoclast-derived complement component 3a stimulates osteoblast differentiation.

    PubMed

    Matsuoka, Kazuhiko; Park, Kyoung-Ae; Ito, Masako; Ikeda, Kyoji; Takeshita, Sunao

    2014-07-01

    Bone remodeling is regulated by a coupling of resorption to subsequent formation; however, the "coupling factor" and underlying mechanism are not fully understood. Here, we found that the condition medium (CM) of mature osteoclasts contains a humoral factor that stimulates the differentiation of primary osteoblasts, as determined by alkaline phosphatase (ALP) activity. We purified osteoblastogenesis-stimulating activity from 3 L of osteoclast CM through successive ion exchange chromatographies by monitoring the ALP activity of osteoblasts and identified complement component 3 (C3). Expression of the C3 gene increased during osteoclastogenesis, and the cleavage product C3a was detected by ELISA in the CM of osteoclasts but not in that of bone marrow macrophages. The osteoblastogenesis-stimulating activity present in osteoclast CM was inhibited by a specific antagonist of the C3a receptor (C3aR), SB290157. Conversely, the retroviral expression of C3a as well as treatment with the C3aR agonist, benzeneacetamide, stimulated osteoblast differentiation. C3 gene expression in bone was increased in the high bone turnover states of ovariectomy (OVX) or a receptor activator of NF-κB ligand (RANKL) injection, and blocking the action of C3a with the daily administration of SB290157 resulted in the attenuation of bone formation elevated by OVX and the exacerbation of bone loss. These results suggest that osteoclast-derived C3a functions in the relay from bone resorption to formation and may be a candidate for a coupling factor.

  11. Proteinase expression during differentiation of human osteoclasts in vitro.

    PubMed

    Blair, H C; Sidonio, R F; Friedberg, R C; Khan, N N; Dong, S S

    2000-06-12

    Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone

  12. Myristoleic acid inhibits osteoclast formation and bone resorption by suppressing the RANKL activation of Src and Pyk2.

    PubMed

    Kwon, Jun-Oh; Jin, Won Jong; Kim, Bongjun; Kim, Hong-Hee; Lee, Zang Hee

    2015-12-01

    Cytoskeletal changes in osteoclasts such as formation of actin ring is required for bone-resorbing activity. The tyrosine kinase Src is a key player in massive cytoskeletal change of osteoclasts, thereby in bone destruction. In order for Src to be activated, trafficking to the inner plasma membrane via myristoylation is of importance. A previous study reported that myristoleic acid derived from myristic acid, inhibited N-myristoyl-transferase, an essential enzyme for myristoylation process. This prompted us to investigate whether myristoleic acid could affect osteoclastogenesis. Indeed, we observed that myristoleic acid inhibited RANKL-induced osteoclast formation in vitro, especially, at later stages of differentiation. Myristoleic acid attenuated the tyrosine phosphorylation of c-Src and Pyk2, which associates with Src, by RANKL. When myristoleic acid was co-administered with soluble RANKL into mice, RANKL-induced bone loss was substantially prevented. Bone dissection clearly revealed that the number of multinucleated osteoclasts was significantly diminished by myristoleic acid. On the other hand, myristoleic acid treatment had little or no influence on early osteoclast differentiation markers, such as c-Fos and NFATc1, and proteins related to cytoskeletal rearrangement, including DC-STAMP, integrin αv and integrin β3 in vitro. Taken together, our data suggest that myristoleic acid is capable of blocking the formation of large multinucleated osteoclasts and bone resorption likely through suppressing activation of Src and Pyk2.

  13. DC-STAMP: A Key Regulator in Osteoclast Differentiation

    PubMed Central

    CHIU, YA-HUI; RITCHLIN, CHRISTOPHER T.

    2016-01-01

    Osteoimmunology research is a new emerging research field that investigates the links between the bone and immune responses. Results from osteoimmunology studies suggest that bone is not only an essential component of the musculoskeletal system, but is also actively involved in immune regulation. Many important factors involved in immune regulation also participate in bone homeostasis. Bone homeostasis is achieved by a coordinated action between bone synthesizing osteoblasts and bone degrading osteoclasts. An imbalanced action between osteoblasts and osteoclasts often results in pathological bone diseases: osteoporosis is caused by an excessive osteoclast activity, whereas osteopetrosis results from an increased osteoblast activity. This review focuses on dendritic cell specific transmembrane protein (DC STAMP), an important protein currently considered as a master regulator of osteoclastogenesis. Of clinical relevance, the frequency of circulating DC STAMPþ cells is elevated during the pathogenesis of psoriatic diseases. Intriguingly, recent results suggest that DC STAMP also plays an imperative role in bone homeostasis by regulating the differentiation of both osteoclasts and osteoblasts. This article summarizes our current knowledge on DC STAMP by focusing on its interacting proteins, its regulation on osteoclastogenesis related genes, its possible involvement in immunoreceptor tyrosine based inhibitory motif (ITIM) mediated signaling cascade, and its potential of developing therapeutics for clinical applications. PMID:27018136

  14. Differentially expressed genes in autosomal dominant osteopetrosis type II osteoclasts reveal known and novel pathways for osteoclast biology.

    PubMed

    Coudert, Amélie E; Del Fattore, Andrea; Baulard, Céline; Olaso, Robert; Schiltz, Corinne; Collet, Corinne; Teti, Anna; de Vernejoul, Marie-Christine

    2014-03-01

    Autosomal dominant osteopetrosis type II (ADO II) is a rare, heritable bone disorder characterized by a high bone mass and insufficient osteoclast activity. Mutations in the CLCN7 gene have been reported to cause ADO II. To gain novel insights into the pathways dysregulated in ADOII osteoclasts, we identified changes in gene expression in osteoclasts from patients with a heterozygous mutation of CLCN7. To do this, we carried out a transcriptomic study comparing gene expression in the osteoclasts of patients with ADO II and healthy donors. Our data show that, according to our selection criteria, 182 genes were differentially expressed in osteoclasts from patients and controls. From the 18 displaying the highest change in microarray, we confirmed differential expression for seven by qPCR. Although two of them have previously been found to be expressed in osteoclasts (ITGB5 and SERPINE2), the other five (CES1 (carboxyl esterase 1), UCHL1 (ubiquitin carboxy-terminal esterase L1, also known as ubiquitin thiolesterase), WARS (tryptophanyl-tRNA synthetase), GBP4 (guanylate-binding protein 4), and PRF1) are not yet known to have a role in this cell type. At the protein level, we confirmed elevated expression of ITGB5 and reduced expression of WARS, PRF1, and SERPINE2. Transfection of ClC-7 harboring the G215R mutation into osteoclasts resulted in an increased ITGB5 and reduced PRF1 expression of borderline significance. Finally, we observed that the ADO II patients presented a normal or increased serum level of bone formation markers, demonstrating a coupling between dysfunctional osteoclasts and osteoblasts. Sphingosine kinase 1 mRNA was expressed at the same level in ADO II and control osteoclasts. In conclusion, these data suggest that in addition to an acidification dysfunction caused by the CLCN7 mutation, a change in ITGB5, PRF1, WARS, and SERPINE2 expression could be part of the osteoclastic phenotype of ADO II.

  15. Decreased Ferroportin Promotes Myeloma Cell Growth and Osteoclast Differentiation

    PubMed Central

    Gu, Zhimin; Wang, He; Xia, Jiliang; Yang, Ye; Jin, Zhendong; Xu, Hongwei; Shi, Jumei; De Domenico, Ivana; Tricot, Guido; Zhan, Fenghuang

    2016-01-01

    Iron homeostasis is disrupted in multiple myeloma, a difficult-to-cure plasma cell malignancy with lytic bone lesions. Here, we systematically analyzed iron gene expression signature and demonstrated that mRNA expression of iron exporter ferroportin (FPN1) is significantly downregulated in myeloma cells and correlates negatively with clinic outcome. Restoring expression of FPN1 reduces intracellular liable iron pool, inhibits STAT3-MCL-1 signaling, and suppresses myeloma cells growth. Furthermore, we demonstrated that mRNA of FPN1 is also downregulated at the initial stages of osteoclast differentiation and suppresses myeloma cell–induced osteoclast differentiation through regulating iron regulator TFRC, NF-κB, and JNK pathways. Altogether, we demonstrated that downregulation of FPN1 plays critical roles in promoting myeloma cell growth and bone resorption in multiple myeloma. PMID:25855377

  16. The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-{kappa}B and MAPK pathways

    SciTech Connect

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit; Pudhom, Khanitha; Palaga, Tanapat

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. Black-Right-Pointing-Pointer Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. Black-Right-Pointing-Pointer The mode of action of this limonoid is by inhibiting activation of the NF-{kappa}B and MAPK pathways which are activated by RANKL. -- Abstract: Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-{kappa}B (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 {mu}M. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-{kappa}B p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

  17. Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α

    PubMed Central

    Wang, Ling; Qiu, Xue-Min; Gui, Yu-Yan; Xu, Ying-Ping; Gober, Hans-Jürgen; Li, Da-Jin

    2015-01-01

    Introduction Bu-Shen-Ning-Xin decoction (BSNXD) is a traditional Chinese medicinal composition that has been used as a remedy for postmenopausal osteoporosis, but the mechanisms affecting bone metabolism are not fully understood. Purpose We investigated the molecular mechanism and signaling pathway underlying the effect of BSNXD on osteoclastogenesis. Materials and methods A postmenopausal osteoporosis animal model generated by ovariectomy was administered BSNXD and drug-derived serum was prepared. An enzyme immunoassay was conducted to measure the 17-β-estradiol (E2) concentration in the drug-derived serum. Bone marrow-derived monocyte/macrophage precursor cells were treated with drug-derived serum, and tartrate-resistance acid phosphatase staining was conducted to observe osteoclastogenesis. A bone resorption assay was performed to analyze the effect on osteoclastic resorptive function. Real-time PCR, flow cytometry, Western blotting, transfection, and luciferase assays were conducted to explore the related mechanism. Results E2 was not elevated in BSNXD-derived serum. BSNXD-derived serum suppressed receptor activation of nuclear factor κB ligand (RANKL)-activated osteoclastogenesis in a dose-dependent manner; this effect could be reversed by estrogen receptor α antagonist methyl-piperidino-pyrazole. The serum suppressed RANKL-induced NF-κB transcription and inhibited the accumulation of nuclear factor of activated T-cells, cytoplasmic 1 in osteoclast precursor cells; the inhibitory effect was abolished by methyl-piperidino-pyrazole but not the estrogen receptor β antagonist or androgen receptor antagonist. Conclusion These results collectively suggest that administration of BSNXD presents inhibitory effects on osteoclast differentiation by abrogating the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 and NF-κB signaling pathways downstream of estrogen receptor α, thereby contributing to the inhibitory effect on bone resorption. PMID

  18. Disulfiram Attenuates Osteoclast Differentiation In Vitro: A Potential Antiresorptive Agent

    PubMed Central

    Cheng, Tak S.; Pavlos, Nathan J.; Rea, Sarah; Dai, Kerong; Zheng, Ming H.

    2015-01-01

    Disulfiram (DSF), a cysteine modifying compound, has long been clinically employed for the treatment of alcohol addiction. Mechanistically, DSF acts as a modulator of MAPK and NF-κB pathways signaling pathways. While these pathways are crucial for osteoclast (OC) differentiation, the potential influence of DSF on OC formation and function has not been directly assessed. Here, we explore the pharmacological effects of DSF on OC differentiation, activity and the modulation of osteoclastogenic signaling cascades. We first analyzed cytotoxicity of DSF on bone marrow monocytes isolated from C57BL/6J mice. Upon the establishment of optimal dosage, we conducted osteoclastogenesis and bone resorption assays in the presence or absence of DSF treatment. Luciferase assays in RAW264.7 cells were used to examine the effects of DSF on major transcription factors activation. Western blot, reverse transcription polymerase chain reaction, intracellular acidification and proton influx assays were employed to further dissect the underlying mechanism. DSF treatment dose-dependently inhibited both mouse and human osteoclastogenesis, especially at early stages of differentiation. This inhibition correlated with a decrease in the expression of key osteoclastic marker genes including CtsK, TRAP, DC-STAMP and Atp6v0d2 as well as a reduction in bone resorption in vitro. Suppression of OC differentiation was found to be due, at least in part, to the blockade of several key receptor activators of nuclear factor kappa-B ligand (RANKL)-signaling pathways including ERK, NF-κB and NFATc1. On the other hand, DSF failed to suppress intracellular acidification and proton influx in mouse and human osteoclasts using acridine orange quenching and microsome-based proton transport assays. Our findings indicate that DSF attenuates OC differentiation via the collective suppression of several key RANKL-mediated signaling cascades, thus making it an attractive agent for the treatment of OC

  19. Smad1/5 and Smad4 expression are important for osteoclast differentiation.

    PubMed

    Tasca, Amy; Stemig, Melissa; Broege, Aaron; Huang, Brandon; Davydova, Julia; Zwijsen, An; Umans, Lieve; Jensen, Eric D; Gopalakrishnan, Raj; Mansky, Kim C

    2015-07-01

    To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP's effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity.

  20. Netrin-1 is a critical autocrine/paracrine factor for osteoclast differentiation.

    PubMed

    Mediero, Aránzazu; Ramkhelawon, Bhama; Perez-Aso, Miguel; Moore, Kathryn J; Cronstein, Bruce N

    2015-05-01

    Bone metabolism is a vital process that involves resorption by osteoclasts and formation by osteoblasts, which is closely regulated by immune cells. The neuronal guidance protein Netrin-1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation, osteoclast precursors increase expression of Netrin-1 and its receptor Unc5b. Netrin-1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro, and absence of Netrin-1 or antibody-mediated blockade of Netrin-1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin-1 in osteoclast differentiation in vivo using Netrin-1-deficient (Ntn1(-/-) ) or wild-type (WT) bone marrow transplanted mice. Notably, Ntn1(-/-) chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared with WT mice. Mechanistic studies revealed that Netrin-1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin-1 increases regulator of Rho-GEF subfamily (LARG) and repulsive guidance molecule (RGMa) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin-1 and its receptor Unc5b likely play a role in fusion of osteoclast precursors because Netrin-1 and DC-STAMP are tightly linked. These results identify Netrin-1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies.

  1. Stochastic differentiation into an osteoclast lineage from cloned macrophage-like cells

    SciTech Connect

    Hayashi, Shin-Ichi; Murata, Akihiko; Okuyama, Kazuki; Shimoda, Yuhki; Hikosaka, Mari; Yasuda, Hisataka; Yoshino, Miya

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer The frequency of C7 differentiation into osteoclast was low and constant. Black-Right-Pointing-Pointer Only extended C7 cell cultures exponentially increased osteoclast+ cultures. Black-Right-Pointing-Pointer C7 cell differentiation into committed osteoclast precursors is on 'autopilot'. Black-Right-Pointing-Pointer The system may maintain the stem cell self-renewal and differentiation. -- Abstract: Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor {kappa}B ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6 days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3 days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on 'autopilot' rather than requiring specific signals to drive this process.

  2. Ethyl-2, 5-dihydroxybenzoate displays dual activity by promoting osteoblast differentiation and inhibiting osteoclast differentiation.

    PubMed

    Kwon, Byeong-Ju; Lee, Mi Hee; Koo, Min-Ah; Kim, Min Sung; Seon, Gyeung Mi; Han, Jae-Jin; Park, Jong-Chul

    2016-03-11

    The interplay between bone-forming osteoblasts and bone-resorbing osteoclasts is essential for balanced bone remodeling. In this study, we evaluate the ability of ethyl-2, 5-dihyrdoxybenzoate (E-2, 5-DHB) to affect both osteoblast and osteoclast differentiation for bone regeneration. Osteogenic differentiation of human mesenchymal stem cells (hMSCs) was quantified by measuring alkaline phosphatase (ALP) activity and calcium deposition. To evaluate osteoclast differentiation, we investigated the effect of E-2, 5-DHB on RANKL-activated osteoclastogenesis in RAW 264.7 cells. E-2, 5-DHB enhanced ALP activity and inhibited RAW 264.7 cell osteoclastogenesis in vitro. To assess the in vivo activity of E-2, 5-DHB, hMSCs were delivered subcutaneosuly alone or in combination with E-2, 5-DHB in an alginate gel into the backs of nude-mice. Histological and immunohistochemical evaluation showed significantly higher calcium deposition in the E-2, 5-DHB group. Osteocalcin (OCN) was highly expressed in cells implanted in the gels containing E-2, 5-DHB. Our results suggest that E-2, 5-DHB can effectively enhance osteoblast differentiation and inhibit osteoclast differentiation both in vitro and in vivo. Understanding the dual function of E-2, 5-DHB on osteoblast and osteoclast differentiation will aid in future development of E-2, 5-DHB as a material for bone tissue engineering.

  3. Sodium hydrosulfide inhibits the differentiation of osteoclast progenitor cells via NRF2-dependent mechanism.

    PubMed

    Gambari, Laura; Lisignoli, Gina; Cattini, Luca; Manferdini, Cristina; Facchini, Andrea; Grassi, Francesco

    2014-09-01

    Hydrogen sulfide (H2S), which recently emerged as a potent regulator of tissues and organs, is broadly produced in mammalian cells but whether it can regulate bone cell function is still elusive. The main objective of this study was to establish the role of H2S in the regulation of human osteoclast differentiation and function. Sodium hydrosulfide (NaHS), a common H2S-donor, was administered in vitro to CD11b+ human monocytes, the pool of circulating osteoclasts precursors which are critically involved in osteoclast development and function in bone. NaHS dose-dependently decreased human osteoclast differentiation at concentrations which did not induce toxicity. The inhibition of human osteoclast differentiation was associated with a down-regulation in RANKL-dependent intracellular ROS levels in human pre-osteoclasts cells. Furthermore, NaHS up-regulated NRF2 protein expression, its nuclear translocation, and the transcription of the two key downstream antioxidant genes Peroxiredoxin-1 and NAD(P)H dehydrogenase quinone 1, suggesting that NRF2 activation may inhibit human osteoclast differentiation by activating a sustained antioxidant response in osteoclast progenitors; furthermore, NRF2 activators Sulforaphane and Tert-butylhydroquinone inhibited in vitro human osteoclast differentiation. Moreover, silencing NRF2 in human pre-osteoclasts totally abolished NaHS-mediated inhibition of osteoclastogenesis, suggesting that NRF2 is essential to the inhibitory function of NaHS in osteoclast development. Finally, we found that NaHS also downregulated the RANKL/OPG mRNA ratio in human mesenchymal stem cells, the key osteoclast-supporting cells. Our results suggest that NaHS shows a potential therapeutical role in erosive diseases of bone by regulating both direct and indirect mechanisms controlling the differentiation of circulating osteoclasts precursors.

  4. Fisetin inhibits osteoclastogenesis through prevention of RANKL-induced ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

    PubMed

    Sakai, Eiko; Shimada-Sugawara, Megumi; Yamaguchi, Yu; Sakamoto, Hiroshi; Fumimoto, Reiko; Fukuma, Yutaka; Nishishita, Kazuhisa; Okamoto, Kuniaki; Tsukuba, Takayuki

    2013-01-01

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2-related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKL-mediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

  5. CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts

    PubMed Central

    Greenblatt, Matthew B.; Park, Kwang Hwan; Oh, Hwanhee; Kim, Jung-Min; Shin, Dong Yeon; Lee, Jae Myun; Lee, Jin Woo; Singh, Anju; Lee, Ki-young; Hu, Dorothy; Xiao, Changchun; Charles, Julia F.; Penninger, Josef M.; Lotinun, Sutada; Baron, Roland; Ghosh, Sankar

    2015-01-01

    Physiological bone remodeling requires that bone formation by osteoblasts be tightly coupled to bone resorption by osteoclasts. However, relatively little is understood about how this coupling is regulated. Here, we demonstrate that modulation of NF-κB signaling in osteoclasts via a novel activity of charged multivesicular body protein 5 (CHMP5) is a key determinant of systemic rates of bone turnover. A conditional deletion of CHMP5 in osteoclasts leads to increased bone resorption by osteoclasts coupled with exuberant bone formation by osteoblasts, resembling an early onset, polyostotic form of human Paget’s disease of bone (PDB). These phenotypes are reversed by haploinsufficiency for Rank, as well as by antiresorptive treatments, including alendronate, zolendronate, and OPG-Fc. Accordingly, CHMP5-deficient osteoclasts display increased RANKL-induced NF-κB activation and osteoclast differentiation. Biochemical analysis demonstrated that CHMP5 cooperates with the PDB genetic risk factor valosin-containing protein (VCP/p97) to stabilize the inhibitor of NF-κBα (IκBα), down-regulating ubiquitination of IκBα via the deubiquitinating enzyme USP15. Thus, CHMP5 tunes NF-κB signaling downstream of RANK in osteoclasts to dampen osteoclast differentiation, osteoblast coupling and bone turnover rates, and disruption of CHMP5 activity results in a PDB-like skeletal disorder. PMID:26195726

  6. The Free Fatty Acid Receptor G Protein-coupled Receptor 40 (GPR40) Protects from Bone Loss through Inhibition of Osteoclast Differentiation*

    PubMed Central

    Wauquier, Fabien; Philippe, Claire; Léotoing, Laurent; Mercier, Sylvie; Davicco, Marie-Jeanne; Lebecque, Patrice; Guicheux, Jérôme; Pilet, Paul; Miot-Noirault, Elisabeth; Poitout, Vincent; Alquier, Thierry; Coxam, Véronique; Wittrant, Yohann

    2013-01-01

    The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40−/− mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/β) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40−/− primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40−/− mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications. PMID:23335512

  7. Effects of Zinc and Strontium Substitution in Tricalcium Phosphate on Osteoclast Differentiation and Resorption

    PubMed Central

    Roy, Mangal; Fielding, Gary; Bandyopadhyay, Amit; Bose, Susmita

    2013-01-01

    Bone replacement materials must be able to regulate both osteoblastic synthesis of new bone and osteoclastic resorption process in order to maintain the balance of bone remodeling. Osteoclasts generate from differentiation of mononuclear cells. In the present study, we have studied the osteoclast-like-cells responses (differentiation from mononuclear cells and resorption) to beta tricalcium phosphate (β-TCP) doped with zinc (Zn) and strontium (Sr). Osteoclast-like-cells differentiation and resorption was studied in vitro using osteoclast-like-cells precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Morphological and immunohistochemical analysis confirmed successful differentiation of osteoclast-like-cells on the doped and undoped β-TCP substrates after 8 days of culture. Cells on the substrate surface expressed specific osteoclast markers such as; actin ring, multiple nucleus, tartrate-resistant acid phosphatase (TRAP) synthesis, and vitronectin receptor. However, quantitative TRAP assay indicated the inhibiting effect of Zn on osteoclast differentiation. Although, Zn doped β-TCP restricted osteoclast-like-cells differentiation, the samples were resorbed much faster. An increased resorption pit volume was noticed on Zn doped β-TCP samples after 28 days of culture compared to pure and Sr doped β-TCP. In this work, we demonstrated that β-TCP bone substitute materials can be successfully resorbed by osteoclast-like-cells, where both osteoclast-like-cells differentiation and resorption were modulated by Zn and/or Sr doping- a much needed property for successful bone remodeling. PMID:24244866

  8. Thymoquinone prevents RANKL-induced osteoclastogenesis activation and osteolysis in an in vivo model of inflammation by suppressing NF-KB and MAPK Signalling.

    PubMed

    Thummuri, Dinesh; Jeengar, Manish Kumar; Shrivastava, Shweta; Nemani, Harishankar; Ramavat, Ravindar Naik; Chaudhari, Pradip; Naidu, V G M

    2015-09-01

    Osteoclasts are multinuclear giant cells responsible for bone resorption in inflammatory bone diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Because of deleterious side effects with currently available drugs the search continues for novel effective and safe therapies. Thymoquinone (TQ), the major bioactive component of Nigella sativa has been investigated for its anti-inflammatory, antioxidant and anticancer activities. However, its effects in osteoclastogenesis have not been reported. In the present study we show for the first time that TQ inhibits nuclear factor-KB ligand (RANKL) induced osteoclastogenesis in RAW 264.7 and primary bone marrow derived macrophages (BMMs) cells. RANKL induced osteoclastogenesis is associated with increased expression of multiple transcription factors via activation of NF-KB, MAPKs signalling and reactive oxygen species (ROS). Mechanistically TQ blocked the RANKL induced NF-KB activation by attenuating the phosphorylation of IkB kinase (IKKα/β). Interestingly, in RAW 264.7 cells TQ inhibited the RANKL induced phosphorylation of MAPKs and mRNA expression of osteoclastic specific genes such as TRAP, DC-STAMP, NFATc1 and c-Fos. In addition, TQ also decreased the RANKL stimulated ROS generation in macropahges (RAW 264.7) and H2O2 induced ROS generation in osteoblasts (MC-3T3-E1). Consistent with in vitro results, TQ inhibited lipopolysaccharide (LPS) induced bone resorption by suppressing the osteoclastogenesis. Indeed, micro-CT analysis showed that bone mineral density (BMD) and bone architecture parameters were positively modulated by TQ. Taken together our data demonstrate that TQ has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-KB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

  9. High Bone Mass in Mice Lacking Cx37 Because of Defective Osteoclast Differentiation*

    PubMed Central

    Pacheco-Costa, Rafael; Hassan, Iraj; Reginato, Rejane D.; Davis, Hannah M.; Bruzzaniti, Angela; Allen, Matthew R.; Plotkin, Lilian I.

    2014-01-01

    Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37−/−) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37−/− mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37−/− mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37+/+ littermates. sRANKL/M-CSF treatment of nonadherent Cx37−/− bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37+/+ cell cultures. Further, Cx37−/− osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37−/− osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37−/− mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo. PMID:24509854

  10. Inhibition of Osteoclast Differentiation and Bone Resorption by Bisphosphonate-conjugated Gold Nanoparticles

    PubMed Central

    Lee, Donghyun; Heo, Dong Nyoung; Kim, Han-Jun; Ko, Wan-Kyu; Lee, Sang Jin; Heo, Min; Bang, Jae Beum; Lee, Jung Bok; Hwang, Deok-Sang; Do, Sun Hee; Kwon, Il Keun

    2016-01-01

    In recent years, gold nanoparticles (GNPs) have been reported to affect the regeneration of bone tissue. The goal of this study was to improve bone tissue regeneration by using targeted GNPs. We fabricated a functionalized GNPs conjugated with alendronate (ALD), of the bisphosphonate group. Subsequently, the ALD, GNPs, and ALD conjugated GNPs (GNPs-ALD) were analyzed by ultraviolet-visible absorbance (UV-vis) spectrophotometer, Attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR), and thermo gravimetric analysis (TGA). The prepared GNPs-ALD were used to investigate their inhibitory effects on the receptor activator of nuclear factor- κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). Additionally, the GNPs-ALD were applied to ovariectomy (OVX)-induced osteoporotic mice and the experiments were evaluated. ALD was found to be successfully conjugated to the GNPs surface, and it displayed significant adhesion onto the bone surface. The in-vitro study indicated that the GNPs, ALD and GNPs-ALD suppressed osteoclast formation in a dose-dependent manner. Furthermore, in the OVX mouse model, the mice treated GNPs-ALD had higher bone density as compared to other OVX mice groups. The results from these tests indicated that GNPs-ALD can be useful agents for preventing and treating osteoporosis. PMID:27251863

  11. Identification of Nedd9 as a TGF-β-Smad2/3 Target Gene Involved in RANKL-Induced Osteoclastogenesis by Comprehensive Analysis

    PubMed Central

    Yasui, Tetsuro; Hirose, Jun; Izawa, Naohiro; Matsumoto, Takumi; Imai, Yuuki; Seo, Sachiko; Kurokawa, Mineo; Tsutsumi, Shuichi; Kadono, Yuho; Morimoto, Chikao; Aburatani, Hiroyuki; Miyamoto, Takeshi; Tanaka, Sakae

    2016-01-01

    TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis. PMID:27336669

  12. Netrin-1 Is a Critical Autocrine/Paracrine Factor for Osteoclast Differentiation

    PubMed Central

    Mediero, Aránzazu; Ramkhelawon, Bhama; Perez-Aso, Miguel; Moore, Kathryn J; Cronstein, Bruce N

    2015-01-01

    Bone metabolism is a vital process that involves resorption by osteoclasts and formation by osteoblasts, which is closely regulated by immune cells. The neuronal guidance protein Netrin-1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation, osteoclast precursors increase expression of Netrin-1 and its receptor Unc5b. Netrin-1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro, and absence of Netrin-1 or antibody-mediated blockade of Netrin-1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin-1 in osteoclast differentiation in vivo using Netrin-1-deficient (Ntn1−/−) or wild-type (WT) bone marrow transplanted mice. Notably, Ntn1−/− chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared with WT mice. Mechanistic studies revealed that Netrin-1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin-1 increases regulator of Rho-GEF subfamily (LARG) and repulsive guidance molecule (RGMa) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin-1 and its receptor Unc5b likely play a role in fusion of osteoclast precursors because Netrin-1 and DC-STAMP are tightly linked. These results identify Netrin-1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies. PMID:25483983

  13. Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone loss.

    PubMed

    Jiang, Hongwei; Wang, Yongqiang; Viniegra, Ana; Sima, Corneliu; McCulloch, Christopher A; Glogauer, Michael

    2015-06-01

    Osteoclast differentiation and function are highly dependent on the assembly and turnover of actin filaments, but little is known about the roles of actin binding proteins in these processes. Adseverin (Ads), a member of the gelsolin superfamily of actin capping and severing proteins, regulates actin filament turnover and can regulate the turnover of cortical actin filaments of chromaffin cells during exocytosis. Using a conditional Ads knockout mouse model, we confirmed our previous finding in cultured cells that Ads plays a role in osteoclastogenesis (OCG) and actin cytoskeletal organization in osteoclasts. Here we show that Ads is required for osteoclast formation and that when alveolar bone resorption is experimentally induced in mice, genetic deletion of Ads prevents osteoclast-mediated bone loss. Further, when Ads-null osteoclasts are cultured, they exhibit defective OCG, disorganized podosome-based actin filament superstructures, and decreased bone resorption. Reintroduction of Ads into Ads-null osteoclast precursor cells restored these osteoclast defects. Collectively, these data demonstrate a unique and osteoclast-specific role for Ads in OCG and osteoclast function.

  14. Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

    PubMed Central

    Weivoda, Megan M; Ruan, Ming; Hachfeld, Christine M; Pederson, Larry; Howe, Alan; Davey, Rachel A; Zajac, Jeffrey D; Kobayashi, Yasuhiro; Williams, Bart O; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of β-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data

  15. Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

    SciTech Connect

    Hong, Huixian; Shi, Zhenqi; Qiao, Ping; Li, Hui; McCoy, Erin M.; Mao, Ping; Xu, Hui; Feng, Xu; Wang, Shunqing

    2013-11-01

    Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We found that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the

  16. Prostate cancer promotes CD11b positive cells to differentiate into osteoclasts

    PubMed Central

    Mizutani, Kosuke; Sud, Sudha; Pienta, Kenneth J

    2009-01-01

    Bone is the preferred site of prostate cancer metastasis, contributing to the morbidity and mortality of this disease. A key step in the successful establishment of prostate cancer bone metastases is activation of osteoclasts with subsequent bone resorption causing the release of several growth factors from the bone matrix. CD11b+ cells in bone marrow are enriched for osteoclast precursors. Conditioned media from prostate cancer PC-3 cells induces CD11b+ cells from human peripheral blood to differentiate into functional osteoclasts with subsequent bone resorption. Analysis of PC-3 conditioned media revealed high amounts of IL-6 and IL-8. CD11b+ cells were cultured with M-CSF and RANKL, IL-6, IL-8 and CCL2, alone or in combination. All of these conditions induced osteoclast fusion, but cells cultured with M-CSF, IL-6, IL-8 and CCL2 were capable of limited bone resorption. Co-incubation with IL-6 and IL-8 and the RANK inhibitor, RANK-Fc, failed to inhibit osteoclast fusion and bone resorption, suggesting a potential RANKL-independent mechanism of functional osteoclast formation. This study demonstrates that functional osteoclasts can be derived from CD11b+ cells derived from human PBMCs. Prostate cancer cells secrete factors, including IL-6 and IL-8, that play an important role in osteoclast fusion by a RANKL-independent mechanism. PMID:19170075

  17. RANKL-induced down-regulation of CX3CR1 via PI3K/Akt signaling pathway suppresses Fractalkine/CX3CL1-induced cellular responses in RAW264.7 cells

    SciTech Connect

    Saitoh, Yurika; Koizumi, Keiichi Sakurai, Hiroaki; Minami, Takayuki; Saiki, Ikuo

    2007-12-21

    The receptor activator of nuclear factor-{kappa}B ligand (RANKL) is essential for osteoclast differentiation. In this study, we examined the effects of RANKL on chemokine receptor expression in osteoclast precursor cells, RAW264.7 cells. CX3CL1 (also called Fractalkine) receptor, CX3CR1 mRNA expression, was rapidly reduced by treatment with RANKL in contrast to the increased expression of CCR1 and tartrate-resistant acid phosphatase (TRAP). This reduction occurred within 12 h and was maintained for 5 days during osteoclastogenesis. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and Akt, but not mitogen-activated protein kinases, restored the RANKL-induced reduction of CX3CR1 mRNA. The stability of CX3CR1 mRNA was not changed, suggesting transcriptional repression by RANKL. The down-regulation of CX3CR1 mRNA correlated with the suppression of CX3CL1-induced activation of Akt and ERK as well as chemotaxis. These results suggest a potential role for decreased CX3CL1-CX3CR1 interaction in osteoclastogenesis.

  18. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    PubMed Central

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  19. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation.

    PubMed

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-05-30

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes.

  20. Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function.

    PubMed

    Miyauchi, A; Notoya, K; Taketomi, S; Takagi, Y; Fujii, Y; Jinnai, K; Takahashi, K; Chihara, K; Fujita, T

    1996-08-01

    Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood. To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+]i) in osteoclasts and their precursors and measured specific binding of 3H-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+]i distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+]i followed by a sustained elevation [EC50 = 5 x 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+]i because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+]i levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7)M) and verapamil (10(-7)M) had no effects on ipriflavone-induced [Ca2+]i responses. [3H]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (Kd), 7.60 x 10(-8)M, 2.67 x 10(-6)M] and in mature osteoclasts (Kd, 4.98 x 10(-8)M, 3.70 x 10(-6)M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-8)M) or retinoic acid (10(-6)M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The

  1. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

    PubMed

    Zach, Frank; Mueller, Alexandra; Gessner, André

    2015-01-01

    In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or

  2. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells

    PubMed Central

    Zach, Frank; Mueller, Alexandra; Gessner, André

    2015-01-01

    In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or

  3. Strontium inhibits titanium particle-induced osteoclast activation and chronic inflammation via suppression of NF-κB pathway

    PubMed Central

    Zhu, Shijun; Hu, Xuanyang; Tao, Yunxia; Ping, Zichuan; Wang, Liangliang; Shi, Jiawei; Wu, Xiexing; Zhang, Wen; Yang, Huilin; Nie, Zhikui; Xu, Yaozeng; Wang, Zhirong; Geng, Dechun

    2016-01-01

    Wear-particle-induced chronic inflammation and osteoclastogenesis have been identified as critical factors of aseptic loosening. Although strontium is known to be involved in osteoclast differentiation, its effect on particle-induced inflammatory osteolysis remains unclear. In this study, we investigate the potential impact and underling mechanism of strontium on particle-induced osteoclast activation and chronic inflammation in vivo and in vitro. As expected, strontium significantly inhibited titanium particle-induced inflammatory infiltration and prevented bone loss in a murine calvarial osteolysis model. Interestingly, the number of mature osteoclasts decreased after treatment with strontium in vivo, suggesting osteoclast formation might be inhibited by strontium. Additionally, low receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-α, interleukin-1β, interleukin-6 and p65 immunochemistry staining were observed in strontium-treatment groups. In vitro, strontium obviously decreased osteoclast formation, osteoclastogenesis-related gene expression, osteoclastic bone resorption and pro-inflammatory cytokine expression in bone-marrow-derived macrophages in a dose-dependent manner. Furthermore, we demonstrated that strontium impaired osteoclastogenesis by blocking RANKL-induced activation of NF-κB pathway. In conclusion, our study demonstrated that strontium can significantly inhibit particle-induced osteoclast activation and inflammatory bone loss by disturbing the NF-κB pathway, and is an effective therapeutic agent for the treatment of wear particle-induced aseptic loosening. PMID:27796351

  4. [Bone and Stem Cells. Molecular mechanisms of the differentiation and activation of osteoclasts derived from hematopoietic cells].

    PubMed

    Hayashi, Mikihito; Nakashima, Tomoki

    2014-04-01

    Mononuclear myeloid lineage cells, which are attracted to bone surfaces by chemokines and other factors, differentiate into multinucleated bone resorbing osteoclasts by cell fusion. Receptor activator of nuclear factor-κB ligand (RANKL) , which is expressed in mesenchymal cells, including osteocytes and hypertrophic chondrocytes, is essential for osteoclast differentiation and function. Osteoclasts have the capacity to resorb bone and impaired osteoclast differentiation and/or function leads to osteopetrosis, a rare disease in which mineralized bone cannot be removed. In contrast, excessive osteoclastogenesis causes diseases such as osteoporosis. Recent findings suggest that osteoclasts can also function as positive and negative regulators of osteoblastic bone formation. Thus, understanding of the molecular mechanisms that regulate osteoclastogenesis is important to develop therapeutic approaches to prevent bone diseases. This paper reviews recent findings of the molecular mechanisms regulating osteoclast differentiation and function.

  5. Prevention of Wear Particle-Induced Osteolysis by a Novel V-ATPase Inhibitor Saliphenylhalamide through Inhibition of Osteoclast Bone Resorption

    PubMed Central

    Lin, Zhen; Cao, Lei; Chim, Shek M.; Pavlos, Nathan J.; Xu, Jiake; Zheng, Ming Hao; Dai, Ke Rong

    2012-01-01

    Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis. PMID:22509274

  6. Glucose-dependent insulinotropic polypeptide (GIP) dose-dependently reduces osteoclast differentiation and resorption.

    PubMed

    Mabilleau, Guillaume; Perrot, Rodolphe; Mieczkowska, Aleksandra; Boni, Sébastien; Flatt, Peter R; Irwin, Nigel; Chappard, Daniel

    2016-10-01

    A role for glucose-dependent insulinotropic polypeptide (GIP) in controlling bone resorption has been suspected. However uncertainty remains to identify whether GIP act directly on osteoclasts. The aim of the present study were (i) to identify in different osteoclast differentiation models (human peripheral blood mononuclear cells-PBMC, murine bone marrow macrophage-BMM and murine Raw 264.7 cells) whether GIP was capable of reducing osteoclast formation and resorption; (ii) ascertain whether the highly potent GIP analogue N-AcGIP was capable of inducing a response at lower concentrations and (iii) to decipher the molecular mechanisms responsible for such effects. [d-Ala(2)]-GIP dose-dependently reduced osteoclast formation at concentration as low as 1nM in human PBMC and 10nM in murine BMM cultures. Furthermore, [d-Ala(2)]-GIP also reduced the extent of osteoclast resorption at concentration as low as 1nM in human PBMC and murine BMM cultures. The mechanism of action of [d-Ala(2)]-GIP appeared to be mediated by reduction in intracellular calcium concentration and oscillation that subsequently inhibited calcineurin activity and NFATc1 nuclear translocation. The potency of the highly potent N-AcGIP was determined and highlighted an effect on osteoclast formation and resorption at concentration ten times lower than observed with [d-Ala(2)]-GIP in vitro. Furthermore, N-AcGIP was also capable of reducing the number of osteoclast in ovariectomized mice as well as the circulating level of type I collagen C-telopeptide. Pharmacological concentrations required for reducing osteoclast formation and resorption provide the impetus to design and exploit enzymatically stable GIP analogues for the treatment of bone resorption disorders in humans. PMID:27451082

  7. Zinc modifies the effect of phyto-oestrogens on osteoblast and osteoclast differentiation in vitro.

    PubMed

    Karieb, Sahar; Fox, Simon W

    2012-11-28

    Osteoblast and osteoclast activity is disrupted in post-menopausal osteoporosis. Thus, to fully address this imbalance, therapies should reduce bone resorption and promote bone formation. Dietary factors such as phyto-oestrogens and Zn have beneficial effects on osteoblast and osteoclast activity. However, the effect of combinations of these factors has not been widely studied. We therefore examined the effect of coumestrol, daidzein and genistein in the presence or absence of zinc sulphate (Zn) on osteoclast and osteoblast activity. Osteoclast differentiation and bone resorption were significantly reduced by coumestrol (10- 7 m), daidzein (10- 5 m) and genistein (10- 7 m); and this direct anti-osteoclastic action was unaffected by Zn (10- 5 m). In addition, Zn augmented the inhibitory effect of phyto-oestrogens on the osteoblast-derived stimulus for osteoclast formation, significantly reducing the ratio of receptor activator of NF-κB ligand (RANKL)-to-osteoprotegerin mRNA expression in human osteoblast. We then examined the effect of these compounds on osteoblast activity. Mineralisation was enhanced by coumestrol (10- 5 to 10- 7 m), daidzein (10- 5 to 10- 6 m) and genistein (10- 5 m); and Zn significantly augmented this response. Zn and phyto-oestrogens also significantly enhanced alkaline phosphatase activity and Runt-related transcription factor 2 (Runx2) mRNA expression. On the other hand, Zn blunted phyto-oestrogen-induced type I collagen and osteocalcin expression and suppressed coumestrol and daidzein-stimulated osterix expression. Zn may therefore modify the anabolic action of phyto-oestrogens, promoting characteristics associated with early rather than late stages of osteoblast differentiation. Our data suggest that while Zn enhances the anti-osteoclastic effect of phyto-oestrogens, it may limit aspects of their anabolic action on bone matrix formation.

  8. Effect of Age on Regulation of Human Osteoclast Differentiation

    PubMed Central

    Chung, Ping-Lin; Zhou, Shuanhu; Eslami, Behnam; Shen, Longxiang; LeBoff, Meryl S.; Glowacki, Julie

    2014-01-01

    Human skeletal aging is characterized as a gradual loss of bone mass due to an excess of bone resorption not balanced by new bone formation. Using human marrow cells, we tested the hypothesis that there is an age-dependent increase in osteoclastogenesis due to intrinsic changes in regulatory factors [macrophage-colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG)] and their receptors [c-fms and RANK]. In bone marrow cells (BMCs), c-fms (r=0.61, p=0.006) and RANK expression (r=0.59, p=0.008) were increased with age (27-82 years, n=19). In vitro generation of osteoclasts was increased with age (r=0.89, p=0.007). In enriched marrow stromal cells (MSCs), constitutive expression of RANKL was increased with age (r=0.41, p=0.049) and expression of OPG was inversely correlated with age (r=-0.43, p=0.039). Accordingly, there was an age-related increase in RANKL/OPG (r=0.56, p=0.005). These data indicate an age-related increase in human osteoclastogenesis that is associated with an intrinsic increase in expression of c-fms and RANK in osteoclast progenitors, and, in the supporting MSCs, an increase in pro-osteoclastogenic RANKL expression and a decrease in anti-osteoclastogenic OPG. These findings support the hypothesis that human marrow cells and their products can contribute to skeletal aging by increasing the generation of bone-resorbing osteoclasts. These findings help to explain underlying molecular mechanisms of progressive bone loss with advancing age in humans. PMID:24700654

  9. Disruption of the dynein-dynactin complex unveils motor-specific functions in osteoclast formation and bone resorption.

    PubMed

    Ng, Pei Ying; Cheng, Tak Sum; Zhao, Haibo; Ye, Shiqiao; Sm Ang, Estabelle; Khor, Ee Cheng; Feng, Hao-Tian; Xu, Jiake; Zheng, Ming H; Pavlos, Nathan J

    2013-01-01

    Osteoclastic bone resorption requires strict interplay between acidified carrier vesicles, motor proteins, and the underlying cytoskeleton in order to sustain the specialized structural and functional polarization of the ruffled border. Cytoplasmic dynein, a large processive mechanochemical motor comprising heavy, intermediate, and light chains coupled to the dynactin cofactor complex, powers unilateral motility of diverse cargos to microtubule minus-ends. We have recently shown that regulators of the dynein motor complex constitute critical components of the osteoclastic bone resorptive machinery. Here, by selectively modulating endogenous dynein activity, we show that the integrity of the dynein-dynactin motor complex is an essential requirement for both osteoclast formation and function. Systematic dissection of the osteoclast dynein-dynactin complex revealed that it is differentially localized throughout RANKL-induced osteoclast formation and activation, undergoing microtubule-coupled reorganization upon the establishment of cellular polarization. In osteoclasts actively resorbing bone, dynein-dynactin intimately co-localizes with the CAP-Gly domain-containing microtubule plus-end protein CLIP-170 at the resorptive front, thus orientating the ruffled border as a microtubule plus-end domain. Unexpectedly, disruption of the dynein-dynactin complex by exogenous p50/dynamitin expression retards osteoclast formation in vitro, owing largely to prolonged mitotic stasis of osteoclast progenitor cells. More importantly, loss of osteoclastic dynein activity results in a drastic redistribution of key intracellular organelles, including the Golgi and lysosomes, an effect that coincides with impaired cathepsin K secretion and diminished bone resorptive function. Collectively, these data unveil a previously unrecognized role for the dynein-dynactin motor complex in osteoclast formation and function, serving not only to regulate their timely maturation but also the delivery

  10. [Research on effect of Sargentodoxae caulis on activity of osteoclasts and proliferation differentiation of osteoblasts].

    PubMed

    Chen, Li-zhen; Zhou, Ying; Huang, Jun-fei; Zhang, Xue; Feng, Ting-ting

    2015-11-01

    Through morphological observation, HE staining, TRAP staining and toluidine blue staining of bone resorption pits to identify osteoclasts which obtained by 1α, 25-(OH)2 VitD3 inducing rabbit bone marrow cells. Three indicators-TRAP staining, TRAP enzyme activity detecting and the number and area of bone resorption pits were adapted to detect the effect of Sargentodoxae caulis on the activity of osteoclasts. Culturing MC3T3-E1 Subclong 14 cells and detecting the effect of S. caulis on differentiation and proliferation of them by MTT and detecting the alkaline phosphatase in cells. The results show that all of the low, middle and high doses of water and alcohol extracts of S. caulis have significant inhibition on osteoclast differentiation and bone resorption ability in a dose-dependent manner. The low and middle doses of water and alcohol extracts of S. caulis can stimulate differentiation and proliferation of MC3T3-ElSubclone 14 cells, which indicates S. caulis can prevent osteoporosis and the function could be achieved by inhibiting osteoclast activity and promoting the proliferation and differentiation of osteoblasts. PMID:27097425

  11. Mutation in Osteoactivin Promotes Receptor Activator of NFκB Ligand (RANKL)-mediated Osteoclast Differentiation and Survival but Inhibits Osteoclast Function.

    PubMed

    Abdelmagid, Samir M; Sondag, Gregory R; Moussa, Fouad M; Belcher, Joyce Y; Yu, Bing; Stinnett, Hilary; Novak, Kimberly; Mbimba, Thomas; Khol, Matthew; Hankenson, Kurt D; Malcuit, Christopher; Safadi, Fayez F

    2015-08-14

    We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb(+)). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts and that survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abrogated by the addition of recombinant OA to cultures, which restored osteoclastogenesis to wild-type levels. Moreover, mix and match co-cultures demonstrated an induction of osteoclastogenesis in D2J osteoblasts co-cultured with osteoclasts of D2J or wild-type. Last, in functional osteo-assays, we show that bone resorption activity of D2J osteoclasts is dramatically reduced, and these osteoclasts present an abnormal ruffled border over the bone surface. Collectively, these data support a model whereby OA/Gpnmb acts as a negative regulator of osteoclast differentiation and survival but not function by inhibiting the ERK/AKT signaling pathways.

  12. V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption

    PubMed Central

    Yang, De-Qin; Feng, Shengmei; Chen, Wei; Zhao, Haibo; Paulson, Christie; Li, Yi-Ping

    2014-01-01

    Lysosomal trafficking and protease exocytosis in osteoclasts are essential for ruffled border formation and bone resorption. Yet, the mechanism underlying lysosomal trafficking and the related process of exocytosis remains largely unknown. We found ATP6ap1 (Ac45), an accessory subunit of vacuolar-type H+-ATPases (V-ATPases), to be highly induced by receptor activator for nuclear factor kappa B ligand (RANKL) in osteoclast differentiation. Ac45 knockdown osteoclasts formed normal actin rings, but had severely impaired extracellular acidification and bone resorption. Ac45 knockdown significantly reduced osteoclast formation. The decrease in the number of osteoclasts does not result from abnormal apoptosis; rather, it results from decreased osteoclast precursor cell proliferation and fusion, which may be partially due to the downregulation of ERK phosphorylation and FBJ osteosarcoma oncogene (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and Tm7sf4 expression. Notably, Ac45 knockdown osteoclasts exhibited impaired lysosomal trafficking and exocytosis, as indicated by the absence of lysosomal trafficking to the ruffled border and a lack of cathepsin K exocytosis into the resorption lacuna. Our data revealed that the impaired exocytosis is specifically due to Ac45 deficiency, and not the general consequence of a defective V-ATPase. Together, our results demonstrate the essential role of Ac45 in osteoclast-mediated extracellular acidification and protease exocytosis, as well as the ability of Ac45 to guide lysosomal intracellular trafficking to the ruffled border, potentially through its interaction with the small GTPase Rab7. Our work indicates that Ac45 may be a novel therapeutic target for osteolytic disease. PMID:22467241

  13. Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation.

    PubMed

    Uchino, Kaho; Okamoto, Kuniaki; Sakai, Eiko; Yoneshima, Erika; Iwatake, Mayumi; Fukuma, Yutaka; Nishishita, Kazuhisa; Tsukuba, Takayuki

    2015-11-01

    Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant-derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin-treated murine osteoblastic MC3T3-E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone-resorption activity through slightly decreased the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and inhibitor of nuclear factor kappa Bα; however, the phosphorylation of Akt and p38 slightly increased in bone marrow-derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T-cell cytoplasmic-1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases.

  14. Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts.

    PubMed

    Zhang, Hui; Huang, Yuefang; Wang, Shuang; Fu, Rong; Guo, Chaohuan; Wang, Hongyue; Zhao, Jijun; Gaskin, Felicia; Chen, Jingxian; Yang, Niansheng; Fu, Shu Man

    2015-12-01

    Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP(+) osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11-7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders. PMID:26318644

  15. Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts.

    PubMed

    Zhang, Hui; Huang, Yuefang; Wang, Shuang; Fu, Rong; Guo, Chaohuan; Wang, Hongyue; Zhao, Jijun; Gaskin, Felicia; Chen, Jingxian; Yang, Niansheng; Fu, Shu Man

    2015-12-01

    Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP(+) osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11-7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.

  16. Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo

    PubMed Central

    Chen, Shuai; Jin, Gu; Huang, Kang-Mao; Ma, Jian-Jun; Wang, Qiang; Ma, Yan; Tang, Xiao-Zhen; Zhou, Zhi-Jie; Hu, Zhi-Jun; Wang, Ji-Ying; Qin, An; Fan, Shun-Wu

    2015-01-01

    Osteoclasts play an important role in diseases involving bone loss. In this study, we assessed the effect of a plant-derived natural alkaloid (lycorine, or LY) on osteoclastogenesis in vitro and in vivo. Our in vitro study showed that receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis could be inhibited by LY; this effect was due to inhibition of mitogen-activated protein kinase (MAPK) signalling via MAP kinase kinases (MKKs). The MAPK agonist anisomycin could partially rescue the inhibitory effect of LY. Furthermore, LY also played a protective role in both a murine ovariectomy (OVX)-induced osteoporosis model and a titanium particle-induced osteolysis model. These results confirmed that LY was effective in preventing osteoclast-related diseases in vivo. In conclusion, our results show that LY is effective in suppressing osteoclastogenesis and therefore could be used to treat OVX-induced osteoporosis and wear particle-induced osteolysis. PMID:26238331

  17. N16, a Nacreous Protein, Inhibits Osteoclast Differentiation and Enhances Osteogenesis.

    PubMed

    Ma, Jie-Yi; Wong, Ka-Lok; Xu, Zhen-Yan; Au, Ka-Yee; Lee, Nga-Lam; Su, Chang; Su, Wei-Wei; Li, Pei-Bo; Shaw, Pang-Chui

    2016-01-22

    N16 is a protein from the nacreous layer of Pinctada fucata, a pearl oyster. It has been found to promote biomineralization, and we hypothesized that it also plays a role in bone metabolism. The cDNA of N16 was cloned and expressed in Escherichia coli to produce N16 protein, which was purified to high homogeneity by ion-exchange and gel filtration columns. The effects of N16 on osteoclast differentiation and osteogenesis were clarified using the murine preosteoclast cell line RAW 264.7 and the preosteoblast cell line MC3T3-E1. Results on preosteoclasts showed that N16 only slightly inhibited cell survival but significantly inhibited differentiation induced by receptor activator of nuclear factor kappa-B ligand (RANKL). Apart from reduced formation of multinucleated osteoclasts, N16-treated cells exhibited lower gene expression and enzymatic activity typical of mature osteoclasts. Actin ring formation and intracellular acidification essential for osteoclastic function were also impaired upon N16 treatment. At concentrations nontoxic to preosteoblasts, N16 strongly up-regulated alkaline phosphatase activity and increased mineralized nodule formation, which are indicative of differentiation into osteoblasts. These effects coincided with an increase in mRNA expression of osteoblast markers osteopotin and osteocalcin. The present study demonstrated that N16 has both anabolic and antiresorptive effects on bone, which makes it potentially useful for treating osteoporosis. PMID:26741297

  18. NDRG2 Expression Decreases Tumor-Induced Osteoclast Differentiation by Down-regulating ICAM1 in Breast Cancer Cells.

    PubMed

    Kim, Bomi; Nam, Sorim; Lim, Ji Hyun; Lim, Jong-Seok

    2016-01-01

    Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression.

  19. TAK1 Is Essential for Osteoclast Differentiation and Is an Important Modulator of Cell Death by Apoptosis and Necroptosis

    PubMed Central

    Lai, YunJu; Xie, Min; Schneider, Michael D.

    2013-01-01

    Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), a mitogen-activated protein 3 (MAP3) kinase, plays an essential role in inflammation by activating the IκB kinase (IKK)/nuclear factor κB (NF-κB) and stress kinase (p38 and c-Jun N-terminal kinase [JNK]) pathways in response to many stimuli. The tumor necrosis factor (TNF) superfamily member receptor activator of NF-κB ligand (RANKL) regulates osteoclastogenesis through its receptor, RANK, and the signaling adaptor TRAF6. Because TAK1 activation is mediated through TRAF6 in the interleukin 1 receptor (IL-1R) and toll-like receptor (TLR) pathways, we sought to investigate the consequence of TAK1 deletion in RANKL-mediated osteoclastogenesis. We generated macrophage colony-stimulating factor (M-CSF)-derived monocytes from the bone marrow of mice with TAK1 deletion in the myeloid lineage. Unexpectedly, TAK1-deficient monocytes in culture died rapidly but could be rescued by retroviral expression of TAK1, inhibition of receptor-interacting protein 1 (RIP1) kinase activity with necrostatin-1, or simultaneous genetic deletion of TNF receptor 1 (TNFR1). Further investigation using TAK1-deficient mouse embryonic fibroblasts revealed that TNF-α-induced cell death was abrogated by the simultaneous inhibition of caspases and knockdown of RIP3, suggesting that TAK1 is an important modulator of both apoptosis and necroptosis. Moreover, TAK1-deficient monocytes rescued from programmed cell death did not form mature osteoclasts in response to RANKL, indicating that TAK1 is indispensable to RANKL-induced osteoclastogenesis. To our knowledge, we are the first to report that mice in which TAK1 has been conditionally deleted in osteoclasts develop osteopetrosis. PMID:23166301

  20. Apolipoprotein E inhibits osteoclast differentiation via regulation of c-Fos, NFATc1 and NF-κB

    SciTech Connect

    Kim, Woo-Shin; Kim, Hyung Joon; Lee, Zang Hee; Lee, Youngkyun; Kim, Hong-Hee

    2013-02-15

    Apolipoprotein E (ApoE) plays a major role in the transport and metabolism of lipid. Other functions of ApoE include modulation of innate and adaptive immune responses. The expression of ApoE in osteoblasts and its relevance with bone formation have also been reported. However, the effect of ApoE on osteoclasts has not yet been examined. Here, we investigated the role of ApoE in osteoclast differentiation using bone marrow-derived macrophages (BMMs) and RAW264.7 cells. We found a down-regulation of ApoE gene expression during osteoclastic differentiation of those cells. Overexpression of ApoE in BMMs and RAW264.7 cells significantly blocked the induction of c-Fos and nuclear factor of activated T cell c1 (NFATc1), transcription factors critical for expression of osteoclast marker genes, by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. ApoE inhibited osteoclast differentiation, as measured by decreased number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs). In addition, ApoE reduced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and ATPase, H{sup +} transporting, lysosomal 38 kDa, V0 subunit d2 (ATP6v0d2), genes involved in cell–cell fusion during osteoclastogenesis. Knock-down of ApoE using a specific siRNA promoted the RANKL-mediated induction of osteoclast differentiation. While ApoE did not affect the activation of ERK, JNK, and p38 MAPK signaling pathways by RANKL, the phosphorylation of p65 trans-activation domain on serine 536 and transcription activity of NF-κB were reduced by ApoE overexpression. These findings suggest that ApoE plays an inhibitory role in osteoclast differentiation via the suppression of RANKL-dependent activation of NF-κB and induction of c-Fos and NFATc1. - Highlights: ► Apolipoprotein E (ApoE) significantly inhibited osteoclast differentiation and activation of NF-κB. ► ApoE decreased the induction of osteoclast marker

  1. Effects of extracellular magnesium on the differentiation and function of human osteoclasts.

    PubMed

    Wu, Lili; Luthringer, Bérengère J C; Feyerabend, Frank; Schilling, Arndt F; Willumeit, Regine

    2014-06-01

    Magnesium-based implants have been shown to influence the surrounding bone structure. In an attempt to partially reveal the cellular mechanisms involved in the remodelling of magnesium-based implants, the influence of increased extracellular magnesium content on human osteoclasts was studied. Peripheral blood mononuclear cells were driven towards an osteoclastogenesis pathway via stimulation with receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor for 28 days. Concomitantly, the cultures were exposed to variable magnesium concentrations (from either magnesium chloride or magnesium extracts). Osteoclast proliferation and differentiation were evaluated based on cell metabolic activity, total protein content, tartrate-resistant acid phosphatase activity, cathepsin K and calcitonin receptor immunocytochemistry, and cellular ability to form resorption pits. While magnesium chloride first enhanced and then opposed cell proliferation and differentiation in a concentration-dependent manner (peaking between 10 and 15mM magnesium chloride), magnesium extracts (with lower magnesium contents) appeared to decrease cell metabolic activity (≈50% decrease at day 28) while increasing osteoclast activity at a lower concentration (twofold higher). Together, the results indicated that (i) variations in the in vitro extracellular magnesium concentration affect osteoclast metabolism and (ii) magnesium extracts should be used preferentially in vitro to more closely mimic the in vivo environment.

  2. Rosmarinic acid and arbutin suppress osteoclast differentiation by inhibiting superoxide and NFATc1 downregulation in RAW 264.7 cells

    PubMed Central

    OMORI, AKINA; YOSHIMURA, YOSHITAKA; DEYAMA, YOSHIAKI; SUZUKI, KUNIAKI

    2015-01-01

    The present study investigated the effect of the natural polyphenols, rosmarinic acid and arbutin, on osteoclast differentiation in RAW 264.7 cells. Rosmarinic acid and arbutin suppressed osteoclast differentiation and had no cytotoxic effect on osteoclast precursor cells. Rosmarinic acid and arbutin inhibited superoxide production in a dose-dependent manner. mRNA expression of the master regulator of osteoclastogenesis, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and the osteoclast marker genes, matrix metalloproteinase-9, tartrate-resistant acid phosphatase and cathepsin-K, decreased following treatments with rosmarinic acid and arbutin. Furthermore, resorption activity decreased with the number of osteoclasts. These results suggest that rosmarinic acid and arbutin may be useful for the prevention and treatment of bone diseases, such as osteoporosis, through mechanisms involving inhibition of superoxide and downregulation of NFATc1. PMID:26171153

  3. Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells

    PubMed Central

    Lee, Sang-Im; Yi, Jin-Kyu; Bae, Won-Jung; Lee, Soojung; Cha, Hee-Jae; Kim, Eun-Cheol

    2016-01-01

    Background Recent reports suggest that thymosin beta-4 (Tβ4) is a key regulator for wound healing and anti-inflammation. However, the role of Tβ4 in osteoclast differentiation remains unclear. Purpose The purpose of this study was to evaluate Tβ4 expression in H2O2-stimulated human periodontal ligament cells (PDLCs), the effects of Tβ4 activation on inflammatory response in PDLCs and osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs), and identify the underlying mechanism. Methods Reverse transcription-polymerase chain reactions and Western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages (BMMs) using conditioned medium (CM) from H2O2-treated PDLCs. Results Tβ4 was down-regulated in H2O2-exposed PDLCs in dose- and time-dependent manners. Tβ4 activation with a Tβ4 peptide attenuated the H2O2-induced production of NO and PGE2 and up-regulated iNOS, COX-2, and osteoclastogenic cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-17) as well as reversed the effect on RANKL and OPG in PDLCs. Tβ4 peptide inhibited the effects of H2O2 on the activation of ERK and JNK MAPK, and NF-κB in PDLCs. Furthermore, Tβ4 peptide inhibited osteoclast differentiation, osteoclast-specific gene expression, and p38, ERK, and JNK phosphorylation and NF-κB activation in RANKL-stimulated BMMs. In addition, H2O2 up-regulated Wnt5a and its cell surface receptors, Frizzled and Ror2 in PDLCs. Wnt5a inhibition by Wnt5a siRNA enhanced the effects of Tβ4 on H2O2-mediated induction of pro-inflammatory cytokines and osteoclastogenic cytokines as well as helping osteoclastic differentiation whereas Wnt5a activation by Wnt5a peptide reversed it. Conclusion In conclusion, this study demonstrated, for the first time, that Tβ4 was down-regulated in ROS-stimulated PDLCs as well as Tβ4 activation exhibited anti-inflammatory effects and anti-osteoclastogenesis in vitro

  4. Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

    PubMed

    Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

    2015-10-01

    Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG.

  5. Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

    PubMed

    Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

    2015-10-01

    Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. PMID:25820496

  6. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    SciTech Connect

    Zawawi, M.S.F.; Dharmapatni, A.A.S.S.K.; Cantley, M.D.; McHugh, K.P.; Haynes, D.R.; Crotti, T.N.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  7. Impaired recruitment and differentiation of osteoclast progenitors by osteocalcin-deplete bone implants.

    PubMed

    Glowacki, J; Lian, J B

    1987-09-01

    This is a report of an experimental system to study differentiation of bone-resorbing osteoclasts and demonstrates that osteocalcin, an extracellular bone-specific component, is necessary for the recruitment of osteoclast progenitor cells. The subcutaneous implantation of devitalized bone particles (BPs) elicits the recruitment and differentiation of osteoclasts that resorb the BPs. In a previous study, we showed by histomorphometric analysis that BPs that were deficient in osteocalcin were resorbed only 60% as well as normal BPs. In this study, the mechanism of this difference was investigated by measurements of recruitment, differentiation and activity of bone resorbing cells by normal and osteocalcin-deficient BP. Mononuclear cells were attracted to control BPs soon after implantation. In dramatic contrast, cellularity was depressed around osteocalcin-deficient BPs with very few mononuclear cells within the implant on day 5 (35% of control cellularity). In implants of normal BPs, tartrate-resistant acid phosphatase-positive multinucleated cells were evident by day 5; very few appeared in implants of osteocalcin-deplete BPs even by day 12. The amount of tartrate-resistant acid phosphatase activity in homogenates of the osteocalcin-deficient bone particle specimens not only lagged behind controls but never reached the maximum activity of control BP specimens. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and/or activation of cells for bone resorption.

  8. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    SciTech Connect

    Takemura, Kenichi; Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji; Suzuki, Hiroshi; Kodama, Tatsuhiko; Mizuta, Hiroshi; Takeya, Motohiro

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  9. Development of 1-Amino-4-(phenylamino)anthraquinone-2-sulfonate Sodium Derivatives as a New Class of Inhibitors of RANKL-Induced Osteoclastogenesis.

    PubMed

    Lee, Chia-Chung; Chen, Chun-Liang; Liu, Fei-Lan; Chiou, Chung-Yu; Chen, Tsung-Chih; Wu, Cheng-Chi; Sun, Wei-Hsin; Chang, Deh-Ming; Huang, Hsu-Shan

    2016-05-01

    A series of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives was synthesized and evaluated for osteoclast inhibition using a TRAP-staining assay. Among them, two compounds, LCCY-13 and LCCY-15, dose-dependently suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Moreover, the cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds was not a result of their cytotoxicity. Further, the inhibitory activities of compounds LCCY-13 and LCCY-15 were further confirmed by including specific inhibition of NFATc1 expression levels in nuclei using an immunofluorescent analysis. In addition, LCCY-13 and LCCY-15 also significantly attenuated the bone resorption activity of osteoclasts according to a pit formation assay. Thus, a new class of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium compounds might be considered as an essential lead structure for the further development of anti-resorptive agents.

  10. Dendritic cells enhance UHMWPE wear particle-induced osteoclast differentiation of macrophages.

    PubMed

    Cang, Dingwei; Guo, Kaijin; Zhao, Fengchao

    2015-10-01

    Ultra-high molecular weight polyethylene (UHMWPE) has been widely used in large joint replacement. Osteolysis induced by the UHMWPE wear particles is one of the main causes of replacement failure. This study aims to elucidate whether dendritic cells play a role in UHMWPE particle-induced osteolysis. An in vitro Raw 264.7 and DC 2.4 coculture system was employed to examine the effects of dendritic cells on the inflammatory and osteoclastogenic responses of Raw 264.7 toward UHMWPE particles. The expression of cytokines, NF-κB, and osteoclast marker genes was analyzed by ELISA, western blot, or quantitative PCR. The osteoclast differentiation was measured by TRAP staining and flow cytometry. UHMWPE particles induced Raw 264.7 cells to differentiate into osteoclasts, which was enhanced by coculturing with DC 2.4 cells. DC 2.4 cells augmented UHMWPE particle-elicited activation of NF-κB signaling, higher levels of TNF-α and MCP-1, and an increased expression of MMP-9, Calcr, and Ctsk, though DC 2.4 coculture alone did not significantly cause the aforementioned changes. These results suggest that dendritic cells, among other immune cells recruited by UHMWPE particle induced inflammation, could further exacerbate inflammation and osteolysis.

  11. Triterpenoid Saponin W3 from Anemone flaccida Suppresses Osteoclast Differentiation through Inhibiting Activation of MAPKs and NF-κB Pathways.

    PubMed

    Kong, Xiangying; Yang, Yue; Wu, Wenbin; Wan, Hongye; Li, Xiaomin; Zhong, Michun; Su, Xiaohui; Jia, Shiwei; Lin, Na

    2015-01-01

    Excessive bone resorption by osteoclasts within inflamed joints is the most specific hallmark of rheumatoid arthritis. A. flaccida has long been used for the treatment of arthritis in folk medicine of China; however, the active ingredients responsible for the anti-arthritis effects of A. flaccida are still elusive. In this study, W3, a saponin isolated from the extract of A. flaccida was identified as the major active ingredient by using an osteoclast formation model induced by receptor activator of nuclear factor kappa-B ligand (RANKL). W3 dose-dependently suppressed the actin ring formation and lacunar resorption. Mechanistic investigation revealed that W3 inhibited the RANKL-induced TRAF6 expression, decreased phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and suppressed NF-κB p65 DNA binding activity. Furthermore, W3 almost abrogated the expression of c-Fos and nuclear factor of activated T cells (NFATc1). Therefore, our results suggest that W3 is a potential agent for treating lytic bone diseases although further evaluation in vivo and in clinical trials is needed. PMID:26327814

  12. Tumor Necrosis Factor α Stimulates Osteoclast Differentiation by a Mechanism Independent of the Odf/Rankl–Rank Interaction

    PubMed Central

    Kobayashi, Kanichiro; Takahashi, Naoyuki; Jimi, Eijiro; Udagawa, Nobuyuki; Takami, Masamichi; Kotake, Shigeru; Nakagawa, Nobuaki; Kinosaki, Masahiko; Yamaguchi, Kyoji; Shima, Nobuyuki; Yasuda, Hisataka; Morinaga, Tomonori; Higashio, Kanji; Martin, T. John; Suda, Tatsuo

    2000-01-01

    Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases. PMID:10637272

  13. Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells

    PubMed Central

    Chen, Shulan; Ye, Xin; Yu, Xinbo; Xu, Quanchen; Pan, Keqing; Lu, Shulai; Yang, Pishan

    2015-01-01

    Objectives: Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro. Materials and methods: Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated. Results: PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7. Conclusions: PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion. PMID:26823783

  14. ATF3 controls proliferation of osteoclast precursor and bone remodeling

    PubMed Central

    Fukasawa, Kazuya; Park, Gyujin; Iezaki, Takashi; Horie, Tetsuhiro; Kanayama, Takashi; Ozaki, Kakeru; Onishi, Yuki; Takahata, Yoshifumi; Yoneda, Yukio; Takarada, Takeshi; Kitajima, Shigetaka; Vacher, Jean; Hinoi, Eiichi

    2016-01-01

    Bone homeostasis is maintained by the sophisticated coupled actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here we identify activating transcription factor 3 (ATF3) as a pivotal transcription factor for the regulation of bone resorption and bone remodeling under a pathological condition through modulating the proliferation of osteoclast precursors. The osteoclast precursor-specific deletion of ATF3 in mice led to the prevention of receptor activator of nuclear factor-κB (RANK) ligand (RANKL)-induced bone resorption and bone loss, although neither bone volume nor osteoclastic parameter were markedly altered in these knockout mice under the physiological condition. RANKL-dependent osteoclastogenesis was impaired in vitro in ATF3-deleted bone marrow macrophages (BMM). Mechanistically, the deficiency of ATF3 impaired the RANKL-induced transient increase in cell proliferation of osteoclast precursors in bone marrow in vivo as well as of BMM in vitro. Moreover, ATF3 regulated cyclin D1 mRNA expression though modulating activator protein-1-dependent transcription in the osteoclast precursor, and the introduction of cyclin D1 significantly rescued the impairment of osteoclastogenesis in ATF3-deleted BMM. Therefore, these findings suggest that ATF3 could have a pivotal role in osteoclastogenesis and bone homeostasis though modulating cell proliferation under pathological conditions, thereby providing a target for bone diseases. PMID:27480204

  15. Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

    SciTech Connect

    Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.; Laurence, Jeffrey

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. Black-Right-Pointing-Pointer Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. Black-Right-Pointing-Pointer Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. Black-Right-Pointing-Pointer Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/{beta}-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of {beta}-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, {beta}-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in

  16. NHE10, a novel osteoclast-specific member of the Na{sup +}/H{sup +} exchanger family, regulates osteoclast differentiation and survival

    SciTech Connect

    Lee, Seoung Hoon; Kim, Taesoo; Park, Eui-Soon; Yang, Sujeong; Jeong, Daewon; Choi, Yongwon; Rho, Jaerang

    2008-05-02

    Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na{sup +}/H{sup +} exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-{kappa}B ligand signaling and is required for OC differentiation and survival.

  17. A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

    PubMed

    Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

    2015-02-01

    Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.

  18. A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

    PubMed

    Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

    2015-02-01

    Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism. PMID:25474166

  19. Murine osteoblastic and osteoclastic differentiation on strontium releasing hydroxyapatite forming cements.

    PubMed

    Singh, Satish S; Roy, Abhijit; Lee, Boeun; Parekh, Shrey; Kumta, Prashant N

    2016-06-01

    Ionic substitutions in hydroxyapatite (HA) scaffolds and self-setting cements containing Sr(2+) ions incorporated are particularly of interest in bone regeneration. To date, the approach widely used to incorporate Sr(2+) ions into HA cements has been the addition of Sr(2+) containing salts, such as SrCO3, SrCl2∙6H2O, or SrHPO4. However, this approach is dependent upon the relative solubility of Sr(2+) containing salts with respect to calcium phosphate (CaP) precursors. Therefore, in the current study Sr(2+) substituted dicalcium phosphate dihydrate (DCPD) was first synthesized and directly reacted with tetracalcium phosphate (TTCP) to form Sr(2+) substituted HA forming cements. Rietveld refinement indicated that after one week of aging in phosphate buffered saline, cements prepared with and without Sr(2+) were composed of 75% HA and 25% unreacted TTCP by weight. Cements prepared with 10% Sr(2+) DCPD exhibited increased compressive strengths in comparison to unsubstituted cements. Increased MC3T3-E1 proliferation and differentiation were also observed on the cements prepared with increasing Sr(2+) content. It was concluded that both the scaffold microstructure and Sr(2+) ion release supported osteogenic differentiation. With respect to osteoclastic differentiation, no statistically significant differences in TRAP activity or cell morphology were observed. This suggests that the amount of Sr(2+) released may have been too low to influence osteoclast formation in comparison to unsubstituted cements. The results obtained herein demonstrate that the use of Sr(2+) substituted DCPD precursors rather than individually separate Sr(2+) containing salts may be a useful approach to prepare Sr(2+) containing HA cements. PMID:27040237

  20. Lysophosphatidic Acid Receptor Type 1 (LPA1) Plays a Functional Role in Osteoclast Differentiation and Bone Resorption Activity*

    PubMed Central

    David, Marion; Machuca-Gayet, Irma; Kikuta, Junichi; Ottewell, Penelope; Mima, Fuka; Leblanc, Raphael; Bonnelye, Edith; Ribeiro, Johnny; Holen, Ingunn; Vales, Rùben Lopez; Jurdic, Pierre; Chun, Jerold; Clézardin, Philippe; Ishii, Masaru; Peyruchaud, Olivier

    2014-01-01

    Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1–6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1−/− mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1−/− mice but not in Lpar2−/− and Lpar3−/− animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1−/− osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP+ osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss. PMID:24429286

  1. T cell costimulation molecules CD80/86 inhibit osteoclast differentiation by inducing the IDO/tryptophan pathway.

    PubMed

    Bozec, Aline; Zaiss, Mario M; Kagwiria, Rosebeth; Voll, Reinhard; Rauh, Manfred; Chen, Zhu; Mueller-Schmucker, Sandra; Kroczek, Richard A; Heinzerling, Lucie; Moser, Muriel; Mellor, Andrew L; David, Jean-Pierre; Schett, Georg

    2014-05-01

    Bone resorption is seminal for the physiological remodeling of bone during life. However, this process needs to be strictly controlled; excessive bone resorption results in pathologic bone loss, osteoporosis, and fracture. We describe a control mechanism of bone resorption by the adaptive immune system. CD80/86, a pair of molecules expressed by antigen-presenting cells and involved in T cell costimulation, act as negative regulator for the generation of bone-resorbing osteoclasts. CD80/86-deficient mice were osteopenic because of increased osteoclast differentiation. CD80/86-deficient osteoclasts escaped physiological inhibition by CTLA-4 or regulatory T cells. Mechanistically, engagement of CD80/86 by CTLA-4 induced activation of the enzyme indoleamine 2,3-dioxygenase (IDO) in osteoclast precursors, which degraded tryptophan and promoted apoptosis. Concordantly, IDO-deficient mice also showed an osteopenic bone phenotype with higher numbers of osteoclast precursors and osteoclasts. Also, IDO-deficient mononuclear cells escaped the anti-osteoclastogenic effect of CTLA-4. This molecular mechanism was also present in humans because targeting CD80/86 by abatacept, a CTLA-4-immunoglobulin fusion protein, reduced, whereas blockade of CTLA-4 by ipilimumab antibody enhanced, the frequency of peripheral osteoclast precursors and osteoclastogenesis. In summary, these data show an important role of the adaptive immune system, in particular T cell CD80/86 costimulation molecules, in the physiological regulation of bone resorption and preservation of bone mass, as well as affect the understanding of the function of current and future drugs fostering or blocking the effects of CTLA-4 in humans. PMID:24807557

  2. Microgravity Induction of TRAIL Expression in Preosteoclast Cells Enhances Osteoclast Differentiation

    PubMed Central

    Sambandam, Yuvaraj; Baird, Kelsey L.; Stroebel, Maxwell; Kowal, Emily; Balasubramanian, Sundaravadivel; Reddy, Sakamuri V.

    2016-01-01

    Evidence indicates that astronauts experience significant bone loss in space. We previously showed that simulated microgravity (μXg) using the NASA developed rotary cell culture system (RCCS) enhanced bone resorbing osteoclast (OCL) differentiation. However, the mechanism by which μXg increases OCL formation is unclear. RANK/RANKL signaling pathway is critical for OCL differentiation. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to increase osteoclastogenesis. We hypothesize that TRAIL may play an important role in μXg enhanced OCL differentiation. In this study, we identified by RT profiler PCR array screening that μXg induces high levels of TRAIL expression in murine preosteoclast cells in the absence of RANKL stimulation compared to ground based (Xg) cultures. We further identified that μXg elevated the adaptor protein TRAF-6 and fusion genes OC-STAMP and DC-STAMP expression in preosteoclast cells. Interestingly, neutralizing antibody against TRAIL significantly reduced μXg induced OCL formation. We further identified that over-expression of pTRAIL in RAW 264.7 cells enhanced OCL differentiation. These results indicate that TRAIL signaling plays an important role in the μXg increased OCL differentiation. Therefore, inhibition of TRAIL expression could be an effective countermeasure for μXg induced bone loss. PMID:27142480

  3. Microgravity Induction of TRAIL Expression in Preosteoclast Cells Enhances Osteoclast Differentiation.

    PubMed

    Sambandam, Yuvaraj; Baird, Kelsey L; Stroebel, Maxwell; Kowal, Emily; Balasubramanian, Sundaravadivel; Reddy, Sakamuri V

    2016-01-01

    Evidence indicates that astronauts experience significant bone loss in space. We previously showed that simulated microgravity (μXg) using the NASA developed rotary cell culture system (RCCS) enhanced bone resorbing osteoclast (OCL) differentiation. However, the mechanism by which μXg increases OCL formation is unclear. RANK/RANKL signaling pathway is critical for OCL differentiation. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to increase osteoclastogenesis. We hypothesize that TRAIL may play an important role in μXg enhanced OCL differentiation. In this study, we identified by RT profiler PCR array screening that μXg induces high levels of TRAIL expression in murine preosteoclast cells in the absence of RANKL stimulation compared to ground based (Xg) cultures. We further identified that μXg elevated the adaptor protein TRAF-6 and fusion genes OC-STAMP and DC-STAMP expression in preosteoclast cells. Interestingly, neutralizing antibody against TRAIL significantly reduced μXg induced OCL formation. We further identified that over-expression of pTRAIL in RAW 264.7 cells enhanced OCL differentiation. These results indicate that TRAIL signaling plays an important role in the μXg increased OCL differentiation. Therefore, inhibition of TRAIL expression could be an effective countermeasure for μXg induced bone loss. PMID:27142480

  4. Microgravity Induction of TRAIL Expression in Preosteoclast Cells Enhances Osteoclast Differentiation

    NASA Astrophysics Data System (ADS)

    Sambandam, Yuvaraj; Baird, Kelsey L.; Stroebel, Maxwell; Kowal, Emily; Balasubramanian, Sundaravadivel; Reddy, Sakamuri V.

    2016-05-01

    Evidence indicates that astronauts experience significant bone loss in space. We previously showed that simulated microgravity (μXg) using the NASA developed rotary cell culture system (RCCS) enhanced bone resorbing osteoclast (OCL) differentiation. However, the mechanism by which μXg increases OCL formation is unclear. RANK/RANKL signaling pathway is critical for OCL differentiation. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to increase osteoclastogenesis. We hypothesize that TRAIL may play an important role in μXg enhanced OCL differentiation. In this study, we identified by RT profiler PCR array screening that μXg induces high levels of TRAIL expression in murine preosteoclast cells in the absence of RANKL stimulation compared to ground based (Xg) cultures. We further identified that μXg elevated the adaptor protein TRAF-6 and fusion genes OC-STAMP and DC-STAMP expression in preosteoclast cells. Interestingly, neutralizing antibody against TRAIL significantly reduced μXg induced OCL formation. We further identified that over-expression of pTRAIL in RAW 264.7 cells enhanced OCL differentiation. These results indicate that TRAIL signaling plays an important role in the μXg increased OCL differentiation. Therefore, inhibition of TRAIL expression could be an effective countermeasure for μXg induced bone loss.

  5. Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice.

    PubMed

    Narahara, Shun; Matsushima, Haruna; Sakai, Eiko; Fukuma, Yutaka; Nishishita, Kazuhisa; Okamoto, Kuniaki; Tsukuba, Takayuki

    2012-04-01

    Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H(2)O(2)) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

  6. Gene Disruption of the Calcium Channel Orai1 Results in Inhibition of Osteoclast and Osteoblast Differentiation and Impairs Skeletal Development

    PubMed Central

    Robinson, Lisa J.; Mancarella, Salvatore; Songsawad, Duangrat; Tourkova, Irina L.; Barnett, John B.; Gill, Donald L.; Soboloff, Jonathan; Blair, Harry C.

    2012-01-01

    Calcium signaling plays a central role in the regulation of bone cells, though uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1−/− mice lacked multinucleated osteoclasts. Yet they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1−/− mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1−/− mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1−/− animals compared to controls; bone deposition was markedly decreased in the knock-out. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1−/− and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation. PMID:22546867

  7. Potent inhibitory effect of Foeniculum vulgare Miller extract on osteoclast differentiation and ovariectomy-induced bone loss.

    PubMed

    Kim, Tae-Ho; Kim, Hyun-Ju; Lee, Sang-Han; Kim, Shin-Yoon

    2012-06-01

    Inhibition of osteoclast differentiation and bone resorption is considered an effective therapeutic approach to the treatment of postmenopausal bone loss. To find natural compounds that may inhibit osteoclastogenesis, we screened herbal extracts on bone marrow cultures. In this study, we found that an aqueous extract of Foeniculum vulgare Miller seed (FvMs) at low concentration, which has traditionally been used as a treatment for a variety of ailments, inhibits the osteoclast differentiation and bone resorptive activity of mature osteoclasts. We further investigated the effects of FvMs on ovariectomy (OVX)-induced bone loss using microcomputed tomography, biomechanical tests and serum marker assays for bone remodeling. Oral administration of FvMs (30 mg or 100 mg/kg/day) for 6 weeks had an intermediary effect on the prevention of femoral bone mineral density (BMD), bone mineral content (BMC), and other parameters compared to OVX controls. In addition, FvMs slightly decreased bone turnover markers that were accelerated by OVX. The bone-protective effects of FvMs may be due to suppression of an OVX-induced increase in bone turnover. Collectively, our findings indicate that FvMs have potential in preventing bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.

  8. Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca(2+) Signaling Pathway and Prevents Inflammation-Mediated Bone Loss.

    PubMed

    Kim, Ju-Young; Park, Sun-Hyang; Baek, Jong Min; Erkhembaatar, Munkhsoyol; Kim, Min Seuk; Yoon, Kwon-Ha; Oh, Jaemin; Lee, Myeung Su

    2015-09-25

    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. PMID:26308264

  9. The Inhibition of RANKL-Induced Osteoclastogenesis through the Suppression of p38 Signaling Pathway by Naringenin and Attenuation of Titanium-Particle-Induced Osteolysis

    PubMed Central

    Wang, Wengang; Wu, Chuanlong; Tian, Bo; Liu, Xuqiang; Zhai, Zanjing; Qu, Xinhua; Jiang, Chuan; Ouyang, Zhengxiao; Mao, Yuanqing; Tang, Tingting; Qin, An; Zhu, Zhenan

    2014-01-01

    The aim of this study was to assess the effect of naringenin on osteoclastogenesis and titanium particle-induced osteolysis. Osteolysis from wear-induced particles and aseptic loosening are the most frequent late complications of total joint arthroplasty leading to revision of the prosthesis. Osteolysis during aseptic loosening is most likely due to increased bone resorption by osteoclasts. Through in vitro studies, we demonstrated that naringenin, a naturally occurring flavanone in grapefruit and tomatoes, exerts potent inhibitory effects on the ligand of the receptor activator of nuclear factor-κB (RANKL)-induced osteoclastogenesis and revealed that the mechanism of action of naringenin, which inhibited osteoclastogenesis by suppression of the p38 signaling pathway. Through in vivo studies, we proved that naringenin attenuated titanium particle-induced osteolysis in a mouse calvarial model. In general, we demonstrated that naringenin inhibited osteoclastogenesis via suppression of p38 signaling in vitro and attenuated titanium particle-induced osteolysis in vivo. This study also suggested that naringenin has significant potential for the treatment of osteolysis-related diseases caused by excessive osteoclast formation and activity. PMID:25464380

  10. Schisantherin A suppresses osteoclast formation and wear particle-induced osteolysis via modulating RANKL signaling pathways

    SciTech Connect

    He, Yi; Zhang, Qing; Shen, Yi; Chen, Xia; Zhou, Feng; Peng, Dan

    2014-07-04

    Highlights: • Schisantherin A suppresses osteoclasts formation and function in vitro. • Schisantherin A impairs RANKL signaling pathway. • Schisantherin A suppresses osteolysis in vivo. • Schisantherin A may be used for treating osteoclast related diseases. - Abstract: Receptor activator of NF-κB ligand (RANKL) plays critical role in osteoclastogenesis. Targeting RANKL signaling pathways has been a promising strategy for treating osteoclast related bone diseases such as osteoporosis and aseptic prosthetic loosening. Schisantherin A (SA), a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been used as an antitussive, tonic, and sedative agent, but its effect on osteoclasts has been hitherto unknown. In the present study, SA was found to inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, SA inhibited OSCAR, cathepsin K and TRAP in a dose dependent manner. Further signal transduction studies revealed that SA down-regulate RANKL-induced nuclear factor-kappaB (NF-κB) signaling activation by suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the NF-κB transcriptional activity. Moreover, SA also decreased the RANKL-induced MAPKs signaling pathway, including JNK and ERK1/2 posphorylation while had no obvious effects on p38 activation. Finally, SA suppressed the NF-κB and MAPKs subsequent gene expression of NFATc1 and c-Fos. In vivo studies, SA inhibited osteoclast function and exhibited bone protection effect in wear-particle-induced bone erosion model. Taken together, SA could attenuate osteoclast formation and wear particle-induced osteolysis by mediating RANKL signaling pathways. These data indicated that SA is a promising therapeutic natural compound for the treatment of osteoclast-related prosthesis loosening.

  11. Manganese superoxide dismutase is required to maintain osteoclast differentiation and function under static force

    PubMed Central

    Guo, Tao; Zhang, Liqiang; Konermann, Anna; Zhou, Hong; Jin, Fang; Liu, Wenjia

    2015-01-01

    Bone homeostasis is maintained by the balance of osteoblasts (OBs) and osteoclasts (OCs). Increased activity of OCs not only contributes to pathological bone resorption, such as osteoporosis and periodontitis, but also is responsible for physiological conditions like orthodontic tooth movement (OTM). However, the detailed mechanism by which orthodontic force promotes the formation of OCs is still poorly understood. In this study, we confirmed that static force promoted the differentiation of human cord monocytes (HMNCs) into OCs depending on loading time and magnitude. Protein expression profiles among HMNCs, HMNCs subjected to static force and mature OCs were established via 2-DE and MALDI-TOF-MS analyses. Total respective protein spot numbers of 549 ± 13, 612 ± 19 and 634 ± 16 were detected in each of the gels by image analysis. The five proteins identified were plasminogen activator inhibitor 2 (PAI-2, Spot 1), peroxiredoxin-6 (PRD-6, Spot 3), manganese superoxide dismutase (SOD2, Spot 6), Rho GDP-dissociation inhibitor 2 (Rho-GDI2, Spot 11) and L-lactate dehydrogenase B chain (L-LDH, Spot 15). More importantly, we revealed that SOD2 was required to maintain monocyte differentiation into functional OCs and may become a potential target for regulating the efficiency of OTM in the future. PMID:25619900

  12. Inhibitory effects of French pine bark extract, Pycnogenol®, on alveolar bone resorption and on the osteoclast differentiation.

    PubMed

    Sugimoto, Hideki; Watanabe, Kiyoko; Toyama, Toshizo; Takahashi, Shun-suke; Sugiyama, Shuta; Lee, Masaichi-Chang-il; Hamada, Nobushiro

    2015-02-01

    Pycnogenol(®) (PYC) is a standardized bark extract from French maritime pine (Pinus pinaster Aiton). We examined the inhibitory effects of PYC on alveolar bone resorption, which is a characteristic feature of periodontitis, induced by Porphyromonas gingivalis (P. gingivalis) and osteoclast differentiation. In rat periodontitis model, rats were divided into four groups: group A served as the non-infected control, group B was infected orally with P. gingivalis ATCC 33277, group C was administered PYC in the diet (0.025%: w/w), and group D was infected with P. gingivalis and administered PYC. Administration of PYC along with P. gingivalis infection significantly reduced alveolar bone resorption. Treatment of P. gingivalis with 1 µg/ml PYC reduced the number of viable bacterial cells. Addition of PYC to epithelial cells inhibited adhesion and invasion by P. gingivalis. The effect of PYC on osteoclast formation was confirmed by tartrate-resistant acid phosphatase staining. PYC treatment significantly inhibited osteoclast formation. Addition of PYC (1-100 µg/ml) to purified osteoclasts culture induced cell apoptosis. These results suggest that PYC may prevent alveolar bone resorption through its antibacterial activity against P. gingivalis and by suppressing osteoclastogenesis. Therefore, PYC may be useful as a therapeutic and preventative agent for bone diseases such as periodontitis.

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

    PubMed

    Deepak, Vishwa; Kasonga, Abe; Kruger, Marlena Cathorina; Coetzee, Magdalena

    2016-07-01

    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. PMID:27170515

  14. Differential expression of chemokines, chemokine receptors and proteinases by foreign body giant cells (FBGCs) and osteoclasts.

    PubMed

    Khan, Usman A; Hashimi, Saeed M; Khan, Shershah; Quan, Jingjing; Bakr, Mahmoud M; Forwood, Mark R; Morrison, Nigel M

    2014-07-01

    Osteoclasts and foreign body giant cells (FBGCs) are both derived from the fusion of macropahges. These cells are seen in close proximity during foreign body reactions, therefore it was assumed that they might interact with each other. The aim was to identify important genes that are expressed by osteoclasts and FBGCs which can be used to understand peri-implantitis and predict the relationship of these cells during foreign body reactions. Bone marrow macrophages (BMM) were treated with receptor activator of nuclear factor kappa B ligand (RANKL) to produce osteoclasts. Quantitative PCR (qPCR) was used to identify the genes that were expressed by osteoclasts and FBGCs compared to macrophage controls. TRAP staining was used to visualise the cells while gelatine zymography and western blots were used for protein expression. Tartrate-resistant acid phosphatase (TRAP), matrix metallo proteinase 9 (MMP9), nuclear factor of activated T cells 1 (NFATc1), cathepsin K (CTSK) and RANK were significantly lower in FBGCs compared to osteoclasts. Inflammation specific chemokines such as monocyte chemotactic protein (MCP1 also called CCL2), macrophage inflammatory protein 1 alpha (MIP1α), MIP1β and MIP1γ, and their receptors CCR1, CCR3 and CCR5, were highly expressed by FBGCs. FBGCs were negative for osteoclast specific markers (RANK, NFATc1, CTSK). FBGCs expressed chemokines such as CCL2, 3, 5 and 9 while osteoclasts expressed the receptors for these chemokines i.e. CCR1, 2 and 3. Our findings show that osteoclast specific genes are not expressed by FBGCs and that FBGCs interact with osteoclasts during foreign body reaction through chemokines.

  15. Rhus javanica Gall Extract Inhibits the Differentiation of Bone Marrow-Derived Osteoclasts and Ovariectomy-Induced Bone Loss

    PubMed Central

    Kim, Tae-Ho; Park, Eui Kyun; Huh, Man-Il; Kim, Hong Kyun; Kim, Shin-Yoon; Lee, Sang-Han

    2016-01-01

    Inhibition of osteoclast differentiation and bone resorption is a therapeutic strategy for the management of postmenopausal bone loss. This study investigated the effects of Rhus javanica (R. javanica) extracts on bone marrow cultures to develop agents from natural sources that may prevent osteoclastogenesis. Extracts of R. javanica (eGr) cocoons spun by Rhus javanica (Bell.) Baker inhibited the osteoclast differentiation and bone resorption. The effects of aqueous extract (aeGr) or 100% ethanolic extract (eeGr) on ovariectomy- (OVX-) induced bone loss were investigated by various biochemical assays. Furthermore, microcomputed tomography (µCT) was performed to study bone remodeling. Oral administration of eGr (30 mg or 100 mg/kg/day for 6 weeks) augmented the inhibition of femoral bone mineral density (BMD), bone mineral content (BMC), and other factors involved in bone remodeling when compared to OVX controls. Additionally, eGr slightly decreased bone turnover markers that were increased by OVX. Therefore, it may be suggested that the protective effects of eGr could have originated from the suppression of OVX-induced increase in bone turnover. Collectively, the findings of this study indicate that eGr has potential to activate bone remodeling by inhibiting osteoclast differentiation and bone loss. PMID:27313644

  16. Rhus javanica Gall Extract Inhibits the Differentiation of Bone Marrow-Derived Osteoclasts and Ovariectomy-Induced Bone Loss.

    PubMed

    Kim, Tae-Ho; Park, Eui Kyun; Huh, Man-Il; Kim, Hong Kyun; Kim, Shin-Yoon; Lee, Sang-Han

    2016-01-01

    Inhibition of osteoclast differentiation and bone resorption is a therapeutic strategy for the management of postmenopausal bone loss. This study investigated the effects of Rhus javanica (R. javanica) extracts on bone marrow cultures to develop agents from natural sources that may prevent osteoclastogenesis. Extracts of R. javanica (eGr) cocoons spun by Rhus javanica (Bell.) Baker inhibited the osteoclast differentiation and bone resorption. The effects of aqueous extract (aeGr) or 100% ethanolic extract (eeGr) on ovariectomy- (OVX-) induced bone loss were investigated by various biochemical assays. Furthermore, microcomputed tomography (µCT) was performed to study bone remodeling. Oral administration of eGr (30 mg or 100 mg/kg/day for 6 weeks) augmented the inhibition of femoral bone mineral density (BMD), bone mineral content (BMC), and other factors involved in bone remodeling when compared to OVX controls. Additionally, eGr slightly decreased bone turnover markers that were increased by OVX. Therefore, it may be suggested that the protective effects of eGr could have originated from the suppression of OVX-induced increase in bone turnover. Collectively, the findings of this study indicate that eGr has potential to activate bone remodeling by inhibiting osteoclast differentiation and bone loss. PMID:27313644

  17. High porous titanium scaffolds showed higher compatibility than lower porous beta-tricalcium phosphate scaffolds for regulating human osteoblast and osteoclast differentiation.

    PubMed

    Hirota, Makoto; Hayakawa, Tohru; Shima, Takaki; Ametani, Akihiro; Tohnai, Iwai

    2015-04-01

    We compared osteoblast and osteoclast differentiation when using beta-tricalcium phosphate (βTCP) and titanium scaffolds by investigating human mesenchymal stem cells (hMSCs) and osteoclast progenitor cell activities. hMSCs were cultured for 7, 14, and 21days on titanium scaffolds with 60%, 73%, and 87% porosity and on βTCP scaffolds with 60% and 75% porosity. Human osteoclast progenitor cells were cultured with osteoblast for 14 and 21days on 87% titanium and 75% βTCP scaffolds. Viable cell numbers with 60% and 73% titanium were higher than with 87% titanium and βTCP scaffolds (P<0.05). An 87% titanium scaffold resulted in the highest osteocalcin production with calcification on day 14 (P<0.01) in titanium scaffolds. All titanium scaffolds resulted in higher osteocalcin production on days 7 and 14 compared to βTCP scaffolds (P<0.01). Osteoblasts cultured on 87% titanium scaffolds suppressed osteoclast differentiation on day 7 but enhanced osteoclast differentiation on day 14 compared to 75% βTCP scaffolds (P<0.01). These findings concluded that high porosity titanium scaffolds could enhance progression of hMSC/osteoblast differentiation and regulated osteoclast differentiation cooperating with osteoblast differentiation for calcification as compared with lower porous βTCP.

  18. Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells to Osteoclasts

    PubMed Central

    Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

    2016-01-01

    Objective Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. Materials and Methods In this experimental study, CD133+ hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Results Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Conclusion Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.

  19. Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells to Osteoclasts

    PubMed Central

    Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

    2016-01-01

    Objective Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. Materials and Methods In this experimental study, CD133+ hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Results Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Conclusion Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast. PMID:27602313

  20. Methanol Extract of Croton Pycnanthus Benth. Inhibits Osteoclast Differentiation by Suppressing the MAPK and NF-κB Signaling Pathways

    PubMed Central

    Lee, Jiyeon

    2014-01-01

    Background Osteoclasts are differentiated from monocytes/macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL). Croton pycnanthus Benth. (CPB) is a herbal plant that belongs to Euphorbiaceae family. The aim of this study was to investigate the effects of CPB on osteoclastogenesis and RANKL-dependent signaling pathways. Methods Methanol extract of CPB was obtained from International Biological Material Research Center. Osteoclast differentiation was achieved by culturing mouse bone marrow-derived macrophages (BMMs) with M-CSF and RANKL. Osteoclast numbers were evaluated by counting multinuclear cells positive for tartrate-resistant acid phosphatase (TRAP). mRNA and protein levels were analyzed by real-time polymerase chain reaction (PCR) and Western blotting, respectively. The activation of signaling molecules were assessed after acute stimulation of cells with high dose of RANKL by Western blotting with phospho-specific antibodies. Results CPB reduced the generation of TRAP-positive multinucleated cells and the activation of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. The induction of the expression of c-Fos, nuclear factor-activated T cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) by RANKL was also suppressed. Conclusions CPB exerts negative effects on osteoclast differentiation in response to the RANKL. The inhibitory mechanism involves the suppression of MAPK and NF-κB signaling pathways and subsequently the down-regulation of c-Fos and NFATc1 transcription factors. PMID:25489576

  1. Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

    SciTech Connect

    Moon, Ho-Jin; Yun, Young-Pil; Han, Choong-Wan; Kim, Min Sung; Kim, Sung Eun; Bae, Min Soo; Kim, Gyu-Tae; Choi, Yong-Suk; Hwang, Eui-Hwan; Lee, Joon Woo; Lee, Jin-Moo; Lee, Chang-Hoon; Kim, Duck-Su; Kwon, Il Keun

    2011-09-23

    Highlights: {yields} We examine bone metabolism of engineered alendronate attached to Ti surfaces. {yields} Alendronate-immobilized Ti enhances activation of osteoblast differentiation. {yields} Alendronate-immobilized Ti inhibits osteoclast differentiation. {yields} Alendronate-immobilized Ti may be a bioactive implant with dual functions. -- Abstract: The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue. In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance

  2. Involvement of Hydrogen Peroxide in the Differentiation of Clonal HD-11EM Cells Into Osteoclast-Like Cells

    PubMed Central

    Steinbeck, Marla J.; Kim, Jung-Keun; Trudeau, Mathew J.; Hauschka, Peter V.; Karnovsky, Morris J.

    2010-01-01

    The present study uses the osteoclast precursor clonal line, HD-11EM, to study the potential of hydrogen peroxide (H2O2) in mediating the differentiation of HD-11EM into osteoclast-like cells. HD-11EM cells are a newly established clonal cell line that, in response to 1α,25-(OH)2D3, differentiate into osteoclast-like cells that are multinucleated (more than three nuclei), express tartrate-resistant acid phosphatase (TRAP), and excavate resorption pits when cultured on dentin slices in the presence of osteoblasts (Hsia et al., 1995, J. Bone Miner. Res., 10(Suppl 1):S424; Hsia, and Hauschka, 1997, unpublished data). Here we demonstrate that HD-11EM express the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase specific cytochrome b558 subunits, and that stimulation of HD-11EM with 1 or 10 nM 1α,25-(OH)2D3 increases the extracellular release of H2O2 within 5–10 min. Ours is the first report that stimulation of a cell with 1α,25-(OH)2D3 enhances the activation of NADPH-oxidase and increases the basal release of superoxide and the formation of its dismutation product, H2O2. To determine the possible involvement of H2O2 in the differentiation of HD-11EM, these cells were exposed to glucose/glucose oxidase. This enzyme system was used to deliver a pure and continuous source of H2O2 in nanomole amounts consistent with quantities produced by HD-11EM in response to 1α,25-(OH)2D3. Both 1α,25-(OH)2D3 and the exogenously generated H2O2 stimulated a dose- and time-dependent increase in TRAP activity/cell and the number of multinucleated cells 24–48 hr after treatment. Northern analysis confirmed an increase in expression of TRAP mRNA in response to either 1α,25-(OH)2D3 or H2O2. Decreases in cell proliferation and v-myc mRNA were also observed in response to these agents. Taken together, our findings indicate that production of H2O2 by HD-11EM is an important local factor involved in differentiation of HD-11EM into osteoclast-like cells, and suggest

  3. Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

    PubMed

    Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

    2012-05-01

    Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis.

  4. Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability, during RANKL-mediated osteoclastogenesis.

    PubMed

    Kim, Ju-Young; Cheon, Yoon-Hee; Yoon, Kwon-Ha; Lee, Myeung Su; Oh, Jaemin

    2014-08-01

    Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of NF-κB ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and IκB, as well as IκB degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption.

  5. NFkappaB decoy oligodeoxynucleotides ameliorates osteoporosis through inhibition of activation and differentiation of osteoclasts.

    PubMed

    Shimizu, H; Nakagami, H; Tsukamoto, I; Morita, S; Kunugiza, Y; Tomita, T; Yoshikawa, H; Kaneda, Y; Ogihara, T; Morishita, R

    2006-06-01

    The transcription factor, nuclear factor-kappa B (NFkappaB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkappaB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkappaB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkappaB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkappaB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkappaB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkappaB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkappaB might be potential therapy in various bone metabolic diseases.

  6. Neuropeptide FF inhibits LPS-mediated osteoclast differentiation of RAW264.7 cells.

    PubMed

    Sun, Yu-Long; Chen, Zhi-Hao; Li, Di-Jie; Zhao, Fan; Ma, Xiao-Li; Shang, Peng; Yang, Tuanming; Qian, Airong

    2014-01-01

    Neuropeptide FF (NPFF) has been implicated in many physiological processes. Previously, we have reported that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. In this study, we investigated the influence of NPFF on lipopolysaccharide (LPS)-mediated osteoclast formation of RAW264.7 cells. Our results suggest that, NPFF dose-dependently (1 nM, 10 nM and 100 nM) inhibited osteoclast formation, TRAP enzyme activity and bone resorption in osteoclasts induced by LPS respectively. Moreover, LPS-provoked NO release was also inhibited by NPFF treatment, indicating a NO-dependent pathway is mainly involved. Furthermore, the alterations of osteoclast marker genes were also assessed including TRAP, Cathepsin K, MMP-9, NFATc1 and Runx2. NPFF downregulated LPS-caused gene augmentations of TRAP, Cathepsin K and MMP-9, whereas showed no influences on NFATc1 and Runx2. In addition, NPFF receptor 2 (NPFFR2) mRNA expression was also augmented in response to NPFF treatment, hinting the involvement of NPFFR2 pathway. It should be mentioned that RF9 (1 µ M), a reported pharmacological inhibitor for NPFF receptors, exerted NPFF-like agonist properties as to attenuate osteoclastogenesis. Collectively, our findings provide new evidence for the in vitro activity of NPFF on osteoclasts, which may be helpful to extend the scope of NPFF functions.

  7. The histamine H2-receptor antagonist, cimetidine, inhibits the articular osteopenia in rats with adjuvant-induced arthritis by suppressing the osteoclast differentiation induced by histamine.

    PubMed

    Yamaura, Katsunori; Yonekawa, Taeko; Nakamura, Tomonori; Yano, Shingo; Ueno, Koichi

    2003-05-01

    The effects of cimetidine on rat adjuvant arthritis (AA) and rat osteoclast differentiation were studied. For the in vivo experiments, AA was induced by injections of Mycobacterium tuberculosis H37RA either subcutaneously into the base of the tail or into the right hind paw. The osteoclast differentiation was assessed by estimating the number of tartrate-resistant acid phosphatase-positive multinuclear cells in the bone marrow culture. Cimetidine, at the dose of 25 mg/kg body weight, reduced the paw swelling by 70% (P<0.01). Cimetidine, at 10 microM concentration, inhibited 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) and histamine mediated osteoclast differentiations by 40% (P<0.01) and 60% (P<0.001), respectively. Dimaprit, at 0.3 microM, stimulated the cell differentiation by 100% (P<0.01). Mepyramine reduced osteoclast differentiation, but the reduction was not statistically significant. Measurements of bone mineral density of the femur indicated that 5 mg/kg of cimetidine treated animals had 30% (P<0.01) higher mineral density in comparison with that of the AA control group that received no cimetidine. These results suggest that histamine is a potent inducer of osteoclast differentiation, at least in part, through the histamine H(2)-receptor, and cimetidine has a preventive effect on articular destruction and accompanying inflammation in arthritic rats. These observations may provide critical insights into the pathogenesis of the bone pathology seen in patients with RA.

  8. Commitment and Differentiation of Osteoclast Precursor Cells by the Sequential Expression of C-Fms and Receptor Activator of Nuclear Factor κb (Rank) Receptors

    PubMed Central

    Arai, Fumio; Miyamoto, Takeshi; Ohneda, Osamu; Inada, Tomohisa; Sudo, Tetsuo; Brasel, Kenneth; Miyata, Takashi; Anderson, Dirk M.; Suda, Toshio

    1999-01-01

    Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor κB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms+RANK−) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms+RANK+) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK–RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation. PMID:10601350

  9. Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

    PubMed

    Kim, Yun-Ho; Kim, Jung-Lye; Lee, Eun-Jung; Park, Sin-Hye; Han, Seon-Young; Kang, Soon Ah; Kang, Young-Hee

    2014-03-01

    Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.

  10. Effects of Brown Rice Extract Treated with Lactobacillus sakei Wikim001 on Osteoblast Differentiation and Osteoclast Formation

    PubMed Central

    Kang, Miran; Song, Jung-Hee; Park, Sung-Hee; Lee, Jong-Hee; Park, Hae Woong; Kim, Tae-Woon

    2014-01-01

    Phytic acid (myo-inositol hexakisphosphate) or phytate is considered an anti-nutrient due to the formation of precipitated complexes that strongly reduces the absorption of essential dietary minerals. In this study, brown rice with reduced phytate was made by inoculation with Lactobacillus sakei Wikim001 having high phytase activity. The effects of brown rice extract treated with L. sakei Wikim001 (BR-WK) on osteoblast differentiation and osteoclast formation were investigated. The proliferation of SaOS-2 cells was measured by the MTT assay. Treatment with BR-WK increased cell proliferation by 136% at a concentration of 100 μg/mL. The Alkaline phosphate activity in SaOS-2 cells was 129% higher when BR-WK was processed at a concentration of 100 μg/mL. The proliferation of bone marrow macrophages decreased by nearly 60% in response to treatment with BR-WK. In addition, BR-WK reduced the number of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from bone marrow macrophages. These results indicate that BR-WK stimulates bone formation through its positive action on osteoblast differentiation and function and furthermore, decreases osteoclast differentiation. PMID:25580402

  11. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    SciTech Connect

    Kaneuji, Takeshi; Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro; Takahashi, Tetsu; Nishihara, Tatsuji

    2011-04-29

    Highlights: {yields} Effect of compressive force on osteoblasts were examined. {yields} Compressive force induced OPG expression and suppressed osteoclastogenesis. {yields} This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm{sup 2}) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-{kappa}B ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of I{kappa}B{alpha}, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca{sup 2+} pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-{kappa}B) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt

  12. MicroRNA-17/20a inhibits glucocorticoid-induced osteoclast differentiation and function through targeting RANKL expression in osteoblast cells.

    PubMed

    Shi, Changgui; Qi, Jin; Huang, Ping; Jiang, Min; Zhou, Qi; Zhou, Hanbing; Kang, Hui; Qian, Niandong; Yang, Qiumeng; Guo, Lei; Deng, Lianfu

    2014-11-01

    Glucocorticoids act on the osteoblasts to up-regulate the expression of RANKL, which is very important in the etiology of glucocorticoid-induced osteoclast differentiation and bone resorption. The mechanisms of this process are still not completely understood. Recent studies have shown that glucocorticoids mediate osteoblast function by decreasing the expression of microRNA-17-92a cluster. Coincidentally, we found that the microRNA-17/20a (microRNA-17, microRNA-20a) seed sequences were also complementary to a sequence conserved in the 3'- untranslated region of RANKL mRNA. Therefore, we hypothesized that glucocorticoids might promote osteoblast-derived RANKL expression by down-regulating microRNA-17/20a, which favors differentiation and function of the osteoclasts. In the present study, Western blot analysis showed that microRNA-17/20a markedly lowered the levels of RANKL protein and attenuated dexamethasone-induced RANKL expression in the osteoblasts. The post-transcriptional repression of RANKL by microRNA-17/20a was further confirmed by the luciferase reporter assay. Furthermore, we found that dexamethasone-induced osteoclast differentiation and function were significantly attenuated in co-culture with osteoblast over-expressed microRNA-17/20a and osteoclast progenitors. These results showed that microRNA-17/20a may play a significant role in glucocorticoid-induced osteoclast differentiation and function by targeting the RANKL expression in osteoblast cells.

  13. Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts

    SciTech Connect

    Koskela, Antti; Viluksela, Matti; Keinänen, Meeri; Tuukkanen, Juha; Korkalainen, Merja

    2012-09-01

    The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6 J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1–10 nM TBT, 0.01–1 nM TCDD or 10 nM TBT + 1 nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption. -- Highlights: ► Combined exposure to TCDD and TBT evoked a unique gene expression profile. ► Osteoblast differentiation was synergistically disturbed after combined exposure. ► Bone resorbing activity was additively decreased after combined exposure.

  14. Carbonic anhydrase II plays a major role in osteoclast differentiation and bone resorption by effecting the steady state intracellular pH and Ca2+.

    PubMed

    Lehenkari, P; Hentunen, T A; Laitala-Leinonen, T; Tuukkanen, J; Väänänen, H K

    1998-07-10

    Carbonic anhydrase II (CA II) expression in characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25 (OH)2D3-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pHi) and Ca2+ (Cai2+) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pHi was dependent neither on the culture substrate nor on the extracellular pH (pHe) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pHi increase significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pHe) of 7.4, but at less extent at pHe 7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pHe 7.4 while inhibition was smaller at pHe 7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pHi regulation.

  15. Triptolide Inhibits Osteoclast Differentiation and Bone Resorption In Vitro via Enhancing the Production of IL-10 and TGF-β1 by Regulatory T Cells

    PubMed Central

    Xu, Huihui; Zhao, Hongyan; Wang, Gui; Huang, Jing; Guo, Minghui; Guo, Baosheng; Tan, Yong

    2016-01-01

    Triptolide, a purified component of Tripterygiumwilfordii Hook F, has been shown to have immunosuppressive and anti-inflammatory properties in rheumatoid arthritis (RA). Although triptolide has demonstrated that it could suppress bone destruction in collagen-induced mice, its therapeutic mechanism remains unclear. Many studies have investigated the effect of triptolide on Tregs and Tregs-related cytokine involved in RA. Additionally, previous studies have implied that Tregs inhibit osteoclast differentiation and bone resorption. Thus, in this study we aimed to explore the regulatory mechanism by which triptolide influences the Treg-mediated production of IL-10 and TGF-β1 to affect osteoclast differentiation and bone resorption. In cocultures system of Tregs and mouse bone marrow macrophages (BMMs), Tregs inhibited the differentiation of osteoclasts and reduced the resorbed areas significantly and the production of both IL-10 and TGF-β1 was upregulated. When the coculture systems were pretreated with triptolide, they produced higher levels of IL-10 and TGF-β1. Our data indicate that triptolide enhances the suppressive effects of Tregs on osteoclast differentiation and bone resorption by enhancing the secretion of IL-10 and TGF-β1. Tregs are most likely involved in the triptolide-mediated regulation of bone metabolism and may provide a potential therapeutic target for the treatment of inflammatory bone destruction. PMID:27413257

  16. Origin of osteoclasts: Mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells

    SciTech Connect

    Udagawa, Nobuyuki; Takahashi, Naoyuki; Akatsu, Takuhiko; Tanaka, Hirofumi; Sasaki, Takahisa; Suda, Tatsuo ); Nishihara, Tatsuji; Koga, Toshihiko ); Martin, T.J. )

    1990-09-01

    The authors previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of 1{alpha},25-dihydroxyvitamin D{sub 3} and dexamethasone. In this study, they developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive monoculear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to 1{alpha},25-dihydroxyvitamin D{sub 3} and dexamethasone. When hemopoietic cells suspended in a collagen-gel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. Salmon {sup 125}I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a suitable microenvironment is provided by bone marrow-derived stromal cells.

  17. Lupeol Isolated from Sorbus commixta Suppresses 1α,25-(OH)2D3-Mediated Osteoclast Differentiation and Bone Loss in Vitro and in Vivo.

    PubMed

    Im, Nam Kyung; Lee, Dong-Sung; Lee, Seong-Ryong; Jeong, Gil Saeng

    2016-02-26

    Lupeol is a lupane-type triterpene isolated from Sorbus commixta, an oriental medicine used to treat arthritis and inflammatory diseases. However, the antiosteoporotic effects of S. commixta or any of its constituents have not been studied yet. In the present study, we have examined the effect of lupeol (a major active triterpenoid isolated from S. commixta) on osteoclastogenesis and sought to elucidate its underlying molecular mechanisms. We evaluated whether lupeol antagonized osteoclast differentiation and bone resorption. Lupeol markedly inhibited osteoclast differentiation and bone resorption activity through its effects on MAP kinases and transcription factors (NF-κB, NFATc1, and c-Fos) downstream of the osteoclast differentiation factor receptor RANK. Furthermore, in vivo efficacy of lupeol was confirmed by using an animal model of hypercalcemic mediated bone loss. Taken together, lupeol showed strong inhibitory effects on osteoclastogenesis. Supplementation with S. commixta and lupeol could be beneficial for bone health or osteoclast-related diseases such as osteoporosis, Paget's disease, osteolysis associated with periodontal disease, and multiple myeloma. PMID:26878936

  18. The polyphenol fisetin protects bone by repressing NF-κB and MKP-1-dependent signaling pathways in osteoclasts.

    PubMed

    Léotoing, Laurent; Wauquier, Fabien; Guicheux, Jérôme; Miot-Noirault, Elisabeth; Wittrant, Yohann; Coxam, Véronique

    2013-01-01

    Osteoporosis is a bone pathology leading to increase fractures risk and challenging quality of life. Since current treatments could exhibit deleterious side effects, the use of food compounds derived from plants represents a promising innovative alternative due to their potential therapeutic and preventive activities against human diseases. In this study, we investigated the ability of the polyphenol fisetin to counter osteoporosis and analyzed the cellular and molecular mechanisms involved. In vivo, fisetin consumption significantly prevented bone loss in estrogen deficiency and inflammation mice osteoporosis models. Indeed, bone mineral density, micro-architecture parameters and bone markers were positively modulated by fisetin. Consistent with in vivo results, we showed that fisetin represses RANKL-induced osteoclast differentiation and activity as demonstrated by an inhibition of multinucleated cells formation, TRAP activity and differentiation genes expression. The signaling pathways NF-κB, p38 MAPK, JNK and the key transcription factors c-Fos and NFATc1 expressions induced by RANKL, were negatively regulated by fisetin. We further showed that fisetin inhibits the constitutive proteasomal degradation of MKP-1, the phosphatase that deactivates p38 and JNK. Consistently, using shRNA stable cell lines, we demonstrated that impairment of MKP-1 decreases fisetin potency. Taken together, these results strongly support that fisetin should be further considered as a bone protective agent.

  19. Effects of JSOG-6 on protection against bone loss in ovariectomized mice through regulation of osteoblast differentiation and osteoclast formation

    PubMed Central

    2014-01-01

    Background JSOG-6 is used as a traditional medicine to relieve the symptoms associated with inflammation, rheumatism, and osteoporosis in Korea. In the present study, we investigated the effects of JSOG-6 on bone loss prevention both in in vitro and in vivo as well as its underlying mechanism of action. Methods Protection against bone loss was assessed in an ovariectomized (OVX) mouse model. Bone microarchitecture was measured using a micro-computed tomography to detect the parameters of three-dimensional structure of a trabecular bone. Serum biomarkers were also evaluated in an OVX-induced model. Osteoclasts derived from mouse bone marrow cells (BMCs) and osteoblastic MC3T3-E1 cells were also employed to investigate the mechanism of action. Results Oral administration of JSOG-6 significantly increased the bone mineral density (BMD) of the femur in OVX mice in vivo. Especially, the reduced Tb.No (trabecular bone number) in the OVX group was significantly recovered by JSOG-6 treatment. The serum levels of alkaline phosphatase (ALP), osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase, biomarkers of bone resorption, were significantly elevated in OVX mice, but JSOG-6 effectively inhibited the increase in OVX mice. JSOG-6 was also found to enhance the osteoblastic differentiation and maturation with the increase of the density and ALP activity, a marker of osteoblastic differentiation, as well as calcium deposition, a marker of osteoblastic maturation in MC3T3-E1 cells. The effects of JSOG-6 on osteoblastic differentiation were also associated in part with the increase of ALP and OPN mRNA expressions and the decrease of RANKL mRNA expression in MC3T3-E1 cells. Conclusions The findings demonstrate that JSOG-6 induced protection against bone loss in OVX mice, and its anti-osteoporotic property might be, in part, a function of the stimulation of osteoblast differentiation and the inhibition of osteoclast formation. These findings suggest that

  20. How the osteoclast degrades bone.

    PubMed

    Blair, H C

    1998-10-01

    Osteoclasts are multinucleated monocyte-macrophage derivatives that degrade bone. Their specialized role is central to a process that continuously removes and replaces segments of the skeleton in the higher vertebrates. Osteoclasts allow skeletal mineral to be used to manage extracellular calcium activity, which is an important adaptation for life on land, and solid skeletal structure to be replaced by hollow architecture that has a superior strength-to-weight ratio. Degrading bone also allows periodic repair and remodeling for ordered growth and efficient response to mechanical loads. A fairly comprehensive view of osteoclastic ontogeny and function is emerging from recent studies. Osteoclasts dissolve bone mineral by massive acid secretion and secrete specialized proteinases that degrade the organic matrix, mainly type I collagen, in this acidic milieu. The site of bone dissolution is a high-calcium environment; removal of degradation products by transcytosis of membrane vesicles allows the osteoclast to maintain a normal intracellular calcium. Osteoclastic differentiation is normally balanced with bone formation, although bone formation is the function of unrelated stromal cell-derived osteoblasts. Interactions between osteoclast precursors and bone-forming cells are believed to control osteoclast differentiation under most circumstances, preserving bone architecture over many cycles of bone replacement. PMID:9819571

  1. Iron Chelation Inhibits Osteoclastic Differentiation In Vitro and in Tg2576 Mouse Model of Alzheimer’s Disease

    PubMed Central

    Guo, Jun-Peng; Pan, Jin-Xiu; Xiong, Lei; Xia, Wen-Fang; Cui, Shun; Xiong, Wen-Cheng

    2015-01-01

    Patients of Alzheimer’s disease (AD) frequently have lower bone mineral density and higher rate of hip fracture. Tg2576, a well characterized AD animal model that ubiquitously express Swedish mutant amyloid precursor protein (APPswe), displays not only AD-relevant neuropathology, but also age-dependent bone deficits. However, the underlying mechanisms remain poorly understood. As APP is implicated as a regulator of iron export, and the metal chelation is considered as a potential therapeutic strategy for AD, we examined iron chelation’s effect on the osteoporotic deficit in Tg2576 mice. Remarkably, in vivo treatment with iron chelator, clinoquinol (CQ), increased both trabecular and cortical bone-mass, selectively in Tg2576, but not wild type (WT) mice. Further in vitro studies showed that low concentrations of CQ as well as deferoxamine (DFO), another iron chelator, selectively inhibited osteoclast (OC) differentiation, without an obvious effect on osteoblast (OB) differentiation. Intriguingly, both CQ and DFO’s inhibitory effect on OC was more potent in bone marrow macrophages (BMMs) from Tg2576 mice than that of wild type controls. The reduction of intracellular iron levels in BMMs by CQ was also more dramatic in APPswe-expressing BMMs. Taken together, these results demonstrate a potent inhibition on OC formation and activation in APPswe-expressing BMMs by iron chelation, and reveal a potential therapeutic value of CQ in treating AD-associated osteoporotic deficits. PMID:26575486

  2. Monocytes/Macrophages Upregulate the Hyaluronidase HYAL1 and Adapt Its Subcellular Trafficking to Promote Extracellular Residency upon Differentiation into Osteoclasts

    PubMed Central

    Puissant, Emeline; Boonen, Marielle

    2016-01-01

    Osteoclasts are giant bone-resorbing cells originating from monocytes/macrophages. During their differentiation, they overexpress two lysosomal enzymes, cathepsin K and TRAP, which are secreted into the resorption lacuna, an acidified sealed area in contact with bone matrix where bone degradation takes place. Here we report that the acid hydrolase HYAL1, a hyaluronidase able to degrade the glycosaminoglycans hyaluronic acid (HA) and chondroitin sulfate, is also upregulated upon osteoclastogenesis. The mRNA expression and protein level of HYAL1 are markedly increased in osteoclasts differentiated from RAW264.7 mouse macrophages or primary mouse bone marrow monocytes compared to these precursor cells. As a result, the HYAL1-mediated HA hydrolysis ability of osteoclasts is strongly enhanced. Using subcellular fractionation, we demonstrate that HYAL1 proteins are sorted to the osteoclast lysosomes even though, in contrast to cathepsin K and TRAP, HYAL1 is poorly mannose 6-phosphorylated. We reported previously that macrophages secrete HYAL1 proforms by constitutive secretion, and that these are recaptured by the cell surface mannose receptor, processed in endosomes and sorted to lysosomes. Present work highlights that osteoclasts secrete HYAL1 in two ways, through lysosomal exocytosis and constitutive secretion, and that these cells promote the extracellular residency of HYAL1 through downregulation of the mannose receptor. Interestingly, the expression of the other main hyaluronidase, HYAL2, and of lysosomal exoglycosidases involved in HA degradation, does not increase similarly to HYAL1 upon osteoclastogenesis. Taken together, these findings point out the predominant involvement of HYAL1 in bone HA metabolism and perhaps bone remodeling via the resorption lacuna. PMID:27755597

  3. Inhibition of inducible nitric oxide synthase and osteoclastic differentiation by Atractylodis Rhizoma Alba extract

    PubMed Central

    Choi, Sung-Ho; Kim, Sung-Jin

    2014-01-01

    Background: Atractylodis Rhizoma Alba (ARA) has been used in Korean folk medicine for constipation, dizziness, and anticancer agent. In the present study, we performed to test whether the methanolic extract of ARA has antioxidant and antiosteoclastogenesis activity in RAW 264.7 macrophage cells. Materials and Methods: Antioxidant capacities were tested by measuring free radical scavenging activity, nitric oxide (NO) levels, reducing power, and inducible nitric oxide synthase (iNOS) expression in response to lipopolysaccharides (LPS). Antiosteoclastogenesis activity was evaluated by performing tartrate-resistant acid phosphatase assay in RAW 264.7 macrophage cells. Results: The extract exerted significant 1,1-diphenyl-2-picrylhydrazyl and NO radical scavenging activity, and it exerted dramatic reducing power. Induction of iNOS and NO by LPS in RAW 264.7 cells was significantly inhibited by the extract, suggesting that the ARA extract inhibits NO production by suppressing iNOS expression. Strikingly, the ARA extracts substantially inhibited the receptor activator of NF-κB ligand-induced osteclastic differentiation of LPS-activated RAW 264.7 cells. The ARA extract contains a significant amount of antioxidant components, including phenolics, flavonoids and anthocyanins. Conclusion: These results suggest that the methanolic extract of ARA exerts significant antioxidant activities potentially via inhibiting free radicals and iNOS induction, thereby leading to the inhibition of osteoclastogenesis. PMID:25298665

  4. Ceylonamides A-F, Nitrogenous Spongian Diterpenes That Inhibit RANKL-Induced Osteoclastogenesis, from the Marine Sponge Spongia ceylonensis.

    PubMed

    El-Desoky, Ahmed H; Kato, Hikaru; Angkouw, Esther D; Mangindaan, Remy E P; de Voogd, Nicole J; Tsukamoto, Sachiko

    2016-08-26

    Seven new spongian diterpenes, ceylonamides A-F (1-6) and 15α,16-dimethoxyspongi-13-en-19-oic acid (7), were isolated from the Indonesian marine sponge Spongia ceylonensis along with eight known spongian diterpenes, 8-15. Compounds 1-6 were determined to be nitrogenous spongian diterpenes. The isolated compounds were examined for the inhibition of RANKL-induced osteoclastogenesis in RAW264 macrophages. Ceylonamide A (1) exhibited the most potent inhibitory activity with an IC50 value of 13 μM, followed by ceylonamide B (2) (IC50, 18 μM). An examination of the structure-activity relationships of the isolated compounds revealed that the position of the carbonyl group of the γ-lactam ring and bulkiness of the substituent at its nitrogen atom were important for inhibitory activity. PMID:27526327

  5. The elementary fusion modalities of osteoclasts.

    PubMed

    Søe, Kent; Hobolt-Pedersen, Anne-Sofie; Delaisse, Jean-Marie

    2015-04-01

    The last step of the osteoclast differentiation process is cell fusion. Most efforts to understand the fusion mechanism have focused on the identification of molecules involved in the fusion process. Surprisingly, the basic fusion modalities, which are well known for fusion of other cell types, are not known for the osteoclast. Here we show that osteoclast fusion partners are characterized by differences in mobility, nuclearity, and differentiation level. Our demonstration was based on time-laps videos of human osteoclast preparations from three donors where 656 fusion events were analyzed. Fusions between a mobile and an immobile partner were most frequent (62%), while fusion between two mobile (26%) or two immobile partners (12%) was less frequent (p<0.001). In general, the immobile fusion partner contained more nuclei than the mobile one (p<0.01). Furthermore, enrichment in nuclei of an osteoclast with three or more nuclei resulted from fusion with a mono-nucleated cell in 67% of the cases (p<0.001), while mono-nucleated cells fused with a multinucleated cell in 61% of the cases (p<0.05). This observation suggested that a more mature osteoclast prefers to fuse with a less mature pre-osteoclast. This hypothesis was supported by a nucleus-tracing approach in a co-culture of more and less differentiated pre-osteoclasts/osteoclasts. Furthermore, we found that osteoclast fusion proceeds through primarily two different types of cell contacts: phagocytic-cup and broad-contact-surfaces (>80% of all fusions). We conclude that osteoclasts most often gain nuclei by addition of one nucleus at a time, and that this nucleus is most often delivered by a moving cell to an immobile cell. These characteristics fit the in vivo observations where mono-nucleated precursors migrating from the bone marrow fuse with more mature osteoclasts sitting on the bone surface. They also fit the fusion modalities of other cell types.

  6. The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function.

    PubMed

    Thirunavukkarasu, K; Halladay, D L; Miles, R R; Yang, X; Galvin, R J; Chandrasekhar, S; Martin, T J; Onyia, J E

    2000-08-18

    Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblast-secreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE(2))), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the beta-galactosidase reporter gene (pOPG5. 9betagal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9betagal reporter construct led to 39-, 7-, and 16-fold increases in beta-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE(2) elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE(2) element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequence-specific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.

  7. Involvement of toll-like receptor 2 and 4 in association between dyslipidemia and osteoclast differentiation in apolipoprotein E deficient rat periodontium

    PubMed Central

    2013-01-01

    Background Dyslipidemia increases circulating levels of oxidized low-density lipoprotein (OxLDL) and this may induce alveolar bone loss through toll-like receptor (TLR) 2 and 4. The purpose of this study was to investigate the effects of dyslipidemia on osteoclast differentiation associated with TLR2 and TLR4 in periodontal tissues using a rat dyslipidemia (apolipoprotein E deficient) model. Methods Levels of plasma OxLDL, and the cholesterol and phospholipid profiles in plasma lipoproteins were compared between apolipoprotein E-deficient rats (16-week-old males) and wild-type (control) rats. In the periodontal tissue, we evaluated the changes in TLR2, TLR4, receptor activator of nuclear factor kappa B ligand (RANKL) and tartrate resistant acid phosphatase (TRAP) expression. Results Apolipoprotein E-deficient rats showed higher plasma levels of OxLDL than control rats (p<0.05), with higher plasma levels of total cholesterol (p<0.05) and LDL-cholesterol (p<0.05) and lower plasma levels of high-density lipoprotein cholesterol (p<0.05). Their periodontal tissue also exhibited a higher ratio of RANKL-positive cells and a higher number of TRAP-positive osteoclasts than control rats (p<0.05). Furthermore, periodontal gene expression of TLR2, TLR4 and RANKL was higher in apolipoprotein E-deficient rats than in control rats (p<0.05). Conclusion These findings underscore the important role for TLR2 and TLR4 in mediating the osteoclast differentiation on alveolar bone response to dyslipidemia. PMID:23295061

  8. Tyrosines 559 and 807 in the cytoplasmic tail of the macrophage colony-stimulating factor receptor play distinct roles in osteoclast differentiation and function.

    PubMed

    Feng, Xu; Takeshita, Sunao; Namba, Noriyuki; Wei, Shi; Teitelbaum, Steven L; Ross, F Patrick

    2002-12-01

    Osteoclast (OC) differentiation requires that precursors, such as macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages, receive signals transduced by receptor activator of nuclear factor kappaB (RANK) and c-Fms, receptors for RANK ligand (RANKL) and M-CSF, respectively. Activated c-Fms autophosphorylates cytoplasmic tail tyrosine residues, which, by recruiting adaptor molecules, initiate specific signaling pathways. To identify which tyrosine residues are involved in c-Fms signaling in primary cells, we retrovirally transduced M-CSF-dependent bone marrow macrophages with a chimera comprising the external domain of the erythropoietin (Epo) receptor linked to the transmembrane and cytoplasmic domains of c-Fms. Transduced cells differentiate into bone-resorbing osteoclasts when treated with RANKL and either M-CSF or Epo, confirming that both endogenous and chimeric receptors transmit osteoclastogenic signals. Cells expressing chimeric receptors with Y(697)F, Y(706)F, Y(721)F, and Y(921)F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo. In contrast, those expressing Y(559)F generate fewer OCs, whereas theY807F mutant is incapable of osteoclastogenesis. Finally, although mature OCs expressing Y(559)F exhibit impaired bone resorption, those bearing Y807F do not. Thus, we have identified specific tyrosine residues in the cytoplasmic tail of c-Fms that are critical for transmitting M-CSF-initiated signals individually required for OC formation or function, respectively.

  9. ABD56 causes osteoclast apoptosis by inhibiting the NF{kappa}B and ERK pathways

    SciTech Connect

    Idris, Aymen; Mrak, Emanuela; Greig, Iain; Guidobono, Francesca; Ralston, Stuart H.; Hof, Rob van 't

    2008-06-20

    We have previously shown that the biphenylcarboxylic acid butanediol ester (ABD56) inhibits osteoclast formation and activity in vitro and in vivo. However, the mechanism of action of this compound is unknown. ABD56 inhibited osteoclast formation and caused osteoclast apoptosis, but had no effects on osteoblasts or macrophages. As the NF{kappa}B and MAPK pathways are essential for osteoclast formation and survival, we studied the effects of ABD56 on these pathways. ABD56 caused phosphorylation of p38, JNK and nuclear translocation of c-jun in osteoclasts. ABD56-induced apoptosis was prevented by the caspase inhibitor zVAD-fmk but was not prevented by the p38- or JNK-inhibitors. ABD56 completely abolished RANKL-induced I{kappa}B and ERK1/2 phosphorylation. Increasing the amount of RANKL partially rescued ABD56-induced apoptosis, indicating that the apoptosis is most probably due to the inhibition of survival signals such as ERK and NF{kappa}B, rather than activation of the p38 or Jnk MAPK pathways.

  10. Strawberry notch homologue 2 regulates osteoclast fusion by enhancing the expression of DC-STAMP.

    PubMed

    Maruyama, Kenta; Uematsu, Satoshi; Kondo, Takeshi; Takeuchi, Osamu; Martino, Mikaël M; Kawasaki, Takumi; Akira, Shizuo

    2013-09-23

    Osteoclasts are multinucleated cells formed by fusion of mononuclear precursors in response to receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). We found that RANKL induced expression of the DExD/H helicase family corepressor strawberry notch homologue 2 (Sbno2). Previous in vitro studies showed that Sbno2 is induced by IL-10 and is involved in NF-κB repression. However, the role of Sbno2 in vivo and its pleiotropic functions are unknown. Sbno2 gene targeting resulted in normal NF-κB activation by TLR ligands. However, Sbno2-deficient mice exhibited increased bone mass due to impaired osteoclast fusion. Expression of dendritic cell-specific transmembrane protein (DC-STAMP), a critical player in osteoclast fusion, was significantly attenuated, and cell fusion of Sbno2-deficient osteoclasts was rescued by DC-STAMP. Sbno2 directly bound to T cell acute lymphocytic leukemia 1 (Tal1) and attenuated its inhibition of DC-STAMP expression, leading to activation of the DC-STAMP promoter by microphthalmia-associated transcription factor (MITF). Thus, Sbno2 plays a pivotal role in bone homeostasis in vivo by fine-tuning osteoclast fusion.

  11. Osteoblastic and osteoclastic differentiation of human mesenchymal stem cells and monocytes in a miniaturized three-dimensional culture with mineral granules.

    PubMed

    Gamblin, Anne Laure; Renaud, Audrey; Charrier, Céline; Hulin, Philippe; Louarn, Guy; Heymann, Dominique; Trichet, Valérie; Layrolle, Pierre

    2014-12-01

    The pathologies of the skeleton have a significant socioeconomic impact on our population. Although therapies have improved the treatment of osteosarcoma and osteoporosis, their efficacy still remains limited. In this context, we developed a miniaturized 3-D culture model of bone cells on calcium phosphate ceramics. Human bone marrow mesenchymal stem cells (MSCs) were three-dimensionally cultured on particles of biphasic calcium phosphate (BCP, 125-200μm) in osteogenic media. The MSCs seeded on the BCP particles adhered and proliferated, producing abundant collagenous extracellular matrix (ECM). Light and confocal laser scanning microscopy showed that the MSCs created bridges between the BCP particles and formed a 3-D structure. Energy dispersive X-ray analysis in a scanning electron microscope confirmed the mineralization of the collagen matrix. The 96-well sized bone constructs were tested by immunohistology and transcription analysis, proving cell differentiation. Both techniques corroborated the osteoblastic differentiation with high production of bone sialoprotein and osteocalcin. Peripheral blood CD14-positive monocytes (MOs) were pre-differentiated into osteoclasts prior to seeding on the 3-D constructs. Multinucleated and tartrate-resistant acid phosphatase-positive cells were also identified at the surface of the 3-D constructs after 90days of culture. In addition, cell viability within these constructs was measured by flow cytometry. In summary, we have developed a miniaturized 3-D culture of bone cell precursors with osteoblasts and osteoclasts. This 3-D culture may make it possible to test the effects of new drugs for bone healing, osteoporosis and osteosarcomas, in more appropriate cell-cell and cell-matrix interactions than conventional 2-D cultures.

  12. Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo

    PubMed Central

    Baek, Jong Min; Kim, Ju-Young; Yoon, Kwon-Ha; Oh, Jaemin; Lee, Myeung Su

    2016-01-01

    Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. However, no reports are available for verifying the pharmacological effects of ebselen on major metabolic bone diseases such as osteoporosis. In this study, we observed that ebselen suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its µ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor. These results indicate that ebselen is a potentially safe drug for treating metabolic bone diseases such as osteoporosis. PMID:27019631

  13. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity

    PubMed Central

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-01-01

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin’s effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity. PMID:27110777

  14. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

    PubMed

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-01-01

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity. PMID:27110777

  15. Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation.

    PubMed

    Tseng, Han-Ching; Kanayama, Keiichi; Kaur, Kawaljit; Park, So-Hyun; Park, Sil; Kozlowska, Anna; Sun, Shuting; McKenna, Charles E; Nishimura, Ichiro; Jewett, Anahid

    2015-08-21

    The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer. PMID:26343372

  16. Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation

    PubMed Central

    Park, So-Hyun; Park, Sil; Kozlowska, Anna; Sun, Shuting; McKenna, Charles E.; Nishimura, Ichiro; Jewett, Anahid

    2015-01-01

    The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing due to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer. PMID:26343372

  17. Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation.

    PubMed

    Tseng, Han-Ching; Kanayama, Keiichi; Kaur, Kawaljit; Park, So-Hyun; Park, Sil; Kozlowska, Anna; Sun, Shuting; McKenna, Charles E; Nishimura, Ichiro; Jewett, Anahid

    2015-08-21

    The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer.

  18. Incorporation of RANKL promotes osteoclast formation and osteoclast activity on β-TCP ceramics.

    PubMed

    Choy, John; Albers, Christoph E; Siebenrock, Klaus A; Dolder, Silvia; Hofstetter, Wilhelm; Klenke, Frank M

    2014-12-01

    β-Tricalcium phosphate (β-TCP) ceramics are approved for the repair of osseous defects. In large defects, however, the substitution of the material by authentic bone is inadequate to provide sufficient long-term mechanical stability. We aimed to develop composites of β-TCP ceramics and receptor activator of nuclear factor κ-B ligand (RANKL) to enhance the formation of osteoclasts and promote cell mediated calcium phosphate resorption. RANKL was adsorbed superficially onto β-TCP ceramics or incorporated into a crystalline layer of calcium phosphate by the use of a co-precipitation technique. Murine osteoclast precursors were seeded onto the ceramics. After 15 days, the formation of osteoclasts was quantified cytologically and colorimetrically with tartrate-resistant acidic phosphatase (TRAP) staining and TRAP activity measurements, respectively. Additionally, the expression of transcripts encoding the osteoclast gene products cathepsin K, calcitonin receptor, and of the sodium/hydrogen exchanger NHA2 were quantified by real-time PCR. The activity of newly formed osteoclasts was evaluated by means of a calcium phosphate resorption assay. Superficially adsorbed RANKL did not induce the formation of osteoclasts on β-TCP ceramics. When co-precipitated onto β-TCP ceramics RANKL supported the formation of mature osteoclasts. The development of osteoclast lineage cells was further confirmed by the increased expression of cathepsin K, calcitonin receptor, and NHA2. Incorporated RANKL stimulated the cells to resorb crystalline calcium phosphate. Our in vitro study shows that RANKL incorporated into β-TCP ceramics induces the formation of active, resorbing osteoclasts on the material surface. Once formed, osteoclasts mediate the release of RANKL thereby perpetuating their differentiation and activation. In vivo, the stimulation of osteoclast-mediated resorption may contribute to a coordinated sequence of material resorption and bone formation. Further in vivo studies

  19. Norisoboldine, an Anti-Arthritis Alkaloid Isolated from Radix Linderae, Attenuates Osteoclast Differentiation and Inflammatory Bone Erosion in an Aryl Hydrocarbon Receptor-Dependent Manner.

    PubMed

    Wei, Zhi-feng; Lv, Qi; Xia, Ying; Yue, Meng-fan; Shi, Can; Xia, Yu-feng; Chou, Gui-xin; Wang, Zheng-tao; Dai, Yue

    2015-01-01

    Norisoboldine (NOR), the primary isoquinoline alkaloid constituent of the root of Lindera aggregata, has previously been demonstrated to attenuate osteoclast (OC) differentiation. Accumulative evidence has shown that aryl hydrocarbon receptor (AhR) plays an important role in regulating the differentiation of various cells, and multiple isoquinoline alkaloids can modulate AhR. In the present study, we explored the role of NOR in the AhR signaling pathway. These data showed that the combination of AhR antagonist resveratrol (Res) or α-naphthoflavone (α-NF) nearly reversed the inhibition of OC differentiation through NOR. NOR could stably bind to AhR, up-regulate the nuclear translocation of AhR, and enhance the accumulation of the AhR-ARNT complex, AhR-mediated reporter gene activity and CYP1A1 expression in RAW 264.7 cells, suggesting that NOR might be an agonist of AhR. Moreover, NOR inhibited the nuclear translocation of NF-κB-p65, resulting in the evident accumulation of the AhR-NF-κB-p65 complex, which could be markedly inhibited through either Res or α-NF. Although NOR only slightly affected the expression of HIF-1α, NOR markedly reduced VEGF mRNA expression and ARNT-HIF-1α complex accumulation. In vivo studies indicated that NOR decreased the number of OCs and ameliorated the bone erosion in the joints of rats with collagen-induced arthritis, accompanied by the up-regulation of CYP1A1 and the down-regulation of VEGF mRNA expression in the synovium of rats. A combination of α-NF nearly completely reversed the effects of NOR. In conclusion, NOR attenuated OC differentiation and bone erosion through the activation of AhR and the subsequent inhibition of both NF-κB and HIF pathways.

  20. Notch signaling promotes osteoclast maturation and resorptive activity.

    PubMed

    Ashley, Jason W; Ahn, Jaimo; Hankenson, Kurt D

    2015-11-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors.

  1. Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics.

    PubMed

    Zaidi, Mone; Blair, Harry C; Moonga, Baltit S; Abe, Etsuko; Huang, Christopher L H

    2003-04-01

    Over the past decade, advances in molecular tools, stem cell differentiation, osteoclast and osteoblast signaling mechanisms, and genetically manipulated mice models have resulted in major breakthroughs in understanding osteoclast biology. This review focuses on key advances in our understanding of molecular mechanisms underlying the formation, function, and survival of osteoclasts. These include key signals mediating osteoclast differentiation, including PU.1, RANK, CSF-1/c-fms, and src, and key specializations of the osteoclast including HCl secretion driven by H+-ATPase and the secretion of collagenolytic enzymes including cathepsin K and matrix metalloproteinases (MMPs). These pathways and highly expressed proteins provide targets for specific therapies to modify bone degradation. The main outstanding issues, basic and translational, will be considered in relation to the osteoclast as a target for antiresorptive therapies.

  2. Aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells.

    PubMed

    Zeng, Yan-Ping; Yang, Chao; Li, Yuan; Fan, Yong; Yang, Hong-Jun; Liu, Bin; Sang, Hong-Xun

    2016-09-01

    Aspirin is a commonly used medicine as an effective antipyretic, analgesic and anti-inflammatory drug. Previous studies have demonstrated its potential effects of anti-postmenopausal osteoporosis, while the molecular mechanisms remain unclear. The effects of aspirin on receptor‑activator of nuclear factor κB (NF‑κB) ligand (RANKL)‑induced osteoclasts were investigated in RAW264.7 cells in the current study. Using tartrate‑resistant acid phosphatase (TRAP) staining, it was observed that aspirin inhibited the differentiation of RANKL‑induced RAW264.7 cells. The mRNA expression of osteoclastic marker genes, including cathepsin K, TRAP, matrix metalloproteinase 9 and calcitonin receptor, were suppressed by aspirin as identified using reverse transcription‑quantitative polymerase chain reaction analysis. The immunofluorescence assay indicated that aspirin markedly inhibited NF‑κB p65 translocation to the nucleus in RANKL‑induced RAW264.7 cells. In addition, aspirin also suppressed the phosphorylation of mitogen‑activated protein kinases (MAPKs), observed by western blot analysis. Taken together, these data identified that aspirin inhibits osteoclastogenesis by suppressing the activation of NF‑κB and MAPKs in RANKL‑induced RAW264.7 cells, implying that aspirin may possess therapeutic potential for use in the prevention and treatment of osteoporosis. PMID:27430169

  3. Sclerostin is expressed in osteoclasts from aged mice and reduces osteoclast-mediated stimulation of mineralization.

    PubMed

    Ota, Kuniaki; Quint, Patrick; Ruan, Ming; Pederson, Larry; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2013-08-01

    Osteoclast-mediated bone resorption precedes osteoblast-mediated bone formation through early adulthood, but formation fails to keep pace with resorption during aging. We previously identified several factors produced by osteoclasts that promote bone formation. In this study, we determined if osteoclast-produced factors contribute to the impaired bone formation with aging. We previously found that mice between the ages of 18 and 22 months develop age-related bone loss. Bone marrow-derived pre-osteoclasts were isolated from 6-week, 12-month, and 18- to 24-month-old mice and differentiated into osteoclasts in vitro. Conditioned media were collected and compared for osteoblast mineralization support. Conditioned medium from osteoclasts from all ages was able to support mineralization of bone marrow stromal cells. Concentrating the conditioned medium from 6-week-old and 12-month-old mouse marrow cells-derived osteoclasts enhanced mineralization support whereas concentrated conditioned medium from 18- to 24-month-old mouse marrow-derived osteoclasts repressed mineralization compared to base medium. This observation suggests that an inhibitor of mineralization was secreted by aged murine osteoclasts. Gene and protein analysis revealed that the Wnt antagonist sclerostin was significantly elevated in the conditioned media from 24-month-old mouse cells compared to 6-week-old mouse cells. Antibodies directed to sclerostin neutralized the influences of the aged mouse cell concentrated conditioned media on mineralization. Sclerostin is primarily produced by osteocytes in young animals. This study demonstrates that osteoclasts from aged mice also produce sclerostin in quantities that may contribute to the age-related impairment in bone formation.

  4. Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease

    PubMed Central

    Ersek, Adel; Xu, Ke; Antonopoulos, Aristotelis; Butters, Terry D.; Santo, Ana Espirito; Vattakuzhi, Youridies; Williams, Lynn M.; Goudevenou, Katerina; Danks, Lynett; Freidin, Andrew; Spanoudakis, Emmanouil; Parry, Simon; Papaioannou, Maria; Hatjiharissi, Evdoxia; Chaidos, Aristeidis; Alonzi, Dominic S.; Twigg, Gabriele; Hu, Ming; Dwek, Raymond A.; Haslam, Stuart M.; Roberts, Irene; Dell, Anne; Rahemtulla, Amin; Horwood, Nicole J.; Karadimitris, Anastasios

    2015-01-01

    Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell–derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM. PMID:25915583

  5. RANKL, osteopontin, and osteoclast homeostasis in a hyperocclusion mouse model

    SciTech Connect

    Walker, Cameron G.; Ito, Yoshihiro; Dangaria, Smit; Luan, Xianghong; Diekwisch, Thomas G.H.

    2009-10-21

    The biological mechanisms that maintain the position of teeth in their sockets establish a dynamic equilibrium between bone resorption and apposition. In order to reveal some of the dynamics involved in the tissue responses towards occlusal forces on periodontal ligament (PDL) and alveolar bone homeostasis, we developed the first mouse model of hyperocclusion. Swiss-Webster mice were kept in hyperocclusion for 0, 3, 6, and 9 d. Morphological and histological changes in the periodontium were assessed using micro-computed tomography (micro-CT) and ground sections with fluorescent detection of vital dye labels. Sections were stained for tartrate-resistant acid phosphatase, and the expression of receptor activator of nuclear factor-{kappa}B ligand (RANKL) and osteopontin (OPN) was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). Traumatic occlusion resulted in enamel surface abrasion, inhibition of alveolar bone apposition, significant formation of osteoclasts at 3, 6 and 9 d, and upregulation of OPN and RANKL. Data from this study suggest that both OPN and RANKL contribute to the stimulation of bone resorption in the hyperocclusive state. In addition, we propose that the inhibition of alveolar bone apposition by occlusal forces is an important mechanism for the control of occlusal height that might work in synergy with RANKL-induced bone resorption to maintain normal occlusion.

  6. Osteoclastogenesis and Osteoclastic Resorption of Tricalcium Phosphate: Effect of Strontium and Magnesium Doping

    PubMed Central

    Roy, Mangal; Bose, Susmita

    2012-01-01

    Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study we have evaluated the effects of 1.0 wt% strontium (Sr) and 1.0 wt% magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr doped β-TCP samples at day 8 which was absent on Mg doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor αvβ3 integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell mediated degradation, however; significantly restricted for Mg doped β-TCP samples. Our present results indicated substrate chemistry controlled osteoclast differentiation and resorptive activity which can be used in designing TCP based resorbable bone substitutes with controlled degradation properties. PMID:22566212

  7. Propolis inhibits osteoclast maturation.

    PubMed

    Pileggi, Roberta; Antony, Kathryn; Johnson, Kristie; Zuo, Jian; Shannon Holliday, L

    2009-12-01

    Propolis, a natural product produced by the honey bee, has been successfully used in medicine as an anti-inflammatory and antimicrobial agent. Traumatic injuries to the teeth, especially avulsion injuries, present a challenging situation for the clinician because of post-treatment complications, such as inflammatory and/or replacement resorption. Agents that reduce osteoclast numbers and activity may be useful in the treatment of traumatic injuries to the teeth. In this study, we evaluated propolis as an anti-resorptive agent. Calcitriol-stimulated mouse marrow cultures, which contain both osteoclasts and osteoblasts, were exposed to the ethanol extracts of propolis or vehicle control and stained for tartrate-resistant acid phosphatase (TRAP)-activity to identify osteoclasts. A significant, dose-dependent reduction in multinuclear TRAP+ cells was demonstrated, although the propolis treatment accommodated cell growth and survival (P < 0.05). Propolis also reduced the formation of actin rings in pure cultures of RAW 264.7 osteoclast-like cells, suggesting that it exerts direct actions on osteoclast maturation. In summary, our data suggest that propolis inhibits late stages of osteoclast maturation including fusion of osteoclasts precursors to form giant cells and formation of actin rings. This supports the hypothesis that it may prove useful as a medicament to reduce resorption associated with traumatic injuries to the teeth. PMID:19843135

  8. Osteoclast Precursor Interaction with Bone Matrix Induces Osteoclast Formation Directly by an Interleukin-1-mediated Autocrine Mechanism*

    PubMed Central

    Yao, Zhenqiang; Xing, Lianping; Qin, Chunlin; Schwarz, Edward M.; Boyce, Brendan F.

    2008-01-01

    Interleukin-1 (IL-1) and tumor necrosis factor (TNF) mediate bone resorption in a variety of diseases affecting bone. Like TNF, IL-1 is secreted by osteoclast precursors (OCPs), but unlike TNF, it does not induce osteoclast formation directly from OCPs in vitro. TNF induces IL-1 expression and activates c-Fos, a transcription factor required in OCPs for osteoclast formation. Here, we examined whether IL-1 can induce osteoclast formation directly from OCPs overexpressing c-Fos and whether interaction with bone matrix affects OCP cytokine expression. We infected OCPs with c-Fos or green fluorescent protein retrovirus, cultured them with macrophage colony-stimulating factor and IL-1 on bone slices or plastic dishes, and assessed osteoclast and resorption pit formation and expression of IL-1 by OCPs. We used a Transwell assay to determine whether OCPs secrete IL-1 when they interact with bone matrix. IL-1 induced osteoclast formation directly from c-Fos-expressing OCPs on plastic. c-Fos-expressing OCPs formed osteoclasts spontaneously on bone slices without addition of cytokines. OCPs on bone secreted IL-1, which induced osteoclast formation from c-Fos-expressing OCPs in the lower Transwell dishes. The bone matrix proteins dentin sialoprotein and osteopontin, but not transforming growth factor-β, stimulated OCP expression of IL-1 and induced c-Fos-expressing OCP differentiation into osteoclasts. Osteoclasts eroding inflamed joints have higher c-Fos expression compared with osteoclasts inside bone. We conclude that OCPs expressing c-Fos may induce their differentiation directly into osteoclasts by an autocrine mechanism in which they produce IL-1 through interaction with bone matrix. TNF could induce c-Fos expression in OCPs at sites of inflammation in bone to promote this autocrine mechanism and thus amplify bone loss. PMID:18250170

  9. Suppressed osteoclast differentiation at the chondro-osseous junction mediates endochondral ossification retardation in long bones of Wistar fetal rats with prenatal ethanol exposure.

    PubMed

    Pan, Zhengqi; Zhang, Xianrong; Shangguan, Yangfan; Hu, Hang; Chen, Liaobin; Wang, Hui

    2016-08-15

    Prenatal ethanol exposure (PEE) inhibits longitudinal growth of fetal bones, but the underlying mechanisms remain unknown. In this study, we aimed to investigate how PEE induces the retardation of long bone development in fetal rats. Pregnant Wistar rats were treated with ethanol or distilled water (control group) by gavage from gestational day (GD) 9 to 20. Fetuses were delivered by cesarean section on GD20. Fetal sera were collected for assessing corticosterone (CORT) level. Fetal long bones were harvested for histochemical, immunohistochemical and gene expression analysis. Primary chondrocytes were treated with ethanol or CORT for analyzing genes expression. PEE fetuses showed a significant reduction in birth weight and body length. The serum CORT concentration in PEE group was significantly increased, while the body weight, body length and femur length all were significantly decreased in the PEE group. The length of the epiphyseal hypertrophy zone was enlarged, whereas the length of the primary ossification center was significantly reduced in PEE fetuses. TUNEL assay showed reduced apoptosis in the PEE group. Further, the gene expression of osteoprotegerin (OPG) was markedly up-regulated. In vitro experiments showed that CORT (but not ethanol) treatment significantly activated the expression of OPG, while the application of glucocorticoid receptor inhibitor, mifepristone, attenuated these change induced by CORT. These results indicated that PEE-induced glucocorticoid over-exposure enhanced the expression of OPG in fetal epiphyseal cartilage and further lead to the suppressed osteoclast differentiation in the chondro-osseous junction and consequently inhibited the endochondral ossification in long bones of fetal rats.

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

    PubMed

    Kasonga, Abe E; Deepak, Vishwa; Kruger, Marlena C; Coetzee, Magdalena

    2015-01-01

    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.

  11. An Osteoclastic Transmembrane Protein-Tyrosine Phosphatase Enhances Osteoclast Activity in Part by Dephosphorylating EphA4 in Osteoclasts.

    PubMed

    Lau, Kin-Hing William; Amoui, Mehran; Stiffel, Virginia; Chen, Shin-Tai; Sheng, Matilda H-C

    2015-08-01

    We have previously shown that PTP-oc is an enhancer of the functional activity of osteoclasts and that EphA4 is a suppressor. Here, we provide evidence that PTP-oc enhances osteoclast activity in part through inactivation of EphA4 by dephosphorylating key phosphotyrosine (pY) residues of EphA4. We show that EphA4 was pulled down by the PTP-oc trapping mutant but not by the wild-type (WT) PTP-oc and that transgenic overexpression of PTP-oc in osteoclasts drastically decreased pY602 and pY779 residues of EphA4. Consistent with the previous findings that EphA4 deficiency increased pY173-Vav3 level (Rac-GTP exchange factor [GEF]) and enhanced bone resorption activity of osteoclasts, reintroduction of WT-Epha4 in Epha4 null osteoclasts led to ∼50% reduction in the pY173-Vav3 level and ∼2-fold increase in bone resorption activity. Overexpression of Y779F-Epha4 mutant in WT osteoclasts markedly increased in pY173-Vav3 and reduced bone resorption activity, but overexpression of Y602F-Epha4 mutant had no effect, suggesting that pY779 residue plays an important role in the EphA4-mediated suppression of osteoclast activity. Deficient EphA4 in osteoclasts has been shown to up-regulate Rac-GTPase and down-regulate Rho-GTPase. PTP-oc overexpression in osteoclasts also increased the GTP-Rac level to 300% of controls, but decreased the GTP-Rho level to ∼50% of controls. PTP-oc overexpression or deficient Epha4 each also reduced pY87-Ephexin level, which is a Rho GEF. Thus, PTP-oc may differentially regulate Rac signaling versus Rho signaling through dephosphorylation of EphA4, which has shown to have opposing effects on Rac-GTPase versus Rho-GTPase through differential regulation of Vav3 versus Ephexin.

  12. Recombinant Human Endostatin Suppresses Mouse Osteoclast Formation by Inhibiting the NF-κB and MAPKs Signaling Pathways

    PubMed Central

    Chen, Nong; Gao, Ru-Feng; Yuan, Feng-Lai; Zhao, Ming-Dong

    2016-01-01

    Rheumatoid arthritis is an autoimmune disease characterized by synovial hyperplasia and progressive joint destruction. As reported previously, recombinant human endostatin (rhEndostatin) is associated with inhibition of joint bone destruction present in rat adjuvant-induced arthritis; however, the effect of rhEndostatin on bone destruction is not known. This study was designed to assess the inhibitory effect and mechanisms of rhEndostatin on formation and function of osteoclasts in vitro, and to gain insight into the mechanism underlying the inhibitory effect of bone destruction. Bone marrow-derived macrophages isolated from BALB/c mice were stimulated with receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor to establish osteoclast formation. Osteoclast formation was determined by TRAP staining. Cell viability of BMMs affected by rhEndostatin was determined using a MTT assay. Bone resorption was examined with a bone resorption pits assay. The expression of osteoclast-specific markers was analyzed using quantitative real-time PCR. The related signaling pathways were examined using a Luciferase reporter assay and western blot analysis. Indeed, rhEndostatin showed a significant reduction in the number of osteoclast-like cells and early-stage bone resorption. Moreover, molecular analysis demonstrated that rhEndostatin attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation of IκBα and NF-κB p65 nuclear translocation. Furthermore, rhEndostatin significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases, such as ERK1/2, JNK, and p38. Hence, we demonstrated for the first time that preventing the formation and function of osteoclasts is an important anti-bone destruction mechanism of rhEndostatin, which might be useful in the prevention and treatment of bone destruction in RA. PMID:27313530

  13. Recombinant Human Endostatin Suppresses Mouse Osteoclast Formation by Inhibiting the NF-κB and MAPKs Signaling Pathways.

    PubMed

    Chen, Nong; Gao, Ru-Feng; Yuan, Feng-Lai; Zhao, Ming-Dong

    2016-01-01

    Rheumatoid arthritis is an autoimmune disease characterized by synovial hyperplasia and progressive joint destruction. As reported previously, recombinant human endostatin (rhEndostatin) is associated with inhibition of joint bone destruction present in rat adjuvant-induced arthritis; however, the effect of rhEndostatin on bone destruction is not known. This study was designed to assess the inhibitory effect and mechanisms of rhEndostatin on formation and function of osteoclasts in vitro, and to gain insight into the mechanism underlying the inhibitory effect of bone destruction. Bone marrow-derived macrophages isolated from BALB/c mice were stimulated with receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor to establish osteoclast formation. Osteoclast formation was determined by TRAP staining. Cell viability of BMMs affected by rhEndostatin was determined using a MTT assay. Bone resorption was examined with a bone resorption pits assay. The expression of osteoclast-specific markers was analyzed using quantitative real-time PCR. The related signaling pathways were examined using a Luciferase reporter assay and western blot analysis. Indeed, rhEndostatin showed a significant reduction in the number of osteoclast-like cells and early-stage bone resorption. Moreover, molecular analysis demonstrated that rhEndostatin attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation of IκBα and NF-κB p65 nuclear translocation. Furthermore, rhEndostatin significantly inhibited the activation of RANKL-dependent mitogen-activated protein kinases, such as ERK1/2, JNK, and p38. Hence, we demonstrated for the first time that preventing the formation and function of osteoclasts is an important anti-bone destruction mechanism of rhEndostatin, which might be useful in the prevention and treatment of bone destruction in RA. PMID:27313530

  14. Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity.

    PubMed

    Irie, Atsushi; Takami, Masamichi; Kubo, Hideo; Sekino-Suzuki, Naoko; Kasahara, Kohji; Sanai, Yutaka

    2007-08-01

    Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFkappaB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D(3) and prostaglandin E(2) on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A (125)I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.

  15. Suppression of T cell-induced osteoclast formation

    SciTech Connect

    Karieb, Sahar; Fox, Simon W.

    2013-07-12

    Highlights: •Genistein and coumestrol prevent activated T cell induced osteoclast formation. •Anti-TNF neutralising antibodies prevent the pro-osteoclastic effect of activated T cells. •Phytoestrogens inhibit T cell derived TNF alpha and inflammatory cytokine production. •Phytoestrogens have a broader range of anti-osteoclastic actions than other anti-resorptives. -- Abstract: Inhibition of T cell derived cytokine production could help suppress osteoclast differentiation in inflammatory skeletal disorders. Bisphosphonates are typically prescribed to prevent inflammatory bone loss but are not tolerated by all patients and are associated with an increased risk of osteonecrosis of the jaw. In light of this other anti-resorptives such as phytoestrogens are being considered. However the effect of phytoestrogens on T cell-induced osteoclast formation is unclear. The effect of genistein and coumestrol on activated T cell-induced osteoclastogenesis and cytokine production was therefore examined. Concentrations of genistein and coumestrol (10{sup −7} M) previously shown to directly inhibit osteoclast formation also suppressed the formation of TRAP positive osteoclast induced by con A activated T cells, which was dependent on inhibition of T cell derived TNF-α. While both reduced osteoclast formation their mechanism of action differed. The anti-osteoclastic effect of coumestrol was associated with a dual effect on con A induced T cell proliferation and activation; 10{sup −7} M coumestrol significantly reducing T cell number (0.36) and TNF-α (0.47), IL-1β (0.23) and IL-6 (0.35) expression, whereas genistein (10{sup −7} M) had no effect on T cell number but a more pronounced effect on T cell differentiation reducing expression of TNF-α (0.49), IL-1β (0.52), IL-6 (0.71) and RANKL (0.71). Phytoestrogens therefore prevent the pro-osteoclastic action of T cells suggesting they may have a role in the control of inflammatory bone loss.

  16. Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques.

    PubMed

    Kotani, Manato; Kikuta, Junichi; Klauschen, Frederick; Chino, Takenao; Kobayashi, Yasuhiro; Yasuda, Hisataka; Tamai, Katsuto; Miyawaki, Atsushi; Kanagawa, Osami; Tomura, Michio; Ishii, Masaru

    2013-01-15

    Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes or from bone tissue-resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation "parabiosis" with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX(3)CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP(+) osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP(+) osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP(+) osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR(+) monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted "red" KikGR(+) osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.

  17. Generation and culture of osteoclasts

    PubMed Central

    Marino, Silvia; Logan, John G; Mellis, David; Capulli, Mattia

    2014-01-01

    Osteoclasts are highly specialized cells of haematopoietic lineage that are uniquely responsible for bone resorption. In the past, osteoclasts were isolated as mature cells from chicken long bones, or were generated using osteoblasts or stromal cells to induce osteoclast formation in total bone marrow from mice or rabbits. The Copernican revolution in osteoclast biology began with the identification of macrophage-colony stimulating factor (M-CSF) and receptor activator NFκB-ligand (RANKL ) as the key regulators of osteoclast formation, fusion and function. The availability of recombinant human and mouse M-CSF and RANKL has enabled researchers to reliably generate osteoclasts from primary monocyte/macrophage cells as well as from cell lines such as RAW 264.7. This article summarizes the most commonly used procedures for the isolation, generation and characterization of human, rodent and chicken osteoclasts in vitro. Lists of further reading and recommendations are included to facilitate a successful application by the reader. PMID:25228983

  18. Generation and culture of osteoclasts.

    PubMed

    Marino, Silvia; Logan, John G; Mellis, David; Capulli, Mattia

    2014-01-01

    Osteoclasts are highly specialized cells of haematopoietic lineage that are uniquely responsible for bone resorption. In the past, osteoclasts were isolated as mature cells from chicken long bones, or were generated using osteoblasts or stromal cells to induce osteoclast formation in total bone marrow from mice or rabbits. The Copernican revolution in osteoclast biology began with the identification of macrophage-colony stimulating factor (M-CSF) and receptor activator NFκB-ligand (RANKL ) as the key regulators of osteoclast formation, fusion and function. The availability of recombinant human and mouse M-CSF and RANKL has enabled researchers to reliably generate osteoclasts from primary monocyte/macrophage cells as well as from cell lines such as RAW 264.7. This article summarizes the most commonly used procedures for the isolation, generation and characterization of human, rodent and chicken osteoclasts in vitro. Lists of further reading and recommendations are included to facilitate a successful application by the reader. PMID:25228983

  19. Citrus nobiletin suppresses bone loss in ovariectomized ddY mice and collagen-induced arthritis in DBA/1J mice: possible involvement of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis regulation.

    PubMed

    Murakami, Akira; Song, Meiyu; Katsumata, Shin-Ichi; Uehara, Mariko; Suzuki, Kazuharu; Ohigashi, Hajime

    2007-01-01

    Bone resorption is known to accelerate during the onset of several disorders, including osteoporosis (OP) and rheumatoid arthritis (RA). Some epidemiological surveys have suggested that a high intake of vegetables and fruits has an inverse relation to such disease incidence, though the number of active constituents elucidated thus far is limited. In the present study, we examined the efficacy of various food phytochemicals using two animal models. First, female ddY mice were ovariectomized (OVX) or sham-operated (sham), after which five different compounds (phenethyl isothiocyanate, zerumbone, auraptene, 1'-acetoxychavicol acetate, and nobiletin) were administered separately to OVX mice with a mini-osmotic pump at doses of 0.25 or 0.5 mg/day for 4 weeks, with 17beta-estradiol (E_{2}, 0.03 microg/day) used as a positive control. Nobiletin, in contrast to the other tested phytochemicals, significantly (P<0.05) suppressed the reduction of whole bone mineral density by 61%, which was comparable to or higher than the efficacy of E_{2}. Next, nobiletin given as an i.p. administration at 20 mg/kg of body weight, but not 2 mg/kg, to male DBA/1J mice every 2 days for 12 days led to a marked decrease in type II collagen-induced arthritis by 45% (P < 0.05). Furthermore, the flavonoid (4-50 microM) attenuated receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells, as detected by tartarate-resistant acid phosphatase activity and microscopic observations. Of note, nobiletin also suppressed RANKL-activated extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2, and p38 mitogen-activated protein kinase activities, and thereby regulated the promoter activation of nuclear factor kappaB (NFkappaB) and activator protein-1, key transcription factors for differentiation. Together, our results suggest that nobiletin is a promising phytochemical for the prevention or treatment of osteoclastogenesis-related disorders, including

  20. Secretion of PDGF isoforms during osteoclastogenesis and its modulation by anti-osteoclast drugs.

    PubMed

    Rahman, M Motiur; Matsuoka, Kazuhiko; Takeshita, Sunao; Ikeda, Kyoji

    2015-06-26

    In an attempt to identify secretory products of osteoclasts that mediate the coupling of bone formation to resorption, we found that along with osteoclast differentiation, PDGF-A gene expression increase occurred first, by 12 h after stimulation of bone marrow macrophages with M-CSF and RANKL, and peaked at 36 h. This was next followed by a progressive increase in PDGF-B gene expression until a peak at 60 h, when mature osteoclasts formed. Isoform-specific ELISA of the conditioned medium collected every 24 h revealed that all three of the isoforms of PDGF-AA, AB and BB were secreted, in this temporal order as differentiation proceeded. Their secretion was enhanced when osteoclasts were activated by placing them on dentin slices. The secretion of all three isoforms was decreased in cathepsin K-deficient osteoclasts compared with wild-type osteoclasts. Pharmacological inhibition of cathepsin K with odanacatib also inhibited the secretion of all three isoforms, as was also the case with alendronate treatment. The secretion of sphingosine-1-phosphate, which increased during osteoclastogenesis, was reduced from cathepsin K-deficient osteoclasts, and was inhibited by treatment with odanacatib more profoundly than with alendronate. Thus, all three isoforms of PDGF, which are secreted at distinct differentiation stages of osteoclasts, appear to have distinct roles in the cell-cell communication that takes place in the microenvironment of bone remodeling, especially from the osteoclast lineage to mesenchymal cells and vascular cells, thereby stimulating osteogenesis and angiogenesis.

  1. Extracellular glutamate alters mature osteoclast and osteoblast functions.

    PubMed

    Seidlitz, Eric P; Sharma, Mohit K; Singh, Gurmit

    2010-09-01

    Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.

  2. Different Blood-Borne Human Osteoclast Precursors Respond in Distinct Ways to IL-17A.

    PubMed

    Sprangers, Sara; Schoenmaker, Ton; Cao, Yixuan; Everts, Vincent; de Vries, Teun J

    2016-06-01

    Osteoclasts are bone-degrading cells that are formed through fusion of their monocytic precursors. Three distinct subsets of monocytes have been identified in human peripheral blood: classical, intermediate, and non-classical monocytes. They are known to play different roles in physiology and pathology, but their capacity to differentiate into osteoclasts and whether inflammatory cytokines influence this differentiation is unknown. We hypothesized that classical, intermediate, and non-classical monocytes generate functionally different osteoclasts and that they respond in different ways to the inflammatory cytokine interleukin-17A (IL-17A). To investigate this, the different monocyte subsets were isolated from human peripheral blood and osteoclastogenesis was induced with the cytokines M-CSF and RANKL, with or without IL-17A. We found that all subsets are able to differentiate into osteoclasts in vitro, and that both osteoclastogenesis and subsequent bone resorption was distinctly affected by IL-17A. Osteoclastogenesis and bone resorption by osteoclasts derived from classical monocytes remained unaffected by IL-17A, while osteoclast formation from intermediate monocytes was inhibited by the cytokine. Surprisingly, bone resorption by osteoclasts derived from intermediate monocytes remained at similar levels as control cultures, indicating an increased bone resorbing activity by these osteoclasts. Limited numbers of osteoclasts were formed from non-classical monocytes on bone and no bone resorption was detected, which suggest that these cells belong to a cell lineage different from the osteoclast. By providing more insight into osteoclast formation from human blood monocytes, this study contributes to the possible targeting of specific osteoclast precursors as a therapeutic approach for diseases associated with inflammatory bone loss.

  3. Regulation of bone mass and osteoclast function depend on the F-actin modulator SWAP-70.

    PubMed

    Garbe, Annette I; Roscher, Anne; Schüler, Christiane; Lutter, Anne-Helen; Glösmann, Martin; Bernhardt, Ricardo; Chopin, Michael; Hempel, Ute; Hofbauer, Lorenz C; Rammelt, Stefan; Egerbacher, Monika; Erben, Reinhold G; Jessberger, Rolf

    2012-10-01

    Bone remodeling involves tightly regulated bone-resorbing osteoclasts and bone-forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F-actin binding and regulatory protein SWAP-70 in osteoclast biology. F-actin ring formation, cell morphology, and bone resorption are impaired in Swap-70(-/-) osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) remains unaffected. Swap-70(-/-) mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP-70 in Swap-70(-/-) osteoclasts in vitro rescues their deficiencies in bone resorption and F-actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP-70, and the F-actin binding domain. Transplantation of SWAP-70-proficient bone marrow into Swap-70(-/-) mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP-70 in promoting osteoclast function through modulating membrane-proximal F-actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis.

  4. RANKL, Osteopontin, and Osteoclast Homeostasis in a Hyper-Occlusion Mouse Model

    SciTech Connect

    Walker, Cameron G.; Ito, Yoshihiro; Dangaria, Smit; Luan, Xianghong; Diekwisch, Thomas G.H.

    2010-11-15

    The biological mechanisms that maintain the position of teeth in their sockets establish a dynamic equilibrium between bone resorption and apposition. In order to reveal some of the dynamics involved in the tissue responses towards occlusal forces on periodontal ligament (PDL) and alveolar bone homeostasis, we developed the first mouse model of hyperocclusion. Swiss-Webster mice were kept in hyperocclusion for 0, 3, 6, and 9 d. Morphological and histological changes in the periodontium were assessed using micro-computed tomography (micro-CT) and ground sections with fluorescent detection of vital dye labels. Sections were stained for tartrate-resistant acid phosphatase, and the expression of receptor activator of nuclear factor-{kappa}B ligand (RANKL) and osteopontin (OPN) was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). Traumatic occlusion resulted in enamel surface abrasion, inhibition of alveolar bone apposition, significant formation of osteoclasts at 3, 6 and 9 d, and upregulation of OPN and RANKL. Data from this study suggest that both OPN and RANKL contribute to the stimulation of bone resorption in the hyperocclusive state. In addition, we propose that the inhibition of alveolar bone apposition by occlusal forces is an important mechanism for the control of occlusal height that might work in synergy with RANKL-induced bone resorption to maintain normal occlusion.

  5. Immunophenotypic differences between osteoclasts and macrophage polykaryons: immunohistological distinction and implications for osteoclast ontogeny and function.

    PubMed Central

    Athanasou, N A; Quinn, J

    1990-01-01

    The antigenic phenotype of human fetal osteoclasts was compared with that of human tissue macrophages and macrophage polykaryons in foreign body lesions using a large number of monoclonal antibodies directed against myeloid (granulocyte/mononuclear phagocyte) antigens. Osteoclasts expressed a restricted range of macrophage-associated antigens including CD13, CD15A, CD44, CD45, CD54, (ICAM-1), CD71 (transferrin receptor), and CD68. These antigens were also present on macrophages and macrophage polykaryons both of which also strongly expressed CD11a,b,c, CD18, (LFA family), CD14, CD31, CD36, CD37, CD39 and CD43 antigens. There was also weak and occasional expression of CD16 (FcRIII), CD25 (interleukin 2 receptor), CD32 (FcRII), CD35 (C3b receptor) and HLA-DR by macrophage polykaryons. The presence of some macrophage associated antigens on osteoclasts is consistent with their originating from cells of the mononuclear phagocyte system. The numerous differences in antigenic phenotype between osteoclasts and macrophage polykaryons, however, suggest that their pathways of development and differentiation are not identical. The differences discerned in antigenic phenotype should also permit distinction between these polykaryons (and possibly their mononuclear precursors) in normal and diseased tissues. Images PMID:2266187

  6. Comprehensive profiling analysis of actively resorbing osteoclasts identifies critical signaling pathways regulated by bone substrate

    PubMed Central

    Purdue, P. Edward; Crotti, Tania N.; Shen, Zhenxin; Swantek, Jennifer; Li, Jun; Hill, Jonathan; Hanidu, Adedayo; Dimock, Janice; Nabozny, Gerald; Goldring, Steven R.; McHugh, Kevin P.

    2014-01-01

    As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis. PMID:25534583

  7. Terpenes and sterols from the fruits of Prunus mume and their inhibitory effects on osteoclast differentiation by suppressing tartrate-resistant acid phosphatase activity.

    PubMed

    Yan, Xi-Tao; Lee, Sang-Hyun; Li, Wei; Jang, Hae-Dong; Kim, Young-Ho

    2015-02-01

    The fruits of Prunus mume are a common commercial product and a valuable source of food and medicinal material in Eastern Asian countries. Our phytochemical investigation of the P. mume fruit led to the isolation of nine terpenes, including three ursane-type triterpenes (1-3), two cycloartane-type triterpenes (4 and 5), and four tocopherols (10-13), as well as four sterols (6-9). Their structures were elucidated based on extensive spectroscopic analysis, including 1D and 2D NMR and ESI-MS, and the majority of these compounds were isolated from this plant for the first time. The anti-osteoporosis activities of 1-13 were evaluated by measuring their inhibitory effects on tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor κB ligand-induced osteoclastic RAW 264.7 macrophage cells. Compounds 2-7 and 9-12 significantly suppressed TRAP activity down to 47.96 ± 2.45-86.45 ± 3.07 % relative to the control at a concentration of 1 μM. These results suggest that the fruits of P. mume could be an excellent source of anti-osteoporosis phytochemicals that may be developed as natural nutraceuticals and functional foods.

  8. Acute hematopoietic stress in mice is followed by enhanced osteoclast maturation in the bone marrow microenvironment.

    PubMed

    Kuzmac, Sania; Grcevic, Danka; Sucur, Alan; Ivcevic, Sanja; Katavic, Vedran

    2014-11-01

    Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220(-)CD3(-)NK1.1(-)CD11b(-/low)CD115(+)CD117(+) cells in BM, and B220(-)CD3(-)NK1.1(-)Gr-1(-)CD11b(+)CD115(+) cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B(+) cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin(-)Sca-1(+)c-kit(+) population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and

  9. M-CSF priming of osteoclast precursors can cause osteoclastogenesis-insensitivity, which can be prevented and overcome on bone.

    PubMed

    De Vries, Teun J; Schoenmaker, Ton; Aerts, David; Grevers, Lilyanne C; Souza, Pedro P C; Nazmi, Kamran; van de Wiel, Mark; Ylstra, Bauke; Lent, Peter L Van; Leenen, Pieter J M; Everts, Vincent

    2015-01-01

    Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.

  10. Regulation of osteoblast and osteoclast functions by FGF-6.

    PubMed

    Bosetti, Michela; Leigheb, Massimiliano; Brooks, Roger A; Boccafoschi, Francesca; Cannas, Mario F

    2010-11-01

    Fibroblast growth factor-6 (FGF-6) is known to be the key ligand for fibroblast growth factor receptor 4 (FGFR4) during muscle regeneration but its role in bone has yet to be verified. FGFR signaling is known to be important in the initiation and regulation of osteogenesis, so in this study the actions of FGF-6 on human osteoblasts and osteoclasts were investigated. Human primary osteoblasts (hOB) were used to study the effect of FGF-6 on proliferation (by ATP quantification), signal transduction (by ERK and AKT phosphorylation), differentiation (by alkaline phosphatase activity, APA), and mineralization (by calcein staining). To study FGF-6 activity on osteoclast differentiation, human bone marrow cells were used and tartrate-resistant acid phosphatase (TRAP) multinucleated cells together with actin filaments arrangements were quantified. Human primary mature osteoclasts were used to evaluate the effect of FGF-6 on osteoclast reabsorbing activity by reabsorbed pit measurements. FGF-6 >10(-9) M as FGF-2 10(-7) M induced hOB proliferation mediated by pERK together with a reduction in APA and reduced mineralization of the treated cells. Moreover FGF-6 increased the formation of TRAP-positive multinucleated cells in a dose-dependent manner (maximal effect at 10(-8) M). FGF-6-treated cells showed also a greater percentage of cells that formed typical osteoclast sealing zones. Mature osteoclasts cultured on dentine slice increased the area of reabsorption with a maximal effect of FGF-6 at 10(-12) M. FGF-6 may be considered a regulator of bone metabolism as shown by its activity on both osteoblasts and osteoclasts.

  11. Advances in the regulation of osteoclasts and osteoclast functions.

    PubMed

    Boyce, B F

    2013-10-01

    Osteoclasts are derived from mononuclear hematopoietic myeloid lineage cells, which are formed in the bone marrow and are attracted to the bloodstream by factors, including sphingsine-1 phosphate. These circulating precursors are attracted to bone surfaces undergoing resorption by chemokines and other factors expressed at these sites, where they fuse to form multinucleated bone-resorbing cells. All aspects of osteoclast formation and functions are regulated by macrophage-colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), cytokines essential for osteoclast formation and expressed by a variety of cell types, including osteoblast lineage cells. Since the discovery of RANKL in the mid-1990s, mouse genetic and molecular studies have revealed numerous signaling pathways activated by RANKL and M-CSF. More recent studies indicate that osteoclasts and their precursors regulate immune responses and osteoblast formation and functions by means of direct cell-cell contact through ligands and receptors, such as ephrins and Ephs, and semaphorins and plexins, and through expression of clastokines. There is also growing recognition that osteoclasts are immune cells with roles in immune responses beyond mediating the bone destruction that can accompany them. This article reviews recent advances in the understanding of the molecular mechanisms regulating osteoclast formation and functions and their interactions with other cells in normal and pathologic states.

  12. Impact of dietary aromatic amino acids on osteoclastic activity.

    PubMed

    Refaey, Mona El; Zhong, Qing; Ding, Ke-Hong; Shi, Xing-Ming; Xu, Jianrui; Bollag, Wendy B; Hill, William D; Chutkan, Norman; Robbins, Richard; Nadeau, Hugh; Johnson, Maribeth; Hamrick, Mark W; Isales, Carlos M

    2014-08-01

    We had shown that aromatic amino acid (phenylalanine, tyrosine, and tryptophan) supplementation prevented bone loss in an aging C57BL/6 mice model. In vivo results from the markers of bone breakdown suggested an inhibition of osteoclastic activity or differentiation. To assess osteoclastic differentiation, we examined the effects of aromatic amino acids on early /structural markers as vitronectin receptor, calcitonin receptor, and carbonic anhydrase II as well as, late/functional differentiation markers; cathepsin K and matrix metalloproteinase 9 (MMP-9). Our data demonstrate that the aromatic amino acids down-regulated early and late osteoclastic differentiation markers as measured by real time PCR. Our data also suggest a link between the vitronectin receptor and the secreted cathepsin K that both showed consistent effects to the aromatic amino acid treatment. However, the non-attachment related proteins, calcitonin receptor, and carbonic anhydrase II, demonstrated less consistent effects in response to treatment. Our data are consistent with aromatic amino acids down-regulating osteoclastic differentiation by suppressing remodeling gene expression thus contributing initially to the net increase in bone mass seen in vivo.

  13. Myeloid-derived suppressor cells function as novel osteoclast progenitors enhancing bone loss in breast cancer

    PubMed Central

    Sawant, Anandi; Deshane, Jessy; Jules, Joel; Lee, Carnella M.; Harris, Brittney A.; Feng, Xu; Ponnazhagan, Selvarangan

    2012-01-01

    Enhanced bone destruction is a hallmark of various carcinomas such as breast cancer, where osteolytic bone metastasis is associated with increased morbidity and mortality. Immune cells contribute to osteolysis in cancer growth but the factors contributing to aggressive bone destruction are not well understood. In this study, we demonstrate the importance of myeloid-derived suppressor cells (MDSC) in this process at bone metastatic sites. Since MDSC originate from the same myeloid lineage as macrophages, which are osteoclast precursors, we hypothesized that MDSC may undergo osteoclast differentiation and contribute to enhanced bone destruction and tumor growth. Using an immunocompetent mouse model of breast cancer bone metastasis, we confirmed that MDSC isolated from the tumor-bone microenvironment differentiated into functional osteoclasts both in vitro and in vivo. Mechanistic investigations revealed that nitric oxide signaling was critical for differentiation of MDSC into osteoclasts. Remarkably, osteoclast differentiation did not occur in MDSC isolated from control or tumor-bearing mice that lacked bone metastasis, signifying the essential cross-talk between tumor cells and myeloid progenitors in the bone microenvironment as a requirement for osteoclast differentiation of MDSC. Overall, our results identify a wholly new facet to the multifunctionality of MDSC in driving tumor progression, in this case as a novel osteoclast progenitor that specifically drives bone metastasis during cancer progression. PMID:23243021

  14. Tensin 3 is a new partner of Dock5 that controls osteoclast podosome organization and activity.

    PubMed

    Touaitahuata, Heiani; Morel, Anne; Urbach, Serge; Mateos-Langerak, Julio; de Rossi, Sylvain; Blangy, Anne

    2016-09-15

    Bone resorption by osteoclasts is mediated by a typical adhesion structure called the sealing zone or actin ring, whose architecture is based on a belt of podosomes. The molecular mechanisms driving podosome organization into superstructures remain poorly understood to date, in particular at the osteoclast podosome belt. We performed proteomic analyses in osteoclasts and found that the adaptor protein tensin 3 is a partner of Dock5, a Rac exchange factor necessary for podosome belt formation and bone resorption. Expression of tensin 3 and Dock5 concomitantly increase during osteoclast differentiation. These proteins associate with the osteoclast podosome belt but not with individual podosomes, in contrast to vinculin. Super-resolution microscopy revealed that, even if they colocalize in the x-y plane of the podosome belt, Dock5 and tensin 3 differentially localize relative to vinculin in the z-axis. Tensin 3 increases Dock5 exchange activity towards Rac, and suppression of tensin 3 in osteoclasts destabilizes podosome organization, leading to delocalization of Dock5 and a severe reduction in osteoclast activity. Our results suggest that Dock5 and tensin 3 cooperate for osteoclast activity, to ensure the correct organization of podosomes. PMID:27505886

  15. Resorption of monetite calcium phosphate cement by mouse bone marrow derived osteoclasts.

    PubMed

    Montazerolghaem, M; Karlsson Ott, M; Engqvist, H; Melhus, H; Rasmusson, A J

    2015-01-01

    Recently the interest for monetite based biomaterials as bone grafts has increased; since in vivo studies have demonstrated that they are degradable, osteoconductive and improve bone healing. So far osteoclastic resorption of monetite has received little attention. The current study focuses on the osteoclastic resorption of monetite cement using primary mouse bone marrow macrophages, which have the potential to differentiate into resorbing osteoclasts when treated with receptor activator NF-κB ligand (RANKL). The osteoclast viability and differentiation were analysed on monetite cement and compared to cortical bovine bone discs. After seven days live/dead stain results showed no significant difference in viability between the two materials. However, the differentiation was significantly higher on the bone discs, as shown by tartrate resistant acid phosphatase (TRAP) activity and Cathepsin K gene expression. Moreover monetite samples with differentiated osteoclasts had a 1.4 fold elevated calcium ion concentration in their culture media compared to monetite samples with undifferentiated cells. This indicates active resorption of monetite in the presence of osteoclasts. In conclusion, this study suggests that osteoclasts have a crucial role in the resorption of monetite based biomaterials. It also provides a useful model for studying in vitro resorption of acidic calcium phosphate cements by primary murine cells. PMID:25953560

  16. Biphasic Effects of Vitamin D and FGF23 on Human Osteoclast Biology.

    PubMed

    Allard, Lise; Demoncheaux, Nathalie; Machuca-Gayet, Irma; Georgess, Dan; Coury-Lucas, Fabienne; Jurdic, Pierre; Bacchetta, Justine

    2015-07-01

    Vitamin D and FGF23 play a major role in calcium/phosphate balance. Vitamin D may control bone resorption but the potential role of FGF23 has never been evaluated. The objective of this study was therefore to compare the effects of vitamin D and FGF23 on osteoclast differentiation and activity in human monocyte-derived osteoclasts. Human monocytes, purified from blood of healthy donors, were incubated with M-CSF and RANKL to obtain mature multinucleated osteoclasts (MNC). Experiments were carried out to assess the effects of FGF23 as compared to native vitamin D (25-D) and active vitamin D (1,25-D) on osteoclast differentiation and on bone-resorbing osteoclast activity. Additional experiments with the pan fibroblast growth factor receptor inhibitor (FGFR-i) were performed. Phosphorylation Akt and Erk pathways were analyzed by Western blot analyses. Both 1,25-D and FGF23, to a lesser extent, significantly inhibited osteoclastogenesis at early stages; when adding FGFR-i, osteoclast formation was restored. Biochemical experiments showed an activation of the Akt and Erk pathways under FGF23 treatment. In contrast, in terms of activity, 1,25-D had no effect on resorption, whereas FGF23 slightly but significantly increased bone resorption; 25-D had no effects on either differentiation or on activity. These data show that 1,25-D inhibits osteoclastogenesis without regulating osteoclast-mediated bone resorption activity; FGF23 has biphasic effects on osteoclast physiology, inhibiting osteoclast formation while stimulating slightly osteoclast activity. These results may be of importance and taken into account in chronic kidney disease when therapies modulating FGF23 are available.

  17. The effect of the degree of sulfation of glycosaminoglycans on osteoclast function and signaling pathways.

    PubMed

    Salbach, Juliane; Kliemt, Stefanie; Rauner, Martina; Rachner, Tilman D; Goettsch, Claudia; Kalkhof, Stefan; von Bergen, Martin; Möller, Stephanie; Schnabelrauch, Matthias; Hintze, Vera; Scharnweber, Dieter; Hofbauer, Lorenz C

    2012-11-01

    To meet the growing need for bone replacement of our aging population, development of new adaptive biomaterials is essential. Collagen and glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major components of the extracellular matrix (ECM) in bone. We manufactured native and sulfate-modified GAG matrices, evaluated how these components modulate different functions of osteoclasts, the cells that resorb bone, and analyzed the underlying mechanisms. GAGs were tested for their effects on osteoclast adhesion, viability, differentiation, morphology, and resorption as well as proteome alterations using murine RAW264.7 cells and primary human osteoclasts. Native and sulfated GAGs were stable and largely non-cytotoxic. Sulfation of GAGs led to a significant inhibition of osteoclast differentiation and resorption, which was largely dependent on the degree of sulfation of GAGs rather than the monosaccharide composition. Sulfation significantly reduced resorptive function by 14% (CS) and 43% (HA). Highly sulfated GAGs dose-dependently suppressed osteoclast differentiation, osteoclast-specific expression of TRAP, cathepsin K, SWAP-70, and OSCAR by 63-95%, and inhibited proteins involved in cytoskeletal rearrangement. In conclusion, highly sulfated GAGs significantly inhibit various functions of bone-resorbing osteoclasts. Whether these properties locally contribute to improved fracture or bone defect healing needs to be validated in vivo.

  18. Screening of Korean medicinal plants for possible osteoclastogenesis effects in vitro

    PubMed Central

    Youn, Yu Na; Lim, Erang; Lee, Nari; Kim, Young Seop; Koo, Min Seon

    2007-01-01

    Bone undergoes continuous remodeling through bone formation and resorption, and maintaining the balance for skeletal rigidity. Bone resorption and loss are generally attributed to osteoclasts. Differentiation of osteoclasts is regulated by receptor activator of nuclear factor NF-kB ligand (RANKL), a member of tumor necrosis factor family. When the balance is disturbed, pathological bone abnormality ensues. Through the screening of traditional Korean medicinal plants, the effective molecules for inhibition and stimulation of RANKL-induced osteoclast differentiation in mouse bone marrow macrophages were identified. Among 222 methanol extracts, of medicinal plants, 10 samples exhibited ability to induce osteoclast differentiation. These include Dryobalanops aromatica, Euphoria longana, Lithospermum erythrorhizon, Prunus mume, Prunus nakaii, and Polygonatum odoratum. In contrast, Ailanthus altissima, Curcuma longa, Solanum nigrum, Taraxacum platycarpa, Trichosanthes kirilowii, and Daphne genkwa showed inhibitory effects in RANKL-induced osteoclast differentiation. PMID:18850234

  19. Deletion of Histone Deacetylase 7 in Osteoclasts Decreases Bone Mass in Mice by Interactions with MITF

    PubMed Central

    Stemig, Melissa; Astelford, Kristina; Emery, Ann; Cho, Jangyeun J.; Allen, Ben; Huang, Tsang-hai; Gopalakrishnan, Rajaram; Mansky, Kim C.; Jensen, Eric D.

    2015-01-01

    Molecular regulators of osteoclast formation and function are an important area of research due to the central role of osteoclasts in bone resorption. Transcription factors such as MITF are essential for osteoclast generation by regulating expression of the genes required for cellular differentiation and resorptive function. We recently reported that histone deacetylase 7 (HDAC7) binds to and represses the transcriptional activity of MITF in osteoclasts, and that loss of HDAC7 in vitro accelerated osteoclastogenesis. In the current study, we extend this initial observation by showing that conditional deletion of HDAC7 in osteoclasts of mice leads to an in vivo enhancement in osteoclast formation, associated with increased bone resorption and lower bone mass. Expression of multiple MITF target genes is increased in bone marrow derived osteoclast cultures from the HDAC7 knockout mice. Interestingly, multiple regions of the HDAC7 amino-terminus can bind to MITF or exert repressive activity. Moreover, mutation or deletion of the HDAC7 conserved deacetylase catalytic domain had little effect on repressive function. These observations identify HDAC7 in osteoclasts as an important molecular regulator of MITF activity and bone homeostasis, but also highlight a gap in our understanding of exactly how HDAC7 functions as a corepressor. PMID:25875108

  20. MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

    SciTech Connect

    Miyamoto, Kana; Ninomiya, Ken; Sonoda, Koh-Hei; Miyauchi, Yoshiteru; Hoshi, Hiroko; Iwasaki, Ryotaro; Miyamoto, Hiroya; and others

    2009-06-05

    Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.

  1. Characterization of Functional Reprogramming during Osteoclast Development Using Quantitative Proteomics and mRNA Profiling*

    PubMed Central

    An, Eunkyung; Narayanan, Manikandan; Manes, Nathan P.; Nita-Lazar, Aleksandra

    2014-01-01

    In addition to forming macrophages and dendritic cells, monocytes in adult peripheral blood retain the ability to develop into osteoclasts, mature bone-resorbing cells. The extensive morphological and functional transformations that occur during osteoclast differentiation require substantial reprogramming of gene and protein expression. Here we employ -omic-scale technologies to examine in detail the molecular changes at discrete developmental stages in this process (precursor cells, intermediate osteoclasts, and multinuclear osteoclasts), quantitatively comparing their transcriptomes and proteomes. The data have been deposited to the ProteomeXchange with identifier PXD000471. Our analysis identified mitochondrial changes, along with several alterations in signaling pathways, as central to the development of mature osteoclasts, while also confirming changes in pathways previously implicated in osteoclast biology. In particular, changes in the expression of proteins involved in metabolism and redirection of energy flow from basic cellular function toward bone resorption appeared to play a key role in the switch from monocytic immune system function to specialized bone-turnover function. These findings provide new insight into the differentiation program involved in the generation of functional osteoclasts. PMID:25044017

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

    PubMed

    Deepak, Vishwa; Kruger, Marlena C; Joubert, Annie; Coetzee, Magdalena

    2015-01-01

    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-κβ 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-κβ 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.. PMID:26627060

  3. IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor γ1

    PubMed Central

    Bendixen, Amy C.; Shevde, Nirupama K.; Dienger, Krista M.; Willson, Timothy M.; Funk, Colin D.; Pike, J. Wesley

    2001-01-01

    IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-κB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor γ1 (PPARγ1) ligands and can be blocked by the irreversible PPARγ antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARγ1, an interpretation strengthened by the observation that IL-4 can activate a PPARγ1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARγ1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARγ1 ligands. Our results reveal that PPARγ1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARγ1 ligands on bone loss in diabetic patients. PMID:11226258

  4. Intracellular calcium puffs in osteoclasts.

    PubMed

    Radding, W; Jordan, S E; Hester, R B; Blair, H C

    1999-12-15

    We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.

  5. Pharmacological blocking of the osteoclastic biocorrosion of surgical stainless steel in vitro.

    PubMed

    Lionetto, S; Little, A; Moriceau, G; Heymann, D; Decurtins, M; Plecko, M; Filgueira, L; Cadosch, D

    2013-04-01

    In vitro studies suggest that human osteoclasts (OC) are able to corrode surgical stainless steel 316L (SS). The aim of this study was to investigate whether osteoclastic biocorrosion can be blocked pharmacologically. Human OCs were generated in vitro from peripheral blood monocytic cells (PBMCs) in the presence of OC differentiation cytokines. The osteoclastic viability, differentiation, and resorptive function (on both bone and SS) were assessed using standard colorimetric cell viability assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt (MTS), fluorescence microscopy, tartrate-resistant acid phosphatase expression (flow cytometry), and scanning electron microscopy. OCs cultured on SS were exposed to nontoxic concentrations of bafilomycin A1, amiloride hydrochloride, or zoledronic acid. The extent of biocorrosion was quantified using atomic emission spectrometry (to measure the concentration of metal ions released into the supernatant) and scanning electron microscopy. PBMCs differentiated into mature and functional OC in the presence of all the drugs used. Osteoclastic resorption of SS was noted with differences in the resorption pattern for all drug treatments. Under the drug treatments, single areas of osteoclastic resorption were larger in size but less abundant when compared with positive controls. None of the drugs used were able to inhibit osteoclastic biocorrosion of SS.

  6. Osteoclasts, mononuclear phagocytes, and c-Fos: new insight into osteoimmunology.

    PubMed

    Matsuo, Koichi; Ray, Neelanjan

    2004-06-01

    Osteoimmunology is the emerging concept that certain molecules link the skeletal and immune systems. The transcription factor c-Fos, a component of activator protein-1 (AP-1), is essential for osteoclast differentiation. Mice lacking c-Fos are osteopetrotic owing to impaired osteoclast development. Recent studies suggest that in contrast to this positive role in osteoclastogenesis, c-Fos expression inhibits differentiation and activation of mononuclear phagocytes. Here, we focus on the contrasting roles of c-Fos in the bone and immune lineages. Both osteoclasts and mononuclear phagocytes are derived from common myeloid precursors. Osteoclasts resorb bone, whereas macrophages and myeloid dendritic cells phagocytose microbial pathogens, initiating innate and adaptive immunity. Differentiation of the common precursors into either bone or immune lineage is determined by ligand binding to cell-surface receptors, particularly receptor activator of NF-kappa B (RANK) for osteoclasts, or Toll-like receptors (TLRs) for mononuclear phagocytes. Both RANK and TLRs activate the dimeric transcription factors NF-kappa B and AP-1. Yet, c-Fos/AP-1 plays a positive role in osteoclasts but a negative role in macrophages and dendritic cells. Further study is necessary to clarify this dual role of c-Fos.

  7. Bone marrow-derived osteoclast-like cells from a patient with craniometaphyseal dysplasia lack expression of osteoclast-reactive vacuolar proton pump.

    PubMed Central

    Yamamoto, T; Kurihara, N; Yamaoka, K; Ozono, K; Okada, M; Yamamoto, K; Matsumoto, S; Michigami, T; Ono, J; Okada, S

    1993-01-01

    Craniometaphyseal dysplasia (CMD) is a rare craniotubular bone dysplasia transmitted in autosomal dominant or recessive form. This disease is characterized by cranial bone hyperostosis and deformity of the metaphyses of the long bones. Using osteoclast-like cells formed from patient bone marrow cells, we investigated the pathophysiology of CMD in a 3-yr-old patient. Untreated bone marrow cells from the patient differentiated into osteoclast-like cells in vitro. These cells were shown to have vitronectin beta-receptors using a specific monoclonal antibody, i.e., 23C6 (CD51), which reacts with osteoclasts in human bone biopsy samples. However, the number of these osteoclast-like cells formed from the patient's bone marrow was only 40% of the normal controls. 1,25-dihydroxyvitamin-D3, bovine 1-34 parathyroid hormone, recombinant human interleukin-1 beta, recombinant human interleukin-6, or recombinant human macrophage colony-stimulating factor significantly increased, while salmon calcitonin significantly inhibited, the number of osteoclast-like cells. However, these cells could not resorb sperm whale dentin slices and lacked the osteoclast-reactive vacuolar proton pump as evidenced by a monoclonal antibody (E11). Western blot analysis using a monoclonal antibody to pp60c-src (327) revealed that protooncogene c-src expression by the platelets of the CMD patient was comparable to the normal control. These data suggest that: (a) the hyperostosis and the metaphyseal long bone deformity in the present CMD patient might be explained by osteoclast dysfunction due to impaired expression of the osteoclast-reactive vacuolar proton pump; and (b) a protooncogene c-src was not associated with the pathogenesis of the present CMD patient. Images PMID:7678608

  8. Electrophysiological responses of osteoclasts to hormones.

    PubMed

    Ferrier, J; Ward, A; Kanehisa, J; Heersche, J N

    1986-07-01

    Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.

  9. Retinoic acid and 1,25-dihydroxyvitamin D3 stimulate osteoclast formation by different mechanisms

    SciTech Connect

    Scheven, B.A.; Hamilton, N.J. )

    1990-01-01

    The effects of retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on osteoclast formation were examined in intact fetal long bones of different ages/developmental stages maintained in organ culture using a chemically defined medium with or without the presence of serum. Besides stimulating bone resorption, RA and 1,25-(OH)2D3 increased the number of osteoclasts in 19-day-old fetal rat tibiae. Likewise, these bone-resorbing agents induced and stimulated osteoclast formation in 19- and 18-day-old metatarsal bones which were osteoclast-free at the beginning of the culture. The response to 1,25-(OH)2D3 was greatly enhanced by 10% fetal bovine serum (FBS) irrespective of the developmental stage of the long bone. The response to RA was not. Light microscopic autoradiography after labeling of the cultures with tritiated thymidine showed that both RA and 1,25-(OH)2D3 induced osteoclast differentiation from proliferating and postmitotic precursors. However, neither agent was able to stimulate proliferation of osteoclast progenitor cells in the older bones (19 days). Studies on the formation of osteoclast-like (tartrate-resistant acid phosphatase positive) cells in bone marrow cultures indicated that FBS was a potent inducer of osteoclast-like cell formation. In the presence of FBS, 1,25-(OH)2D3 significantly stimulated this response, but RA did not. The results demonstrate that although both RA and 1,25-(OH)2D3 stimulate osteoclast formation from proliferating and postmitotic precursors in long bones in vitro, they do so by different mechanisms.

  10. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling.

    PubMed

    Ahn, Heejin; Lee, Kyunghee; Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  11. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  12. Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects

    PubMed Central

    Kenner, Lukas; Hoebertz, Astrid; Beil, Timo; Keon, Niamh; Karreth, Florian; Eferl, Robert; Scheuch, Harald; Szremska, Agnieszka; Amling, Michael; Schorpp-Kistner, Marina; Angel, Peter; Wagner, Erwin F.

    2004-01-01

    Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBΔ/Δ mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBΔ/Δ osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16INK4a levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage–osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity. PMID:14769860

  13. Identification of enoxacin as an inhibitor of osteoclast formation and bone resorption by structure-based virtual screening.

    PubMed

    Ostrov, David A; Magis, Andrew T; Wronski, Thomas J; Chan, Edward K L; Toro, Edgardo J; Donatelli, Richard E; Sajek, Kristen; Haroun, Ireni N; Nagib, Michael I; Piedrahita, Ana; Harris, Ashley; Holliday, L Shannon

    2009-08-27

    An interaction between the B2 subunit of vacuolar H(+)-ATPase (V-ATPase) and microfilaments is required for osteoclast bone resorption. An atomic homology model of the actin binding site on B2 was generated and molecular docking simulations were performed. Enoxacin, a fluoroquinolone antibiotic, was identified and in vitro testing demonstrated that enoxacin blocked binding between purified B2 and microfilaments. Enoxacin dose dependently reduced the number of osteoclasts differentiating in mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D(3), as well as markers of osteoclast activity, and the number of resorption lacunae formed on bone slices. Enoxacin inhibited osteoclast formation at concentrations where osteoblast formation was not altered. In summary, enoxacin is a novel small molecule inhibitor of osteoclast bone resorption that acts by an unique mechanism and is therefore an attractive lead molecule for the development of a new class of antiosteoclastic agents.

  14. Osteoprotegerin Induces Apoptosis of Osteoclasts and Osteoclast Precursor Cells via the Fas/Fas Ligand Pathway.

    PubMed

    Liu, Wei; Xu, Chao; Zhao, Hongyan; Xia, Pengpeng; Song, Ruilong; Gu, Jianhong; Liu, Xuezhong; Bian, Jianchun; Yuan, Yan; Liu, Zongping

    2015-01-01

    Osteoprotegerin (OPG) is known to inhibit differentiation and activation of osteoclasts (OCs) by functioning as a decoy receptor blocking interactions between RANK and RANKL. However, the exact role of OPG in the survival/apoptosis of OCs remains unclear. OPG caused increased rates of apoptosis of both OCs and osteoclast precursor cells (OPCs). The expression of Fas and activated caspase-8 was increased by both 20 ng/mL and 40 ng/mL of OPG, but was markedly decreased at 80 ng/mL. Interestingly, we noted that while levels of Fas ligand (FasL) increased with increasing doses of OPG, the soluble form of FasL in the supernatant decreased. The results of a co-immunoprecipitation assay suggested that the decrease of sFasL might be caused by the binding of OPG. This would block the inhibition of the apoptosis of OCs and OPCs. Furthermore, changes in expression levels of Bax/Bcl-2, cleaved-caspase-9, cleaved-caspased-3 and the translocation of cytochrome c, illustrated that OPG induced apoptosis of OCs and OPCs via the classic Fas/FasL apoptosis pathway, and was mediated by mitochondria. Altogether, our results demonstrate that OPG induces OCs and OPCs apoptosis partly by the Fas/FasL signaling pathway.

  15. Biocorrosion and uptake of titanium by human osteoclasts.

    PubMed

    Cadosch, Dieter; Al-Mushaiqri, Mohamed S; Gautschi, Oliver P; Meagher, James; Simmen, Hans-Peter; Filgueira, Luis

    2010-12-15

    All metals in contact with a biological system undergo corrosion through an electrochemical redox reaction. This study investigated whether human osteoclasts (OC) are able to grow on titanium and aluminum, and directly corrode the metals leading to the release of corresponding metal ions, which are believed to cause inflammatory reactions and activate osteoclastic differentiation. Scanning electron microscopy analysis demonstrated long-term viable OC cultures on the surface of titanium and aluminum foils. Atomic emission spectrometry investigations showed significantly increased levels of aluminum in the supernatant of OC cultured on aluminum; however, all measurements in the supernatants of cell cultures on titanium were below detection limits. Despite this, confocal microscopy analysis with Newport Green DCF diacetate ester staining depicted intense fluorescence throughout the cytoplasm and nucleolus of OC cultured on titanium foils. Comparable fluorescence intensities were not observed in monocytes and control cells cultured on glass. The present study demonstrated that human osteoclast precursors are able to grow and differentiate toward mature OC on titanium and aluminum. Furthermore, it established that the mature cells are able to directly corrode the metal surface and take up corresponding metal ions, which subsequently may be released and thereby induce the formation of osteolytic lesions in the periprosthetic bone, contributing to the loosening of the implant.

  16. Obatoclax regulates the proliferation and fusion of osteoclast precursors through the inhibition of ERK activation by RANKL.

    PubMed

    Oh, Ju Hee; Lee, Jae Yoon; Park, Jin Hyeong; No, Jeong Hyeon; Lee, Na Kyung

    2015-03-01

    Obatoclax, a pan-Bcl2 inhibitor, shows antitumor activities in various solid malignancies. Bcl2-deficient mice have shown the importance of Bcl2 in osteoclasts, as the bone mass of the mice was increased by the induced apoptosis of osteoclasts. Despite the importance of Bcl2, the effects of obatoclax on the proliferation and differentiation of osteoclast precursors have not been studied extensively. Here, we describe the anti-proliferative effects of obatoclax on osteoclast precursors and its negative role on fusion of the cells. Stimulation with low doses of obatoclax significantly suppressed the proliferation of osteoclast precursors in a dose-dependent manner while the apoptosis was markedly increased. Its stimulation was sufficient to block the activation of ERK MAP kinase by RANKL. The same was true when PD98059, an ERK inhibitor, was administered to osteoclast precursors. The activation of JNK1/2 and p38 MAP kinase, necessary for osteoclast differentiation, by RANKL was not affected by obatoclax. Interestingly, whereas the number of TRAP-positive mononuclear cells was increased by both obatoclax and PD98059, fused, multinucleated cells larger than 100 μm in diameter containing more than 20 nuclei were completely reduced. Consistently, obatoclax failed to regulate the expression of osteoclast marker genes, including c-Fos, TRAP, RANK and CtsK. Instead, the expression of DC-STAMP and Atp6v0d2, genes that regulate osteoclast fusion, by RANKL was significantly abrogated by both obatoclax and PD98059. Taken together, these results suggest that obatoclax down-regulates the proliferation and fusion of osteoclast precursors through the inhibition of the ERK1/2 MAP kinase pathway.

  17. The Great Beauty of the osteoclast.

    PubMed

    Cappariello, Alfredo; Maurizi, Antonio; Veeriah, Vimal; Teti, Anna

    2014-09-15

    Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an "orchestrator" implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the '00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting "clastokines" that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast. PMID:24976175

  18. The Roles of Acidosis in Osteoclast Biology

    PubMed Central

    Yuan, Feng-Lai; Xu, Ming-Hui; Li, Xia; Xinlong, He; Fang, Wei; Dong, Jian

    2016-01-01

    The adverse effect of acidosis on the skeletal system has been recognized for almost a century. Although the underlying mechanism has not been fully elucidated, it appears that acidosis acts as a general stimulator of osteoclasts derived from bone marrow precursors cells and enhances osteoclastic resorption. Prior work suggests that acidosis plays a significant role in osteoclasts formation and activation via up-regulating various genes responsible for its adhesion, migration, survival and bone matrix degradation. Understanding the role of acidosis in osteoclast biology may lead to development of novel therapeutic approaches for the treatment of diseases related to low bone mass. In this review, we aim to discuss the recent investigations into the effects of acidosis in osteoclast biology and the acid-sensing molecular mechanism. PMID:27445831

  19. Bisphosphonate-functionalized hyaluronic acid showing selective affinity for osteoclasts as a potential treatment for osteoporosis.

    PubMed

    Kootala, Sujit; Ossipov, Dmitri; van den Beucken, Jeroen J J P; Leeuwenburgh, Sander; Hilborn, Jöns

    2015-08-01

    Current treatments for osteoporosis involve the administration of high doses of bisphosphonates (BPs) over a number of years. However, the efficiency of the absorption of these drugs and specificity towards targeted osteoclastic cells is still suboptimal. In this study, we have exploited the natural affinity of high (H) and low (L) molecular-weight hyaluronic acid (HA) towards a cluster of differentiation 44 (CD44) receptors on osteoclasts to use it as a biodegradable targeting vehicle. We covalently bonded BP to functionalised HA (HA-BP) and found that HA-BP conjugates were highly specific to osteoclastic cells and reduced mature osteoclast numbers significantly more than free BP. To study the uptake of HA-BP, we fluorescently derivatised the polymer-drug with fluorescein B isothiocyanate (FITC) and found that L-HA-BP could seamlessly enter osteoclastic cells. Alternatively, we tested polyvinyl alcohol (PVA) as a synthetic polymer delivery vehicle using similar chemistry to link BP and found that osteoclast numbers did not reduce in the same way. These findings could pave the way for biodegradable polymers to be used as vehicles for targeted delivery of anti-osteoporotic drugs. PMID:26222035

  20. Knee loading inhibits osteoclast lineage in a mouse model of osteoarthritis

    PubMed Central

    Li, Xinle; Yang, Jing; Liu, Daquan; Li, Jie; Niu, Kaijun; Feng, Shiqing; Yokota, Hiroki; Zhang, Ping

    2016-01-01

    Osteoarthritis (OA) is a whole joint disorder that involves cartilage degradation and periarticular bone response. Changes of cartilage and subchondral bone are associated with development and activity of osteoclasts from subchondral bone. Knee loading promotes bone formation, but its effects on OA have not been well investigated. Here, we hypothesized that knee loading regulates subchondral bone remodeling by suppressing osteoclast development, and prevents degradation of cartilage through crosstalk of bone-cartilage in osteoarthritic mice. Surgery-induced mouse model of OA was used. Two weeks application of daily dynamic knee loading significantly reduced OARSI scores and CC/TAC (calcified cartilage to total articular cartilage), but increased SBP (subchondral bone plate) and B.Ar/T.Ar (trabecular bone area to total tissue area). Bone resorption of osteoclasts from subchondral bone and the differentiation of osteoclasts from bone marrow-derived cells were completely suppressed by knee loading. The osteoclast activity was positively correlated with OARSI scores and negatively correlated with SBP and B.Ar/T.Ar. Furthermore, knee loading exerted protective effects by suppressing osteoclastogenesis through Wnt signaling. Overall, osteoclast lineage is the hyper responsiveness of knee loading in osteoarthritic mice. Mechanical stimulation prevents OA-induced cartilage degeneration through crosstalk with subchondral bone. Knee loading might be a new potential therapy for osteoarthritis patients. PMID:27087498

  1. Pro-osteoclastic in vitro effect of Polyethylene-like nanoparticles: Involvement in the pathogenesis of implant aseptic loosening.

    PubMed

    Brulefert, Kevin; Córdova, Luis A; Brulin, Bénédicte; Faucon, Adrien; Hulin, Philipe; Nedellec, Steven; Gouin, François; Passuti, Norbert; Ishow, Eléna; Heymann, Dominique

    2016-11-01

    Polyethylene micro-sized wear particles released from orthopedic implants promote inflammation and osteolysis; however, less is known about the bioactivity of polyethylene nanosized wear particles released from the last generation of polymer-bearing surfaces. We aim to assess the internalization of fluorescent polyethylene-like nanoparticles by both human macrophages and osteoclasts and also, to determine their effects in osteoclastogenesis in vitro. Human macrophages and osteoclasts were incubated with several ratios of fluorescent polyethylene-like nanoparticles between 1 and 72 h, and 4 h, 2, 4, 6, and 9 days, respectively. The internalization of nanoparticles was quantified by flow cytometry and followed by both confocal and video time-lapse microscopy. Osteoclast differentiation and activity was semiquantified by tartrate-resistant acid phosphatase (TRAP) staining, TRAP mRNA relative expression, and pit resorption assay, respectively. Macrophages, osteoclast precursors and mature osteoclasts internalized nanoparticles in a dose- and time-dependent manner and maintained their resorptive activity. In addition, nanoparticles significantly increased the osteoclastogenesis as shown by upregulation of the TRAP expressing cell number. We conclude that polyethylene-like nanosized wear particles promote osteoclast differentiation without alteration of bone resorptive activity of mature osteoclasts and they could be considered as important actors in periprosthetic osteolysis of the last new generation of polymer-bearing surfaces. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2649-2657, 2016.

  2. Osteoclast-independent bone resorption by fibroblast-like cells

    PubMed Central

    Pap, Thomas; Claus, Anja; Ohtsu, Susumu; Hummel, Klaus M; Schwartz, Peter; Drynda, Susanne; Pap, Géza; Machner, Andreas; Stein, Bernhard; George, Michael; Gay, Renate E; Neumann, Wolfram; Gay, Steffen; Aicher, Wilhelm K

    2003-01-01

    To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders. PMID:12723988

  3. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts.

    PubMed

    Burgess, T L; Qian, Y; Kaufman, S; Ring, B D; Van, G; Capparelli, C; Kelley, M; Hsu, H; Boyle, W J; Dunstan, C R; Hu, S; Lacey, D L

    1999-05-01

    Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects. PMID:10225954

  4. Different cysteine proteinases involved in bone resorption and osteoclast formation.

    PubMed

    Brage, M; Abrahamson, M; Lindström, V; Grubb, A; Lerner, U H

    2005-06-01

    Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

  5. The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen.

    PubMed

    Boraschi-Diaz, Iris; Komarova, Svetlana V

    2016-01-01

    Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm(2) for spleen, as compared to 50,000/cm(2) for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70-80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2-5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.

  6. Modeled microgravity and hindlimb unloading sensitize osteoclast precursors to RANKL-mediated osteoclastogenesis.

    PubMed

    Saxena, Ritu; Pan, George; Dohm, Erik D; McDonald, Jay M

    2011-01-01

    Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLCγ2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3-4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (>30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing

  7. Osteoprotegerin induces podosome disassembly in osteoclasts through calcium, ERK, and p38 MAPK signaling pathways.

    PubMed

    Zhao, Hongyan; Liu, Xuezhong; Zou, Hui; Dai, Nannan; Yao, Lulian; Gao, Qian; Liu, Wei; Gu, Jianhong; Yuan, Yan; Bian, Jianchun; Liu, Zongping

    2015-02-01

    Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca(2+) concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30ng/mL) and M-CSF (25ng/mL). The cells were pretreated with Bapta-AM (5μM), U0126 (5μM), or SB202190 (10μM) for 30min, followed by 40ng/mL OPG for 3h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca(2+) concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca(2+) concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling

  8. Double Stranded RNA-Dependent Protein Kinase is Necessary for TNF-α-Induced Osteoclast Formation In Vitro and In Vivo.

    PubMed

    Shinohara, Hiroki; Teramachi, Jumpei; Okamura, Hirohiko; Yang, Di; Nagata, Toshihiko; Haneji, Tatsuji

    2015-09-01

    Double-stranded RNA-dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF-α plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF-α-induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF-α-induced osteoclastogenesis. The TNF-α-induced osteoclast differentiation in bone marrow-derived macrophages and RAW264.7 cells was markedly suppressed by the pretreatment of PKR inhibitor, 2-aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K, and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF-α-induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF-α-induced activation of NF-κB and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF-κB and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF-α-induced expression of NFATc1 and c-fos, master transcription factors in osteoclastogenesis. TNF-α-induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF-α-induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF-α-induced osteoclast differentiation in vitro and in vivo.

  9. Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification.

    PubMed

    Tonna, Stephen; Poulton, Ingrid J; Taykar, Farzin; Ho, Patricia W M; Tonkin, Brett; Crimeen-Irwin, Blessing; Tatarczuch, Liliana; McGregor, Narelle E; Mackie, Eleanor J; Martin, T John; Sims, Natalie A

    2016-02-15

    The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.

  10. Macrophage Migration Inhibitory Factor (MIF) Supports Homing of Osteoclast Precursors to Peripheral Osteolytic Lesions.

    PubMed

    Movila, Alexandru; Ishii, Takenobu; Albassam, Abdullah; Wisitrasameewong, Wichaya; Howait, Mohammed; Yamaguchi, Tsuguno; Ruiz-Torruella, Montserrat; Bahammam, Laila; Nishimura, Kazuaki; Van Dyke, Thomas; Kawai, Toshihisa

    2016-09-01

    By binding to its chemokine receptor CXCR4 on osteoclast precursor cells (OCPs), it is well known that stromal cell-derived factor-1 (SDF-1) promotes the chemotactic recruitment of circulating OCPs to the homeostatic bone remodeling site. However, the engagement of circulating OCPs in pathogenic bone resorption remains to be elucidated. The present study investigated a possible chemoattractant role of macrophage migration inhibitory factor (MIF), another ligand for C-X-C chemokine receptor type 4 (CXCR4), in the recruitment of circulating OCPs to the bone lytic lesion. To accomplish this, we used Csf1r-eGFP-knock-in (KI) mice to establish an animal model of polymethylmethacrylate (PMMA) particle-induced calvarial osteolysis. In the circulating Csf1r-eGFP+ cells of healthy Csf1r-eGFP-KI mice, Csf1r+/CD11b+ cells showed a greater degree of RANKL-induced osteoclastogenesis compared to a subset of Csf1r+/RANK+ cells in vitro. Therefore, Csf1r-eGFP+/CD11b+ cells were targeted as functionally relevant OCPs in the present study. Although expression of the two cognate receptors for MIF, CXCR2 and CXCR4, was elevated on Csf1r+/CD11b+ cells, transmigration of OCPs toward recombinant MIF in vitro was facilitated by ligation with CXCR4, but not CXCR2. Meanwhile, the level of PMMA-induced bone resorption in calvaria was markedly greater in wild-type (WT) mice compared to that detected in MIF-knockout (KO) mice. Interestingly, in contrast to the elevated MIF, diminished SDF-1 was detected in a particle-induced bone lytic lesion of WT mice in conjunction with an increased number of infiltrating CXCR4+ OCPs. However, such diminished SDF-1 was not found in the PMMA-injected calvaria of MIF-KO mice. Furthermore, stimulation of osteoblasts with MIF in vitro suppressed their production of SDF-1, suggesting that MIF can downmodulate SDF-1 production in bone tissue. Systemically administered anti-MIF neutralizing monoclonal antibody (mAb) inhibited the homing of CXCR4+ OCPs, as well as

  11. Impairment of osteoclastic bone resorption in rapidly growing female p47phox knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bone formation is dependent on the activity and differentiation of osteoblasts; whereas resorption of preexisting mineralized bone matrix by osteoclasts is necessary not only for bone development but also for regeneration and remodeling. Bone remodeling is a process in which osteoblasts and osteocla...

  12. The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts

    PubMed Central

    ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I.; Davison, Noel L.; de Vries, Teun J.; Everts, Vincent

    2015-01-01

    Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones—cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme

  13. Biphasic influence of PGE2 on the resorption activity of osteoclast-like cells derived from human peripheral blood monocytes and mouse RAW264.7 cells.

    PubMed

    Lutter, Anne-Helen; Hempel, Ute; Anderer, Ursula; Dieter, Peter

    2016-08-01

    Osteoclasts are large bone-resorbing cells of hematopoietic origin. Their main function is to dissolve the inorganic component hydroxyapatite and to degrade the organic bone matrix. Prostaglandin E2 (PGE2) indirectly affects osteoclasts by stimulating osteoblasts to release factors that influence osteoclast activity. The direct effect of PGE2 on osteoclasts is still controversial. To study the influence of PGE2 on osteoclast activity, human peripheral blood monocytes (hPBMC) and mouse RAW264.7 cells were cultured on osteoblast-derived extracellular matrix. hPBMC and RAW264.7 cells were differentiated by the addition of macrophage colony-stimulation factor and receptor activator of NFκB ligand and treated with PGE2 before and after differentiation induction. The pit area, an indicator of resorption activity, and the activity of tartrate-resistant acid phosphatase were dose-dependently inhibited when PGE2 was present ab initio, whereas the resorption activity remained unchanged when the cells were exposed to PGE2 from day 4 of culture. These results lead to the conclusion that PGE2 treatment inhibits only the differentiation of precursor osteoclasts whereas differentiated osteoclasts are not affected. PMID:27499447

  14. Approximating bone ECM: Crosslinking directs individual and coupled osteoblast/osteoclast behavior.

    PubMed

    Hwang, Mintai P; Subbiah, Ramesh; Kim, In Gul; Lee, Kyung Eun; Park, Jimin; Kim, Sang Heon; Park, Kwideok

    2016-10-01

    Osteoblast and osteoclast communication (i.e. osteocoupling) is an intricate process, in which the biophysical profile of bone ECM is an aggregate product of their activities. While the effect of microenvironmental cues on osteoblast and osteoclast maturation has been resolved into individual variables (e.g. stiffness or topography), a single cue can be limited with regards to reflecting the full biophysical scope of natural bone ECM. Additionally, the natural modulation of bone ECM, which involves collagenous fibril and elastin crosslinking via lysyl oxidase, has yet to be reflected in current synthetic platforms. Here, we move beyond traditional substrates and use cell-derived ECM to examine individual and coupled osteoblast and osteoclast behavior on a physiological platform. Specifically, preosteoblast-derived ECM is crosslinked with genipin, a biocompatible crosslinker, to emulate physiological lysyl oxidase-mediated ECM crosslinking. We demonstrate that different concentrations of genipin yield changes to ECM density, stiffness, and roughness while retaining biocompatibility. By approximating various bone ECM profiles, we examine how individual and coupled osteoblast and osteoclast behavior are affected. Ultimately, we demonstrate an increase in osteoblast and osteoclast differentiation on compact and loose ECM, respectively, and identify ECM crosslinking density as an underlying force in osteocoupling behavior. PMID:27376556

  15. Characterization of two types of osteoclasts from human peripheral blood monocytes

    SciTech Connect

    Yuasa, Kimitaka . E-mail: yuasa911@doc.medic.mie-u.ac.jp; Mori, Kouki; Ishikawa, Hitoshi; Sudo, Akihiro; Uchida, Atsumasa; Ito, Yasuhiko

    2007-05-04

    The two osteoclastogenesis pathways, receptor activator nuclear factor (NF)-{kappa}B ligand (RANKL)-mediated and fusion regulatory protein-1 (FRP-1)-mediated osteoclastogenesis, have recently been reported. There were significant differences in differentiation and activation mechanisms between the two pathways. When monocytes were cultured with FRP-1 without adding M-CSF, essential for the RANKL system, TRAP-positive polykaryocyte formation occurred. FRP-1-mediated osteoclasts formed larger pits on mineralized calcium phosphate plates than RANKL+M-CSF-mediated osteoclasts did. Lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were inclined to be single and isolated. However, osteoclasts induced by RANKL+M-CSF made many connected pits on dentin surfaces as if they crawled on there. Interestingly, FRP-1 osteoclastogenesis was enhanced by M-CSF/IL-1{alpha}, while chemotactic behavior to the dentin slices was not effected. There were differences in pH and concentration of HCO3- at culture endpoint and in adherent feature to dentin surfaces. Our findings indicate there are two types of osteoclasts with distinct properties.

  16. Choline Kinase β Mutant Mice Exhibit Reduced Phosphocholine, Elevated Osteoclast Activity, and Low Bone Mass*

    PubMed Central

    Kular, Jasreen; Tickner, Jennifer C.; Pavlos, Nathan J.; Viola, Helena M.; Abel, Tamara; Lim, Bay Sie; Yang, Xiaohong; Chen, Honghui; Cook, Robert; Hool, Livia C.; Zheng, Ming Hao; Xu, Jiake

    2015-01-01

    The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase β (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase β mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5′-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis. PMID:25451916

  17. Osteoclasts, key players in skeletal health and disease

    PubMed Central

    Novack, Deborah Veis; Mbalaviele, Gabriel

    2016-01-01

    Summary The differentiation of osteoclasts (OC) from early myeloid progenitors is a tightly regulated process that is modulated by a variety of mediators present in the bone microenvironment. Once generated, the function of mature OC depends on cytoskeletal features controlled by an αvβ3-containing complex at the bone-apposed membrane, and the secretion of protons and acid-protease cathepsin K. OC also have important interactions with other cells in the bone microenvironment including osteoblasts and immune cells. Dysregulation of OC differentiation and/or function can cause bone pathology. In fact, many components of OC differentiation and activation have been targeted therapeutically with great success. However, questions remain about the identity and plasticity of OC precursors, and the interplay between essential networks that control OC fate. In this review, we summarize the key principles of OC biology, and highlight recently uncovered mechanisms regulating OC development and function in homeostatic and disease states. PMID:27337470

  18. Phosphatidylinositol 3-kinase association with the osteoclast cytoskeleton, and its involvement in osteoclast attachment and spreading.

    PubMed

    Lakkakorpi, P T; Wesolowski, G; Zimolo, Z; Rodan, G A; Rodan, S B

    1997-12-15

    Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts. PMID:9434625

  19. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  20. Inhibition of osteoclast bone resorption activity through osteoprotegerin-induced damage of the sealing zone.

    PubMed

    Song, Ruilong; Gu, Jianhong; Liu, Xuezhong; Zhu, Jiaqiao; Wang, Qichao; Gao, Qian; Zhang, Jiaming; Cheng, Laiyang; Tong, Xishuai; Qi, Xinyi; Yuan, Yan; Liu, Zongping

    2014-09-01

    Bone remodeling is dependent on the dynamic equilibrium between osteoclast-mediated bone resorption and osteoblast-mediated osteogenesis. The sealing zone is an osteoclast-specific cytoskeletal structure, the integrity of which is critical for osteoclast-mediated bone resorption. To date, studies have focused mainly on the osteoprotegerin (OPG)‑induced inhibition of osteoclast differentiation through the OPG/receptor activator of the nuclear factor kappa-B ligand (RANKL)/RANK system, which affects the bone resorption of osteoclasts. However, the effects of OPG on the sealing zone have not been reported to date. In this study, the formation of the sealing zone was observed by Hoffman modulation contrast (HMC) microscopy and confocal laser scanning microscopy. The effects of OPG on the existing sealing zone and osteoclast-mediated bone resorption activity, as well as the regulatory role of genes involved in the formation of the sealing zone were examined by immunofluorescence staining, HMC microscopy, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis and scanning electron microscopy. The sealing zone was formed on day 5, with belt-like protuberances at the cell edge and scattered distribution of cell nuclei, but no filopodia. The sealing zone was intact in the untreated control group. However, defects in the sealing zone were observed in the OPG-treated group (20 ng/ml) and the structure was absent in the groups treated with 40 and 80 ng/ml OPG. The podosomes showed a scattered or clustered distribution between the basal surface of the osteoclasts and the well surface. Furthermore, resorption lacunae were not detected in the 20 ng/ml OPG-treated group, indicating the loss of osteoclast-mediated bone resorption activity. Treatment with OPG resulted in a significant decrease in the expression of Arhgef8/Net1 and DOCK5 Rho guanine nucleotide exchange factors (RhoGEFs), 10 of 18 RhoGTPases (RhoA, RhoB, cdc42v1, cdc42v2

  1. Molecular Profiling of Giant Cell Tumor of Bone and the Osteoclastic Localization of Ligand for Receptor Activator of Nuclear Factor κB

    PubMed Central

    Morgan, Teresa; Atkins, Gerald J.; Trivett, Melanie K.; Johnson, Sandra A.; Kansara, Maya; Schlicht, Stephen L.; Slavin, John L.; Simmons, Paul; Dickinson, Ian; Powell, Gerald; Choong, Peter F.M.; Holloway, Andrew J.; Thomas, David M.

    2005-01-01

    Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor κB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease. PMID:15972958

  2. How are osteoclasts induced to resorb bone?

    PubMed

    Chambers, T J; Fuller, K

    2011-12-01

    Although much is known about how osteoclasts are formed, we know little about how they are activated, or how they recognize bone as the substrate appropriate for resorption. Bone mineral is considered to be essential to this recognition process, but a "mineral receptor" has never been identified. Recently, we found that resorptive behavior, as judged by the formation of ruffled borders and actin rings, occurs on ordinary tissue culture substrates if they are first coated with vitronectin. Similarly, vitronectin-coated substrates induce osteoclasts to secrete tartrate-resistant acid phosphatase and to form podosome belts, and to make resorption trails in the protein that coat the substrate. The same applies to bone mineral, which only induces resorptive behavior if coated with vitronectin. In contrast, fibronectin has none of these effects, despite inducing adhesion and spreading. It appears that osteoclasts recognize bone as the substrate appropriate for resorption through the high affinity of vitronectin-receptor ligands for bone mineral. PMID:22172032

  3. Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1.

    PubMed

    Riihonen, Riikka; Nielsen, Søren; Väänänen, H Kalervo; Laitala-Leinonen, Tiina; Kwon, Tae-Hwan

    2010-05-01

    Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.

  4. Human iPSC-derived osteoblasts and osteoclasts together promote bone regeneration in 3D biomaterials

    PubMed Central

    Jeon, Ok Hee; Panicker, Leelamma M.; Lu, Qiaozhi; Chae, Jeremy J.; Feldman, Ricardo A.; Elisseeff, Jennifer H.

    2016-01-01

    Bone substitutes can be designed to replicate physiological structure and function by creating a microenvironment that supports crosstalk between bone and immune cells found in the native tissue, specifically osteoblasts and osteoclasts. Human induced pluripotent stem cells (hiPSC) represent a powerful tool for bone regeneration because they are a source of patient-specific cells that can differentiate into all specialized cell types residing in bone. We show that osteoblasts and osteoclasts can be differentiated from hiPSC-mesenchymal stem cells and macrophages when co-cultured on hydroxyapatite-coated poly(lactic-co-glycolic acid)/poly(L-lactic acid) (HA–PLGA/PLLA) scaffolds. Both cell types seeded on the PLGA/PLLA especially with 5% w/v HA recapitulated the tissue remodeling process of human bone via coupling signals coordinating osteoblast and osteoclast activity and finely tuned expression of inflammatory molecules, resulting in accelerated in vitro bone formation. Following subcutaneous implantation in rodents, co-cultured hiPSC-MSC/-macrophage on such scaffolds showed mature bone-like tissue formation. These findings suggest the importance of coupling matrix remodeling through osteoblastic matrix deposition and osteoclastic tissue resorption and immunomodulation for tissue development. PMID:27225733

  5. Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption.

    PubMed

    Su, Nan; Li, Xiaogang; Tang, Yubin; Yang, Jing; Wen, Xuan; Guo, Jingyuan; Tang, Junzhou; Du, Xiaolan; Chen, Lin

    2016-09-01

    Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled-hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts derived from wild-type and Fgfr3-deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3-deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3-deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research.

  6. Targeted overexpression of osteoactivin in cells of osteoclastic lineage promotes osteoclastic resorption and bone loss in mice.

    PubMed

    Sheng, Matilda H-C; Wergedal, Jon E; Mohan, Subburaman; Amoui, Mehran; Baylink, David J; Lau, K-H William

    2012-01-01

    This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.

  7. Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption.

    PubMed

    Su, Nan; Li, Xiaogang; Tang, Yubin; Yang, Jing; Wen, Xuan; Guo, Jingyuan; Tang, Junzhou; Du, Xiaolan; Chen, Lin

    2016-09-01

    Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled-hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts derived from wild-type and Fgfr3-deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3-deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3-deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research. PMID:26990430

  8. Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo

    PubMed Central

    Zhu, Meiling; Sun, Ben-hua; Saar, Katarzyna; Simpson, Christine; Troiano, Nancy; Dallas, Sarah L; Tiede-Lewis, LeAnn M; Nevius, Erin; Pereira, João P; Weinstein, Robert S; Tommasini, Steven M; Insogna, Karl L

    2016-01-01

    Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1flox/flox mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2−/− mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism. PMID:26496249

  9. Nano-topography sensing by osteoclasts.

    PubMed

    Geblinger, Dafna; Addadi, Lia; Geiger, Benjamin

    2010-05-01

    Bone resorption by osteoclasts depends on the assembly of a specialized, actin-rich adhesive 'sealing zone' that delimits the area designed for degradation. In this study, we show that the level of roughness of the underlying adhesive surface has a profound effect on the formation and stability of the sealing zone and the associated F-actin. As our primary model substrate, we use 'smooth' and 'rough' calcite crystals with average topography values of 12 nm and 530 nm, respectively. We show that the smooth surfaces induce the formation of small and unstable actin rings with a typical lifespan of approximately 8 minutes, whereas the sealing zones formed on the rough calcite surfaces are considerably larger, and remain stable for more than 6 hours. It was further observed that steps or sub-micrometer cracks on the smooth surface stimulate local ring formation, raising the possibility that similar imperfections on bone surfaces may stimulate local osteoclast resorptive activity. The mechanisms whereby the physical properties of the substrate influence osteoclast behavior and their involvement in osteoclast function are discussed.

  10. Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass

    PubMed Central

    Hirozane, Toru; Tohmonda, Takahide; Yoda, Masaki; Shimoda, Masayuki; Kanai, Yae; Matsumoto, Morio; Morioka, Hideo; Nakamura, Masaya; Horiuchi, Keisuke

    2016-01-01

    Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption. PMID:27677594

  11. Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

    PubMed

    Carron, C P; Meyer, D M; Engleman, V W; Rico, J G; Ruminski, P G; Ornberg, R L; Westlin, W F; Nickols, G A

    2000-06-01

    Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

  12. Divergent resorbability and effects on osteoclast formation of commonly used bone substitutes in a human in vitro-assay.

    PubMed

    Keller, Johannes; Brink, Silja; Busse, Björn; Schilling, Arndt F; Schinke, Thorsten; Amling, Michael; Lange, Tobias

    2012-01-01

    Bioactive bone substitute materials are a valuable alternative to autologous bone transplantations in the repair of skeletal defects. However, clinical studies have reported varying success rates for many commonly used biomaterials. While osteoblasts have traditionally been regarded as key players mediating osseointegration, increasing evidence suggests that bone-resorbing osteoclasts are of crucial importance for the longevity of applied biomaterials. As no standardized data on the resorbability of biomaterials exists, we applied an in vitro-assay to compare ten commonly used bone substitutes. Human peripheral blood mononuclear cells (PBMCs) were differentiated into osteoclasts in the co-presence of dentin chips and biomaterials or dentin alone (control) for a period of 28 days. Osteoclast maturation was monitored on day 0 and 14 by light microscopy, and material-dependent changes in extracellular pH were assessed twice weekly. Mature osteoclasts were quantified using TRAP stainings on day 28 and their resorptive activity was determined on dentin (toluidin blue staining) and biomaterials (scanning electron microscopy, SEM). The analyzed biomaterials caused specific changes in the pH, which were correlated with osteoclast multinuclearity (r = 0.942; p = 0.034) and activity on biomaterials (r = 0.594; p = 0.041). Perossal led to a significant reduction of pH, nuclei per osteoclast and dentin resorption, whereas Tutogen bovine and Tutobone human strikingly increased all three parameters. Furthermore, natural biomaterials were resorbed more rapidly than synthetic biomaterials leading to differential relative resorption coefficients, which indicate whether bone substitutes lead to a balanced resorption or preferential resorption of either the biomaterial or the surrounding bone. Taken together, this study for the first time compares the effects of widely used biomaterials on osteoclast formation and resorbability in an unbiased approach that may now aid in improving

  13. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

    PubMed

    Kulkarni, Rishikesh N; Voglewede, Philip A; Liu, Dawei

    2013-12-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP) and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1h of mechanical vibration with 20μm displacement at a frequency of 4Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells was determined after 1h of mechanical vibration, while protein production of the DC-STAMP was determined after 6h of postincubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduces DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation.

  14. Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

    PubMed

    Lymperi, Stefania; Ersek, Adel; Ferraro, Francesca; Dazzi, Francesco; Horwood, Nicole J

    2011-02-01

    Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

  15. The role of osteoclasts in bone tissue engineering.

    PubMed

    Detsch, Rainer; Boccaccini, Aldo R

    2015-10-01

    The success of scaffold-based bone regeneration approaches strongly depends on the performance of the biomaterial utilized. Within the efforts of regenerative medicine towards a restitutio ad integrum (i.e. complete reconstruction of a diseased tissue), scaffolds should be completely degraded within an adequate period of time. The degradation of synthetic bone substitute materials involves both chemical dissolution (physicochemical degradation) and resorption (cellular degradation by osteoclasts). Responsible for bone resorption are osteoclasts, cells of haematopoietic origin. Osteoclasts play also a crucial role in bone remodelling, which is essential for the regeneration of bone defects. There is, however, surprisingly limited knowledge about the detailed effects of osteoclasts on biomaterials degradation behaviour. This review covers the relevant fundamental knowledge and progress made in the field of osteoclast activity related to biomaterials used for bone regeneration. In vitro studies with osteoclastic precursor cells on synthetic bone substitute materials show that there are specific parameters that inhibit or enhance resorption. Moreover, analyses of the bone-material interface reveal that biomaterials composition has a significant influence on their degradation in contact with osteoclasts. Crystallinity, grain size, surface bioactivity and density of the surface seem to have a less significant effect on osteoclastic activity. In addition, the topography of the scaffold surface can be tailored to affect the development and spreading of osteoclast cells. The present review also highlights possible areas on which future research is needed and which are relevant to enhance our understanding of the complex role of osteoclasts in bone tissue engineering.

  16. Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages-An in vitro Study.

    PubMed

    Visagie, Amcois; Kasonga, Abe; Deepak, Vishwa; Moosa, Shaakirah; Marais, Sumari; Kruger, Marlena C; Coetzee, Magdalena

    2015-10-28

    Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.

  17. Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages—An in vitro Study

    PubMed Central

    Visagie, Amcois; Kasonga, Abe; Deepak, Vishwa; Moosa, Shaakirah; Marais, Sumari; Kruger, Marlena C.; Coetzee, Magdalena

    2015-01-01

    Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone. PMID:26516894

  18. Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures

    SciTech Connect

    Collins, D.A.; Chambers, T.J. )

    1991-02-01

    Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10{sup {minus} 6}-10{sup {minus} 9} M) and PGE1 (10{sup {minus} 6} - 10{sup {minus} 7} M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Bone resorption was increased (10{sup {minus} 7} M PGE2 and 10{sup {minus} 6} M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.

  19. Impaired signaling through the Fms-like tyrosine kinase 3 receptor increases osteoclast formation and bone damage in arthritis.

    PubMed

    Svensson, Mattias N D; Erlandsson, Malin C; Jonsson, Ing-Marie; Andersson, Karin M E; Bokarewa, Maria I

    2016-03-01

    Osteoclasts are bone-resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms-like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms-like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms-like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms-like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms-like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin-induced arthritis using mice that lack the gene for Flt3l (Flt3L(-/-)) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L(-/-) mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L(-/-) mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L(-/-) mice with Fms-like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L(-/-) mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms-like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

  20. Nicotine Affects Bone Resorption and Suppresses the Expression of Cathepsin K, MMP-9 and Vacuolar-Type H+-ATPase d2 and Actin Organization in Osteoclasts

    PubMed Central

    Tanaka, Hideki; Tanabe, Natsuko; Kawato, Takayuki; Nakai, Kumiko; Kariya, Taro; Matsumoto, Sakurako; Zhao, Ning; Motohashi, Masafumi; Maeno, Masao

    2013-01-01

    Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization. PMID

  1. MiR-142-3p is a RANKL-dependent inducer of cell death in osteoclasts

    PubMed Central

    Fordham, Jezrom B.; Guilfoyle, Katherine; Naqvi, Afsar Raza; Nares, Salvador

    2016-01-01

    MicroRNA are small, non-coding, single-stranded RNAs that are estimated to regulate ~60% of the human genome. MiRNA profiling of monocyte-to-osteoclast differentiation identified miR-142-3p as a miRNA that is significantly, differentially expressed throughout osteoclastogenesis. Enforced expression of miR-142-3p via transient transfection with miR-142-3p mimic inhibited cell-to-cell contact and fusion, decreased protein kinase C alpha expression, and ultimately reduced cell viability. miR-142-3p was also identified as significantly differentially expressed during monocyte-to-macrophage differentiation and overexpression of miR-142-3p prevented their conversion to osteoclasts. Furthermore, the inhibitory effect of miR-142-3p on osteoclastogenesis extended to the conversion of a third osteoclast precursor cell type- dendritic cells. These results demonstrate miR-142-3p to be a negative regulator of osteoclastogenesis from the 3 main precursor cell types: monocytes, macrophages and dendritic cells. Importantly, decreased survival was dependent upon both miR-142-3p expression and RANK-signaling, with no harmful effects detected in the absence of this combination. As such, miR-142-3p represents a novel target for the selective removal of osteoclasts by targeting of osteoclastogenic pathways. PMID:27113904

  2. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    SciTech Connect

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa . E-mail: baekjh@snu.ac.kr

    2006-08-18

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.

  3. Potential of Resveratrol Analogues as Antagonists of Osteoclasts and Promoters of Osteoblasts

    PubMed Central

    Boissy, Patrice; Abdallah, Basem M.; Hansen, Frederik Dagnaes; Erben, Reinhold G.; Savouret, Jean-Francois; Søe, Kent; Andersen, Thomas L.; Plesner, Torben; Delaisse, Jean-Marie

    2010-01-01

    The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations. In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss could not be detected. Based on their powerful antiresorptive activity in vitro, resveratrol analogues might be attractive modulators of bone remodeling. However, further studies are required to establish their efficacy in vivo. Electronic supplementary material The online version of this article (doi:10.1007/s00223-010-9399-3) contains supplementary material, which is available to authorized users. PMID:20842496

  4. Joint erosion in rheumatoid arthritis: interactions between tumour necrosis factor α, interleukin 1, and receptor activator of nuclear factor κB ligand (RANKL) regulate osteoclasts

    PubMed Central

    O', G; Ireland, D; Bord, S; Compston, J

    2004-01-01

    Background: Osteoclasts, specialised bone resorbing cells regulated by RANKL and M-CSF, are implicated in rheumatoid joint erosion. Lymphocyte-monocyte interactions activate bone resorption, this being attributed to tumour necrosis factor α (TNFα) and interleukin 1 ß (IL1ß) enhanced osteoblast expression of RANKL. In animal studies, TNF potently increases osteoclast formation in the presence of RANKL. RANKL-independent osteoclastogenesis also occurs, though IL1 is required for resorptive function in most studies. These inflammatory cytokines have a pivotal role in rheumatoid arthritis, Objective: To study the interactions of TNFα and IL1ß with RANKL, particularly the time course of the interactions and the role of lymphocytes. Method: Cultures of lymphocytes and monocytes (osteoclast precursors) or of purified CD14+ cells alone (osteoclast precursors) were exposed to various combinations of TNFα, RANKL, and IL1ß or the inhibitors osteoprotegerin, IL1 receptor antagonist, or neutralising antibodies to RANKL or to IL1. Osteoclastogenesis and resorptive activity were assessed on microscopy of dentine slices. Results: TNFα potently increased osteoclast proliferation/differentiation in the presence of RANKL. This effect was greatest when RANKL was present before but not after exposure of osteoclast precursor cells to TNFα. The resorptive activity of osteoclasts generated by TNFα in the absence of RANKL was critically dependent upon IL1, which was expressed by lymphocyte-monocyte interaction. Conclusion: TNFα potently enhances RANKL mediated osteoclast activity. Interactions between TNFα and IL1 also result in osteoclastic activity independently of RANKL. These findings will inform therapeutic approaches to the prevention of joint erosion in rheumatoid arthritis. PMID:15020327

  5. Acredinone C and the Effect of Acredinones on Osteoclastogenic and Osteoblastogenic Activity.

    PubMed

    Yeon, Jeong-Tae; Kim, Hiyoung; Kim, Kwang-Jin; Lee, Jihye; Won, Dong Hwan; Nam, Sang-Jip; Kim, Seong Hwan; Kang, Heonjoong; Son, Young-Jin

    2016-07-22

    A new inhibitor, acredinone C (1), of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was isolated from the culture broth of the fungus Acremonium sp. (F9A015) along with acredinones A (2) and B (3). The structure of acredinone C (1), which incorporates benzophenone and xanthone moieties, was established by the analyses of combined spectroscopic data including 1D and 2D NMR and MS. All of the acredinones studied efficiently inhibited the RANKL-induced formation of TRAP(+)-MNCs in a dose-dependent manner without any cytotoxicity up to 10 μM. Acredinone A showed dual activity in both osteoclast and osteoblast differentiation in vitro and good efficacy in an animal disease model of bone formation. PMID:27356092

  6. Calebin A downregulates osteoclastogenesis through suppression of RANKL signalling.

    PubMed

    Tyagi, Amit K; Prasad, Sahdeo; Majeed, Muhammed; Aggarwal, Bharat B

    2016-03-01

    Osteoporosis is a bone disease that is exacerbated by aging and age-associated chronic diseases such as cancer. Cancer-induced bone loss is usually treated with bisphosphonates or denosumab, an antibody against receptor activator of nuclear factor (NF)-κB ligand (RANKL). Because these drugs are expensive and have numerous side effects and high rates of toxicity, safer, more effective, and more affordable therapies for osteoporosis are still needed. We identified a compound, calebin A (CA), derived from turmeric (Curcuma longa) that affects osteoclastogenesis through modulation of the RANKL signalling pathway. The CA's effect on NF-κB activation was examined by electrophoretic mobility shift assay. Using mouse macrophages in vitro model, we found that CA suppressed RANKL-induced osteoclast differentiation of macrophages into osteoclasts, and downregulate RANKL-induced osteoclastogenesis-related marker gene expression, including NFATc-1, TRAP, CTR, and cathepsin K. CA also suppressed the osteoclastogenesis induced by multiple myeloma and breast cancer cells. This effect of CA was correlated with suppression of the phosphorylation and degradation of inhibitor of κB and, thus, inhibition of NF-κB activation. Furthermore, we found that an NF-κB-specific inhibitory peptide blocked RANKL-induced osteoclastogenesis, demonstrating that the NF-κB signalling pathway is mandatory for RANKL-induced osteoclastogenesis. Our results conclusively indicate that CA downmodulates the osteoclastogenesis induced by RANKL and by tumour cells through suppression of NF-κB pathway.

  7. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling

    PubMed Central

    ZHANG, YI; WANG, XI-CHAO; BAO, XING-FU; HU, MIN; YU, WEI-XIAN

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites. PMID:24348768

  8. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling.

    PubMed

    Zhang, Yi; Wang, Xi-Chao; Bao, Xing-Fu; Hu, Min; Yu, Wei-Xian

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites.

  9. Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

    PubMed

    Inoue, D; Santiago, P; Horne, W C; Baron, R

    1997-10-01

    Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

  10. Bisphosphonate-osteoclasts: changes in osteoclast morphology and function induced by antiresorptive nitrogen-containing bisphosphonate treatment in osteoporosis patients.

    PubMed

    Jobke, Björn; Milovanovic, Petar; Amling, Michael; Busse, Björn

    2014-02-01

    Osteoclasts are unique cells capable of bone resorption and therefore have become a major target in osteoporosis treatment strategies. Bisphosphonates suppress bone turnover via interference with the internal enzymatic cell system of osteoclasts leading to cytoskeletal disruption. This mechanism found its clinical relevance in reducing bone resorption, stabilizing bone mass and reducing fracture risk in osteoporosis patients. However, knowledge about specific in vivo changes in osteoclast cell morphology and function is still insufficient. We examined osteoclasts in 23 paired bone biopsies from osteoporosis patients (18 males, 5 females; age: 52.6±11.5yrs) under nitrogen-containing bisphosphonate administration with a mean treatment duration of three years. Formalin-fixed, undecalcified sections were assessed by qualitative and quantitative bone histomorphometry, where the osteoclast morphology, nuclei, distribution, location as well as resorption parameters were investigated to obtain information about cell function and viability. After three years of treatment, resorption parameters decreased significantly while the number of osteoclasts remained unchanged. Out of 23 patients, nine developed previously termed "giant-osteoclasts" with increased size, numerous nuclei (>10 nuclei/Oc) and oftentimes detachment from the bone surface. These cells frequently had pycnotic nuclei and other morphological signs suggestive of osteoclast apoptosis. Characteristic large-sized osteoclasts were uniquely found in patients treated with nitrogen-containing bisphosphonates, thus being clearly distinguishable from giant-osteoclasts in other bone disorders such as Paget disease, secondary hyperparathyroidism or osteopetrosis. The resorption indices of large-sized osteoclasts, specifically the eroded perimeter and erosion depth, revealed significantly reduced values but not an entirely inhibited resorption capability. Bisphosphonate-osteoclasts' viability and affinity to bone seem

  11. A Comprehensive Review of Immunoreceptor Regulation of Osteoclasts.

    PubMed

    Humphrey, Mary Beth; Nakamura, Mary C

    2016-08-01

    Osteoclasts require coordinated co-stimulation by several signaling pathways to initiate and regulate their cellular differentiation. Receptor activator for NF-κB ligand (RANKL or TNFSF11), a tumor necrosis factor (TNF) superfamily member, is the master cytokine required for osteoclastogenesis with essential co-stimulatory signals mediated by immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptors, DNAX-associated protein 12 kDa size (DAP12) and FcεRI gamma chain (FcRγ). The ITAM-signaling adaptors do not have an extracellular ligand-binding domain and, therefore, must pair with ligand-binding immunoreceptors to interact with their extracellular environment. DAP12 pairs with a number of different immunoreceptors including triggering receptor expressed on myeloid cells 2 (TREM2), myeloid DAP12-associated lectin (MDL-1), and sialic acid-binding immunoglobulin-type lectin 15 (Siglec-15); while FcRγ pairs with a different set of receptors including osteoclast-specific activating receptor (OSCAR), paired immunoglobulin receptor A (PIR-A), and Fc receptors. The ligands for many of these receptors in the bone microenvironment remain unknown. Here, we will review immunoreceptors known to pair with either DAP12 or FcRγ that have been shown to regulate osteoclastogenesis. Co-stimulation and the effects of ITAM-signaling have turned out to be complex, and now include paradoxical findings that ITAM-signaling adaptor-associated receptors can inhibit osteoclastogenesis and immunoreceptor tyrosine-based inhibitory motif (ITIM) receptors can promote osteoclastogenesis. Thus, co-stimulation of osteoclastogenesis continues to reveal additional complexities that are important in the regulatory mechanisms that seek to maintain bone homeostasis.

  12. Dihydroartemisinin prevents breast cancer-induced osteolysis via inhibiting both breast caner cells and osteoclasts

    PubMed Central

    Feng, Ming-Xuan; Hong, Jian-Xin; Wang, Qiang; Fan, Yong-Yong; Yuan, Chi-Ting; Lei, Xin-Huan; Zhu, Min; Qin, An; Chen, Hai-Xiao; Hong, Dun

    2016-01-01

    Bone is the most common site of distant relapse in breast cancer, leading to severe complications which dramatically affect the patients’ quality of life. It is believed that the crosstalk between metastatic breast cancer cells and osteoclasts is critical for breast cancer-induced osteolysis. In this study, the effects of dihydroartemisinin (DHA) on osteoclast formation, bone resorption, osteoblast differentiation and mineralization were initially assessed in vitro, followed by further investigation in a titanium-particle-induced osteolysis model in vivo. Based on the proved inhibitory effect of DHA on osteolysis, DHA was further applied to MDA-MB-231 breast cancer-induced mouse osteolysis model, with the underlying molecular mechanisms further investigated. Here, we verified for the first time that DHA suppressed osteoclast differentiation, F-actin ring formation and bone resorption through suppressing AKT/SRC pathways, leading to the preventive effect of DHA on titanium-particle-induced osteolysis without affecting osteoblast function. More importantly, we demonstrated that DHA inhibited breast tumor-induced osteolysis through inhibiting the proliferation, migration and invasion of MDA-MB-231 cells via modulating AKT signaling pathway. In conclusion, DHA effectively inhibited osteoclastogenesis and prevented breast cancer-induced osteolysis. PMID:26743690

  13. New methodology for evaluating osteoclastic activity induced by orthodontic load.

    PubMed

    Araújo, Adriele Silveira; Fernandes, Alline Birra Nolasco; Maciel, José Vinicius Bolognesi; Netto, Juliana de Noronha Santos; Bolognese, Ana Maria

    2015-01-01

    Orthodontic tooth movement (OTM) is a dynamic process of bone modeling involving osteoclast-driven resorption on the compression side. Consequently, to estimate the influence of various situations on tooth movement, experimental studies need to analyze this cell. Objectives The aim of this study was to test and validate a new method for evaluating osteoclastic activity stimulated by mechanical loading based on the fractal analysis of the periodontal ligament (PDL)-bone interface. Material and Methods The mandibular right first molars of 14 rabbits were tipped mesially by a coil spring exerting a constant force of 85 cN. To evaluate the actual influence of osteoclasts on fractal dimension of bone surface, alendronate (3 mg/Kg) was injected weekly in seven of those rabbits. After 21 days, the animals were killed and their jaws were processed for histological evaluation. Osteoclast counts and fractal analysis (by the box counting method) of the PDL-bone interface were performed in histological sections of the right and left sides of the mandible. Results An increase in the number of osteoclasts and in fractal dimension after OTM only happened when alendronate was not administered. Strong correlation was found between the number of osteoclasts and fractal dimension. Conclusions Our results suggest that osteoclastic activity leads to an increase in bone surface irregularity, which can be quantified by its fractal dimension. This makes fractal analysis by the box counting method a potential tool for the assessment of osteoclastic activity on bone surfaces in microscopic examination. PMID:25760264

  14. Reprint of: The Great Beauty of the osteoclast.

    PubMed

    Cappariello, Alfredo; Maurizi, Antonio; Veeriah, Vimal; Teti, Anna

    2014-11-01

    Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an "orchestrator" implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the '00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting "clastokines" that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast. PMID:25282390

  15. Regulation of osteoclast structure and function by FAK family kinases

    PubMed Central

    Ray, Brianne J.; Thomas, Keena; Huang, Cynthia S.; Gutknecht, Michael F.; Botchwey, Edward A.; Bouton, Amy H.

    2012-01-01

    Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAKΔmyeloid), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAKΔmyeloid mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells. PMID:22941736

  16. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    SciTech Connect

    Choi, Bongkun; Kang, Soon-Suk; Kang, Sang-Wook; Min, Bon-Hong; Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min; Song, Youngsup; Yoon, Seung-Yong; Chang, Eun-Ju

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  17. Osteoprotective Effects of IL-33/ST2 Link to Osteoclast Apoptosis.

    PubMed

    Lima, Izabella L A; Macari, Soraia; Madeira, Mila F M; Rodrigues, Letícia F D; Colavite, Priscila M; Garlet, Gustavo P; Soriani, Frederico M; Teixeira, Mauro M; Fukada, Sandra Y; Silva, Tarcília A

    2015-12-01

    The relevance of IL-33 and its receptor ST2 for bone remodeling is not well-defined. Our aim was to assess the role and underlying mechanisms of IL-33/ST2 in mechanically induced bone remodeling. BALB/c (wild type) and ST2 deficient (St2(-/-)) mice were subjected to mechanical loading in alveolar bone. Microtomography, histology, and real-time quantitative PCR were performed to analyze bone parameters, apoptosis and bone cell counts, and expression of bone remodeling markers, respectively. MC3T3-E1 osteoblastic cells and bone marrow cells were used to verify if mechanical force triggered IL-33 and ST2 expression as well as the effects of IL-33 on osteoclast differentiation and activity. Mechanical loading increased the expression of IL-33 and ST2 in alveolar bone in vivo and in osteoblastic cells in vitro. St2(-/-) mice had increased mechanical loading-induced bone resorption, number of osteoclasts, and expression of proresorptive markers. In contrast, St2(-/-) mice exhibited reduced numbers of osteoblasts and apoptotic cells in periodontium and diminished expression of osteoblast signaling molecules. In vitro, IL-33 treatment inhibited osteoclast differentiation and activity even in the presence of receptor activator of NF-κB ligand. IL-33 also increased the expression of pro-apoptotic molecules, including Bcl-2-associated X protein (BAX), cell-surface Fas receptor (FAS), FASL, FAS-associated death domain, tumor necrosis factor-related apoptosis-inducing ligand, and BH3 interacting-domain death (BID). Overall, these findings suggest that IL-33/ST2 have anti-osteoclastogenic effects and reduce osteoclast formation and activity by inducing their apoptosis.

  18. Advanced glycation end products biphasically modulate bone resorption in osteoclast-like cells.

    PubMed

    Li, Ziqing; Li, Chaohong; Zhou, Yuhuan; Chen, Weishen; Luo, Guotian; Zhang, Ziji; Wang, Haixing; Zhang, Yangchun; Xu, Dongliang; Sheng, Puyi

    2016-03-01

    Advanced glycation end products (AGEs) disturb bone remodeling during aging, and this process is accelerated in diabetes. However, their role in modulation of osteoclast-induced bone resorption is controversial, with some studies indicating that AGEs enhance bone resorption and others showing the opposite effect. We determined whether AGEs present at different stages of osteoclast differentiation affect bone resorption differently. Based on increased levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), we identified day 4 of induction as the dividing time of cell fusion stage and mature stage in RAW264.7 cell-derived osteoclast-like cells (OCLs). AGE-modified BSA (50-400 μg/ml) or control BSA (100 μg/ml) was then added at the beginning of each stage. Results showed that the presence of AGEs at the cell fusion stage reduced pit numbers, resorption area, and CTSK expression. Moreover, expression of receptor activator of nuclear factor-κB (RANK) as well as the number of TRAP-positive cells, nuclei per OCL, actin rings, and podosomes also decreased. However, the presence of AGEs at the mature stage enlarged the resorption area markedly and increased pit numbers slightly. Intriguingly, only the number of nuclei per OCL and podosomes increased. These data indicate that AGEs biphasically modulate bone resorption activity of OCLs in a differentiation stage-dependent manner. AGEs at the cell fusion stage reduce bone resorption dramatically, mainly via suppression of RANK expression in osteoclast precursors, whereas AGEs at the mature stage enhance bone resorption slightly, most likely by increasing the number of podosomes in mature OCLs.

  19. Effects of Silicon on Osteoclast Cell Mediated Degradation, In Vivo Osteogenesis and Vasculogenesis of Brushite Cement

    PubMed Central

    Vahabzadeh, Sahar; Roy, Mangal; Bose, Susmita

    2015-01-01

    Calcium phosphate cements (CPCs) are being widely used for treating small scale bone defects. Among the various CPCs, brushite (dicalcium phosphate dihydrate, DCPD) cement is widely used due to its superior solubility and ability to form new bone. In the present study, we have studied the physical, mechanical, osteoclast-like-cells differentiation and in vivo osteogenic and vasculogenic properties of silicon (Si) doped brushite cements. Addition of Si did not alter the phase composition of final product and regardless of Si level, all samples included β-tricalcium phosphate (β-TCP) and DCPD. 1.1 wt. % Si addition increased the compressive strength of undoped brushite cement from 4.78±0.21 MPa to 5.53±0.53 MPa, significantly. Cellular activity was studied using receptor activator of nuclear factor κβ ligand (RANKL) supplemented osteoclast-like-cells precursor RAW 264.7 cell. Phenotypic expressions of the cells confirmed successful differentiation of RAW264.7 monocytes to osteoclast-like-cells on undoped and doped brushite cements. An increased activity of osteoclast-like cells was noticed due to Si doping in the brushite cement. An excellent new bone formation was found in all cement compositions, with significant increase in Si doped brushite samples as early as 4 weeks post implantation in rat femoral model. After 4 weeks of implantation, no significant difference was found in blood vessel formation between the undoped and doped cements, however, a significant increase in vasculgenesis was found in 0.8 and 1.1 wt. % Si doped brushite cements after 8 weeks. These results show the influence of Si dopant on physical, mechanical, in vitro osteoclastogenesis and in vivo osteogenic and vasculogenic properties of brushite cements. PMID:26855779

  20. Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT

    PubMed Central

    Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Bueno-Silva, Bruno; DiRienzo, Joseph M.; Mayer, Marcia P. A.

    2016-01-01

    The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP+ cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP+ cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP+ cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis. PMID:27064424

  1. Substrate influences rat osteoclast morphology and expression of potassium conductances.

    PubMed

    Arkett, S A; Dixon, S J; Sims, S M

    1992-12-01

    1. We studied the electrophysiological properties of freshly isolated rat osteoclasts using the whole-cell configuration of the patch-clamp technique. Membrane currents were recorded from cells plated on three substates: dentine, type I collagen and glass. 2. Based on their morphology, we defined two categories of osteoclasts. 'Rounded' osteoclasts were dome-shaped and lacked lamellipodia. 'Spread' osteoclasts were flattened and had lamellipodia. The proportion of 'rounded' osteoclasts was significantly greater when cells were plated on dentine or type I collagen than when cells were plated on glass. 3. 'Spread' osteoclasts expressed an inwardly rectifying K+ conductance regardless of the substrate on which they were plated. 4. 'Rounded' osteoclasts, on all substrates, expressed a transient, outwardly rectifying conductance that was selective for K+ based on: reversal of deactivation tail currents at -74 mV; a 60 mV shift in tail current reversal potential for 10-fold change in [K+]o; and blockade of outward current by extracellular 4-aminopyridine, charybdotoxin, and intracellular Cs+. The outward K+ current had an activation threshold of approximately -50 mV, with half-activation at -29 mV. The current also exhibited voltage-dependent inactivation, with half-inactivation at approximately -40 mV. 5. Outward K+ current in 'rounded' osteoclasts was reduced when extracellular Ca2+ was removed and upon addition of Ni2+, but was unaffected by Cd2+ or nifedipine. 6. 'Rounded' osteoclasts had large whole-cell capacitance for their apparent surface area. Capacitance was positively correlated with K+ conductance. The additional surface membrane we detected through capacitance measurements may be the 'ruffled border' of actively resorbing osteoclasts. 7. We conclude that substrate influences the expression of osteoclast phenotype, as defined by morphology and K+ conductances. 'Rounded' osteoclasts express an outwardly rectifying K+ conductance, with no apparent inwardly

  2. Mycobacterium tuberculosis escapes from the phagosomes of infected human osteoclasts reprograms osteoclast development via dysregulation of cytokines and chemokines.

    PubMed

    Hoshino, Akiyoshi; Hanada, Sanshiro; Yamada, Hiroyuki; Mii, Shinji; Takahashi, Masahide; Mitarai, Satoshi; Yamamoto, Kenji; Manome, Yoshinobu

    2014-02-01

    Spinal tuberculosis is a condition characterized by massive resorption of the spinal vertebrae due to the infection with Mycobacterium tuberculosis (Mtb). However, the pathogenesis of spinal tuberculosis has not been established because it was almost completely eradicated by the establishment of antibiotic treatment in the mid-20th century. In this study, we investigated the inflammatory responses of human multinucleated osteoclasts infected with virulent Mtb strain. We found that the intracellular Mtb infection of multinuclear osteoclasts resulted in the rapid growth of Mtb and an osteolytic response, rather than inflammation. In response to Mtb infection, the mononuclear osteoclast precursors produced proinflammatory cytokines including tumor necrosis factor (TNF)-α, an intrinsic characteristic they share with macrophages. In contrast, highly fused multinucleated osteoclasts incapacitated the production of these cytokines. Instead, the intracellular Mtb inside multinuclear osteoclasts escaped from the endosome/phagosome, leading to a different pattern of osteoclast activation, with the production of chemokines such as CCL5, CCL17, CCL20, CCL22, CCL24, and CCL25. Moreover, intracellular infection with an avirulent Mtb strain resulted in diminished production of these chemokines. These findings indicate that intracellular Mtb infection in multinuclear osteoclasts reprograms osteoclast development via the dysregulation of cytokines and chemokines.

  3. Sulfated glycosaminoglycans support osteoblast functions and concurrently suppress osteoclasts.

    PubMed

    Salbach-Hirsch, Juliane; Ziegler, Nicole; Thiele, Sylvia; Moeller, Stephanie; Schnabelrauch, Matthias; Hintze, Vera; Scharnweber, Dieter; Rauner, Martina; Hofbauer, Lorenz C

    2014-06-01

    In order to improve bone regeneration, development and evaluation of new adaptive biomaterials is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major extracellular matrix (ECM) components of bone, and display osteogenic properties that are potentially useful for biomaterial applications. Using native and synthetic sulfate-modified GAGs, we manufactured artificial collagen/GAG ECM (aECMs) coatings, and evaluated how the presence of GAGs and their degree of sulfation affects the differentiation of murine mesenchymal stem cells to osteoblasts (OB) cultivated on these aECMs. GAG sulfation regulated osteogenesis at all key steps of OB development. Adhesion, but not migration, was diminished by 50% (P < 0.001). Proliferation and metabolic activity were slightly (P < 0.05) and cell death events strongly (P < 0.001) down-regulated due to a switch from proliferative to matrix synthesis state. When exposed to sulfated GAGs, OB marker genes, such as alkaline phosphatase, osteoprotegerin (OPG), and osteocalcin increased by up to 28-fold (P < 0.05) and calcium deposition up to 4-fold (P < 0.05). Furthermore, GAG treatment of OBs suppressed their ability to support osteoclast (OC) differentiation and resorption. In conclusion, GAG sulfation controls bone cell homeostasis by concurrently promoting osteogenesis and suppressing their paracrine support of OC functions, thus displaying a favorable profile on bone remodeling. Whether these cellular properties translate into improved bone regeneration needs to be validated in vivo.

  4. 5-Azacytidine-induced protein 2 (AZI2) regulates bone mass by fine-tuning osteoclast survival.

    PubMed

    Maruyama, Kenta; Fukasaka, Masahiro; Uematsu, Satoshi; Takeuchi, Osamu; Kondo, Takeshi; Saitoh, Tatsuya; Martino, Mikaël M; Akira, Shizuo

    2015-04-10

    5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival.

  5. 5-Azacytidine-induced Protein 2 (AZI2) Regulates Bone Mass by Fine-tuning Osteoclast Survival*

    PubMed Central

    Maruyama, Kenta; Fukasaka, Masahiro; Uematsu, Satoshi; Takeuchi, Osamu; Kondo, Takeshi; Saitoh, Tatsuya; Martino, Mikaël M.; Akira, Shizuo

    2015-01-01

    5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival. PMID:25691576

  6. Lysosomal pH Plays a Key Role in Regulation of mTOR Activity in Osteoclasts.

    PubMed

    Hu, Yingwei; Carraro-Lacroix, Luciene R; Wang, Andrew; Owen, Celeste; Bajenova, Elena; Corey, Paul N; Brumell, John H; Voronov, Irina

    2016-02-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation.

  7. Loss of Protein Kinase C-δ Protects against LPS-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption

    PubMed Central

    Khor, Ee Cheng; Abel, Tamara; Tickner, Jennifer; Chim, Shek Man; Wang, Cathy; Cheng, Taksum; Ng, Benjamin; Ng, Pei Ying; Teguh, Dian Astari; Kenny, Jacob; Yang, Xiaohong; Chen, Honghui; Nakayama, Keiichi I.; Nakayama, Keiko; Pavlos, Nathan; Zheng, Ming H.; Xu, Jiake

    2013-01-01

    Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (−α, −β and −γ), novel PKCs (−δ, −ε, −η and −θ) and atypical PKCs (−ι/λ and −ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC −α, −γ and −ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis. PMID:23951014

  8. Glucocorticoids mediate circadian timing in peripheral osteoclasts resulting in the circadian expression rhythm of osteoclast-related genes.

    PubMed

    Fujihara, Yuko; Kondo, Hisataka; Noguchi, Toshihide; Togari, Akifumi

    2014-04-01

    Circadian rhythms are prevalent in bone metabolism. However, the molecular mechanisms involved are poorly understood. Recently, we suggested that output signals from the suprachiasmatic nucleus (SCN) are transmitted from the master circadian rhythm to peripheral osteoblasts through β-adrenergic and glucocorticoid signaling. In this study, we examined how the master circadian rhythm is transmitted to peripheral osteoclasts and the role of clock gene in osteoclast. Mice were maintained under 12-hour light/dark periods and sacrificed at Zeitgeber times 0, 4, 8, 12, 16 and 20. mRNA was extracted from femur (cancellous bone) and analyzed for the expression of osteoclast-related genes and clock genes. Osteoclast-related genes such as cathepsin K (CTSK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) showed circadian rhythmicity like clock genes such as period 1 (PER1), PER2 and brain and muscle Arnt-like protein 1 (BMAL1). In an in vitro study, not β-agonist but glucocorticoid treatment remarkably synchronized clock and osteoclast-related genes in cultured osteoclasts. Chromatin immunoprecipitation (ChIP) assay showed the interaction between BMAL1 proteins and promoter region of CTSK and NFATc1. To examine whether endogenous glucocorticoids influence the osteoclast circadian rhythms, mice were adrenalectomized (ADX) and maintained under 12-hour light/dark periods at least two weeks before glucocorticoid injection. A glucocorticoid injection restarted the circadian expression of CTSK and NFATc1 in ADX mice. These results suggest that glucocorticoids mediate circadian timing to peripheral osteoclasts and osteoclast clock contributes to the circadian expression of osteoclast-related genes such as CTSK and NFATc1.

  9. Late stages of the synchronized macrophage fusion in osteoclast formation depend on dynamin.

    PubMed

    Verma, Santosh K; Leikina, Evgenia; Melikov, Kamran; Chernomordik, Leonid V

    2014-12-15

    Macrophage fusion that leads to osteoclast formation is one of the most important examples of cell-cell fusion in development, tissue homoeostasis and immune response. Protein machinery that fuses macrophages remains to be identified. In the present study, we explored the fusion stage of osteoclast formation for RAW macrophage-like murine cells and for macrophages derived from human monocytes. To uncouple fusion from the preceding differentiation processes, we accumulated fusion-committed cells in the presence of LPC (lysophosphatidylcholine) that reversibly blocks membrane merger. After 16 h, we removed LPC and observed cell fusion events that would normally develop within 16 h develop instead within 30-90 min. Thus, whereas osteoclastogenesis, generally, takes several days, our approach allowed us to focus on an hour in which we observe robust fusion between the cells. Complementing syncytium formation assay with a novel membrane merger assay let us study the synchronized fusion events downstream of a local merger between two plasma membranes, but before expansion of nascent membrane connections and complete unification of the cells. We found that the expansion of membrane connections detected as a growth of multinucleated osteoclasts depends on dynamin activity. In contrast, a merger between the plasma membranes of the two cells was not affected by inhibitors of dynamin GTPase. Thus dynamin that was recently found to control late stages of myoblast fusion also controls late stages of macrophage fusion, revealing an intriguing conserved mechanistic motif shared by diverse cell-cell fusion processes. PMID:25336256

  10. Generation of avian cells resembling osteoclasts from mononuclear phagocytes

    NASA Technical Reports Server (NTRS)

    Alvarez, J. I.; Teitelbaum, S. L.; Blair, H. C.; Greenfield, E. M.; Athanasou, N. A.; Ross, F. P.

    1991-01-01

    Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.

  11. Sclerostin stimulates osteocyte support of osteoclast activity by a RANKL-dependent pathway.

    PubMed

    Wijenayaka, Asiri R; Kogawa, Masakazu; Lim, Hui Peng; Bonewald, Lynda F; Findlay, David M; Atkins, Gerald J

    2011-01-01

    Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL) and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL) mRNA and down-regulated that of osteoprotegerin (OPG) mRNA, causing an increase in the RANK:OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC) were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold) compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKL-dependent manner.

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

    PubMed

    Kusakabe, A; Francis, M J

    1984-03-01

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

  13. [The role of osteoclasts in maintaining calcium homeostasis in newts].

    PubMed

    Berezovskaia, O P; Rodionova, N V; Grigorian, E N; Mitashov, V I

    1997-01-01

    The intensity of osteoclastic resorption and calcium contact were investigated in intact limb bones of the newts flown onboard of biosatellite Cosmos-2229 after amputation of their forelimbs and tail. X-ray microanalysis has shown an increase of calcium content in the bones on the 20th day after operation. Histological study revealed activated osteoclastic resorption on the inner surface of long bones. The newts exposed to weightlessness after the operation had the same level of bone mineralization as the operated ground control ones, but the number of polynuclear osteoclasts increased to a lesser extent.

  14. A comparison of osteoclast-rich and osteoclast-poor osteopetrosis in adult mice sheds light on the role of the osteoclast in coupling bone resorption and bone formation.

    PubMed

    Thudium, Christian S; Moscatelli, Ilana; Flores, Carmen; Thomsen, Jesper S; Brüel, Annemarie; Gudmann, Natasja Stæhr; Hauge, Ellen-Margrethe; Karsdal, Morten A; Richter, Johan; Henriksen, Kim

    2014-07-01

    Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts.

  15. Enhanced osteoclast-like cell functions on nanophase ceramics.

    PubMed

    Webster, T J; Ergun, C; Doremus, R H; Siegel, R W; Bizios, R

    2001-06-01

    Synthesis of tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits by osteoclast-like cells, the bone-resorbing cells, on nanophase (that is, material formulations with grain sizes less than 100nm) alumina and hydroxyapatite (HA) were investigated in the present in vitro study. Compared to conventional (that is, grain sizes larger than 100 nm) ceramics, synthesis of TRAP was significantly greater in osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 10 and 13 days, respectively. In addition, compared to conventional ceramics, formation of resorption pits was significantly greater by osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 7, 10, and 13 days, respectively. The present study, therefore, demonstrated, for the first time, enhanced osteoclast-like cell function on ceramic surfaces with nanometer-size surface topography.

  16. Aggressive Metaplastic Carcinoma of the Breast with Osteoclastic Giant Cells.

    PubMed

    Khong, Kathleen; Zhang, Yanhong; Tomic, Mary; Lindfors, Karen; Aminololama-Shakeri, Shadi

    2015-09-01

    Metaplastic carcinoma of the breast is an uncommon type of malignancy that is aggressive but can mimic other benign breast neoplastic processes on imaging. We present a case of a young female patient who presented with a rapidly progressing metaplastic carcinoma with osteoclastic giant cells subtype. There have been only very rare published reports of this pathologic subtype of metaplastic carcinoma containing osteoclastic giant cells.

  17. Aggressive Metaplastic Carcinoma of the Breast with Osteoclastic Giant Cells

    PubMed Central

    Khong, Kathleen; Zhang, Yanhong; Tomic, Mary; Lindfors, Karen; Aminololama-Shakeri, Shadi

    2015-01-01

    Metaplastic carcinoma of the breast is an uncommon type of malignancy that is aggressive but can mimic other benign breast neoplastic processes on imaging. We present a case of a young female patient who presented with a rapidly progressing metaplastic carcinoma with osteoclastic giant cells subtype. There have been only very rare published reports of this pathologic subtype of metaplastic carcinoma containing osteoclastic giant cells. PMID:26629304

  18. Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

    PubMed

    Rieman, D J; McClung, H A; Dodds, R A; Hwang, S M; Holmes, M W; James, I E; Drake, F H; Gowen, M

    2001-03-01

    Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae. PMID:11248658

  19. Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases.

    PubMed

    Endo-Munoz, Liliana; Cumming, Andrew; Rickwood, Danny; Wilson, Danielle; Cueva, Claudia; Ng, Charlotte; Strutton, Geoffrey; Cassady, A Ian; Evdokiou, Andreas; Sommerville, Scott; Dickinson, Ian; Guminski, Alexander; Saunders, Nicholas A

    2010-09-15

    We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.

  20. The "love-hate" relationship between osteoclasts and bone matrix.

    PubMed

    Rucci, Nadia; Teti, Anna

    2016-01-01

    Osteoclasts are unique cells that destroy the mineralized matrix of the skeleton. There is a "love-hate" relationship between the osteoclasts and the bone matrix, whereby the osteoclast is stimulated by the contact with the matrix but, at the same time, it disrupts the matrix, which, in turn, counteracts this disruption by some of its components. The balance between these concerted events brings about bone resorption to be controlled and to contribute to bone tissue integrity and skeletal health. The matrix components released by osteoclasts are also involved in the local regulation of other bone cells and in the systemic control of organismal homeostasis. Disruption of this regulatory loop causes bone diseases, which may end up with either reduced or increased bone mass, often associated with poor bone quality. Expanding the knowledge on osteoclast-to-matrix interaction could help to counteract these diseases and improve the human bone health. In this article, we will present evidence of the physical, molecular and regulatory relationships between the osteoclasts and the mineralized matrix, discussing the underlying mechanisms as well as their pathologic alterations and potential targeting.

  1. TRAP-positive osteoclast precursors mediate ROS/NO-dependent bactericidal activity via TLR4.

    PubMed

    Nishimura, Kazuaki; Shindo, Satoru; Movila, Alexandru; Kayal, Rayyan; Abdullah, Albassam; Savitri, Irma Josefina; Ikeda, Atsushi; Yamaguchi, Tsuguno; Howait, Mohammed; Al-Dharrab, Ayman; Mira, Abdulghani; Han, Xiaozhe; Kawai, Toshihisa

    2016-08-01

    Osteoclastogenesis was induced by RANKL stimulation in mouse monocytes to examine the possible bactericidal function of osteoclast precursors (OCp) and mature osteoclasts (OCm) relative to their production of NO and ROS. Tartrate-resistant acid phosphatase (TRAP)-positive OCp, but few or no OCm, phagocytized and killed Escherichia coli in association with the production of reactive oxygen species (ROS) and nitric oxide (NO). Phagocytosis of E. coli and production of ROS and NO were significantly lower in TRAP+ OCp derived from Toll-like receptor (TLR)-4 KO mice than that derived from wild-type (WT) or TLR2-KO mice. Interestingly, after phagocytosis, TRAP+ OCp derived from wild-type and TLR2-KO mice did not differentiate into OCm, even with continuous exposure to RANKL. In contrast, E. coli-phagocytized TRAP+ OCp from TLR4-KO mice could differentiate into OCm. Importantly, neither NO nor ROS produced by TRAP+ OCp appeared to be engaged in phagocytosis-induced suppression of osteoclastogenesis. These results suggested that TLR4 signaling not only induces ROS and NO production to kill phagocytized bacteria, but also interrupts OCm differentiation. Thus, it can be concluded that TRAP+ OCp, but not OCm, can mediate bactericidal activity via phagocytosis accompanied by the production of ROS and NO via TLR4-associated reprograming toward phagocytic cell type. PMID:27343691

  2. Osteoclast fusion is initiated by a small subset of RANKL-stimulated monocyte progenitors, which can fuse to RANKL-unstimulated progenitors.

    PubMed

    Levaot, Noam; Ottolenghi, Aner; Mann, Mati; Guterman-Ram, Gali; Kam, Zvi; Geiger, Benjamin

    2015-10-01

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

  3. Regulation of human umbilical cord blood-derived multi-potent stem cells by autogenic osteoclast-based niche-like structure

    SciTech Connect

    Sun, Bo; Jeong, Yun-Hyeok; Jung, Ji-Won; Seo, Kwangwon; Lee, Yong-Soon ||; Kang, Kyung-Sun ||. E-mail: kangpub@snu.ac.kr

    2007-05-25

    Stem cell niches provide the micro-environment for the development of stem cells. Under our culturing regimen, a kind of osteoclast-centralized structure supports the proliferation of MSCs, derived from human cord blood, once they reside on osteoclasts. MSCs in this structure expressed Oct4 which is a marker of embryonic stem cells. Floating daughter cells of MSCs colony showed abilities to differentiate into osteocyte, adipocyte, and neuronal progenitor cells. Compared with the easy senescence of MSCs without this niche-like structure in vitro, these results suggested that osteoclasts might play an important role the development and maintenance of Umbilical cord blood (UCB)-derived MSCs and might provide a means to expand UCB-MSCs in vitro, more easily, through a stem cell niche-like structure.

  4. Osteoactivin inhibition of osteoclastogenesis is mediated through CD44-ERK signaling

    PubMed Central

    Sondag, Gregory R; Mbimba, Thomas S; Moussa, Fouad M; Novak, Kimberly; Yu, Bing; Jaber, Fatima A; Abdelmagid, Samir M; Geldenhuys, Werner J; Safadi, Fayez F

    2016-01-01

    Osteoactivin is a heavily glycosylated protein shown to have a role in bone remodeling. Previous studies from our lab have shown that mutation in Osteoactivin enhances osteoclast differentiation but inhibits their function. To date, a classical receptor and a signaling pathway for Osteoactivin-mediated osteoclast inhibition has not yet been characterized. In this study, we examined the role of Osteoactivin treatment on osteoclastogenesis using bone marrow-derived osteoclast progenitor cells and identify a signaling pathway relating to Osteoactivin function. We reveal that recombinant Osteoactivin treatment inhibited osteoclast differentiation in a dose-dependent manner shown by qPCR, TRAP staining, activity and count. Using several approaches, we show that Osteoactivin binds CD44 in osteoclasts. Furthermore, recombinant Osteoactivin treatment inhibited ERK phosphorylation in a CD44-dependent manner. Finally, we examined the role of Osteoactivin on receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteolysis in vivo. Our data indicate that recombinant Osteoactivin inhibits RANKL-induced osteolysis in vivo and this effect is CD44-dependent. Overall, our data indicate that Osteoactivin is a negative regulator of osteoclastogenesis in vitro and in vivo and that this process is regulated through CD44 and ERK activation. PMID:27585719

  5. Osteoactivin inhibition of osteoclastogenesis is mediated through CD44-ERK signaling.

    PubMed

    Sondag, Gregory R; Mbimba, Thomas S; Moussa, Fouad M; Novak, Kimberly; Yu, Bing; Jaber, Fatima A; Abdelmagid, Samir M; Geldenhuys, Werner J; Safadi, Fayez F

    2016-01-01

    Osteoactivin is a heavily glycosylated protein shown to have a role in bone remodeling. Previous studies from our lab have shown that mutation in Osteoactivin enhances osteoclast differentiation but inhibits their function. To date, a classical receptor and a signaling pathway for Osteoactivin-mediated osteoclast inhibition has not yet been characterized. In this study, we examined the role of Osteoactivin treatment on osteoclastogenesis using bone marrow-derived osteoclast progenitor cells and identify a signaling pathway relating to Osteoactivin function. We reveal that recombinant Osteoactivin treatment inhibited osteoclast differentiation in a dose-dependent manner shown by qPCR, TRAP staining, activity and count. Using several approaches, we show that Osteoactivin binds CD44 in osteoclasts. Furthermore, recombinant Osteoactivin treatment inhibited ERK phosphorylation in a CD44-dependent manner. Finally, we examined the role of Osteoactivin on receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteolysis in vivo. Our data indicate that recombinant Osteoactivin inhibits RANKL-induced osteolysis in vivo and this effect is CD44-dependent. Overall, our data indicate that Osteoactivin is a negative regulator of osteoclastogenesis in vitro and in vivo and that this process is regulated through CD44 and ERK activation. PMID:27585719

  6. GDF11 decreases bone mass by stimulating osteoclastogenesis and inhibiting osteoblast differentiation

    PubMed Central

    Liu, Weiqing; Zhou, Liyan; Zhou, Chenchen; Zhang, Shiwen; Jing, Junjun; Xie, Liang; Sun, Ningyuan; Duan, Xiaobo; Jing, Wei; Liang, Xing; Zhao, Hu; Ye, Ling; Chen, Qianming; Yuan, Quan

    2016-01-01

    Osteoporosis is an age-related disease that affects millions of people. Growth differentiation factor 11 (GDF11) is a secreted member of the transforming growth factor beta (TGF-β) superfamily. Deletion of Gdf11 has been shown to result in a skeletal anterior–posterior patterning disorder. Here we show a role for GDF11 in bone remodelling. GDF11 treatment leads to bone loss in both young and aged mice. GDF11 inhibits osteoblast differentiation and also stimulates RANKL-induced osteoclastogenesis through Smad2/3 and c-Fos-dependent induction of Nfatc1. Injection of GDF11 impairs bone regeneration in mice and blocking GDF11 function prevents oestrogen-deficiency-induced bone loss and ameliorates age-related osteoporosis. Our data demonstrate that GDF11 is a previously unrecognized regulator of bone remodelling and suggest that GDF11 is a potential target for treatment of osteoporosis. PMID:27653144

  7. Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

    PubMed

    Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

    2016-09-01

    Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

  8. Pyrroloquinoline quinine inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos in mouse bone marrow cells and inhibits wear particle-induced osteolysis in mice.

    PubMed

    Kong, Lingbo; Yang, Chongfei; Yu, Lifeng; Smith, Wanli; Zhu, Shu; Zhu, Jinyu; Zhu, Qingsheng

    2013-01-01

    The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.

  9. Osteopontin Deficiency Produces Osteoclast Dysfunction Due to Reduced CD44 Surface Expression

    PubMed Central

    Chellaiah, M. A.; Kizer, N.; Biswas, R.; Alvarez, U.; Strauss-Schoenberger, J.; Rifas, L.; Rittling, S. R.; Denhardt, D. T.; Hruska, K. A.

    2003-01-01

    Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN−/− osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the αvβ3 integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling. PMID:12529435

  10. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression.

    PubMed

    Chellaiah, M A; Kizer, N; Biswas, R; Alvarez, U; Strauss-Schoenberger, J; Rifas, L; Rittling, S R; Denhardt, D T; Hruska, K A

    2003-01-01

    Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

  11. Megakaryocyte-mediated inhibition of osteoclast development.

    PubMed

    Kacena, Melissa A; Nelson, Tracy; Clough, Mary E; Lee, Sun-Kyeong; Lorenzo, Joseph A; Gundberg, Caren M; Horowitz, Mark C

    2006-11-01

    A growing body of evidence indicates that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. We previously identified a novel regulatory pathway that controls bone formation, which is mediated by MK. In vivo megakaryocytosis resulted in massive bone formation. The co-culture of MK with osteoblasts (OB) resulted in increased OB proliferation in vitro, by a mechanism that required direct cell-to-cell contact. Here, we examined a second MK-mediated pathway that regulates osteoclast (OC) development. We have begun examining the unique inhibitory effect of MK on OC development. Spleen or bone marrow (BM) cells from C57BL/6 mice, as a source of OC precursors, were cultured with M-CSF and RANKL to induce OC development. MK were prepared by culturing fetal liver cells with thrombopoietin and separating cells into MK and non-MK populations. MK were titrated into spleen cell cultures and OC were identified as tartrate-resistant acid phosphatase-positive giant cells with >3 nuclei. There was a significant, P < 0.001, up to 10-fold reduction in OC formed when MK were added to the spleen cell cultures. We determined that 30% (vol:vol) MK conditioned media (CM) were able to completely block OC development from precursors, whereas 3% MK CM resulted in up to a 10-fold reduction in OC development, P < 0.001. These data indicate that a soluble factor(s) was responsible, at least in part, for the inhibition. We examined MK CM for known inhibitors of OC formation, using ELISAs. IL-4 was undetectable in MK CM, whereas IL-10 and IFN-gamma levels were similar in MK and non-MK CM. TGFbeta-1 levels were increased 2-fold in MK CM compared to control CM but were not responsible for the inhibition in OC development. Although, we found a significant increase in the levels of osteoprotegerin (OPG) in MK CM, antibody neutralization studies, MK derived from OPG-deficient mice, and tandem mass spectrophotometry, all confirm that OPG was not responsible for the MK

  12. Osteoclast precursors in murine bone marrow express CD27 and are impeded in osteoclast development by CD70 on activated immune cells.

    PubMed

    Xiao, Yanling; Song, Ji-Ying; de Vries, Teun J; Fatmawati, Christien; Parreira, Diana B; Langenbach, Geerling E J; Babala, Nikolina; Nolte, Martijn A; Everts, Vincent; Borst, Jannie

    2013-07-23

    Osteoclasts (OCs) are bone-resorbing cells that are formed from hematopoietic precursors. OCs ordinarily maintain bone homeostasis, but they can also cause major pathology in autoimmune and inflammatory diseases. Under homeostatic conditions, receptor activator of nuclear factor kappa-B (RANK) ligand on osteoblasts drives OC differentiation by interaction with its receptor RANK on OC precursors. During chronic immune activation, RANK ligand on activated immune cells likewise drives pathogenic OC differentiation. We here report that the related TNF family member CD70 and its receptor CD27 can also mediate cross-talk between immune cells and OC precursors. We identified CD27 on a rare population (0.3%) of B220(-)c-Kit(+)CD115(+)CD11b(low) cells in the mouse bone marrow (BM) that are highly enriched for osteoclastogenic potential. We dissected this population into CD27(high) common precursors of OC, dendritic cells (DCs) and macrophages and CD27(low/neg) downstream precursors that could differentiate into OC and macrophages, but not DC. In a recombinant mouse model of chronic immune activation, sustained CD27/CD70 interactions caused an accumulation of OC precursors and a reduction in OC activity. These events were due to a CD27/CD70-dependent inhibition of OC differentiation from the OC precursors by BM-infiltrating, CD70(+)-activated immune cells. DC numbers in BM and spleen were increased, suggesting a skewing of the OC precursors toward DC differentiation. The impediment in OC differentiation culminated in a high trabecular bone mass pathology. Mice additionally presented anemia, leukopenia, and splenomegaly. Thus, under conditions of constitutive CD70 expression reflecting chronic immune activation, the CD27/CD70 system inhibits OC differentiation and favors DC differentiation.

  13. Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

    PubMed

    Swamy, N; Ghosh, S; Schneider, G B; Ray, R

    2001-01-01

    conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf. PMID:11255236

  14. Synergistic toxicity of gentamicin- and nanosilver-doped polymethylmethacrylate bone cement on primary human osteoclasts.

    PubMed

    Pauksch, Linda; Franke, Jörg; Schnettler, Reinhard; Lips, Katrin S

    2014-01-01

    Bacterial colonization of implant surfaces is a feared complication in surgery and orthopedics. Due to the increasing number of periprosthetic infections caused by multidrug-resistant microorganisms, new antibacterial coatings for biomaterials must be developed. The excellent antibacterial properties of silver nanoparticles (AgNPs) against multidrug-resistant bacteria, for example, have been repeatedly described. For this reason, we tested a nanosilver-doped polymethylmethacrylate (PMMA) bone cement and a nanosilver-coated titanium alloy regarding their influence on osteoclastogenesis of primary human peripheral blood mononuclear cells. Both implant variants did not inhibit osteoclast differentiation. Excellent cell attachment and unaltered podosomal structures were confirmed. Additionally, no induction of oxidative or endoplasmic reticulum stress could be observed. However, PMMA loaded with gentamicin and nanosilver inhibited preosteoclast fusion and further osteoclastogenesis. The material also led to decreased clathrin-dependent endocytosis as well as decreased levels of endoplasmic reticulum stress. Therefore, biomaterial functionalization with AgNPs did not disturb osteoclastogenesis, while addition of gentamicin reduced the cytocompatibility of nanosilver-doped materials towards human osteoclasts.

  15. Osteoclastic giant cell rich squamous cell carcinoma of the uterine cervix: a case report and review of the literature.

    PubMed

    Alemán-Meza, Lucía; Gómez-Macías, Gabriela Sofía; Barboza-Quintana, Oralia; Garza-Guajardo, Raquel; Loya-Solis, Abelardo

    2014-01-01

    Cervical carcinoma is the most common malignancy of the female genital tract and represents the second most common malignancy in women worldwide. Histologically 85 to 90% of cervical cancers are squamous cell carcinoma. Osteoclastic giant cell rich squamous cell carcinoma is an unusual histological variant of which only 4 cases have been reported. We present the case of a 49-year-old woman with a 6-month history of irregular vaginal bleeding. Examination revealed a 2.7 cm polypoid mass in the anterior lip of the uterine cervix. The patient underwent hysterectomy with bilateral salpingo-oophorectomy. Microscopically the tumor was composed of infiltrative nests of poorly differentiated nonkeratinizing squamous cell carcinoma. Interspersed in between these tumor cells were numerous osteoclastic giant cells with abundant eosinophilic cytoplasm devoid of nuclear atypia, hyperchromatism, or mitotic activity. Immunohistochemistry was performed; CK and P63 were strongly positive in the squamous component and negative in the osteoclastic giant cells, while CD68 and Vimentin were strongly positive in the giant cell population and negative in the squamous component. The patient received chemo- and radiotherapy for recurrent disease identified 3 months later on a follow-up CT scan; 7 months after the surgical procedure the patient is clinically and radiologically disease-free.

  16. Osteoclastic Giant Cell Rich Squamous Cell Carcinoma of the Uterine Cervix: A Case Report and Review of the Literature

    PubMed Central

    Alemán-Meza, Lucía; Gómez-Macías, Gabriela Sofía; Barboza-Quintana, Oralia; Garza-Guajardo, Raquel; Loya-Solis, Abelardo

    2014-01-01

    Cervical carcinoma is the most common malignancy of the female genital tract and represents the second most common malignancy in women worldwide. Histologically 85 to 90% of cervical cancers are squamous cell carcinoma. Osteoclastic giant cell rich squamous cell carcinoma is an unusual histological variant of which only 4 cases have been reported. We present the case of a 49-year-old woman with a 6-month history of irregular vaginal bleeding. Examination revealed a 2.7 cm polypoid mass in the anterior lip of the uterine cervix. The patient underwent hysterectomy with bilateral salpingo-oophorectomy. Microscopically the tumor was composed of infiltrative nests of poorly differentiated nonkeratinizing squamous cell carcinoma. Interspersed in between these tumor cells were numerous osteoclastic giant cells with abundant eosinophilic cytoplasm devoid of nuclear atypia, hyperchromatism, or mitotic activity. Immunohistochemistry was performed; CK and P63 were strongly positive in the squamous component and negative in the osteoclastic giant cells, while CD68 and Vimentin were strongly positive in the giant cell population and negative in the squamous component. The patient received chemo- and radiotherapy for recurrent disease identified 3 months later on a follow-up CT scan; 7 months after the surgical procedure the patient is clinically and radiologically disease-free. PMID:25587478

  17. High-fat diet-induced obesity stimulates ketone body utilization in osteoclasts of the mouse bone.

    PubMed

    Yamasaki, Masahiro; Hasegawa, Shinya; Imai, Masahiko; Takahashi, Noriko; Fukui, Tetsuya

    2016-04-29

    Previous studies have shown that high-fat diet (HFD)-induced obesity increases the acetoacetyl-CoA synthetase (AACS) gene expression in lipogenic tissue. To investigate the effect of obesity on the AACS gene in other tissues, we examined the alteration of AACS mRNA levels in HFD-fed mice. In situ hybridization revealed that AACS was observed in several regions of the embryo, including the backbone region (especially in the somite), and in the epiphysis of the adult femur. AACS mRNA expression in the adult femur was higher in HFD-fed mice than in normal-diet fed mice, but this increase was not observed in high sucrose diet (HSD)-induced obese mice. In addition, HFD-specific increases were observed in the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and interleukin (IL)-6 genes. Moreover, we detected higher AACS mRNA expression in the differentiated osteoclast cells (RAW 264), and found that AACS mRNA expression was significantly up-regulated by IL-6 treatment only in osteoclasts. These results indicate the novel function of the ketone body in bone metabolism. Because the abnormal activation of osteoclasts by IL-6 induces bone resorption, our data suggest that AACS and ketone bodies are important factors in the relationship between obesity and osteoporosis.

  18. Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption.

    PubMed

    Sun, Li; Iqbal, Jameel; Dolgilevich, Svetlana; Yuen, Tony; Wu, Xue-Bin; Moonga, Baljit S; Adebanjo, Olugbenga A; Bevis, Peter J R; Lund, Frances; Huang, Christopher L-H; Blair, Harry C; Abe, Etsuko; Zaidi, Mone

    2003-03-01

    We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions

  19. Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts.

    PubMed

    Nakayama, Takahiro; Mizoguchi, Toshihide; Uehara, Shunsuke; Yamashita, Teruhito; Kawahara, Ichiro; Kobayashi, Yasuhiro; Moriyama, Yoshinori; Kurihara, Saburo; Sahara, Noriyuki; Ozawa, Hidehiro; Udagawa, Nobuyuki; Takahashi, Naoyuki

    2011-12-01

    Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts. PMID:21983021

  20. Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

    PubMed Central

    Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-an; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

    2016-01-01

    Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation. PMID:26947250

  1. Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation.

    PubMed

    Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-An; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

    2016-01-01

    Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation.

  2. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro.

    PubMed

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N; Neutzsky-Wulff, Anita V; Geisler, Carsten; Karlsson, Lars; Söderström, Kalle

    2015-07-01

    Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions.

  3. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro

    PubMed Central

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N; Neutzsky-Wulff, Anita V; Geisler, Carsten; Karlsson, Lars; Söderström, Kalle

    2015-01-01

    Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions. PMID:25684021

  4. Calcium signal induced by mechanical perturbation of osteoclasts.

    PubMed

    Xia, S L; Ferrier, J

    1995-06-01

    Multinucleated osteoclasts from rabbit long bone, 1-6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca2+]i), as measured with the fluorescent indicator fluo-3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium-free), the incidence, the magnitude, and the duration of [Ca2+]i responses decreases with decreasing bathing [Ca2+]. Following mechanical perturbation, a thapsigargin-induced [Ca2+]i response has a lower magnitude than the thapsigargin-induced response without mechanical perturbation. In thapsigargin-pretreated osteoclasts the mechanical perturbation-induced rise in [Ca2+]i is larger and longer than in control cells. Ni2+ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd3+ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca2+]i signal via both a calcium influx and an internal calcium release.

  5. Inhibition of microtubule dynamics affects podosome belt formation during osteoclast induction.

    PubMed

    Ti, Yunfan; Zhou, Lingjun; Wang, Rui; Zhao, Jianning

    2015-03-01

    Osteoclast is the only cell that can degrade bone tissue in vivo. Recent studies have shown the important role of cytoskeleton dynamics in osteolysis and the formation of podosome belt in osteoclasts. This process is regulated by the dynamic microtubule (MT) network. We treated osteoclast precursor cells Raw264.7 with low concentration of nocodazole (10 nM) and antineoplastic drug taxol (10 nM) to block MT turnover, and used end binding protein 1 fused to GFP to track the movement of microtubules in induced osteoclasts. We show that low concentrations of nocodazole and taxol interfere with the formation of podosome belt, and reduce TRAP activity of induced osteoclasts. These results suggest that the effect of taxol on MT dynamics may be used clinically to reduce osteoclast activity and potentially prevent development of osteoporosis and other metabolic bone diseases.

  6. Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts.

    PubMed

    Kanehisa, J

    1989-01-01

    The reversible calcitonin (CT)-induced inhibition of osteoclastic activity has been studied to clarify the mechanisms responsible for the so-called "escape phenomenon." Osteoclasts disaggregated from neonatal rabbits were cultured on glass coverslips or thin bovine bone slices. Resorption activity was evaluated by using time-lapse recording and scanning electron microscopy. Addition of CT to the cultures caused most osteoclasts on glass surfaces to be immotile and contracted. From 1.5 h onward, in cultures with CT, osteoclasts started to escape from CT-induced quiescence independently of other cells. CT also prevented osteoclasts on bone slices from excavating bone while concomitant cell immobility occurred. Inhibited osteoclasts were able to regain apparent bone-resorbing potency only after resumption of cytoplasmic immobility. The resumption of bone resorption could begin as early as 9.7 h after CT addition. The observations indicate that CT-induced inhibition of osteoclastic bone resorption is associated with inhibition of cytoplasmic motility and that the "escape" phenomenon reflects resumption of activity of osteoclasts that were previously inhibited by CT action rather than the resportive activity of newly formed osteoclasts. PMID:2765310

  7. siRNA Knock-Down of RANK Signaling to Control Osteoclast-Mediated Bone Resorption

    PubMed Central

    Wang, Yuwei; Grainger, David W.

    2010-01-01

    Purpose To demonstrate the ability of small interfering (si)RNA targeting the cell receptor, RANK, to control osteoclast function in cultures of both primary and secondary osteoclasts and their precursor cells. Methods siRNA targeting RANK was transfected into both RAW264.7 and primary bone marrow cell cultures. RANK knock-down by siRNA and functional inhibition were assessed in both mature osteoclast and their precursor cell cultures. RANK mRNA message and protein expression after the transfections were analyzed by PCR and Western blot, respectively. Off-target effects were assessed. The inhibition of osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) assay, and subsequent bone resorption was determined by resorption pit assay. Results Both osteoclasts and osteoclast precursors can be targeted by siRNA in serum-containing media. Delivery of siRNA targeting RANK to both RAW 264.7 and primary bone marrow cell cultures produces short term repression of RANK expression without off-targeting effects, and significantly inhibits both osteoclast formation and bone resorption. Moreover, data support successful RANK knock-down by siRNA specifically in mature osteoclast cultures. Conclusions RANK is demonstrated to be an attractive target for siRNA control of osteoclast activity, with utility for development of new therapeutics for low bone mass pathologies or osteoporosis. PMID:20333451

  8. Correlating RANK ligand/RANK binding kinetics with osteoclast formation and function.

    PubMed

    Warren, Julia T; Zou, Wei; Decker, Corinne E; Rohatgi, Nidhi; Nelson, Christopher A; Fremont, Daved H; Teitelbaum, Steven L

    2015-11-01

    The interaction between Receptor Activator of NF-κB Ligand (RANKL) and its receptor RANK is essential for the differentiation and bone resorbing capacity of the osteoclast. Osteoprotegerin (OPG), a soluble homodimer, acts as a decoy receptor for RANKL and thus inhibits osteoclastogenesis. An imbalance in the RANKL/RANK/OPG axis, with decreased OPG and/or increased RANKL, is associated with diseases that favor bone loss, including osteoporosis. Recently, we established a yeast surface display system and screened libraries of randomly mutated RANKL proteins to identify mutations that abolish binding to OPG while preserving recognition of RANK. These efforts yielded several RANKL variants possessing substantially higher affinity for RANK compared to their wild-type (WT) counterpart. Using recombinant RANKL mutant proteins, we find those with increased affinity for RANK produce more robust signaling in osteoclast lineage cells and have greater osteoclastogenic potential. Our results are the first to document gain of function RANKL mutations. They indicate that the physiological RANKL/RANK interaction is not optimized for maximal signaling and function, perhaps reflecting the need to maintain receptor specificity within the tumor necrosis factor superfamily (TNFSF). Instead, we find, a biphasic relationship exists between RANKL/RANK affinity and osteoclastogenic capacity. In our panel of RANKL variants, this relationship is driven entirely by manipulation of the kinetic off-rate. Our structure-based and yeast surface display-derived insights into manipulating this critical signaling axis may aid in the design of novel anti-resorptive therapies as well as provide a paradigm for design of other receptor-specific TNF superfamily ligand variants. PMID:25864714

  9. Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis.

    PubMed

    Sućur, Alan; Katavić, Vedran; Kelava, Tomislav; Jajić, Zrinka; Kovačić, Natasa; Grčević, Danka

    2014-09-01

    The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes. The frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220(-)CD3(-)CD11b(-/lo)CD115(+)CD117(+)CX3CR1(+) and possibly also Ter119(-)CD11c(-)CD135(lo)Ly6C(+)RANK(-). In peripheral blood the OCP population bears the monocytoid phenotype B220(-)CD3(-)NK1.1(-)CD11b(+)Ly6C(hi)CD115(+)CX3CR1(+), presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3(-)CD19(-)CD56(-)), within immature monocyte subset (CD11b(+)CD14(+)CD16(-)), expressing receptors for M-CSF and RANKL (CD115(+)RANK(+)). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3(-)CD19(-)CD56(-)CD11b(+)CD14(+), and the disease activity score (DAS28) in the follow-up samples from patients with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption. Control of the activity and migratory behaviour of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.

  10. Identification of the Common Origins of Osteoclasts, Macrophages, and Dendritic Cells in Human Hematopoiesis.

    PubMed

    Xiao, Yanling; Zijl, Sebastiaan; Wang, Liqin; de Groot, Daniel C; van Tol, Maarten J; Lankester, Arjan C; Borst, Jannie

    2015-06-01

    Osteoclasts (OCs) originate from the myeloid cell lineage, but the successive steps in their lineage commitment are ill-defined, especially in humans. To clarify OC origin, we sorted cell populations from pediatric bone marrow (BM) by flow cytometry and assessed their differentiation potential in vitro. Within the CD11b(-)CD34(+)c-KIT(+) BM cell population, OC-differentiation potential was restricted to FLT3(+) cells and enriched in an IL3 receptor (R)α(high) subset that constituted less than 0.5% of total BM. These IL3Rα(high) cells also generated macrophages (MΦs) and dendritic cells (DCs) but lacked granulocyte (GR)-differentiation potential, as demonstrated at the clonal level. The IL3Rα(low) subset was re-defined as common progenitor of GR, MΦ, OC, and DC (GMODP) and gave rise to the IL3Rα(high) subset that was identified as common progenitor of MΦ, OC, and DC (MODP). Unbiased transcriptome analysis of CD11b(-)CD34(+)c-KIT(+)FLT3(+) IL3Rα(low) and IL3Rα(high) subsets corroborated our definitions of the GMODP and MODP and their developmental relationship.

  11. Novel immunostimulatory effects of osteoclasts and macrophages on human γδ T cells.

    PubMed

    Pappalardo, Angela; Thompson, Keith

    2015-02-01

    It has been widely reported that T cells are capable of influencing osteoclast formation and bone remodelling, yet relatively little is known of the reciprocal effects of osteoclasts for affecting T cell function and/or activity. In this study we investigated the effects of human osteoclasts on the function of γδ T cells, a subset of non-CD4(+) T cells implicated in a variety of inflammatory disease states. γδ T cells and CD4(+) T cells were isolated from peripheral blood of healthy volunteers and were co-cultured with autologous mature osteoclasts (generated by treatment with M-CSF and RANKL) before phenotypical and functional changes in the T cell populations were assessed. Macrophages, osteoclasts, and conditioned medium derived from macrophages or osteoclasts induced activation of γδ T cells, as determined by the expression of the early activation marker CD69. TNFα was a major mediator of this stimulatory effect on γδ T cells. Consistent with this stimulatory effect, osteoclasts augmented proliferation of IL-2-stimulated γδ T cells and also supported the survival of unstimulated γδ and CD4(+) T cells, although these effects required co-culture with osteoclasts. Co-culture with osteoclasts also increased the proportion of γδ T cells producing IFNγ, but did not modulate IFNγ or IL-17 production by CD4(+) T cells. We provide new insights into the in vitro interactions between human γδ T cells and osteoclasts/macrophages, and demonstrate that osteoclasts or their precursors are capable of influencing γδ T function both via the release of soluble factors and also through direct cell-cell interactions.

  12. IL-1β differently stimulates proliferation and multinucleation of distinct mouse bone marrow osteoclast precursor subsets.

    PubMed

    Cao, Yixuan; Jansen, Ineke D C; Sprangers, Sara; Stap, Jan; Leenen, Pieter J M; Everts, Vincent; de Vries, Teun J

    2016-09-01

    Osteoclasts are bone-resorbing cells and targets for treating bone diseases. Previously, we reported that distinct murine osteoclast precursor subsets, such as early blasts (CD31(hi) Ly-6C(-)), myeloid blasts (CD31(+) Ly-6C(+)), and monocytes (CD31(-) Ly-6C(hi)), respond differently to the osteoclastogenesis-inducing cytokines, macrophage colony-stimulating factor, and receptor activator for nuclear factor κB ligand. It is unknown, however, how these cell types respond to the osteoclast-stimulating inflammatory cytokine interleukin 1β. This study aims to investigate the effect of interleukin 1β on osteoclastogenesis derived from different mouse bone marrow precursors. Early blasts, myeloid blasts, and monocytes were sorted from mouse bone marrow cells using flow cytometry. Cells were cultured on plastic or on bone slices in the presence of macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand, without or with interleukin 1β (0.1-10 ng/ml). We found that interleukin 1β stimulated multinucleation and bone resorption of osteoclasts derived from the 3 precursors at different rates. The most large osteoclasts (>20 nuclei) and highest level of bone resorption (16.3%) was by myeloid blast-derived osteoclasts. Interleukin 1β particularly accelerated proliferation of early blasts and the most small osteoclasts (3-5 nuclei) formed on plastic. Life span varied among osteoclasts derived from different precursors: large osteoclasts (>2400 µm(2)) formed most rapidly (75 h) from myeloid blasts but had a short life span (30 h). Monocytes needed the longest time (95 h) for the generation of such large osteoclasts, but these cells had a longer life span (50 h). Our results indicate that the different bone marrow osteoclast precursors are differently stimulated by interleukin 1β with respect to proliferation, multinucleation, life span, and bone resorption. PMID:26957213

  13. Versatile Roles of V-ATPases Accessory Subunit Ac45 in Osteoclast Formation and Function

    PubMed Central

    Lin, Zhen; Pavlos, Nathan J.; Jiang, Qing; Xu, Jiake; Dai, Ke R.; Zheng, Ming H.

    2011-01-01

    Vacuolar-type H+-ATPases (V-ATPases) are macromolecular proton pumps that acidify intracellular cargos and deliver protons across the plasma membrane of a variety of specialized cells, including bone-resorbing osteoclasts. Extracellular acidification is crucial for osteoclastic bone resorption, a process that initiates the dissolution of mineralized bone matrix. While the importance of V-ATPases in osteoclastic resorptive function is well-defined, whether V-ATPases facilitate additional aspects of osteoclast function and/or formation remains largely obscure. Here we report that the V-ATPase accessory subunit Ac45 participates in both osteoclast formation and function. Using a siRNA-based approach, we show that targeted suppression of Ac45 impairs intracellular acidification and endocytosis, both are prerequisite for osteoclastic bone resorptive function in vitro. Interestingly, we find that knockdown of Ac45 also attenuates osteoclastogenesis owing to a reduced fusion capacity of osteoclastic precursor cells. Finally, in an effort to gain more detailed insights into the functional role of Ac45 in osteoclasts, we attempted to generate osteoclast-specific Ac45 conditional knockout mice using a Cathepsin K-Cre-LoxP system. Surprisingly, however, insertion of the neomycin cassette in the Ac45-FloxNeo mice resulted in marked disturbances in CNS development and ensuing embryonic lethality thus precluding functional assessment of Ac45 in osteoclasts and peripheral bone tissues. Based on these unexpected findings we propose that, in addition to its canonical function in V-ATPase-mediated acidification, Ac45 plays versatile roles during osteoclast formation and function. PMID:22087256

  14. Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

    PubMed

    Li, Jing; Zeng, Li; Xie, Juan; Yue, Zhiying; Deng, Huayun; Ma, Xueyun; Zheng, Chunbing; Wu, Xiushan; Luo, Jian; Liu, Mingyao

    2015-01-01

    Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis. PMID:26620037

  15. Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops

    PubMed Central

    Li, Jing; Zeng, Li; Xie, Juan; Yue, Zhiying; Deng, Huayun; Ma, Xueyun; Zheng, Chunbing; Wu, Xiushan; Luo, Jian; Liu, Mingyao

    2015-01-01

    Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca2+/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis. PMID:26620037

  16. Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus

    PubMed Central

    2010-01-01

    Background The purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts. Results Differentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP) activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP) activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively. Conclusions In conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells. PMID:20969794

  17. The association of bone and osteoclasts with vascular calcification.

    PubMed

    Han, Kum Hyun; Hennigar, Randolph A; O'Neill, W Charles

    2015-12-01

    The presence of bone tissue in calcified arteries may provide insights into the pathophysiology and potential reversibility of calcification, but the prevalence, distribution, and determinants of bone and osteoclasts in calcified arteries are unknown. Specimens of 386 arteries from lower limb amputations in 108 patients were examined retrospectively. Calcification was present in 282 arteries from 89 patients, which was medial in 64%, intimal in 9%, and both in 27%. Bone was present in 6% of arteries, essentially all of which were heavily calcified. Multiple sampling revealed that the true prevalence of bone in heavily calcified arteries was 25%. Bone was more common in medial rather than intimal calcifications (10% vs 3%, p=0.03) but did not vary with artery location (above vs below the knee). Heavily calcified arteries with bone were more likely to come from patients who were older (p=0.04), had diabetes (p=0.06), or were receiving warfarin (p=0.06), but there was no association with gender or renal failure. Bone was almost always adjacent to calcifications, along the periphery, but never within. Staining for the bone-specific proteins osteocalcin and osterix was noted in 20% and 45% of heavily calcified arteries without visible bone. Osteoclasts were present in 4.9% of arteries, all of which were heavily calcified and most of which contained bone. The frequent absence of bone in heavily calcified vessels and the histologic pattern strongly suggests a secondary rather than primary event. Recruitment of osteoclasts to vascular calcifications can occur but is rare, suggesting a limited capacity to reverse calcifications.

  18. Effect of calcitonin treatment on osteoclast counts in Paget's disease of bone.

    PubMed

    Williams, C P; Meachim, G; Taylor, W H

    1978-12-01

    Histological and biochemical changes during calcitonin treatment have been studied in 15 patients with Paget's disease of bone. For each pa