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Sample records for rankl-induced osteoclast differentiation

  1. Regulation of RANKL-induced osteoclastic differentiation by vascular cells.

    PubMed

    Tintut, Yin; Abedin, Moeen; Cho, John; Choe, Andrea; Lim, Jina; Demer, Linda L

    2005-08-01

    Vascular calcification is a regulated process of biomineralization resembling osteogenesis. Many bone-related factors, including resorptive osteoclast-like cells, although in low abundance, have been found in calcified atherosclerotic lesions. The regulatory mechanisms governing them in the vasculature, however, are not clear. Previously, we found that calcifying vascular cells (CVC), a subpopulation of bovine aortic smooth muscle cells (BASMC), undergo osteoblastic differentiation and form mineralized nodules. Since osteoblasts and marrow stromal preosteoblasts regulate osteoclastic differentiation in bone, we hypothesized that vascular cells also regulate differentiation of osteoclastic precursors in the artery wall. Peripheral blood monocytes, which are osteoclast precursors, were co-cultured with CVC or BASMC. Results showed that monocytes co-cultured with both of the vascular cells yielded fewer resorption pits than monocytes cultured alone. Furthermore, monocytes co-cultured with CVC had fewer resorption pits than those co-cultured with BASMC. Conditioned media from the vascular cells also inhibited resorptive activity of monocytes suggesting that the inhibitory effect was mediated in part by soluble factors. Compared with BASMC, CVC had lower mRNA expression for osteopontin, which promotes osteoclast attachment, but greater mRNA expression for the soluble inhibitory cytokine, IL-18. Increased osteoclastic differentiation was observed when neutralizing antibody to IL-18 receptor was added to the cultures of preosteoclasts with CVC conditioned media. Osteoprotegerin, another osteoclast inhibitory cytokine, was expressed at similar levels in both cultures. These results suggest that vascular cells inhibit osteoclastic differentiation, and that CVC have greater inhibitory effects than BASMC.

  2. The Transmembrane Adaptor Protein, Linker for Activation of T cells (LAT), Regulates RANKL-Induced Osteoclast Differentiation

    PubMed Central

    Kim, Kabsun; Kim, Jung Ha; Moon, Jang Bae; Lee, Jongwon; Kwak, Han bok; Park, Yong-Wook; Kim, Nacksung

    2012-01-01

    RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein we investigate the role of the transmembrane adaptor proteins in RANKL-induced osteoclastogenesis. LAT positively regulates osteoclast differentiation and is up-regulated by RANKL via c-Fos and NFATc1, whereas LAB and LIME act as negative modulators of osteoclastogenesis. In addition, silencing of LAT by RNA interference or overexpression of a LAT dominant negative in bone marrow-derived macrophage cells attenuates RANKL-induced osteoclast formation. Furthermore, LAT is involved in RANKL-induced PLCγ activation and NFATc1 induction. Thus, our data suggest that LAT acts as a positive regulator of RANKL-induced osteoclastogenesis. PMID:22382685

  3. 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.

  4. 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

  5. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26580592

  7. NADPH oxidase gp91phox contributes to RANKL-induced osteoclast differentiation by upregulating NFATc1

    PubMed Central

    Kang, In Soon; Kim, Chaekyun

    2016-01-01

    Bone-marrow derived monocyte-macrophages (BMMs) differentiate into osteoclasts by M-CSF along subsequent RANKL stimulation possibly in collaboration with many other unknown cytokines released by pre- or mature osteoblasts. The differentiation process requires receptor activator of nuclear factor kappa-B ligand (RANKL)/RANK signaling and reactive oxygen species (ROS) such as superoxide anion (O2•−). Gp91phox, a plasma membrane subunit of NADPH oxidase (Nox), is constitutively expressed in BMMs and plays a major role in superoxide anion production. In this study, we found that mice deficient in gp91phox (gp91phox−/−) showed defects in osteoclast differentiation. Femurs of these mice produced osteoclasts at about 70% of the levels seen in femurs from wild-type mice, and accordingly exhibited excessive bone density. This abnormal bone growth in the femurs of gp91phox−/− mice resulted from impaired osteoclast differentiation. In addition, gp91phox−/− mice were defective for RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). However, H2O2 treatment compensated for gp91phox deficiency in BMMs, almost completely rescuing osteoclast differentiation. Treating wild-type BMMs with antioxidants and superoxide inhibitors resulted in a differentiation defect resembling the phenotype of gp91phox−/− BMMs. Therefore, our results demonstrate that gp91phox-derived superoxide is important for promoting efficient osteoclast differentiation by inducing NFATc1 as a downstream signaling mediator of RANK. PMID:27897222

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

    PubMed Central

    2011-01-01

    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. PMID:21777482

  9. 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

  10. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation

    PubMed Central

    Kim, Jung Young; Kim, Yun-Kyung; Choi, Min Kyu; Oh, Jaemin; Kwak, Han Bok

    2012-01-01

    Objectives Osteoporosis is a disease of bones that is thought to result from an imbalance between bone resorption and bone formation. Although osteoporosis itself has no symptoms, osteoporosis caused by osteoclasts leads to an increased risk of fracture. Here we examined the effects of cornus officinalis on receptor activator of nuclear factor-kappaB ligand (RANKL)-mediated osteoclast differentiation. Methods We evaluated the effects of cornus officinalis on RANKL-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs) and performed a cytotoxicity assay, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot analysis. Results Cornus officinalis significantly inhibits RANKL-mediated osteoclast differentiation in a dose-dependent manner, but without cytotoxicity against BMMs. The mRNA expression of tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in BMMs treated with RANKL was considerably inhibited by cornus officinalis treatment. Also, cornus officinalis inhibits the protein expression of c-Fos and NFATc1. Cornus officinalis greatly inhibits RANKL-induced phosphorylation of p38 and c-JUN N-terminal kinase (JNK). Also, cornus officinalis significantly suppresses RANKL-induced degradation of I-κB. Conclusions Taken together, our results suggest that cornus officinalis may be a useful the treatment of osteoporosis. PMID:24524042

  12. 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

  13. 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.

  14. Alternative NF-κB Regulates RANKL-induced Osteoclast Differentiation and Mitochondrial Biogenesis via Independent Mechanisms

    PubMed Central

    Zeng, Rong; Faccio, Roberta; Novack, Deborah V

    2016-01-01

    Mitochondrial biogenesis, the generation of new mitochondrial DNA and proteins, has been linked to osteoclast (OC) differentiation and function. In this study we used mice with mutations in key alternative NF-κB pathway proteins, RelB and NIK, to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. OC precursors lacking either NIK or RelB, RANKL were unable to increase mitochondrial DNA or OxPhos protein expression, associated with lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL-induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. To deduce the mechanism for mitochondrial biogenesis defects in NIK- and RelB-deficient precursors, we examined expression of genes known to control this process. PGC-1β (Ppargc1b) expression, but not PGC-1α, PPRC1 or ERRα, was significantly reduced in RelB−/− and NIK−/− OCs. Because PGC-1β has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC-1β in RelB−/− cells, but surprisingly found that it did not affect differentiation, nor restore RANKL-induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB-deficient cells precludes mitochondrial biogenesis, we rescued RelB−/− differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB-deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB −/− was not restored. Furthermore, NFATc1 co-overexpression with PGC-1β, while allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB−/− OCs, by CTX-I levels. Thus, our results indicate that the alternative NF-κB pathway plays dual, but

  15. 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

  16. Inhibition of osteoclast differentiation and bone resorption by rotenone, through down-regulation of RANKL-induced c-Fos and NFATc1 expression.

    PubMed

    Kwak, Han Bok; Lee, Byeong Ki; Oh, Jaemin; Yeon, Jeong-Tae; Choi, Sik-Won; Cho, Hae Joong; Lee, Myeung Su; Kim, Jeong-Joong; Bae, Ji-Myung; Kim, Seong Hwan; Kim, Hun Soo

    2010-03-01

    Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease. The effect of rotenone in RANKL-induced osteoclast differentiation was examined in this study. Rotenone 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, TRAP, and OSCAR in RANKL-treated BMMs was inhibited by rotenone treatment. Rotenone strongly inhibited p38 and ERK phosphorylation and I-kappaB degradation in RANKL-stimulated BMMs, and did not inhibit JNK phosphorylation. Further, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by rotenone. Rotenone additionally inhibited the bone resorptive activity of differentiated osteoclasts. A lipopolysaccharide (LPS)-induced bone erosion study was also performed to assess the effects of rotenone in vivo. Mice treated with rotenone demonstrated marked attenuation of bone erosion based on Micro CT and histologic analysis of femurs. These results collectively suggested that rotenone demonstrated inhibitory effects on osteoclast differentiation in vitro and suppressed inflammatory bone loss in vivo. Rotenone may therefore serve as a useful drug in the prevention of bone loss.

  17. 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.

  18. 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.

  19. Treatment with hydrogen molecules prevents RANKL-induced osteoclast differentiation associated with inhibition of ROS formation and inactivation of MAPK, AKT and NF-kappa B pathways in murine RAW264.7 cells.

    PubMed

    Li, Dong-Zhu; Zhang, Qing-Xiang; Dong, Xiao-Xian; Li, Huai-Dong; Ma, Xin

    2014-09-01

    The bone protective effects of the hydrogen molecule (H2) have been demonstrated in several osteoporosis models while the underlying molecular mechanism has remained unclear. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. In this work, we evaluated the effects of incubation with H2 on receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation. We found that treatment with H2 prevented RANKL-induced osteoclast differentiation in RAW264.7 cells and BMMs. Treatment with H2 inhibits the ability to form resorption pits of BMMs stimulated by RANKL. Treatment with H2 reduced mRNA levels of osteoclast-specific markers including tartrate resistant acid phosphatase, calcitonin receptor, cathepsin K, metalloproteinase-9, carbonic anhydrase typeII, and vacuolar-type H(+)-ATPase. Treatment with H2 decreased intracellular reactive oxygen species (ROS) formation, suppressed NADPH oxidase activity, down-regulated Rac1 activity and Nox1 expression, reduced mitochondrial ROS formation, and enhanced nuclear factor E2-related factor 2 nuclear translocation and heme oxygenase-1 activity. In addition, treatment with H2 suppressed RANKL-induced expression of nuclear factor of activated T cells c1 and c-Fos. Furthermore, treatment with H2 suppressed NF-κB activation and reduced phosphorylation of p38, extracellular signal-regulated kinase, c-Jun-N-terminal kinase, and protein kinases B (AKT) stimulated with RANKL. In conclusion, hydrogen molecules prevented RANKL-induced osteoclast differentiation associated with inhibition of reactive oxygen species formation and inactivation of NF-κB, mitogen-activated protein kinase and AKT pathways.

  20. 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.

  1. 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

    2016-03-05

    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.

  2. 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.

  3. The Paired-box Homeodomain Transcription Factor Pax6 Binds to the Upstream Region of the TRAP Gene Promoter and Suppresses Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation*

    PubMed Central

    Kogawa, Masakazu; Hisatake, Koji; Atkins, Gerald J.; Findlay, David M.; Enoki, Yuichiro; Sato, Tsuyoshi; Gray, Peter C.; Kanesaki-Yatsuka, Yukiko; Anderson, Paul H.; Wada, Seiki; Kato, Naoki; Fukuda, Aya; Katayama, Shigehiro; Tsujimoto, Masafumi; Yoda, Tetsuya; Suda, Tatsuo; Okazaki, Yasushi; Matsumoto, Masahito

    2013-01-01

    Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption. PMID:23990468

  4. Beta-glycerophosphate accelerates RANKL-induced osteoclast formation in the presence of ascorbic acid.

    PubMed

    Noh, A Long Sae Mi; Yim, Mijung

    2011-03-01

    Despite numerous reports of the synergistic effects of beta-glycerophosphate and ascorbic acid in inducing the differentiation of osteoblasts, little is known about their roles in osteoclastic differentiation. Therefore, we investigated the effect of beta-glycerophosphate on osteoclastogenesis in the presence of ascorbic acid using primary mouse bone marrow cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). Beta-Glycerophosphate dose-dependently increased RANKL-induced osteoclast formation in the presence of ascorbic acid. This stimulatory effect was apparent when beta-glycerophosphate and ascorbic acid were only added during the late stages of the culture period, indicating that they influence later events in osteoclastic differentiation. While the combination of beta-glycerophosphate and ascorbic acid inhibited RANKL-stimulated activation of ERK and p38, and degradation of IkappaB, it increased the induction of c-Fos and NFATc1. In addition, beta-glycerophosphate and ascorbic acid together enhanced the induction of COX-2 following RANKL stimulation. Taken together, our data suggest that beta-glycerophosphate and ascorbic acid have synergistic effects on osteoclast formation, increasing RANKL-mediated induction of c-Fos, NFATc1 and COX-2 in osteoclast precursors.

  5. 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. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Differential intensity-dependent effects of pulsed electromagnetic fields on RANKL-induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells.

    PubMed

    Wang, Pan; Liu, Juan; Yang, Yuefan; Zhai, Mingming; Shao, Xi; Yan, Zedong; Zhang, Xuhui; Wu, Yan; Cao, Lu; Sui, Bingdong; Luo, Erping; Jing, Da

    2017-07-24

    Pulsed electromagnetic fields (PEMF) have been proven to be effective for promoting bone mass and regulating bone turnover both experimentally and clinically. However, the exact mechanisms for the regulation of PEMF on osteoclastogenesis as well as optical exposure parameters of PEMF on inhibiting osteoclastic activities and functions remain unclear, representing significant limitations for extensive scientific application of PEMF in clinics. In this study, RAW264.7 cells incubated with RANKL were exposed to 15 Hz PEMF (2 h/day) at various intensities (0.5, 1, 2, and 3 mT) for 7 days. We demonstrate that bone resorbing capacity was significantly decreased by 0.5 mT PEMF mainly by inhibiting osteoclast formation and maturation, but enhanced at 3 mT by promoting osteoclast apoptosis. Moreover, gene expression of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 was significantly decreased by 0.5 mT PEMF, but increased by 3 mT. Our findings reveal a significant intensity window for low-intensity PEMF in regulating bone resorption with diverse nature for modulating osteoclastogenesis and apoptosis. This study not only enriches our basic knowledge for the regulation of PEMF in osteoclastogenesis, but also may lead to more efficient and scientific clinical application of PEMF in regulating bone turnover and inhibiting osteopenia/osteoporosis. Bioelectromagnetics. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. 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.

  8. 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

  9. Alpha-1 antitrypsin inhibits RANKL-induced osteoclast formation and functions.

    PubMed

    Akbar, Mohammad Ahsanul; Nardo, David; Chen, Mong-Jen; Elshikha, Ahmed S; Ahamed, Rubina; Elsayed, Eslam M; Bigot, Claire; Holliday, Lexie Shannon; Song, Sihong

    2017-03-21

    Osteoporosis is a global public health problem affecting more than 200 million people worldwide. We previously showed that treatment with alpha-1 antitrypsin (AAT), a multifunctional protein with anti-inflammatory properties, mitigated bone loss in an ovariectomized mouse model. However, the underlying mechanisms of the protective effect of AAT on bone tissue are largely unknown. In this study, we investigated the effect of AAT on osteoclast formation and function in vitro. Our results showed that AAT dose-dependently inhibited the formation of RANKL (receptor activator of nuclear factor κB ligand) induced osteoclasts derived from mouse bone marrow macrophages/monocyte (BMM) lineage cells and the murine macrophage cell line, RAW 264.7 cells. In order to elucidate the possible mechanisms underlying this inhibition, we tested the effect of AAT on the gene expression of cell surface molecules, transcription factors, and cytokines associated with osteoclast formation. We showed that AAT inhibited M-CSF (macrophage colony-stimulating factor) induced cell surface RANK expression in osteoclast precursor cells. In addition, AAT inhibited RANKL-induced TNF-α production, cell surface CD9 expression, and dendritic cell-specific transmembrane protein (DC-STAMP) gene expression. Importantly, AAT treatment significantly inhibited osteoclast-associated mineral resorption. Together, these results uncovered new mechanisms for the protective effects of AAT and strongly support the notion that AAT has therapeutic potential for the treatment of osteoporosis.

  10. Attenuation of RANKL-induced Osteoclast Formation via p38-mediated NFATc1 Signaling Pathways by Extract of Euphorbia Lathyris L

    PubMed Central

    Kang, Ju-Hee; Lim, Hyojung; Jeong, Ji-Eun

    2016-01-01

    Background Osteoclasts are the only cell type capable of breaking down bone matrix, and its excessive activation is responsible for the development of bone-destructive diseases. Euphorbia lathyris L. (ELL) is an herbal plant that belongs to the Euphorbiaceae family. This study investigated the effects of the methanol extract of the aerial part of ELL on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and signaling pathways. Methods Osteoclasts were formed by co-culturing mouse bone marrow with osteoblasts or by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The expression level of mRNA was analyzed by real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR. Western blotting assays were performed to detect the expression or activation level of proteins. Results ELL inhibited RANKL-induced osteoclast formation without cytotoxicity. Furthermore, the RANKL-stimulated bone resorption was diminished by ELL. Mechanistically, ELL blocked the RANKL-triggered p38 mitogen-activated protein kinase (MAPK) phosphorylation, which resulted in the suppression of the expression of c-Fos and nuclear factor of activated T cells (NFATc1). In osteoblasts, ELL had little effect on the mRNA expression of RANKL and osteoprotegerin (OPG). Conclusions The present data suggest that ELL has an inhibitory effect on osteoclast differentiation and function via downregulation of the p38/c-Fos/NFATc1 signaling pathways. Thus, ELL could be useful for the treatment of bone diseases associated with excessive bone resorption. PMID:27965942

  11. 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.

  12. Water extract of the fruits of Alpinia oxyphylla inhibits osteoclast differentiation and bone loss.

    PubMed

    Ha, Hyunil; Shim, Ki-Shuk; Kim, Taesoo; Lee, Chung-Jo; Park, Ji Hyung; Kim, Han Sung; Ma, Jin Yeul

    2014-09-23

    Excessive bone resorption by osteoclasts causes pathological bone destruction, seen in various bone diseases. There is accumulating evidence that certain herbal extracts have beneficial effects on bone metabolism. The fruits of Alpinia oxyphylla has been traditionally used for the treatment of diarrhea and enuresis. In this study, we investigated the effects of water extract of the fruits of Alpinia oxyphylla (WEAO) on osteoclast differentiation and osteoclast-mediated bone destruction. For osteoclast differentiation assay, mouse bone marrow-derived macrophages (BMMs) were cultured in the presence of RANKL and M-CSF. RANKL signaling pathways and gene expression of transcription factors regulating osteoclast differentiation were investigated by real-time PCR and Western blotting. A constitutively active form of NFATc1 was retrovirally transduced into BMMs. Bone resorbing activity of mature osteoclast was examined on a plate coated with an inorganic crystalline calcium phosphate. The in vivo effect against bone destruction was assessed in a murine model of RANKL-induced osteoporosis by micro-computed tomography and bone metabolism marker analyses. WEAO dose-dependently inhibited RANKL-induced osteoclast differentiation from BMMs by targeting the early stages of osteoclast differentiation. WEAO inhibited RANKL-induced expression of NFATc1, the master regulator of osteoclast differentiation. Overexpression of a constitutively active form of NFATc1 blunted the inhibitory effect of WEAO on osteoclast differentiation, suggesting that NFATc1 is a critical target of the inhibitory action of WEAO. WEAO inhibited RANKL-induced expression of c-Fos, an upstream activator of NFATc1, by suppressing the classical NF-κB signaling pathway. WEAO also inhibited RANKL-induced down-regulation of Id2 and MafB, negative regulators of NFATc1. WEAO does not directly affect bone resorbing activity of mature osteoclasts. In accordance with the in vitro results, WEAO attenuated RANKL-induced

  13. 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

  14. Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-κB, ERK and JNK signaling pathways

    PubMed Central

    Feng, Wei; Liu, Hongrui; Luo, Tingting; Liu, Di; Du, Juan; Sun, Jing; Wang, Wei; Han, Xiuchun; Yang, Kaiyun; Guo, Jie; Amizuka, Norio; Li, Minqi

    2017-01-01

    Interleukin (IL)-6 is known to indirectly enhance osteoclast formation by promoting receptor activator of nuclear factor kappa-B ligand (RANKL) production by osteoblastic/stromal cells. However, little is known about the direct effect of IL-6 on osteoclastogenesis. Here, we determined the direct effects of IL-6 and its soluble receptor (sIL-6R) on RANKL-induced osteoclast formation by osteoclast precursors in vitro. We found IL-6/sIL-6R significantly promoted and suppressed osteoclast differentiation induced by low- (10 ng/ml) and high-level (50 ng/ml) RANKL, respectively. Using a bone resorption pit formation assay, expression of osteoclastic marker genes and transcription factors confirmed differential regulation of RANKL-induced osteoclastogenesis by IL-6/sIL-6R. Intracellular signaling transduction analysis revealed IL-6/sIL-6R specifically upregulated and downregulated the phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), ERK (extracellular signal–regulated kinase) and JNK (c-Jun N-terminal kinase) induced by low- and high level RANKL, respectively. Taken together, our findings demonstrate that IL-6/sIL-6R differentially regulate RANKL-induced osteoclast differentiation and activity through modulation of NF-κB, ERK and JNK signaling pathways. Thus, IL-6 likely plays a dual role in osteoclastogenesis either as a pro-resorption factor or as a protector of bone, depending on the level of RANKL within the local microenvironment. PMID:28128332

  15. 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

  16. RANKL induces heterogeneous DC-STAMP(lo) and DC-STAMP(hi) osteoclast precursors of which the DC-STAMP(lo) precursors are the master fusogens.

    PubMed

    Mensah, Kofi A; Ritchlin, Christopher T; Schwarz, Edward M

    2010-04-01

    Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL-induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC-STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation-Western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC-STAMP in non-denaturing and denaturing conditions, respectively, with greater sensitivity versus rabbit anti-sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100-fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC-STAMP(lo) and DC-STAMP(hi) cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC-STAMP(hi) cells to purified DC-STAMP(lo) cultures produced larger, more nucleated OC vs. pure DC-STAMP(lo) cultures. RT-qPCR analysis of these two populations showed that OC markers (Trap and Oc-stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC-STAMP(lo) vs. DC-STAMP(hi) cells. Collectively, these results demonstrate that DC-STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC-STAMP internalization in some OCP, and these DC-STAMP(lo) cells display the "master fusogen" phenotype. In contrast, DC-STAMP(hi) OCP can only act as mononuclear donors. J. Cell. Physiol. 223: 76-83, 2010. (c) 2009 Wiley-Liss, Inc.

  17. RANKL induces heterogeneous DC-STAMPlo and DC-STAMPhi osteoclast precursors of which the DC-STAMPlo precursors are the master fusogens

    PubMed Central

    Mensah, Kofi A.; Ritchlin, Christopher T.; Schwarz, Edward M.

    2009-01-01

    Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL-induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC-STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation-western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC-STAMP in non-denaturing and denaturing conditions respectively, with greater sensitivity vs. rabbit anti-sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100-fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC-STAMPlo and DC-STAMPhi cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC-STAMPhi cells to purified DC-STAMPlo cultures produced larger, more nucleated OC vs. pure DC-STAMPlo cultures. RT-qPCR analysis of these two populations showed that OC markers (Trap and Oc-stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC-STAMPlo vs. DC-STAMPhi cells. Collectively, these results demonstrate that DC-STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC-STAMP internalization in some OCP, and these DC-STAMPlo cells display the “master fusogen” phenotype. In contrast, DC-STAMPhi OCP can only act as mononuclear donors. PMID:20039274

  18. 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

    2014-03-07

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Ginsenoside Re Inhibits Osteoclast Differentiation in Mouse Bone Marrow-Derived Macrophages and Zebrafish Scale Model

    PubMed Central

    Park, Chan-Mi; Kim, Hye-Min; Kim, Dong Hyun; Han, Ho-Jin; Noh, Haneul; Jang, Jae-Hyuk; Park, Soo-Hyun; Chae, Han-Jung; Chae, Soo-Wan; Ryu, Eun Kyoung; Lee, Sangku; Liu, Kangdong; Liu, Haidan; Ahn, Jong-Seog; Kim, Young Ock; Kim, Bo-Yeon; Soung, Nak-Kyun

    2016-01-01

    Ginsenosides, which are the active materials of ginseng, have biological functions that include anti-osteoporotic effects. Aqueous ginseng extract inhibits osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL). Aqueous ginseng extract produces chromatography peaks characteristic of ginsenosides. Among these peaks, ginsenoside Re is a major component. However, the preventive effects of ginsenoside Re against osteoclast differentiation are not known. We studied the effect of ginsenoside Re on osteoclast differentiation, RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, and formation of multinucleated osteoclasts in vitro. Ginsenoside Re hampered osteoclast differentiation in a dose-dependent manner. In an in vivo zebrafish model, aqueous ginseng extract and ginsenoside Re had anti-osteoclastogenesis effects. These findings suggest that both aqueous ginseng extract and ginsenoside Re prevent bone resorption by inhibiting osteoclast differentiation. Ginsenoside Re could be important for promoting bone health. PMID:27927007

  20. Ginsenoside Re Inhibits Osteoclast Differentiation in Mouse Bone Marrow-Derived Macrophages and Zebrafish Scale Model.

    PubMed

    Park, Chan-Mi; Kim, Hye-Min; Kim, Dong Hyun; Han, Ho-Jin; Noh, Haneul; Jang, Jae-Hyuk; Park, Soo-Hyun; Chae, Han-Jung; Chae, Soo-Wan; Ryu, Eun Kyoung; Lee, Sangku; Liu, Kangdong; Liu, Haidan; Ahn, Jong-Seog; Kim, Young Ock; Kim, Bo-Yeon; Soung, Nak-Kyun

    2016-12-01

    Ginsenosides, which are the active materials of ginseng, have biological functions that include anti-osteoporotic effects. Aqueous ginseng extract inhibits osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL). Aqueous ginseng extract produces chromatography peaks characteristic of ginsenosides. Among these peaks, ginsenoside Re is a major component. However, the preventive effects of ginsenoside Re against osteoclast differentiation are not known. We studied the effect of ginsenoside Re on osteoclast differentiation, RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity, and formation of multinucleated osteoclasts in vitro. Ginsenoside Re hampered osteoclast differentiation in a dose-dependent manner. In an in vivo zebrafish model, aqueous ginseng extract and ginsenoside Re had anti-osteoclastogenesis effects. These findings suggest that both aqueous ginseng extract and ginsenoside Re prevent bone resorption by inhibiting osteoclast differentiation. Ginsenoside Re could be important for promoting bone health.

  1. 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.

  2. Echinocystic acid inhibits RANKL-induced osteoclastogenesis by regulating NF-κB and ERK signaling pathways

    SciTech Connect

    Yang, Jian-hui; Li, Bing; Wu, Qiong; Lv, Jian-guo; Nie, Hui-Yong

    2016-09-02

    Receptor activator of nuclear factor-κB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Echinocystic acid (EA), a pentacyclic triterpene isolated from the fruits of Gleditsia sinensis Lam, was reported to prevent reduction of bone mass and strength and improve the cancellous bone structure and biochemical properties in ovariectomy rats. However, the molecular mechanism of EA on the osteoclast formation has not been reported. The purpose of this study was to investigate the effects and mechanism of EA on RANKL-induced osteoclastogenesis. Our results showed that EA inhibited the formation of osteoclast, as well as the expression of osteoclastogenesis-related marker proteins in bone marrow macrophages (BMMs). At molecular levels, EA inhibited RANKL-induced NF-κB activation and ERK phosphorylation in BMMs. In conclusion, the present study demonstrated that EA can suppress osteoclastogenesis in vitro. Moreover, we clarified that these inhibitory effects of EA occur through suppression of NF-κB and ERK activation. Therefore, EA may be a potential agent in the treatment of osteoclast-related diseases such as osteoporosis. - Highlights: • EA inhibited the formation of osteoclast in BMMs. • EA inhibits the expression of osteoclastogenesis-related marker proteins in BMMs. • EA inhibits RANKL-induced NF-κB activation in BMMs. • EA inhibits RANKL-induced ERK phosphorylation in BMMs.

  3. 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.

  4. 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.

  5. The protein kinase 2 inhibitor CX-4945 regulates osteoclast and osteoblast differentiation in vitro.

    PubMed

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

    2013-11-01

    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.

  6. Bajijiasu Abrogates Osteoclast Differentiation via the Suppression of RANKL Signaling Pathways through NF-κB and NFAT

    PubMed Central

    Hong, Guoju; Zhou, Lin; Shi, Xuguang; He, Wei; Wang, Haibin; Wei, Qiushi; Chen, Peng; Qi, Longkai; Tickner, Jennifer; Lin, Li; Xu, Jiake

    2017-01-01

    Pathological osteolysis is commonly associated with osteoporosis, bone tumors, osteonecrosis, and chronic inflammation. It involves excessive resorption of bone matrix by activated osteoclasts. Suppressing receptor activator of NF-κB ligand (RANKL) signaling pathways has been proposed to be a good target for inhibiting osteoclast differentiation and bone resorption. Bajijiasu—a natural compound derived from Morinda officinalis F. C. How—has previously been shown to have anti-oxidative stress property; however, its effect and molecular mechanism of action on osteoclastogenesis and bone resorption remains unclear. In the present study, we found that Bajijiasu dose-dependently inhibited RANKL-induced osteoclast formation and bone resorption from 0.1 mM, and reached half maximal inhibitory effects (IC50) at 0.4 mM without toxicity. Expression of RANKL-induced osteoclast specific marker genes including cathepsin K (Ctsk), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP), vacuolar-type H+-ATPase V0 subunit D2 (V-ATPase d2), and (matrix metalloproteinase-2 (MMP2) was inhibited by Bajijiasu treatment. Luciferase reporter gene studies showed that Bajijiasu could significantly reduce the expression and transcriptional activity of NFAT as well as RANKL-induced NF-κB activation in a dose-dependent manner. Further, Bajijiasu was found to decrease the RANKL-induced phosphorylation of extracellular signal-regulated kinases (ERK), inhibitor of κB-α (IκB-α), NFAT, and V-ATPase d2. Taken together, this study revealed Bajijiasu could attenuate osteoclast formation and bone resorption by mediating RANKL signaling pathways, indicative of a potential effect of Bajijiasu on osteolytic bone diseases. PMID:28106828

  7. Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

    PubMed Central

    Herbert, Bethany A.; Valerio, Michael S.; Gaestel, Matthias; Kirkwood, Keith L.

    2015-01-01

    Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220-Gr1-CD11blo/-CD115+ (dOCPlo/-). In this study, we isolated dOCPs from male and female mice to determine sex-specific effects of MK2 signaling in osteoclastogenesis (OCgen). Male Mk2-/- mice display an increase in the dOCPlo cell population when compared to Mk2+/+ mice, while female Mk2-/- and Mk2+/+ mice exhibit no difference. Defined OCPs from male and female Mk2+/+ and Mk2-/- bone marrow were treated with macrophage colony stimulation factor (M-CSF) and RANKL cytokines to promote OCgen. RANKL treatment of dOCPlo cells stimulated p38 and MK2 phosphorylation. Tartrate-resistant acid phosphatase (TRAP) assays were used to quantify OC number, size, and TRAP enzyme activity post-RANKL stimulation. MK2 signaling was critical for male dOCPlo OCgen, yet MK2 signaling regulated OCgen from female dOCP- and CD11bhi subpopulations as well. The functional gene, Ctsk, was attenuated in both male and female Mk2-/- dOCPlo-derived OCs. Conversely, MK2 signaling was only critical for gene expression of pre-OC fusion genes, Oc-stamp andTm7sf4, in male OCgen. Therefore, these data suggest there is a sexual dimorphism in MK2 signaling of OCP subpopulations. PMID:25946081

  8. High D(+)glucose concentration inhibits RANKL-induced osteoclastogenesis

    PubMed Central

    Wittrant, Y; Gorin, Y; Woodruff, K; Horn, D; Abboud, HE; Mohan, S; Abboud-Werner, SL

    2009-01-01

    Diabetes is a chronic disease associated with hyperglycemia and altered bone metabolism that may lead to complications including osteopenia, increased risk of fracture and osteoporosis. Hyperglycemia has been implicated in the pathogenesis of diabetic bone disease; however, the biologic effect of glucose on osteoclastogenesis is unclear. In the present study, we examined the effect of high D(+)glucose (D-Glc) and L(−)glucose (L-Glc; osmotic control) on RANKL-induced osteoclastogenesis using RAW264.7 cells and Bone Marrow Macrophages (BMM) as models. Cells were exposed to sustained high glucose levels to mimic diabetic conditions. Osteoclast formation was analyzed using tartrate resistant acid phosphatase (TRACP) assay, expression of calcitonin receptor (CTR) and cathepsin K mRNAs, and cultures were examined for reactive oxygen species (ROS) using dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, caspase-3 and Nuclear Factor kappaB (NF-κB) activity. Cellular function was assessed using a migration assay. Results show, for the first time, that high D-Glc inhibits osteoclast formation, ROS production, caspase-3 activity and migration in response to RANKL through a metabolic pathway. Our findings also suggest that high D-Glc may alter RANKL-induced osteoclast formation by inhibiting redox-sensitive NF-κB activity through an anti-oxidative mechanism. This study increases our understanding of the role of glucose in diabetes-associated bone disease. Our data suggest that high glucose levels may alter bone turnover by decreasing osteoclast differentiation and function in diabetes and provide new insight into the biologic effects of glucose on osteoclastogenesis. PMID:18378205

  9. 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.

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

    SciTech Connect

    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. - Highlights: • Salicortin suppresses osteoclastogenesis in vitro. • Salicortin impairs the JNK and NF-κB/NFATc1 signaling pathway. • Salicortin may be of interest in developments of osteoporosis treatment.

  11. (-)-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.

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

    SciTech Connect

    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. - Highlights: • Tenuigenin suppresses osteoclasts formation, survival and function in vitro. • Tenuigenin impairs NF-κB activation. • Tenuigenin suppresses RANKL-induced bone lose in vivo. • Tenuigenin may be used for treating osteoclast related diseases.

  13. 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.

  14. 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

  15. 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.

  16. Osteoporosis regulation by salubrinal through eIF2α mediated differentiation of osteoclast and osteoblast

    PubMed Central

    He, Long; Lee, Junwon; Jang, Jae Hyuk; Sakchaisri, Krisada; Hwang, Joonsung; Cha-Molstad, Hyun Joo; Kim, Kyung A; Ryoo, In Ja; Lee, Hee Gu; Kim, Sun Ok; Soung, Nak Kyun; Lee, Kyung Sang; Kwon, Yong Tae; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon

    2013-01-01

    Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis. PMID:23178987

  17. Osteoporosis regulation by salubrinal through eIF2α mediated differentiation of osteoclast and osteoblast.

    PubMed

    He, Long; Lee, Junwon; Jang, Jae Hyuk; Sakchaisri, Krisada; Hwang, Joonsung; Cha-Molstad, Hyun Joo; Kim, Kyung A; Ryoo, In Ja; Lee, Hee Gu; Kim, Sun Ok; Soung, Nak Kyun; Lee, Kyung Sang; Kwon, Yong Tae; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon

    2013-02-01

    Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Stachybotrysin, an Osteoclast Differentiation Inhibitor from the Marine-Derived Fungus Stachybotrys sp. KCB13F013.

    PubMed

    Kim, Jong Won; Ko, Sung-Kyun; Kim, Hye-Min; Kim, Gun-Hee; Son, Sangkeun; Kim, Gil Soo; Hwang, Gwi Ja; Jeon, Eun Soo; Shin, Kee-Sun; Ryoo, In-Ja; Hong, Young-Soo; Oh, Hyuncheol; Lee, Kyung Ho; Soung, Nak-Kyun; Hashizume, Daisuke; Nogawa, Toshihiko; Takahashi, Shunji; Kim, Bo Yeon; Osada, Hiroyuki; Jang, Jae-Hyuk; Ahn, Jong Seog

    2016-10-28

    Two new phenylspirodrimane derivatives, stachybotrysin (1) and stachybotrylactone B (2), were isolated from the cultures of the marine-derived fungus Stachybotrys sp. KCB13F013. The structures were determined by analyzing the spectroscopic data (1D and 2D NMR and MS) and chemical transformation, including the modified Mosher's method and single-crystal X-ray structure analysis. Compound 1 exhibited an inhibitory effect on osteoclast differentiation in bone marrow macrophage cells via suppressing the RANKL-induced activation of p-ERK, p-JNK, p-p38, c-Fos, and NFATc1.

  19. Antiosteoclastogenic activity of silicate-based materials antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine marcophages.

    PubMed

    Hung, Chi-Jr; Kao, Chia-Tze; Chen, Yi-Jyun; Shie, Ming-You; Huang, Tsui-Hsien

    2013-12-01

    This study investigated whether calcium silicate cement extract exerted antiosteoclastogenic actions in murine RAW 264.7 macrophages cultured with receptor activator for nuclear factor kappaB (RANKL). The RAW 264.7 macrophage cell was treated with RANKL to osteoclastogenesis. Then, cell viability, cell death, and cathepsin K expression were examined. The silicon (Si)-inhibited RANKL-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that ≥4 mmol/L Si reduced RANKL-enhanced tartrate-resistant acid phosphatase (TRAP) activity in a dose-dependent manner. Furthermore, Si diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and nuclear factor kappaB activation. The current report shows that silicate abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The Si can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts, and the function of osteoclasts. Therefore, silicate-based materials may be a potential therapeutic agent targeting osteoclast differentiation in bone defects. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  20. 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

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

    PubMed

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

    2015-01-01

    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. 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). 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. The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.

  2. 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.

  3. 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.

  4. Glycosaminoglycans modulate RANKL induced osteoclastogenesis

    PubMed Central

    Ling, Ling; Murali, Sadasivam; Stein, Gary S.; van Wijnen, Andre J.; Cool, Simon M.

    2011-01-01

    Skeletal integrity is tightly regulated by the activity of osteoblasts and osteoclasts that are both under the control of extracellular glycosaminoglycans (GAGs) through their interactions with endogenous growth factors and differentiation-promoting ligands. Receptor Activator of NF-kappa-B Ligand (RANKL), which is a Tumor Necrosis Factor (TNF)-related protein that is critical for osteoclast formation, is produced by osteoblasts and further modulated by certain types of GAGs. Using unfractionated osteoblast-derived GAGs that reflect the complex tissue microenvironment within which osteoclasts reside, we demonstrate that these GAGs block the osteoclastogenic activity of RANKL. Furthermore, RANKL significantly reduces ERK activity, a putative suppressor of osteoclastogenesis, but osteoblast-derived GAGs eliminate the inhibitory effects of RANKL on ERK activity. Notably, while imposing an anti-osteoclastic affect, these GAGs also enhanced the proliferation of osteoblasts. Thus, the osteoblast microenvironment is a potent source of GAGs that promote bone anabolic activities. The anti-osteoclastogenic and osteoblast-related mitogenic activities of these GAGs together may provide a key starting point for the development of selective sugar-based therapeutic compounds for the treatment of osteopenic disorders. PMID:20135643

  5. 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

  6. Herba epimedii flavonoids suppress osteoclastic differentiation and bone resorption by inducing G2/M arrest and apoptosis.

    PubMed

    Zhang, Dawei; Zhang, Jinchao; Fong, Chichun; Yao, Xinsheng; Yang, Mengsu

    2012-12-01

    Accumulating evidences suggest that Herba epimedii has the potential benefits against osteoporosis. However, previous studies were focused on the crude extract, total flavonoids (TF) and icariin (ICA), and the detailed molecular mechanisms of action and structure-activity relationship (SAR) remain unclear. Herein we aimed to systematically investigate the effects of Herba epimedii flavonoids (HEF) on the activity of osteoclasts, and explore the potential SAR. Both ICA and baohuoside-1 (BS) significantly inhibited the proliferation of RAW 264.7 cells (IC(50) 25 μM and 67 μM, respectively). Treatment of ICA resulted in G2/M arrest and apoptosis in RAW 264.7 cells as early as 12 h. Besides, HEF remarkably suppressed vitamin D-induced differentiation of osteoclasts in rabbit bone marrow cells and the bone resorption of rabbit mature osteoclasts in vitro. It is notable that the inhibitory effect of 100 μM ICA and BS on osteoclast formation is almost 90%; and the inhibition rate on bone resorption is 50% and 80%, respectively. Besides, RANKL-induced osteoclast formation from RAW 264.7 cells and the expression of TRAP, CA II, CTSK and MMP-9 was significantly reduced by the treatment of 25 μM HEF and 17β-estradiol (ES), and the inhibitory strength increases in the order TF < ES < ICA < BS, which was blocked by ICI182780 suggesting that the regulation of osteoclast activity might be ER dependent. Furthermore, the free hydroxyl group at C-7 of BS played an important role in the SAR for anti-osteoclast action. To conclude, HEF could regulate the formation and activity of osteoclasts by inhibiting the proliferation and differentiation, inducing apoptosis and cell cycle arrest and suppressing bone resorption of osteoclasts. Changes in osteoclast activity are probably mediated predominantly by interaction with nuclear estrogen receptors and via mitochondrial pathway. HEF, especially BS, has great potential for the prevention and treatment of osteoporosis.

  7. 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.

  8. Nicotinamide phosphoribosyltransferase inhibits receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation in vitro

    PubMed Central

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

    2017-01-01

    The adipokine nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B-cell colony-enhancing factor or the insulin-mimetic hormone visfatin, has a crucial role in the conversion of nicotinamide to nicotinamide mononucleotide during biosynthesis of the coenzyme nicotinamide adenine dinucleotide. Previous reports have demonstrated the inhibitory effects of Nampt on osteoclast formation from human peripheral blood mononuclear cells and CD14+ monocytes. However, the effects of Nampt on bone marrow macrophage (BMM)-derived osteoclastogenesis and its precise role in the process remain unclear. The present in vitro study used recombinant Nampt and BMMs as osteoclast precursors demonstrated that Nampt suppresses receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis by decreasing the phosphorylation of various early signal transducers, including c-Jun N-terminal kinase, Akt, glycogen synthase kinase-3 β, Bruton's tyrosine kinase and phospholipase C γ-2. In addition, western blotting and reverse transcription-quantitative polymerase chain reaction analysis indicated that Nampt downregulates the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1, leading to a decrease in the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase, osteoclast-associated receptor and cathepsin K. However, the bone-resorbing activity of mature osteoclasts treated with Nampt was similar to untreated control osteoclasts. This finding indicates that Nampt exerts its anti-osteoclastogenic activity by targeting osteoclast precursor cells rather than mature osteoclasts. Consequently, the present study demonstrated that Nampt acts as a negative regulator of RANKL-mediated differentiation of BMMs into osteoclasts, suggesting the potential therapeutic targets to treat bone-related disorders such as osteoporosis. PMID:28035412

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

    SciTech Connect

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

    2016-02-05

    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{sup 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. - Highlights: • We first investigated the anti-osteoclastogenic effects of niclosamide in vitro and in vivo. • Niclosamide impairs the activation of the Akt-IκB-STAT3 ser{sup 727} signaling axis. • Niclosamide acts a negative regulator of actin ring formation during osteoclast differentiation. • Niclosamide suppresses LPS-induced bone loss in vivo. • Niclosamide deserves new evaluation as a potential treatment target in various bone diseases.

  10. 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.

  11. WHI-131 Promotes Osteoblast Differentiation and Prevents Osteoclast Formation and Resorption in Mice.

    PubMed

    Cheon, Yoon-Hee; Kim, Ju-Young; Baek, Jong Min; Ahn, Sung-Jun; Jun, Hong Young; Erkhembaatar, Munkhsoyol; Kim, Min Seuk; Lee, Myeung Su; Oh, Jaemin

    2016-02-01

    The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K (CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca(2+) oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)-Bruton agammaglobulinemia tyrosine kinase (Btk)-phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression

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

    SciTech Connect

    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. - Highlights: • CD147 expression was markedly upregulated during osteoclast differentiation. • Downregulation of CD147 expression inhibited osteoclastgenesis and bone resorption. • Decreased CD147 expression inhibited RANKL-induced nuclear translocation of NFATc1.

  13. Nitidine chloride prevents OVX-induced bone loss via suppressing NFATc1-mediated osteoclast differentiation

    PubMed Central

    Liu, Qian; Wang, Tao; Zhou, Lin; Song, Fangming; Qin, An; Feng, Hao Tian; Lin, Xi Xi; Lin, Zhen; Yuan, Jin Bo; Tickner, Jennifer; Liu, Hua Gang; Zheng, Ming Hao; Xu, Jiake; Zhao, Jin Min

    2016-01-01

    Nitidine chloride (NC), a bioactive alkaloid isolated from Zanthoxylum nitidum, has been used as a herbal ingredient in toothpaste that prevents cavities for decades. It also displays potential antitumor and anti-inflammation properties. However, its anticatabolic effect on bone is not known. We investigated the effect of NC on osteoclastogenesis, bone resorption and RANKL-induced NF-κB and NFATc1 signalling. In mouse-derived bone marrow monocytes (BMMs), NC suppressed RANKL-induced multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation and bone resorption in a dose dependent manner. NC attenuated the expression of osteoclast marker genes including cathepsin K, D2, calcitonin receptor, NFATc1, and TRAP. Further, NC inhibited RANKL-activated NF-κB and NFATc1 signalling pathways. In vivo study revealed that NC abrogated oestrogen deficiency-induced bone loss in ovariectomized mice. Histological analysis showed that the number of osteoclasts was significantly lower in NC-treated groups. Collectively, our data demonstrate that NC suppressed osteoclastogenesis and prevented OVX-induced bone loss by inhibiting RANKL-induced NF-κB and NFATc1 signalling pathways. NC may be a natural and novel treatment for osteoclast-related bone lytic diseases. PMID:27821837

  14. Effect of radiation on the expression of osteoclast marker genes in RAW264.7 cells.

    PubMed

    Yang, Bing; Zhou, Hui; Zhang, Xiao-Dong; Liu, Zheng; Fan, Fei-Yue; Sun, Yuan-Ming

    2012-04-01

    Cancer radiation therapy can cause skeletal complications, such as osteopenia and osteoporosis. To understand the mechanism responsible for the skeletal complications, the expression profiles of osteoclast marker genes in RAW264.7 cells were observed. Osteoclast formation was established by RAW264.7 cells that were treated with the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and detected using immunochemistry and morphological observations. Quantitative real-time polymerase chain reaction was used to assess the expression of a panel of osteoclast markers, including the receptor activator of NF-κB (RANK), tartrate-resistant acid phosphatase (TRAP), integrin β3 and the calcitonin receptor (CTR). RANKL-induced osteoclasts were TRAP-positive and multinucleated, and displayed a distinct morphology. RANKL-induced osteoclast precursor cells had increased TRAP and RANK expression and decreased CTR expression compared to the control cells not treated with RANKL. RAW264.7 cells irradiated with 2-Gy γ-rays had upregulated integrin β3 and RANK expression and downregulated CTR expression compared to the control RAW264.7 cells. The effect of radiation on RANKL-induced osteoclast differentiation enhanced the expression of CTR and inhibited the expression of RANK and TRAP. Therefore, radiation damage from 2-Gy γ-rays can promote the activities of osteoclast precursor cells, but not those of osteoclasts.

  15. Effect of radiation on the expression of osteoclast marker genes in RAW264.7 cells

    PubMed Central

    YANG, BING; ZHOU, HUI; ZHANG, XIAO-DONG; LIU, ZHENG; FAN, FEI-YUE; SUN, YUAN-MING

    2012-01-01

    Cancer radiation therapy can cause skeletal complications, such as osteopenia and osteoporosis. To understand the mechanism responsible for the skeletal complications, the expression profiles of osteoclast marker genes in RAW264.7 cells were observed. Osteoclast formation was established by RAW264.7 cells that were treated with the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and detected using immunochemistry and morphological observations. Quantitative real-time polymerase chain reaction was used to assess the expression of a panel of osteoclast markers, including the receptor activator of NF-κB (RANK), tartrate-resistant acid phosphatase (TRAP), integrin β3 and the calcitonin receptor (CTR). RANKL-induced osteoclasts were TRAP-positive and multinucleated, and displayed a distinct morphology. RANKL-induced osteoclast precursor cells had increased TRAP and RANK expression and decreased CTR expression compared to the control cells not treated with RANKL. RAW264.7 cells irradiated with 2-Gy γ-rays had upregulated integrin β3 and RANK expression and downregulated CTR expression compared to the control RAW264.7 cells. The effect of radiation on RANKL-induced osteoclast differentiation enhanced the expression of CTR and inhibited the expression of RANK and TRAP. Therefore, radiation damage from 2-Gy γ-rays can promote the activities of osteoclast precursor cells, but not those of osteoclasts. PMID:22294242

  16. Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro

    PubMed Central

    Wang, Liping; Zhao, Rong; Shi, Xiaoyou; Wei, Tzuping; Halloran, Bernard P.; Clark, David J.; Jacobs, Christopher R.; Kingery, Wade S.

    2009-01-01

    Introduction SP is a neuropeptide distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum. Previously we demonstrated that inhibition of neuropeptide signaling after capsaicin treatment resulted in a loss of bone mass and we hypothesized that SP contributes to bone integrity by stimulating osteogenesis. Materials and Methods Osteoblast precursors (bone marrow stromal cells, BMSCs) and osteoclast precursors (bone marrow macrophages, BMMs) derived from C57BL/6 mice were cultured. Expression of the SP receptor (NK1) was detected by using immunocytochemical staining and PCR. Effects of SP on proliferation and differentiation of BMSCs were studied by measuring BrdU incorporation, gene expression, alkaline phosphatase activity, and osteocalcin and Runx2 protein levels with EIA and western blot assays, respectively. Effects of SP on BMMs were determined using a BrdU assay, counting multinucleated cells staining positive for tartrate-resistant acid phosphatase (TRAP+), measuring pit erosion area, and evaluating RANKL protein production and NF-κB activity with ELISA and western blot. Results The NK1 receptor was expressed in both BMSCs and BMMs. SP stimulated the proliferation of BMSCs in a concentration-dependent manner. Low concentrations (10−12 M) of SP stimulated alkaline phosphatase and osteocalcin expression, increased alkaline phosphatase activity, and up-regulated Runx2 protein levels, and higher concentrations of SP (10−8 M) enhanced mineralization in differentiated BMSCs. SP also stimulated BMSCs to produce RANKL, but at concentrations too low to evoke osteoclastogenesis in co-culture with macrophages in the presence of SP. SP also activated NF-κB in BMMs and directly facilitate RANKL induced macrophage osteoclastogenesis and bone resorption activity. Conclusions NK1 receptors are expressed by osteoblast and osteoclast precursors and SP stimulates osteoblast and osteoclast differentiation and function in

  17. Mutan: A mixed linkage α-[(1,3)- and (1,6)]-d-glucan from Streptococcus mutans, that induces osteoclast differentiation and promotes alveolar bone loss.

    PubMed

    Kwon, Hyun-Jung; Kim, Jung Min; Han, Kook-Il; Jung, Eui-Gil; Kim, Yong Hyun; Patnaik, Bharat Bhusan; Yoon, Mi Sook; Chung, Sung Kyun; Kim, Wan Jong; Han, Man-Deuk

    2016-02-10

    Mutan is an extracellular polysaccharide of Streptococcus mutans (S. mutans) that consists of α-(1,3)-linked glucose residues in main chains and α-(1,6) bonds in side chains. In the present study, mutan was isolated from S. mutans, and its structural characteristics were determined using Fourier-transform infrared spectroscopy (FT-IR) and (13)C nuclear magnetic resonance (NMR) spectroscopy. The effects of mutan on RANKL-induced osteoclast differentiation in RAW 264.7 cells were examined. Furthermore, microCT and morphometric analyses were used to determine the contribution of mutan to alveolar bone loss in the maxilla of a rat periodontitis model. Mutan increased (more than 2-fold) RANKL-induced osteoclast differentiation in a dose-dependent manner. Mutan also enhanced the alveolar bone loss in the rat maxilla 2.3-fold. In mutan-treated rats, the bone mineral density, bone volume, trabecular number, and trabecular thickness decreased, whereas trabecular separation significantly increased. In addition, mutan and lipopolysaccharide (LPS) induced similar microarray profiles in RAW 264.7 cells. A total of 43 genes related to osteoclastogenesis were differentially expressed after either mutan or LPS treatment. Five-fold increases in the expression of several genes, including IL-1β, IL-1α, IL-6, and chemokine ligands, were observed in mutan-treated RAW 264.7 cells. These results suggest a molecular mechanism for the inflammation induced by S. mutans during the establishment of periodontal disease.

  18. NADPH oxidase-derived reactive oxygen species are essential for differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts.

    PubMed

    Sasaki, Hideyuki; Yamamoto, Hironori; Tominaga, Kumiko; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

    2009-02-01

    Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) homologues have been suggested to regulate osteoclast differentiation. However, no bone abnormalities have been documented in Nox1 deficient, Nox2 deficient, or Nox3 mutant mice. During receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts, mRNA levels of Nox enzymes (Nox1-4) and their adaptor proteins were monitored by real-time reverse transcriptase PCR. RAW264.7 cells constitutively expressed abundant Nox2 mRNA and small amounts of Nox1 and Nox3 transcripts. RANKL markedly attenuated Nox2 mRNA expression in association with reciprocal up-regulation of Nox1 and Nox3 transcripts. Introduction of small interference RNA targeting p67(phox) or p22(phox) into RAW264.7 cells effectively down-regulated ROS generation and significantly suppressed the RANKL-stimulated differentiation, which was assessed by appearance of tartrate resistant acid phosphatase (TRAP)-positive, multinucleated cells having an ability to form resorption pits on calcium phosphate thin film-coated disks, and by expression of osteoclast marker genes (TRAP, cathepsin K, Atp6i, ClC-7, and NFATc1). Our results suggest that RANKL may stimulate switching between Nox homologues during osteoclast differentiation, and Nox-derived ROS may be crucial for RANKL-induced osteoclast differentiation.

  19. 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

  20. Arctigenin Inhibits Osteoclast Differentiation and Function by Suppressing Both Calcineurin-Dependent and Osteoblastic Cell-Dependent NFATc1 Pathways

    PubMed Central

    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

  1. Salt-inducible kinases (SIK) inhibition reduces RANKL-induced osteoclastogenesis.

    PubMed

    Lombardi, Maria Stella; Gilliéron, Corine; Berkelaar, Majoska; Gabay, Cem

    2017-01-01

    Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-κB activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus

  2. 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

  3. 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.

  4. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. ERK5 Activation Is Essential for Osteoclast Differentiation

    PubMed Central

    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. PMID:25885811

  6. 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

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

    SciTech Connect

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

    2015-11-06

    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 D{sub 3}. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D{sub 3}. 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.

  8. 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

  9. Extracts of marine algae show inhibitory activity against osteoclast differentiation.

    PubMed

    Koyama, Tomoyuki

    2011-01-01

    Osteoclasts are multinucleated cells that play a crucial role in bone resorption. The imbalance between bone resorption and bone formation results in osteoporosis. Therefore, substances that can suppress osteoclast formation are potential candidate materials for drug development or functional foods. There have been reports that extracts or purified compounds from marine micro- and macroalgae can suppress osteoclast differentiation. Symbioimine, isolated from the cultured dinoflagellate Symbiodinium sp., had suppressive effects against osteoclast differentiation in osteoclast-like cells. Norzoanthamine, isolated from the colonial zoanthid Zoanthas sp., has been shown to have antiosteoporosis activity in ovariectomized mice. With regard to marine extracts, the fucoxanthin-rich component from brown algae has been shown to have suppressive effects against osteoclast differentiation. An extract of Sargassum fusiforme has recently been shown to have antiosteoporosis activity. This extract suppressed both osteoclast differentiation and accelerated osteoblast formation in separate in vitro experiments. It also showed antiosteoporosis activity in ovariectomized mice by regulating the balance between bone resorption and bone formation. These marine algae and their extracts may be sources of marine medicinal foods for the prevention of osteoporosis.

  10. 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.

  11. The Study of Mechanisms of Protective Effect of Rg1 against Arthritis by Inhibiting Osteoclast Differentiation and Maturation in CIA Mice

    PubMed Central

    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. PMID:25214714

  12. Stimulation of Osteoclast Formation by RANKL Requires Interferon Regulatory Factor-4 and Is Inhibited by Simvastatin in a Mouse Model of Bone Loss

    PubMed Central

    Nakashima, Yoshiki; Haneji, Tatsuji

    2013-01-01

    Diseases of bone loss are a major public health problem. Here, we report the novel therapeutic action of simvastatin in osteoclastogenesis and osteoprotection, demonstrated by the ability of simvastatin to suppress osteoclast formation in vitro and in vivo. We found that in vitro, IRF4 expression is upregulated during osteoclast differentiation induced by RANKL (receptor activator of nuclear factor-κB ligand), while simvastatin blocks RANKL-induced osteoclastogenesis and decreases expression of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1), IRF4 and osteoclast markers. We also show that IRF4 acts in cooperation with NFATc2 and NF-κB on the promoter region of NFATc1 to accelerate its initial transcription during the early stage of osteoclastogenesis. Moreover, our study using IRF4 siRNA knockdown directly demonstrates the requirement for IRF4 in NFATc1 mRNA transcription and its necessity in RANKL-induced osteoclast differentiation. Our results suggest that the reduction in osteoclastogenesis is partly due to the inhibition of IRF4 production in RANKL-induced osteoclast differentiation. To investigate the in vivo effects of simvastatin in RANKL-treated mice, we examined the bone mineral density (BMD) of a mouse model of bone loss, and found that simvastatin significantly reduced bone loss by suppressing osteoclast numbers in vivo, even in the presence of high concentrations of RANKL. These results suggest that the depletion of osteoclasts is not due to the reduction in RANKL produced by osteoblasts in vivo. The results are consistent with the hypothesis that simvastatin blocks RANKL-induced IRF4 expression in osteoclastogenesis. We propose that the expression of IRF4 by osteoclasts could be a promising new therapeutic target in bone-loss diseases. PMID:24039733

  13. Runx1-Mediated Regulation of Osteoclast Differentiation and Function

    PubMed Central

    Soung, Do Y.; Kalinowski, Judith; Baniwal, Sanjeev K.; Jacome-Galarza, Christian E.; Frenkel, Baruch

    2014-01-01

    Excessive bone resorption is the cause of several metabolic bone diseases including osteoporosis. Thus, identifying factors that can inhibit osteoclast formation and/or activity may define new drug targets that can be used to develop novel therapies for these conditions. Emerging evidence demonstrates that the master regulator of hematopoiesis, Runx1, is expressed in preosteoclasts and may influence skeletal health. To examine the potential role of Runx1 in osteoclast formation and function, we deleted its expression in myeloid osteoclast precursors by crossing Runx1 floxed mice (Runx1F/F) with CD11b-Cre transgenic mice. Mice lacking Runx1 in preosteoclasts (CD11b-Cre;Runx1F/F) exhibited significant loss of femoral trabecular and cortical bone mass compared with that in Cre-negative mice. In addition, serum levels of collagen type 1 cross-linked C-telopeptide, a biomarker of osteoclast-mediated bone resorption, were significantly elevated in CD11b-Cre;Runx1F/F mice compared with those in Runx1F/F mice. Tartrate-resistant acid phosphatase–positive osteoclasts that differentiated from bone marrow cells of CD11b-Cre;Runx1F/F mice in vitro were larger, were found in greater numbers, and had increased bone resorbing activity than similarly cultured cells from Runx1F/F mice. CD11b-Cre;Runx1F/F bone marrow cells that were differentiated into osteoclasts in vitro also had elevated mRNA levels of osteoclast-related genes including vacuolar ATPase D2, cathepsin K, matrix metalloproteinase 9, calcitonin receptor, osteoclast-associated receptor, nuclear factor of activated T cells cytoplasmic 1, and cFos. These data indicate that Runx1 expression in preosteoclasts negatively regulates osteoclast formation and activity and contributes to overall bone mass. PMID:24606124

  14. 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.

  15. 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.

  16. 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.

  17. Alpinia officinarum Stimulates Osteoblast Mineralization and Inhibits Osteoclast Differentiation.

    PubMed

    Shim, Ki-Shuk; Lee, Chung-Jo; Yim, Nam-Hui; Gu, Min Jung; Ma, Jin Yeul

    2016-01-01

    Alpinia officinarum rhizome has been used as a traditional herbal remedy to treat inflammatory and internal diseases. Based on the previously observed inhibitory effect of A. officinarum rhizome in an arthritis model, we evaluated whether a water extract of A. officinarum rhizome (WEAO) would enhance in vitro osteoblast mineralization using calvarial osteoblast precursor cells or would inhibit in vitro osteoclast differentiation and bone resorption using bone marrow derived macrophages. In osteoblasts, WEAO enhanced the mRNA levels of transcription factor (runt-related transcription factor 2, smad1, smad5, and junB) and marker (bone morphogenetic protein-2, collagen type 1alpha1, and osteocalcin) genes related to osteoblast mineralization, consistent with increased alizarin red S staining intensity. WEAO markedly inhibited osteoclast differentiation by suppressing the receptor activator for nuclear factor-[Formula: see text]B ligand-induced downregulation of inhibitor of DNA binding 2 and V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and the phosphorylation of c-Jun N-terminal kinase, p38, nuclear factor-[Formula: see text]B, c-Src, and Bruton's tyrosine kinase to induce nuclear factor of activated T cells cytoplasmic 1 expression. WEAO also suppressed the resorbing activity of mature osteoclasts by altering actin ring formation. Therefore, the results of this study demonstrate that WEAO stimulates osteoblast mineralization and inhibits osteoclast differentiation. Thus, WEAO may be a promising herbal candidate to treat or prevent pathological bone diseases by regulating the balance between osteoclast and osteoblast activity.

  18. Chlorogenic acid inhibits osteoclast differentiation and bone resorption by down-regulation of receptor activator of nuclear factor kappa-B ligand-induced nuclear factor of activated T cells c1 expression.

    PubMed

    Kwak, Sung Chul; Lee, Cheol; Kim, Ju-Young; Oh, Hyun Mee; So, Hong-Seob; Lee, Myeung Su; Rho, Mun Chual; Oh, Jaemin

    2013-01-01

    Excessive osteoclastic bone resorption plays a critical role in inflammation-induced bone loss such as rheumatoid arthritis and periodontal bone erosion. Therefore, identification of osteoclast targeted-agents may be a therapeutic approach to the treatment of pathological bone loss. In this study, we isolated chlorogenic acid (CGA) from fructus of Gardenia jasminoides to discover anti-bone resorptive agents. CGA is a polyphenol with anti-inflammatory and anti-oxidant activities, however, its effects on osteoclast differentiation is unknown. Thus, we investigated the effect of CGA in receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation and RANKL signaling. CGA dose-dependently inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. CGA inhibited the phosphorylation of p38, Akt, extracellular signal-regulated kinase (ERK), and inhibitor of nuclear factor-kappa B (IκB), and IκB degradation by RANKL treatment. CGA suppressed the mRNA expression of nuclear factor of activated T cells c1 (NFATc1), TRAP and OSCAR in RANKL-treated bone marrow macrophages (BMMs). Also, overexpression of NFATc1 in BMMs blocked the inhibitory effect of CGA on RANKL-mediated osteoclast differentiation. Furthermore, to evaluate the effects of CGA in vivo, lipopolysaccharide (LPS)-induced bone erosion study was carried out. CGA remarkably attenuated LPS-induced bone loss based on micro-computed tomography and histologic analysis of femurs. Taken together, our findings suggest that CGA may be a potential treatment option for osteoclast-related diseases with inflammatory bone destruction.

  19. KMUP-1 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss: Roles of MAPKs, Akt, NF-κB and Calcium/Calcineurin/NFATc1 Pathways

    PubMed Central

    Lin, I-Ling; Ho, Mei-Ling; Hsu, Pei-Chuan; Chen, Li-Wen; Chen, Ing-Jun; Yeh, Jwu-Lai

    2013-01-01

    Background KMUP-1 is a xanthine derivative with inhibitory activities on the phosphodiesterase (PDE) 3,4 and 5 isoenzymes to suppress the degradation of cyclic AMP and cyclic GMP. However, the effects of KMUP-1 on osteoclast differentiation are still unclear. In this study, we investigated whether KMUP-1 inhibits osteoclastogenesis induced by RANKL in RAW 264.7 cells and bone loss induced by ovariectomy in mice, and the underlying mechanisms. Principal Findings In vitro, KMUP-1 inhibited RANKL-induced TRAP activity, the formation of multinucleated osteoclasts and resorption-pit formation. It also inhibited key mediators of osteoclastogenesis including IL-1β, IL-6, TNF-α and HMGB1. In addition, KMUP-1 inhibited RANKL-induced activation of signaling molecules (Akt, MAPKs, calcium and NF-κB), mRNA expression of osteoclastogensis-associated genes (TRAP, MMP-9, Fra-1, and cathepsin K) and transcription factors (c-Fos and NFATc1). Furthermore, most inhibitory effects of KMUP-1 on RANKL-mediated signal activations were reversed by a protein kinase A inhibitor (H89) and a protein kinase G inhibitor (KT5823). In vivo, KMUP-1 prevented loss of bone mineral content, preserved serum alkaline phosphate and reduced serum osteocalcin in ovariectomized mice. Conclusions KMUP-1 inhibits RANKL-induced osteoclastogenesis in vitro and protects against ovariectomy-induced bone loss in vivo. These effects are mediated, at least in part, by cAMP and cGMP pathways. Therefore, KMUP-1 may have a role in pharmacologic therapy of osteoporosis. PMID:23936022

  20. 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

  1. 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

  2. Inhibition of the classical NF-kappaB pathway prevents osteoclast bone-resorbing activity.

    PubMed

    Soysa, Niroshani S; Alles, Neil; Shimokawa, Hitoyata; Jimi, Eijiro; Aoki, Kazuhiro; Ohya, Keiichi

    2009-01-01

    The classical NF-kappaB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-kappaB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-kappaB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-kappaB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-kappaB pathway plays a pivotal role in osteoclast bone-resorbing activity.

  3. Artesunate Inhibits RANKL-induced Osteoclastogenesis and Bone Resorption In Vitro and Prevents LPS-induced Bone Loss In Vivo.

    PubMed

    Wei, Cheng-Ming; Liu, Qian; Song, Fang-Ming; Lin, Xi-Xi; Su, Yi-Ji; Xu, Jiake; Huang, Lin; Zong, Shao-Hui; Zhao, Jin-Min

    2017-03-15

    Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side-effects caused by the currently available drugs, a continuous search for novel bone-protective therapies is essential. Artesunate (Art), the water-soluble derivative of artemisinin has been investigated owing to its anti-malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, the mRNA expression of osteoclastic-specific genes, and resorption pit formation in a dose-dependent manner in primary bone marrow-derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL-induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF-κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)-induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL-induced osteoclastogenesis by suppressing the NF-κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases. This article is protected by copyright. All rights reserved.

  4. Disulfiram attenuates osteoclast differentiation in vitro: a potential antiresorptive agent.

    PubMed

    Ying, Hua; Qin, An; 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Artemisia capillaris Alleviates Bone Loss by Stimulating Osteoblast Mineralization and Suppressing Osteoclast Differentiation and Bone Resorption.

    PubMed

    Lee, Chung-Jo; Shim, Ki-Shuk; Ma, Jin Yeul

    2016-01-01

    Artemisia capillaris has been used to treat jaundice and relieve high liver-heat in traditional medicine. In this study, we found that the administration of a water extract from A. capillaris (WEAC) to the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced bone loss model significantly prevents osteoporotic bone loss, increasing bone volume/trabecular volume by 22% and trabecular number by 24%, and decreasing trabecular separation by 29%. WEAC stimulated in vitro osteoblast mineralization from primary osteoblasts in association with increasing expression of osterix, nuclear factor of activated T cells cytoplasmic 1, and activator protein-1, as well as phosphorylation of extracellular signal-regulated kinase. In contrast to the anabolic effect of WEAC, WEAC significantly suppressed in vitro osteoclast formation from bone marrow macrophages by inhibiting the RANKL signaling pathways and bone resorption by downregulating the expression of resorption markers. Therefore, this study demonstrated that WEAC has a beneficial effect on bone loss through the regulation of osteoblast mineralization, as well as osteoclast formation and bone resorption. These results suggest that A. capillaris may be a promising herbal candidate for therapeutic agents to treat or prevent osteoporotic bone diseases.

  10. Glow discharge plasma pretreatment enhances osteoclast differentiation and survival on titanium plates.

    PubMed

    Kawai, Hiroyuki; Shibata, Yo; Miyazaki, Takashi

    2004-05-01

    Despite the fact that several reports have demonstrated osteoclast activity on various bioactive ceramics, osteoclast functions on surface-modified titanium have not come under focus. This study aimed to examine whether the increasing surface energy of glow discharge plasma (GDP) involved in protein adhesion containing the RGD (Arg-Gly-Asp) sequence affects osteoclast responses on titanium plates. We examined osteoclast differentiation and survival rates on titanium plates with and without GDP. The amounts of osteoclasts on titanium plates were not increased by GDP after 1 week. However, osteoclast differentiation was greatly activated by GDP pretreatment, as tartrate-resistant acid phosphatase synthesis significantly increased on the titanium plates with GDP. Additionally, since the presence of osteoclasts was detected only on the titanium plates with GDP, even after 4h cultivation in a coculture test, the osteoclasts survival rate was increased by GDP pretreatment. As osteoclast responses were affected even on surface modified metallic materials, we concluded that novel approaches are needed not only for osteoclastic resorption on ceramic materials but also for osteoclast responses on surface-modified metallic materials.

  11. 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.

  12. RIP140 in monocytes/macrophages regulates osteoclast differentiation and bone homeostasis

    PubMed Central

    Lee, Bomi; Iwaniec, Urszula T.; Turner, Russell T.; Lin, Yi-Wei; Clarke, Bart L.; Gingery, Anne

    2017-01-01

    Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage–specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases. PMID:28405613

  13. 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

  14. Anti-Osteoporotic Activity of Harpagoside by Upregulation of the BMP2 and Wnt Signaling Pathways in Osteoblasts and Suppression of Differentiation in Osteoclasts.

    PubMed

    Chung, Hwa-Jin; Kim, Won Kyung; Oh, Jedo; Kim, Me-Riong; Shin, Joon-Shik; Lee, Jinho; Ha, In-Hyuk; Lee, Sang Kook

    2017-02-24

    Harpagoside (1) is an iridoid glycoside isolated from the radix of Harpagophytum procumbens var. sublobatum, commonly called Devil's claw. The anti-osteoporotic effect of 1 was investigated in both in vitro cell cultures and in vivo using an ovariectomized (OVX) mouse model. Compound 1 induced bone formation by stimulating osteoblast proliferation, alkaline phosphatase activity, and mineralization in osteoblastic MC3T3-E1 cells. Treatment with 1 increased the mRNA and protein expression of bone formation biomarkers through regulation of the BMP2 and Wnt signaling pathway in MC3T3-E1 cells. Compound 1 also suppressed the RANKL-induced osteoclastogenesis of cultured mouse bone marrow cells. Oral administration of 1 restored the OVX-induced destruction of trabecular bone. The bone mineral density of the femur was also increased significantly by 1. The elevated serum levels of osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase in the OVX mice were decreased by treatment with 1. These findings suggest that compound 1 may protect against bone loss induced by OVX in mice by regulating stimulation of osteoblast differentiation and inhibition of osteoclast resorption. Therefore, harpagoside (1) is a potential candidate for management of postmenopausal osteoporosis.

  15. High throughput, quantitative analysis of human osteoclast differentiation and activity.

    PubMed

    Diepenhorst, Natalie A; Nowell, Cameron J; Rueda, Patricia; Henriksen, Kim; Pierce, Tracie; Cook, Anna E; Pastoureau, Philippe; Sabatini, Massimo; Charman, William N; Christopoulos, Arthur; Summers, Roger J; Sexton, Patrick M; Langmead, Christopher J

    2017-02-15

    Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an 'osteoclast population' is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.

  16. Wogonin inhibits osteoclast differentiation by inhibiting NFATc1 translocation into the nucleus

    PubMed Central

    GENG, XIAOLIN; YANG, LIBIN; ZHANG, CHAO; QIN, HUA; LIANG, QIUDONG

    2015-01-01

    The aim of the present study was to identify a natural product with the ability to inhibit nuclear factor of activated T cells c1 (NFATc1) translocation from the cytoplasm to the nucleus by high-throughput screening, and to investigate the effect of the natural product upon osteoclast differentiation and its underlying mechanism. An NFATc1 antagonist redistribution assay was performed in U2OS-NFATc1 cells against a natural product library, and Wogonin was found to have the ability to inhibit the NFATc1 translocation from the cytoplasm to the nucleus. The effect of Wogonin on NFATc1 transcription activation was further determined by luciferase assay. An osteoclast differentiation assay was executed to evaluate the effect of Wogonin on osteoclast differentiation. The effect of Wogonin upon the vital genes in osteoclast differentiation was investigated using fluorescent quantitative polymerase chain reaction analysis. The natural product Wogonin significantly inhibited the translocation of NFATc1 from the cytoplasm to the nucleus and its transcriptional activation activity. Wogonin also significantly inhibited osteoclast differentiation and decreased the transcription of osteoclast-associated immunoglobulin-like receptor, tartrate-resistant acid phosphatase and calcitonin receptor. In conclusion, the natural product Wogonin inhibited osteoclast differentiation through the inhibition of NFATc1 translocation from the cytoplasm to the nucleus, and thus the downregulation of genes associated with osteoclast differentiation, which marked Wogonin as a potential treatment for osteoporosis. PMID:26622440

  17. High bone mass in mice lacking Cx37 because of defective osteoclast differentiation.

    PubMed

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

    2014-03-21

    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.

  18. 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

  19. 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

  20. 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.

  1. Calmodulin interacts with Rab3D and modulates osteoclastic bone resorption

    PubMed Central

    Zhu, Sipin; Chim, Shek Man; Cheng, Taksum; Ang, Estabelle; Ng, Benjamin; Lim, Baysie; Chen, Kai; Qiu, Heng; Tickner, Jennifer; Xu, Huazi; Pavlos, Nathan; Xu, Jiake

    2016-01-01

    Calmodulin is a highly versatile protein that regulates intracellular calcium homeostasis and is involved in a variety of cellular functions including cardiac excitability, synaptic plasticity and signaling transduction. During osteoclastic bone resorption, calmodulin has been reported to concentrate at the ruffled border membrane of osteoclasts where it is thought to modulate bone resorption activity in response to calcium. Here we report an interaction between calmodulin and Rab3D, a small exocytic GTPase and established regulator osteoclastic bone resorption. Using yeast two-hybrid screening together with a series of protein-protein interaction studies, we show that calmodulin interacts with Rab3D in a calcium dependent manner. Consistently, expression of a calcium insensitive form of calmodulin (i.e. CaM1234) perturbs calmodulin-Rab3D interaction as monitored by bioluminescence resonance energy transfer (BRET) assays. In osteoclasts, calmodulin and Rab3D are constitutively co-expressed during RANKL-induced osteoclast differentiation, co-occupy plasma membrane fractions by differential gradient sedimentation assay and colocalise in the ruffled border as revealed by confocal microscopy. Further, functional blockade of calmodulin-Rab3D interaction by calmidazolium chloride coincides with an attenuation of osteoclastic bone resorption. Our data imply that calmodulin- Rab3D interaction is required for efficient bone resorption by osteoclasts in vitro. PMID:27897225

  2. Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration.

    PubMed

    Voronov, I; Heersche, J N M; Casper, R F; Tenenbaum, H C; Manolson, M F

    2005-07-15

    We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

  3. 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

  4. Positive regulation of osteoclastic differentiation by growth differentiation factor 15 upregulated in osteocytic cells under hypoxia.

    PubMed

    Hinoi, Eiichi; Ochi, Hiroki; Takarada, Takeshi; Nakatani, Eri; Iezaki, Takashi; Nakajima, Hiroko; Fujita, Hiroyuki; Takahata, Yoshifumi; Hidano, Shinya; Kobayashi, Takashi; Takeda, Shu; Yoneda, Yukio

    2012-04-01

    Osteocytes are thought to play a role as a mechanical sensor through their communication network in bone. Although osteocytes are the most abundant cells in bone, little attention has been paid to their physiological and pathological functions in skeletogenesis. Here, we have attempted to delineate the pivotal functional role of osteocytes in regulation of bone remodeling under pathological conditions. We first found markedly increased osteoclastic differentiation by conditioned media (CM) from osteocytic MLO-Y4 cells previously exposed to hypoxia in vitro. Using microarray and real-time PCR analyses, we identified growth differentiation factor 15 (GDF15) as a key candidate factor secreted from osteocytes under hypoxia. Recombinant GDF15 significantly promoted osteoclastic differentiation in a concentration-dependent manner, with concomitant facilitation of phosphorylation of both p65 and inhibitory-κB in the presence of receptor activator of nuclear factor-κB ligand. To examine the possible functional significance of GDF15 in vivo, mice were subjected to ligation of the right femoral artery as a hypoxic model. A significant increase in GDF15 expression was specifically observed in tibias of the ligated limb but not in tibias of the normally perfused limb. Under these experimental conditions, in cancellous bone of proximal tibias in the ligated limb, a significant reduction was observed in bone volume, whereas a significant increase was seen in the extent of osteoclast surface/bone surface when determined by bone histomorphometric analysis. Finally, the anti-GDF15 antibody prevented bone loss through inhibiting osteoclastic activation in tibias from mice with femoral artery ligation in vivo, in addition to suppressing osteoclastic activity enhanced by CM from osteocytes exposed to hypoxia in vitro. These findings suggest that GDF15 could play a pivotal role in the pathogenesis of bone loss relevant to hypoxia through promotion of osteoclastogenesis after

  5. Inhibition of osteoclast differentiation by tanshinones from the root of Salvia miltiorrhiza bunge.

    PubMed

    Lee, Song-Yi; Choi, Doo-Youn; Woo, Eun-Rhan

    2005-08-01

    We screened natural products to find compounds with anti-osteoporotic potential using a coculture-based system by which osteoclast differentiation is effectively achieved. We found that methylene chloride soluble fraction of the root of Salvia miltiorrhiza Bunge (Labiatae) suppressed osteoclast differentiation. Five tanshinones, tanshinone IIA (1), tanshinone 1 (2), cryptotanshinone (3), 15,16-dihydrotanshinone I (4), and ferruginol (5) were subsequently isolated from fraction. Among the five compounds, compounds 1-4 reduced the formation of TRAP-positive multinuclear osteoclasts. These results suggest that the identified tanshinones may be useful candidates for development of therapeutic agents to treat osteoporosis and other bone-resorptive diseases.

  6. Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss

    PubMed Central

    Harre, Ulrike; Lang, Stefanie C.; Pfeifle, René; Rombouts, Yoann; Frühbeißer, Sabine; Amara, Khaled; Bang, Holger; Lux, Anja; Koeleman, Carolien A.; Baum, Wolfgang; Dietel, Katharina; Gröhn, Franziska; Malmström, Vivianne; Klareskog, Lars; Krönke, Gerhard; Kocijan, Roland; Nimmerjahn, Falk; Toes, René E. M.; Herrmann, Martin; Scherer, Hans Ulrich; Schett, Georg

    2015-01-01

    Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss. PMID:25825024

  7. Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway

    PubMed Central

    Iezaki, Takashi; Fukasawa, Kazuya; Park, Gyujin; Horie, Tetsuhiro; Kanayama, Takashi; Ozaki, Kakeru; Onishi, Yuki; Takahata, Yoshifumi; Nakamura, Yukari; Takarada, Takeshi; Yoneda, Yukio; Nakamura, Takashi; Vacher, Jean

    2016-01-01

    Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we show that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) is expressed in osteoclast lineages and represents a component of the machinery that regulates bone homeostasis. Ifrd1 expression was transcriptionally regulated in preosteoclasts by receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) through activator protein 1. Global deletion of murine Ifrd1 increased bone formation and decreased bone resorption, leading to a higher bone mass. Deletion of Ifrd1 in osteoclast precursors prevented RANKL-induced bone loss, although no bone loss was observed under normal physiological conditions. RANKL-dependent osteoclastogenesis was impaired in vitro in Ifrd1-deleted bone marrow macrophages (BMMs). Ifrd1 deficiency increased the acetylation of p65 at residues K122 and K123 via the inhibition of histone deacetylase-dependent deacetylation in BMMs. This repressed the NF-κB-dependent transcription of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), an essential regulator of osteoclastogenesis. These findings suggest that an Ifrd1/NF-κB/NFATc1 axis plays a pivotal role in bone remodeling in vivo and represents a therapeutic target for bone diseases. PMID:27381458

  8. Characterization of bone mineral-resembling biomaterials for optimizing human osteoclast differentiation and resorption.

    PubMed

    Nakamura, Miho; Hentunen, Teuvo; Salonen, Jukka; Nagai, Akiko; Yamashita, Kimihiro

    2013-11-01

    Bioresorption is a biological mechanism by which biomaterials are resorbed and thereby disappear from implantation sites partially or completely over a period of time. Osteoclast-medicated bioresorption is a possible new advantage to incorporate material degradation into remodeling in bone metabolism process. The purpose of this study was to investigate the osteoclastogenesis and bioresorption of synthesized calcium phosphate materials. Differentiation into mature human osteoclasts on carbonated hydroxyapatite (CA) was significantly enhanced compared to hydroxyapatite (HA) and β-tricalcium phosphate, based on the quantitative gene expressions of molecular markers for osteoclast differentiation. Osteoclasts adhered and differentiated into giant multinuclear TRAP-positive cells on every type of synthesized sample based on the histological analysis. Morphological observations using fluorescence and quantitative analysis revealed that the actin rings of osteoclasts on CA were thick, small in diameter and co-localized with vinculin, similar to the rings found on bone slices. In contrast, the actin rings of osteoclasts on HA and culture dishes were thin and large in diameter. Scanning electron microscopic images and quantitative analysis indicated that the resorption pits on CA were significantly deeper than those on HA due to the enhanced tight sealing ability between osteoclasts and their substrate. Copyright © 2013 Wiley Periodicals, Inc.

  9. Tanshinone IIA inhibits osteoclast differentiation through down-regulation of c-Fos and NFATc1.

    PubMed

    Kwak, Han Bok; Yang, Daum; Ha, Hyunil; Lee, Jong Ho; Kim, Ha Neui; Woo, Eun Ran; Lee, Seungbok; Kim, Hong Hee; Lee, Zang Hee

    2006-06-30

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

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

    PubMed

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

    2012-11-16

    Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor κB 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.

  11. Role of FK506 binding protein 5 (FKBP5) in osteoclast differentiation.

    PubMed

    Kimura, Miho; Nagai, Tatsuo; Matsushita, Reiko; Hashimoto, Atsushi; Miyashita, Toshiyuki; Hirohata, Shunsei

    2013-11-01

    We previously disclosed the enhanced expression of FK506 binding protein 5 (FKBP5) messenger RNA (mRNA) in bone marrow (BM) CD34(+) cells in rheumatoid arthritis (RA), in which systemic osteoporosis takes place. Since BM CD34(+) cells are precursors of osteoclasts, it is possible that FKBP5 overexpression might lead to osteoporosis by affecting osteoclastogenesis. We therefore explore the influences of FKBP5 in osteoclast differentiation. Stable transfectants of RAW264.7 overexpressing murine FKBP5 gene were established. Osteoclast differentiation was induced by receptor activator of nuclear factor kappa B (NF-κB) ligand and was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay. FKBP5 transfectants of RAW264.7 generated higher numbers of TRAP-positive multinucleated cells with increased activity of pit formation on calcium phosphate-coated culture slides than mock transfectants. The enhancement of osteoclast differentiation of FKBP5 transfectants was only partially inhibited by N-acetyl L-cysteine. Finally, glucocorticoid enhanced FKBP5 mRNA expression as well as osteoclast differentiation of RAW264.7 cells in a dose-dependent manner. These results indicate that FKBP5 promotes osteoclast differentiation by a mechanism distinct from NF-κB activation. Moreover, the data suggest that FKBP5 might play a role in bone destruction and development of osteoporosis in RA as well as in glucocorticoid-induced osteoporosis.

  12. 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

  13. Regulation of osteoclast differentiation and actin ring formation by the cytolinker protein plectin.

    PubMed

    Matsubara, Takuma; Kinbara, Masayuki; Maeda, Toshihiro; Yoshizawa, Mitsuhiro; Kokabu, Shoichiro; Takano Yamamoto, Teruko

    2017-08-05

    Osteoclasts are cells that resorb the bone matrix and maintain bone and calcium homeostasis. An actin ring is a characteristic actin structure that is essential for bone resorption by osteoclasts. Tyrosine kinase Src deficient osteoclasts do not form actin rings; thus, Src is a key molecule for actin ring formation in osteoclasts. However, how Src regulates actin ring formation is not fully understood. We identified the cytolinker protein plectin as a Src-binding protein by immunoprecipitation and liquid chromatography tandem mass spectrometry. Plectin is a huge protein (>500 kDa) and regulates the cytoskeleton by binding to actin and tubulin. We assessed the expression and role of plectin in osteoclasts. Plectin was expressed and co-localized with Src close to the actin ring in osteoclasts. Moreover, plectin was tyrosine-phosphorylated by Src. Differentiation and actin ring formation were inhibited by downregulation of plectin. These results suggest an important role for plectin in osteoclast differentiation and actin ring formation through Src binding. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes

    SciTech Connect

    Kang, Kyeongah; Nam, Sorim; Kim, Bomi; Lim, Ji Hyun; Yang, Young; Lee, Myeong-Sok; Lim, Jong-Seok

    2015-12-25

    N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34{sup +} progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. - Highlights: • The expression of NDRG2 significantly impairs osteoclast differentiation. • PU.1 and p38 MAPK inhibitions by NDRG2 are critical for the inhibition of osteoclastogenesis. • Knockdown of NDRG2 rescues the ability of monocytes to differentiate into osteoclasts. • NDRG2 expression in BM and primary macrophages also impairs osteoclast differentiation. • This study implies the potential of NDRG2 expression in the inhibition of osteoclastogenesis.

  15. 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

  16. Macrophage-osteoclast differentiation and bone resorption in osteoarthrotic subchondral acetabular cysts.

    PubMed

    Sabokbar, A; Crawford, R; Murray, D W; Athanasou, N A

    2000-06-01

    A macrophage infiltrate is commonly found in enlarging subchondral cysts in osteoarthrosis (OA) and the surrounding bone. To determine whether osteoclast differentiation by these cells contributes to the increase in the number of osteoclasts and bone resorption that accompanies OA cyst enlargement, we isolated macrophages from the wall of OA cysts and co-cultured them with osteoblast-like UMR106 cells in the presence or absence of 1,25(OH)2D3 and M-CSE After 14 days of incubation, co-cultures of UMR106 cells and cyst-derived macrophages showed evidence of osteoclast differentiation by expression of TRAP, VNR and formation of numerous lacunar pits. We found that, unlike osteoclast precursors in monocyte and other tissue macrophage populations, the addition of M-CSF to medium is not required for osteoclast differentiation. Our findings suggest that macrophage-osteoclast differentiation is one means whereby the osteolysis associated with the enlargement of OA cysts could be effected.

  17. Cdc42 regulates bone modeling and remodeling in mice by modulating RANKL/M-CSF signaling and osteoclast polarization

    PubMed Central

    Ito, Yuji; Teitelbaum, Steven L.; Zou, Wei; Zheng, Yi; Johnson, James F.; Chappel, Jean; Ross, F. Patrick; Zhao, Haibo

    2010-01-01

    The modeling and remodeling of bone requires activation and polarization of osteoclasts, achieved by reorganization of the cytoskeleton. Members of the Rho subfamily of small GTPases, including Cdc42, are known regulators of cytoskeletal components, but the role of these proteins in bone physiology and pathophysiology remains unclear. Here, we examined loss-of-function mice in which Cdc42 was selectively ablated in differentiated osteoclasts and gain-of-function animals wherein Cdc42Gap, a protein that inactivates the small GTPase, was deleted globally. Cdc42 loss-of-function mice were osteopetrotic and resistant to ovariectomy-induced bone loss, while gain-of-function animals were osteoporotic. Isolated Cdc42-deficient osteoclasts displayed suppressed bone resorption, while osteoclasts with increased Cdc42 activity had enhanced resorptive capacity. We further demonstrated that Cdc42 modulated M-CSF–stimulated cyclin D expression and phosphorylation of Rb and induced caspase 3 and Bim, thus contributing to osteoclast proliferation and apoptosis rates. Furthermore, Cdc42 was required for multiple M-CSF– and RANKL-induced osteoclastogenic signals including activation and expression of the differentiation factors MITF and NFATc1 and was a component of the Par3/Par6/atypical PKC polarization complex in osteoclasts. These data suggest that Cdc42 regulates osteoclast formation and function and may represent a promising therapeutic target for prevention of pathological bone loss. PMID:20501942

  18. Osteoblastic and osteoclastic differentiation on SLA and hydrophilic modified SLA titanium surfaces.

    PubMed

    Bang, Sung-Moon; Moon, Ho-Jin; Kwon, Yong-Dae; Yoo, Ji-Yeon; Pae, Ahran; Kwon, Il Keun

    2014-07-01

    We evaluated the activities of both osteoblastic and osteoclastic differentiation on sandblasted/acid etched (SLA), hydrophilic SLA surfaces (modSLA) and pretreatment titanium (PT). The osteoblastic differentiation was evaluated by alkaline phosphatase analysis and Alizarin Red S staining, and the expression of bone-related proteins, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN), was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Primary mice monocytes were expanded and differentiated in the presence of macrophage-colony stimulating factor (M-CSF), and osteoclastic differentiation was evaluated by actin ring formation assay and tartrate-resistant acid phosphatase (TRAP) activity assay. Real-time PCR tests were performed to investigate the expression of gene mRNA expression levels in osteoclast cells. Differentiation of osteoblasts in the Alizarin Red S test staining and ALP assay was significantly increased in the modSLA surface. The preceding results were supported by the result of RT-PCR for the expression of Runx2, OPN, and OCN. As for osteoclastic activity, differentiated osteoclasts rarely existed on the SLA and modSLA surface with actin ring. The results of real-time PCR and TRAP activity supported the preceding results. It may be concluded that the modSLA surface promotes osteogenic effect and prevents osteoclastic differentiation. Promotion of osteoblastic proliferation after a short-term cell culture might be responsible for stimulated bone regeneration implying that early loading may be possible. Also, the anti-osteoclastic effect of the modSLA surface may contribute to maintenance of the marginal bone level of dental implants, implying long-term stability would be provided by this surface technology. The modSLA surface may not only make early loading possible but possibly reduce marginal bone loss during the maintenance phase. © 2013 John Wiley & Sons A

  19. Serotype b of Aggregatibacter actinomycetemcomitans increases osteoclast and memory T-lymphocyte activation.

    PubMed

    Melgar-Rodríguez, S; Díaz-Zúñiga, J; Alvarez, C; Rojas, L; Monasterio, G; Carvajal, P; Escobar, A; Sanz, M; Vernal, R

    2016-04-01

    During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor-κB ligand (RANKL) -induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17-type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17-associated RANKL production, RANKL-induced osteoclast activation, and antigen-specific memory T lymphocyte proliferation. On naive CD4(+) T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T-bet, GATA-3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double-expression, TRAP(+) osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4(+) T lymphocytes stimulated by serotype b-primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP(+) osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co-expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption.

  20. 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.

  1. Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival.

    PubMed

    Kim, Hyung Joon; Prasad, Vikram; Hyung, Seok-Won; Lee, Zang Hee; Lee, Sang-Won; Bhargava, Aditi; Pearce, David; Lee, Youngkyun; Kim, Hong-Hee

    2012-12-24

    The precise regulation of Ca(2+) dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca(2+) ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand-induced Ca(2+) oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca(2+) efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca(2+) signaling in osteoclasts.

  2. 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

  3. In Vitro and In Vivo Effects of Gracilaria verrucosa Extracts on Osteoclast Differentiation

    PubMed Central

    Kim, Kwang-Jin; Lee, Yong-Jin; Hwang, Yun-Ho; Kang, Kyung-Yun; Yee, Sung-Tae; Son, Young-Jin

    2017-01-01

    Bone remodeling, a physiological process characterized by bone formation by osteoblasts and bone resorption by osteoclasts, is important for the maintenance of healthy bone in adult humans. Osteoclasts play a critical role in bone erosion and osteoporosis and are bone-specific multinucleated cells generated through differentiation of monocyte/macrophage lineage precursors. Receptor activator of NF-κB ligand (RANKL) has been reported to induce osteoclast differentiation. In this study, we explored whether Gracilaria verrucosa extracts (GE) could affect RANKL-mediated osteoclast differentiation. GE significantly inhibited RANKL-activated osteoclast differentiation by inhibiting protein expression of c-fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), vital factors in RANKL-mediated osteoclastogenesis. In addition, GE attenuated ovariectomy-induced bone loss in mice. In summary, GE can prevent osteoclastogenesis and hormone-related bone loss via blockage of c-fos-NFATc1 signaling. Our results suggest that GE may have therapeutic potential in the treatment of postmenopausal osteoporosis. PMID:28335442

  4. Non-resorbing osteoclasts induce migration and osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Kreja, L; Brenner, R E; Tautzenberger, A; Liedert, A; Friemert, B; Ehrnthaller, C; Huber-Lang, M; Ignatius, A

    2010-02-01

    Osteoclast activity has traditionally been regarded as restricted to bone resorption but there is some evidence that also non-resorbing osteoclasts might influence osteoblast activity. The aim of the present study was to further investigate the hypothesis of an anabolic function of non-resorbing osteoclasts by investigating their capability to recruit mesenchymal stem cells (MSC) and to provoke their differentiation toward the osteogenic lineage. Bone-marrow-derived human MSC were exposed to conditioned media (CM) derived from non-resorbing osteoclast cultures, which were generated from human peripheral blood monocytes. Osteogenic marker genes (transcription factor Runx2, bone sialoprotein, alkaline phosphatase (AP), and osteopontin) were significantly increased. Osteogenic differentiation (OD) was also proved by von Kossa and AP staining occurred in the same range as in MSC cultures stimulated with osteogenic supplements. Chemotactic responses of MSC were measured with a modified Boyden chamber assay. CM from osteoclast cultures induced a strong migratory response in MSC, which was greatly reduced in the presence of an anti-human platelet-derived growth factor (PDGF) receptor beta antibody. Correspondingly, significantly increased PDGF-BB concentrations were measured in the CM using a PDGF-BB immunoassay. CM derived from mononuclear cell cultures did not provoke MSC differentiation and had a significantly lower migratory effect on MSC suggesting that the effects were specifically mediated by osteoclasts. In conclusion, it can be suggested that human non-resorbing osteoclasts induce migration and OD of MSC. While effects on MSC migration might be mainly due to PDGF-BB, the factors inducing OD remain to be elucidated. (c) 2009 Wiley-Liss, Inc.

  5. Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.

    PubMed

    Ciapetti, Gabriela; Di Pompo, Gemma; Avnet, Sofia; Martini, Desirée; Diez-Escudero, Anna; Montufar, Edgar B; Ginebra, Maria-Pau; Baldini, Nicola

    2017-03-01

    The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca(++)) and phosphorus (P(5+)) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca(++). Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.

  6. Differentiation of osteoblast and osteoclast precursors on pure and silicon-substituted synthesized hydroxyapatites.

    PubMed

    Lehmann, Giorgia; Cacciotti, Ilaria; Palmero, Paola; Montanaro, Laura; Bianco, Alessandra; Campagnolo, Luisa; Camaioni, Antonella

    2012-10-01

    Calcium phosphate-based materials should show excellent bone-bonding and cell-mediated resorption characteristics at the same time, in order to be employed for bone replacement. In this perspective, pure (HAp) and silicon-substituted hydroxyapatite (Si-HAp, 1.4% wt) porous cylinders were prepared starting from synthesized powders and polyethylene spheres used as porogens, and investigated as supports for osteoblast and osteoclast progenitor differentiation. A systematic and detailed biological characterization is reported, in terms of cell adhesion, viability, proliferation, differentiation and bioresorption, aimed at proposing a complete and reliable picture of bone cell in vitro behavior, comprehensive of both the osteogenesis and the bone resorption processes. In order to achieve this purpose, cytocompatibility, differentiation and gene expression by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were carried out using parietal bone-derived pre-osteoblasts obtained from neonatal mice and the bioresorption capability was assessed by seeding human peripheral blood monocytes, as osteoclast precursors. It resulted that both pure and Si-substituted HAps were able to promote differentiation of precursor cells in mature osteoblasts and osteoclasts. In particular, the Si-HAps enhanced the pre-osteoblast proliferation and showed higher osteoclast-mediated bioresorption capability, as supported by the presence of larger and more numerous resorption lacunae, whereas HAps promoted a more robust cell differentiation in terms of both osteocalcin gene expression by qRT-PCR and cell morphological evaluation by SEM analysis.

  7. Sodium hydrogen sulfide inhibits nicotine and lipopolysaccharide-induced osteoclastic differentiation and reversed osteoblastic differentiation in human periodontal ligament cells.

    PubMed

    Lee, Sun-Kyung; Chung, Jong-Hyuk; Choi, Sung-Chul; Auh, Q-Schick; Lee, Young-Man; Lee, Sang-Im; Kim, Eun-Cheol

    2013-05-01

    Although previous studies have demonstrated that hydrogen sulfide (H(2)S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H(2)S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H(2)S on bone metabolism, we investigated the in vitro effects of H(2)S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine-stimulated human periodontal ligament cells (hPDLCs). The H(2)S donor, NaHS, protected hPDLCs from nicotine and LPS-induced cytotoxicity and recovered nicotine- and LPS-downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in mouse bone marrow cells and blocked nicotine- and LPS-induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M-CSF, MMP-9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS-induced activation of p38, ERK, MKP-1, PI3K, PKC, and PKC isoenzymes, and NF-κB. The effects of H(2)S on nicotine- and LPS-induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP-1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H(2)S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine- and periodontopathogen-stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases.

  8. Chrotacumines G-J, chromone alkaloids from Dysoxylum acutangulum with osteoclast differentiation inhibitory activity.

    PubMed

    Morita, Hiroshi; Nugroho, Alfarius Eko; Nagakura, Yuta; Hirasawa, Yusuke; Yoshida, Haruka; Kaneda, Toshio; Shirota, Osamu; Ismail, Intan Safinar

    2014-06-01

    Four new chromone alkaloids, chrotacumines G-J (1-4), have been isolated from the barks of Dysoxylum acutangulum. Their structures and absolute configurations were elucidated on the basis of NMR and CD data. Chrotacumines G and J (1 and 4) showed osteoclast differentiation inhibitory activity in a dose dependent manner. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Silk fibroin hydrolysate inhibits osteoclastogenesis and induces apoptosis of osteoclasts derived from RAW 264.7 cells.

    PubMed

    Chon, Jeon-Woo; Kim, Hyeryeon; Jeon, Ha-Na; Park, Kyungho; Lee, Kwang-Gill; Yeo, Joo-Hong; Kweon, Haeyong; Lee, Heui-Sam; Jo, You-Young; Park, Yoo Kyoung

    2012-11-01

    Bone disease can be associated with bone resorption by osteoclasts, and interest in the development of antiresorptive agents has recently increased. The hydrolysate of silk fibroin has been studied with respect to such biomedical applications. In a previous study, silk fibroin showed indirect inhibitory effects on the differentiation of osteoclasts. To further evaluate the effect of a hydrolysate of silk fibroin on osteoclasts, we investigated the direct effects of the silk fibroin hydrolysate on osteoclastogenesis and apoptosis of osteoclasts induced by receptor activation of nuclear factor κB ligand (RANKL). The silk fibroin hydrolysate inhibited RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP) in RAW 264.7 cells. The inhibitory effect of the silk fibroin hydrolysate resulted in the decreased expression of osteoclast marker genes, such as matrix metalloproteinase-9 (MMP-9), cathepsin-K and calcitonin receptor (CTR). In addition, the silk fibroin hydrolysate blocked the signaling pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and expression of transcription factors, such as nuclear factor of activated T cells c1 (NFATc1) and NF-κB. Finally, the silk fibroin hydrolysate induced apoptosis signaling cascades. Taken together, the present results indicate that silk fibroin hydrolysate has antiresorptive activity by both inhibiting osteoclastogenesis and inducing osteoclast apoptosis.

  10. 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

  11. Inhibitory regulation of osteoclast differentiation by interleukin-3 via regulation of c-Fos and Id protein expression.

    PubMed

    Oh, Jaemin; Lee, Myeung Su; Yeon, Jeong-Tae; Choi, Sik-Won; Kim, Hun Soo; Shim, Hyeok; Lee, Sam Youn; Youn, Byung Soo; Yokota, Yoshifumi; Kim, Jung Ha; Kwak, Han Bok

    2012-05-01

    Interleukin-3 (IL-3) is produced under various pathological conditions and is thought to be involved in the pathogenesis of inflammatory diseases; however, its function in bone homeostasis under normal conditions or nature of the downstream molecular targets remains unknown. Here we examined the effect of IL-3 on osteoclast differentiation from mouse and human bone marrow-derived macrophages (BMMs). Although IL-3 can induce osteoclast differentiation of multiple myeloma bone marrow cells, IL-3 greatly inhibited osteoclast differentiation of human BMMs isolated from healthy donors. These inhibitory effects of IL-3 were only observed at early time points (days 0 and 1). IL-3 inhibited the expression of c-Fos and NFATc1 in BMMs treated with RANKL. However, IL-3-mediated inhibition of osteoclast differentiation was not completely reversed by ectopic expression of c-Fos or NFATc1. Importantly, IL-3 induced inhibitor of DNA binding/differentiation (Id)1 in hBMMs, while Id2 were sustained during osteoclast differentiation of mBMMs treated with IL-3. Ectopic expression of NFATc1 in Id2-deficient BMMs completely reversed the inhibitory effect of IL-3 on osteoclast differentiation. Furthermore, inflammation-induced bone erosion was markedly inhibited by IL-3 administration. Taken together, our results suggest that IL-3 plays an inhibitory role in osteoclast differentiation by regulating c-Fos and Ids, and also exerts anti-bone erosion effects.

  12. Clodronate acts on human osteoclastic cell proliferation, differentiation and function in a bioreversible manner

    PubMed Central

    Recenti, Raffaella; Leone, Giuseppe; Simi, Lisa; Orfei, Marco; Pinzani, Pamela; Pieraccini, Giuseppe; Moneti, Gloriano; Carossino, Anna Maria; Franchi, Alessandro; Bartolucci, Gianluca; Carbonell Sala, Silvia; Ginanneschi, Mauro; Tanini, Annalisa; Brandi, Maria Luisa

    2007-01-01

    Background. Clodronate is used in high bone resorption diseases. Its action was defined as “cytotoxic” based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As “in vitro” bioeffects of clodronate are reversible, this compound should not be defined as “cytotoxic”. Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R2side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a β-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase. Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex “matrix metalloproteinase 9/tissue inhibitor of metalloproteinase” (MMP9/TIMP1) by RT-PCR analysis based on “TaqMan” technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5’-[ β - γ -dichloromethylene] triphosphate (AppCCl2p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of

  13. Enhancing Osteoclastic Resorption for the Prevention and Treatment of Heterotopic Ossification

    DTIC Science & Technology

    2015-03-01

    titration of OPG for inhibition of RANKL-induced osteoclast formation by RAW264.7 cells. TRAP stain (violet) for osteoclast formation. Multinuclear...caALK2 HO model (Figures 2A & B and Figures 3A & B). We have adopted a sensitive passive range-of-motion assay based on one presented by Paul Yu2...orthotopic bone), were stained for the osteoclast marker tartrate-resistant acid phosphatase (TRAP) (Figure 4

  14. Inhibition of osteoclast formation by 3-methylcholanthrene, a ligand for arylhydrocarbon receptor: suppression of osteoclast differentiation factor in osteogenic cells.

    PubMed

    Naruse, M; Otsuka, E; Naruse, M; Ishihara, Y; Miyagawa-Tomita, S; Hagiwara, H

    2004-01-01

    We investigated the effects of 3-methylcholanthrene (3MC), a ligand for arylhydrocarbon receptor (AhR), on osteoclastogenesis. Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) produced by ST2 cells in response to 1alpha,25(OH)(2) Vitamin D(3). 3MC dose-dependently inhibited the formation of mono- and multinuclear osteoclast-like cells. However, 3MC did not inhibit the formation of osteoclast-like cells from mouse spleen cells which was supported by the exogenous soluble RANKL and M-CSF. 3MC did not affect the formation of an actin ring and pits on slices of dentine by osteoclast-like cells, both of which are typical indices of osteoclast activity. These results suggest that 3MC affects osteoclast-supporting cells such as ST2 cells but not osteoclast precursor cells and mature osteoclastic cells. When we measured the expression levels of RANKL mRNA in ST2 cells, 3MC dose-dependently decreased the level of this mRNA. However, 3MC did not affect levels of mRNAs for osteoprotegerin (OPG), M-CSF, and the receptor of 1alpha,25(OH)(2) Vitamin D(3) in ST2 cells. Furthermore, soluble RANKL was able to counteract the inhibitory effect of 3MC on the formation of osteoclast-like cells. Our findings indicate that 3MC inhibits osteoclastogenesis via the inhibition of RANKL expression in osteoblastic cells.

  15. Increased levels of sodium chloride directly increase osteoclastic differentiation and resorption in mice and men.

    PubMed

    Wu, L; Luthringer, B J C; Feyerabend, F; Zhang, Z; Machens, H G; Maeda, M; Taipaleenmäki, H; Hesse, E; Willumeit-Römer, R; Schilling, A F

    2017-08-29

    To better understand the association between high salt intake and osteoporosis, we investigated the effect of sodium chloride (NaCl) on mice and human osteoclastogenesis. The results suggest a direct, activating role of NaCl supplementation on bone resorption. High NaCl intake is associated with increased urinary calcium elimination and parathyroid hormone (PTH) secretion which in turn stimulates the release of calcium from the bone, resulting in increased bone resorption. However, while calciuria after NaCl loading could be shown repeatedly, several studies failed to reveal a significant increase in PTH in response to a high-sodium diet. Another possible explanation that we investigated here could be a direct effect of high-sodium concentration on bone resorption. Mouse bone marrow macrophage and human peripheral blood mononuclear cells (PBMC) driven towards an osteoclastogenesis pathway were cultivated under culture conditions mimicking hypernatremia environments. In this study, a direct effect of increased NaCl concentrations on mouse osteoclast differentiation and function was observed. Surprisingly, in a human osteoclast culture system, significant increases in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, calcitonin receptor (CTR)-positive osteoclasts, nuclear factor-activated T cells c1 (NFATc1) gene expression, and areal and volumetric resorptions were observed for increasing concentrations of NaCl. This suggests a direct, activating, cell-mediated effect of increased concentrations of NaCl on osteoclasts. The reported that enhanced bone resorption after high-sodium diets may not only be secondary to the urinary calcium loss but may also be a direct, cell-mediated effect on osteoclastic resorption. These findings allow us to suggest an explanation for the clinical findings independent of a PTH-mediated regulation.

  16. 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. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  17. 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

  18. Regulation of matrix metalloproteinase-9 protein expression by 1α, 25-(OH)₂D₃ during osteoclast differentiation.

    PubMed

    Gu, Jian-Hong; Tong, Xi-Shuai; Chen, Guo-Hong; Liu, Xue-Zhong; Bian, Jian-Chun; Yuan, Yan; Liu, Zong-Ping

    2014-01-01

    To investigate 1α,25-(OH)₂D₃ regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)₂D₃ during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)₂D₃ inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)₂D₃ administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.

  19. Rab13 Is Upregulated During Osteoclast Differentiation and Associates with Small Vesicles Revealing Polarized Distribution in Resorbing Cells

    PubMed Central

    Mulari, Mika T. K.; Büki, Kálmán G.; Vihko, Pirkko; Härkönen, Pirkko L.; Väänänen, H. Kalervo

    2012-01-01

    Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts. PMID:22562557

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

    PubMed

    Omori, Akina; Yoshimura, Yoshitaka; Deyama, Yoshiaki; Suzuki, Kuniaki

    2015-07-01

    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.

  1. 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

  2. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis.

    PubMed

    Rho, Jaerang; Altmann, Curtis R; Socci, Nicholas D; Merkov, Lubomir; Kim, Nacksung; So, Hongseob; Lee, Okbok; Takami, Masamichi; Brivanlou, Ali H; Choi, Yongwon

    2002-08-01

    Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte-macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans.

  3. The collection of NFATc1-dependent transcripts in the osteoclast includes numerous genes non-essential to physiologic bone resorption

    PubMed Central

    Charles, Julia F.; Coury, Fabienne; Sulyanto, Rosalyn; Sitara, Despina; Wu, Jing; Brady, Nicholas; Tsang, Kelly; Sigrist, Kirsten; Tollefsen, Douglas M.; He, Li; Storm, Daniel; Aliprantis, Antonios O.

    2012-01-01

    Osteoclasts are specialized secretory cells of the myeloid lineage important for normal skeletal homeostasis as well as pathologic conditions of bone including osteoporosis, inflammatory arthritis and cancer metastasis. Differentiation of these multinucleated giant cells from precursors is controlled by the cytokine RANKL, which through its receptor RANK initiates a signaling cascade culminating in the activation of transcriptional regulators which induce the expression of the bone degradation machinery. The transcription factor nuclear factor of activated T-cells c1 (NFATc1) is the master regulator of this process and in its absence osteoclast differentiation is aborted both in vitro and in vivo. Differential mRNA expression analysis by microarray is used to identify genes of potential physiologic relevance across nearly all biologic systems. We compared the gene expression profile of murine wild-type and NFATc1-deficient osteoclast precursors stimulated with RANKL and identified that the majority of the known genes important for osteoclastic bone resorption require NFATc1 for induction. Here, five novel RANKL-induced, NFATc1-dependent transcripts in the osteoclast are described: Nhedc2, Rhoc, Serpind1, Adcy3 and Rab38. Despite reasonable hypotheses for the importance of these molecules in the bone resorption pathway and their dramatic induction during differentiation, the analysis of mice with mutations in these genes failed to reveal a function in osteoclast biology. Compared to littermate controls, none of these mutants demonstrated a skeletal phenotype in vivo or alterations in osteoclast differentiation or function in vitro. These data highlight the need for rigorous validation studies to complement expression profiling results before functional importance can be assigned to highly regulated genes in any biologic process. PMID:22985540

  4. Anti-Osteoclastic Activity of Artemisia capillaris Thunb. Extract Depends upon Attenuation of Osteoclast Differentiation and Bone Resorption-Associated Acidification Due to Chlorogenic Acid, Hyperoside, and Scoparone

    PubMed Central

    Lee, Sang-Hyun; Lee, Jung-Yun; Kwon, Young-In; Jang, Hae-Dong

    2017-01-01

    The present study attempts to elucidate the anti-osteoporotic activity of Artemisia capillaris Thunb. in the form of anti-osteoclastic effect and responsible bioactive compounds. The contents of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid A, and scoparone in Artemisia capillaris hydroethanolic extract (ACHE) were 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively. ACHE diminished osteoclast differentiation and bone resorption due to chlorogenic acid, hyperoside, and scoparone. In addition, ACHE attenuated acidification as well as reducing tumor necrosis factor receptor-associated factor 6 (TRAF6) expression and its association with vacuolar H+-adenosine triphosphatase (V-ATPase). Furthermore, chlorogenic acid, hyperoside, and scoparone from A. capillaris abrogated the association of V-ATPase with TRAF6, suggesting that the blockage of bone resorption by A. capillaris was partially mediated by reducing acidification through down-regulating interaction of V-ATPase with TRAF6 due to scoparone as well as chlorogenic acid and hyperoside. These results imply that the anti-osteoclastic effect of A. capillaris through down-regulating osteoclast differentiation and bone resorption may contribute to its anti-osteoporotic effect. PMID:28165389

  5. Anti-Osteoclastic Activity of Artemisia capillaris Thunb. Extract Depends upon Attenuation of Osteoclast Differentiation and Bone Resorption-Associated Acidification Due to Chlorogenic Acid, Hyperoside, and Scoparone.

    PubMed

    Lee, Sang-Hyun; Lee, Jung-Yun; Kwon, Young-In; Jang, Hae-Dong

    2017-02-04

    The present study attempts to elucidate the anti-osteoporotic activity of Artemisia capillaris Thunb. in the form of anti-osteoclastic effect and responsible bioactive compounds. The contents of chlorogenic acid, caffeic acid, hyperoside, isoquercitrin, isochlorogenic acid A, and scoparone in Artemisia capillaris hydroethanolic extract (ACHE) were 38.53, 0.52, 4.07, 3.03, 13.90, and 6.59 mg/g, respectively. ACHE diminished osteoclast differentiation and bone resorption due to chlorogenic acid, hyperoside, and scoparone. In addition, ACHE attenuated acidification as well as reducing tumor necrosis factor receptor-associated factor 6 (TRAF6) expression and its association with vacuolar H⁺-adenosine triphosphatase (V-ATPase). Furthermore, chlorogenic acid, hyperoside, and scoparone from A. capillaris abrogated the association of V-ATPase with TRAF6, suggesting that the blockage of bone resorption by A. capillaris was partially mediated by reducing acidification through down-regulating interaction of V-ATPase with TRAF6 due to scoparone as well as chlorogenic acid and hyperoside. These results imply that the anti-osteoclastic effect of A. capillaris through down-regulating osteoclast differentiation and bone resorption may contribute to its anti-osteoporotic effect.

  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. 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.

  8. 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.

  9. Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    PubMed Central

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

    2014-01-01

    The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE) on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation. PMID:25530776

  10. Suppressive effects of the leaf of Terminalia catappa L. on osteoclast differentiation in vitro and bone weight loss in vivo.

    PubMed

    Koyama, Tomoyuki; Nakajima, Chie; Nishimoto, Sogo; Takami, Masamichi; Woo, Je-Tae; Yazawa, Kazunaga

    2012-01-01

    Oral administration of Terminalia catappa extract (TCE; 1,000 mg/kg) for 5 wk suppressed bone weight loss and trabecular bone loss in ovariectomized mice. An in vitro experiment showed that TCE (1.3-20 µg/mL) did not increase alkaline phosphatase activity, which would indicate osteoclast formation, in osteoblast-like 3T3-L1 cells. On the other hand, TCE (12.5 µg/mL) markedly decreased the number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells, which would indicate osteoclast formation, in a co-culture system (bone marrow cells/osteoblastic UAMS-32 cells). A detailed analysis of the stages of osteoclast differentiation revealed that TCE mainly suppressed the differentiation of bone marrow mononuclear cells into osteoclast progenitor cells in the presence of M-CSF and TGF-β. An additional experiment using fractionated TCE revealed that the water-soluble fraction suppressed the bone weight loss in OVX-mice and osteoclast differentiation in vitro. Therefore, the suppressive effects of TCE on bone weight loss in mice might be due to the suppressive effects of highly polar components on the early stage of osteoclast differentiation.

  11. Earthworm (Pheretima aspergillum) extract stimulates osteoblast activity and inhibits osteoclast differentiation.

    PubMed

    Fu, Yuan-Tsung; Chen, Kuo-Yu; Chen, Yueh-Sheng; Yao, Chun-Hsu

    2014-11-11

    The potential benefits of earthworm (Pheretima aspergillum) for healing have received considerable attention recently. Osteoblast and osteoclast activities are very important in bone remodeling, which is crucial to repair bone injuries. This study investigated the effects of earthworm extract on bone cell activities. Osteoblast-like MG-63 cells and RAW 264.7 macrophage cells were used for identifying the cellular effects of different concentrations of earthworm extract on osteoblasts and osteoclasts, respectively. The optimal concentration of earthworm extract was determined by mitochondrial colorimetric assay, alkaline phosphatase activity, matrix calcium deposition, Western blotting and tartrate-resistant acid phosphatase activity. Earthworm extract had a dose-dependent effect on bone cell activities. The most effective concentration of earthworm extract was 3 mg/ml, significantly increasing osteoblast proliferation and differentiation, matrix calcium deposition and the expression levels of alkaline phosphatase, osteopontin and osteocalcin. Conversely, 3 mg/ml earthworm extract significantly reduced the tartrate-resistant acid phosphatase activity of osteoclasts without altering cell viability. Earthworm extract has beneficial effects on bone cell cultures, indicating that earthworm extract is a potential agent for use in bone regeneration.

  12. Psoriatic cutaneous inflammation promotes human monocyte differentiation into active osteoclasts, facilitating bone damage.

    PubMed

    Raimondo, Annunziata; Lembo, Serena; Di Caprio, Roberta; Donnarumma, Giovanna; Monfrecola, Giuseppe; Balato, Nicola; Ayala, Fabio; Balato, Anna

    2017-06-01

    Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy that can be associated with focal bone erosions. Psoriasis usually precedes the psoriatic arthritis onset by an average of 10 years, but this relation is not yet fully elucidated. Pro-inflammatory cytokines, such as IL-33, OPN, IL-17, and TNF-α are involved in both psoriasis and PsA pathogenesis as well as in bone homeostasis. In this study, we have demonstrated that IL-33, OPN, IL-17, and TNF-α induced the release of a wide range of pro-osteoclastogenic factors from the skin, such as RANKL, that promote monocyte differentiation in osteoclasts. The addition of osteoprotegerin, a RANKL inhibitor, to monocyte cultures treated with supernatant from stimulated skin did not completely deplete osteoclast formation, suggesting that skin produced several additional pro-osteoclastogenic mediators, which could act in a RANKL-independent manner. Moreover, we have found that RANKL serum levels as well as osteoclast number and activity in psoriatic patients with and without arthritis, was influenced by severity of cutaneous disease. Our data demonstrate that psoriatic cutaneous inflammation contributes to bone damage. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. α-Tocotrienol inhibits osteoclastic bone resorption by suppressing RANKL expression and signaling and bone resorbing activity.

    PubMed

    Ha, Hyunil; Lee, Jong-Ho; Kim, Ha-Neui; Lee, Zang Hee

    2011-03-25

    Vitamin E, an essential nutrient with powerful antioxidant activity, is the mixture of two classes of compounds, tocopherols (TPs) and tocotrienols (TTs). Although TTs exhibit better bone protective activity than α-TP, the underlying mechanism is poorly understood. In this study, we investigated whether α-TT and α-TP can modulate osteoclastic bone resorption. We found that α-TT but not α-TP inhibits osteoclastogenesis in coculture of osteoblasts and bone marrow cells induced by either IL-1 or combined treatment with 1α,25(OH)(2) vitamin D(3) and prostaglandin E(2). In accordance with this, only α-TT inhibited receptor activator of NF-κB ligand (RANKL) expression in osteoblasts. In addition, α-TT but not α-TP inhibited RANKL-induced osteoclast differentiation from precursors by suppression of c-Fos expression, possibly through inhibiting ERK and NF-κB activation. This anti-osteoclastogenic effect was reversed when c-Fos or an active form of NFATc1, a critical downstream of c-Fos during osteoclastogenesis, was overexpressed. Furthermore, only α-TT reduced bone resorbing activity of mature osteoclasts without affecting their survival. Overall, our results demonstrate that α-TT but not α-TP has anti-bone resorptive properties by inhibiting osteoclast differentiation and activation, suggesting that α-TT may have therapeutic value for treating and preventing bone diseases characterized by excessive bone destruction.

  14. Cadmium stimulates the osteoclastic differentiation of RAW264.7 cells in presence of osteoblasts.

    PubMed

    Chen, Xiao; Zhu, Guoying; Jin, Taiyi; Zhou, Zhijun; Gu, Shuzhu; Qiu, Jing; Xiao, Hanfang

    2012-06-01

    Low level of cadmium exposure may have direct effects on bone. But the probable mechanism is far from clarified. Using a co-culture system, the present study investigated the effects of low level of cadmium exposure on osteoclast differentiation in the presence of osteoblasts. Primary osteoblasts were isolated from calvarial bone of newborn Sprague Dawley rats. Primary osteoblasts and RAW264.7 cells were exposed to cadmium (0-60 nmol/l) in a co-culture system. Then, osteoblast viability was observed by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Osteoclast formation and tartrate-resistant acid phosphatase 5b levels were determined by tartrate-resistant acid phosphatase staining and enzyme-linked immunosorbent assay. Osteoprotegerin and receptor activator of NF-kB ligand mRNA expression in osteoblasts were studied via reverse transcription polymerase chain reaction. Viability of osteoblast was obviously decreased by Cd exposure (P < 0.05). Cadmium significantly stimulated the formation of osteoclasts in co-culture system (7.5-60 nmol/l) compared with the control. The levels of tartrate-resistant acid phosphatase 5b in RAW264.7 cells co-cultured with osteoblasts were significantly enhanced by cadmium exposure compared with that without cadmium. The mRNA expression of receptor activator of NF-kB ligand was upregulated by cadmium at 15 and 60 nmol/l. But cadmium had no obvious influence on osteoprotegerin mRNA expression. This data suggested that osteoblasts might be involved in the progress of cadmium effects on osteoclasts.

  15. Gadolinium inhibits prostate cancer PC3 cell migration and suppresses osteoclast differentiation in vitro.

    PubMed

    Wang, Peng; Zou, Xiao-Min; Huang, Jian; Zhang, Tian-Lan; Wang, Kui

    2011-11-01

    This study examined whether Gd (gadolinium) could suppress prostate cancer cell migration and prostate cancer cell-induced osteoclast differentiation. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and colony forming assay showed that GdCl3 treatment inhibited both cell viability and colony forming ability in PC3 cells more significantly than that in DU145 cells. Annexin/PI (propidium iodide) staining showed an increase in apoptotic death of PC3 cells in the presence of GdCl3. Wound healing and adhesion assay indicated that GdCl3 suppressed PC3 cell migration. Western-blot analysis demonstrated that GdCl3 treatment inhibited phosphorylation of ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase). Pretreatment with PTx (pertussis toxin), a Gi protein inhibitor, conferred resistance to GdCl3-induced colony formation, ERK and p38 phosphorylation in PC3 cells. Moreover, GdCl3 inhibited PC3 cell-induced osteoclast differentiation. RT-PCR (reverse transcription-PCR) indicated that GdCl3 decreased the expression of RANKL (receptor activator of nuclear factor-κB ligand) in PC3 cells, whereas it increased the expression of OPG (osteoprotegerin) in PC3 and DU145 cells. In conclusion, the present study indicated that GdCl3 inhibited PC3 cell migration mediated by the inactivation of both ERK and p38 MAPK pathways via PTx-sensitive G proteins, and also suppressed PC3 cell-induced osteoclast differentiation via regulating the mRNA expression of OPG and RANKL.

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

    PubMed Central

    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

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

    PubMed

    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

    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 (FcRγ) and DNAX-activating protein 12kDa (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 (β3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10days 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-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p<0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p<0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the

  18. Thyroid-stimulating hormone maintains bone mass and strength by suppressing osteoclast differentiation.

    PubMed

    Zhang, Wenwen; Zhang, Yanling; Liu, Yuantao; Wang, Jie; Gao, Ling; Yu, Chunxiao; Yan, Huili; Zhao, Jiajun; Xu, Jin

    2014-04-11

    It has been suggested that pituitary hormone might be associated with bone metabolism. To investigate the role of thyroid-stimulating hormone (TSH) in bone metabolism, we designed the present study as follows. After weaning, TSH receptor (TSHR) null mice (Tshr(-/-)) were randomly divided into a thyroxine treatment group (n=10) or non-treatment group (n=10); the treatment group received a dose of desiccated thyroid extract at 100 ppm daily for 5 weeks. Age-matched wild-type (Tshr(+/+), n=10) and heterozygote mice (Tshr(+/-), n=10) served as controls. After 5 weeks, the animals were sacrificed, and the femurs were collected for histomorphometrical and biomechanical analyses. In addition, the effect of TSH on osteoclastogenesis was examined in the RAW264.7 osteoclast cell line. We found that compared with Tshr(+/+) mice, Tshr(-/-) and Tshr(+/-) mice had lower bone strength. The histomorphometric results showed that trabecular bone volume, osteoid surface, osteoid thickness and osteoblast surface were significantly decreased, whereas the osteoclast surface was significantly increased in both Tshr(-/-) and Tshr(+/-) mice compared with Tshr(+/+) mice. Bone resorption and formation in Tshr(-/-) mice were further enhanced by thyroxine replacement. bTSH inhibited osteoclast differentiation in vitro, as demonstrated by reduced development of TRAP-positive cells and down-regulation of differentiation markers, including tartrate-resistant acid phosphatase, matrix metallo-proteinase-9 and cathepsin K in RAW264.7 cells. Our results confirm that TSH increased bone volume and improved bone microarchitecture and strength at least partly by inhibiting osteoclastogenesis.

  19. 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

  20. Icariin influences adipogenic differentiation of stem cells affected by osteoblast-osteoclast co-culture and clinical research adipogenic.

    PubMed

    Zhang, Shuncong; Feng, Pengbo; Mo, Guoye; Li, Daxing; Li, Yongxian; Mo, Ling; Yang, Zhidong; Liang, De

    2017-04-01

    To build mouse osteoblast MC3T3-E1 and mouse osteoclast RAW264.7 co-culture system and to study the effect of icariin on the activity of osteoblasts and osteoclasts in the co-culture system. In vitro acquisition and cultivation of mouse osteoblasts MC3T3-E1 and mouse RAW264.7 cells were conducted. Osteoblast and osteoclast activities of cells were detected by CCK-8 staining experiment, alizarin red staining and tartaric-resistant acid phosphatase (TRAP) staining. We used different concentrations of icariin to interfere in osteoblast-osteoclast co-culture system. The effects of icariin on various genes were detected by PCR and Western blot methods The correction between the expression of PPARγ and BMD was analyzed in patients with osteoporosis. Mouse osteoblast-osteoclast co-culture system was built, and the osteogenic differentiation effect was enhanced. Icariin can improve the MC3T3-E1 osteogenic differentiation activity, enhance the expression of OPG and RANKL gene protein, reduce the NF-κb gene and protein expression, increase of ALP, TGF-b1 and RANKL gene expression level and reduce RANK gene expression. Icariin can act on MC3T3-E1 cells-RAW264.7 cells co-culture system, and promote the osteogenic activity of MC3T3-E1 cells, inhibit the osteoclast activity of RAW264.7 cells and reduce the level of BMSCs adipogenic differentiation. The expression level of PPAR-γ gene was negatively correlated with the level of BMD. Mouse MC3T3-E1 cells and mouse RAW264.7 cells could be co-cultured in vitro, and icariin could improve the osteogenic activity of MC3T3 cells-RAW264.7 cells and decrease the osteoclast activity. Icariin could inhibit adipogenic differentiation of BMSCs in the osteoblast-osteoclast co-culture, promoting osteogenic differentiation and inhibiting osteoclast differentiation. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

    PubMed

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2013-01-01

    Fewer molecules have been identified on human than murine osteoclasts, the former differing from murine osteoclasts in many ways. We show that voltage-dependent anion channels (VDACs, porin) are expressed in the plasma membrane of human osteoclasts. A search for novel proteins expressed in the plasma membrane of human osteoclasts identified VDAC. Anti-VDAC antibodies inhibited human osteoclastogenesis in vitro. VDAC expression was detected in membranes by immunoelectron microscopy and immunocytochemical double staining. The VDAC protein functions as a Cl(-) channel. VDACs regulate bone resorption, which show using Osteologic™ plates. The epitope of the antibody lay within a 10-amino acid sequence in the VDAC. The findings suggest that the VDAC is, at least partly, a novel Cl(-) channel regulating the differentiation and function of human osteoclasts. VDACs may play a crucial role in acidifying the resorption lacunae between osteoclasts and bone. Inhibitors of VDACs could be used to treat diseases involving increased resorption, such as osteoporosis, rheumatoid arthritis, and Paget's disease. © 2012 International Federation for Cell Biology.

  2. Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo.

    PubMed

    Baek, Jong Min; Kim, Ju-Young; Ahn, Sung-Jun; Cheon, Yoon-Hee; Yang, Miyoung; Oh, Jaemin; Choi, Min Kyu

    2016-03-01

    Dendrobium moniliforme (DM) is a well-known plant-derived extract that is widely used in Oriental medicine. DM and its chemical constituents have been reported to have a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor activities; however, no reports discuss the beneficial effects of DM on bone diseases such as osteoporosis. Thus, we investigated the relationship between DM and osteoclasts, cells that function in bone resorption. We found that DM significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation; DM directly induced the down-regulation of c-Fos and nuclear factor of activated T cells c1 (NFATc1) without affecting other RANKL-dependent transduction pathways. In the later stages of osteoclast maturation, DM negatively regulated the organization of filamentous actin (F-actin), resulting in impaired bone-resorbing activity by the mature osteoclasts. In addition, micro-computed tomography (μ-CT) analysis of the murine model revealed that DM had a beneficial effect on lipopolysaccharide (LPS)-mediated bone erosion. Histological analysis showed that DM attenuated the degradation of trabecular bone matrix and formation of TRAP-positive osteoclasts in bone tissues. These results suggest that DM is a potential candidate for the treatment of metabolic bone disorders such as osteoporosis.

  3. Transforming Growth Factor β1/Smad4 Signaling Affects Osteoclast Differentiation via Regulation of miR-155 Expression

    PubMed Central

    Zhao, Hongying; Zhang, Jun; Shao, Haiyu; Liu, Jianwen; Jin, Mengran; Chen, Jinping; Huang, Yazeng

    2017-01-01

    Transforming growth factor β1 (TGFβ1)/Smad4 signaling plays a pivotal role in maintenance of the dynamic balance between bone formation and resorption. The microRNA miR-155 has been reported to exert a significant role in the differentiation of macrophage and dendritic cells. The goal of this study was to determine whether miR-155 regulates osteoclast differentiation through TGFβ1/Smad4 signaling. Here, we present that TGFβ1 elevated miR-155 levels during osteoclast differentiation through the stimulation of M-CSF and RANKL. Additionally, we found that silencing Smad4 attenuated the upregulation of miR-155 induced by TGFβ1. The results of luciferase reporter experiments and ChIP assays demonstrated that TGFβ1 promoted the binding of Smad4 to the miR-155 promoter at a site located in 454 bp from the transcription start site in vivo, further verifying that miR-155 is a transcriptional target of the TGFβ1/Smad4 pathway. Subsequently, TRAP staining and qRT-PCR analysis revealed that silencing Smad4 impaired the TGFβ1-mediated inhibition on osteoclast differentiation. Finally, we found that miR-155 may target SOCS1 and MITF to suppress osteoclast differentiation. Taken together, we provide the first evidence that TGFβ1/Smad4 signaling affects osteoclast differentiation by regulation of miR-155 expression and the use of miR-155 as a potential therapeutic target for osteoclast-related diseases shows great promise. PMID:28359146

  4. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

    PubMed

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

    2016-06-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction

    PubMed Central

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M.; Katayama, Ichiro; Inohara, Hidenori

    2016-01-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. PMID:27001307

  6. 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

  7. 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.

  8. 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.

  9. Runx1 Regulates Myeloid Precursor Differentiation Into Osteoclasts Without Affecting Differentiation Into Antigen Presenting or Phagocytic Cells in Both Males and Females

    PubMed Central

    Paglia, David N.; Yang, Xiaochuan; Kalinowski, Judith; Jastrzebski, Sandra

    2016-01-01

    Runt-related transcription factor 1 (Runx1), a master regulator of hematopoiesis, is expressed in preosteoclasts. Previously we evaluated the bone phenotype of CD11b-Cre Runx1fl/fl mice and demonstrated enhanced osteoclasts and decreased bone mass in males. However, an assessment of the effects of Runx1 deletion in female osteoclast precursors was impossible with this model. Moreover, the role of Runx1 in myeloid cell differentiation into other lineages is unknown. Therefore, we generated LysM-Cre Runx1fl/fl mice, which delete Runx1 equally (∼80% deletion) in myeloid precursor cells from both sexes and examined the capacity of these cells to differentiate into osteoclasts and phagocytic and antigen-presenting cells. Both female and male LysM-Cre Runx1fl/fl mice had decreased trabecular bone mass (72% decrease in bone volume fraction) and increased osteoclast number (2–3 times) (P < .05) without alteration of osteoblast histomorphometric indices. We also demonstrated that loss of Runx1 in pluripotential myeloid precursors with LysM-Cre did not alter the number of myeloid precursor cells in bone marrow or their ability to differentiate into phagocytizing or antigen-presenting cells. This study demonstrates that abrogation of Runx1 in multipotential myeloid precursor cells significantly and specifically enhanced the ability of receptor activator of nuclear factor-κB ligand to stimulate osteoclast formation and fusion in female and male mice without affecting other myeloid cell fates. In turn, increased osteoclast activity in LysM-Cre Runx1fl/fl mice likely contributed to a decrease in bone mass. These dramatic effects were not due to increased osteoclast precursors in the deleted mutants and argue that inhibition of Runx1 in multipotential myeloid precursor cells is important for osteoclast formation and function. PMID:27267711

  10. Lysophosphatidic acid receptor type 1 (LPA1) plays a functional role in osteoclast differentiation and bone resorption activity.

    PubMed

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

    2014-03-07

    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.

  11. 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

  12. 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

  13. Deltamethrin inhibits osteoclast differentiation via regulation of heme oxygenase-1 and NFATc1.

    PubMed

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

    2012-09-01

    Deltamethrin is a widely used pyrethroid pesticide. Although the cytotoxicity of deltamethrin has been reported, especially in neuronal cells, there is no information concerning the effects of deltamethrin on osteoclasts (OCLs). In this study, we showed that deltamethrin inhibited OCL differentiation in vitro. The effects of deltamethrin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated in bone marrow-derived macrophages (BMMs) or the murine monocytic cell line RAW-D. Treatment with deltamethrin inhibited OCL formation and bone resorption and up-regulated expression of heme oxygenase-1 (HO-1), an anti-oxidative stress enzyme. Deltamethrin also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator for OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of deltamethrin on intracellular signaling during the OCL differentiation of BMMs indicated that deltamethrin-treated OCLs displayed impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Jun N-terminal kinase, and Akt, and slightly delayed phosphorylation of inhibitor of nuclear factor kappa B alpha (IκBα) compared with untreated OCLs. Thus, deltamethrin possibly affects bone metabolism by inhibiting OCL differentiation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. 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.

  15. 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

    2016-05-04

    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.

  16. CCN3 Impairs Osteoblast and Stimulates Osteoclast Differentiation to Favor Breast Cancer Metastasis to Bone

    PubMed Central

    Ouellet, Véronique; Tiedemann, Kerstin; Mourskaia, Anna; Fong, Jenna E.; Tran-Thanh, Danh; Amir, Eitan; Clemons, Mark; Perbal, Bernard; Komarova, Svetlana V.; Siegel, Peter M.

    2011-01-01

    Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases. PMID:21514448

  17. 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.

  18. 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.

  19. A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation.

    PubMed

    Min, Seung-Ki; Kang, Hyun Ki; Jung, Sung Youn; Jang, Da Hyun; Min, Byung-Moo

    2017-09-22

    Osteoporosis affects millions of people worldwide by promoting bone resorption and impairing bone formation. Bisphosphonates, commonly used agents to treat osteoporosis, cannot reverse the substantial bone loss that has already occurred by the time of diagnosis. Moreover, their undesirable side-effects, including osteonecrosis of the jaw, have been reported. Here, we demonstrated that a new bioactive core vitronectin-derived peptide (VnP-16) promoted bone formation by accelerating osteoblast differentiation and activity through direct interaction with β1 integrin followed by FAK activation. Concomitantly, VnP-16 inhibited bone resorption by restraining JNK-c-Fos-NFATc1-induced osteoclast differentiation and αvβ3 integrin-c-Src-PYK2-mediated resorptive function. Moreover, VnP-16 decreased the bone resorbing activity of pre-existing mature osteoclasts without changing their survival rate. Furthermore, VnP-16 had a strong anabolic effect on bone regeneration by stimulating osteoblast differentiation and increasing osteoblast number, and significantly alleviated proinflammatory cytokine-induced bone resorption by restraining osteoclast differentiation and function in murine models. Moreover, VnP-16 could reverse ovariectomy-induced bone loss by both inhibiting bone resorption and promoting bone formation. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that VnP-16 could be an attractive therapeutic agent for treating osteoporosis.Cell Death and Differentiation advance online publication, 22 September 2017; doi:10.1038/cdd.2017.153.

  20. Xanthohumol modulates the expression of osteoclast-specific genes during osteoclastogenesis in RAW264.7 cells.

    PubMed

    Suh, Kwang Sik; Rhee, Sang Youl; Kim, Young Seol; Lee, Young Soon; Choi, Eun Mi

    2013-12-01

    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 study, we examined the ability of xanthohumol, a structurally simple prenylated chalcone, to suppress RANKL signaling during osteoclastogenesis in RAW264.7 cells. Xanthohumol markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclasts formation, and resorption-pit formation. In experiments to elucidate its mechanism of action, xanthohumol was found to suppress RANKL-induced expression of TRAF6, GAB2, ERK, c-Src, PI3K, and Akt genes. Moreover, RANKL-induced expressions of c-Fos and NFATc1, which are crucial transcription factors for osteoclastogenesis, were reduced by treatment with xanthohumol. Xanthohumol also inhibited RANKL-induced expression of bone-resorption related osteoclast-specific genes (carbonic anhydrase II, TCIRG, CLCN7, OSTM1, cathepsin K, and MMP-9). These data demonstrate that xanthohumol inhibits osteoclastogenesis by modulating RANKL signaling and may be useful for the prevention of bone-destructive diseases such as osteoporosis, arthritis and periodontitis.

  1. 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.

  2. Effects of carboxymethylcellulose and hydroxypropylmethylcellulose on the differentiation and activity of osteoclasts and osteoblasts.

    PubMed

    Agis, Hermann; Beirer, Benedict; Watzek, Georg; Gruber, Reinhard

    2010-11-01

    Carboxymethylcellulose (CMC) and hydroxypropylmethylcellulose (HPMC) serve as carriers for growth factors and bone substitutes. Although both carriers are placed into the defect sites, their impacts on bone regeneration are unclear. Herein, we examined whether CMC and HPMC affect the differentiation of bone marrow progenitors into osteoclasts and osteoblasts. We therefore induced osteoclastogenesis and osteoblastogenesis in murine bone marrow progenitors in the presence of CMC and HPMC, respectively. Measures of osteoclastogesis were based on the number and activity of tartrate-resistant acid-phosphatase-positive (TRAP(+)) multinucleated cells and expression of marker genes. Osteoblastogenesis was determined by the number and activity of alkaline-phosphatase-positive (AP(+)) colonies and relevant marker genes. Viability was assessed by colorimetric measurement of formazan formation. We report that CMC at 1% caused a significant reduction in the number and activity of TRAP(+) multinucleated cells. Changes in viability were not responsible for the observed effects. HPMC showed no remarkable impact on osteoclastogenesis; however, the concentration was limited to 0.5% because of the high viscosity. The ability of bone marrow progenitors to form AP(+) colonies was not affected by either of the two carriers. Together, these results suggest that CMC and possibly also HPMC can decrease osteoclastogenesis while osteoblastogenesis remains unchanged in vitro. These observations raise the possibility that these carriers might affect the cellular process of bone regeneration.

  3. Osteoblast protects osteoclast devoid of sodium-dependent vitamin C transporters from oxidative cytotoxicity of ascorbic acid.

    PubMed

    Takarada, Takeshi; Hinoi, Eiichi; Kambe, Yuki; Sahara, Koichi; Kurokawa, Shintaro; Takahata, Yoshifumi; Yoneda, Yukio

    2007-12-01

    The view that ascorbic acid indirectly benefits osteoclastogenesis through expression of receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) by osteoblasts is prevailing. In this study, we have examined the direct effect of ascorbic acid on osteoclastogenesis in cultured mouse osteoclasts differentiated from bone marrow precursors. The absence of alkaline phosphatase and osteoblastic marker genes validated the usefulness of isolation procedures. Sustained exposure to ascorbic acid, but not to dehydroascorbic acid, significantly reduced the number of multinucleated cells positive to tartrate-resistant acid phosphatase (TRAP) staining. In cultured osteoclasts, mRNA expression was seen for glucose transporter-1 involved in membrane transport of dehydroascorbic acid, but not for sodium-dependent vitamin C transporters-1 and -2 that are both responsible for the transport of ascorbic acid. The inhibition by ascorbic acid was completely prevented by catalase, while ascorbic acid or hydrogen peroxide drastically increased the number of cells stained with propidium iodide and the generation of reactive oxygen species, in addition to inducing mitochondrial membrane depolarization in cultured osteoclasts. In pre-osteoclastic cell line RAW264.7 cells, ascorbic acid similarly inhibited the formation of TRAP-positive multinucleated cells, with a significant decrease in RANKL-induced NF-kappaB transactivation. Moreover, co-culture with osteoblastic MC3T3-E1 cells significantly prevented the ascorbic acid-induced decrease in the number of TRAP-positive multinucleated cells in RAW264.7 cells. These results suggest that ascorbic acid may play a dual repulsive role in osteoclastogenesis toward bone remodeling through the direct cytotoxicity mediated by oxidative stress to osteoclasts, in addition to the indirect trophism mediated by RANKL from osteoblasts.

  4. 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

  5. Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin.

    PubMed

    Yasuhara, Rika; Miyamoto, Yoichi; Takami, Masamichi; Imamura, Takahisa; Potempa, Jan; Yoshimura, Kentaro; Kamijo, Ryutaro

    2009-04-01

    Porphyromonas gingivalis is one of the major pathogens of periodontitis, a condition characterized by excessive alveolar bone resorption by osteoclasts. The bacterium produces cysteine proteases called gingipains, which are classified according to their cleavage-site specificity into Kgps (lysine-specific gingipains) and Rgps (arginine-specific gingipains). In the present study we examined the effects of gingipains on osteoclast differentiation. In co-cultures of mouse bone-marrow cells and osteoblasts, formation of multinucleated osteoclasts induced by 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxyvitamin D(3)) was augmented by Kgp but not by RgpB. A physiological concentration (0.1 nM) of 1alpha,25(OH)(2)D(3) induced the osteoclast formation in the presence of 100 nM Kgp to an extent comparable with that induced by 10 nM 1alpha,25(OH)(2)D(3). Kgp also enhanced osteoclastogenesis induced by various microbial components, including lipopolysaccharide. Combined use of Kgp and 1alpha,25(OH)(2)D(3) or lipopolysaccharide also increased the number of resorption pits developed on dentin slices, indicating that the osteoclasts formed in the presence of Kgp possess bone-resorbing activity. The enhanced osteoclastogenesis by Kgp was correlated with a depletion of osteoprotegerin in co-culture medium and was proteolytic-activity-dependent, since benzyloxycarbonyl-L-phenylalanyl-L-lysylacycloxyketone, an inhibitor of Kgp, completely abolished osteoclastogenesis induced by Kgp. Kgp digested osteoprotegerin, since its recombinant protein was susceptible to degradation by Kgp in the presence of serum. As a result, Kgp did not augment osteoclastogenesis in co-cultures of osteoprotegerin-deficient osteoblasts and bone-marrow cells. In addition, enhanced osteoclastogenesis by Kgp was abolished by an excess amount of recombinant osteoprotegerin. These findings suggest that degradation of osteoprotegerin is one of the mechanisms underlying promotion of osteoclastogenesis by Kgp.

  6. Effects of extracellular magnesium extract on the proliferation and differentiation of human osteoblasts and osteoclasts in coculture.

    PubMed

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

    2015-11-01

    Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment. An attractive human osteoblasts and osteoclasts cocultivation regime was

  7. Osteoclasts differentiate from resident precursors in an in vivo model of synchronized resorption: a temporal and spatial study in rats.

    PubMed

    Baroukh, B; Cherruau, M; Dobigny, C; Guez, D; Saffar, J L

    2000-11-01

    Osteoclasts differentiate from mononucleated precursors expressing monocyte markers, which gradually evolve to preosteoclasts expressing the osteoclast phenotype. Although the role of osteogenic cells in these changes has been well documented in vitro, their contribution in vivo has not been established. In this study, a synchronized wave of resorption was activated along the mandibular periosteum. The periosteum adjacent to the bone surface studied was separated by a computer-assisted technique into an osteogenic alkaline phosphatase-positive compartment and an outer nonosteogenic compartment. Specific markers (nonspecific esterase [NSE], tartrate-resistant acid phosphatase [TRAP], and ED1 antibody, a marker of the monocyte-macrophage lineage) were used to follow osteoclast differentiation quantitatively as a function of time after activation of resorption, from day 0 to day 4 (peak of resorption in this model). Local cell proliferation was assessed in parallel. Between day 0 and day 3, the thickness of the osteogenic compartment decreased by 50% (p < 0.0002). In the osteogenic compartment, proliferating cell numbers fell by 80% at 12 day, NSE(+) cells (located farthest from the bone surface) increased 3. 9-fold on day 4 vs. day 0 (p < 0.005), ED1(+) cells decreased between day 0 and day 2 (p < 0.02) before returning to their initial value, and TRAP(+) cells increased 2.7-fold between day 1 and day 3 (p < 0.0005). Resorption was absent in the site studied on day 0, but on day 4 there were 20.5 osteoclast nuclei per millimeter of bone surface. The cell ratio changed from 30.3 NSE(+) and ED1(+) (some of which were also TRAP(+)) cells per millimeter on day 0 to 37.6 mononucleated cells plus 20.5 osteoclast nuclei on day 4. In the nonosteogenic compartment, an entry of ED1(+)/NSE(-) was observed on 12 day (+23 cells, p < 0.02 vs. day 0). This was followed by a return of ED1(+) cell numbers to the control level on day 1, and a transient increase in NSE(+) cells (+47

  8. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-{alpha} with BCAR1 and Traf6

    SciTech Connect

    Robinson, Lisa J.; Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L.; Blair, Harry C.

    2009-04-15

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at {approx} 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-{beta}-estradiol. Estrogen receptor-{alpha} (ER{alpha}) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ER{alpha}. However, ER{alpha} was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ER{alpha} in the presence of estrogen, was abundant. Immunoprecipitation showed rapid ({approx} 5 min) estrogen-dependent formation of ER{alpha}-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-{kappa}B activity, precipitated with this complex. Reduction of NF-{kappa}B nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of I{kappa}B in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ER{alpha}.

  9. 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.

  10. 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.

  11. 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

  12. Gsα Controls Cortical Bone Quality by Regulating Osteoclast Differentiation via cAMP/PKA and β-Catenin Pathways

    PubMed Central

    Ramaswamy, Girish; Kim, Hyunsoo; Zhang, Deyu; Lounev, Vitali; Wu, Joy Y.; Choi, Yongwon; Kaplan, Frederick S.; Pignolo, Robert J.; Shore, Eileen M.

    2017-01-01

    Skeletal bone formation and maintenance requires coordinate functions of several cell types, including bone forming osteoblasts and bone resorbing osteoclasts. Gsα, the stimulatory subunit of heterotrimeric G proteins, activates downstream signaling through cAMP and plays important roles in skeletal development by regulating osteoblast differentiation. Here, we demonstrate that Gsα signaling also regulates osteoclast differentiation during bone modeling and remodeling. Gnas, the gene encoding Gsα, is imprinted. Mice with paternal allele deletion of Gnas (Gnas+/p−) have defects in cortical bone quality and strength during early development (bone modeling) that persist during adult bone remodeling. Reduced bone quality in Gnas+/p− mice was associated with increased endosteal osteoclast numbers, with no significant effects on osteoblast number and function. Osteoclast differentiation and resorption activity was enhanced in Gnas+/p− cells. During differentiation, Gnas+/p− cells showed diminished pCREB, β-catenin and cyclin D1, and enhanced Nfatc1 levels, conditions favoring osteoclastogenesis. Forskolin treatment increased pCREB and rescued osteoclast differentiation in Gnas+/p− by reducing Nfatc1 levels. Cortical bone of Gnas+/p− mice showed elevated expression of Wnt inhibitors sclerostin and Sfrp4 consistent with reduced Wnt/β-catenin signaling. Our data identify a new role for Gsα signaling in maintaining bone quality by regulating osteoclast differentiation and function through cAMP/PKA and Wnt/β-catenin pathways. PMID:28338087

  13. 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

  14. 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

  15. p38α MAPK regulates proliferation and differentiation of osteoclast progenitors and bone remodeling in an aging-dependent manner

    PubMed Central

    Cong, Qian; Jia, Hao; Li, Ping; Qiu, Shoutao; Yeh, James; Wang, Yibin; Zhang, Zhen-Lin; Ao, Junping; Li, Baojie; Liu, Huijuan

    2017-01-01

    Bone mass is determined by the balance between bone formation, carried out by mesenchymal stem cell-derived osteoblasts, and bone resorption, carried out by monocyte-derived osteoclasts. Here we investigated the potential roles of p38 MAPKs, which are activated by growth factors and cytokines including RANKL and BMPs, in osteoclastogenesis and bone resorption by ablating p38α MAPK in LysM+monocytes. p38α deficiency promoted monocyte proliferation but regulated monocyte osteoclastic differentiation in a cell-density dependent manner, with proliferating p38α−/− cultures showing increased differentiation. While young mutant mice showed minor increase in bone mass, 6-month-old mutant mice developed osteoporosis, associated with an increase in osteoclastogenesis and bone resorption and an increase in the pool of monocytes. Moreover, monocyte-specific p38α ablation resulted in a decrease in bone formation and the number of bone marrow mesenchymal stem/stromal cells, likely due to decreased expression of PDGF-AA and BMP2. The expression of PDGF-AA and BMP2 was positively regulated by the p38 MAPK-Creb axis in osteoclasts, with the promoters of PDGF-AA and BMP2 having Creb binding sites. These findings uncovered the molecular mechanisms by which p38α MAPK regulates osteoclastogenesis and coordinates osteoclastogenesis and osteoblastogenesis. PMID:28382965

  16. Anti–IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss

    PubMed Central

    Hsu, Yu-Hsiang; Chen, Wei-Yu; Chan, Chien-Hui; Wu, Chih-Hsing; Sun, Zih-Jie

    2011-01-01

    IL-20 is a proinflammatory cytokine of the IL-10 family that is involved in psoriasis, rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about the role of IL-20 in bone destruction. We explored the function of IL-20 in osteoclastogenesis and the therapeutic potential of anti–IL-20 monoclonal antibody 7E for treating osteoporosis. Higher serum IL-20 levels were detected in patients with osteopenia and osteoporosis and in ovariectomized (OVX) mice. IL-20 mediates osteoclastogenesis by up-regulating the receptor activator of NF-κB (RANK) expression in osteoclast precursor cells and RANK ligand (RANKL) in osteoblasts. 7E treatment completely inhibited osteoclast differentiation induced by macrophage colony-stimulating factor (M-CSF) and RANKL in vitro and protected mice from OVX-induced bone loss in vivo. Furthermore, IL-20R1–deficient mice had significantly higher bone mineral density (BMD) than did wild-type controls. IL-20R1 deficiency also abolished IL-20–induced osteoclastogenesis and increased BMD in OVX mice. We have identified a pivotal role of IL-20 in osteoclast differentiation, and we conclude that anti–IL-20 monoclonal antibody is a potential therapeutic for protecting against osteoporotic bone loss. PMID:21844205

  17. 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

    2012-05-05

    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. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. TRPV1 deletion impaired fracture healing and inhibited osteoclast and osteoblast differentiation.

    PubMed

    He, Lin-Hai; Liu, Meng; He, Yang; Xiao, E; Zhao, Lu; Zhang, Ting; Yang, Hua-Qian; Zhang, Yi

    2017-02-22

    Fracture healing, in which osteoclasts and osteoblasts play important roles, has drawn much clinical attention. Osteoclast deficiency or decreased osteoblast activity will impair fracture healing. TRPV1 is a member of the Ca(2+) permeable cation channel subfamily, and pharmacological inhibition of TRPV1 prevents ovariectomy-induced bone loss, which makes TRPV1 a potential target for osteoporosis. However, whether long term TRPV1 inhibition or TRPV1 deletion will affect the fracture healing process is unclear. In this study, we found that the wild-type mice showed a well-remodeled fracture callus, whereas TRPV1 knockout mice still had an obvious fracture gap with unresorbed soft-callus 4 weeks post-fracture. The number of osteoclasts was reduced in the TRPV1 knockout fracture callus, and osteoclast formation and resorption activity were also impaired in vitro. TRPV1 deletion decreased the calcium oscillation frequency and peak cytoplasmic concentration in osteoclast precursors, subsequently reducing the expression and nuclear translocation of NFATc1 and downregulating DC-stamp, cathepsin K, and ATP6V. In addition, TRPV1 deletion caused reduced mRNA and protein expression of Runx2 and ALP in bone marrow stromal cells (BMSCs) and reduced calcium deposition in vitro. Our results suggest that TRPV1 deletion impairs fracture healing, and inhibited osteoclastogenesis and osteogenesis.

  19. 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.

  20. TRPV1 deletion impaired fracture healing and inhibited osteoclast and osteoblast differentiation

    PubMed Central

    He, Lin-Hai; Liu, Meng; He, Yang; Xiao, E.; Zhao, Lu; Zhang, Ting; Yang, Hua-Qian; Zhang, Yi

    2017-01-01

    Fracture healing, in which osteoclasts and osteoblasts play important roles, has drawn much clinical attention. Osteoclast deficiency or decreased osteoblast activity will impair fracture healing. TRPV1 is a member of the Ca2+ permeable cation channel subfamily, and pharmacological inhibition of TRPV1 prevents ovariectomy-induced bone loss, which makes TRPV1 a potential target for osteoporosis. However, whether long term TRPV1 inhibition or TRPV1 deletion will affect the fracture healing process is unclear. In this study, we found that the wild-type mice showed a well-remodeled fracture callus, whereas TRPV1 knockout mice still had an obvious fracture gap with unresorbed soft-callus 4 weeks post-fracture. The number of osteoclasts was reduced in the TRPV1 knockout fracture callus, and osteoclast formation and resorption activity were also impaired in vitro. TRPV1 deletion decreased the calcium oscillation frequency and peak cytoplasmic concentration in osteoclast precursors, subsequently reducing the expression and nuclear translocation of NFATc1 and downregulating DC-stamp, cathepsin K, and ATP6V. In addition, TRPV1 deletion caused reduced mRNA and protein expression of Runx2 and ALP in bone marrow stromal cells (BMSCs) and reduced calcium deposition in vitro. Our results suggest that TRPV1 deletion impairs fracture healing, and inhibited osteoclastogenesis and osteogenesis. PMID:28225019

  1. Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation

    PubMed Central

    Rodrigues, WF; Madeira, MFM; da Silva, TA; Clemente-Napimoga, JT; Miguel, CB; Dias-da-Silva, VJ; Barbosa-Neto, O; Lopes, AH; Napimoga, MH

    2012-01-01

    BACKGROUND AND PURPOSE Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease. EXPERIMENTAL APPROACH Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg−1 propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1β, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro. RESULTS Propranolol at 0.1 and 5 mg·kg−1 reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg−1 reduced IL-1β, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9. CONCLUSIONS AND IMPLICATIONS Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters. PMID:21950592

  2. Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation.

    PubMed

    Rodrigues, W F; Madeira, M F M; da Silva, T A; Clemente-Napimoga, J T; Miguel, C B; Dias-da-Silva, V J; Barbosa-Neto, O; Lopes, A H; Napimoga, M H

    2012-04-01

    Bones are widely innervated, suggesting an important role for the sympathetic regulation of bone metabolism, although there are controversial studies. We investigated the effects of propranolol in a model of experimental periodontal disease. Rats were assigned as follows: animals without ligature; ligated animals receiving vehicle and ligated animals receiving 0.1, 5 or 20 mg·kg(-1) propranolol. After 30 days, haemodynamic parameters were measured by cardiac catheterization. Gingival tissues were removed and assessed for IL-1β, TNF-α and cross-linked carboxyterminal telopeptides of type I collagen (CTX) by elisa, or intercellular adhesion molecule 1 (ICAM-1), receptor activator of NF-κ B ligand (RANKL) and osteoprotegerin (OPG) by Western blot analysis. Sections from the mandibles were evaluated for bone resorption. Also, we analysed the ability of propranolol to inhibit osteoclastogenesis in vitro. Propranolol at 0.1 and 5 mg·kg(-1) reduced the bone resorption as well as ICAM-1 and RANKL expression. However, only 0.1 mg·kg(-1) reduced IL-1β, TNF-α and CTX levels as well as increased the expression of OPG, but did not alter any of the haemodynamic parameters. Propranolol also suppressed in vitro osteoclast differentiation and resorptive activity by inhibiting the nuclear factor of activated T cells (NFATc)1 pathway and the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K and MMP-9. Low doses of propranolol suppress bone resorption by inhibiting RANKL-mediated osteoclastogenesis as well as inflammatory markers without affecting haemodynamic parameters. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  3. 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

  4. Insulin enhances RANKL-induced osteoclastogenesis via ERK1/2 activation and induction of NFATc1 and Atp6v0d2.

    PubMed

    Oh, Ju Hee; Lee, Jae Yoon; Joung, Seung Hee; Oh, Yoon Taek; Kim, Hong Sung; Lee, Na Kyung

    2015-12-01

    Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 μm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin.

  5. Inhibition of CaMKK2 Stimulates Osteoblast Formation and Inhibits Osteoclast Differentiation

    PubMed Central

    Cary, Rachel L.; Waddell, Seid; Racioppi, Luigi; Long, Fanxin; Novack, Deborah V.; Voor, Michael J.; Sankar, Uma

    2013-01-01

    Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OB) and resorption of pre-existing bone matrix by osteoclasts (OC), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulates bone accrual is in high clinical demand. Here we identify Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics, as its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. Whereas Camkk2−/− MSCs yield significantly higher numbers of OBs, bone marrow cells from Camkk2−/− mice produce fewer multinuclear OCs, in vitro. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser133 phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells c1 (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic

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

    PubMed Central

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

    2014-01-01

    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. [BMB Reports 2014; 47(8): 451-456] PMID:24314143

  7. Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates Osteoclast Differentiation via the Ca(2+) /NFATc1 Axis.

    PubMed

    Chiu, Ya-Hui; Schwarz, Edward; Li, Dongge; Xu, Yuexin; Sheu, Tzong-Ren; Li, Jinbo; de Mesy Bentley, Karen L; Feng, Changyong; Wang, Baoli; Wang, Jhih-Cheng; Albertorio-Saez, Liz; Wood, Ronald; Kim, Minsoo; Wang, Wensheng; Ritchlin, Christopher T

    2017-09-01

    DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion between osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function. The absence of a known DC-STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca(2+) signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca(2+) flux. Decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that the ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca(2+) axis. J. Cell. Physiol. 232: 2538-2549, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. PSTPIP2 deficiency in mice causes osteopenia and increased differentiation of multipotent myeloid precursors into osteoclasts

    PubMed Central

    Nacu, Viorel; Charles, Julia F.; Henne, William M.; McMahon, Harvey T.; Nandi, Sayan; Ketchum, Halley; Harris, Renee; Nakamura, Mary C.

    2012-01-01

    Missense mutations that reduce or abrogate myeloid cell expression of the F-BAR domain protein, proline serine threonine phosphatase-interacting protein 2 (PSTPIP2), lead to autoinflammatory disease involving extramedullary hematopoiesis, skin and bone lesions. However, little is known about how PSTPIP2 regulates osteoclast development. Here we examined how PSTPIP2 deficiency causes osteopenia and bone lesions, using the mouse PSTPIP2 mutations, cmo, which fails to express PSTPIP2 and Lupo, in which PSTPIP2 is dysfunctional. In both models, serum levels of the pro-osteoclastogenic factor, MIP-1α, were elevated and CSF-1 receptor (CSF-1R)–dependent production of MIP-1α by macrophages was increased. Treatment of cmo mice with a dual specificity CSF-1R and c-Kit inhibitor, PLX3397, decreased circulating MIP-1α and ameliorated the extramedullary hematopoiesis, inflammation, and osteopenia, demonstrating that aberrant myelopoiesis drives disease. Purified osteoclast precursors from PSTPIP2-deficient mice exhibit increased osteoclastogenesis in vitro and were used to probe the structural requirements for PSTPIP2 suppression of osteoclast development. PSTPIP2 tyrosine phosphorylation and a functional F-BAR domain were essential for PSTPIP2 inhibition of TRAP expression and osteoclast precursor fusion, whereas interaction with PEST-type phosphatases was only required for suppression of TRAP expression. Thus, PSTPIP2 acts as a negative feedback regulator of CSF-1R signaling to suppress inflammation and osteoclastogenesis. PMID:22923495

  9. The controlled release of simvastatin from TiO2 nanotubes to promote osteoblast differentiation and inhibit osteoclast resorption

    NASA Astrophysics Data System (ADS)

    Lai, Min; Jin, Ziyang; Yang, Xinyi; Wang, Huaying; Xu, Kui

    2017-02-01

    The aim of this study was to fabricate a novel drug-releasing bioactive platform that has excellent potential for improving osteoblast differentiation and inhibiting osteoclast resorption. TiO2 nanotubes (TNTs) with an outer diameter of around 70 nm were prepared by an anodization method. TNTs were filled with simvastatin (SV) and then coated using chitosan/gelatin multilayers (TNT-SV-LBL). The successful fabrication of TNT-SV-LBL substrates was confirmed by field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurement, respectively. The in vitro release behavior of simvastatin from TNT-SV-LBL substrates showed a sustained release as compared to the uncoated group. Osteoblasts adhering to TNT-SV-LBL substrates attached well and displayed significantly higher (p < 0.01) cell viability compared with the other substrates. More importantly, osteoblasts grown on TNT-SV-LBL substrates displayed a statistically significant (p < 0.01 or p < 0.05) increase in protein production levels of alkaline phosphatase (ALP), osteocalcin (OC) and mRNA expression of runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN), OC and osteoprotegerin (OPG) compared to the other groups after 4, 7 and 14 days of culture, respectively. Additionally, multinuclear osteoclastic differentiation of RAW264.7 cells grown on TNT-SV-LBL substrates was inhibited as confirmed by tartrate-resistant acid phosphatase (TRAP) analysis. These results demonstrated that bio-functionalized substrates with SV and chitosan/gelatin multilayers have great potential for improving osteoblast differentiation, as well as inhibiting osteoclast formation. Therefore, these advanced surface and chemical capabilities make this substrate well suited for the development of a drug-releasing Ti implant for bone regeneration.

  10. 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

  11. MHC class II transactivator is an in vivo regulator of osteoclast differentiation and bone homeostasis co-opted from adaptive immunity.

    PubMed

    Benasciutti, Elisa; Mariani, Elisabetta; Oliva, Laura; Scolari, Maria; Perilli, Egon; Barras, Emmanuele; Milan, Enrico; Orfanelli, Ugo; Fazzalari, Nicola L; Campana, Lara; Capobianco, Annalisa; Otten, Luc; Particelli, Francesca; Acha-Orbea, Hans; Baruffaldi, Fabio; Faccio, Roberta; Sitia, Roberto; Reith, Walter; Cenci, Simone

    2014-02-01

    The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation

  12. The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis

    PubMed Central

    Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji

    2016-01-01

    The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)–mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow–derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos–dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. PMID:27849171

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

    PubMed

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

    2014-12-01

    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.

  14. 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

  15. The natural flavonoid galangin inhibits osteoclastic bone destruction and osteoclastogenesis by suppressing NF-κB in collagen-induced arthritis and bone marrow-derived macrophages.

    PubMed

    Huh, Jeong-Eun; Jung, In-Tae; Choi, Junyoung; Baek, Yong-Hyeon; Lee, Jae-Dong; Park, Dong-Suk; Choi, Do-Young

    2013-01-05

    We investigated the effect of galangin, a natural flavonoid, on osteoclastic bone destruction in collagen-induced arthritis and examined the molecular mechanisms by which galangin affects osteoclastogenesis in bone marrow derived macrophages. In mice with collagen-induced arthritis, administration of galangin significantly reduced the arthritis clinical score, edema and severity of disease without toxicity. Interestingly, galangin treatment during a later stage of collagen-induced arthritis, using mice with a higher clinical arthritis score, still significantly slowed the progression of the disease. Extensive cartilage and bone erosive changes as well as synovial inflammation, synovial hyperplasia and pannus formation were dramatically inhibited in arthritic mice treated with galangin. Furthermore, galangin-treated arthritic mice showed a significant reduction in the concentrations of IL-1β, TNF-α and IL-17. We found that galangin inhibited osteoclastogenic factors and osteoclast formation in bone marrow-derived macrophages and osteoblast co-cultured cells, and increased osteoprotegerin (OPG) levels in osteoblasts. Galangin and NF-κB siRNA suppressed RANKL-induced phosphorylation of the c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Also, the JNK inhibitor SP600125 and p38 inhibitor SB203580 reduced RANKL-induced expressions of phospho-c-Jun, c-fos and NFATc1 genes during osteoclast development. In addition, galangin suppressed RANKL-induced phosphorylation of NF-κB, phospho-IκBα, inflammatory cytokines and osteoclast formation in bone marrow-derived macrophages. Our data suggest that galangin prevented osteoclastic bone destruction and osteoclastogenesis in osteoclast precursors as well as in collagen-induced arthritis mice without toxicity via attenuation of RANKL-induced activation of JNK, p38 and NF-κB pathways.

  16. Transcriptomic meta-analysis reveals up-regulation of gene expression functional in osteoclast differentiation in human septic shock

    PubMed Central

    Mukhopadhyay, Samanwoy; Thatoi, Pravat K.; Pandey, Abhay D.; Das, Bidyut K.; Ravindran, Balachandran; Bhattacharjee, Samsiddhi; Mohapatra, Saroj K.

    2017-01-01

    Septic shock is a major medical problem with high morbidity and mortality and incompletely understood biology. Integration of multiple data sets into a single analysis framework empowers discovery of new knowledge about the condition that may have been missed by individual analysis of each of these datasets. Electronic search was performed on medical literature and gene expression databases for selection of transcriptomic studies done in circulating leukocytes from human subjects suffering from septic shock. Gene-level meta-analysis was conducted on the six selected studies to identify the genes consistently differentially expressed in septic shock. This was followed by pathway-level analysis using three different algorithms (ORA, GSEA, SPIA). The identified up-regulated pathway, Osteoclast differentiation pathway (hsa04380) was validated in two independent cohorts. Of the pathway, 25 key genes were selected that serve as an expression signature of Septic Shock. PMID:28199355

  17. Transcriptomic meta-analysis reveals up-regulation of gene expression functional in osteoclast differentiation in human septic shock.

    PubMed

    Mukhopadhyay, Samanwoy; Thatoi, Pravat K; Pandey, Abhay D; Das, Bidyut K; Ravindran, Balachandran; Bhattacharjee, Samsiddhi; Mohapatra, Saroj K

    2017-01-01

    Septic shock is a major medical problem with high morbidity and mortality and incompletely understood biology. Integration of multiple data sets into a single analysis framework empowers discovery of new knowledge about the condition that may have been missed by individual analysis of each of these datasets. Electronic search was performed on medical literature and gene expression databases for selection of transcriptomic studies done in circulating leukocytes from human subjects suffering from septic shock. Gene-level meta-analysis was conducted on the six selected studies to identify the genes consistently differentially expressed in septic shock. This was followed by pathway-level analysis using three different algorithms (ORA, GSEA, SPIA). The identified up-regulated pathway, Osteoclast differentiation pathway (hsa04380) was validated in two independent cohorts. Of the pathway, 25 key genes were selected that serve as an expression signature of Septic Shock.

  18. Sinomenine Suppresses Osteoclast Formation and Mycobacterium tuberculosis H37Ra-Induced Bone Loss by Modulating RANKL Signaling Pathways

    PubMed Central

    Li, Xiaojuan; He, Longgang; Hu, Yiping; Duan, Heng; Li, Xianglian; Tan, Suiyi; Zou, Min; Gu, Chunping; Zeng, Xiangzhou; Yu, Le; Xu, Jiake; Liu, Shuwen

    2013-01-01

    Receptor activator of NF-κB ligand (RANKL) is essential for osteoclastogenesis. Targeting RANKL signaling pathways has been an encouraging strategy for treating lytic bone diseases such as osteoporosis and rheumatoid arthritis (RA). Sinomenine (SIN), derived from Chinese medicinal plant Sinomenioumacutum, is an active compound to treat RA, but its effect on osteoclasts has been hitherto unknown. In the present study, SIN was found to ameliorate M. tuberculosis H37Ra (Mt)-induced bone loss in rats with a decreased serum level of TRACP5b and RANKL, and an increased level of osteoprotegerin (OPG). In vitro study also showed that SIN could inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, MMP-9, TRACP were inhibited by SIN in a dose dependent manner. Signal transduction studies showed that SIN could obviously reduce the expression of RANK adaptor molecule TRAF6 and down-regulate RANKL-induced NF-κB activation. It decreased the RANKL-induced p38, JNK posphorylation but not ERK1/2 posphorylation. SIN could also reduce RANKL-mediated calcium influx which is associated with TRAF6/c-Src complex. Finally, SIN suppressed RANKL induced AP-1 and NFAT transcription, as well as the gene expression of NFATc1 and AP-1 components (Fra-1, Fra-2, c-Fos). The protein expression of c-Fos and TRAF6 were also inhibited by SIN after RANKL stimulation. Taken together, SIN could attenuate osteoclast formation and Mt-induced bone loss by mediating RANKL signaling pathways. PMID:24066131

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

    PubMed

    Patel, Jessal J; Butters, Oliver R; Arnett, Timothy R

    2014-06-01

    Drugs used in the treatment of type 2 diabetes and cardiovascular disease, specifically peroxisome proliferator-activated receptor (PPAR) agonists, have been reported to affect bone cell function and fracture risk. In this study, we assessed the direct effects of PPAR-γ agonists (rosiglitazone and troglitazone), used in the treatment of diabetes, and a PPAR-α agonist (fenofibrate), used to treat hyperlipidaemia, on the function of primary osteoblasts and osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by up to 85% in cultures treated with rosiglitazone and by 45% in troglitazone-treated or fenofibrate-treated cultures; at the same time, lipid droplet formation was increased by 40-70%. The expression of key osteogenic markers was similarly downregulated in cultures treated with PPAR agonists, whereas adipogenesis markers were upregulated. Formation of osteoclasts in cultures derived from mouse marrow diminished with fenofibrate treatment, whereas both glitazones reduced resorptive activity without affecting osteoclast number. Metformin, although not a PPAR agonist, is also commonly used in the treatment of type 2 diabetes. Here, metformin was found to have no effect on bone cell function. Taken together, these data suggest that PPAR-γ agonists may enhance bone loss via increased adipogenesis at the expense of osteoblast formation. In contrast, PPAR-α agonists may prevent bone loss. Given that the prevalence of diabetes and cardiovascular disease is expected to rise significantly, greater attention may need to be paid to the effects of PPAR agonists on bone homeostasis. Copyright © 2014 John Wiley & Sons, Ltd.

  20. High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation.

    PubMed

    Maycas, Marta; Portolés, María Teresa; Matesanz, María Concepción; Buendía, Irene; Linares, Javier; Feito, María José; Arcos, Daniel; Vallet-Regí, María; Plotkin, Lilian I; Esbrit, Pedro; Gortázar, Arancha R

    2017-12-01

    Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and β-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1β MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication. © 2017 Wiley Periodicals, Inc.

  1. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. 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.

  3. 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.

  4. Nardosinone Suppresses RANKL-Induced Osteoclastogenesis and Attenuates Lipopolysaccharide-Induced Alveolar Bone Resorption

    PubMed Central

    Niu, Chenguang; Xiao, Fei; Yuan, Keyong; Hu, XuChen; Lin, Wenzhen; Ma, Rui; Zhang, Xiaoling; Huang, Zhengwei

    2017-01-01

    Periodontitis is a chronic inflammatory disease that damages the integrity of the tooth-supporting tissues, known as the periodontium, and comprising the gingiva, periodontal ligament and alveolar bone. In this study, the effects of nardosinone (Nd) on bone were tested in a model of lipopolysaccharide (LPS)-induced alveolar bone loss, and the associated mechanisms were elucidated. Nd effectively suppressed LPS-induced alveolar bone loss and reduced osteoclast (OC) numbers in vivo. Nd suppressed receptor activator of nuclear factor-κB ligand (RANKL)-mediated OC differentiation, bone resorption, and F-actin ring formation in a dose-dependent manner. Further investigation revealed that Nd suppressed osteoclastogenesis by suppressing the ERK and JNK signaling pathways, scavenging reactive oxygen species, and suppressing the activation of PLCγ2 that consequently affects the expression and/or activity of the OC-specific transcription factors, c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In addition, Nd significantly reduced the expression of OC-specific markers in mouse bone marrow-derived pre-OCs, including c-Fos, cathepsin K (Ctsk), VATPase d2, and Nfatc1. Collectively, these findings suggest that Nd has beneficial effects on bone, and the suppression of OC number implies that the effect is exerted directly on osteoclastogenesis. PMID:28955231

  5. Receptor activator of nuclear factor-kappaB ligand-induced mouse osteoclast differentiation is associated with switching between NADPH oxidase homologues.

    PubMed

    Sasaki, Hideyuki; Yamamoto, Hironori; Tominaga, Kumiko; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Matsuno, Kuniharu; Yabe-Nishimura, Chihiro; Rokutan, Kazuhito

    2009-07-15

    Reactive oxygen species (ROS) have been suggested to regulate receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated osteoclast differentiation. Stimulation of wild-type mouse bone marrow monocyte/macrophage lineage (BMM) cells by RANKL down-regulated NADPH oxidase 2 (Nox2) mRNA expression by half. RANKL reciprocally increased Nox1 mRNA levels and newly induced Nox4 transcript expression. BMM cells from Nox1 knockout (Nox1(-/-)) as well as Nox2(-/-) mice generated ROS in response to RANKL and differentiated into osteoclasts in the same way as wild-type BMM cells, which was assessed by the appearance of tartrate-resistant acid phosphatase-positive, multinucleated cells having the ability to form resorption pits and by the expression of osteoclast marker genes. A small interfering RNA (siRNA) targeting Nox1 or Nox2 failed to inhibit the RANKL-stimulated ROS generation and osteoclast formation in wild-type cells, whereas Nox1 and Nox2 siRNAs significantly suppressed the ROS generation and osteoclast formation in Nox2(-/-) and Nox1(-/-) cells, respectively. We also confirmed that Nox4 siRNA did not affect the RANKL-dependent events in Nox2(-/-) cells, whereas p22(phox) siRNA suppressed the events in both wild-type and Nox1(-/-) cells. Collectively, our results suggest that there may be a flexible compensatory mechanism between Nox1 and Nox2 for RANKL-stimulated ROS generation to facilitate osteoclast differentiation.

  6. Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

    PubMed Central

    Sakai, H; Jingushi, S; Shuto, T; Urabe, K; Ikenoue, T; Okazaki, K; Kukita, T; Kukita, A; Iwamoto, Y

    2002-01-01

    Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1α,25(OH)2D3 (10-8 M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFκB ligand (RANKL) was investigated by immunohistochemistry. Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1α,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening. PMID:11796394

  7. Regulation of osteoclast homeostasis and inflammatory bone loss by MFG-E81

    PubMed Central

    Abe, Toshiharu; Shin, Jieun; Hosur, Kavita; Udey, Mark C.; Chavakis, Triantafyllos; Hajishengallis, George

    2014-01-01

    The glycoprotein milk fat globule-EGF factor 8 (MFG-E8) is expressed in several tissues and mediates diverse homeostatic functions. However, whether MFG-E8 plays a role in bone homeostasis has not been established. Here we show for the first time that osteoclasts express and are regulated by MFG-E8. Bone marrow-derived osteoclast precursors (OCPs) from MFG-E8–deficient (Mfge8−/−) mice underwent increased RANKL-induced osteoclastogenesis leading to enhanced resorption pit formation as compared with wild-type controls. Consistently, exogenously added MFG-E8 inhibited RANKL-induced osteoclastogenesis from mouse or human OCPs. Upon induction of experimental periodontitis, an oral inflammatory disease characterized by loss of bone support of the dentition, Mfge8−/− mice exhibited higher numbers of osteoclasts and more bone loss than wild-type controls. Accordingly, local microinjection of anti-MFG-E8 mAb exacerbated periodontal bone loss in wild-type mice. Conversely, microinjection of MFG-E8 inhibited bone loss in experimental mouse periodontitis. In comparison to wild-type controls, Mfge8−/− mice also experienced >60% more naturally occurring chronic periodontal bone loss. In conclusion, MFG-E8 is a novel homeostatic regulator of osteoclasts and could be exploited therapeutically to treat periodontitis and perhaps other immunological disorders associated with inflammatory bone loss. PMID:24958900

  8. Scopoletin and scopolin isolated from Artemisia iwayomogi suppress differentiation of osteoclastic macrophage RAW 264.7 cells by scavenging reactive oxygen species.

    PubMed

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

    2013-04-26

    Artemisia iwayomogi has been used as a folk medicine for treating various diseases including inflammatory and immune-related diseases. Scopoletin (1) and scopolin (2) were isolated from this species. Scopoletin (1) showed more potent peroxyl radical-scavenging capacity, reducing capacity, and cellular antioxidant capacity compared to scopolin (2). The inhibitory effect of 1 on the receptor activator of nuclear factor κB ligand-induced osteoclastic differentiation of RAW 264.7 macrophage cells was also more potent than that of 2. The production of general reactive oxygen species (ROS) and superoxide anions during differentiation of preosteoclastic RAW 264.7 cells into osteoclasts was attenuated by compounds 1 and 2. These findings indicate that the suppressive effects of 1 and 2 on the differentiation of preosteoclastic RAW 264.7 cells is partially due to their intracellular antioxidant capacity, as they can scavenge ROS and play an important signaling role in the differentiation process.

  9. Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca(2+)-calcineurin-NFATc1 signaling pathway.

    PubMed

    Zhang, Jie; Xu, Haixia; Han, Zhongyu; Chen, Ping; Yu, Qiang; Lei, Yutian; Li, Zongze; Zhao, Ming; Tian, Jing

    2017-01-08

    Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca(2+)-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca(2+) in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca(2+) channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca(2+)-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca(2+)-calcineurin-NFATc1 signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. 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

  11. Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-α-induced osteoclast formation in vivo.

    PubMed

    Sakai, Eiko; Aoki, Yuri; Yoshimatsu, Masako; Nishishita, Kazuhisa; Iwatake, Mayumi; Fukuma, Yutaka; Okamoto, Kuniaki; Tanaka, Takashi; Tsukuba, Takayuki

    2016-07-15

    Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated. To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption. Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-α (TNF-α)-mediated osteoclastogenesis was examined in vivo. R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-κB alpha (IκBα) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-κB in vitro, as well as TNF-α-mediated osteoclastogenesis in vivo. Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. The Polyphenol Fisetin Protects Bone by Repressing NF-κB and MKP-1-Dependent Signaling Pathways in Osteoclasts

    PubMed Central

    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. PMID:23861901

  13. 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.

  14. PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation

    PubMed Central

    2013-01-01

    Background DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved. Results Here we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation. Conclusions The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process. PMID:24028770

  15. F-spondin inhibits differentiation of clastic precursors via lipoprotein receptor-related protein 8 (LRP8).

    PubMed

    Oka, Hiroko; Kitagawa, Masae; Takata, Takashi

    2015-03-01

    F-spondin, known to be a secreted neuronal glycoprotein, is highly expressed on the tooth root surface. The authors previously reported that F-spondin is one of the specific markers of cementoblasts in periodontal tissue. In chronic periodontitis, significant cemental resorption rarely occurs on the root side, although alveolar bone resorption by osteoclasts is one of the major pathologic changes. Thus, it was hypothesized that secretory F-spondin from cementoblasts might be involved in differentiation of clastic cells on the root surface. The authors studied effects of secretory F-spondin from F-spondin-expressing cells and its pathway on receptor activator of nuclear factor-κB ligand (RANKL)-mediated differentiation of clastic cells. Osteoclast precursors were used in this study. With a chamber assay, the authors examined effects of secretory molecules from F-spondin-expressing cells of transgenic mice on RANKL-induced clastic cell differentiation. Secretory molecules from F-spondin-overexpressing cells significantly inhibited the RANKL-mediated tartrate-resistant acid phosphatase (TRAP)-positive cells from primary progenitor cells with the chamber system. F-spondin suppressed RANKL-mediated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1); TRAP; cathepsin K; and dendritic cell-specific transmembrane protein (DC-STAMP) expression in the cells. The suppressive effect of F-spondin on RANKL-induced differentiation of clastic cells was partially blocked by knockdown of low-density lipoprotein receptor-related protein 8 (LRP8). These findings indicate that secretory factors from F-spondin-expressing cells, including F-spondin, downregulate differentiation of clastic precursors. Moreover, F-spondin inhibits RANKL-mediated differentiation of clastic cells partially via LRP8. It is suggested that secretory F-spondin may act protectively from cemental resorption partially via LRP8 in periodontal tissue.

  16. 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

  17. Rapamycin and the transcription factor C/EBPbeta as a switch in osteoclast differentiation: implications for lytic bone diseases.

    PubMed

    Smink, Jeske J; Leutz, Achim

    2010-03-01

    Lytic bone diseases and in particular osteoporosis are common age-related diseases characterized by enhanced bone fragility due to loss of bone density. Increasingly, osteoporosis poses a major global health-care problem due to the growth of the elderly population. Recently, it was found that the gene regulatory transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) is involved in bone metabolism. C/EBPbeta occurs as different protein isoforms of variable amino terminal length, and regulation of the C/EBPbeta isoform ratio balance was found to represent an important factor in osteoclast differentiation and bone homeostasis. Interestingly, adjustment of the C/EBPbeta isoform ratio by the process of translational control is downstream of the mammalian target of rapamycin kinase (mTOR), a sensor of the nutritional status and a target of immunosuppressive and anticancer drugs. The findings imply that modulating the process of translational control of C/EBPbeta isoform expression could represent a novel therapeutic approach in osteolytic bone diseases, including cancer and infection-induced bone loss.

  18. RANKL-induced schlafen2 is a positive regulator of osteoclastogenesis.

    PubMed

    Lee, Na Kyung; Choi, Han Kyung; Yoo, Hyun Joo; Shin, Jihye; Lee, Soo Young

    2008-12-01

    Osteoclasts are hematopoietic lineage derived-multinucleated cells that resorb bone. Their activity in balance with that of osteoblast is essential for bone homeostasis. Receptor activator of NF-kappaB ligand (RANKL) is known as an essential cytokine for the osteoclastogenesis, and c-Jun signaling in cooperation with NFAT family is crucial for RANKL-regulated osteoclastogenesis. We show here that schlafen2 (Slfn2), a member of a new family of growth regulatory genes involved in thymocyte development, is critical for osteoclastogenesis. RANKL selectively induces Slfn2 expression in osteoclast precursors via Rac1 signaling pathway. Targeted inhibition of Slfn2 by small interfering RNAs (siRNAs) markedly inhibits the formation of osteoclasts by diminishing the activation of c-Jun and the expression of c-Jun and NFATc1. In contrast, the overexpression of Slfn2 markedly increased phosphorylation and transactivation of c-Jun by RANKL. Together, these results indicate that Slfn2 has an essential role in osteoclastogenesis, functioning upstream of c-Jun and NFATc1.

  19. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase 2 stimulates osteoblast formation and inhibits osteoclast differentiation.

    PubMed

    Cary, Rachel L; Waddell, Seid; Racioppi, Luigi; Long, Fanxin; Novack, Deborah V; Voor, Michael J; Sankar, Uma

    2013-07-01

    Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. In vitro, although Camkk2⁻/⁻ mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2⁻/⁻ mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells, cytoplasmic (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and

  20. 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

  1. MiR-7b directly targets DC-STAMP causing suppression of NFATc1 and c-Fos signaling during osteoclast fusion and differentiation.

    PubMed

    Dou, Ce; Zhang, Chengcheng; Kang, Fei; Yang, Xiaochao; Jiang, Hong; Bai, Yan; Xiang, Junyu; Xu, Jianzhong; Dong, Shiwu

    2014-11-01

    DC-STAMP is a key regulating molecule of osteoclastogenesis and osteoclast precursor (OCP) fusion. Emerging lines of evidence showed that microRNAs play crucial roles in bone metabolism and osteoclast differentiation, but no microRNA has yet been reported to be directly related to OCPs fusion. Through a microarray, we found that the expression of miR-7b in RAW264.7 cells was significantly decreased after induction with M-CSF and RANKL. The overexpression of miR-7b in RAW264.7 cells attenuated the number of TRAP-positive cells number and the formation of multinucleated cells, whereas the inhibition of miR-7b enhanced osteoclastogenesis. Through a dual luciferase reporter assay, we confirmed that miR-7b directly targets DC-STAMP. Other fusogenic molecules, such as CD47, ATP6v0d2, and OC-STAMP, were detected to be down-regulated in accordance with the inhibition of DC-STAMP. Because DC-STAMP also participates in osteoclast differentiation through the ITAM-ITIM network, multiple osteoclast-specific genes in the ITAM-ITIM network were detected to identify how DC-STAMP is involved in this process. The results showed that molecules associated with the ITAM-ITIM network, such as NFATc1 and OSCAR, which are crucial in osteoclastogenesis, were consistently altered due to DC-STAMP inhibition. These findings suggest that miR-7b inhibits osteoclastogenesis and cell-cell fusion by directly targeting DC-STAMP. In addition, the inhibition of DC-STAMP and its downstream signals changed the expression of other fusogenic genes and key regulating genes, such as Nfatc1, c-fos, Akt, Irf8, Mapk1, and Traf6. In conclusion, our findings indicate that miR-7b may be a potential therapeutic target for the treatment of osteoclast-related bone disorders. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Contributions to osteoclast biology from Japan

    PubMed Central

    Suda, Tatsuo; Takahashi, Naoyuki

    2008-01-01

    Bone is a dynamic tissue, in which bone formation by osteoblasts and bone resorption by osteoclasts continue throughout life. In 1998, we molecularly cloned osteoclast differentiation factor (ODF), a long-thought factor responsible for osteoclast formation. This review article describes how Japanese scientists contributed to osteoclast biology before and after the discovery of ODF. This review article is based on the Louis V. Avioli Memorial Lecture of the American Society for Bone and Mineral Research (ASBMR) held in Honolulu in September, 2007. PMID:19075515

  3. Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation.

    PubMed

    Schröder, H C; Wang, X H; Wiens, M; Diehl-Seifert, B; Kropf, K; Schloßmacher, U; Müller, W E G

    2012-10-01

    It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS-2 sarcoma cells in vitro. In contrast, silicate does not affect the steady-state gene expression level of the osteoclastogenic ligand receptor activator of NF-κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS-2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10-100 µM. Applying a co-cultivation assay system, using SaOS-2 cells and RAW 264.7 cells, it is shown that in the presence of 10 µM silicate the number of RAW 264.7 cells in general, and the number of TRAP(+) RAW 264.7 cells in particular markedly decreases. The SaOS-2 cells retain their capacity of differential gene expression of OPG and RANKL in favor of OPG after exposure to silicate. It is concluded that after exposure of the cells to silicate a factor(s) is released from SaOS-2 cells that causes a significant inhibition of osteoclastogenesis of RAW 264.7 cells. It is assumed that it is an increased secretion of the cytokine OPG that is primarily involved in the reduction of the osteoclastogenesis of the RAW 264.7 cells. It is proposed that silicate might have the potential to stimulate osteogenesis in vivo and perhaps to ameliorate osteoporotic disorders. Copyright © 2012 Wiley Periodicals, Inc.

  4. The role of microRNAs in osteoclasts and osteoporosis.

    PubMed

    Tang, Peifu; Xiong, Qi; Ge, Wei; Zhang, Lihai

    2014-01-01

    Osteoclasts are the exclusive cells of bone resorption. Abnormally activating osteoclasts can lead to low bone mineral density, which will cause osteopenia, osteoporosis, and other bone disorders. To date, the mechanism of how osteoclast precursors differentiate into mature osteoclasts remains elusive. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in numerous cellular processes, including cell differentiation and apoptosis, by post-transcriptional regulation of genes. Recently, a number of studies have revealed that miRNAs participate in bone homeostasis, including osteoclastic bone resorption, which sheds light on the mechanisms underlying osteoclast differentiation. In this review, we highlight the miRNAs involved in regulating osteoclast differentiation and bone resorption, and their roles in osteoporosis.

  5. Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha.

    PubMed

    Kim, Hyon Jong; Chang, Eun-Ju; Kim, Hyun-Man; Lee, Seung Bok; Kim, Hyun-Duck; Su Kim, Ghi; Kim, Hong-Hee

    2006-05-01

    The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.

  6. 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.

  7. Overexpression of prohibitin-1 inhibits RANKL-induced activation of p38-Elk-1-SRE signaling axis blocking MKK6 activity.

    PubMed

    Lee, Chang Hoon; Choi, Sik-Won; Kim, Ju-Young; Kim, Seong Hwan; Yoon, Kwon-Ha; Oh, Jaemin; Lee, Myeung Su

    2015-08-07

    Prohibitin-1 (PHB) regulates diverse cellular processes by controlling several signaling pathways. In this study, we investigated the functional involvement of PHB in osteoclast differentiation. PHB expression was time-dependently increased by RANKL in BMMs. However, the retroviral over-expression of PHB strongly inhibited the expression of c-Fos and NFATc1, and activation of p38-Elk-1-SRE signaling pathway. Anti-osteoclastogenic action of PHB was significantly inhibited by constitutively active forms of MKK6, but not Elk-1. Collectively, PHB negatively regulates the formation of mature osteoclasts via inhibition of MKK6 activity that affects the activation of the p38-Elk-1 signaling axis required for the expression of c-Fos and NFATc1.

  8. Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells.

    PubMed

    Konermann, A; Staubwasser, M; Dirk, C; Keilig, L; Bourauel, C; Götz, W; Jäger, A; Reichert, C

    2014-04-01

    The aim of this study was to determine the material composition and cell-mediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure. Copyright © 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  9. 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.

  10. A novel 11β-HSD1 inhibitor improves diabesity and osteoblast differentiation.

    PubMed

    Park, Ji Seon; Bae, Su Jung; Choi, Sik-Won; Son, You Hwa; Park, Sung Bum; Rhee, Sang Dal; Kim, Hee Youn; Jung, Won Hoon; Kang, Seung Kyu; Ahn, Jin Hee; Kim, Seong Hwan; Kim, Ki Young

    2014-04-01

    Selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential as treatment for osteoporosis as well as metabolic syndrome including type 2 diabetes mellitus. Here, we investigated the anti-diabetic, anti-adipogenic, and anti-osteoporotic activity of KR-67500, as a novel selective 11β-HSD1 inhibitor. Cellular 11β-HSD1 activity was tested based on a homogeneous time-resolved fluorescence method. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) levels were measured in diet-induced obese (DIO)-C57BL/6 mice administered KR-67500 (50  mg/kg per day, p.o.) for 28 days and, additionally, its anti-diabetic effect was evaluated by OGTT and ITT. The in vitro anti-adipogenic effect of KR-67500 was determined by Oil Red O Staining. The in vitro anti-osteoporotic activity of KR-67500 was evaluated using bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation model systems. KR-67500 improved the in vivo glucose tolerance and insulin sensitivity in DIO-C57BL/6 mice. KR-67500 suppressed cortisone-induced differentiation of 3T3-L1 cells into adipocytes. KR-67500 enhanced BMP2-induced osteoblastogenesis in C2C12 cells and inhibited RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages. KR-67500, a new selective 11β-HSD1 inhibitor, may provide a new therapeutic window in the prevention and/or treatment of type 2 diabetes, obesity, and/or osteoporosis.

  11. Role of myeloid early endothelial progenitor cells in bone formation and osteoclast differentiation in tissue construct based on hydroxyapatite poly(ester-urethane) scaffolds.

    PubMed

    Shi, Yang; Wang, Fanlu; Tiwari, Sanjay; Yesilbas, Meran; Steubesand, Nadine; Weitkamp, Jan-Tobias; Klüter, Tim; Lippross, Sebastian; Eglin, David; Seekamp, Andreas; Fuchs, Sabine

    2016-11-01

    Engineering of a vascularized bone construct is a highly challenging task which needs to take into account the impact of different components on the bone regeneration process. Bone repair influencing factors in such constructs range from the material properties and scaffold design, to the interaction of different cell types contributing to bone formation and remodeling or neovascularization, respectively. In this context, early endothelial progenitor cells (EPC), mononuclear cells isolated from the peripheral blood, express the endothelial marker CD31 but also a series of myeloid markers and have been shown to support the formation of vessel-like structures. These cells are also characterized by a highly adaptable phenotype influenced by other cells creating an instructive niche. The present study was designed to investigate the impact of EPC on bone formation or remodeling using a co-culture system of outgrowth endothelial cells, mature endothelial cells isolated from the peripheral blood cell cultures, and mesenchymal stem cells grown on hydroxyapatite poly(ester-urethane) scaffolds. The formation of vessel-like structures in these constructs was shown by CLSM and immunohistochemistry and further evaluated by real time RT-PCR. Osteogenic differentiation in these constructs was investigated by von Kossa, Alizarin Red, and real time PCR. Data indicated that osteogenic differentiation occurred within the constructs after 14 days of culture but without a direct influence by EPC in this process. Finally, although we observed a series of osteoclast related makers in the constructs when EPC were included, no indications for an increased osteoclast-like activity, which might lead to increased bone resorption, were observed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1922-1932, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  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. IL-37 inhibits lipopolysaccharide-induced osteoclast formation and bone resorption in vivo.

    PubMed

    Saeed, Jafari; Kitaura, Hideki; Kimura, Keisuke; Ishida, Masahiko; Sugisawa, Haruki; Ochi, Yumiko; Kishikawa, Akiko; Takano-Yamamoto, Teruko

    2016-07-01

    IL-37 is a newly defined member of the IL-1 cytokine family. It has been reported that IL-37 inhibited innate immunity and inflammatory responses in autoimmune diseases and tumors. IL-37 also inhibited Lipopolysaccharide (LPS)-induced immunological reaction. LPS is a bacterial cell wall component that is capable of inducing osteoclast formation and pathological bone resorption. However, there is no study to investigate the effect of IL-37 on LPS-induced osteoclast formation and bone resorption. The purpose of this study is to investigate the effect of IL-37 in LPS-induced osteoclast formation and bone resorption. LPS was administrated with or without IL-37 by subcutaneous injection on mice calvariae. The number of osteoclasts, the level of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA, the ratio of the bone resorption pits and the level of C-terminal telopeptide fragments of type I collagen cross-Links as a marker of bone resorption in mice administrated both LPS and IL-37 were lower than that in mice administrated LPS alone. Real-time RT-PCR was performed to analyze osteoclast related cytokines RANKL, TNF-α and IL-1β mRNA levels in vivo. RANKL, TNF-α and IL-1β mRNAs were increased in the LPS alone administrated mice as compared with PBS administrated groups. On the other hand, RANKL, TNF-α and IL-1β mRNAs were inhibited in the IL-37 and LPS administrated mice as compared with LPS alone administrated group. In vitro analysis, there was no effect of IL-37 in RANKL-induced osteoclast formation, TNF-α-induced osteoclast formation and cell viability from bone marrow macrophages as osteoclast precursor and LPS-induced RANKL expression from stromal cells. These results indicated that IL-37 inhibited LPS-induced osteoclast formation and bone resorption via inhibition of LPS-induced osteoclast related cytokines, but might not directly inhibit osteoclast formation on osteoclast precursor and RANKL expression on stromal cells. Copyright © 2016

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

    PubMed

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

    2016-04-22

    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.

  15. DMSO regulates osteoclast development in vitro

    PubMed Central

    Lemieux, Justin M.; Wu, Gary; Morgan, Joseph A.

    2011-01-01

    Dimethyl sulfoxide (DMSO) is routinely used in the laboratory as a solvent and vehicle for organic molecules. Although it has been used in previous studies involving myeloid cells and macrophages, we are unaware of data demonstrating the effects of DMSO alone on osteoclast development. Recently, we were using DMSO as a vehicle and included a non-vehicle control. Surprisingly, we observed a marked change in osteoclast development, and therefore designed this study to examine the effects of DMSO on osteoclast development. Osteoclasts were generated from two sources: bone marrow macrophages and an osteoclast progenitor cell line. Cells were cultured with DMSO for various durations and at differing concentrations and mature, multinucleated (>3 nuclei) TRAP+ cells were assessed in terms of cell number, cell surface area, and number of nuclei/cell. Osteoclast surface area increased in 5 μM DMSO to a mean of 156,422 pixels from a mean of 38,510 pixels in control culture, and subsequently decreased in 10 μM DMSO to a mean of 18,994 pixels. With serial addition of DMSO over 5 d, a significant increase in mean surface area, and number of nuclei/cell was also observed, while the opposite was true when DMSO was serially removed from culture. These findings show that DMSO exerts a marked effect on osteoclast differentiation. Since many investigators use DMSO to solubilize compounds for treatment of osteoclasts, caution is warranted as altering DMSO concentrations may have a profound effect on the final data, especially if osteoclast differentiation is being assessed. PMID:21359822

  16. 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

  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. Immature dendritic cells in multiple myeloma are prone to osteoclast-like differentiation through interleukin-17A stimulation.

    PubMed

    Tucci, Marco; Stucci, Stefania; Savonarola, Annalisa; Ciavarella, Sabino; Cafforio, Paola; Dammacco, Franco; Silvestris, Franco

    2013-06-01

    Interleukin 17A (IL17A), a cytokine involved in allergy, inflammation and osteoclastogenesis, was investigated in multiple myeloma (MM) to assess its role in the osteoclast (OC)-like activity of marrow immature dendritic cells (iDCs). Comparing nine MM patients with control subjects affected by monoclonal gammopathy of undetermined significance, we found high IL17A expression in the marrow plasma of MM patients in parallel with its deposits within the stromal matrix. Increased expression of the IL17A receptor (IL17RA) was also found in primary myeloma iDCs, which underwent OC-like transdifferentiation after IL17A stimulation. To assess the role of IL17A, we measured the activity of the IL17/IL17RA pathway in IL17A-transdifferentiated iDCs and the expression of functional OC genes by Western blotting and real-time polymerase chain reaction. These cells showed increased RNA transcription of genes enrolled in the maturation of OCs, while NFATC1 and FOS were induced by IL17A, independently of NFKB1 phosphorylation. Moreover, the concurrent phosphorylation of the Lip isoform of CEBPB and the down-regulation of MAFB supported the activation of IL17RA pathway in OC-like transdifferentiated iDCs that was apparently unrelated to TNFRSF11A signalling. These data emphasize the involvement of iDCs in MM hyperactive osteoclastogenesis and suggest that their bone resorption activity is also regulated, at least in vitro, by IL17RA. © 2013 John Wiley & Sons Ltd.

  19. Notch signaling promotes osteoclast maturation and resorptive activity

    PubMed Central

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

    2015-01-01

    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. PMID:25914241

  20. 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.

  1. 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

  2. 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.

  3. Osteoclast precursor interaction with bone matrix induces osteoclast formation directly by an interleukin-1-mediated autocrine mechanism.

    PubMed

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

    2008-04-11

    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-beta, 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.

  4. c-Src Links a RANK/αvβ3 Integrin Complex to the Osteoclast Cytoskeleton

    PubMed Central

    Izawa, Takashi; Zou, Wei; Chappel, Jean C.; Ashley, Jason W.; Feng, Xu

    2012-01-01

    RANK ligand (RANKL), by mechanisms unknown, directly activates osteoclasts to resorb bone. Because c-Src is key to organizing the cell's cytoskeleton, we asked if the tyrosine kinase also mediates RANKL-stimulated osteoclast activity. RANKL induces c-Src to associate with RANK369–373 in an αvβ3-dependent manner. Furthermore, RANK369–373 is the only one of six putative TRAF binding motifs sufficient to generate actin rings and activate the same cytoskeleton-organizing proteins as the integrin. While c-Src organizes the cell's cytoskeleton in response to the cytokine, it does not participate in RANKL-stimulated osteoclast formation. Attesting to their collaboration, αvβ3 and activated RANK coprecipitate, but only in the presence of c-Src. c-Src binds activated RANK via its Src homology 2 (SH2) domain and αvβ3 via its SH3 domain, suggesting the kinase links the two receptors. Supporting this hypothesis, deletion or inactivating point mutation of either the c-Src SH2 or SH3 domain obviates the RANK/αvβ3 association. Thus, activated RANK prompts two distinct signaling pathways; one promotes osteoclast formation, and the other, in collaboration with c-Src-mediated linkage to αvβ3, organizes the cell's cytoskeleton. PMID:22615494

  5. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    PubMed Central

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

  6. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Cytoskeletal dysfunction dominates in DAP12-deficient osteoclasts

    PubMed Central

    Zou, Wei; Zhu, Tingting; Craft, Clarissa S.; Broekelmann, Thomas J.; Mecham, Robert P.; Teitelbaum, Steven L.

    2010-01-01

    Despite evidence that DAP12 regulates osteoclasts, mice lacking the ITAM-bearing protein exhibit only mild osteopetrosis. Alternatively, Dap12−/− mice, also lacking FcRγ, are severely osteopetrotic, suggesting that FcRγ compensates for DAP12 deficiency in the bone-resorbing polykaryons. Controversy exists, however, as to whether these co-stimulatory molecules regulate differentiation of osteoclasts or the capacity of the mature cell to degrade bone. We find that Dap12−/− osteoclasts differentiate normally when generated on osteoblasts but have a dysfunctional cytoskeleton, impairing their ability to transmigrate through the osteoblast layer and resorb bone. To determine whether the FcRγ co-receptor, OSCAR mediates osteoclast function in the absence of DAP12, we overexpressed OSCAR fused to FLAG (OSCAR-FLAG), in Dap12−/− osteoclasts. OSCAR-FLAG partially rescues the abnormal cytoskeleton of Dap12−/− osteoclasts grown on bone, but not those grown on osteoblasts. Thus, cytoskeletal dysfunction, and not arrested differentiation, is the dominant consequence of DAP12 deficiency in osteoclasts. The failure of osteoblasts to normalize Dap12−/− osteoclasts indicates that functionally relevant quantities of OSCAR ligand do not reside in bone-forming cells. PMID:20720152

  8. 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.

  9. Response of human rheumatoid arthritis osteoblasts and osteoclasts to adiponectin.

    PubMed

    Krumbholz, Grit; Junker, Susann; Meier, Florian M P; Rickert, Markus; Steinmeyer, Jürgen; Rehart, Stefan; Lange, Uwe; Frommer, Klaus W; Schett, Georg; Müller-Ladner, Ulf; Neumann, Elena

    2017-01-01

    Adiponectin is an effector molecule in the pathophysiology of rheumatoid arthritis, e.g. by inducing cytokines and matrix degrading enzymes in synovial fibroblasts. There is growing evidence that adiponectin affects osteoblasts and osteoclasts although the contribution to the aberrant bone metabolism in rheumatoid arthritis is unclear. Therefore, the adiponectin effects on rheumatoid arthritis-derived osteoblasts and osteoclasts were evaluated. Adiponectin and its receptors were examined in bone tissue. Primary human osteoblasts and osteoclasts were stimulated with adiponectin and analysed using realtime polymerase chain-reaction and immunoassays. Effects on matrix-production by osteoblasts and differentiation and resorptive activity of osteoclasts were examined. Immunohistochemistry of rheumatoid arthritis bone tissue showed adiponectin expression in key cells of bone remodelling. Adiponectin altered gene expression and cytokine release in osteoblasts and increased IL-8 secretion by osteoclasts. Adiponectin inhibited osterix and induced osteoprotegerin mRNA in osteoblasts. In osteoclasts, MMP-9 and tartrate resistant acid phosphatase expression was increased. Accordingly, mineralisation capacity of osteoblasts decreased whereas resorptive activity of osteoclasts increased. The results confirm the proinflammatory potential of adiponectin and support the idea that adiponectin influences rheumatoid arthritis bone remodelling through alterations in osteoblast and osteoclast.

  10. The pathobiology of the osteoclast.

    PubMed Central

    Chambers, T J

    1985-01-01

    This article reviews recent information concerning the origin of osteoclasts and the local and systemic regulation of their activity. It appears that much of the environmental responsiveness of osteoclasts is mediated by cells of the osteoblastic lineage, which exert a major influence on the localisation, induction, stimulation, and inhibition of osteoclastic bone resorption. Some of the mechanisms by which osteoclast function may be disturbed by inflammatory and neoplastic diseases are discussed, and it is suggested that many pathological disturbances of osteoclastic bone resorption may be explicable as mimicry of physiological regulatory mechanisms by local hormones introduced into bone as the local regulators of the diseased tissue. Images PMID:2982920

  11. TULA-2, a novel histidine phosphatase regulates bone remodeling by modulating osteoclast function

    PubMed Central

    Back, Steven H.; Adapala, Naga Suresh; Barbe, Mary F.; Carpino, Nick C.; Tsygankov, Alexander Y.; Sanjay, Archana

    2013-01-01

    Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The novel protein T-cell Ubiquitin Ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO Mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of Immune-receptor-Tyrosine-based-Activation-Motif (ITAM)–mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function. PMID:23149425

  12. The roles of interferons in osteoclasts and osteoclastogenesis.

    PubMed

    Xiong, Qi; Zhang, Lihai; Ge, Wei; Tang, Peifu

    2016-05-01

    Interferons (IFNs) play essential roles in regulating osteoclast differentiation and bone resorption. Over the last decade, we have seen tremendous developments in our understanding of the mechanisms by which interferons regulate osteoclastogenesis. Of the type I interferons, IFN-β inhibits osteoclastogenesis via autoregulatory or exogenous regulatory mechanisms, while IFN-α was recently shown to participate in regulating osteoclast formation. And the only member of type II interferons, IFN-γ, has biphasic effects on osteoclastogenesis. Type III interferons have also been shown to be involved in osteoclast bone resorption, although no direct regulatory mechanism has been demonstrated. In this review, we provide an update account of the current knowledge on these recently revealed novel roles of interferons in the regulation of a variety of signaling pathways in osteoclast differentiation and function. The potential clinical applications are also discussed. Copyright © 2015 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

  13. Augmented LPS Responsiveness in Type 1 Diabetes-Derived Osteoclasts

    PubMed Central

    Catalfamo, Dana L.; Calderon, Nadia L.; Harden, Scott W.; Sorenson, Heather L.; Neiva, Kathleen G.; Wallet, Shannon M.

    2012-01-01

    Bone abnormalities are frequent co-morbidities of type 1 diabetes [T1D] and are principally mediated by osteoblasts and osteoclasts which in turn are regulated by immunologic mediators. While decreased skeletal health in T1D involves alterations in osteoblast maturation and function, the affect of altered immune function on osteoclasts in T1D-associated bone and joint pathologies is less understood. Here T1D-associated osteoclast-specific differentiation and function in the presence and absence of inflammatory mediators was characterized utilizing bone marrow-derived osteoclasts [BM-OCs] isolated from non-obese diabetic [NOD] mice, a model for spontaneous autoimmune diabetes with pathology similar to individuals with T1D. Differentiation and osteoclast-mediated bone resorption were evaluated along with cathepsin K, MMP-9, and immune soluble mediator expression. The affect of LPS, a pro-inflammatory cytokine cocktail, and NOD-derived conditioned supernatants on BM-OC function was also determined. Although NOD BM-OCs cultures contained smaller osteoclasts, they resorbed more bone concomitant with increased cathepsin K, MMP-9 and pro-osteoclastogenic mediator expression. NOD BM-OCs also displayed an inhibition of LPS-induced deactivation that was not a result of soluble mediators produced by NOD BM-OCs, although a pro-inflammatory milieu did enhance NOD BM-OCs bone resorption. Together these data indicate that osteoclasts from a T1D mouse model hyper-respond to RANK-L resulting in excessive bone degradation via enhanced cathepsin K and MMP-9 secretion concomitant with an increased expression of pro-osteoclastic soluble mediators. Our data also suggest that inhibition of LPS-induced deactivation in NOD-derived BM-OC cultures is most likely due to NOD osteoclast responsiveness rather than LPS-induced expression of soluble mediators. PMID:22718269

  14. [Frontiers in Live Bone Imaging Researches. Intravital imaging of osteoclast dynamics].

    PubMed

    Kikuta, Junichi; Ishii, Masaru

    2015-06-01

    Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. Upon the stimulation of essential factors such as M-CSF and RANKL, osteoclast precursor monocytes attach to the bone surface ( "migration" ), fuse with each other to form giant cells ( "differentiation" ) and mediate bone resorption ( "function" ). To reveal the regulatory mechanism of these three dynamic steps of osteoclastic activity, we have originally established an advanced imaging system for visualizing living bone tissues with intravital multiphoton microscopy. By means of the system, we have recently succeeded in visualization of osteoclast migration, differentiation, and function in living bone tissues in vivo. In this review we summarize the latest data of intravital imaging of osteoclast dynamics, and discuss novel lines of osteoclast-targeted therapies that will impact future treatment of bone destructive diseases.

  15. Inhibitory effects of tert-butylhydroquinone on osteoclast differentiation via up-regulation of heme oxygenase-1 and down-regulation of HMGB1 release and NFATc1 expression.

    PubMed

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

    2014-01-01

    Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Our recent studies have shown that heme-oxygenase-1 (HO-1), a stress-induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert-butylhydroquinone (tBHQ), a pharmacological HO-1 inducer, on in vitro differentiation of bone marrow-derived macrophages (BMMs) or murine monocytic cell line RAW-D into OCLs. tBHQ inhibited the formation and the bone-resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (IκBα). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO-1/HMGB1 pathways. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Inhibitor of DASH proteases affects expression of adhesion molecules in osteoclasts and reduces myeloma growth and bone disease.

    PubMed

    Pennisi, Angela; Li, Xin; Ling, Wen; Khan, Sharmin; Gaddy, Dana; Suva, Larry J; Barlogie, Bart; Shaughnessy, John D; Aziz, Nazneen; Yaccoby, Shmuel

    2009-06-01

    Dipeptidyl peptidase (DPP) IV activity and/or structure homologues (DASH) are serine proteases implicated in tumourigenesis. We previously found that a DASH protease, fibroblast activation protein (FAP), was involved in osteoclast-induced myeloma growth. Here we further demonstrated expression of various adhesion molecules in osteoclasts cultured alone or cocultured with myeloma cells, and tested the effects of DASH inhibitor, PT-100, on myeloma cell growth, bone disease, osteoclast differentiation and activity, and expression of adhesion molecules in osteoclasts. PT-100 had no direct effects on viability of myeloma cells or mature osteoclasts, but significantly reduced survival of myeloma cells cocultured with osteoclasts. Real-time PCR array for 85 adhesion molecules revealed upregulation of 17 genes in osteoclasts after coculture with myeloma cells. Treatment of myeloma/osteoclast cocultures with PT-100 significantly downregulated 18 of 85 tested genes in osteoclasts, some of which are known to play roles in tumourigenesis and osteoclastogenesis. PT-100 also inhibited osteoclast differentiation and subsequent pit formation. Resorption activity of mature osteoclasts and differentiation of osteoblasts were not affected by PT-100. In primary myelomatous severe combined immunodeficient (SCID)-hu mice PT-100 reduced osteoclast activity, bone resorption and tumour burden. These data demonstrated that DASH proteases are involved in myeloma bone disease and tumour growth.

  17. Minireview: Nuclear Receptor Regulation of Osteoclast and Bone Remodeling

    PubMed Central

    Jin, Zixue; Li, Xiaoxiao

    2015-01-01

    Osteoclasts are bone-resorbing cells essential for skeletal remodeling and regeneration. However, excessive osteoclasts often contribute to prevalent bone degenerative diseases such as osteoporosis, arthritis, and cancer bone metastasis. Osteoclast dysregulation is also associated with rare disorders such as osteopetrosis, pycnodysostosis, Paget's disease, and Gorham-Stout syndrome. The nuclear receptor (NR) family of transcription factors functions as metabolic sensors that control a variety of physiological processes including skeletal homeostasis and serves as attractive therapeutic targets for many diseases. In this review, we highlight recent findings on the new players and the new mechanisms for how NRs regulate osteoclast differentiation and bone resorption. An enhanced understanding of NR functions in osteoclastogenesis will facilitate the development of not only novel osteoprotective medicine but also prudent strategies to minimize the adverse skeletal effects of certain NR-targeting drugs for a better treatment of cancer and metabolic diseases. PMID:25549044

  18. 25-hydroxycholesterol promotes RANKL-induced osteoclastogenesis through coordinating NFATc1 and Sp1 complex in the transcription of miR-139-5p.

    PubMed

    Zhang, Lishan; Lv, Yinping; Xian, Guozhe; Lin, Yanliang

    2017-04-15

    25-hydroxycholesterol (25-HC) is implicated in many processes, including lipid metabolism and the immune response. However, the role of 25-HC in RANKL-induced osteoclastogenesis remains largely unknown. Our results showed that 25-HC inhibited miR-139-5p expression in mouse bone marrow macrophages (BMMs) cultured in receptor activator of NF-κB ligand (RANKL) and monocyte macrophage colony-stimulating factor (M-CSF). Further investigation suggested that 25-HC promoted the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and Sp1, especially in the presence of RANKL and M-CSF. Meanwhile, 25-HC induced nuclear translocation of NFATc1, resulting in the interaction between NFATc1 and Sp1 that was confirmed by co-immunoprecipitation. Chromatin immunoprecipitation assay indicated that Sp1 could bind to miR-139-5p promoter, but NFATc1 had no binding capacity. Although forming NFATc1/Sp1 complex increased its binding to miR-139-5p promoter, the complex inhibited the transcriptional activity of Sp1. Inhibition of NFATc1 increase the expression of miR-139-5p, which might be due to the release of free Sp1 that could bind to the promoter of miR-139-5p. Enforced expression of miR-139-5p impaired osteoclastogenesis induced by co-treatment with 25-HC and RANKL. These results suggested that 25-HC induced the interaction between NFATc1 and Sp1, reducing the level of free Sp1 to inhibit miR-139-5p expression and promote osteoclastogenesis.

  19. Estrogens maintain bone mass by regulating expression of genes controlling function and life span in mature osteoclasts.

    PubMed

    Imai, Yuuki; Youn, Ming-Young; Kondoh, Shino; Nakamura, Takashi; Kouzmenko, Alexander; Matsumoto, Takahiro; Takada, Ichiro; Takaoka, Kunio; Kato, Shigeaki

    2009-09-01

    Estrogens play a key role in regulation of bone mass and strength by controlling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. Cellular effects of estrogens are mediated predominantly by the action of estrogen receptor alpha (ERalpha). In earlier studies, ablation of the ERalpha gene in mice did not result in osteoporotic phenotypes due to systemic endocrine disturbance and compensatory effects of elevated levels of testosterone. Despite the relatively well-established effects in osteoblasts, little is known about the direct action of estrogen in osteoclasts. Development in the last decade of more sophisticated genetic manipulation approaches opened new possibilities to explore cell-specific roles of nuclear receptors in bone tissue. Recently, we have generated osteoclast-specific ERalpha gene knockout mice and shown that in vivo estrogens directly regulate the life span of mature osteoclasts by inducing the expression of pro-apoptotic Fas ligand (FasL). Inhibitory effects of estrogens on osteoclast function were further studied in vitro. We observed sufficiently detectable ERalpha expression in osteoclasts differentiating from primary bone marrow cells or RAW264 cells, although levels of ERalpha were decreasing during progression of the differentiation into mature osteoclasts. Treatment with estrogens led to reduction in expression of osteoclast-specific genes controlling bone resorption activity. However, estrogens did not affect the size of multinucleated osteoclasts or number of nuclei in a mature osteoclast. In conclusion, in osteoclasts, estrogens function to inhibit bone resorption activity and vitality rather than differentiation.

  20. Oxygen ultra-fine bubbles water administration prevents bone loss of glucocorticoid-induced osteoporosis in mice by suppressing osteoclast differentiation.

    PubMed

    Noguchi, T; Ebina, K; Hirao, M; Morimoto, T; Koizumi, K; Kitaguchi, K; Matsuoka, H; Iwahashi, T; Yoshikawa, H

    2017-03-01

    Oxygen ultra-fine bubbles (OUB) saline injection prevents bone loss of glucocorti\\coid-induced osteoporosis in mice, and OUB inhibit osteoclastogenesis via RANK-TRAF6-c-Fos-NFATc1 signaling and RANK-p38 MAPK signaling in vitro. Ultra-fine bubbles (<200 nm in diameter) have several unique properties, and they are tested in various medical fields. The purpose of this study was to investigate the effects of oxygen ultra-fine bubbles (OUB) on glucocorticoid-induced osteoporosis (GIO) model mice. Prednisolone (PSL, 5 mg) was subcutaneously inserted in 6-month-old male C57BL/6J mice, and 200 μl of saline, OUB-diluted saline, or nitrogen ultra-fine bubbles (NUB)-diluted saline was intraperitoneally injected three times per week for 8 weeks the day after operations. Mice were divided into four groups; (1) control, sham-operation + saline; (2) GIO, PSL + saline; (3) GIO + OUB, PSL + OUB saline; (4) GIO + NUB, PSL + NUB saline. The effects of OUB on osteoblasts and osteoclasts were examined by serially diluted OUB medium in vitro. Bone mass was significantly decreased in GIO [bone volume/total volume (%): control vs. GIO 12.6 vs. 7.9; p < 0.01] while significantly preserved in GIO + OUB (GIO vs. GIO + OUB 7.9 vs. 12.9; p < 0.05). In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the distal femur [mean osteoclasts number/bone surface (mm(-1))] was significantly increased in GIO (control vs. GIO 6.8 vs. 11.6; p < 0.01) while suppressed in GIO + OUB (GIO vs. GIO + OUB 11.6 vs. 7.5; p < 0.01). NUB did not affect these parameters. In vitro experiments revealed that OUB significantly inhibited osteoclastogenesis by inhibiting RANK-TRAF6-c-Fos-NFATc1 signaling, RANK-p38 MAPK signaling, and TRAP/Cathepsin K/DC-STAMP mRNA expression in a concentration-dependent manner. OUB did not affect osteoblastogenesis in vitro. OUB prevent bone loss in GIO mice by inhibiting osteoclastogenesis.

  1. Secretion of L-glutamate from osteoclasts through transcytosis

    PubMed Central

    Morimoto, Riyo; Uehara, Shunsuke; Yatsushiro, Shouki; Juge, Narinobu; Hua, Zhaolin; Senoh, Shigenori; Echigo, Noriko; Hayashi, Mitsuko; Mizoguchi, Toshihide; Ninomiya, Tadashi; Udagawa, Nobuyuki; Omote, Hiroshi; Yamamoto, Akitsugu; Edwards, Robert H; Moriyama, Yoshinori

    2006-01-01

    Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1−/− knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1−/− mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis. PMID:16957773

  2. Talin1 and Rap1 are critical for osteoclast function.

    PubMed

    Zou, Wei; Izawa, Takashi; Zhu, Tingting; Chappel, Jean; Otero, Karel; Monkley, Susan J; Critchley, David R; Petrich, Brian G; Morozov, Alexei; Ginsberg, Mark H; Teitelbaum, Steven L

    2013-02-01

    To determine talin1's role in osteoclasts, we mated TLN1(fl/fl) mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.

  3. 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.

  4. Low Molecular Weight Protein Tyrosine Phosphatase Slow Isoform Knockdown in MDA-MB-435 Cells Decreases RAW 264.7 Osteoclastic Differentiation.

    PubMed

    Alho, Irina; Costa, Luis; Bicho, Manuel; Coelho, Constança

    2016-05-01

    During the bone metastatic process, tumor cells and bone cells drive a vicious cycle stimulating growth and activity of each other. We here address how low molecular weight protein tyrosine phosphatase (LMW-PTP) could be involved in this process. We targeted LMW-PTP by siRNA and evaluated the effect of various soluble factors released to the culture medium by the MDA-MB-435 breast cancer cell line, in RAW 264.7 osteoclastogenesis. We showed that these soluble factors did not change RAW 264.7 osteoclastogenic potential. The knockdown of the LMW-PTP slow isoform decreased osteoclastogenesis of RAW 264.7, showing less active Src. The knockdown of LMW-PTP and its slow isoform decreased the release of IL-8 but not IL-6 in MDA-MB-435. The LMW-PTP slow isoform can be an important protein in bone metastatic disease, with a fundamental role in the interplay between tumor cells and osteoclasts, through the regulation of Src activity and IL-8 secretion. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  5. 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.

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

    PubMed Central

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

    2008-01-01

    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 toward occlusal forces on periodontal ligament and alveolar bone homeostasis, we have developed the first mouse model of hyper-occlusion. Swiss Webster mice were kept in hyper-occlusion for 0, 3, 6, and 9 d. Morphological and histological changes in the periodontium were assessed using micro CT and ground sections with fluorescent detection of vital dye labels. Sections were stained for tartrate-resistant acid phosphatase (TRAP), and expression of RANKL and OPN was analyzed by immunohistochemistry and real-time PCR. Traumatic occlusion resulted in enamel surface abrasion, inhibition of alveolar bone apposition, significant formation of osteoclasts at 3 d, 6 d, 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. PMID:18705798

  7. Lutein, a carotenoid, suppresses osteoclastic bone resorption and stimulates bone formation in cultures.

    PubMed

    Tominari, Tsukasa; Matsumoto, Chiho; Watanabe, Kenta; Hirata, Michiko; Grundler, Florian M W; Inada, Masaki; Miyaura, Chisato

    2017-02-01

    Lutein, a member of the xanthophyll family of carotenoids, suppressed IL-1-induced osteoclast differentiation and bone resorption. The survival of mature osteoclasts was also suppressed by lutein in cultures. When lutein was added to the cultures of osteoblasts, lutein enhanced the formation of mineralized bone nodules by elevating BMP2 expression and inhibiting sclerostin expression. Lutein may be beneficial for bone health.

  8. Hyperglycemia Induced and Intrinsic Alterations in Type 2 Diabetes-Derived Osteoclast Function

    PubMed Central

    Catalfamo, Dana L.; Britten, Todd M.; Storch, Douglas I.; Calderon, Nadia L.; Sorenson, Heather L.; Wallet, Shannon M.

    2012-01-01

    Periodontal disease-associated alveolar bone loss is a co-morbidity of type-2-diabetes, where the roles of osteoclasts are poorly understood. Objective To evaluate osteoclast differentiation and function in the context of type-2-diabetes. Materials/Methods Bone marrow-derived osteoclasts from db/db mice, a model of type-2-diabetes, as well as human osteoclasts derived from peripheral blood of individuals with type-2-diabetes were evaluated for differentiation, resorption, and soluble mediator expression. Results While db/db mice were hyperglycemic at time of cell harvest, human participants were glycemically controlled. Although db/db cultures resulted in a higher number of larger osteoclasts, individual cell RANKL-mediated bone resorption was similar to that observed in diabetes-free OCs. Osteoclasts derived from individuals with type-2-diabetes differentiated similarly to controls with again no difference in bone resorbing capacity. Murine and human type-2-diabetes cultures both displayed inhibition of LPS-induced deactivation and increased pro-osteoclastogenic mediator expression. Conclusions Hyperglycemia plays a role in aberrant osteoclast differentiation leading to an increased capacity for bone resorption. Osteoclasts derived from murine models of and individuals with type-2-diabetes are unable to be inhibited by LPS, again leading to increased capacity for bone resorption. Here environmental and intrinsic mechanisms associated with the increased alveolar bone loss observed in periodontal patients with type-2-diabetes are described. PMID:24079914

  9. Gallium modulates osteoclastic bone resorption in vitro without affecting osteoblasts

    PubMed Central

    Verron, Elise; Masson, Martial; Khoshniat, Solmaz; Duplomb, Laurence; Wittrant, Yohann; Baud'huin, Marc; Badran, Zahi; Bujoli, Bruno; Janvier, Pascal; Scimeca, Jean-Claude; Bouler, Jean-Michel; Guicheux, Jérôme

    2010-01-01

    Background and purpose: Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. Experimental approach: In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. Key results: Gallium dose-dependently (0–100 µM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. Conclusions and implications: Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation. PMID:20397300

  10. Paxillin Contracts the Osteoclast Cytoskeleton

    PubMed Central

    Zou, Wei; DeSelm, Carl J.; Broekelmann, Thomas J.; Mecham, Robert P.; Pol, Scott Vande; Choi, Kyunghee; Teitelbaum, Steven L.

    2012-01-01

    Osteoclastic bone resorption depends upon the cell’s ability to organize its cytoskeleton via the αvβ3 integrin and osteoclastogenic cytokines. Since paxillin associates with αvβ3, we asked if it participates in skeletal degradation. Unlike deletion of other αvβ3-associated cytoskeleton-regulating molecules, which impairs the cell’s ability to spread, paxillin-deficient (Pax−/−) osteoclasts, generated from embryonic stem cells, “superspread” in response to RANK ligand (RANKL) and form large, albeit dynamically atypical, actin bands. Despite their increased size, Pax−/− osteoclasts resorb bone poorly, excavating pits approximately 1/3 normal depth. Ligand-occupied αvβ3 or RANKL promotes paxillin serine and tyrosine phosphorylation, the latter via c-Src. The abnormal Pax−/− phenotype is rescued by WT paxillin but not that lacking its LD4 domain. In keeping with the appearance of mutant osteoclasts, WT paxillin, overexpressed in WT cells, contracts the cytoskeleton. Most importantly, the abnormal phenotype of Pax−/− osteoclasts likely represents failed RANKL-mediated delivery of myosin IIA to the actin cytoskeleton via the paxillin LD4 domain but is independent of tyrosine phosphorylation. Thus, in response to RANKL, paxillin associates with myosin IIA to contract the osteoclast cytoskeleton thereby promoting its bone-degrading capacity. PMID:22807029

  11. Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

    PubMed Central

    Muschter, Dominique; Schäfer, Nicole; Stangl, Hubert; Straub, Rainer H.; Grässel, Susanne

    2015-01-01

    Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors. PMID:26431344

  12. 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

  13. Defective co-activator recruitment in osteoclasts from microphthalmia-oak ridge mutant mice.

    PubMed

    Sharma, Sudarshana M; Sif, Said; Ostrowski, Michael C; Sankar, Uma

    2009-07-01

    The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.

  14. Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology.

    PubMed

    Di Ceglie, Irene; van den Akker, Guus G H; Ascone, Giuliana; Ten Harkel, Bas; Häcker, Hans; van de Loo, Fons A J; Koenders, Marije I; van der Kraan, Peter M; de Vries, Teun J; Vogl, Thomas; Roth, Johannes; van Lent, Peter L E M

    2017-04-01

    Osteoclasts are cells specialized in bone resorption. Currently, studies on murine osteoclasts are primarily performed on bone marrow-derived cells with the use of many animals and limited cells available. ER-Hoxb8 cells are conditionally immortalized monocyte/macrophage murine progenitor cells, recently described to be able to differentiate toward functional osteoclasts. Here, we produced an ER-Hoxb8 clonal cell line from C57BL/6 bone marrow cells that strongly resembles phenotype and function of the conventional bone marrow-derived osteoclasts. We then used CRISPR/Cas9 technology to specifically inactivate genes by biallelic mutation. The CRISPR/Cas9 system is an adaptive immune system in Bacteria and Archaea and uses small RNAs and Cas nucleases to degrade foreign nucleic acids. Through specific-guide RNAs, the nuclease Cas9 can be redirected toward any genomic location to genetically modify eukaryotic cells. We genetically modified ER-Hoxb8 cells with success, generating NFATc1(-/-) and DC-STAMP(-/-) ER-Hoxb8 cells that lack the ability to differentiate into osteoclasts or to fuse into multinucleated osteoclasts, respectively. In conclusion, this method represents a markedly easy highly specific and efficient system for generating potentially unlimited numbers of genetically modified osteoclast precursors. © Society for Leukocyte Biology.

  15. A study of osteoclasts on calvaria of normal and osteopetrotic (mi/mi) mice by vital staining with acridine orange.

    PubMed Central

    Green, P. M.; Marshall, M. J.; Nisbet, N. W.

    1986-01-01

    A novel staining procedure for enumerating osteoclasts on neonatal mouse calvaria with the vital fluorescent dye acridine orange is described. It has the advantage over Barnicot's neutral-red method in that the nuclei and cytoplasm of the osteoclast are stained differentially. The osteopetrotic calvarium (mi/mi) has fewer multinucleate osteoclasts than its normal counterpart (mi/+) and they are differently distributed. The osteopetrotic calvarium has more mononucleate cells which stain like osteoclasts with acridine orange than the normal calvarium and these cells also are differently distributed. These mononuclear cells may be mononuclear osteoclasts or their precursors. These observations suggest that the defect resulting in this osteopetrosis lies with osteoclast differentiation. Images Fig. 1 p[88]-a PMID:2418863

  16. Selective Estrogen Receptor Modulators Suppress Hif1α Protein Accumulation in Mouse Osteoclasts

    PubMed Central

    Iwasaki, Ryotaro; Kobayashi, Tami; Watanabe, Ryuichi; Oike, Takatsugu; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Kawana, Hiromasa; Nakagawa, Taneaki; Miyamoto, Takeshi

    2016-01-01

    Anti-bone resorptive drugs such as bisphosphonates, the anti-RANKL antibody (denosumab), or selective estrogen receptor modulators (SERMs) have been developed to treat osteoporosis. Mechanisms underlying activity of bisphosphonates or denosumab in this context are understood, while it is less clear how SERMs like tamoxifen, raloxifene, or bazedoxifene inhibit bone resorption. Recently, accumulation of hypoxia inducible factor 1 alpha (Hif1α) in osteoclasts was shown to be suppressed by estrogen in normal cells. In addition, osteoclast activation and decreased bone mass seen in estrogen-deficient conditions was found to require Hif1α. Here, we used western blot analysis of cultured osteoclast precursor cells to show that tamoxifen, raloxifene, or bazedoxifene all suppress Hif1α protein accumulation. The effects of each SERM on osteoclast differentiation differed in vitro. Our results suggest that interventions such as the SERMs evaluated here could be useful to inhibit Hif1α and osteoclast activity under estrogen-deficient conditions. PMID:27802325

  17. Age-Related Effects of Advanced Glycation End Products (Ages) in Bone Matrix on Osteoclastic Resorption.

    PubMed

    Yang, Xiao; Gandhi, Chintan; Rahman, Md Mizanur; Appleford, Mark; Sun, Lian-Wen; Wang, Xiaodu

    2015-12-01

    Advanced glycation end products (AGEs) accumulate in bone extracellular matrix as people age. Previous studies have shown controversial results regarding the role of in situ AGEs accumulation in osteoclastic resorption. To address this issue, this study cultured human osteoclast cells directly on human cadaveric bone slices from different age groups (young and elderly) to warrant its relevance to in vivo conditions. The cell culture was terminated on the 3rd, 7th, and 10th day, respectively, to assess temporal changes in the number of differentiated osteoclasts, the number and size of osteoclastic resorption pits, the amount of bone resorbed, as well as the amount of matrix AGEs released in the medium by resorption. In addition, the in situ concentration of matrix AGEs at each resorption pit was also estimated based on its AGEs autofluorescent intensity. The results indicated that (1) osteoclastic resorption activities were significantly correlated with the donor age, showing larger but shallower resorption pits on the elderly bone substrates than on the younger ones; (2) osteoclast resorption activities were not significantly dependent on the in situ AGEs concentration in bone matrix, and (3) a correlation was observed between osteoclast activities and the concentration of AGEs released by the resorption. These results suggest that osteoclasts tend to migrate away from initial anchoring sites on elderly bone substrate during resorption compared to younger bone substrates. However, such behavior is not directly related to the in situ concentration of AGEs in bone matrix at the resorption sites.

  18. Characterization of inorganic phosphate transport in osteoclast-like cells.

    PubMed

    Ito, Mikiko; Matsuka, Naoko; Izuka, Michiyo; Haito, Sakiko; Sakai, Yuko; Nakamura, Rie; Segawa, Hiroko; Kuwahata, Masashi; Yamamoto, Hironori; Pike, Wesley J; Miyamoto, Ken-ichi

    2005-04-01

    Osteoclasts possess inorganic phosphate (Pi) transport systems to take up external Pi during bone resorption. In the present study, we characterized Pi transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-kappaB ligand (RANKL). In undifferentiated RAW264.7 cells, Pi transport into the cells was Na+ dependent, but after treatment with RANKL, Na+-independent Pi transport was significantly increased. In addition, compared with neutral pH, the activity of the Na+-independent Pi transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na+-independent system consisted of two components with Km of 0.35 mM and 7.5 mM. The inhibitors of Pi transport, phosphonoformic acid, and arsenate substantially decreased Pi transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K+ ionophore, valinomycin, significantly suppressed Pi transport activity. Analysis of BCECF fluorescence indicated that Pi transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K+ ion stimulated Pi transport, suggesting that membrane voltage is involved in the regulation of Pi transport activity. Finally, bone particles significantly increased Na+-independent Pi transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a Pi transport system with characteristics that are different from those of other Na+-dependent Pi transporters. We conclude that stimulation of Pi transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.

  19. Regulation of Osteoclast Growth and Fusion by mTOR/raptor and mTOR/rictor/Akt.

    PubMed

    Tiedemann, Kerstin; Le Nihouannen, Damien; Fong, Jenna E; Hussein, Osama; Barralet, Jake E; Komarova, Svetlana V

    2017-01-01

    Osteoclasts are giant bone cells formed by fusion from monocytes and uniquely capable of a complete destruction of mineralized tissues. Previously, we have demonstrated that in energy-rich environment not only osteoclast fusion index (the number of nuclei each osteoclast contains), but also cytoplasm volume per single nucleus was increased. The goal of this study was to investigate the regulation of metabolic sensor mTOR during osteoclast differentiation in energy-rich environment simulated by addition of pyruvate. We have found that in the presence of pyruvate, the proportion of mTOR associated with raptor increased, while mTOR-rictor-mediated Akt phosphorylation decreased. Inhibition of mTOR with rapamycin (10 nM) significantly interfered with all aspects of osteoclastogenesis. However, rapamycin at 1 nM, which preferentially targets mTOR-raptor complex, was only effective in control cultures, while in the presence of pyruvate osteoclast fusion index was successfully increased. Inhibition of Akt drastically reduced osteoclast fusion, however in energy-rich environment, osteoclasts of comparable size were formed through increased cytoplasm growth. These data suggest that mTOR-rictor mediated Akt signaling regulates osteoclast fusion, while mTOR-raptor regulation of protein translation contributes to fusion-independent cytoplasm growth. We demonstrate that depending on the bioenergetics microenvironment osteoclastogenesis can adjust to occur through preferential multinucleation or through cell growth, implying that attaining large cell size is part of the osteoclast differentiation program.

  20. The transient appearance of zipper-like actin superstructures during the fusion of osteoclasts.

    PubMed

    Takito, Jiro; Nakamura, Masanori; Yoda, Masaki; Tohmonda, Takahide; Uchikawa, Shinichi; Horiuchi, Keisuke; Toyama, Yoshiaki; Chiba, Kazuhiro

    2012-02-01

    Multinucleated osteoclasts are responsible for bone resorption. Hypermultinucleated osteoclasts are often observed in some bone-related diseases such as Paget's disease and cherubism. The cellular mechanics controlling the size of osteoclasts is poorly understood. We introduced EGFP-actin into RAW 264.7 cells to monitor actin dynamics during osteoclast differentiation. Before their terminal differentiation into osteoclasts, syncytia displayed two main types of actin assembly, podosome clusters and clusters of zipper-like structures. The zipper-like structures morphologically resembled the adhesion zippers found at the initial stage of cell-cell adhesion in keratinocytes. In the zipper-like structure, Arp3 and cortactin overlapped with the distribution of dense F-actin, whereas integrin β3, paxillin and vinculin were localized to the periphery of the structure. The structure was negative for WGA-lectin staining and biotin labeling. The zipper-like structure broke down and transformed into a large actin ring, called a podosome belt. Syncytia containing clusters of zipper-like structures had more nuclei than those with podosome clusters. Differentiated osteoclasts with a podosome belt also formed the zipper-like structure at the cell contact site during cell fusion. The breakdown of the cell contact site resulted in the fusion of the podosome belts following plasma membrane fusion. Additionally, osteoclasts in mouse calvariae formed the zipper-like structure in the sealing zone. Therefore, we propose that the zipper-like actin superstructures might be involved in cell-cell interaction to achieve efficient multinucleation of osteoclasts. Understanding of the zipper-like structure might lead to selective therapeutics for bone diseases caused by hypermultinucleated 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. Baicalin positively regulates osteoclast function by activating MAPK/Mitf signalling.

    PubMed

    Lu, Li; Rao, Li; Jia, Huanhuan; Chen, Jun; Lu, Xingyan; Yang, Guozhu; Li, Qingnan; Lee, Kenneth Ka Ho; Yang, Li

    2017-02-03

    Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose-dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up-regulation of downstream osteoclast-related target genes expression. The baicalin-induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p-ERK (U0126) and the Mitf-specific siRNA, respectively. Protein-ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p-ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p-ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.

  3. Slight changes in the mechanical stimulation affects osteoblast- and osteoclast-like cells in co-culture.

    PubMed

    Kadow-Romacker, Anke; Duda, Georg N; Bormann, Nicole; Schmidmaier, Gerhard; Wildemann, Britt

    2013-12-01

    Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1-5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells.

  4. Impaired osteoclast homeostasis in the cystatin B-deficient mouse model of progressive myoclonus epilepsy.

    PubMed

    Manninen, Otto; Puolakkainen, Tero; Lehto, Jemina; Harittu, Elina; Kallonen, Aki; Peura, Marko; Laitala-Leinonen, Tiina; Kopra, Outi; Kiviranta, Riku; Lehesjoki, Anna-Elina

    2015-12-01

    Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by incapacitating stimulus-sensitive myoclonus and tonic-clonic epileptic seizures with onset at the age of 6 to 16 years. EPM1 patients also exhibit a range of skeletal changes, e.g., thickened frontal cranial bone, arachnodactyly and scoliosis. Mutations in the gene encoding cystatin B (CSTB) underlie EPM1. CSTB is an inhibitor of cysteine cathepsins, including cathepsin K, a key enzyme in bone resorption by osteoclasts. CSTB has previously been shown to protect osteoclasts from experimentally induced apoptosis and to modulate bone resorption in vitro. Nevertheless, its physiological function in bone and the cause of the bone changes in patients remain unknown. Here we used the CSTB-deficient mouse (Cstb(-/-)) model of EPM1 to evaluate the contribution of defective CSTB protein function on bone pathology and osteoclast differentiation and function. Micro-computed tomography of hind limbs revealed thicker trabeculae and elevated bone mineral density in the trabecular bone of Cstb(-/-) mice. Histology from Cstb(-/-) mouse bones showed lower osteoclast count and thinner growth plates in long bones. Bone marrow-derived osteoclast cultures revealed lower osteoclast number and size in the Cstb(-/-) group. Cstb(-/-) osteoclasts formed less and smaller resorption pits in an in vitro assay. This impaired resorptive capacity was likely due to a decrease in osteoclast numbers and size. These data imply that the skeletal changes in Cstb(-/-) mice and in EPM1 patients are a result of CSTB deficiency leading to impaired osteoclast formation and consequently compromised resorptive capacity. These results suggest that the role of CSTB in osteoclast homeostasis and modulation of bone metabolism extends beyond cathepsin K regulation.

  5. Emodin suppresses cadmium-induced osteoporosis by inhibiting osteoclast formation.

    PubMed

    Chen, Xiao; Ren, Shuai; Zhu, Guoying; Wang, Zhongqiu; Wen, Xiaolin

    2017-09-01

    Environmental level of cadmium (Cd) exposure can induce bone loss. Emodin, a naturally compound found in Asian herbal medicines, could influence osteoblast/osteoclast differentiation. However, the effects of emodin on Cd-induced bone damage are not clarified. The aim of this study was to investigate the role of emodin on Cd-induced osteoporosis. Sprague-Dawley male rats were divided into three groups which were given 0mg/L, 50mg Cd/L and 50mg Cd/L plus emodin (50mg/kg body weight). Bone histological investigation, microCT analysis, metabolic biomarker determination and immunohistochemical staining were performed at the 12th week. The bone mass and bone microstructure index of rats treated with Cd were obviously lower than in control. Cd markedly enhanced the osteoclast formation compared with control. Emodin significantly abolished the Cd-induced bone microstructure damage (p<0.05), osteoclast formation and increase of tartrate-resistant acid phosphatase 5b level (p<0.05). Our data further showed that emodin attenuated the Cd-induced inhibition of osteoprotegerin expression and stimulation of receptor activator for nuclear factor-κ B ligand expression. Our data show that emodin suppresses the Cd-induced osteoporosis by inhibiting osteoclast formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. 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

  7. Osteoclast progenitors from cats with and without tooth resorption respond differently to 1,25-dihydroxyvitamin D and interleukin-6.

    PubMed

    Booij-Vrieling, H E; de Vries, T J; Schoenmaker, T; Tryfonidou, M A; Penning, L C; Hazewinkel, H A W; Everts, V

    2012-04-01

    Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)(2)D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR(+) and TR(-)) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14 days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR(-) PBMCs than from TR(+) PBMCs on plastic. More osteoclasts were formed from TR(+) PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)(2)D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR(+) osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR(+) PBMCs when cultured on bone are sensitive to 1,25(OH)(2)D, whereas the differentiation of TR(-) PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.

  8. Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts

    SciTech Connect

    Kwon, Jun-Oh; Lee, Yong Deok; Kim, Haemin; Kim, Min Kyung; Song, Min-Kyoung; Lee, Zang Hee; Kim, Hong-Hee

    2016-09-02

    Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules. - Highlights: • Tspan7 expression is up-regulated during osteoclastogenesis. • Tspan7 regulates podosome belt organization in osteoclasts. • Tspan7 is crucial for sealing zone formation and bone-resorption by osteoclasts. • Src and Pyk2 phosphorylation and microtubule acetylation mediate Tspan7 function.

  9. Mice lacking β3 integrins are osteosclerotic because of dysfunctional osteoclasts

    PubMed Central

    McHugh, Kevin P.; Hodivala-Dilke, Kairbaan; Zheng, Ming-Hao; Namba, Noriyuki; Lam, Jonathan; Novack, Deborah; Feng, Xu; Ross, F. Patrick; Hynes, Richard O.; Teitelbaum, Steven L.

    2000-01-01

    Osteoclasts express the αvβ3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the β3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that αvβ3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, αvβ5, does not substitute for αvβ3 during cytokine stimulation or authentic osteoclastogenesis. β3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, β3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in β3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin αvβ3 is essential for normal osteoclast function. PMID:10683372

  10. 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. Copyright © 2012 Wiley Periodicals, Inc.

  11. Engineered Osteoclasts for the Treatment and Prevention of Heterotopic Ossification

    DTIC Science & Technology

    2014-10-01

    stereotaxic microinjection protocol that will allow us to induce HO lesions in the calf midbelly in a highly reproducible manner. The stereotaxic... protocol for the collection and storage of human HO specimens. During the next year we will begin collecting human HO samples. HO samples will be fixed...treated with AP20187 or 0.1% EtOH for 5 days. Cells were then fixed and stained for TRAP. Validation of osteoclast differentiation protocol for

  12. Engineered Osteoclasts for the Treatment and Prevention of Heterotopic Ossification

    DTIC Science & Technology

    2014-10-01

    stereotaxic microinjection protocol that will allow us to induce HO lesions in the calf midbelly in a highly reproducible manner. The stereotaxic...traumatic bone injuries. Subtask 1: Collection of human HO samples (PI: Sangeorzan, Harborview) We have currently established a protocol for...with AP20187 or 0.1% EtOH for 5 days. Cells were then fixed and stained for TRAP. Validation of osteoclast differentiation protocol for human iPS

  13. Glucocorticoids suppress bone formation via the osteoclast.

    PubMed

    Kim, Hyun-Ju; Zhao, Haibo; Kitaura, Hideki; Bhattacharyya, Sandip; Brewer, Judson A; Muglia, Louis J; Ross, F Patrick; Teitelbaum, Steven L

    2006-08-01

    The pathogenesis of glucocorticoid-induced (GC-induced) bone loss is unclear. For example, osteoblast apoptosis is enhanced by GCs in vivo, but they stimulate bone formation in vitro. This conundrum suggests that an intermediary cell transmits a component of the bone-suppressive effects of GCs to osteoblasts in the intact animal. Bone remodeling is characterized by tethering of the activities of osteoclasts and osteoblasts. Hence, the osteoclast is a potential modulator of the effect of GCs on osteoblasts. To define the direct impact of GCs on bone-resorptive cells, we compared the effects of dexamethasone (DEX) on WT osteoclasts with those derived from mice with disruption of the GC receptor in osteoclast lineage cells (GRoc-/- mice). While the steroid prolonged longevity of osteoclasts, their bone-degrading capacity was suppressed. The inhibitory effect of DEX on bone resorption reflects failure of osteoclasts to organize their cytoskeleton in response to M-CSF. DEX specifically arrested M-CSF activation of RhoA, Rac, and Vav3, each of which regulate the osteoclast cytoskeleton. In all circumstances GRoc-/- mice were spared the impact of DEX on osteoclasts and their precursors. Consistent with osteoclasts modulating the osteoblast-suppressive effect of DEX, GRoc-/- mice are protected from the steroid's inhibition of bone formation.

  14. The 19S proteasomal lid subunit POH1 enhances the transcriptional activation by Mitf in osteoclasts.

    PubMed

    Schwarz, Toni; Sohn, Chee; Kaiser, Bria; Jensen, Eric D; Mansky, Kim C

    2010-04-01

    The microphthalmia-associated transcription factor (Mitf) regulates gene expression required for osteoclast differentiation. Genes regulated by Mitf have been previously identified. However, proteins that interact and regulate Mitf's activity in osteoclasts are not well known. Here, we report that POH1, a subunit of the 19S proteasome lid is a regulator of Mitf. We show that POH1 and Mitf interact in osteoclasts and that this interaction is dependent on RANKL signaling. Overexpression of POH1 increased Mitf's activation of 5XGal4-TK and Acp5 promoters. The amino terminus of POH1 mediates the binding to Mitf and is sufficient to increase Mitf's transcriptional activity. Finally, we show that mutations in the JAMM motif of POH1 reduced Mitf activation of promoters. In summary, our results identify a novel mechanism of Mitf regulation in osteoclasts by POH1.

  15. Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects.

    PubMed

    Kenner, Lukas; Hoebertz, Astrid; Beil, F Timo; Beil, Timo; Keon, Niamh; Karreth, Florian; Eferl, Robert; Scheuch, Harald; Szremska, Agnieszka; Amling, Michael; Schorpp-Kistner, Marina; Angel, Peter; Wagner, Erwin F

    2004-02-16

    Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta 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, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) 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.

  16. 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. Copyright © 2010 Wiley Periodicals, Inc.

  17. Sinomenine induces apoptosis in RAW 264.7 cell-derived osteoclasts in vitro via caspase-3 activation

    PubMed Central

    He, Long-gang; Li, Xiang-lian; Zeng, Xiang-zhou; Duan, Heng; Wang, Song; Lei, Lin-sheng; Li, Xiao-juan; Liu, Shu-wen

    2014-01-01

    Aim: Sinomenine (SIN) is an alkaloid found in the roots and stems of Sinomenium acutum, which has been used to treat rheumatic arthritis in China and Japan. In this study we investigated the effects of SIN on osteoclast survival in vitro and the mechanisms of the actions. Methods: Mature osteoclasts were differentiated from murine monocyte/macrophage cell line RAW264.7 through incubation in the presence of receptor activator of NF-κB ligand (RANKL, 100 ng/mL) for 4 d. The cell viability was detected using the CCK-8 method. The survival and actin ring construction of the osteoclasts were scored using TRACP staining and phalloidin-FITC staining, respectively. The apoptosis of the osteoclasts was detected by DNA fragmentation and Hoechst 33258 staining, and the cell necrosis was indicated by LDH activity. The activation of caspase-3 in osteoclasts was measured using Western blotting and the caspase-3 activity colorimetric method. Results: SIN (0.25–2 mmol/L) inhibited the viability of mature osteoclasts in dose-dependent and time-dependent manners, but did not affect that of RAW264.7 cells. Consistently, SIN dose-dependently suppressed the survival of mature osteoclasts. The formation of actin ring, a marker associated with actively resorbing osteoclasts, was also impaired by the alkaloid. SIN (0.5 mmol/L) induced the apoptosis of mature osteoclasts, which was significantly attenuated in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. SIN increased the cleavage of caspase-3 in mature osteoclasts in dose-dependent and time-dependent manners. Furthermore, SIN dose-dependently enhanced caspase-3 activity, which was blocked in the presence of Ac-DEVD-CHO. Conclusion: Sinomenine inhibits osteoclast survival in vitro through caspase-3-mediated apoptosis, thus it is a potential agent for treating excessive bone resorption diseases. PMID:24362325

  18. The generation of osteoclasts from RAW 264.7 precursors in defined, serum-free conditions.

    PubMed

    Vincent, Cristina; Kogawa, Masakazu; Findlay, David M; Atkins, Gerald J

    2009-01-01

    Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete alpha-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1alpha,25-dihydroxyvitamin D(3) (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.

  19. Eos, MITF, and PU.1 recruit corepressors to osteoclast-specific genes in committed myeloid progenitors.

    PubMed

    Hu, Rong; Sharma, Sudarshana M; Bronisz, Agnieszka; Srinivasan, Ruchika; Sankar, Uma; Ostrowski, Michael C

    2007-06-01

    Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.

  20. Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration.

    PubMed

    Kim, Jin-Man; Kim, Mi Yeong; Lee, Kyunghee; Jeong, Daewon

    2016-12-05

    Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses.

  1. Osteoclast-gene expression profiling reveals osteoclast-derived CCR2 chemokines promoting myeloma cell migration

    PubMed Central

    Moreaux, Jérôme; Hose, Dirk; Kassambara, Alboukadel; Rème, Thierry; Moine, Philippe; Requirand, Guilhem; Goldschmidt, Hartmut; Klein, Bernard

    2011-01-01

    Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells (multiple myeloma cells, MMC), primarily in the bone marrow (BM). Osteolytic bone lesions are detected in 80% of patients, due to increased osteoclastic bone resorption and reduced osteoblastic bone formation. MMC are found closely associated to sites of increased bone resorption. Osteoclasts strongly support MMC survival and vice versa in vitro. To further elucidate the mechanisms involved in osteoclast/MMC interaction, we have identified 552 genes overexpressed in osteoclasts compared to other BM cell subpopulations. Osteoclasts express specifically genes coding for four CCR2-targeting chemokines, and genes coding for MMC growth factors (IGF-1, APRIL). An anti-CCR2 MoAb blocked osteoclast chemoattractant activity for MMC and CCR2-chemokines are also MMC growth factors, promoting MAPK activation in MMC. An anti-IGF-1 receptor MoAb completely blocked the osteoclast-induced survival of MMC suppressing both osteoclast and MMC survival. Specific APRIL or IL-6 inhibitors partially blocked osteoclast-induced MMC survival. These in-vitro data may explain why newly-diagnosed patients whose MMC express high levels of CCR2 present numerous bone lesions. Taken together, this study displays additional mechanisms involved in osteoclast/MMC interaction and suggests using CCR2 and/or IGF-1 targeting strategies to block this interaction and prevent drug resistance. PMID:21097672

  2. FSH-Receptor Isoforms and FSH-dependent Gene Transcription in Human Monocytes and Osteoclasts

    PubMed Central

    Robinson, Lisa J; Tourkova, Irina; Wang, Yujuan; Sharrow, Allison C; Landau, Michael S; Yaroslavskiy, Beatrice B; Li, Sun; Zaidi, Mone; Blair, Harry C

    2010-01-01

    Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8–10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes or osteoclasts transcribed TNFα in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2 hours in 25 ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFα and TSG-6, increased 2–3 fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms. PMID:20171950

  3. 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

  4. 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.

  5. Inhibition of Osteoclast Formation and Function by Bicarbonate: Role of Soluble Adenylyl Cyclase

    PubMed Central

    Geng, Weidong; Hill, Kathy; Zerwekh, Joseph; Kohler, Thomas; Müller, Ralph; Moe, Orson W.

    2010-01-01

    High [HCO3−] inhibits and low [HCO3−] stimulates bone resorption which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, 24 mM HCO3− at pH 7.4 for 7–8 days and assayed for tartrate-resistant acid phosphatase (TRAP) and vacuolar-ATPase expression, and H+ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by HCO3− in a dose-dependent manner. V-ATPase expression and H+ accumulation normalized to cell cross-sectional area or protein were not significantly changed. The HCO3−-induced inhibition of osteoclast growth and differentiation was blocked by either 2-hydroxyestradiol, an inhibitor of sAC or sAC knock-down by sAC specific siRNA. The model of HCO3− inhibiting osteoclast via sAC was further supported by the fact that the HCO3− dose-response on osteoclasts is flat when cells were saturated with 8-bromo-cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a one-week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro-computed tomography (µCT), was higher in 24mM HCO3− compared to 12 mM HCO3− treated calvaria. This HCO3− effect on BV/TV was blocked by 2-hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by HCO3−, by acting as a HCO3− sensor. PMID:19360717

  6. Inhibition of osteoclast formation and function by bicarbonate: role of soluble adenylyl cyclase.

    PubMed

    Geng, Weidong; Hill, Kathy; Zerwekh, Joseph E; Kohler, Thomas; Müller, Ralph; Moe, Orson W

    2009-08-01

    High [HCO(3)(-)] inhibits and low [HCO(3)(-)] stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM HCO(3)(-) at pH 7.4 for 7-8 days and assayed for tartrate-resistant acid phosphatase (TRAP) and vacuolar-ATPase expression, and H+ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by HCO(3)(-) in a dose-dependent manner. V-ATPase expression and H+ accumulation normalized to cell cross-sectional area or protein were not significantly changed. The HCO(3)(-) -induced inhibition of osteoclast growth and differentiation was blocked by either 2-hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of HCO(3)(-) inhibiting osteoclast via sAC was further supported by the fact that the HCO(3)(-) dose-response on osteoclasts is flat when cells were saturated with 8-bromo-cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1-week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro-computed tomography (microCT), was higher in 24 mM HCO(3)(-) compared to 12 mM HCO(3)(-) treated calvaria. This HCO(3)(-) effect on BV/TV was blocked by 2-hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by HCO(3)(-), by acting as a HCO(3)(-) sensor.

  7. 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

  8. Osteoclast-like cells in soft tissue leiomyosarcomas.

    PubMed

    Gibbons, C L M H; Sun, S G; Vlychou, M; Kliskey, K; Lau, Y S; Sabokbar, A; Athanasou, N A

    2010-03-01

    Giant cell-rich leiomyosarcoma of soft tissues is an unusual variant of malignant smooth muscle tumor characterized by the presence of numerous multinucleated giant cells (MNGCs). The nature of MNGCs and the cellular mechanisms underlying their accumulation in this tumor are poorly understood. Analysis of the expression of osteoclast, macrophage, and smooth muscle markers in two cases of giant cell-rich leiomyosarcoma revealed that the MNGCs in giant cell-rich leiomyosarcoma were negative for smooth muscle markers and that these cells expressed an osteoclast-like phenotype, being positive for CD45, CD68, tartrate-resistant acid phosphatase, and CD51 but negative for CD14 and HLA-DR. Scattered tumor-associated macrophages (TAMs) also expressed this phenotype. Leiomyosarcoma tumor cells strongly reacted for CD51 but were negative for CD14, CD45, and CD68. An analysis of 25 conventional (nongiant cell-containing) leiomyosarcomas found isolated CD68(+) MNGCs in three cases (12%), all of which were grade II/III leiomyosarcomas containing a prominent TAM infiltrate. Leiomyosarcoma-derived TAMs in the presence of receptor activator for nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor were capable of differentiating into osteoclast-like cells capable of resorbing bone. Reverse transcription polymerase chain reaction studies showed that RANKL, osteoprotegerin, and TNF-related apoptosis-inducing ligand were expressed by leiomyosarcoma cells. Our findings indicate that the giant cells found in leiomyosarcomas are osteoclast-like and that they are formed from TAMs by a RANKL-dependent mechanism.

  9. OCLI-023, a Novel Pyrimidine Compound, Suppresses Osteoclastogenesis In Vitro and Alveolar Bone Resorption In Vivo

    PubMed Central

    Kim, Ju Ang; Lee, Doohyun; Kim, Nam Doo; Shin, Hong-In; Bae, Yong Chul; Park, Eui Kyun

    2017-01-01

    An abnormal increase in osteoclast differentiation and activation results in various bone-resorptive diseases, including periodontitis, rheumatoid arthritis, and osteoporosis. Chemical compounds containing pyrimidine ring have been shown to regulate a variety of biological processes. Therefore, in order to identify an antiresorptive agent, we synthesized a series of pyrimidine ring-containing chemical compounds, and found that OCLI-023 suppressed the differentiation and activation of osteoclasts in vitro. OCLI-023 directly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced differentiation of bone marrow macrophages into osteoclasts, without a cytotoxic response. OCLI-023 also downregulated the RANKL-induced mRNA expression of osteoclast markers as well as inhibited the formation of actin rings and resorption pits. OCLI-023 attenuated the RANKL-induced activation of c-Jun N-terminal kinase and nuclear factor kappa-light-chain-enhancer of activated B cell signaling pathways. In a mouse model of periodontitis, ligature induced an increase of distance between cementoenamel junction (CEJ) and alveolar bone crest (ABC) in the second molar, and OCLI-023 significantly reduced it. Histological analysis showed ligature-induced increase of osteoclast numbers was also significantly reduced by OCLI-023. These data demonstrated the inhibitory effect of OCLI-023 on osteoclast differentiation and activity of osteoclasts in vitro, as well as on ligature-induced bone loss in vivo, and OCLI-023 can be proposed as a novel anti-resorptive compound. PMID:28085946

  10. 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.

  11. Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro

    PubMed Central

    Sipola, Annina; Nelo, Katri; Hautala, Timo; Ilvesaro, Joanna; Tuukkanen, Juha

    2006-01-01

    Background Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. Methods Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. Results Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. Conclusion Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption. PMID:16839420

  12. Osteoinductive LIM Mineralization Protein-1 Suppresses Activation of NF-κB and Selectively Regulates MAPK Pathways in Pre-osteoclasts

    PubMed Central

    Liu, Hui; Bargouti, Maggie; Zughaier, Susu; Zheng, Zhaomin; Liu, Yunshan; Sangadala, Sreedhara; Boden, Scott D.; Titus, Louisa

    2009-01-01

    LIM Mineralization Protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible Nitric Oxide Synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-κB), and prevented the phosphorylation of inhibitor of kappa B (IκB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-κB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-κB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways. PMID:19931434

  13. Osteoinductive LIM mineralization protein-1 suppresses activation of NF-kappaB and selectively regulates MAPK pathways in pre-osteoclasts.

    PubMed

    Liu, Hui; Bargouti, Maggie; Zughaier, Susu; Zheng, Zhaomin; Liu, Yunshan; Sangadala, Sreedhara; Boden, Scott D; Titus, Louisa

    2010-05-01

    LIM mineralization protein-1 (LMP-1) is an intracellular regulator of bone formation and has been shown to be osteoinductive in vitro and in vivo. The effect of LMP-1 on other aspects of bone homeostasis has not been previously studied. In a pilot study we observed that LMP-1 decreased nitric oxide (NO) production in pre-osteoclasts. Here we report a new anti-inflammatory effect of LMP-1 and define its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 pre-osteoclasts. We found that LMP-1 significantly inhibited LPS-induced NO production. LMP-1 also effectively inhibited the expression of inducible nitric oxide synthase (iNOS), potently suppressed the transcriptional activity and nuclear translocation of nuclear factor kappa B (NF-kappaB), and prevented the phosphorylation of inhibitor of kappa B (IkappaB). Interestingly, LMP-1 had no effect on Receptor-Activator of Nuclear Factor B Ligand (RANKL)-induced activation of NF-kappaB. Furthermore, LMP-1 had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it did attenuate the phosphorylation of c-Jun NH2-terminal kinase (JNK) while enhancing phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK). These results suggest that LMP-1 has an anti-inflammatory effect, and this effect is, at least in part, due to the inhibition of NO production by the suppression of NF-kappaB activation and selective regulation of mitogen-activated protein kinase (MAPK) pathways.

  14. C3 Rho-Inhibitor for Targeted Pharmacological Manipulation of Osteoclast-Like Cells

    PubMed Central

    Tautzenberger, Andrea; Förtsch, Christina; Zwerger, Christian; Dmochewitz, Lydia; Kreja, Ludwika; Ignatius, Anita; Barth, Holger

    2013-01-01

    The C3 toxins from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) as well as C3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the C3-catalyzed ADP-ribosylation of Rho results in inhibition of Rho-signalling and characteristic morphological changes. Since the fusion toxin C2IN-C3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like RAW 264.7 cells, its effects on RAW 264.7-derived osteoclasts were investigated. C2IN-C3lim was taken up into differentiated osteoclasts and decreased their resorption activity. In undifferentiated RAW 264.7 cells, C2IN-C3lim-treatment significantly decreased their differentiation into osteoclasts as determined by counting the multi-nucleated, TRAP-positive cells. This inhibitory effect was concentration- and time-dependent and most efficient when C2IN-C3lim was applied in the early stage of osteoclast-formation. A single-dose application of C2IN-C3lim at day 0 and its subsequent removal at day 1 reduced the number of osteoclasts in a comparable manner while C2IN-C3lim-application at later time points did not reduce the number of osteoclasts to a comparable degree. Control experiments with an enzymatically inactive C3 protein revealed that the ADP-ribosylation of Rho was essential for the observed effects. In conclusion, the results indicate that Rho-activity is crucial during the early phase of osteoclast-differentiation. Other bone cell types such as pre-osteoblastic cells were not affected by C2IN-C3lim. Due to their cell-type selective and specific mode of action, C3 proteins and C3-fusions might be valuable tools for targeted pharmacological manipulation of osteoclast formation and activity, which could lead to development of novel therapeutic strategies against osteoclast-associated diseases. PMID:24386487

  15. 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

  16. The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts.

    PubMed

    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

  17. Impairment of osteoclastic bone resorption in rapidly growing female p47phox knockout mice

    USDA-ARS?s Scientific Manuscript database

    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...

  18. 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

  19. Osteoclastic miR-214 targets TRAF3 to contribute to osteolytic bone metastasis of breast cancer

    PubMed Central

    Liu, Jin; Li, Defang; Dang, Lei; Liang, Chao; Guo, Baosheng; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Y. S.; He, Bing; Liu, Biao; Li, Fangfei; Lu, Jun; Wang, Luyao; Shaikh, Atik Badshah; Jiang, Feng; Lu, Changwei; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Xiao, Lianbo; Lu, Aiping; Zhang, Ge

    2017-01-01

    The role of osteoclastic miRNAs in regulating osteolytic bone metastasis (OBM) of breast cancer is still underexplored. Here, we examined the expression profiles of osteoclastogenic miRNAs in human bone specimens and identified that miR-214-3p was significantly upregulated in breast cancer patients with OBM. Consistently, we found increased miR-214-3p within osteoclasts, which was associated with the elevated bone resorption, during the development of OBM in human breast cancer xenografted nude mice (BCX). Furthermore, genetic ablation of osteoclastic miR-214-3p in nude mice prevent the development of OBM. Conditioned medium from MDA-MB-231 cells dramatically stimulated miR-214-3p expression to promote osteoclast differentiation. Mechanistically, a series of in vitro study showed that miR-214-3p directly targeted Traf3 to promote osteoclast activity and bone-resorbing activity. In addition, osteoclast-specific miR-214-3p knock-in mice showed remarkably increased bone resorption when compared to the littermate controls, which was attenuated after osteoclast-targeted treatment with Traf3 3′UTR-containing plasmid. In BCX nude mice, osteoclast-targeted antagomir-214-3p delivery could recover the TRAF3 protein expression and attenuate the development of OBM, respectively. Collectively, inhibition of osteoclastic miR-214-3p may be a potential therapeutic strategy for breast cancer patients with OBM. Meanwhile, the intraosseous TRAF3 could be a promising biomarker for evaluation of the treatment response of antagomir-214-3p. PMID:28071724

  20. [Separation of osteoclasts by lectin affinity chromatography].

    PubMed

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  1. 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

  2. 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.

  3. Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite.

    PubMed

    Casarrubios, Laura; Matesanz, María Concepción; Sánchez-Salcedo, Sandra; Arcos, Daniel; Vallet-Regí, María; Portolés, María Teresa

    2016-11-15

    Silicon substituted hydroxyapatites (SiHA) are highly crystalline bioceramics treated at high temperatures (about 1200°C) which have been approved for clinical use with spinal, orthopedic, periodontal, oral and craniomaxillofacial applications. The preparation of SiHA with lower temperature methods (about 700°C) provides nanocrystalline SiHA (nano-SiHA) with enhanced bioreactivity due to higher surface area and smaller crystal size. The aim of this study has been to know the nanocrystallinity effects on the response of both osteoblasts and osteoclasts (the two main cell types involved in bone remodelling) to silicon substituted hydroxyapatite. Saos-2 osteoblasts and osteoclast-like cells (differentiated from RAW-264.7 macrophages) have been cultured on the surface of nano-SiHA and SiHA disks and different cell parameters have been evaluated: cell adhesion, proliferation, viability, intracellular content of reactive oxygen species, cell cycle phases, apoptosis, cell morphology, osteoclast-like cell differentiation and resorptive activity. This comparative in vitro study evidences that nanocrystallinity of SiHA affects the cell/biomaterial interface inducing bone cell apoptosis by loss of cell anchorage (anoikis), delaying osteoclast-like cell differentiation and decreasing the resorptive activity of this cell type. These results suggest the potential use of nano-SiHA biomaterial for preventing bone resorption in treatment of osteoporotic bone. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Osteopontin regulates macrophage activation and osteoclast formation in hypertensive patients with vascular calcification

    PubMed Central

    Ge, Qian; Ruan, Cheng-Chao; Ma, Yu; Tang, Xiao-Feng; Wu, Qi-Hong; Wang, Ji-Guang; Zhu, Ding-Liang; Gao, Ping-Jin

    2017-01-01

    Vascular calcification (VC) is a highly regulated ectopic mineral deposition process involving immune cell infiltration in the vasculatures, which has been recognized to be promoted by hypertension. The matricellular glycoprotein osteopontin (OPN) is strongly induced in myeloid cells as a potential inflammatory mediator of vascular injury. This study aims to examine whether OPN is involved in the regulation of macrophage activation and osteoclast formation in hypertensive subjects with VC. We firstly found an increased proportion of CD11c+CD163- pro-inflammatory peripheral monocytes in hypertensive subjects with VC compared to those without VC by flow cytometric analysis. Primary cultured macrophages from hypertensive subjects with VC also showed altered expression profile of inflammatory factors and higher serum OPN level. Exogenous OPN promoted the differentiation of peripheral monocytes into an alternative, anti-inflammatory phenotype, and inhibited macrophage-to-osteoclast differentiation from these VC patients. In addition, calcified vessels showed increased osteoclasts accumulation accompanied with decreased macrophages infiltration in the of hypertensive subjects. Taken together, these demonstrated that OPN exerts an important role in the monocytes/macrophage phenotypic differentiation from hypertensive patients with VC, which includes reducing inflammatory factor expression and attenuating osteoclast formation. PMID:28091516

  5. 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.

  6. 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

  7. 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

  8. Nano-topography sensing by osteoclasts

    PubMed Central

    Geblinger, Dafna; Addadi, Lia; Geiger, Benjamin

    2010-01-01

    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 ~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. PMID:20375065

  9. Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages.

    PubMed

    Choe, Jung-Yoon; Kim, Seong-Kyu

    2017-03-01

    Melittin is a major toxic component of bee venom (Apis mellifera). It is not known whether melittin is involved in bone metabolism and osteoclastogenesis. The aim of this study was to determine the role of melittin in the regulation of osteoclastogenesis. In vitro osteoclastogenesis assays were performed using mouse RAW 264.7 cells and bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Morphologic and functional analyses for osteoclast-like multinucleated cells (MNCs) were performed by tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and pit formation methods. The gene expression of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II was measured by reverse transcription-quantitative PCR. The protein expression levels of mitogen-activated protein kinases (MAPKs), the p65 subunit of nuclear factor-κB (NF-κB), c-Fos, c-Jun, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TNF receptor-associated factor-6 (TRAF6), and interleukin-1β (IL-1β) were assessed by western blot analysis. Melittin inhibited the mRNA expression of TRAP, cathepsin K, MMP-9 and carbonic anhydrase II in RANKL-stimulated RAW 264.7 cells. The increased protein expression of TRAF6, p-extracellular signal-regulated kinase (ERK), p-JNK, p-p65, p-c-Fos and NFATc1 induced by RANKL was significantly suppressed in the RAW 264.7 cells treated with melittin. A synergistic effect of IL-1β on the formation of RANKL-induced osteoclast-like MNCs was found in two experimental cells. The increased expression of IL-1β following the stimulation of RAW 264.7 cells with RANKL activated TRAF6, p-ERK, p-JNK, p-p65, p-c-Fos and NFATc1. These effects were attenuated by the downregulation of IL-1β using siRNA against IL-1β, and also by treatment with melittin. On the whole, the findings of this study demonstrate that melittin

  10. Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages

    PubMed Central

    Choe, Jung-Yoon; Kim, Seong-Kyu

    2017-01-01

    Melittin is a major toxic component of bee venom (Apis mellifera). It is not known whether melittin is involved in bone metabolism and osteoclastogenesis. The aim of this study was to determine the role of melittin in the regulation of osteoclastogenesis. In vitro osteoclastogenesis assays were performed using mouse RAW 264.7 cells and bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Morphologic and functional analyses for osteoclast-like multinucleated cells (MNCs) were performed by tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and pit formation methods. The gene expression of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II was measured by reverse transcription-quantitative PCR. The protein expression levels of mitogen-activated protein kinases (MAPKs), the p65 subunit of nuclear factor-κB (NF-κB), c-Fos, c-Jun, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TNF receptor-associated factor-6 (TRAF6), and interleukin-1β (IL-1β) were assessed by western blot analysis. Melittin inhibited the mRNA expression of TRAP, cathepsin K, MMP-9 and carbonic anhydrase II in RANKL-stimulated RAW 264.7 cells. The increased protein expression of TRAF6, p-extracellular signal-regulated kinase (ERK), p-JNK, p-p65, p-c-Fos and NFATc1 induced by RANKL was significantly suppressed in the RAW 264.7 cells treated with melittin. A synergistic effect of IL-1β on the formation of RANKL-induced osteoclast-like MNCs was found in two experimental cells. The increased expression of IL-1β following the stimulation of RAW 264.7 cells with RANKL activated TRAF6, p-ERK, p-JNK, p-p65, p-c-Fos and NFATc1. These effects were attenuated by the downregulation of IL-1β using siRNA against IL-1β, and also by treatment with melittin. On the whole, the findings of this study demonstrate that melittin inhibits the formation

  11. The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic.

    PubMed

    Ramaswamy, Yogambha; Wu, Chengtie; Van Hummel, Annika; Combes, Valery; Grau, Georges; Zreiqat, Hala

    2008-11-01

    In this study we have developed Ca(3)ZrSi(2)O(9) (Baghdadite) ceramics by incorporating Zirconium in Ca-Si system and determined their biological properties. Ca(3)ZrSi(2)O(9) ceramics possess apatite-formation ability in simulated body fluid, indicating their potential bioactivity. The response of human osteoblast like cells (HOB), osteoclast and endothelial cells when cultured on Ca(3)ZrSi(2)O(9) ceramics was investigated. Scanning electron microscopy and immunofluorescence studies demonstrated that this material supports HOB cell attachment with organized cytoskeleton structure. Compared to CaSiO(3), Ca(3)ZrSi(2)O(9) ceramics induced increased HOB proliferation and differentiation as shown by increased methyltetrazidium salt (MTS), alkaline phosphatase activity, and mRNA expression levels of bone-related genes (Collagen type I, alkaline phosphatase, Bone Sialoprotein, receptor activator of NF-kappaB ligand and osteoprotegerin). Ca(3)ZrSi(2)O(9) ceramics supported the fusion of monocytes to form functional osteoclasts with their characteristic features of f-actin ring structures and the expression of alpha(v)beta(3) integrin consistent with functional activity. Osteoclasts cultured on Ca(3)ZrSi(2)O(9) expressed increased levels of osteoclast-related genes; Cathepsin K, Carbonic Anhydrase II, Matrix metalloproteinase-9, receptor activator of NF-kappaB and Calcitonin Receptor, consistent with the formation of functional osteoclasts. In addition to HOB and osteoclasts, Ca(3)ZrSi(2)O(9) supported the attachment of endothelial cells, which expressed the endothelial cell markers; ZO-1 and VE-Cadherin. Results presented here indicate that Ca(3)ZrSi(2)O(9) ceramics have the potential for applications in bone tissue regeneration.

  12. Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.

    PubMed

    Hirozane, Toru; Tohmonda, Takahide; Yoda, Masaki; Shimoda, Masayuki; Kanai, Yae; Matsumoto, Morio; Morioka, Hideo; Nakamura, Masaya; Horiuchi, Keisuke

    2016-09-28

    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.

  13. 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

  14. Deletion of Orai1 alters expression of multiple genes during osteoclast and osteoblast maturation.

    PubMed

    Hwang, Sung-Yong; Foley, Julie; Numaga-Tomita, Takuro; Petranka, John G; Bird, Gary S; Putney, James W

    2012-12-01

    Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1(-/-)). Orai1(-/-) mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1(-/-) mice were examined. Orai1-mediated SOCE was completely abolished in Orai1(-/-) osteoclast precursor cells and osteoclastogenesis in vitro from Orai1(-/-) mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1(-/-) osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1(-/-) mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1(-/-) osteoblast precursor cells and osteoblastogenesis in vitro from Orai1(-/-) stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts. Published by Elsevier India Pvt Ltd.

  15. Deletion of Orai1 Alters Expression of Multiple Genes during Osteoclast and Osteoblast Maturation

    PubMed Central

    Hwang, Sung-Yong; Foley, Julie; Numaga-Tomita, Takuro; Petranka, John G.; Bird, Gary S.; Putney, James W.

    2012-01-01

    Summary Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1−/−). Orai1−/− mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1−/− mice were examined. Orai1-mediated SOCE was completely abolished in Orai1−/− osteoclast precursor cells and osteoclastogenesis in vitro from Orai1−/− mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1−/− osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1−/− mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1−/− osteoblast precursor cells and osteoblastogenesis in vitro from Orai1−/− stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts. PMID:23122304

  16. 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

  17. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    PubMed Central

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2014-01-01

    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 20 ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1 hour of mechanical vibration with 20 µm displacement at a frequency of 4 Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5 days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells were determined after 1 hour mechanical vibration, while protein production of the DC-STAMP was determined after 6 hours of post incubation 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 reduce DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. PMID:23994170

  18. 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. © 2013 Elsevier Inc. All rights reserved.

  19. Imatinib mesylate suppresses bone metastases of breast cancer by inhibiting osteoclasts through the blockade of c-Fms signals.

    PubMed

    Hiraga, Toru; Nakamura, Hiroaki

    2009-01-01

    Imatinib mesylate (imatinib) is a potent and selective inhibitor of the tyrosine kinases, Bcr-Abl, c-Kit and platelet-derived growth factor receptors (PDGFRs). Recently, it has been reported that imatinib also targets the macrophage colony-stimulating factor (M-CSF) receptor c-Fms. M-CSF signals are essential for the differentiation of osteoclasts. Bone metastases of breast cancer are frequently associated with osteoclastic bone destruction. Furthermore, several lines of evidence suggest that osteoclasts play central roles in the development and progression of bone metastases. Thus, in the present study, we examined the effects of imatinib on bone metastases of breast cancer. Coimmunoprecipitation assays showed that imatinib inhibited the M-CSF-induced phosphorylation of c-Fms in osteoclast precursor cells as well as the PDGF-induced PDGFR phosphorylation in MDA-MB-231 human breast cancer cells. Imatinib also markedly reduced osteoclast formation in vitro. In contrast, those concentrations of imatinib did not affect osteoblast differentiation. We then examined the effects of imatinib on bone metastases of MDA-MB-231 cells in a nude mouse model. Radiographic and histomorphometric analyses demonstrated that imatinib significantly decreased bone metastases associated with the reduced number of osteoclasts. In support of the notion that the inhibition of c-Fms acts to suppress the development of bone metastases, we found that a specific inhibitor of c-Fms Ki20227 also decreased bone metastases. In conclusion, these results collectively suggest that imatinib reduced bone metastases, at least in part, by inhibiting osteoclastic bone destruction through the blockade of c-Fms signals. Our results also suggest that imatinib may have a protective effect against cancer treatment-induced bone loss.

  20. Osteoclasts and giant cells: macrophage–macrophage fusion mechanism

    PubMed Central

    Vignery, Agnès

    2000-01-01

    Membrane fusion is a ubiquitous event that occurs in a wide range of biological processes. While intracellular membrane fusion mediating organelle trafficking is well understood, much less is known about cell–cell fusion mediating sperm cell–oocyte, myoblast–myoblast and macrophage–macrophage fusion. In the case of mononuclear phagocytes, their fusion is not only associated with the differentiation of osteoclasts, cells which play a key role in the pathogenesis of osteoporosis, but also of giant cells that are present in chronic inflammatory reactions and in tumours. Despite the biological and pathophysiological importance of intercellular fusion events, the actual molecular mechanism of macrophage fusion is still unclear. One of the main research themes in my laboratory has been to investigate the molecular mechanism of mononuclear phagocyte fusion. Our hypothesis has been that macrophage–macrophage fusion, similar to virus–cell fusion, is mediated by specific cell surface proteins. But, in contrast with myoblasts and sperm cells, macrophage fusion is a rare event that occurs in specific instances. To test our hypothesis, we established an in vitro cell–cell fusion assay as a model system which uses alveolar macrophages. Upon multinucleation, these macrophages acquire the osteoclast phenotype. This indicates that multinucleation of macrophages leads to a specific and novel functional phenotype in macrophages. To identify the components of the fusion machinery, we generated four monoclonal antibodies (mAbs) which block the fusion of alveolar macrophages and purified the unique antigen recognized by these mAbs. This led us to the cloning of MFR (Macrophage Fusion Receptor). MFR was cloned simultaneously as P84/SHPS-1/SIRPα/BIT by other laboratories. We subsequently showed that the recombinant extracellular domain of MFR blocks fusion. Most recently, we identified a lower molecular weight form of MFR that is missing two extracellular immunoglobulin (Ig

  1. Polarity and membrane transport in osteoclasts.

    PubMed

    Baron, R

    1989-01-01

    The osteoclast is a highly polarized non-epithelial cell. The apical pole of the cell is determined by the cell's attachment to the extracellular matrix. This attachment forms the sealing zone, delimiting the subosteoclastic bone resorbing compartment. The apical membrane of the cell forms the ruffled-border, which contains some specific membrane proteins and a proton pump ATPase, which acidifies the apical compartment. Newly synthesized lysosomal enzymes are vectorially transported into this apical compartment bound to mannose-6-phosphate receptors. The basolateral membrane is highly enriched in sodium pumps with beta and alpha 1 subunits. Associated with the acidification process is the carbonic anhydrase found in the cytoplasm and membrane-associated and a bicarbonate-chloride exchanger in the membrane.2 These features put the osteoclast in the same functional category as the kidney tubule intercalated cell and the gastric oxyntic cell, both of epithelial origin, which secrete acid in a polarized fashion.

  2. Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts.

    PubMed

    Boyce, Brendan F

    2013-04-01

    Osteoclasts are multinucleated myeloid lineage cells formed in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) by fusion of bone marrow-derived precursors that circulate in the blood and are attracted to sites of bone resorption in response to factors, such as sphingosine-1 phosphate signaling. Major advances in understanding of the molecular mechanisms regulating osteoclast functions have been made in the past 20 years, mainly from mouse and human genetic studies. These have revealed that osteoclasts express and respond to proinflammatory and anti-inflammatory cytokines. Some of these cytokines activate NF-κB and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling to induce osteoclast formation and activity and also regulate communication with neighboring cells through signaling proteins, including ephrins and semaphorins. Osteoclasts also positively and negatively regulate immune responses and osteoblastic bone formation. These advances have led to development of new inhibitors of bone resorption that are in clinical use or in clinical trials; and more should follow, based on these advances. This article reviews current understanding of how bone resorption is regulated both positively and negatively in normal and pathologic states. Copyright © 2013 American Society for Bone and Mineral Research.

  3. Force-Induced H2S by PDLSCs Modifies Osteoclastic Activity during Tooth Movement.

    PubMed

    Liu, F; Wen, F; He, D; Liu, D; Yang, R; Wang, X; Yan, Y; Liu, Y; Kou, X; Zhou, Y

    2017-02-01

    Hydrogen sulfide (H2S), a gasotransmitter, has been recently linked to mesenchymal stem cell (MSC) function and bone homeostasis. Periodontal ligament stem cells (PDLSCs) are the main MSCs in PDL, which respond to mechanical force to induce physiological activities during orthodontic tooth movement (OTM). However, it is unknown whether mechanical force might induce endogenous H2S production by PDLSCs to regulate alveolar bone homeostasis. Here, we used a mouse OTM model to demonstrate that orthodontic force-induced endogenous H2S production in PDL tissue was associated with macrophage accumulation and osteoclastic activity in alveolar bone. Then, we showed that mechanical force application induced cystathionine β-synthase (CBS) expression and endogenous H2S production by PDLSCs. Moreover, blocking endogenous H2S or systemically increasing H2S levels could decrease or enhance force-induced osteoclastic activities to control tooth movement. We further revealed how force-induced H2S production by PDLSCs contributed to the secretion of monocyte chemoattractant protein-1 (MCP-1) and the expression of receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) system by PDLSCs. The secretion and expression of these factors controlled macrophage migration and osteoclast differentiation. This study demonstrated that PDLSCs produced H2S to respond to and transduce force signals. Force-induced gasotransmitter H2S production in PDLSCs therefore regulated osteoclastic activities in alveolar bone and controlled the OTM process through the MCP-1 secretion and RANKL/OPG system.

  4. Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis

    PubMed Central

    Rodrigues, Ana Maria; Campanilho-Marques, Raquel; Ponte, Cristina; Canhão, Helena; Ainola, Mari

    2017-01-01

    Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16− monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6. PMID:28286757

  5. The immunomodulatory adapter proteins DAP12 and Fc receptor γ-chain (FcRγ) regulate development of functional osteoclasts through the Syk tyrosine kinase

    PubMed Central

    Mócsai, Attila; Humphrey, Mary Beth; Van Ziffle, Jessica A. G.; Hu, Yongmei; Burghardt, Andrew; Spusta, Steven C.; Majumdar, Sharmila; Lanier, Lewis L.; Lowell, Clifford A.; Nakamura, Mary C.

    2004-01-01

    Osteoclasts, the only bone-resorbing cells, are central to the pathogenesis of osteoporosis, yet their development and regulation are incompletely understood. Multiple receptors of the immune system use a common signaling paradigm whereby phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within receptor-associated adapter proteins recruit the Syk tyrosine kinase. Here we demonstrate that a similar mechanism is required for development of functional osteoclasts. Mice lacking two ITAM-bearing adapters, DAP12 and the Fc receptor γ-chain (FcRγ), are severely osteopetrotic. DAP12-/-FcRγ-/- bone marrow cells fail to differentiate into multinucleated osteoclasts or resorb bone in vitro and show impaired phosphorylation of the Syk tyrosine kinase. syk-/- progenitors are similarly defective in osteoclast development and bone resorption. Intact SH2-domains of Syk, introduced by retroviral transduction, are required for functional reconstitution of syk-/- osteoclasts, whereas intact ITAM-domains on DAP12 are required for reconstitution of DAP12-/- FcRγ-/- cells. These data indicate that recruitment of Syk to phosphorylated ITAMs is critical for osteoclastogenesis. Although DAP12 appears to be primarily responsible for osteoclast differentiation in cultures directly stimulated with macrophage-colony stimulating factor and receptor activator of NF-κB ligand cytokines, DAP12 and FcRγ have overlapping roles in supporting osteoclast development in osteoblast–osteoclast cocultures, which mirrors their overlapping functions in vivo. These results provide new insight into the biology of osteoclasts and suggest novel therapeutic targets in diseases of bony remodeling. PMID:15073337

  6. ADAM8 Enhances Osteoclast Precursor Fusion and Osteoclast Formation In Vitro and In Vivo

    PubMed Central

    Ishizuka, Hisako; García-Palacios, Verónica; Lu, Ganwei; Subler, Mark A; Zhang, Heju; Boykin, Christina S; Choi, Sun Jin; Zhao, Liena; Patrene, Kenneth; Galson, Deborah L; Blair, Harry C; Hadi, Tamer M; Windle, Jolene J; Kurihara, Noriyoshi; Roodman, G David

    2011-01-01

    ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate-resistant acid phosphatase (TRAP)–ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP-ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone-resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF-κB, Erk, and Akt signaling compared with wild-type (WT) littermates. This increased bone-resorbing capacity per OCL was associated with increased levels of p-Pyk2 and p-Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF-α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF-α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease. © 2011 American Society for Bone and Mineral Research. PMID:20683884

  7. Cytokine expression in feline osteoclastic resorptive lesions.

    PubMed

    DeLaurier, A; Allen, S; deFlandre, C; Horton, M A; Price, J S

    2002-01-01

    Feline osteoclastic resorptive lesions (FORL) of the teeth are common in cats, and lead to pain, destruction of the periodontal ligament, and tooth loss. The expression of interleukin (IL)-1 beta and IL-6 mRNA was higher in teeth with FORL than in normal teeth (P<0.01 and P<0.001, respectively), but no such differences were found between pathological and normal gingival tissue samples. There were no differences between teeth affected with FORL and normal teeth in respect of the expression of receptor activator of NF kappa B ligand (RANKL) mRNA or osteoprotegerin (OPG) mRNA. However, OPG mRNA expression was higher in gingival tissue associated with teeth affected with FORL than in normal gingival tissue (P<0.05), whereas the reverse was true of RANKL mRNA expression (P<0.05). OPG mRNA expression was significantly higher in teeth than in femoral and alveolar bone (P<0.001). RANKL and OPG mRNAs were detected in all tissues examined. The data suggest that the elevated expression of IL-l beta and IL-6 mRNA plays a role in the mediation of osteoclast activity in advanced FORL. In contrast, OPG and RANKL do not appear to regulate osteoclasts in advanced disease. The results also suggest that OPG and RANKL mRNA play a role in mediating inflammatory responses in gingival cells, and that OPG has an inhibiting effect on tooth resorption.

  8. Intravenous Immunoglobulin (IVIG) Attenuates TNF-induced Pathologic Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20 Expression

    PubMed Central

    Mun, Sehwan; Bae, Seyeon; Murata, Koichi; Ivashkiv, Lionel B.; Park-Min, Kyung-Hyun

    2016-01-01

    Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody- and immune complex-mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone-resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast-mediated pathologic bone resorption. IVIG or cross-linking of Fcγ receptors with plate-bound IgG suppressed receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and expression of osteoclast-related genes such as integrin β3 and cathepsin K in a dose-dependent manner. Mechanistically, IVIG or plate-bound IgG suppressed osteoclastogenesis by downregulating RANKL-induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL-induced NF-κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)-induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders. PMID:26189496

  9. Reactive oxygen species are required for zoledronic acid-induced apoptosis in osteoclast precursors and mature osteoclast-like cells

    PubMed Central

    Tai, Ta-Wei; Chen, Ching-Yu; Su, Fong-Chin; Tu, Yuan-Kun; Tsai, Tsung-Ting; Lin, Chiou-Feng; Jou, I.-Ming

    2017-01-01

    Inhibiting osteoclasts and osteoclast precursors to reduce bone resorption is an important strategy to treat osteoclast-related diseases, such as osteoporosis, inflammatory bone loss, and malignant bone metastasis. However, the mechanism by which apoptosis is induced in the osteoclasts and their precursors are not completely understood. Here, we used nitrogen-containing bisphosphonate zoledronic acid (ZA) to induce cell apoptosis in human and murine osteoclast precursors and mature osteoclast-like cells. Caspase-3-mediated cell apoptosis occurred following the ZA (100 μM) treatment. Reactive oxygen species (ROS) were also generated in a time-dependent manner. Following knock-down of the p47phox expression, which is required for ROS activation, or co-treatment with the ROS inhibitor, N-acetyl-L-cysteine, ZA-induced apoptosis was significantly suppressed in both osteoclast precursors and mature osteoclast-like cells. The ROS-activated mitogen-activated protein kinases pathways did not trigger cell apoptosis. However, a ROS-regulated Mcl-1 decrease simultaneously with glycogen synthase kinase (GSK)-3β promoted cell apoptosis. These findings show that ZA induces apoptosis in osteoclast precursors and mature osteoclast-like cells by triggering ROS- and GSK-3β-mediated Mcl-1 down-regulation. PMID:28281643

  10. 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

  11. Osteoclast Inhibitory Peptide-1 Therapy for Paget’s Disease

    DTIC Science & Technology

    2012-08-01

    1 (SQSTM1/p62) gene have been widely identified in PDB patients. We previously detected expression of measles virus nucleocapsid (MVNP) transcripts...high bone turnover in PDB. 15. SUBJECT TERMS Paget’s Disease, measles virus nucleocapsid, sequestosome1 , osteoclast, osteoclast inhibitory peptide...detected expression of measles virus nucleocapsid (MVNP) transcripts in osteoclasts from patients with PDB. Also, we have shown that MVNP gene

  12. Macrophage inflammatory protein-1 delta: a novel osteoclast stimulating factor secreted by renal cell carcinoma bone metastasis.

    PubMed

    Kominsky, Scott L; Abdelmagid, Samir M; Doucet, Michele; Brady, Kelly; Weber, Kristy L

    2008-03-01

    Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1 delta (MIP-1 delta), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1 delta expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1 delta, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1 delta stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1 delta treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1 delta significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1 delta expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.

  13. The Effect of Everolimus in an In Vitro Model of Triple Negative Breast Cancer and Osteoclasts.

    PubMed

    Mercatali, Laura; Spadazzi, Chiara; Miserocchi, Giacomo; Liverani, Chiara; De Vita, Alessandro; Bongiovanni, Alberto; Recine, Federica; Amadori, Dino; Ibrahim, Toni

    2016-11-01

    Metastatic bone disease has a major impact on morbidity of breast cancer (BC) patients. Alterations in mTOR signaling are involved both in cancer progression and in osteoclast differentiation. The purpose of this study was to assess the role of mTOR inhibitor Everolimus (Eve) on osteoclastogenesis induced by triple negative BC cells. To this aim, we developed an in vitro human model of osteoclastogenesis from peripheral blood monocytes co-cultured with the triple negative SCP2 and the hormonal receptor positive MCF7 cell lines. Osteoclastogenesis was evaluated by TRAP staining, evaluation of F actin rings and Calcitonin Receptor expression. Eve significantly reduced differentiation induced by cancer cells and resulted more effective when evaluated in combination with Denosumab and Zoledronic Acid (Zol). Combination with Zol showed a total abrogation of osteoclast differentiation induced by the triple negative cell line, not by MCF7. Finally, we observed that Eve was active in the inhibition of the crosstalk between cancer cells and osteoclasts reproduced by our model, highlighting a new therapeutic choice for the subsetting of triple negative BC patients. We observed a difference in the response to bone-targeted therapy with respect to BC subtypes. Our model may represent a valid platform for preclinical trials on bone-targeted drugs and for the study of the interplay of BC with bone stromal cells.

  14. The Effect of Everolimus in an In Vitro Model of Triple Negative Breast Cancer and Osteoclasts

    PubMed Central

    Mercatali, Laura; Spadazzi, Chiara; Miserocchi, Giacomo; Liverani, Chiara; De Vita, Alessandro; Bongiovanni, Alberto; Recine, Federica; Amadori, Dino; Ibrahim, Toni

    2016-01-01

    Metastatic bone disease has a major impact on morbidity of breast cancer (BC) patients. Alterations in mTOR signaling are involved both in cancer progression and in osteoclast differentiation. The purpose of this study was to assess the role of mTOR inhibitor Everolimus (Eve) on osteoclastogenesis induced by triple negative BC cells. To this aim, we developed an in vitro human model of osteoclastogenesis from peripheral blood monocytes co-cultured with the triple negative SCP2 and the hormonal receptor positive MCF7 cell lines. Osteoclastogenesis was evaluated by TRAP staining, evaluation of F actin rings and Calcitonin Receptor expression. Eve significantly reduced differentiation induced by cancer cells and resulted more effective when evaluated in combination with Denosumab and Zoledronic Acid (Zol). Combination with Zol showed a total abrogation of osteoclast differentiation induced by the triple negative cell line, not by MCF7. Finally, we observed that Eve was active in the inhibition of the crosstalk between cancer cells and osteoclasts reproduced by our model, highlighting a new therapeutic choice for the subsetting of triple negative BC patients. We observed a difference in the response to bone-targeted therapy with respect to BC subtypes. Our model may represent a valid platform for preclinical trials on bone-targeted drugs and for the study of the interplay of BC with bone stromal cells. PMID:27809291

  15. The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

    PubMed Central

    Huang, Su; Eleniste, Pierre P.; Wayakanon, Kornchanok; Mandela, Prashant; Eipper, Betty A.; Mains, Richard E.; Allen, Matthew R.; Bruzzaniti, Angela

    2014-01-01

    Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass. PMID:24380811

  16. Reduced RANKL expression impedes osteoclast activation and tooth eruption in alendronate-treated rats.

    PubMed

    Bradaschia-Correa, Vivian; Moreira, Mariana M; Arana-Chavez, Victor E

    2013-07-01

    The creation of the eruption pathway requires the resorption of the occlusal alveolar bone by osteoclasts and signaling events between bone and dental follicle are necessary. The aim of the present study has been to evaluate the effect of alendronate on osteoclastogenesis and the expression of the regulator proteins of osteoclast activation, namely RANK, RANKL and OPG, in the bone that covers the first molar germ. Newborn Wistar rats were treated daily with 2.5 mg/kg alendronate for 4, 8, 14, 21 and 28 days, whereas controls received sterile saline solution. At the time points cited, maxillae were fixed, decalcified and processed for light and electron microscopic analysis. TRAP histochemistry was performed on semi-serial sections and the osteoclasts in the occlusal half of the bony crypt surface were counted. TUNEL analysis was carried out on paraffin sections. The occlusal bone that covers the upper first molar was removed in additional 4- and 8-day-old alendronate-treated and control rats in which the expression of RANK, RANKL and OPG was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. TRAP-positive osteoclasts were more numerous in the alendronate group at all time points, despite their unactivated phenotype and the presence of apoptotic cells. RANKL expression in the alendronate specimens was inhibited at all time points, unlike in controls. Our findings indicate that the expression of RANKL in the occlusal portion of the bony crypt is unrelated to osteoclast recruitment and differentiation but is crucial to their activation during the creation of the eruption pathway.

  17. Effects of bleaching on osteoclast activity and their modulation by osteostatin and fibroblast growth factor 2.

    PubMed

    Torres-Rodríguez, Carolina; Portolés, M Teresa; Matesanz, M Concepción; Linares, Javier; Feito, M José; Izquierdo-Barba, Isabel; Esbrit, Pedro; Vallet-Regí, María

    2016-01-01

    Dental bleaching with H2O2 is a common daily practice in dentistry to correct discoloration of anterior teeth. The aim of this study has been to determine whether this treatment of human teeth affects growth, differentiation and activity of osteoclast-like cells, as well as the putative modulatory action of osteostatin and fibroblast growth factor 2 (FGF-2). Previously to the in vitro assays, structural, physical-chemical and morphological features of teeth after bleaching were studied. Osteoclast-like cells were cultured on human dentin disks, pre-treated or not with 38% H2O2 bleaching gel, in the presence or absence of osteostatin (100 nM) or FGF-2 (1 ng/ml). Cell proliferation and viability, intracellular content of reactive oxygen species (ROS), pro-inflammatory cytokine (IL-6 and TNFα) secretion and resorption activity were evaluated. Bleaching treatment failed to affect either the structural or the chemical features of both enamel and dentin, except for slight morphological changes, increased porosity in the most superficial parts (enamel), and a moderate increase in the wettability degree. In this scenario, bleaching produced an increased osteoclast-like cell proliferation but decreased cell viability and cytokine secretion, while it augmented resorption activity on dentin. The presence of either osteostatin or FGF-2 reduced the osteoclast-like cell proliferation induced by bleaching. FGF-2 enhanced ROS content, whereas osteostatin decreased ROS but increased TNFα secretion. The bleaching effect on resorption activity was increased by osteostatin, but this effect was less evident with FGF-2. These findings further confirm the deleterious effects of tooth bleaching by affecting osteoclast growth and function as well as different modulatory actions of osteostatin and FGF-2. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts.

    PubMed

    Huang, Su; Eleniste, Pierre P; Wayakanon, Kornchanok; Mandela, Prashant; Eipper, Betty A; Mains, Richard E; Allen, Matthew R; Bruzzaniti, Angela

    2014-03-01

    Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass.

  19. 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 recept