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Sample records for suppress osteoclast activity

  1. Osteoclast activated FoxP3+ CD8+ T-cells suppress bone resorption in vitro.

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    Zachary S Buchwald

    Full Text Available BACKGROUND: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG. The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption. PRINCIPAL FINDINGS: To determine the net effect of Tc(REG on osteoclast activity we used a number of in vitro assays. We found that Tc(REG can potently and directly suppress bone resorption by osteoclasts. Tc(REG could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG and osteoclasts. SIGNIFICANCE: We have determined that osteoclast-induced Tc(REG can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology.

  2. Dihydromyricetin Protects against Bone Loss in Ovariectomized Mice by Suppressing Osteoclast Activity

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    Libo Zhao

    2017-12-01

    Full Text Available Dihydromyricetin (DMY, the main flavonoid component of Ampelopsis grossedentata, possesses pharmacological activities useful for treatment of diseases associated with inflammation and oxidative damage. Because osteoclasts are often involved in chronic low-grade systemic inflammation and oxidative damage, we hypothesized that DMY may be an effective treatment for osteoclast-related diseases. The effects of DMY on osteoclast formation and activity were examined in vitro. Female C57BL/6 mice were ovariectomized to mimic menopause-induced bone loss and treated with DMY, and femur samples were subjected to bone structure and histological analysis, serum biochemical indicators were also measured. DMY suppressed the activation of nuclear factor-κB, c-Fos and mitogen-activated protein kinase, and prevented production of reactive oxygen species. DMY decreased expression of osteoclast-specific genes, including Trap, Mmp-9, Cathepsin K, C-Fos, Nfatc1, and Rank. In addition, DMY prevented bone loss and decreased serum levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6, and with a decrease in the ratio between receptor activator of nuclear factor-κB (RANK ligand (RANKL and osteoprotegerin (OPG in vivo. These findings demonstrate that DMY attenuates bone loss and inhibits osteoclast formation and activity through modulation of multiple pathways both upstream and downstream of RANKL signaling. DMY may thus be a useful option for treatment of osteoclast-related diseases such as rheumatoid arthritis and osteoporosis.

  3. Kaempferide Prevents Titanium Particle Induced Osteolysis by Suppressing JNK Activation during Osteoclast Formation.

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    Jiao, Zixian; Xu, Weifeng; Zheng, Jisi; Shen, Pei; Qin, An; Zhang, Shanyong; Yang, Chi

    2017-11-30

    Kaempferide (KF) is an O-methylated flavonol, a natural plant extract, which is often found in Kaempferia galanga. It has a variety of effects including anti-carcinogenic, anti-inflammatory, anti-oxidant, anti-bacterial and anti-viral properties. In this study, we aimed to investigate whether KF effectively inhibits titanium particle induced calvarial bone loss via down regulation of the JNK signaling pathway. In the mice with titanium particle induced calvarial osteolysis, the Low dose of KF mildly reduced the resorption pits while in the high dose group, fewer scattered pits were observed on the surface of calvarium. Histological examination showed fewer osteoclasts formation in the KF group. In mouse bone marrow macrophages (BMMs) and RAW264.7 cells, KF significantly inhibited the osteoclast formation and bone resorption at 12.5 μM. However, KF does not affect the mature osteoclast F-actin ring formation. But when being co-treated with KF and anisomycin, BMMs differentiated into mature osteoclasts. At the molecular levels, the JNK phosphorylation was inhibited and the osteoclastogenesis-related specific gene expression including V-ATPase d2, TRAP, calcitonin receptor (CTR), c-Fos and NFATc1 was markedly suppressed. In conclusion, these results indicated that KF is a promising agent in the treatment of osteoclast-related diseases.

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

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    Kim, Hyun-Ju, E-mail: biohjk@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Hye-Jin [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Kyung-Ae [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Gwon, Mi-Ri; Jin Seong, Sook [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Shin-Yoon [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Young-Ran, E-mail: yry@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    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.

  5. Dextromethorphan inhibits osteoclast differentiation by suppressing RANKL-induced nuclear factor-κB activation.

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    Wu, Karl; Lin, Tzu-Hung; Liou, Houng-Chi; Lu, Dai-Hua; Chen, Yi-Ru; Fu, Wen-Mei; Yang, Rong-Sen

    2013-08-01

    Dextromethorphan (DXM), a commonly used antitussive, is a dextrorotatory morphinan. Here, we report that DXM inhibits the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption by abrogating the activation of NF-κB signalling in vitro. Oral administration of DXM ameliorates ovariectomy (OVX)-induced osteoporosis in vivo. DXM was reported to possess anti-inflammatory properties through inhibition of the release of pro-inflammatory factors. However, the potential role and action mechanism of DXM on osteoclasts and osteoblasts remain unclear. In this study, in vitro and in vivo studies were performed to investigate the potential effects of DXM on osteoclastogenesis and OVX-induced bone loss. Osteoclastogenesis was examined by the TRAP staining, pit resorption, TNF-α release, and CCR2 and CALCR gene expression. Osteoblast differentiation was analyzed by calcium deposition. Osteogenic and adipogenic genes were measured by real-time PCR. Signaling pathways were explored using Western blot. ICR mice were used in an OVX-induced osteoporosis model. Tibiae were measured by µCT and serum markers were examined with ELISA kits. DXM inhibited RANKL-induced osteoclastogenesis. DXM mainly inhibited osteoclastogenesis via abrogation of IKK-IκBα-NF-κB pathways. However, a higher dosage of DXM antagonized the differentiation of osteoblasts via the inhibition of osteogenic signals and increase of adipogenic signals. Oral administration of DXM (20 mg/kg/day) partially reduced trabecular bone loss in ovariectomized mice. DXM inhibits osteoclast differentiation and activity by affecting NF-κB signaling. Therefore, DXM at suitable doses may have new therapeutic applications for the treatment of diseases associated with excessive osteoclastic activity.

  6. Suppression of T cell-induced osteoclast formation

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    Karieb, Sahar; Fox, Simon W., E-mail: Simon.fox@plymouth.ac.uk

    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.

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

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    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.

  8. Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

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    Park, Eui Kyun; Kim, Myung Sunny; Lee, Seung Ho; Kim, Kyung Hee; Park, Ju-Young; Kim, Tae-Ho; Lee, In-Seon; Woo, Je-Tae; Jung, Jae-Chang; Shin, Hong-In; Choi, Je-Yong; Kim, Shin-Yoon

    2004-01-01

    Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation

  9. Ethanol Extracts of Fresh Davallia formosana (WL1101) Inhibit Osteoclast Differentiation by Suppressing RANKL-Induced Nuclear Factor- ? B Activation

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    Lin, Tzu-Hung; Yang, Rong-Sen; Wang, Kuan-Chin; Lu, Dai-Hua; Liou, Houng-Chi; Ma, Yun; Chang, Shao-Han; Fu, Wen-Mei

    2013-01-01

    The rhizome of Davallia formosana is commonly used to treat bone disease including bone fracture, arthritis, and osteoporosis in Chinese herbal medicine. Here, we report the effects of WL1101, the ethanol extracts of fresh rhizomes of Davallia formosana on ovariectomy-induced osteoporosis. In addition, excess activated bone-resorbing osteoclasts play crucial roles in inflammation-induced bone loss diseases, including rheumatoid arthritis and osteoporosis. In this study, we examined the effect...

  10. Ethanol Extracts of Fresh Davallia formosana (WL1101 Inhibit Osteoclast Differentiation by Suppressing RANKL-Induced Nuclear Factor-κB Activation

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    Tzu-Hung Lin

    2013-01-01

    Full Text Available The rhizome of Davallia formosana is commonly used to treat bone disease including bone fracture, arthritis, and osteoporosis in Chinese herbal medicine. Here, we report the effects of WL1101, the ethanol extracts of fresh rhizomes of Davallia formosana on ovariectomy-induced osteoporosis. In addition, excess activated bone-resorbing osteoclasts play crucial roles in inflammation-induced bone loss diseases, including rheumatoid arthritis and osteoporosis. In this study, we examined the effects of WL1101 on receptor activator of nuclear factor-κB ligand (RANKL-induced osteoclastogenesis. Treatment with WL1101 significantly inhibited RANKL-stimulated osteoclastogenesis. Two isolated active compounds, ((−-epicatechin or WL14 (4-hydroxy-3-aminobenzoic acid could also inhibit RANKL-induced osteoclastogenesis. WL1101 suppressed the RANKL-induced nuclear factor-κB (NF-κB activation and nuclear translocation, which is the key process during osteoclastogenesis, by inhibiting the activation of IκB kinase (IKK and IκBα. In animal model, oral administration of WL1101 (50 or 200 mg/kg/day effectively decreased the excess bone resorption and significantly antagonized the trabecular bone loss in ovariectomized rats. Our results demonstrate that the ethanol extracts of fresh rhizomes of Davallia formosana inhibit osteoclast differentiation via the inhibition of NF-κB activation and effectively ameliorate ovariectomy-induced osteoporosis. WL1101 may thus have therapeutic potential for the treatment of diseases associated with excessive osteoclastic activity.

  11. Ethanol Extracts of Fresh Davallia formosana (WL1101) Inhibit Osteoclast Differentiation by Suppressing RANKL-Induced Nuclear Factor- κ B Activation.

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    Lin, Tzu-Hung; Yang, Rong-Sen; Wang, Kuan-Chin; Lu, Dai-Hua; Liou, Houng-Chi; Ma, Yun; Chang, Shao-Han; Fu, Wen-Mei

    2013-01-01

    The rhizome of Davallia formosana is commonly used to treat bone disease including bone fracture, arthritis, and osteoporosis in Chinese herbal medicine. Here, we report the effects of WL1101, the ethanol extracts of fresh rhizomes of Davallia formosana on ovariectomy-induced osteoporosis. In addition, excess activated bone-resorbing osteoclasts play crucial roles in inflammation-induced bone loss diseases, including rheumatoid arthritis and osteoporosis. In this study, we examined the effects of WL1101 on receptor activator of nuclear factor- κ B ligand (RANKL)-induced osteoclastogenesis. Treatment with WL1101 significantly inhibited RANKL-stimulated osteoclastogenesis. Two isolated active compounds, ((-)-epicatechin) or WL14 (4-hydroxy-3-aminobenzoic acid) could also inhibit RANKL-induced osteoclastogenesis. WL1101 suppressed the RANKL-induced nuclear factor- κ B (NF- κ B) activation and nuclear translocation, which is the key process during osteoclastogenesis, by inhibiting the activation of I κ B kinase (IKK) and I κ B α . In animal model, oral administration of WL1101 (50 or 200 mg/kg/day) effectively decreased the excess bone resorption and significantly antagonized the trabecular bone loss in ovariectomized rats. Our results demonstrate that the ethanol extracts of fresh rhizomes of Davallia formosana inhibit osteoclast differentiation via the inhibition of NF- κ B activation and effectively ameliorate ovariectomy-induced osteoporosis. WL1101 may thus have therapeutic potential for the treatment of diseases associated with excessive osteoclastic activity.

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

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    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. Copyright © 2011 Elsevier Inc. All rights reserved.

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

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    Rantlha, Mpho; Sagar, Travers; Kruger, Marlena C; Coetzee, Magdalena; Deepak, Vishwa

    2017-01-01

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

  14. Norisoboldine suppresses osteoclast differentiation through preventing the accumulation of TRAF6-TAK1 complexes and activation of MAPKs/NF-κB/c-Fos/NFATc1 Pathways.

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    Zhi-Feng Wei

    Full Text Available Norisoboldine (NOR is the main alkaloid constituent in the dry root of Lindera aggregata (Sims Kosterm. (L. strychnifolia Vill.. As reported previously, orally administered NOR displayed a robust inhibition of joint bone destruction present in both mouse collagen-induced arthritis and rat adjuvant-induced arthritis with lower efficacious doses than that required for ameliorating systemic inflammation. This attracted us to assess the effects of NOR on differentiation and function of osteoclasts, primary effector cells for inflammatory bone destruction, to get insight into its anti-rheumatoid arthritis mechanisms. Both RAW264.7 cells and mouse bone marrow-derived macrophages (BMMs were stimulated with RANKL (100 ng/mL to establish osteoclast differentiation models. ELISA, RT-PCR, gelatin zymography, western blotting, immunoprecipitation and EMSA were used to reveal related signalling pathways. NOR (10 and 30 µM, without significant cytotoxicity, showed significant reduction of the number of osteoclasts and the resorption pit areas, and it targeted osteoclast differentiation at the early stage. In conjunction with the anti-resorption effect of NOR, mRNA levels of cathepsin K and MMP-9 were decreased, and the activity of MMP-9 was attenuated. Furthermore, our mechanistic studies indicated that NOR obviously suppressed the ubiquitination of TRAF6, the accumulation of TRAF6-TAK1 complexes and the activation of ERK and p38 MAPK, and reduced the nuclear translocation of NF-κB-p65 and DNA-binding activity of NF-κB. However, NOR had little effect on expressions of TRAF6 or the phosphorylation and degradation of IκBα. Moreover, NOR markedly inhibited expressions of transcription factor NFATc1, but not c-Fos. Intriguingly, the subsequent nuclear translocations of c-Fos and NFATc1 were substantially down-regulated. Hence, we demonstrated for the first time that preventing the differentiation and function of osteoclasts at the early stage was an

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

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    He, Yi; Zhang, Qing; Shen, Yi; Chen, Xia; Zhou, Feng; Peng, Dan, E-mail: xyeypd@163.com

    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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

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

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

    2014-01-01

    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

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

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    Yu, Mingxiang, E-mail: yu.mingxiang@zs-hospital.sh.cn [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Chen, Xianying [Department of Endocrinology and Metabolism, Hainan Provincial Nong Ken Hospital, Hainan (China); Lv, Chaoyang [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Yi, Xilu [Department of Endocrinology and Metabolism, Shanghai Songjiang District Central Hospital, Shanghai (China); Zhang, Yao; Xue, Mengjuan; He, Shunmei [Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai (China); Zhu, Guoying [Institute of Radiation Medicine, Fudan University, Shanghai (China); Wang, Hongfu, E-mail: hfwang@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, Shanghai (China)

    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. 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.

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    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.

  20. Diphlorethohydroxycarmalol from Ishige okamurae Suppresses Osteoclast Differentiation by Downregulating the NF-κB Signaling Pathway

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    Hye Jung Ihn

    2017-12-01

    Full Text Available Marine algae possess a variety of beneficial effects on human health. In this study, we investigated whether diphlorethohydroxycarmalol (DPHC, isolated from Ishige okamurae, a brown alga, suppresses receptor activator of nuclear factor-κB ligand (RANKL-induced osteoclast differentiation. DPHC significantly suppressed RANKL-induced osteoclast differentiation and macrophage-colony stimulating factor (M-CSF expression in a dose-dependent manner. In addition, it significantly inhibited actin ring formation, the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (TRAP, nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1, cathepsin K (Ctsk, and dendritic cell-specific transmembrane protein (Dcstamp, and osteoclast-induced bone resorption. Analysis of the RANKL-mediated signaling pathway showed that the phosphorylation of both IκB and p65 was specifically inhibited by DPHC. These results suggest that DPHC substantially suppresses osteoclastogenesis by downregulating the RANK-NF-κB signaling pathway. Thus, it holds significant potential for the treatment of skeletal diseases associated with an enhanced osteoclast activity.

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

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    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.

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

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    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.

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

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    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

  4. Activation of dimeric glucocorticoid receptors in osteoclast progenitors potentiates RANKL induced mature osteoclast bone resorbing activity.

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    Conaway, H Herschel; Henning, Petra; Lie, Anita; Tuckermann, Jan; Lerner, Ulf H

    2016-12-01

    Glucocorticoid (GC) therapy is the greatest risk factor for secondary osteoporosis. Pathogenic mechanisms involve an initial increase in bone resorption followed by decreased bone formation. To gain a better understanding of the resorptive activity of GCs, we have used mouse bone marrow macrophages (BMM) to determine if GCs can directly modulate RANKL stimulated osteoclast formation and/or activity. In agreement with previous studies, experiments performed in plastic wells showed that GCs (dexamethasone, hydrocortisone, and prednisolone) inhibited osteoclast number and size during the initial phases of RANKL stimulated osteoclastogenesis; however, in prolonged cultures, decreased apoptosis was observed and escape from GC induced inhibition occurred with an enhanced number of osteoclasts formed, many with an increased area. When BMM cells were seeded on bone slices, GCs robustly enhanced RANKL stimulated formation of resorption pits and release of CTX without affecting the number or size of osteoclasts formed and with no effect on apoptosis. Stimulation of pit formation was not associated with increased life span of osteoclasts or an effect on mRNA expression of several osteoclastic or osteoclastogenic genes. The potentiation of RANKL induced CTX release by dexamethasone was significantly less in BMM cells from mice with conditional knockout of the osteoclastic glucocorticoid receptor and completely absent in cells from GR dim mice, which carry a point mutation in one dimerizing interface of the GC receptor. These data suggest that: 1. Plastic is a poor medium to use for studying direct effects of GCs on osteoclasts 2. GCs can enhance bone resorption without decreasing apoptosis, and 3. A direct enhancement of RANKL mediated resorption is stimulated by the dimeric GC-receptor. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Sitagliptin, An Anti-diabetic Drug, Suppresses Estrogen Deficiency-Induced OsteoporosisIn Vivo and Inhibits RANKL-Induced Osteoclast Formation and Bone Resorption In Vitro

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    Chuandong Wang

    2017-06-01

    Full Text Available Postmenopausal osteoporosis is a disease characterized by excessive osteoclastic bone resorption. Some anti-diabetic drugs were demonstrated for anti-osteoclastic bone-loss effects. The present study investigated the skeletal effects of chronic administration of sitagliptin, a dipeptidyl peptidase IV (DPP IV inhibitor that is increasingly used for type 2 diabetes treatments, in an estrogen deficiency-induced osteoporosis and elucidated the associated mechanisms. This study indicated that sitagliptin effectively prevented ovariectomy-induced bone loss and reduced osteoclast numbers in vivo. It was also indicated that sitagliptin suppressed receptor activator of nuclear factor-κB ligand (RANKL-mediated osteoclast differentiation, bone resorption, and F-actin ring formation in a manner of dose-dependence. In addition, sitagliptin significantly reduced the expression of osteoclast-specific markers in mouse bone-marrow-derived macrophages, including calcitonin receptor (Calcr, dendrite cell-specific transmembrane protein (Dc-stamp, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1. Further study indicated that sitagliptin inhibited osteoclastogenesis by suppressing AKT and ERK signaling pathways, scavenging ROS activity, and suppressing the Ca2+ oscillation that consequently affects the expression and/or activity of the osteoclast-specific transcription factors, c-Fos and NFATc1. Collectively, these findings suggest that sitagliptin possesses beneficial effects on bone and the suppression of osteoclast number implies that the effect is exerted directly on osteoclastogenesis.

  6. Sitagliptin, An Anti-diabetic Drug, Suppresses Estrogen Deficiency-Induced OsteoporosisIn Vivo and Inhibits RANKL-Induced Osteoclast Formation and Bone Resorption In Vitro.

    Science.gov (United States)

    Wang, Chuandong; Xiao, Fei; Qu, Xinhua; Zhai, Zanjing; Hu, Guoli; Chen, Xiaodong; Zhang, Xiaoling

    2017-01-01

    Postmenopausal osteoporosis is a disease characterized by excessive osteoclastic bone resorption. Some anti-diabetic drugs were demonstrated for anti-osteoclastic bone-loss effects. The present study investigated the skeletal effects of chronic administration of sitagliptin, a dipeptidyl peptidase IV (DPP IV) inhibitor that is increasingly used for type 2 diabetes treatments, in an estrogen deficiency-induced osteoporosis and elucidated the associated mechanisms. This study indicated that sitagliptin effectively prevented ovariectomy-induced bone loss and reduced osteoclast numbers in vivo . It was also indicated that sitagliptin suppressed receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation, bone resorption, and F-actin ring formation in a manner of dose-dependence. In addition, sitagliptin significantly reduced the expression of osteoclast-specific markers in mouse bone-marrow-derived macrophages, including calcitonin receptor ( Calcr ), dendrite cell-specific transmembrane protein ( Dc-stamp ), c-Fos , and nuclear factor of activated T-cells cytoplasmic 1 ( Nfatc1 ). Further study indicated that sitagliptin inhibited osteoclastogenesis by suppressing AKT and ERK signaling pathways, scavenging ROS activity, and suppressing the Ca 2+ oscillation that consequently affects the expression and/or activity of the osteoclast-specific transcription factors, c-Fos and NFATc1. Collectively, these findings suggest that sitagliptin possesses beneficial effects on bone and the suppression of osteoclast number implies that the effect is exerted directly on osteoclastogenesis.

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

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    Teruhito Yamashita

    Full Text Available 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

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

    Science.gov (United States)

    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

  9. New methodology for evaluating osteoclastic activity induced by orthodontic load.

    Science.gov (United States)

    Araújo, Adriele Silveira; Fernandes, Alline Birra Nolasco; Maciel, José Vinicius Bolognesi; Netto, Juliana de Noronha Santos; Bolognese, Ana Maria

    2015-01-01

    Orthodontic tooth movement (OTM) is a dynamic process of bone modeling involving osteoclast-driven resorption on the compression side. Consequently, to estimate the influence of various situations on tooth movement, experimental studies need to analyze this cell. Objectives The aim of this study was to test and validate a new method for evaluating osteoclastic activity stimulated by mechanical loading based on the fractal analysis of the periodontal ligament (PDL)-bone interface. Material and Methods The mandibular right first molars of 14 rabbits were tipped mesially by a coil spring exerting a constant force of 85 cN. To evaluate the actual influence of osteoclasts on fractal dimension of bone surface, alendronate (3 mg/Kg) was injected weekly in seven of those rabbits. After 21 days, the animals were killed and their jaws were processed for histological evaluation. Osteoclast counts and fractal analysis (by the box counting method) of the PDL-bone interface were performed in histological sections of the right and left sides of the mandible. Results An increase in the number of osteoclasts and in fractal dimension after OTM only happened when alendronate was not administered. Strong correlation was found between the number of osteoclasts and fractal dimension. Conclusions Our results suggest that osteoclastic activity leads to an increase in bone surface irregularity, which can be quantified by its fractal dimension. This makes fractal analysis by the box counting method a potential tool for the assessment of osteoclastic activity on bone surfaces in microscopic examination.

  10. Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo

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    Yun-Ho Hwang

    2018-03-01

    Full Text Available Osteoporosis is characterized by a reduction of the bone mineral density (BMD and microarchitectural deterioration of the bone, which lead to bone fragility and susceptibility to fracture. Astaxanthin (AST has a variety of biological activities, such as a protective effect against asthma or neuroinflammation, antioxidant effect, and decrease of the osteoclast number in the right mandibles in the periodontitis model. Although treatment with AST is known to have an effect on inflammation, no studies on the effect of AST exposure on bone loss have been performed. Thus, in the present study, we examined the antiosteoporotic effect of AST on bone mass in ovariectomized (OVX mice and its possible mechanism of action. The administration of AST (5, 10 mg/kg for 6 weeks suppressed the enhancement of serum calcium, inorganic phosphorus, alkaline phosphatase, total cholesterol, and tartrate-resistant acid phosphatase (TRAP activity. The bone mineral density (BMD and bone microarchitecture of the trabecular bone in the tibia and femur were recovered by AST exposure. Moreover, in the in vitro experiment, we demonstrated that AST inhibits osteoclast formation through the expression of the nuclear factor of activated T cells (NFAT c1, dendritic cell-specific transmembrane protein (DC-STAMP, TRAP, and cathepsin K without any cytotoxic effects on bone marrow-derived macrophages (BMMs. Therefore, we suggest that AST may have therapeutic potential for the treatment of postmenopausal osteoporosis.

  11. Zoledronate suppressed angiogenesis and osteogenesis by inhibiting osteoclasts formation and secretion of PDGF-BB.

    Science.gov (United States)

    Gao, Si-Yong; Zheng, Guang-Sen; Wang, Lin; Liang, Yu-Jie; Zhang, Si-En; Lao, Xiao-Mei; Li, Kan; Liao, Gui-Qing

    2017-01-01

    Bisphosphonates related osteonecrosis of jaw (BRONJ) is a severe complication of systemic BPs administration, the mechanism of which is still unclarified. Recently, platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts was reported to promote angiogenesis and osteogenesis. This study aimed to clarify whether bisphosphonates suppressed preosteoclasts releasing PDGF-BB, and whether the suppression harmed coupling of angiogenesis and osteogenesis, which could contribute to BRONJ manifestation. Zoledronate significantly inhibited osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining and PDGF-BB secretion tested by ELISA. In line with decreasing secretion of PDGF-BB by preosteoclasts exposed to zoledronate, conditioned medium (CM) from the cells significantly induced less migration of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) compared to CM from unexposed preosteoclasts. Meanwhile, angiogenic function of EPCs and osteoblastic differentiation of MSCs also declined when culturing with CM from preosteoclasts treated by zoledronate (PZ-CM), evidenced by tube formation assay of EPCs and alkaline phosphatase activity of MSCs. Western blot assay showed that the expression of VEGF in EPCs and OCN, RUNX2 in MSCs declined when culturing with PZ-CM compared to CM from preostoeclasts without exposure of zoledronate. Our study found that zoledronate was able to suppress preosteoclasts releasing PDGF-BB, resulting in suppression of angiogenesis and osteogenesis. Our study may partly contributed to the mechanism of BRONJ.

  12. BMP-2 and titanium particles synergistically activate osteoclast formation

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    Sun, S.X.; Guo, H.H.; Zhang, J.; Yu, B.; Sun, K.N.; Jin, Q.H.

    2014-01-01

    A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation. PMID:24820069

  13. Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway.

    Science.gov (United States)

    Chawalitpong, Supatta; Chokchaisiri, Ratchanaporn; Suksamrarn, Apichart; Katayama, Shigeru; Mitani, Takakazu; Nakamura, Soichiro; Athamneh, Ahmad Ai; Ritprajak, Patcharee; Leelahavanichkul, Asada; Aeimlapa, Ratchaneevan; Charoenphandhu, Narattaphol; Palaga, Tanapat

    2018-04-04

    Cyperenoic acid is a terpenoid isolated from the root of a medicinal plant Croton crassifolius with a wide range of biological activities. In this study, the effects of cyperenoic acid on osteoclast differentiation were investigated both in vitro and in vivo using receptor activator of nuclear factor-κB ligand (RANKL)-induced bone marrow-derived osteoclasts and senescence-accelerated mouse prone 6 (SAMP6). Cyperenoic acid significantly suppressed RANKL-induced osteoclast differentiation at the concentrations with no apparent cytotoxicity. The half maximum inhibitory concentration (IC 50 ) for osteoclast differentiation was 36.69 μM ± 1.02. Cyperenoic acid treatment evidently reduced the expression of two key transcription factors in osteoclast differentiation, NFATc1 and c-Fos. Detailed signaling analysis revealed that cyperenoic acid did not affect MAPK pathways and canonical NF-κB pathway but impaired activation of p100/p52 in the non-canonical NF-κB pathway upon RANKL stimulation. Moreover, the expression of osteoclast-related genes, nfatc1, ctsk, irf8, acp5 and cfos were disrupted by cyperenoic acid treatment. The bone resorption activity by cyperenoic acid-treated osteoclasts were impaired. In a senile osteoporosis mouse model SAMP6, mice fed on diet supplemented with cyperenoic acid showed delay in bone loss, compared to the control. Taken together, plant-derived cyperenoic acid shows great potential as therapeutic agent for osteoporosis.

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

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    Guoju Hong

    2017-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-03-15

    We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in osteoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-κB ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow macrophages and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre(+)FOXO1(L/L)) compared with matched controls (LyzM.Cre(-)FOXO1(L/L)). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream factors, including dendritic cell-specific transmembrane protein, ATP6vod2, cathepsin K, and integrin αv. Lastly, FOXO1 deletion reduced M-CSF-induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions. Copyright © 2015 by The American Association of Immunologists, Inc.

  16. Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative, on Osteoclast Differentiation, Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway.

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    Hye Jung Ihn

    Full Text Available Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs, but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF and receptor activator of nuclear factor-κB ligand (RANKL. KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5, cathepsin K (Ctsk, dendritic cell-specific transmembrane protein (Dcstamp, matrix metallopeptidase 9 (Mmp9, and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1. Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and mitogen-activated protein kinase kinase1/2 (MEK1/2. In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.

  17. Cofilin activation during podosome belt formation in osteoclasts.

    Directory of Open Access Journals (Sweden)

    Anne Blangy

    Full Text Available Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.

  18. Stem cell factor stimulates chicken osteoclast activity in vitro

    NARCIS (Netherlands)

    van't Hof, R. J.; von Lindern, M.; Nijweide, P. J.; Beug, H.

    1997-01-01

    Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally

  19. The RhoGAP activity of myosin IXB is critical for osteoclast podosome patterning, motility, and resorptive capacity.

    Directory of Open Access Journals (Sweden)

    Brooke K McMichael

    Full Text Available Osteoclasts are large, multinucleated cells of the monocyte-macrophage lineage that generate specialized substrate adhesion complexes to facilitate their function as bone-degrading cells. The patterning and function of these actin-based complexes, podosomes and sealing zones, are regulated by the small GTPase Rho. Myosin IXB (Myo9b is a unique actin-based motor protein that contains a RhoGAP domain, which, like other RhoGAPs, is inhibitory to Rho signaling. In this study, Myo9b is shown to be expressed in osteoclasts and act as a critical regulator of podosome patterning and osteoclast function. SiRNA-mediated knockdown of Myo9b results in increased activity of Rho but not Rac in osteoclasts. Knockdown in osteoclasts on glass results in altered podosome patterning and decreased motility, and this effect is reversed by addition of a Rho inhibitor. SiRNA-mediated suppression of Myo9b expression in osteoclasts on bone results in a dramatic loss of resorptive capacity even though sealing zones appear normal. This loss of resorption is also reversible with addition of a Rho inhibitor. Cells with diminished Myo9b levels display mislocalization and suppressed activation of Src, a tyrosine kinase with critical effects on osteoclast actin cytoskeletal rearrangement and function. In addition, siRNA-treated cells display poorly formed microtubule networks and a lack of tubulin acetylation, a marker of microtubule stability. However, short-term addition of TNFα to cells with suppressed Myo9b levels overcomes or circumvents these defects and causes increased sealing zone size and resorptive capacity. These results indicate that the RhoGAP activity of Myo9b plays a key role in regulating the actin-based structures necessary for osteoclast motility and resorption, and confirms that Myo9b can act as a motorized signaling molecule that links Rho signaling to the dynamic actin cytoskeleton.

  20. Effect of vibration on osteoblastic and osteoclastic activities: Analysis of bone metabolism using goldfish scale as a model for bone

    Science.gov (United States)

    Suzuki, N.; Kitamura, K.; Nemoto, T.; Shimizu, N.; Wada, S.; Kondo, T.; Tabata, M. J.; Sodeyama, F.; Ijiri, K.; Hattori, A.

    In osteoclastic activity during space flight as well as hind limb unloading by tail suspension, inconsistent results have been reported in an in vivo study. The bone matrix plays an important role in the response to physical stress. However, there is no suitable in vitro co-culture system of osteoblasts and osteoclasts including bone matrix. On the other hand, fish scale is a calcified tissue that contains osteoblasts, osteoclasts, and bone matrix, all of which are similar to those found in human bones. Recently, we developed a new in vitro model system using goldfish scale. This system can detect the activities of osteoclasts and osteoblasts with tartrate-resistant acid phosphatase and alkaline phosphatase as the respective markers and precisely analyze the co-relationship between osteoblasts and osteoclasts. Using this system, we analyzed the bone metabolism under various degrees of acceleration (0.5-, 1-, 2-, 4-, and 6-G) by vibration with a G-load apparatus. After loading for 5 and 10 min, the scales were incubated for 6 and 24 h. The osteoblastic and osteoclastic activities were then measured. The osteoblastic activities gradually increased corresponding to 1-G to 6-G acceleration. In addition, ER mRNA expression was the highest under 6-G acceleration. On the other hand, the osteoclastic activity decreased at 24 h of incubation under low acceleration (0.5- and 1-G). This change coincided with TRAP mRNA expression. Under 2-G acceleration, the strength of suppression in osteoclastic activity was the highest. The strength of the inhibitory action under 4- and 6-G acceleration was lower than that under 2-G acceleration. In our co-culture system, osteoblasts and osteoclasts in the scale sensitively responded to several degrees of acceleration. Therefore, we strongly believe that our in vitro co-culture system is useful for the analysis of bone metabolism under loading or unloading.

  1. Sulfated glycosaminoglycans support osteoblast functions and concurrently suppress osteoclasts.

    Science.gov (United States)

    Salbach-Hirsch, Juliane; Ziegler, Nicole; Thiele, Sylvia; Moeller, Stephanie; Schnabelrauch, Matthias; Hintze, Vera; Scharnweber, Dieter; Rauner, Martina; Hofbauer, Lorenz C

    2014-06-01

    In order to improve bone regeneration, development and evaluation of new adaptive biomaterials is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major extracellular matrix (ECM) components of bone, and display osteogenic properties that are potentially useful for biomaterial applications. Using native and synthetic sulfate-modified GAGs, we manufactured artificial collagen/GAG ECM (aECMs) coatings, and evaluated how the presence of GAGs and their degree of sulfation affects the differentiation of murine mesenchymal stem cells to osteoblasts (OB) cultivated on these aECMs. GAG sulfation regulated osteogenesis at all key steps of OB development. Adhesion, but not migration, was diminished by 50% (P osteogenesis and suppressing their paracrine support of OC functions, thus displaying a favorable profile on bone remodeling. Whether these cellular properties translate into improved bone regeneration needs to be validated in vivo. © 2014 Wiley Periodicals, Inc.

  2. C/ebpα controls osteoclast terminal differentiation, activation, function, and postnatal bone homeostasis through direct regulation of Nfatc1.

    Science.gov (United States)

    Chen, Wei; Zhu, Guochun; Tang, Jun; Zhou, Hou-De; Li, Yi-Ping

    2018-03-01

    Osteoclast lineage commitment and differentiation have been studied extensively, although the mechanism by which transcription factor(s) control osteoclast terminal differentiation, activation, and function remains unclear. CCAAT/enhancer-binding protein α (C/ebpα) has been reported to be a key regulator of osteoclast cell lineage commitment, yet C/ebpα's roles in osteoclast terminal differentiation, activation and function, and bone homeostasis, under physiological or pathological conditions, have not been studied because newborn C/ebpα-null mice die within several hours after birth. Furthermore, the function of C/ebpα in osteoclast terminal differentiation, activation, and function is largely unknown. Herein, we generated and analyzed an osteoclast-specific C/ebpα conditional knockout (CKO) mouse model via Ctsk-Cre mice and found that C/ebpα-deficient mice exhibited a severe osteopetrosis phenotype due to impaired osteoclast terminal differentiation, activation, and function, including mildly reduced osteoclast number, impaired osteoclast polarization, actin formation, and bone resorption, which demonstrated the novel function of C/ebpα in cell function and terminal differentiation. Interestingly, C/ebpα deficiency did not affect bone formation or monocyte/macrophage development. Our results further demonstrated that C/ebpα deficiency suppressed the expression of osteoclast functional genes, e.g. encoding cathepsin K (Ctsk), Atp6i (Tcirg1), and osteoclast regulator genes, e.g. encoding c-fos (Fos), and nuclear factor of activated T-cells 1 (Nfatc1), while having no effect on Pu.1 (Spi1) expression. Promoter activity mapping and ChIP assay defined the critical cis-regulatory element (CCRE) in the promoter region of Nfatc1, and also showed that the CCREs were directly associated with C/ebpα, which enhanced the promoter's activity. The deficiency of C/ebpα in osteoclasts completely blocked ovariectomy-induced bone loss, indicating that C/ebpα is a

  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

    Energy Technology Data Exchange (ETDEWEB)

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Myeung Su [Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young, E-mail: kimjy1014@gmail.com [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    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. Delayed tooth eruption and suppressed osteoclast number in the eruption pathway of heterozygous Runx2/Cbfa1 knockout mice.

    Science.gov (United States)

    Yoda, Shuichi; Suda, Naoto; Kitahara, Yutaka; Komori, Toshihisa; Ohyama, Kimie

    2004-06-01

    Genetic studies have recently identified a mutation of one allele of runt-related gene 2 (RUNX2/CBFA1) as the cause for an autosomal-dominant skeletal disorder, cleidocranial dysplasia (CCD), which is characterised by hypoplasia of the clavicles and calvariae and widened sutures and fontanelles. In addition, CCD is frequently affected with multiple supernumerary teeth and the impaction and delayed eruption of teeth, the causes of all these dental abnormalities are still unknown. To clarify the cellular mechanism of the delayed tooth eruption in CCD, the process of tooth eruption was examined in heterozygous Runx2/Cbfa1 (mouse homolog of RUNX2/CBFA1) knockout mice, known to mimic most of the bone abnormalities of CCD. The timing of the appearance of maxillary and mandibular teeth into the oral cavity was significantly delayed in heterozygous mutant mice compared with wild-type mice. From postnatal days 8 to 10, an active alveolar bone resorption and a marked increase of the osteoclast surfaces was observed in the eruption pathway of both genotypes, but this increase was significantly suppressed in the mutant mice. In contrast, the osteoclast surfaces did not show a significant difference between the two genotypes in the future cortical area of femora. These results suggest that haploinsufficiency of Runx2/Cbfa1 does not effect the femoral bone remodelling but is insufficient for the active alveolar bone resorption essential for the prompt timing of tooth eruption. These results also suggest the possibility that impaired recruitment of osteoclasts is one of the cellular mechanisms of delayed tooth eruption in CCD patients.

  5. Predictors of osteoclast activity in patients with sickle cell disease

    Science.gov (United States)

    Nouraie, Mehdi; Cheng, Kevin; Niu, Xiaomei; Moore-King, Evadne; Fadojutimi-Akinsi, Margaret F.; Minniti, Caterina P.; Sable, Craig; Rana, Sohail; Dham, Niti; Campbell, Andrew; Ensing, Gregory; Kato, Gregory J.; Gladwin, Mark T.; Castro, Oswaldo L.; Gordeuk, Victor R.

    2011-01-01

    Background Bone changes are common in sickle cell disease, but the pathogenesis is not fully understood. Tartrate-resistant acid phosphatase (TRACP) type 5b is produced by bone-resorbing osteoclasts. In other forms of hemolytic anemia, increased iron stores are associated with osteoporosis. We hypothesized that transfusional iron overload would be associated with increased osteoclast activity in patients with sickle cell disease. Design and Methods We examined tartrate-resistant acid phosphatase 5b concentrations in patients with sickle cell disease and normal controls of similar age and sex distribution at steady state. Serum tartrate-resistant acid phosphatase 5b concentration was measured using an immunocapture enzyme assay and plasma concentrations of other cytokines were assayed using the Bio-Plex suspension array system. Tricuspid regurgitation velocity, an indirect measure of systolic pulmonary artery pressure, was determined by echocardiography. Results Tartrate-resistant acid phosphatase 5b concentrations were higher in 58 adults with sickle cell disease than in 22 controls (medians of 4.4 versus 2.4 U/L, respectively; P=0.0001). Among the patients with sickle cell disease, tartrate-resistant acid phosphatase 5b independently correlated with blood urea nitrogen (standardized beta=0.40, P=0.003), interleukin-8 (standardized beta=0.30, P=0.020), and chemokine C-C motif ligand 5 (standardized beta=−0.28, P=0.031) concentrations, but not with serum ferritin concentration. Frequent blood transfusions (>10 units in life time) were not associated with higher tartrate-resistant acid phosphatase 5b levels in multivariate analysis. There were strong correlations among tartrate-resistant acid phosphatase 5b, alkaline phosphatase and tricuspid regurgitation velocity (r>0.35, Psickle cell disease have increased osteoclast activity as reflected by serum tartrate-resistant acid phosphatase 5b concentrations. Our results may support a potential role of inflammation

  6. Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2017-03-01

    Full Text Available In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR. Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca2+ influx intensity (staining with Fluo-3/AM. Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

  7. Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss.

    Science.gov (United States)

    Baek, Jong Min; Kim, Ju-Young; Lee, Chang Hoon; Yoon, Kwon-Ha; Lee, Myeung Su

    2017-03-07

    In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP) staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca 2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca 2+ influx intensity (staining with Fluo-3/AM). Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

    2016-03-01

    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.

  10. Medaka Osteoclast

    Data.gov (United States)

    National Aeronautics and Space Administration — During space flight bone mineral density is decreased by the influence of osteoclast activation which molecular mechanism is expectantly investigated. In the study...

  11. CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity.

    Science.gov (United States)

    Li, Yuheng; Zhong, Guohui; Sun, Weijia; Zhao, Chengyang; Zhang, Pengfei; Song, Jinping; Zhao, Dingsheng; Jin, Xiaoyan; Li, Qi; Ling, Shukuan; Li, Yingxian

    2015-11-04

    The CD44 is cellular surface adhesion molecule that is involved in physiological processes such as hematopoiesis, lymphocyte homing and limb development. It plays an important role in a variety of cellular functions including adhesion, migration, invasion and survival. In bone tissue, CD44 is widely expressed in osteoblasts, osteoclasts and osteocytes. However, the mechanisms underlying its role in bone metabolism remain unclear. We found that CD44 expression was upregulated during osteoclastogenesis. CD44 deficiency in vitro significantly inhibited osteoclast activity and function by regulating the NF-κB/NFATc1-mediated pathway. In vivo, CD44 mRNA levels were significantly upregulated in osteoclasts isolated from the hindlimb of tail-suspended mice. CD44 deficiency can reduce osteoclast activity and counteract cortical bone loss in the hindlimb of unloaded mice. These results suggest that therapeutic inhibition of CD44 may protect from unloading induced bone loss by inhibiting osteoclast activity.

  12. Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

    DEFF Research Database (Denmark)

    Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

    2002-01-01

    that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

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

    Directory of Open Access Journals (Sweden)

    Non eChen

    2016-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Jun-Oh; Lee, Yong Deok; Kim, Haemin; Kim, Min Kyung; Song, Min-Kyoung; Lee, Zang Hee; Kim, Hong-Hee, E-mail: hhbkim@snu.ac.kr

    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.

  15. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro

    DEFF Research Database (Denmark)

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N

    2015-01-01

    as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte......Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow...... decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance...

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

    OpenAIRE

    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 osteoblasti...

  17. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R

    2007-01-01

    The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganizat......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...... reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express u......PAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. MATERIALS AND METHODS: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected...

  18. Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

    DEFF Research Database (Denmark)

    Delaissé, Jean-Marie; Andersen, Thomas L; Engsig, Michael T

    2003-01-01

    The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute...... significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone...... in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations...

  19. Pulse treatment with the proteasome inhibitor bortezomib inhibits osteoclast resorptive activity in clinically relevant conditions

    DEFF Research Database (Denmark)

    Boissy, P; Levin Andersen, Thomas; Lund, T

    2008-01-01

    Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib...... on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3h pulse with 25nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone...... cells drastically reduced their survival. We measured next the levels of two bone resorption markers in patients during the 3 days following five and seven therapeutic bortezomib administrations, respectively. These levels decreased significantly already 1-2 days after injection, and then increased...

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

    Science.gov (United States)

    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.

  1. Cell fusion in osteoclasts plays a critical role in controlling bone mass and osteoblastic activity

    International Nuclear Information System (INIS)

    Iwasaki, Ryotaro; Ninomiya, Ken; Miyamoto, Kana; Suzuki, Toru; Sato, Yuiko

    2008-01-01

    The balance between osteoclast and osteoblast activity is central for maintaining the integrity of bone homeostasis. Here we show that mice lacking dendritic cell specific transmembrane protein (DC-STAMP), an essential molecule for osteoclast cell-cell fusion, exhibited impaired bone resorption and upregulation of bone formation by osteoblasts, which do not express DC-STAMP, which led to increased bone mass. On the contrary, DC-STAMP over-expressing transgenic (DC-STAMP-Tg) mice under the control of an actin promoter showed significantly accelerated cell-cell fusion of osteoclasts and bone resorption, with decreased osteoblastic activity and bone mass. Bone resorption and formation are known to be regulated in a coupled manner, whereas DC-STAMP regulates bone homeostasis in an un-coupled manner. Thus our results indicate that inhibition of a single molecule provides both decreased osteoclast activity and increased bone formation by osteoblasts, thereby increasing bone mass in an un-coupled and a tissue specific manner.

  2. Osteoclast Fusion

    DEFF Research Database (Denmark)

    Marie Julie Møller, Anaïs; Delaissé, Jean-Marie; Søe, Kent

    2017-01-01

    Investigations addressing the molecular keys of osteoclast fusion are primarily based on end-point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi-nucleated cells, the number of nuclei per...... multinucleated cell or TRAcP activity. But end-point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts was a random process and occurred by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data...... suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed...

  3. Antiresorptive Activity of Bacillus-Fermented Antler Extracts: Inhibition of Osteoclast Differentiation

    Directory of Open Access Journals (Sweden)

    Sik-Won Choi

    2013-01-01

    Full Text Available Antlers have been traditionally used for thousands of years as a natural product with medicinal and pharmaceutical properties. In developing healthy foods, Bacillus-mediated fermentation is widely used to enhance the biological activity of nutrients in foods. Recently, fermentation was shown to enhance the osteogenic activity of antlers. This study aimed to elucidate the antiresorptive activity of Bacillus-fermented antler and its mode of action. We found that Bacillus-fermented antler extract strongly inhibited osteoclast differentiation by downregulating the expression and activity of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1. This extract also inhibited the activation of phospholipase Cγ2 (PLCγ2, a signaling molecule that could regulate NFATc1 transcriptional activity. This suggested that Bacillus-fermented antler extract could inhibit PLCγ2-NFATc1 signaling required for bone resorption and cell fusion. Consequently, Bacillus-fermented antler extract might benefit osteoclast-related disorders, including osteoporosis; furthermore, it may improve gastrointestinal activity.

  4. Antiresorptive Activity of Bacillus-Fermented Antler Extracts: Inhibition of Osteoclast Differentiation

    Science.gov (United States)

    Choi, Sik-Won; Moon, Seong-Hee; Yang, Hye Jeong; Kwon, Dae Young; Son, Young-Jin; Yu, Ri; Kim, Young Su; Kim, So I.; Chae, Eun Jeong; Park, Sang-Joon; Kim, Seong Hwan

    2013-01-01

    Antlers have been traditionally used for thousands of years as a natural product with medicinal and pharmaceutical properties. In developing healthy foods, Bacillus-mediated fermentation is widely used to enhance the biological activity of nutrients in foods. Recently, fermentation was shown to enhance the osteogenic activity of antlers. This study aimed to elucidate the antiresorptive activity of Bacillus-fermented antler and its mode of action. We found that Bacillus-fermented antler extract strongly inhibited osteoclast differentiation by downregulating the expression and activity of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). This extract also inhibited the activation of phospholipase Cγ2 (PLCγ2), a signaling molecule that could regulate NFATc1 transcriptional activity. This suggested that Bacillus-fermented antler extract could inhibit PLCγ2-NFATc1 signaling required for bone resorption and cell fusion. Consequently, Bacillus-fermented antler extract might benefit osteoclast-related disorders, including osteoporosis; furthermore, it may improve gastrointestinal activity. PMID:23509596

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

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Hiroaki [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Mochizuki, Ayako [Department of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Yoshimura, Kentaro; Miyamoto, Yoichi [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Kaneko, Kotaro [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo 160-0023 (Japan); Inoue, Tomio [Department of Oral Physiology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Chikazu, Daichi [Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo 160-0023 (Japan); Takami, Masamichi [Department of Pharmacology, Showa University School of Dentistry, Tokyo 142-8555 (Japan); Kamijo, Ryutaro, E-mail: kamijor@dent.showa-u.ac.jp [Department of Biochemistry, Showa University School of Dentistry, Tokyo 142-8555 (Japan)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D 3 . Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D 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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Xinhua; Zhai, Zanjing; Liu, Xuqiang; Li, Haowei [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Ouyang, Zhengxiao [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Department of Orthopaedics, Hunan Provincial Tumor Hospital and Tumor Hospital of Xiangya School of Medicine, Central South University, Changsha (China); Wu, Chuanlong [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Guangwang [Department of Orthopaedic Surgery, The Central Hospital of Xuzhou, Affiliated Hospital of Medical Collage of Southeast University, Xuzhou (China); Fan, Qiming; Tang, Tingting [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Qin, An, E-mail: dr.qinan@gmail.com [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Dai, Kerong, E-mail: krdai@163.com [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

    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.

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

    Science.gov (United States)

    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.

  10. Sclerostin stimulates osteocyte support of osteoclast activity by a RANKL-dependent pathway.

    Directory of Open Access Journals (Sweden)

    Asiri R Wijenayaka

    Full Text Available Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL mRNA and down-regulated that of osteoprotegerin (OPG mRNA, causing an increase in the RANK:OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKL-dependent manner.

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

    International Nuclear Information System (INIS)

    Franco, Gilson C.N.; Kajiya, Mikihito; Nakanishi, Tadashi; Ohta, Kouji; Rosalen, Pedro L.; Groppo, Francisco C.; Ernst, Cory W.O.; Boyesen, Janie L.; Bartlett, John D.; Stashenko, Philip; Taubman, Martin A.; Kawai, Toshihisa

    2011-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Franco, Gilson C.N. [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Pharmacology, FOP/UNICAMP, Piracicaba, SP (Brazil); Kajiya, Mikihito [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA (United States); Nakanishi, Tadashi [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Ohta, Kouji [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA (United States); Rosalen, Pedro L.; Groppo, Francisco C. [Department of Pharmacology, FOP/UNICAMP, Piracicaba, SP (Brazil); Ernst, Cory W.O.; Boyesen, Janie L. [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Bartlett, John D.; Stashenko, Philip [Department of Cytokine Biology, Forsyth Institute, Cambridge, MA (United States); Taubman, Martin A. [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Kawai, Toshihisa, E-mail: tkawai@forsyth.org [Department of Immunology, Forsyth Institute, Cambridge, MA (United States); Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA (United States)

    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.

  13. Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.

    Science.gov (United States)

    Zeng, Canjun; Goodluck, Helen; Qin, Xuezhong; Liu, Bo; Mohan, Subburaman; Xing, Weirong

    2016-10-01

    Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains. Copyright © 2016 the American Physiological Society.

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

    OpenAIRE

    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-menopaus...

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    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.

  17. Role of Muramyl Dipeptide in Lipopolysaccharide-Mediated Biological Activity and Osteoclast Activity

    Directory of Open Access Journals (Sweden)

    Hideki Kitaura

    2018-01-01

    Full Text Available Lipopolysaccharide (LPS is an endotoxin and bacterial cell wall component that is capable of inducing inflammation and immunological activity. Muramyl dipeptide (MDP, the minimal essential structural unit responsible for the immunological activity of peptidoglycans, is another inflammation-inducing molecule that is ubiquitously expressed by bacteria. Several studies have shown that inflammation-related biological activities were synergistically induced by interactions between LPS and MDP. MDP synergistically enhances production of proinflammatory cytokines that are induced by LPS exposure. Injection of MDP induces lethal shock in mice challenged with LPS. LPS also induces osteoclast formation and pathological bone resorption; MDP enhances LPS induction of both processes. Furthermore, MDP enhances the LPS-induced receptor activator of NF-κB ligand (RANKL expression and toll-like receptor 4 (TLR4 expression both in vivo and in vitro. Additionally, MDP enhances LPS-induced mitogen-activated protein kinase (MAPK signaling in stromal cells. Taken together, these findings suggest that MDP plays an important role in LPS-induced biological activities. This review discusses the role of MDP in LPS-mediated biological activities, primarily in relation to osteoclastogenesis.

  18. In vitro studies of human and rat osteoclast activity on hydroxyapatite, beta-tricalcium phosphate, calcium carbonate.

    Science.gov (United States)

    Monchau, F; Lefèvre, A; Descamps, M; Belquin-myrdycz, A; Laffargue, P; Hildebrand, H F

    2002-08-01

    Investigations on the ceramic degradation caused by osteoclasts are designed to assess osteoclast-ceramic interactions and to determine which ceramics are more suitable for use as bone substitute. This study investigated the resorptive activity of osteoclasts on ceramics presenting different solubility rates. Osteoclasts isolated from new-born rat and from human giant cell tumour were cultured on different bioceramics: hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and calcium carbonate (calcite). Cytoskeletal was revealed by actin labelling and ceramic surfaces were observed by scanning electron microscopy (SEM). On all materials, the distribution of actin in typical ring was revealed. SEM examinations showed a clear difference in the shape and the depth of resorption lacunae on different ceramics. On pure HA, a superficial attack, clearly visible but very little extended. Numerous resorption lacunae, deep and well-delimited were observed on pure beta-TCP, but attacks less punctually were detected too. On pure calcite, an attack with form of spikes, very widespread but superficial was revealed. Degradation measurements revealed a significant increase of P release from the phosphocalcic ceramics and of Ca from all ceramics in the presence of osteoclasts. The both cell models found these characteristics, the rat osteoclasts were also an excellent model to study the ceramic resorption.

  19. Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

    DEFF Research Database (Denmark)

    Bi, Yanming; Nielsen, Karina L; Kilts, Tina M

    2006-01-01

    Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated...... the effects of Bgn on 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate...

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

    Science.gov (United States)

    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.

  1. Proteomic Analysis of Estrogen-Mediated Signal Transduction in Osteoclasts Formation

    Directory of Open Access Journals (Sweden)

    Qi Xiong

    2015-01-01

    Full Text Available Estrogen plays an important role in inhibiting osteoclast differentiation and protecting against bone loss from osteoporosis, especially in postmenopausal women. However, the precise mechanisms underlying the effect of estrogen on osteoclasts are not well known. In the present study, we performed proteomics analysis and bioinformatics analysis to comprehensively compare the differential expression of proteins in receptor activator of nuclear factor-κB ligand RANKL-induced osteoclasts in the presence and absence of estrogen. We identified 6403 proteins, of which 124 were upregulated and 231 were downregulated by estrogen. Bioinformatics analysis showed that estrogen treatment interfered with 77 intracellular pathways, including both confirmed canonical and unconfirmed pathways of osteoclast formation. Our findings validate the inhibitory effect of estrogen on osteoclasts via the promotion of apoptosis and suppression of differentiation and polarization and suggest that estrogen might inhibit osteoclast formation via other pathways, which requires further investigation and verification.

  2. Interleukin-2 stimulates osteoclastic activity: Increased acid production and radioactive calcium release

    International Nuclear Information System (INIS)

    Ries, W.L.; Seeds, M.C.; Key, L.L.

    1989-01-01

    Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption. (author)

  3. Disturbed MEK/ERK signaling increases osteoclast activity via the Hedgehog-Gli pathway in postmenopausal osteoporosis.

    Science.gov (United States)

    Li, Xiaojie; Jie, Qiang; Zhang, Hongyang; Zhao, Yantao; Lin, Yangjing; Du, Junjie; Shi, Jun; Wang, Long; Guo, Kai; Li, Yong; Wang, Chunhui; Gao, Bo; Huang, Qiang; Liu, Jian; Yang, Liu; Luo, Zhuojing

    2016-11-01

    Postmenopausal osteoporosis is a worldwide health problem and is characterized by increased and activated osteoclasts. However, the mechanism by which osteoclasts are dysregulated in postmenopausal osteoporosis is not fully understood. In this study, we found that the Hedgehog-Gli pathway was upregulated in postmenopausal osteoporotic osteoclasts and that 17β-estradiol both inhibited osteoclastogenesis and induced osteoclast apoptosis by downregulating Hedgehog-Gli signaling. Furthermore, we demonstrated that the Hedgehog-Gli pathway was negatively regulated by MEK/ERK signaling and that this effect was Sonic Hedgehog (SHH)-dependent and was partially blocked by an anti-SHH antibody. Moreover, we found that the stimulatory effect of Hedgehog signaling on osteoclastogenesis and the inhibitory effect on osteoclast apoptosis were dependent on the Gli family of transcription factors. The pathways and molecules that contribute to the regulation of osteoclastogenesis and apoptosis represent potential new strategies for designing molecular drugs for the treatment of postmenopausal osteoporosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. The Src inhibitor AZD0530 reversibly inhibits the formation and activity of human osteoclasts

    NARCIS (Netherlands)

    de Vries, T.J.; Mullender, M.G.; van Duin, M.A.; Semeins, C.M.; James, N.; Green, T.P.; Everts, V.; Klein Nulend, J.

    2009-01-01

    Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src

  5. Osteoprotegerin disrupts peripheral adhesive structures of osteoclasts by modulating Pyk2 and Src activities.

    Science.gov (United States)

    Zhao, Hongyan; Liu, Xuezhong; Zou, Hui; Dai, Nannan; Yao, Lulian; Zhang, Xiao; Gao, Qian; Liu, Wei; Gu, Jianhong; Yuan, Yan; Bian, Jianchun; Liu, Zongping

    2016-05-03

    Osteoprotegerin has previously been shown to modulate bone mass by blocking osteoclast maturation and function. The detailed mechanisms of osteoprotegerin-induced disassembly of podosomes, disruption of adhesive structures and modulation of adhesion-related proteins in osteoclasts, however, are not well characterized. In this study, tartrate-resistant acidic phosphatase staining demonstrated that osteoprotegerin inhibited differentiation of osteoclasts. The use of scanning electron microscopy, real-time cell monitoring and confocal microscopy indicated that osteoclasts responded in a time and dose-dependent manner to osteoprotegerin treatments with retraction of peripheral adhesive structures and detachment from the extracellular substrate. Combined imaging and Western blot studies showed that osteoprotegerin induced dephosphorylation of Tyr 402 in Pyk2 and decreased its labeling in peripheral adhesion regions. osteoprotegerin induced increased intracellular labeling of Tyr 402 in Pyk2, Tyr 416 in Src, increased dephosphorylation of Tyr 527 in Src, and increased Pyk2/Src association in the central region of osteoclasts. This evidence suggests that Src may function as an adaptor protein that competes for Pyk2 and relocates it from the peripheral adhesive zone to the central region of osteoclasts in response to osteoprotegerin treatment. Osteoprotegerin may induce podosome reassembly and peripheral adhesive structure detachment by modulating phosphorylation of Pyk2 and Src and their intracellular distribution in osteoclasts.

  6. Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone

    Directory of Open Access Journals (Sweden)

    MacMillan AK

    2014-12-01

    Full Text Available Adam K MacMillan,1 Francis V Lamberti,1 Julia N Moulton,2 Benjamin M Geilich,2 Thomas J Webster2,3 1RTI Surgical, Alachua, FL, USA; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: While there have been numerous studies to determine osteoblast (bone forming cell functions on nanocrystalline compared to micron crystalline ceramics, there have been few studies which have examined osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and receptor activator of nuclear factor κB [RANK]. This is despite the fact that osteoclasts are an important part of maintaining healthy bone since they resorb bone during the bone remodeling process. Moreover, while it is now well documented that bone formation is enhanced on nanoceramics compared to micron ceramics, some have pondered whether osteoblast functions (such as osteoprotegerin and RANK ligand [RANKL] are normal (ie, non-diseased on such materials compared to natural bone. For these reasons, the objective of the present in vitro study was to determine various functions of osteoclasts and osteoblasts on nanocrystalline and micron crystalline hydroxyapatite as well as tri-calcium phosphate materials and compare such results to cortical and cancellous bone. Results showed for the first time similar osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and RANK and osteoblast activity (osteoprotegerin and RANKL on nanocrystalline hydroxyapatite compared to natural bone, whereas osteoclast and osteoblast functions on micron crystalline versions of these ceramics were much different than natural bone. In this manner, this study provides additional evidence that nanocrystalline calcium phosphates can serve as suitable synthetic

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shuo [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China); Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012 (China); Li, Xianan [Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012 (China); Cheng, Liang [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China); Wu, Hongwei [Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012 (China); Zhang, Can [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China); Li, Kanghua, E-mail: lkh8738@sina.com [Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008 (China)

    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.

  10. Evidence that activation of ASIC1a by acidosis increases osteoclast migration and adhesion by modulating integrin/Pyk2/Src signaling pathway.

    Science.gov (United States)

    Li, X; Ye, J-X; Xu, M-H; Zhao, M-D; Yuan, F-L

    2017-07-01

    Activated acid-sensing ion channel 1a (ASIC1a) is involved in acid-induced osteoclastogenesis by regulating activation of the transcription factor NFATc1. These results indicated that ASIC1a activation by extracellular acid may cause osteoclast migration and adhesion through Ca 2+ -dependent integrin/Pyk2/Src signaling pathway. Osteoclast adhesion and migration are responsible for osteoporotic bone loss. Acidic conditions promote osteoclastogenesis. ASIC1a in osteoclasts is associated with acid-induced osteoclastogenesis through modulating transcription factor NFATc1 activation. However, the influence and the detailed mechanism of ASIC1a in regulating osteoclast adhesion and migration, in response to extracellular acid, are not well characterized. In this study, knockdown of ASIC1a was achieved in bone marrow macrophage cells using small interfering RNA (siRNA). The adhesion and migration abilities of osteoclast precursors and osteoclasts were determined by adhesion and migration assays, in vitro. Bone resorption was performed to measure osteoclast function. Cytoskeletal changes were assessed by F-actin ring formation. αvβ3 integrin expression in osteoclasts was measured by flow cytometry. Western blotting and co-immunoprecipitation were performed to measure alterations in integrin/Pyk2/Src signaling pathway. Our results showed that blockade of ASIC1a using ASIC1a-siRNA inhibited acid-induced osteoclast precursor migration and adhesion, as well as osteoclast adhesion and bone resorption; we also demonstrated that inhibition of ASIC1a decreased the cell surface αvβ3 integrin and β3 protein expression. Moreover, blocking of ASIC1a inhibited acidosis-induced actin ring formation and reduced Pyk2 and Src phosphorylation in osteoclasts and also inhibited the acid-induced association of the αvβ3 integrin/Src/Pyk2. Together, these results highlight a key functional role of ASIC1a/αvβ3 integrin/Pyk2/Src signaling pathway in migration and adhesion of osteoclasts.

  11. Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

    Directory of Open Access Journals (Sweden)

    Ilaria Roato

    Full Text Available BACKGROUND: Phenylketonuria (PKU is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. METHODOLOGY: Peripheral blood mononuclear cell (PBMC cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF and receptor activator of NFκB ligand (RANKL. Flow cytometry was utilized to analyze osteoclast precursors (OCPs and T cell phenotype. Tumour necrosis factor α (TNF-α, RANKL and osteoprotegerin (OPG were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. PRINCIPAL FINDINGS: Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS. This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. CONCLUSIONS: Our results indicate that PKU spontaneous osteoclastogenesis

  12. Inhibitory effects of methyl-3,5-di-O-caffeoyl-epi-quinate on RANKL-induced osteoclast differentiation.

    Science.gov (United States)

    Kim, Tae Hoon; Ihn, Hye Jung; Kim, Kiryeong; Cho, Hye-Sung; Shin, Hong-In; Bae, Yong Chul; Park, Eui Kyun

    2018-04-09

    In this study, we have shown that methyl-3,5-di-O-caffeoyl-epi-quinate, a naturally occurring compound isolated from Ainsliaea acerifolia, inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and the expression of osteoclast marker genes. Methyl-3,5-di-O-caffeoyl-epi-quinate also inhibited RANKL-induced activation of p38, Akt and extracellular signal-regulated kinase (ERK) as well as the expression of nuclear factor of activated T-cell (NFATc1), the key regulator of osteoclast differentiation. Negative regulators for osteoclast differentiation was upregulated by methyl-3,5-di-O-caffeoyl-epi-quinate. Collectively, our results suggested that methyl-3,5-di-O-caffeoyl-epi-quinate suppresses osteoclast differentiation via downregulation of RANK signaling pathways and NFATc1. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Human scaphoid non-unions exhibit increased osteoclast activity compared to adjacent cancellous bone.

    Science.gov (United States)

    Schira, Jessica; Schulte, Matthias; Döbele, Carmen; Wallner, Christoph; Abraham, Stephanie; Daigeler, Adrien; Kneser, Ulrich; Lehnhardt, Marcus; Behr, Björn

    2015-12-01

    Scaphoid bones have a high prevalence for non-union. Even with adequate treatment, bone regeneration may not occur in certain instances. Although this condition is well described, the molecular pathology of scaphoid non-unions is still poorly defined. In this study, gene expression of osteogenic and angiogenic growth and transcription factors as well as inflammatory mediators were analysed in human scaphoid non-unions and intraindividually compared to adjacent autologous cancellous bone from the distal radius. In addition, histology and immunohistochemical stainings were performed to verify qRT-PCR data. Gene expression analysis revealed a significant up-regulation of RANKL, ALP, CYCLIN D1, MMP-13, OPG, NFATc1, TGF-β and WNT5A in scaphoid non-unions. Interestingly, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in non-unions. Moreover, WNT5A was highly up-regulated in all non-union samples. TRAP staining confirmed the observation of induced osteoclastogenesis in non-unions. With respect to genes related to osteogenesis, alkaline phosphatase was significantly up-regulated in scaphoid non-unions. No differences were detectable for other osteogenic genes such as RUNX-2 or BMP-2. Importantly, we did not detect differences in angiogenesis between scaphoid non-unions and controls in both gene expression and immunohistochemistry. Summarized, our data indicate increased osteoclast activity in scaphoid non-unions possibly as a result of the alterations in RANKL, TGF-β and WNT5A expression levels. These data increase our understanding for the reduced bone regeneration capacity present in scaphoid non-unions and may translate into the identification of new therapeutic targets to avoid secondary damages and prevent occurrence of non-unions to scaphoid bones. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  14. Diclofenac sodium inhibits NFkappaB transcription in osteoclasts.

    Science.gov (United States)

    Karakawa, A; Fukawa, Y; Okazaki, M; Takahashi, K; Sano, T; Amano, H; Yamamoto, M; Yamada, S

    2009-11-01

    A non-steroidal anti-inflammatory drug, diclofenac, acts efficiently against inflammation; however, down-regulation of diclofenac on bone remodeling has raised concerns. The inhibitory mechanisms of diclofenac are poorly understood. We hypothesized that diclofenac down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFkappaB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrateresistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner. Pit formation assay revealed the abolition of osteoclastic bone resorption; levels of cathepsin K transcripts, an osteoclastic resorption marker, were down-regulated time-dependently. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFkappaB and phosphorylated NFkappaB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFkappaB reduction, which regulates osteoclast differentiation and activation.

  15. Donepezil prevents RANK-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase.

    Science.gov (United States)

    Sato, Tsuyoshi; Enoki, Yuichiro; Sakamoto, Yasushi; Yokota, Kazuhiro; Okubo, Masahiko; Matsumoto, Masahito; Hayashi, Naoki; Usui, Michihiko; Kokabu, Shoichiro; Mimura, Toshihide; Nakazato, Yoshihiko; Araki, Nobuo; Fukuda, Toru; Okazaki, Yasushi; Suda, Tatsuo; Takeda, Shu; Yoda, Tetsuya

    2015-09-01

    Donepezil, an inhibitor of acetylcholinesterase (AChE) targeting the brain, is a common medication for Alzheimer's disease. Interestingly, a recent clinical study found that administration of this agent is associated with lower risk of hip fracture independently of falling, suggesting its direct effect on bone tissues as well. AChE has been reported to be involved in osteoblast function, but the role of AChE on osteoclastogenesis still remains unclear. We analyzed the effect of AChE and donepezil on osteoclastogenesis in vivo and in vitro. Cell-based assays were conducted using osteoclasts generated in cultures of murine bone marrow macrophages (BMMs) with receptor activator of nuclear factor-kappa B ligand (RANKL). The effect of donepezil was also determined in vivo using a mouse model of RANKL-induced bone loss. Recombinant AChE in BMMs cultured with RANKL further promoted RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation. RANKL also upregulated AChE expression in BMMs. RNA interference-mediated knockdown of AChE significantly inhibited RANKL-induced osteoclast differentiation and suppressed gene expression specific for osteoclasts. AChE upregulated expression of RANK, the receptor of RANKL, in BMMs. Donepezil decreased cathepsin K expression in BMMs and the resorptive function of osteoclasts on dentine slices. Donepezil decreased RANK expression in BMMs, resulting in the inhibition of osteoclast differentiation with downregulation of c-Fos and upregulation of Id2. Moreover, administration of donepezil prevented RANKL-induced bone loss in vivo, which was associated with the inhibition of bone resorption by osteoclasts. AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

  16. Iguratimod prevents ovariectomy‑induced bone loss and suppresses osteoclastogenesis via inhibition of peroxisome proliferator‑activated receptor‑γ.

    Science.gov (United States)

    Wu, Ying-Xing; Sun, Yue; Ye, Ya-Ping; Zhang, Peng; Guo, Jia-Chao; Huang, Jun-Ming; Jing, Xing-Zhi; Xiang, Wei; Yu, Shi-Ying; Guo, Feng-Jing

    2017-12-01

    Iguratimod is known for its anti‑inflammatory activities and therapeutic effects in patients with rheumatoid arthritis. It has previously been demonstrated that iguratimod attenuates bone destruction and osteoclast formation in the Walker 256 rat mammary gland carcinoma cell‑induced bone cancer pain model. Therefore, it was hypothesized that iguratimod may additionally exhibit therapeutic effects on benign osteoclast‑associated diseases including postmenopausal osteoporosis. In the present study, ovariectomized mice were used to investigate the effects of iguratimod in vivo. Bone marrow mononuclear cells were cultured to detect the effects of iguratimod on receptor activator of nuclear factor‑κB ligand (RANKL)‑induced osteoclastogenesis in vitro and the molecular mechanisms involved. It was demonstrated that iguratimod may prevent ovariectomy‑induced bone loss by suppressing osteoclast activity in vivo. Consistently, iguratimod may inhibit RANKL‑induced osteoclastogenesis and bone resorption in primary bone marrow mononuclear cells. At the molecular level, peroxisome proliferator‑activated receptor‑γ (PPAR‑γ)/c‑Fos pathway, which is essential in RANKL‑induced osteoclast differentiation, was suppressed by iguratimod. Subsequently, iguratimod decreased the expression of nuclear factor of activated T cells c1 and downstream osteoclast marker genes. The results of the present study demonstrated that iguratimod may inhibit ovariectomy‑induced bone loss and osteoclastogenesis by modulating RANKL signaling. Therefore, iguratimod may act as a novel therapeutic to prevent postmenopausal osteoporosis.

  17. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R

    2007-01-01

    PAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. MATERIALS AND METHODS: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...... of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation. RESULTS: pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load-sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed...

  18. Cancer cell expression of autotaxin controls bone metastasis formation in mouse through lysophosphatidic acid-dependent activation of osteoclasts.

    Directory of Open Access Journals (Sweden)

    Marion David

    2010-03-01

    Full Text Available Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2 is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD activity, ATX controls the level of lysophosphatidic acid (LPA in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models.Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis.Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and

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

    Science.gov (United States)

    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 (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 suppressed in GIO + OUB (GIO vs. GIO + OUB 11.6 vs. 7.5; p 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.

  20. Hydroxyapatite nanocrystals functionalized with alendronate as bioactive components for bone implant coatings to decrease osteoclastic activity

    Science.gov (United States)

    Bosco, Ruggero; Iafisco, Michele; Tampieri, Anna; Jansen, John A.; Leeuwenburgh, Sander C. G.; van den Beucken, Jeroen J. J. P.

    2015-02-01

    The integration of bone implants within native bone tissue depends on periprosthetic bone quality, which is severely decreased in osteoporotic patients. In this work, we have synthesized bone-like hydroxyapatite nanocrystals (nHA) using an acid-base neutralization reaction and analysed their physicochemical properties. Subsequently, we have functionalized the nHA with alendronate (nHAALE), a well-known bisphosphonate drug used for the treatment of osteoporosis. An in vitro osteoclastogenesis test was carried out to evaluate the effect of nHAALE on the formation of osteoclast-like cells from monocytic precursor cells (i.e. RAW264.7 cell line) showing that nHAALE significantly promoted apoptosis of osteoclast-like cells. Subsequently, nHA and nHAALE were deposited on titanium disks using electrospray deposition (ESD), for which characterisation of the deposited coatings confirmed the presence of alendronate in nHAALE coatings with nanoscale thickness of about 700 nm. These results indicate that alendronate linked to hydroxyapatite nanocrystals has therapeutic potential and nHAALE can be considered as an appealing coating constituent material for orthopaedic and oral implants for application in osteoporotic patients.

  1. Celecoxib treatment does not alter recruitment and activation of osteoclasts in the initial phase of experimental tooth movement

    Science.gov (United States)

    Carvalho-Filho, E.P.; Stabile, A.C.; Ervolino, E.; Stuani, M.B.S.; Iyomasa, M.M.; Rocha, M.J.A.

    2012-01-01

    In a previous study, we reported that the short-term treatment with celecoxib, a non-steroidal anti-inflammatory drug (NSAID) attenuates the activation of brain structures related to nociception and does not interfere with orthodontic incisor separation in rats. The conclusion was that celecoxib could possibly be prescribed for pain in orthodontic patients. However, we did not analyze the effects of this drug in periodontium. The aim of this follow-up study was to analyze effects of celecoxib treatment on recruitment and activation of osteoclasts and alveolar bone resorption after inserting an activated orthodontic appliance between the incisors in our rat model. Twenty rats (400–420 g) were pretreated through oral gavage with celecoxib (50 mg/kg) or vehicle (carboxymethyl-cellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance, set not to cause any palate disjunction. In sham animals, the appliance was immediately removed after introduction. All animals received ground food and, every 12 h, celecoxib or vehicle. After 48 h, they were anesthetized and transcardiacally perfused through the aorta with 4% formaldehyde. Subsequently, maxillae were removed, post-fixed and processed for histomorphometry or immunohistochemical analyses. As expected, incisor distalization induced an inflammatory response with certain histological changes, including an increase in the number of active osteoclasts at the compression side in group treated with vehicle (appliance:32.2±2.49 vs sham: 4.8±1.79, P<0.05) and celecoxib (appliance: 31.0±1.45 vs sham: 4.6±1.82, P<0.05). The treatment with celecoxib did not modify substantially the histological alterations and the number of active osteoclasts after activation of orthodontic appliance. Moreover, we did not see any difference between the groups with respect to percentage of bone resorption area. Taken together with our previous results we conclude that short-term treatment with celecoxib can indeed be

  2. Celecoxib treatment does not alter recruitment and activation of osteoclasts in the initial phase of experimental tooth movement

    Directory of Open Access Journals (Sweden)

    E.P. Carvalho-Filho

    2012-10-01

    Full Text Available In a previous study, we reported that the short-term treatment with celecoxib, a non-steroidal anti-inflammatory drug (NSAID attenuates the activation of brain structures related to nociception and does not interfere with orthodontic incisor separation in rats. The conclusion was that celecoxib could possibly be prescribed for pain in orthodontic patients. However, we did not analyze the effects of this drug in periodontium. The aim of this follow-up study was to analyze effects of celecoxib treatment on recruitment and activation of osteoclasts and alveolar bone resorption after inserting an activated orthodontic appliance between the incisors in our rat model. Twenty rats (400-420 g were pretreated through oral gavage with celecoxib (50 mg/kg or vehicle (carboxymethylcellulose 0.4%. After 30 min, they received an activated (30 g orthodontic appliance, set not to cause any palate disjunction. In sham animals, the appliance was immediately removed after introduction. All animals received ground food and, every 12 h, celecoxib or vehicle. After 48 h, they were anesthetized and transcardiacally perfused through the aorta with 4% formaldehyde. Subsequently, maxillae were removed, post-fixed and processed for histomorphometry or immunohistochemical analyses. As expected, incisor distalization induced an inflammatory response with certain histological changes, including an increase in the number of active osteoclasts at the compression side in group treated with vehicle (appliance: 32.2±2.49 vs sham: 4.8 ± 1.79, P<0.05 and celecoxib (appliance: 31.0±1.45 vs sham: 4.6±1.82, P<0.05. The treatment with celecoxib did not modify substantially the histological alterations and the number of active osteoclasts after activation of orthodontic appliance. Moreover, we did not see any difference between the groups with respect to percentage of bone resorption area. Taken together with our previous results we conclude that short-term treatment with celecoxib

  3. Stattic inhibits RANKL-mediated osteoclastogenesis by suppressing activation of STAT3 and NF-κB pathways.

    Science.gov (United States)

    Li, Chang-Hong; Xu, Lin-Lin; Jian, Lei-Lei; Yu, Ruo-Han; Zhao, Jin-Xia; Sun, Lin; Du, Guo-Hong; Liu, Xiang-Yuan

    2018-03-24

    Tofacitinib, a small molecule JAK inhibitor, has been widely used to reduce inflammation and inhibit progression of bone destruction in rheumatoid arthritis. STAT3, a downstream signaling molecule of JAK, plays a key role in the activation of signaling in response to inflammatory cytokines. Thus, targeting STAT3 may be an inspiring strategy for treating osteoclast-related diseases such as rheumatoid arthritis. In this study, we first investigated the effects of Stattic, a STAT3 inhibitor, on receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. Stattic inhibited osteoclast differentiation and bone resorption in RANKL-induced RAW264.7 cells in a dose-dependent manner. Stattic also suppressed RANKL-induced upregulation of osteoclast-related genes tartrate-resistant acid phosphatase, matrix metalloproteinase 9, cathepsin K, RANK, tumor necrosis factor receptor-associated factor 6, and osteoclast-associated receptor in RAW264.7 cells. Moreover, Stattic exhibited an inhibitory effect on cell proliferation and cell cycle progression at higher dosages. At the molecular level, Stattic inhibited RANKL-induced activation of STAT3 and NF-κB pathways, without significantly affecting MAPK signaling. In addition, Stattic inhibited RANKL-induced expression of osteoclast-related transcription factors c-Fos and NFATc1. Importantly, Stattic also prevented bone loss caused by ovariectomy. Together, our data confirm that Stattic restricts osteoclastogenesis and bone loss by disturbing RANKL-induced STAT3 and NF-κB signaling. Thus, Stattic represents a novel type of osteoclast inhibitor that could be useful for conditions such as osteoporosis and rheumatoid arthritis. Copyright © 2018. Published by Elsevier B.V.

  4. Glucocorticoids maintain human osteoclasts in the active mode of their resorption cycle

    DEFF Research Database (Denmark)

    Søe, Kent; Delaissé, Jean-Marie

    2010-01-01

    that glucocorticoids deeply modify this resorptive behavior. First, glucocorticoids gradually induce excavations with a trenchlike morphology while reducing the time-dependent increase in excavation numbers. This indicates that glucocorticoids make osteoclasts elongate the excavations they initiated rather than...... migrating to a new resorption site, as in control conditions. Second, the round excavations in control conditions contain undegraded demineralized collagen as repeatedly reported earlier, whereas the excavations with a trenchlike morphology generated under glucocorticoid exposure appear devoid of leftovers...... of demineralized collagen. This indicates that collagenolysis proceeds generally at a lower rate than demineralization under control conditions, whereas collagenolysis rates are increased up to the level of demineralization rates in the presence of glucocorticoids. Taking these observations together leads...

  5. Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

    Science.gov (United States)

    Chellaiah, Meenakshi A; Schaller, Michael D

    2009-08-01

    PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

  6. Germline deletion of AMP-activated protein kinase β subunits reduces bone mass without altering osteoclast differentiation or function

    Science.gov (United States)

    Quinn, Julian M. W.; Tam, Shanna; Sims, Natalie A.; Saleh, Hasnawati; McGregor, Narelle E.; Poulton, Ingrid J.; Scott, John W.; Gillespie, Matthew T.; Kemp, Bruce E.; van Denderen, B. J. W.

    2010-01-01

    Since AMP-activated protein kinase (AMPK) plays important roles in modulating metabolism in response to diet and exercise, both of which influence bone mass, we examined the influence of AMPK on bone mass in mice. AMPK is an αβγ heterotrimer where the β subunit anchors the α catalytic and γ regulatory subunits. Germline deletion of either AMPK β1 or β2 subunit isoforms resulted in reduced trabecular bone density and mass, but without effects on osteoclast (OC) or osteoblast (OB) numbers, as compared to wild-type littermate controls. We tested whether activating AMPK in vivo would enhance bone density but found AICA-riboside treatment caused a profound loss of trabecular bone volume (49.5%) and density and associated increased OC numbers. Consistent with this, AICA-riboside strongly stimulated OC differentiation in vitro, in an adenosine kinase-dependent manner. OCs and macrophages (unlike OBs) lacked AMPK β2 subunit expression, and when generated from AMPK β1−/− mice displayed no detectable AMPK activity. Nevertheless, AICA-riboside was equally effective at stimulating OC differentiation from wild-type or β1−/− progenitors, indicating that AMPK is not essential for OC differentiation or the stimulatory action of AICA-riboside. These results show that AMPK is required to maintain normal bone density, but not through bone cell differentiation, and does not mediate powerful osteolytic effects of AICA-riboside.—Quinn, J. M. W., Tam, S., Sims, N. A., Saleh, H., McGregor, N. E., Poulton, I. J., Scott, J. W., Gillespie, M. T., Kemp, B. E., van Denderen, B. J. W. Germline deletion of AMP-activated protein kinase β subunits reduces bone mass without altering osteoclast differentiation or function. PMID:19723702

  7. The intact strontium ranelate complex stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-κB activation.

    Science.gov (United States)

    Yamaguchi, Masayoshi; Weitzmann, M Neale

    2012-01-01

    Strontium ranelate, a pharmaceutical agent shown in clinical trials to be effective in managing osteoporosis and reducing fracture risk in postmenopausal women, is relatively unique in its ability to both blunt bone resorption and stimulate bone formation. However, its mechanisms of action are largely unknown. As the nuclear factor-kappa B (NF-κB) activation antagonists both stimulate osteoblastic bone formation and repress osteoclastic bone resorption, we hypothesized that strontium ranelate may achieve its anabolic and anti-catabolic activities by modulating NF-κB activation in bone cells. In this study, osteoclast and osteoblast precursors were treated with intact strontium ranelate or its individual components sodium ranelate and/or strontium chloride, and its effect on in vitro osteoclastogenesis and osteoblastogenesis and on NF-κB activation quantified. Although the activity of strontium ranelate has been attributed to the release of strontium ions, low dose intact strontium ranelate complex, but not sodium ranelate and/or strontium chloride, potently antagonized NF-κB activation in osteoclasts and osteoblasts in vitro, and promoted osteoblast differentiation while suppressing osteoclast formation. Taken together, our data suggest a novel centralized mechanism by which strontium ranelate promotes osteoblast activity and suppresses osteoclastogenesis, based on suppression of NF-κB signal transduction. We further demonstrate that the biological actions of strontium ranelate may be related to low dose of the intact molecule rather than dissociation and release of strontium ions, as previously thought. These data may facilitate the development of additional novel pharmacological agents for the amelioration of osteoporosis, based on NF-κB blockade.

  8. Donepezil prevents RANK-induced bone loss via inhibition of osteoclast differentiation by downregulating acetylcholinesterase

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Sato

    2015-09-01

    Conclusions: AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

  9. Matrix metalloproteinase-12 (MMP-12) in osteoclasts

    DEFF Research Database (Denmark)

    Hou, Peng; Troen, Tine; Ovejero, Maria C

    2004-01-01

    Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called...

  10. Constitutively Active Parathyroid Hormone Receptor Signaling in Cells in Osteoblastic Lineage Suppresses Mechanical Unloading-induced Bone Resorption*

    Science.gov (United States)

    Ono, Noriaki; Nakashima, Kazuhisa; Schipani, Ernestina; Hayata, Tadayoshi; Ezura, Yoichi; Soma, Kunimichi; Kronenberg, Henry M.; Noda, Masaki

    2013-01-01

    Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity. PMID:17500070

  11. Antiosteoclastic Activity of Milk Thistle Extract after Ovariectomy to Suppress Estrogen Deficiency-Induced Osteoporosis

    Science.gov (United States)

    Kim, Jung-Lye; Kim, Yun-Ho; Kang, Min-Kyung; Gong, Ju-Hyun; Han, Seoung-Jun; Kang, Young-Hee

    2013-01-01

    Bone integrity abnormality and imbalance between bone formation by osteoblasts and bone resorption by osteoclasts are known to result in metabolic bone diseases such as osteoporosis. Silymarin-rich milk thistle extract (MTE) and its component silibinin enhanced alkaline phosphatase activity of osteoblasts but reduced tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts. The osteoprotective effects of MTE were comparable to those of estrogenic isoflavone. Low-dose combination of MTE and isoflavone had a pharmacological synergy that may be useful for osteogenic activity. This study attempted to reveal the suppressive effects of MTE on bone loss. C57BL/6 female mice were ovariectomized (OVX) as a model for postmenopausal osteopenia and orally administered 10 mg/kg MTE or silibinin for 8 weeks. The sham-operated mice served as estrogen controls. The treatment of ovariectomized mice with nontoxic MTE and silibinin improved femoral bone mineral density and serum receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio, an index of osteoclastogenic stimulus. In addition, the administration of MTE or silibinin inhibited femoral bone loss induced by ovariectomy and suppressed femoral TRAP activity and cathepsin K induction responsible for osteoclastogenesis and bone resorption. Collectively, oral dosage of MTE containing silibinin in the preclinical setting is effective in preventing estrogen deficiency-induced bone loss. PMID:23781510

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

    Energy Technology Data Exchange (ETDEWEB)

    Kiyomiya, Hiroyasu [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Ariyoshi, Wataru; Okinaga, Toshinori [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Kaneuji, Takeshi [Division of Oral Medicine, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Mitsugi, Sho [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Sakurai, Takuma [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Habu, Manabu [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Yoshioka, Izumi [Division of Oral Medicine, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); Tominaga, Kazuhiro [Division of Oral and Maxillofacial Surgery, Department of Science of Physical Functions, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580 (Japan); 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nie, Shaobo [Department of Orthopaedics, PLA General Hospital, Beijing 100853 (China); Xu, Jiawei [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Zhang, Chenghua [Department of Orthopaedics, Changle County Hospital of Traditional Chinese Medicine, Weifang 262400 (China); Xu, Chen [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Liu, Ming, E-mail: ming_li4717@sina.com [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Yu, Degang, E-mail: ydg163@126.com [Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China)

    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.

  15. Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation

    Directory of Open Access Journals (Sweden)

    Dong Ryun Gu

    2013-01-01

    Full Text Available Chrysanthemum zawadskii Herbich var. latilobum Kitamura, known as “Gujulcho” in Korea, has been used in traditional medicine to treat various inflammatory diseases, including rheumatoid arthritis. However, these effects have not been tested on osteoclasts, the bone resorbing cells that regulate bone metabolism. Here, we investigated the effects of C. zawadskii Herbich var. latilobum Kitamura ethanol extract (CZE on osteoclast differentiation induced by treatment with the receptor activator of NF-κB ligand (RANKL. CZE inhibited osteoclast differentiation and formation in a dose-dependent manner. The inhibitory effect of CZE on osteoclastogenesis was due to the suppression of ERK activation and the ablation of RANKL-stimulated Ca2+-oscillation via the inactivation of PLCγ2, followed by the inhibition of CREB activation. These inhibitory effects of CZE resulted in a significant repression of c-Fos expression and a subsequent reduction of NFATc1, a key transcription factor for osteoclast differentiation, fusion, and activation in vitro and in vivo. These results indicate that CZE negatively regulates osteoclast differentiation and may be a therapeutic candidate for the treatment of various bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Kyeongah; Nam, Sorim; Kim, Bomi; Lim, Ji Hyun; Yang, Young; Lee, Myeong-Sok; Lim, Jong-Seok, E-mail: jslim@sookmyung.ac.kr

    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.

  17. Osteoclasts prefer aged bone

    DEFF Research Database (Denmark)

    Henriksen, K; Leeming, Diana Julie; Byrjalsen, I

    2007-01-01

    We investigated whether the age of the bones endogenously exerts control over the bone resorption ability of the osteoclasts, and found that osteoclasts preferentially develop and resorb bone on aged bone. These findings indicate that the bone matrix itself plays a role in targeted remodeling...... of aged bones....

  18. Parathyroid hormone binding to cultured avian osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Teti, A.; Rizzoli, R.; Zambonin Zallone, A. (Univ. of Bari (Italy))

    1991-02-14

    Parathyroid hormone (PTH) increases serum calcium concentration via a controversial cellular mechanism. We investigated whether PTH binds avian osteoclasts. Isolated hypocalcaemic hen osteoclasts were incubated with ({sup 125}I)--bovine PTH (1-84). Specific binding of the hormone to the cells, which reached the equilibrium within 60 min, was observed. Half maximal binding was reached by 10 min. Binding was competitively inhibited by increasing doses of unlabeled PTH, and was about 55% displaced by adding, at the equilibrium, 10(-6) M unlabeled PTH. Autoradiography demonstrated specific label on the osteoclast. The cellular mechanism activated by the hormone remains to be elucidated.

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

    Directory of Open Access Journals (Sweden)

    Hua Ying

    Full Text Available 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

  20. Conditional deletion of CD98hc inhibits osteoclast development

    Directory of Open Access Journals (Sweden)

    Hideki Tsumura

    2016-03-01

    Full Text Available The CD98 heavy chain (CD98hc regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hcflox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages. Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH2 vitamin D3, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc. In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport.

  1. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

    International Nuclear Information System (INIS)

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

    2009-01-01

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 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-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB 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 ERα.

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

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Lisa J., E-mail: robinsonlj@msx.upmc.edu [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Blair, Harry C. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Veteran' s Affairs Medical Center, Pittsburgh, PA 15243 (United States)

    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}.

  3. TGF-β inducible early gene 1 regulates osteoclast differentiation and survival by mediating the NFATc1, AKT, and MEK/ERK signaling pathways.

    Directory of Open Access Journals (Sweden)

    Muzaffer Cicek

    Full Text Available TGF-β Inducible Early Gene-1 (TIEG1 is a Krüppel-like transcription factor (KLF10 that was originally cloned from human osteoblasts as an early response gene to TGF-β treatment. As reported previously, TIEG1(-/- mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1(-/- osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1(-/- precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1(-/- osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1(-/- osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1 to TIEG1(-/- cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1(-/- precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.

  4. Sargachromanol G inhibits osteoclastogenesis by suppressing the activation NF-κB and MAPKs in RANKL-induced RAW 264.7 cells.

    Science.gov (United States)

    Yoon, Weon-Jong; Kim, Kil-Nam; Heo, Soo-Jin; Han, Sang-Chul; Kim, Jihyeon; Ko, Yeong-Jong; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2013-05-17

    Inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with osteoporosis. Sargachromanol G (SG), isolated from the brown alga Sargassum siliquastrum, inhibits the production of inflammatory cytokines. In the present study, we determined the effect of SG on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. SG inhibited RANKL-induced osteoclast differentiation from RAW264.7 cells without signs of cytotoxicity. Additionally, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase 9 (MMP9), and calcitonin receptor (CTR), was strongly inhibited. SG inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. Furthermore, SG inhibited RANKL-induced phosphorylation of mitogen activated protein kinases (p38, JNK, and ERK). This study identified SG as an inhibitor for osteoclast formation and provided evidence that natural compounds, such as SG, are an alternative medicines for preventing and treating osteolysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Suppression of FRP-1/CD98-mediated multinucleated giant cell and osteoclast formation by an anti-FRP-1/CD98 mAb, HBJ 127, that inhibits c-src expression.

    Science.gov (United States)

    Tajima, M; Higuchi, S; Higuchi, Y; Miyamoto, N; Uchida, A; Ito, M; Nishio, M; Komada, H; Kawano, M; Kusagawa, S; Tsurudome, M; Ito, Y

    1999-05-01

    When anti-CD98 mAb 6-1-13, 4-5-1, or 38-2-2 was added to the culture fluids of monocytes, extensive cell aggregation and polykaryocyte formation were induced. These multinucleated giant cells were tartrate-resistant acid phosphatase (TRAP) positive. On the other hand, when monocytes were incubated with another anti-CD98 mAb, HBJ 127, polykaryocyte formation was not detected, although extensive cell aggregation was induced. When HBJ 127 and 6-1-13 were simultaneously added to the culture fluids, anti-CD98 mAb-induced cell fusion was inhibited almost completely. HBJ 127 suppressed formation of 6-1-13-induced cell fusion in a dose-dependent manner. If, however, HBJ 127 was added after incubation of monocytes with mAb 6-1-13 for 6 h, an appreciable degree of TRAP-positive polykaryocyte formation was found. The bindings of 6-1-13 and HBJ 127 were not mutually competed. When monocytes were incubated with 6-1-13 or HBJ 127, 6-1-13 induced c-src mRNA, while HBJ 127 did not. Furthermore, when monocytes were incubated with both 6-1-13 and HBJ 127, c-src mRNA could not be detected, showing that HBJ 127 suppresses c-src expression. Therefore, CD98-mediated osteoclast formation can be regulated by modification of CD98 system. Copyright 1999 Academic Press.

  6. The Effects of Aronia melanocarpa ‘Viking’ Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling

    Directory of Open Access Journals (Sweden)

    Mithun Ghosh

    2018-03-01

    Full Text Available This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa ‘Viking’ (AM and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK, c-Jun-N-terminal kinase (JNK and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1 protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β3. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB-mediated c-Fos and NFATc1 signaling pathway.

  7. Extracellular phosphates enhance activities of voltage-gated proton channels and production of reactive oxygen species in murine osteoclast-like cells.

    Science.gov (United States)

    Li, Guangshuai; Miura, Katsuyuki; Kuno, Miyuki

    2017-02-01

    Osteoclasts are highly differentiated bone-resorbing cells and play a significant role in bone remodelling. In the resorption pit, inorganic phosphate (Pi) concentrations increase because of degradation of hydroxyapatite. We studied effects of extracellular Pi on voltage-gated H + channels in osteoclast-like cells derived from a macrophage cell line (RAW264). Extracellular Pi (1.25-20 mM) increased the H + channel currents dose dependently and reversibly. The Pi-induced increases were attenuated by removal of extracellular Na + and by phosphonoformic acid, a blocker of Na + -dependent Pi transporters. Pi increased the maximal conductance, decreased activation time constant, increased deactivation time constant, and shifted the conductance-voltage relationship to more negative voltages. The most marked change was enhanced gating which was mainly caused by elevation of intracellular Pi levels. The Pi-induced enhanced gating was partially inhibited by protein kinase C (PKC) inhibitors, GF109203X and staurosporine, indicating that PKC-mediated phosphorylation was involved in part. The increase in the maximal conductance was mainly due to accompanying decrease in intracellular pH. These effects of Pi were not affected by intracellular Mg 2+ , bafilomycin A 1 (V-ATPase inhibitor) and removal of intracellular ATP. Extracellular Pi also upregulated reactive oxygen species (ROS). Diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, decreased ROS production and partially attenuated the enhanced gating. In the cells during later passages where osteoclastogenesis declined, H + channel activities and ROS production were both modest. These results suggest that, in osteoclasts, ambient Pi is a common enhancer for H + channels and ROS production and that potentiation of H + channels may help ROS production.

  8. Daily administration of eldecalcitol (ED-71), an active vitamin D analog, increases bone mineral density by suppressing RANKL expression in mouse trabecular bone.

    Science.gov (United States)

    Harada, Suguru; Mizoguchi, Toshihide; Kobayashi, Yasuhiro; Nakamichi, Yuko; Takeda, Satoshi; Sakai, Sadaoki; Takahashi, Fumiaki; Saito, Hitoshi; Yasuda, Hisataka; Udagawa, Nobuyuki; Suda, Tatsuo; Takahashi, Naoyuki

    2012-02-01

    Eldecalcitol (ED-71) is a new vitamin D₃ derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED-71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED-71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED-71 (50 ng/kg body weight) to 8-week-old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED-71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle-arrested receptor activator of NF-κB (RANK)-positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED-71 affected neither the number of RANK-positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED-71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL-positive cell surface around the trabecular bone was significantly reduced in ED-71-treated mice than in the control mice. ED-71 administration also increased BMD in 12-week-old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED-71 increases BMD by suppressing RANKL expression in trabecular bone in vivo.

  9. A comparison of osteoclast-rich and osteoclast-poor osteopetrosis in adult mice sheds light on the role of the osteoclast in coupling bone resorption and bone formation

    DEFF Research Database (Denmark)

    Thudium, Christian S; Moscatelli, Ilana; Flores, Carmen

    2014-01-01

    that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult...... osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone...

  10. The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

    OpenAIRE

    Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek; De Camilli, Pietro; Baron, Roland

    2008-01-01

    Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and ...

  11. Seawater Polluted with Highly Concentrated Polycyclic Aromatic Hydrocarbons Suppresses Osteoblastic Activity in the Scales of Goldfish, Carassius auratus.

    Science.gov (United States)

    Suzuki, Nobuo; Sato, Masayuki; Nassar, Hossam F; Abdel-Gawad, Fagr Kh; Bassem, Samah M; Yachiguchi, Koji; Tabuchi, Yoshiaki; Endo, Masato; Sekiguchi, Toshio; Urata, Makoto; Hattori, Atsuhiko; Mishima, Hiroyuki; Shimasaki, Youhei; Oshima, Yuji; Hong, Chun-Sang; Makino, Fumiya; Tang, Ning; Toriba, Akira; Hayakawa, Kazuichi

    2016-08-01

    We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-κB ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.

  12. Revival of nitrogen-containing bisphosphonate-induced inhibition of osteoclastogenesis and osteoclast function by water-soluble microfibrous borate glass.

    Science.gov (United States)

    Yuan, He; Niu, Li-Na; Jiao, Kai; Pei, Dan-Dan; Pramanik, Chandrani; Li, Ji-Yao; Messer, Regina; Kumar, Satish; Pashley, David H; Tay, Franklin R

    2016-02-01

    Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious skeletal complication associated with the long-term oral or intravenous use of nitrogen-containing bisphosphonates (N-BPs). Here, we investigated the effects of an ionic cocktail prepared from water-soluble microfibrous borate glass on neutralizing the inhibitory effects of two heterocyclic N-BPs, risedronate or zoledronic acid, on osteoclastogenesis, apoptosis of differentiated osteoclasts and osteoclast function. Cell growth and proliferation assays were first performed on RAW 264.7 cells to optimize the concentrations of the ionic cocktail and N-BPs to be used for static cell culture. The pre-osteoclasts were then stimulated with RANKL to differentiate into osteoclasts. The effects of the ionic cocktail and N-BPs on osteoclast differentiation, apoptosis and function were subsequently examined using 3 series of experiments conducted at the gene, protein, morphological and functional levels. After concentration optimization, the ionic cocktail was found to partially reverse N-BP-induced inhibition of osteoclastogenesis, stimulation of osteoclasts apoptosis and reduction of osteoclast resorptive activity. Ultrastructural examination of osteoclasts that had been exposed to either N-BP identified classical features of late apoptosis and secondary necrosis, while osteoclasts exposed simultaneously to the concentration-optimized ionic cocktail and N-BPs exhibited only signs of early apoptosis that were possibly reversible. Taken together, the results of the 4 series of experiments indicate that the ionic cocktail produced from dissolution of borate glass dressings has the potential to rescue the adverse effects of heterocyclic N-BPs on osteoclast differentiation and function. These results warrant further confirmation using dynamic cell culture and small animal BRONJ models. Long-term oral and intravenous use of nitrogen-containing bisphosphonates (N-BPs) may result in bisphosphonate

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

    Science.gov (United States)

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

    2011-11-18

    Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

    DEFF Research Database (Denmark)

    Henriksen, Kim; Andreassen, Kim V; Thudium, Christian S

    2012-01-01

    Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium wa...

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

    Energy Technology Data Exchange (ETDEWEB)

    Wisutsitthiwong, Chonnaree; Buranaruk, Chayanit [Graduate Program in Industrial Microbiology, Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand); Pudhom, Khanitha [Department of Chemistry, Faculty of Science and Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand); Palaga, Tanapat, E-mail: tanapat.p@chula.ac.th [Graduate Program in Industrial Microbiology, Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330 (Thailand)

    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.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    Highlights: ► A gedunin type limonoid from seeds of mangroves, 7-oxo-7-deacetoxygedunin, exhibits strong anti-osteoclastogenic activity. ► Treatment with this limonoid results in significant decrease in expression of NFATc1 and osteoclast-related genes. ► The mode of action of this limonoid is by inhibiting activation of the NF-κB 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-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

    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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. ► Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. ► Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. ► Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/β-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 β-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, β-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 regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies

  19. Amlexanox Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss.

    Science.gov (United States)

    Zhang, Yong; Guan, Hanfeng; Li, Jing; Fang, Zhong; Chen, Wenjian; Li, Feng

    2015-09-04

    The activity of protein kinases IKK-ε and TANK-binding kinase 1 (TBK1) has been shown to be associated with inflammatory diseases. As an inhibitor of IKK-ε and TBK1, amlexanox is an anti-inflammatory, anti-allergic, immunomodulator and used for treatment of ulcer, allergic rhinitis and asthma in clinic. We hypothesized that amlexanox may be used for treatment of osteoclast-related diseases which frequently associated with a low grade of systemic inflammation. In this study, we investigated the effects of amlexanox on RANKL-induced osteoclastogenesis in vitro and ovariectomy-mediated bone loss in vivo. In primary bone marrow derived macrophages (BMMs), amlexanox inhibited osteoclast formation and bone resorption. At the molecular level, amlexanox suppressed RANKL-induced activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPKs), c-Fos and NFATc1. Amlexanox decreased the expression of osteoclast-specific genes, including TRAP, MMP9, Cathepsin K and NFATc1. Moreover, amlexanox enhanced osteoblast differentiation of BMSCs. In ovariectomized (OVX) mouse model, amlexanox prevented OVX-induced bone loss by suppressing osteoclast activity. Taken together, our results demonstrate that amlexanox suppresses osteoclastogenesis and prevents OVX-induced bone loss. Therefore, amlexanox may be considered as a new therapeutic candidate for osteoclast-related diseases, such as osteoporosis and rheumatoid arthritis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Fei; Zhai, Zanjing; Jiang, Chuan; Liu, Xuqiang; Li, Haowei; Qu, Xinhua [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Ouyang, Zhengxiao [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Department of Orthopaedics, Hunan Provincial Tumor Hospital and Tumor Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410013 (China); Fan, Qiming; Tang, Tingting [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Qin, An, E-mail: dr.qinan@gmail.com [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Gu, Dongyun, E-mail: dongyungu@gmail.com [Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People' s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Engineering Research Center of Digital Medicine and Clinical Translation, Ministry of Education of PR China (China); School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030 (China)

    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.

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

    Science.gov (United States)

    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

  2. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

    Science.gov (United States)

    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.

  3. Microbial enrichment to enhance the disease suppressive activity of compost

    NARCIS (Netherlands)

    Postma, J.; Montenari, M.; Boogert, van den P.H.J.F.

    2003-01-01

    Compost amended soil has been found to be suppressive against plant diseases in various cropping systems. The level and reproducibility of disease suppressive properties of compost might be increased by the addition of antagonists. In the present study, the establishment and suppressive activity of

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

    Directory of Open Access Journals (Sweden)

    Hye Jung Ihn

    Full Text Available 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.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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 + /H + 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-κB ligand signaling and is required for OC differentiation and survival

  6. The vitamin D analogue ED71 but Not 1,25(OH2D3 targets HIF1α protein in osteoclasts.

    Directory of Open Access Journals (Sweden)

    Yuiko Sato

    Full Text Available Although both an active form of the vitamin D metabolite, 1,25(OH2D3, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH2D3 -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH2D3, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH2D3 or ED71 function in osteoclasts requires the vitamin D receptor (VDR. ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnβ mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH2D3 in vitro, were both significantly higher following treatment with 1,25(OH2D3 than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH2D3, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.

  7. Platelet-derived growth factor BB enhances osteoclast formation and osteoclast precursor cell chemotaxis.

    Science.gov (United States)

    Li, Dian-Qi; Wan, Qi-Long; Pathak, Janak L; Li, Zu-Bing

    2017-07-01

    Enhanced osteoclast formation increases bone resorption, which triggers bone remodeling. Platelet-derived growth factor BB (PDGF-BB) enhances precursor cell homing, angiogenesis, and bone healing, and thereby could also treat osteoporosis. However, the effect of PDGF-BB on osteoclast formation is not fully understood. We investigated whether exogenous recombinant PDGF-BB directly affects osteoclast formation and osteoclast precursor cell chemotaxis. The murine monocyte-macrophage cell line RAW264.7 and bone-marrow-derived macrophages were cultured with recombinant mouse PDGF-BB with or without a platelet-derived growth factor receptor β inhibitor (AG-1295) or a Janus kinase 2 inhibitor (AG-490) to analyze the effect on osteoclastogenesis in vitro. PDGF-BB with or without AG-490 or AG-1295 was locally administrated in the mandibular fracture of 16-week-old Sprague Dawley rats (n = 18) for 1-2 weeks to analyze the effect on osteoclastogenesis in vivo. The effect of the treatments on osteoclast formation, osteoclast precursor cell migration, and expression of osteoclastogenic signaling molecules was analyzed. PDGF-BB enhanced osteoclast formation both in vitro and in vivo, but AG-490 and AG-1295 inhibited this effect. PDGF-BB enhanced phosphorylation of extracellular-signal-regulated kinase 1/2 (ERK1/2), Akt, and signal transducer and activator of transcription 3 (STAT3) in RAW264.7 cells. AG-490 inhibited PDGF-BB-induced STAT3 phosphorylation. PDGF-BB enhanced RAW264.7 cell migration and gene expression of osteoclastogenic signaling molecules (i.e., nuclear factor of activated T cells 1, dendrocyte-expressed seven transmembrane protein, and B-cell lymphoma 2), and treatment with AG-1295, AG-490, or S3I-201 (a STAT3 inhibitor) reduced this effect. PDGF-BB enhanced osteoclast formation, osteoclast precursor cell chemotaxis, and phosphorylation of STAT3, Akt, and ERK1/2. but AG-1295 and AG-490 reduced this effect. These findings reflect the complexity of

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

    Science.gov (United States)

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

    2016-09-01

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

  9. Stauntonia hexaphylla (Lardizabalaceae) leaf methanol extract inhibits osteoclastogenesis and bone resorption activity via proteasome-mediated degradation of c-Fos protein and suppression of NFATc1 expression.

    Science.gov (United States)

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

    2015-08-14

    Natural plants, including common vegetables and fruits, have been recognized as essential sources for drug discovery and the development of new, safe, and economical medicaments. Stauntonia hexaphylla (Lardizabalaceae) is widely distributed in Korea, Japan, and China, and is a popular herbal supplement in Korean and Chinese folk medicine owing to its analgesic, sedative, and diuretic properties. However, the exact pharmacological effects of S. hexaphylla extract, particularly its effect on osteoclastogenesis, are not known. Osteoclast differentiation and function were identified with tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay, and the underling mechanisms were determined by real-time RT-PCR and western blot analysis. S. hexaphylla was found to inhibit early-stage receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without cytotoxicity and bone-resorbing activity in mature osteoclasts in a dose-dependent manner. This S. hexaphylla-mediated blockade of osteoclastogenesis involved abrogation of the NF-κB, ERK, and c-Src-Btk-PLCγ2 calcium signal pathways. Interestingly, we found that S. hexaphylla down-regulated RANKL-associated c-Fos protein induction by suppressing its translation. Furthermore, ectopic overexpression of c-Fos and NFATc1 rescued the inhibition of osteoclast differentiation by S. hexaphylla. Furthermore, S. hexaphylla inhibited the c-Fos- and NFATc1-regulated expression of genes required for osteoclastogenesis, such as TRAP, OSCAR, β3-integrin, ATP6v0d2, and CtsK. These findings suggest that S. hexaphylla might be useful for the development of new anti-osteoporosis agents.

  10. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Bongkun; Kang, Soon-Suk [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Kang, Sang-Wook [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Min, Bon-Hong [Department of Pharmacology, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Song, Youngsup [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Yoon, Seung-Yong [Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Chang, Eun-Ju, E-mail: ejchang@amc.seoul.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Department of Anatomy and Cell Biology, Cell Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736 (Korea, Republic of)

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  11. Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases.

    Science.gov (United States)

    Fukada, S Y; Silva, T A; Garlet, G P; Rosa, A L; da Silva, J S; Cunha, F Q

    2009-02-01

    Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CKbeta8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

  12. Interferon regulatory factor-8 regulates bone metabolism by suppressing osteoclastogenesis.

    Science.gov (United States)

    Zhao, Baohong; Takami, Masamichi; Yamada, Atsushi; Wang, Xiaogu; Koga, Takako; Hu, Xiaoyu; Tamura, Tomohiko; Ozato, Keiko; Choi, Yongwon; Ivashkiv, Lionel B; Takayanagi, Hiroshi; Kamijo, Ryutaro

    2009-09-01

    Bone metabolism results from a balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases such as periodontitis and rheumatoid arthritis are characterized by increased bone destruction due to enhanced osteoclastogenesis. Here we report that interferon regulatory factor-8 (IRF-8), a transcription factor expressed in immune cells, is a key regulatory molecule for osteoclastogenesis. IRF-8 expression in osteoclast precursors was downregulated during the initial phase of osteoclast differentiation induced by receptor activator of nuclear factor-kappaB ligand (RANKL), which is encoded by the Tnfsf11 gene. Mice deficient in Irf8 showed severe osteoporosis, owing to increased numbers of osteoclasts, and also showed enhanced bone destruction after lipopolysaccharide (LPS) administration. Irf8-/- osteoclast precursors underwent increased osteoclastogenesis in response to RANKL and tumor necrosis factor-alpha (TNF-alpha). IRF-8 suppressed osteoclastogenesis by inhibiting the function and expression of nuclear factor of activated T cells c1 (NFATc1). Our results show that IRF-8 inhibits osteoclast formation under physiological and pathological conditions and suggest a model where downregulation of inhibitory factors such as IRF-8 contributes to RANKL-mediated osteoclastogenesis.

  13. Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II

    DEFF Research Database (Denmark)

    Henriksen, Kim; Gram, Jeppe; Schaller, Sophie

    2004-01-01

    Autosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts...... osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption....

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway.

    Science.gov (United States)

    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.

  16. Evidence of a correlation of estrogen receptor level and avian osteoclast estrogen responsiveness.

    Science.gov (United States)

    Pederson, L; Kremer, M; Foged, N T; Winding, B; Ritchie, C; Fitzpatrick, L A; Oursler, M J

    1997-05-01

    Isolated osteoclasts from 5-week-old chickens respond to estradiol treatment in vitro with decreased resorption activity, increased nuclear proto-oncogene expression, and decreased lysosomal enzyme secretion. This study examines osteoclasts from embryonic chickens and egg-laying hens for evidence of estrogen responsiveness. Although osteoclasts from both of these sources express estrogen receptor mRNA and protein, estradiol treatment had no effect on resorption activity. In contrast to the lack of effect on resorption, estradiol treatment for 30 minutes resulted in steady-state mRNA levels of c-fos and c-jun increasing in osteoclasts from embryonic chickens and decreasing in osteoclasts from egg-laying hens. These data suggest that a nuclear proto-oncogene response may not be involved in estradiol-mediated decreased osteoclast resorption activity. To examine the influence of circulating estrogen on osteoclast estrogen responsiveness, 5-week-old chickens were injected with estrogen for 4 days prior to sacrifice. Estradiol treatment of osteoclasts from these chickens did not decrease resorption activity in vitro. Transfection of an estrogen receptor expression vector into osteoclasts from the estradiol-injected chickens and egg-laying hens restored estrogen responsiveness. Osteoclasts from 5-week-old chickens and estradiol treated 5-week-old chickens transfected with the estrogen receptor expression vector contained significantly higher levels of estrogen receptor protein and responded to estradiol treatment by decreasing secretion of cathepsins B and L and tartrate-resistant acid phosphatase. In contrast, osteoclasts from embryonic chickens, egg-laying hens, and estradiol-treated 5-week-old chickens either untransfected or transfected with an empty expression vector did not respond similarly. These data suggest that modulation of osteoclast estrogen responsiveness may be controlled by changes in the osteoclast estrogen receptor levels.

  17. Emotion suppression reduces hippocampal activity during successful memory encoding.

    Science.gov (United States)

    Binder, Julia; de Quervain, Dominique J-F; Friese, Malte; Luechinger, Roger; Boesiger, Peter; Rasch, Björn

    2012-10-15

    People suppressing their emotions while facing an emotional event typically remember it less well. However, the neural mechanisms underlying the impairing effect of emotion suppression on successful memory encoding are not well understood. Because successful memory encoding relies on the hippocampus and the amygdala, we hypothesized that memory impairments due to emotion suppression are associated with down-regulated activity in these brain areas. 59 healthy females were instructed either to simply watch the pictures or to down-regulate their emotions by using a response-focused emotion suppression strategy. Brain activity was recorded using functional magnetic resonance imaging (fMRI), and free recall of pictures was tested afterwards. As expected, suppressing one's emotions resulted in impaired recall of the pictures. On the neural level, the memory impairments were associated with reduced activity in the right hippocampus during successful encoding. No significant effects were observed in the amygdala. In addition, functional connectivity between the hippocampus and the right dorsolateral prefrontal cortex was strongly reduced during emotion suppression, and these reductions predicted free-recall performance. Our results indicate that emotion suppression interferes with memory encoding on the hippocampal level, possibly by decoupling hippocampal and prefrontal encoding processes, suggesting that response-focused emotion suppression might be an adaptive strategy for impairing hippocampal memory formation in highly arousing situations. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

    International Nuclear Information System (INIS)

    Burger, E.H.; Van der Meer, J.W.; van de Gevel, J.S.; Gribnau, J.C.; Thesingh, G.W.; van Furth, R.

    1982-01-01

    The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45 Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes

  19. Andrographolide suppresses RANKL-induced osteoclastogenesis in vitro and prevents inflammatory bone loss in vivo.

    Science.gov (United States)

    Zhai, Z J; Li, H W; Liu, G W; Qu, X H; Tian, B; Yan, W; Lin, Z; Tang, T T; Qin, A; Dai, K R

    2014-02-01

    Osteoclasts play a pivotal role in diseases such as osteoporosis, rheumatoid arthritis and tumour bone metastasis. Thus, searching for natural compounds that may suppress osteoclast formation and/or function is promising for the treatment of osteoclast-related diseases. Here, we examined changes in osteoclastogenesis and LPS-induced osteolysis in response to andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata. Effects of AP on osteoclast differentiation and bone resorption were measured in vitro. Western blots and RT-PCR techniques were used to examine the underlying molecular mechanisms. The bone protective activity of AP in vivo was assessed in a mouse model of osteolysis. AP concentration-dependently suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro and reduced the expression of osteoclast-specific markers, including tartrate-resistant acid phosphatase, calcitonin receptors and cathepsin K. Further molecular analysis revealed that AP impaired RANKL-induced NF-κB signalling by inhibiting the phosphorylation of TGF-β-activated kinase 1, suppressing the phosphorylation and degradation of IκBα, and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. AP also inhibited the ERK/MAPK signalling pathway without affecting p38 or JNK signalling. AP suppressed RANKL-induced osteoclastogenesis through attenuating NF-κB and ERK/MAPK signalling pathways in vitro, thus preventing bone loss in vivo. These data indicated that AP is a promising natural compound for the treatment of osteoclast-related bone diseases. © 2013 The British Pharmacological Society.

  20. The Effects of Kaempferol-Inhibited Autophagy on Osteoclast Formation.

    Science.gov (United States)

    Kim, Chang-Ju; Shin, Sang-Hun; Kim, Bok-Joo; Kim, Chul-Hoon; Kim, Jung-Han; Kang, Hae-Mi; Park, Bong-Soo; Kim, In-Ryoung

    2018-01-02

    Kaempferol, a flavonoid compound, is derived from the rhizome of Kaempferia galanga L ., which is used in traditional medicine in Asia. Autophagy has pleiotropic functions that are involved in cell growth, survival, nutrient supply under starvation, defense against pathogens, and antigen presentation. There are many studies dealing with the inhibitory effects of natural flavonoids in bone resorption. However, no studies have explained the relationship between the autophagic and inhibitory processes of osteoclastogenesis by natural flavonoids. The present study was undertaken to investigate the inhibitory effects of osteoclastogenesis through the autophagy inhibition process stimulated by kaempferol in murin macrophage (RAW 264.7) cells. The cytotoxic effect of Kaempferol was investigated by MTT assay. The osteoclast differentiation and autophagic process were confirmed via tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, western blot, and real-time PCR. Kaempferol controlled the expression of autophagy-related factors and in particular, it strongly inhibited the expression of p62/SQSTM1. In the western blot and real time-PCR analysis, when autophagy was suppressed with the application of 3-Methyladenine (3-MA) only, osteoclast and apoptosis related factors were not significantly affected. However, we found that after cells were treated with kaempferol, these factors inhibited autophagy and activated apoptosis. Therefore, we presume that kaempferol-inhibited autophagy activated apoptosis by degradation of p62/SQSTM1. Further study of the p62/SQSTM1 gene as a target in the autophagy mechanism, may help to delineate the potential role of kaempferol in the treatment of bone metabolism disorders.

  1. Role of the osteoclast in cranial suture waveform patterning.

    Science.gov (United States)

    Byron, Craig Daniel

    2006-05-01

    This study investigates the role of bone resorption in defining interdigitations characteristic of cranial suture waveform. Male mice from the CD-1 (ICR) background were analyzed at six age groups (n = 5 mice per group) in order to study the ontogenetic changes of osteoclast counts using tartrate-resistant acid phosphatase-stained histological sections of sagittal sutures. Additionally, the complexity of suture lines were measured ectocranially from the same age groups (n = 5 per group) using image capture and fractal geometry (ruler dimension method). The results suggest that osteoclast resorption is a contributor to suture patterning. Specifically, osteoclasts show the greatest activity along concave suture regions at 42 and 84 days (Kruskal-Wallis test statistic = 14.9; P weaning and the transition to a hard chow diet. These data demonstrate osteoclasts along the bone margin of the cranial suture and also indicate that sutures attain their complex shape at the same age when osteoclast number is highest along concave suture margins, underscoring the role of osteoclasts in generating the suture waveform pattern. 2006 Wiley-Liss, Inc.

  2. Cold suppresses agonist-induced activation of TRPV1.

    Science.gov (United States)

    Chung, M-K; Wang, S

    2011-09-01

    Cold therapy is frequently used to reduce pain and edema following acute injury or surgery such as tooth extraction. However, the neurobiological mechanisms of cold therapy are not completely understood. Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated nociceptive ion channel implicated in thermosensation and pathological pain under conditions of inflammation or injury. Although capsaicin-induced nociception, neuropeptide release, and ionic currents are suppressed by cold, it is not known if cold suppresses agonist-induced activation of recombinant TRPV1. We demonstrate that cold strongly suppressed the activation of recombinant TRPV1 by multiple agonists and capsaicin-evoked currents in trigeminal ganglia neurons under normal and phosphorylated conditions. Cold-induced suppression was partially impaired in a TRPV1 mutant that lacked heat-mediated activation and potentiation. These results suggest that cold-induced suppression of TRPV1 may share a common molecular basis with heat-induced potentiation, and that allosteric inhibition may contribute, in part, to the cold-induced suppression. We also show that combination of cold and a specific antagonist of TRPV1 can produce an additive suppression. Our results provide a mechanistic basis for cold therapy and may enhance anti-nociceptive approaches that target TRPV1 for managing pain under inflammation and tissue injury, including that from tooth extraction.

  3. Mitochondria related peptide MOTS-c suppresses ovariectomy-induced bone loss via AMPK activation

    Energy Technology Data Exchange (ETDEWEB)

    Ming, Wei, E-mail: weiming@xiyi.edu.cn [State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi’an 710032 (China); Department of Pharmacology, Xi’an Medical University, Xi’an 710021 (China); Lu, Gan, E-mail: leonming99@163.com [Department of Gynecology of Shaanxi Provincial People’s Hospital, Xi’an, 710068 (China); Xin, Sha, E-mail: 248967979@qq.com [Institute of Orthopedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032 (China); Huanyu, Lu, E-mail: 2366927258@qq.com [Department of Occupational and Environmental Health and the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, School of Public Health, Fourth Military Medical University, Xi’an 710032 (China); Yinghao, Jiang, E-mail: jiangyh@fmmu.edu.cn [State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi’an 710032 (China); Xiaoying, Lei, E-mail: leixiaoy@fmmu.edu.cn [State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi’an 710032 (China); Chengming, Xu, E-mail: chengmingxu@yeah.net [State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi’an 710032 (China); Banjun, Ruan, E-mail: running@163.com [State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi’an 710032 (China); Li, Wang, E-mail: wanglifw@fmmu.edu.cn [State Key Laboratory of Cancer Biology, Department of Pharmacogenomics, Fourth Military Medical University, Xi’an 710032 (China); and others

    2016-08-05

    Therapeutic targeting bone loss has been the focus of the study in osteoporosis. The present study is intended to evaluate whether MOTS-c, a novel mitochondria related 16 aa peptide, can protect mice from ovariectomy-induced osteoporosis. After ovary removal, the mice were injected with MOTS-c at a dose of 5 mg/kg once a day for 12 weeks. Our results showed that MOTS-c treatment significantly alleviated bone loss, as determined by micro-CT examination. Mechanistically, we found that the receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclast differentiation was remarkably inhibited by MOTS-c. Moreover, MOTS-c increased phosphorylated AMPK levels, and compound C, an AMPK inhibitor, could partially abrogate the effects of the MOTS-c on osteoclastogenesis. Thus, our findings provide evidence that MOTS-c may exert as an inhibitor of osteoporosis via AMPK dependent inhibition of osteoclastogenesis. -- Highlights: •MOTS-c decreases OVX-induced bone loss in vivo. •MOTS-c inhibits RANKL-induced osteoclast formation. •MOTS-c inhibits RANKL-induced osteoclast-specific gene expression. •MOTS-c represses osteoclast differentiation via the activation of AMPK.

  4. Mitochondria related peptide MOTS-c suppresses ovariectomy-induced bone loss via AMPK activation

    International Nuclear Information System (INIS)

    Ming, Wei; Lu, Gan; Xin, Sha; Huanyu, Lu; Yinghao, Jiang; Xiaoying, Lei; Chengming, Xu; Banjun, Ruan; Li, Wang

    2016-01-01

    Therapeutic targeting bone loss has been the focus of the study in osteoporosis. The present study is intended to evaluate whether MOTS-c, a novel mitochondria related 16 aa peptide, can protect mice from ovariectomy-induced osteoporosis. After ovary removal, the mice were injected with MOTS-c at a dose of 5 mg/kg once a day for 12 weeks. Our results showed that MOTS-c treatment significantly alleviated bone loss, as determined by micro-CT examination. Mechanistically, we found that the receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclast differentiation was remarkably inhibited by MOTS-c. Moreover, MOTS-c increased phosphorylated AMPK levels, and compound C, an AMPK inhibitor, could partially abrogate the effects of the MOTS-c on osteoclastogenesis. Thus, our findings provide evidence that MOTS-c may exert as an inhibitor of osteoporosis via AMPK dependent inhibition of osteoclastogenesis. -- Highlights: •MOTS-c decreases OVX-induced bone loss in vivo. •MOTS-c inhibits RANKL-induced osteoclast formation. •MOTS-c inhibits RANKL-induced osteoclast-specific gene expression. •MOTS-c represses osteoclast differentiation via the activation of AMPK.

  5. Key Triggers of Osteoclast-Related Diseases and Available Strategies for Targeted Therapies: A Review

    Directory of Open Access Journals (Sweden)

    Haidi Bi

    2017-12-01

    Full Text Available Osteoclasts, the only cells with bone resorption functions in vivo, maintain the balance of bone metabolism by cooperating with osteoblasts, which are responsible for bone formation. Excessive activity of osteoclasts causes many diseases such as osteoporosis, periprosthetic osteolysis, bone tumors, and Paget’s disease. In contrast, osteopetrosis results from osteoclast deficiency. Available strategies for combating over-activated osteoclasts and the subsequently induced diseases can be categorized into three approaches: facilitating osteoclast apoptosis, inhibiting osteoclastogenesis, and impairing bone resorption. Bisphosphonates are representative molecules that function by triggering osteoclast apoptosis. New drugs, such as tumor necrosis factor and receptor activator of nuclear factor kappa-B ligand (RANKL inhibitors (e.g., denosumab have been developed for targeting the receptor activator of nuclear factor kappa-B /RANKL/osteoprotegerin system or CSF-1/CSF-1R axis, which play critical roles in osteoclast formation. Furthermore, vacuolar (H+-ATPase inhibitors, cathepsin K inhibitors, and glucagon-like peptide 2 impair different stages of the bone resorption process. Recently, significant achievements have been made in this field. The aim of this review is to provide an updated summary of the current progress in research involving osteoclast-related diseases and of the development of targeted inhibitors of osteoclast formation.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Shin-Ichi, E-mail: shayashi@med.tottori-u.ac.jp [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan); Murata, Akihiko; Okuyama, Kazuki; Shimoda, Yuhki; Hikosaka, Mari [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan); Yasuda, Hisataka [Planning and Development, Bioindustry Division, Oriental Yeast Co., Ltd, Itabashi-Ku, Tokyo 174-8505 (Japan); Yoshino, Miya [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan)

    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.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► The frequency of C7 differentiation into osteoclast was low and constant. ► Only extended C7 cell cultures exponentially increased osteoclast+ cultures. ► C7 cell differentiation into committed osteoclast precursors is on ‘autopilot’. ► 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 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.

  9. Picrasidine I from Picrasma Quassioides Suppresses Osteoclastogenesis via Inhibition of RANKL Induced Signaling Pathways and Attenuation of ROS Production

    Directory of Open Access Journals (Sweden)

    Lingbo Kong

    2017-10-01

    Full Text Available Background/Aims: Osteoporosis is a metabolic bone disorder that tortures about millions of people worldwide. Recent study demonstrated agents derived from picrasma quassioides is a promising drug for targets multiple signaling pathways. However its potential in treatment of bone loss has not been fully understood. Methods: The bone marrow macrophages (BMMs were cultured and induced with M-CSF and RANKL followed by picrasidine I (PI treatment. Then the effects of PI on osteoclast formation were evaluated by counting tartrate-resistant acid phosphatase (TRAP-positive multinucleated cells. Moreover, effects of PI on bone resorption activity of mature osteoclast were studied through bone resorption pit counting and actin ring structure analysis. Further, the involved potential signaling pathways cross-talking were investigated by performed Western blotting and quantitative real-time PCR examination. Results: Results demonstrated PI strongly inhibited RANKL induced osteoclast formation from its precursors. Mechanistically, the inhibitory effect of PI on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and NFATc1. Moreover, PI markedly blocked the RANKL-induced osteoclastogenesis by attenuating MAPKs and NF-κB signaling pathways. In addition, PI decreased the ROS generation in osteoclast and osteoblast. Conclusion: Taken together our data demonstrate that PI has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-κB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

  10. Picrasidine I from Picrasma Quassioides Suppresses Osteoclastogenesis via Inhibition of RANKL Induced Signaling Pathways and Attenuation of ROS Production.

    Science.gov (United States)

    Kong, Lingbo; Wang, Biao; Yang, Xiaobin; Guo, Hua; Zhang, Ke; Zhu, Ziqi; Liu, Jijun; Hao, Dingjun

    2017-01-01

    Osteoporosis is a metabolic bone disorder that tortures about millions of people worldwide. Recent study demonstrated agents derived from picrasma quassioides is a promising drug for targets multiple signaling pathways. However its potential in treatment of bone loss has not been fully understood. The bone marrow macrophages (BMMs) were cultured and induced with M-CSF and RANKL followed by picrasidine I (PI) treatment. Then the effects of PI on osteoclast formation were evaluated by counting tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Moreover, effects of PI on bone resorption activity of mature osteoclast were studied through bone resorption pit counting and actin ring structure analysis. Further, the involved potential signaling pathways cross-talking were investigated by performed Western blotting and quantitative real-time PCR examination. Results demonstrated PI strongly inhibited RANKL induced osteoclast formation from its precursors. Mechanistically, the inhibitory effect of PI on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and NFATc1. Moreover, PI markedly blocked the RANKL-induced osteoclastogenesis by attenuating MAPKs and NF-κB signaling pathways. In addition, PI decreased the ROS generation in osteoclast and osteoblast. Taken together our data demonstrate that PI has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-κB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors. © 2017 The Author(s). Published by S. Karger AG, Basel.

  11. Salvia plebeia R.Br. inhibits signal transduction of IL-6 and prevents ovariectomy-induced bone loss by suppressing osteoclastogenesis.

    Science.gov (United States)

    Kim, Mi-Hwa; Jung, Kyungsook; Nam, Ki-Hoan; Jang, Hyun-Jae; Lee, Seung Woong; Kim, Yesol; Park, Chan Sun; Lee, Tae-Hoon; Park, Jee Hun; Choi, Jung Ho; Rho, Mun-Chual; Oh, Hyun-Mee

    2016-12-01

    The interleukin-6 (IL-6) family of cytokines plays a key role in the pathogenesis of rheumatoid arthritis and osteoporosis through the regulation of bone formation and resorption. In this study, it was observed that ethanol extract of Salvia plebeia R.Br. (S.P-EE) inhibited IL-6-induced signaling cascade including phosphorylation of JAK2/STAT3 and ERK. Subsequently, it was examined whether S.P-EE treatment could recover bone loss in ovariectomized (OVX) mice. Indeed, S.P-EE exhibited both preventive and therapeutic effect on OVX-induced bone loss in trabecular microarchitecture along with significant increase in bone mineral density and content. To understand the mechanism of action of S.P-EE in bone metabolism, the effect of S.P-EE on osteoclast differentiation and activity was investigated. S.P-EE significantly inhibited RANKL-induced osteoclast differentiation by suppressing phosphorylation of MAPK and Akt, and expression of NFATc1 and osteoclast marker genes. S.P-EE also inhibited bone-resorbing activity of osteoclasts. Furthermore, isolation and identification of the active compounds which are responsible for the inhibitory effect of S.P-EE on osteoclast differentiation was carried out. Six major flavonoids and plebeiolide A-C were isolated and examined their effects on osteoclast differentiation. Luteolin and hispidulin, and plebeiolide A and C, not B exhibited potent inhibitory activity on RANKL-induced osteoclast formation.

  12. Mevalonate-suppressive dietary isoprenoids for bone health.

    Science.gov (United States)

    Mo, Huanbiao; Yeganehjoo, Hoda; Shah, Anureet; Mo, Warren K; Soelaiman, Ima Nirwana; Shen, Chwan-Li

    2012-12-01

    Osteoclastogenesis and osteoblastogenesis, the balancing acts for optimal bone health, are under the regulation of small guanosine triphosphate-binding proteins (GTPases) including Ras, Rac, Rho and Rab. The activities of GTPases require post-translational modification with mevalonate-derived prenyl pyrophosphates. Mevalonate deprivation induced by competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (e.g., statins) prevents the activation of GTPases, suppresses the expression of the receptor for activation of nuclear factor kappa B (NFκB) ligand (RANKL) and activation of NFκB and, consequently, inhibits osteoclast differentiation and induces osteoclast apoptosis. In contrast, statin-mediated inactivation of GTPases enhances alkaline phosphatase activity and the expression of bone morphogenetic protein-2, vascular epithelial growth factor, and osteocalcin in osteoblasts and induces osteoblast proliferation and differentiation. Animal studies show that statins inhibit bone resorption and increase bone formation. The anabolic effect of statins and other mevalonate pathway-suppressive pharmaceuticals resembles the anti-osteoclastogenic and bone-protective activities conferred by dietary isoprenoids, secondary products of plant mevalonate metabolism. The tocotrienols, vitamin E molecules with HMG CoA reductase-suppressive activity, induce mevalonate deprivation and concomitantly suppress the expression of RANKL and cyclooxygenase-2, the production of prostaglandin E2 and the activation of NFκB. Accordingly, tocotrienols inhibit osteoclast differentiation and induce osteoclast apoptosis, impacts reminiscent of those of statins. In vivo studies confirm the bone protective activity of tocotrienols at nontoxic doses. Blends of tocotrienols, statins and isoprenoids widely found in fruits, vegetables, grains, herbs, spices, and essential oils may synergistically suppress osteoclastogenesis while promoting osteoblastogenesis, offering a novel

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

    Directory of Open Access Journals (Sweden)

    Yanqing Gu

    2014-01-01

    Full Text Available 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.

  14. Proton concentrations can be a major contributor to the modification of osteoclast and osteoblast differentiation, working independently of extracellular bicarbonate ions.

    Science.gov (United States)

    Kato, Kohtaro; Matsushita, Misao

    2014-01-01

    We established a system to separately analyze the role of protons and bicarbonate ions in vitro in which the pH of the medium was controlled by HEPES at various concentrations of sodium bicarbonate (NaHCO3) in the absence of carbon dioxide (CO2). Using this system, we demonstrated that acidosis promoted osteoclast formation independently of extracellular NaHCO3 in a short-term culture. Protons and bicarbonate ions acted on osteoclast differentiation with opposite effects, the former positively and the latter negatively. The HEPES-based system maintained pH in the absence of extracellular NaHCO3 without CO2. Therefore, we could demonstrate that osteoblast differentiation was promoted at higher pH in a long-term culture system without NaHCO3 in which ALP activity and nodule mineralization were enhanced. This finding indicates that protons negatively control osteoblast differentiation independently of extracellular bicarbonate ions. However, the difference in the concentration of NaHCO3 did not have any influence on nodule mineralization. The opposite effects of protons, the promotion of osteoclast formation and the inhibition of osteoblast differentiation, were suppressed in the presence of 5 mM N-acetyl cysteine, a reagent activating the scavenging of reactive oxygen species (ROS), implying that ROS act on both systems, the promotion of large osteoclast formation and the deterioration of osteoblast formation under acidosis.

  15. Next Generation Active Buffet Suppression System

    Science.gov (United States)

    Galea, Stephen C.; Ryall, Thomas G.; Henderson, Douglas A.; Moses, Robert W.; White, Edward V.; Zimcik, David G.

    2003-01-01

    Buffeting is an aeroelastic phenomenon that is common to high performance aircraft, especially those with twin vertical tails like the F/A-18, at high angles of attack. These loads result in significant random stresses, which may cause fatigue damage leading to restricted capabilities and availability of the aircraft. This paper describes an international collaborative research activity among Australia, Canada and the United States involving the use of active structural control to alleviate the damaging structural response to these loads. The research program is being co-ordinated by the Air Force Research Laboratory (AFRL) and is being conducted under the auspices of The Technical Cooperative Program (TTCP). This truly unique collaborative program has been developed to enable each participating country to contribute resources toward a program that coalesces a broad range of technical knowledge and expertise into a single investigation. This collaborative program is directed toward a full-scale test of an F/A-18 empennage, which is an extension of an earlier initial test. The current program aims at applying advanced directional piezoactuators, the aircraft rudder, switch mode amplifiers and advanced control strategies on a full-scale structure to demonstrate the enhanced performance and capability of the advanced active BLA control system in preparation for a flight test demonstration.

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

    Directory of Open Access Journals (Sweden)

    Frank Zach

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

  17. Vitamin E decreases bone mass by stimulating osteoclast fusion.

    Science.gov (United States)

    Fujita, Koji; Iwasaki, Makiko; Ochi, Hiroki; Fukuda, Toru; Ma, Chengshan; Miyamoto, Takeshi; Takitani, Kimitaka; Negishi-Koga, Takako; Sunamura, Satoko; Kodama, Tatsuhiko; Takayanagi, Hiroshi; Tamai, Hiroshi; Kato, Shigeaki; Arai, Hiroyuki; Shinomiya, Kenichi; Itoh, Hiroshi; Okawa, Atsushi; Takeda, Shu

    2012-03-04

    Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells that are formed by mononuclear preosteoclast fusion. Fat-soluble vitamins such as vitamin D are pivotal in maintaining skeletal integrity. However, the role of vitamin E in bone remodeling is unknown. Here, we show that mice deficient in α-tocopherol transfer protein (Ttpa(-/-) mice), a mouse model of genetic vitamin E deficiency, have high bone mass as a result of a decrease in bone resorption. Cell-based assays indicated that α-tocopherol stimulated osteoclast fusion, independent of its antioxidant capacity, by inducing the expression of dendritic-cell-specific transmembrane protein, an essential molecule for osteoclast fusion, through activation of mitogen-activated protein kinase 14 (p38) and microphthalmia-associated transcription factor, as well as its direct recruitment to the Tm7sf4 (a gene encoding DC-STAMP) promoter. Indeed, the bone abnormality seen in Ttpa(-/-) mice was rescued by a Tm7sf4 transgene. Moreover, wild-type mice or rats fed an α-tocopherol-supplemented diet, which contains a comparable amount of α-tocopherol to supplements consumed by many people, lost bone mass. These results show that serum vitamin E is a determinant of bone mass through its regulation of osteoclast fusion.

  18. Binding of PICK1 PDZ domain with calcineurin B regulates osteoclast differentiation.

    Science.gov (United States)

    Kamano, Yuya; Watanabe, Jun; Iida, Tsutomu; Kondo, Takeru; Okawa, Hiroko; Yatani, Hirofumi; Saeki, Makio; Egusa, Hiroshi

    2018-01-29

    The calcineurin/nuclear factor of activated T cell (NFAT) signaling pathway plays a major role in osteoclast differentiation; however, the proteins that react with the calcineurin-NFAT complex in osteoclasts to regulate osteoclastogenesis remain unclear. Here, we present evidence that PICK1 also positively regulates calcineurin B in osteoclasts to activate NFAT to promote osteoclastogenesis. mRNA and protein expression of PICK1 in murine primary bone marrow macrophages (BMMs) was significantly increased during RANKL-induced osteoclast differentiation. The interaction of PICK1 with calcineurin B in BMMs was confirmed by co-immunoprecipitation. An inhibitor of the PICK1 PDZ domain significantly decreased osteoclastogenesis marker gene expression and the number of TRAP-positive multinucleated cells among RAW264.7 osteoclast progenitor cells. Overexpression of PICK1 in RAW264.7 cells significantly increased the number of TRAP-positive mature osteoclasts. Increased NFAT activation with transcriptional activation of PICK1 during RAW264.7 osteoclastogenesis was also confirmed in a tetracycline-controlled PICK1 expression system. These results suggest that the PDZ domain of PICK1 directly interacts with calcineurin B in osteoclast progenitor cells and promotes osteoclast differentiation through activation of calcineurin-NFAT signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Regulation of apoptosis in osteoclasts and osteoblastic cells

    International Nuclear Information System (INIS)

    Xing Lianping; Boyce, Brendan F.

    2005-01-01

    In postnatal life, the skeleton undergoes continuous remodeling in which osteoclasts resorb aged or damaged bone, leaving space for osteoblasts to make new bone. The balance of proliferation, differentiation, and apoptosis of bone cells determines the size of osteoclast or osteoblast populations at any given time. Bone cells constantly receive signals from adjacent cells, hormones, and bone matrix that regulate their proliferation, activity, and survival. Thus, the amount of bone and its microarchitecture before and after the menopause or following therapeutic intervention with drugs, such as sex hormones, glucocorticoids, parathyroid hormone, and bisphosphonates, is determined in part by effects of these on survival of osteoclasts, osteoblasts, and osteocytes. Understanding the mechanisms and regulation of bone cell apoptosis will enhance our knowledge of bone cell function and help us to develop better therapeutics for the management of osteoporosis and other bone diseases

  20. Kinetics of the osteoclast cytoskeleton during the resorption cycle in vitro.

    Science.gov (United States)

    Lakkakorpi, P T; Väänänen, H K

    1991-08-01

    Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and F-actin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With alpha-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.

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

    Directory of Open Access Journals (Sweden)

    Tae-Ho Kim

    2016-01-01

    Full Text Available 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.

  2. Active Suppression of Rotating Stall Inception with Distributed Jet Actuation

    Directory of Open Access Journals (Sweden)

    Huu Duc Vo

    2007-01-01

    Full Text Available An analytical and experimental investigation of the effectiveness of full-span distributed jet actuation for active suppression of long length-scale rotating stall inception is carried out. Detailed modeling and experimental verification highlight the important effects of mass addition, discrete injectors, and feedback dynamics, which may be overlooked in preliminary theoretical studies of active control with jet injection. A model of the compression system incorporating nonideal injection and feedback dynamics is verified with forced response measurements to predict the right trends in the movement of the critical pole associated with the stall precursor. Active control experiments with proportional feedback control show that the predicted stall precursors are suppressed to give a 5.5% range extension in compressor flow coefficient. In addition, results suggest that the proposed model could be used to design a more sophisticated controller to further improve performance while reducing actuator bandwidth requirements.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    International Nuclear Information System (INIS)

    Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae

    2007-01-01

    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-κB 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

  5. Adaptive Active Noise Suppression Using Multiple Model Switching Strategy

    Directory of Open Access Journals (Sweden)

    Quanzhen Huang

    2017-01-01

    Full Text Available Active noise suppression for applications where the system response varies with time is a difficult problem. The computation burden for the existing control algorithms with online identification is heavy and easy to cause control system instability. A new active noise control algorithm is proposed in this paper by employing multiple model switching strategy for secondary path varying. The computation is significantly reduced. Firstly, a noise control system modeling method is proposed for duct-like applications. Then a multiple model adaptive control algorithm is proposed with a new multiple model switching strategy based on filter-u least mean square (FULMS algorithm. Finally, the proposed algorithm was implemented on Texas Instruments digital signal processor (DSP TMS320F28335 and real time experiments were done to test the proposed algorithm and FULMS algorithm with online identification. Experimental verification tests show that the proposed algorithm is effective with good noise suppression performance.

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

    Directory of Open Access Journals (Sweden)

    Young Woo Kim

    2014-10-01

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

  7. Global suppression of electrocortical activity in unilateral perinatal thalamic stroke.

    LENUS (Irish Health Repository)

    Kharoshankaya, Liudmila

    2014-07-01

    We present an unusual case of persistent generalized electroencephalography (EEG) suppression and right-sided clonic seizures in a male infant born at 40(+2) weeks\\' gestation, birthweight 3240g, with an isolated unilateral thalamic stroke. The EEG at 13 hours after birth showed a generalized very low amplitude background pattern, which progressed to frequent electrographic seizures over the left hemisphere. The interictal background EEG pattern remained grossly abnormal over the next 48 hours, showing very low background amplitudes (<10μV). Magnetic resonance imaging revealed an isolated acute left-sided thalamic infarction. This is the first description of severe global EEG suppression caused by an isolated unilateral thalamic stroke and supports the role of the thalamus as the control centre for cortical electrical activity.

  8. Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model.

    Science.gov (United States)

    Yu, Tingsheng; Witten, Paul Eckhard; Huysseune, Ann; Buettner, Anita; To, Thuy Thanh; Winkler, Christoph

    2016-02-01

    Osteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl) under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs. © 2016. Published by The Company of Biologists Ltd.

  9. Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model

    Directory of Open Access Journals (Sweden)

    Tingsheng Yu

    2016-02-01

    Full Text Available Osteoclasts are bone-resorbing cells derived from the monocyte/macrophage lineage. Excess osteoclast activity leads to reduced bone mineral density, a hallmark of diseases such as osteoporosis. Processes that regulate osteoclast activity are therefore targeted in current osteoporosis therapies. To identify and characterize drugs for treatment of bone diseases, suitable in vivo models are needed to complement cell-culture assays. We have previously reported transgenic medaka lines expressing the osteoclast-inducing factor receptor activator of nuclear factor κB ligand (Rankl under control of a heat shock-inducible promoter. Forced Rankl expression resulted in ectopic osteoclast formation, as visualized by live imaging in fluorescent reporter lines. This led to increased bone resorption and a dramatic reduction of mineralized matrix similar to the situation in humans with osteoporosis. In an attempt to establish the medaka as an in vivo model for osteoporosis drug screening, we treated Rankl-expressing larvae with etidronate and alendronate, two bisphosphonates commonly used in human osteoporosis therapy. Using live imaging, we observed an efficient, dose-dependent inhibition of osteoclast activity, which resulted in the maintenance of bone integrity despite an excess of osteoclast formation. Strikingly, we also found that bone recovery was efficiently promoted after inhibition of osteoclast activity and that osteoblast distribution was altered, suggesting effects on osteoblast-osteoclast coupling. Our data show that transgenic medaka lines are suitable in vivo models for the characterization of antiresorptive or bone-anabolic compounds by live imaging and for screening of novel osteoporosis drugs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nishioku, Tsuyoshi, E-mail: nishiokut@niu.ac.jp [Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298 (Japan); Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi [Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan)

    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.

  11. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

    International Nuclear Information System (INIS)

    Islam, Shamima; Hassan, Ferdaus; Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Koide, Naoki; Naiki, Yoshikazu; Mori, Isamu; Yoshida, Tomoaki; Yokochi, Takashi

    2007-01-01

    Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-α antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL). TNF-α might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-κB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed

  12. Bone degradation machinery of osteoclasts: An HIV-1 target that contributes to bone loss.

    Science.gov (United States)

    Raynaud-Messina, Brigitte; Bracq, Lucie; Dupont, Maeva; Souriant, Shanti; Usmani, Shariq M; Proag, Amsha; Pingris, Karine; Soldan, Vanessa; Thibault, Christophe; Capilla, Florence; Al Saati, Talal; Gennero, Isabelle; Jurdic, Pierre; Jolicoeur, Paul; Davignon, Jean-Luc; Mempel, Thorsten R; Benichou, Serge; Maridonneau-Parini, Isabelle; Vérollet, Christel

    2018-03-13

    Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.

  13. Osteoclastic finger arthrosis - a subtype of polyarthrosis of the hand

    International Nuclear Information System (INIS)

    Dihlmann, W.; Dihlmann, A.

    1998-01-01

    Aim: Description of a subtype of arthrosis deformans of the hand which is characterised as osteoclastic arthrosis. Patients and methods: Retrospective analysis of radiographs of the hands of 150 women and 100 men with radiological findings of arthrosis deformans. Results: 5% of women and 2% of men showed at least one digital joint with subchondral osteolysis of one or both articulating bones involving at least a third of the phalanx. This subchondral osteolysis far exceeds the cysts which are situated in the epiphyseal part of the articular region. It may develop within a year. Conclusion: Osteoclastic arthrosis of the finger is a subtype of polyarthrosis of the hand. Serial observations suggest that an osteoclast stimulating substance is produced by the cysts or arises directly from the synovial fluid; this enters the subchondral part of the bone through clefts which may or may not be visible radiologically and that this produces osteoclastic activity. The most important differential diagnoses are chronic tophacious gout and a benign tumor. (orig.) [de

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

    Science.gov (United States)

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Cheon, Yoon-Hee [Center for Metabolic Function Regulation, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Baek, Jong Min; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); So, Hong-Seob, E-mail: jeanso@wku.ac.kr [Center for Metabolic Function Regulation, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Department of Anatomy, School of Medicine, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    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.

  17. Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis

    NARCIS (Netherlands)

    Everts, V.; Aronson, D. C.; Beertsen, W.

    1985-01-01

    Electron microscopic examination of bone biopsies obtained from two patients suffering from pycnodysostosis revealed that osteoclasts contained (sometimes large) cytoplasmic vacuoles filled with bone collagen fibrils. These vacuoles stained positive for acid phosphatase activity, thereby suggesting

  18. Potential of Resveratrol Analogues as Antagonists of Osteoclasts and Promoters of Osteoblasts

    DEFF Research Database (Denmark)

    Kupisiewicz, Katarzyna; Boissy, Patrice; Abdallah, Basem M

    2010-01-01

    The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations...

  19. Optimal Active Vibration Suppression of Smart Composite Wind Turbine Blades

    Science.gov (United States)

    Abd El-Maksoud Mohamed, Sherif Ibrahim

    The purpose of this study is to apply active vibration control technique numerically for suppressing the vibrational level of a horizontal axis wind turbine blade. Two systems are studied to apply active vibration control on the wind turbine blade model, the first is a uniform cantilever beam and the other system is a non-uniform (tapered) cantilever beam. A single piezoelectric actuator and sensor are bonded on the upper and lower surface of the systems, respectively. The vibration analysis and dynamic characteristics of smart systems are obtained using approximate analytical methods. The entire structure is modeled in the state space form using the state space method, generalized coordinates and piezoelectric theory. Two types of controllers are designed to study the performance of the piezoelectric active controller. The first is a Proportional-Derivative (PD) controller and the other type is a Linear Quadratic Regulator (LQR). The Linear Quadratic Regulator (LQR) demonstrates better results for vibration suppression. The MATLAB code Simulink is used to simulate the different cases.

  20. Apamin suppresses biliary fibrosis and activation of hepatic stellate cells.

    Science.gov (United States)

    Kim, Jung-Yeon; An, Hyun-Jin; Kim, Woon-Hae; Park, Yoon-Yub; Park, Kyung Duck; Park, Kwan-Kyu

    2017-05-01

    Cholestatic liver disease is characterized by the progressive destruction of biliary epithelial cells (BECs) followed by fibrosis, cirrhosis and liver failure. Activated hepatic stellate cells (HSCs) and portal fibroblasts are the major cellular effectors of enhanced collagen deposition in biliary fibrosis. Apamin, an 18 amino acid peptide neurotoxin found in apitoxin (bee venom), is known to block Ca2+-activated K+ channels and prevent carbon tetrachloride-induced liver fibrosis. In the present study, we aimed to ascertain whether apamin inhibits biliary fibrosis and the proliferation of HSCs. Cholestatic liver fibrosis was established in mouse models with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Cellular assays were performed on HSC-T6 cells (rat immortalized HSCs). DDC feeding led to increased hepatic damage and proinflammtory cytokine levels. Notably, apamin treatment resulted in decreased liver injury and proinflammatory cytokine levels. Moreover, apamin suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice. In HSCs, apamin suppressed activation of HSCs by inhibiting the Smad signaling pathway. These data suggest that apamin may be a potential therapeutic target in cholestatic liver disease.

  1. Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function

    International Nuclear Information System (INIS)

    Hattersley, G.; Chambers, T.J.

    1991-01-01

    Transforming growth factor (TGF) beta 1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-beta 1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-beta 1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-beta 1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-beta 1 in osteoclast generation

  2. Osteoclast cell-surface specializations and nuclear kinetics during egg-laying in Japanese quail

    International Nuclear Information System (INIS)

    Miller, S.C.

    1981-01-01

    Medullary bone deposits serve as a reservoir of labile calcium for egg-shell calcification in birds. Quantitative transmission-electron-microscope methods and light-microscope autoradiographic cell-population-kinetic analyses were used to determine changes in cell-surface specializations and population dynamics of medullary bone osteoclasts during egg-laying in Japanese quail. Prior to egg-shell formation, from 0 to about 8 hours after the previous oviposition, very few osteoclast profiles had ruffled borders. The appearance of ruffled borders coincided with the beginning of egg-shell calcification, about 9-10 hours after the previous oviposition. During egg-shell calcification, about 10-21 hours after the previous oviposition, most osteoclast profiles had ruffled borders. Ruffled borders disappeared at the completion of egg-shell calcification and commencement of egg-shell pigmentation. Thus, functional activities of medullary bone osteoclasts appear to be closely synchronized with egg-shell calcification during egg-laying. From 1 to 48 hours after a single injection of 3H-thymidine (3H-TdR), very few labeled osteoclast nuclei were seen during egg-laying. Following multiple injections of 3H-TdR, the percentage of labeled nuclei reached a peak at about 170 hours after the first injection. At this peak-labeling time, relatively few of the osteoclast profiles that had labeled nuclei had two or more; although the average number of nuclei per osteoclast profile was about 3.6. These kinetic data suggest that the medullary bone osteoclast population has a prolonged rate of turnover compared to rapid changes in cell activities associated with each 24-hour egg-laying cycle; and collectively they would suggest that rapid changes in osteoclast functions occur independently of changes in cell-population dynamics

  3. [Suppression of cycling activity in sheep using parenteral progestagen treatment].

    Science.gov (United States)

    Janett, F; Camponovo, L; Lanker, U; Hässig, M; Thun, R

    2004-03-01

    The objective of this study was to evaluate the effect of two synthetic progestagen preparations Chlormadinone acetate (CAP, Chronosyn, Veterinaria AG Zürich) and Medroxyprogesterone acetate (MPA, Nadigest, G Streuli & Co. Uznach) on cycling activity and fertility in sheep. A flock of 28 non pregnant white alpine sheep was randomly divided into three groups, A (n = 10), B (n = 9) and C (n = 9). During a period of 4 weeks the cycling activity was confirmed by blood progesterone analysis. Thereafter, the animals of group A were treated with 50 mg CAP, those of group B with 140 mg MPA and those of group C with physiological saline solution. All injections were given intramuscularly. Suppression of endogenous progesterone secretion lasted from 28 to 49 days (mean = 39 days) in group A and from 42 to 70 days (mean = 50 days) in group B. The synchronization effect of both preparations was unsatisfactory as the occurrence of first estrus was distributed over a period of 3 weeks in group A and 4 weeks in group B. These findings could also be confirmed by the lambing period which lasted 52 days in group A and 36 days in group B. Control animals lambed within 9 days due to the synchronizing effect of the ram. The first fertile estrus was observed 36 days (group A) and 45 days (group B) after the treatment. In group A all 10 animals and in groups B and C 8 of 9 ewes each became pregnant. Parenteral progestagen application with CAP and MPA is a simple, safe and reversible method of estrus suppression in the sheep. The minimal suppressive duration of 4 (CAP) and 5 weeks (MPA) is not sufficient when a period of 3 months (alpine pasture period) is desired.

  4. NF-κB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

    Science.gov (United States)

    Kubota, Tetsuo; Hoshino, Machiko; Aoki, Kazuhiro; Ohya, Keiichi; Komano, Yukiko; Nanki, Toshihiro; Miyasaka, Nobuyuki; Umezawa, Kazuo

    2007-01-01

    Inhibition of NF-κB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-κB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-κB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-κB inhibitors to rheumatoid arthritis therapy. PMID

  5. TNF-α contributes to postmenopausal osteoporosis by synergistically promoting RANKL-induced osteoclast formation.

    Science.gov (United States)

    Zha, Li; He, Li; Liang, Yijian; Qin, Hui; Yu, Bin; Chang, Linli; Xue, Li

    2018-03-20

    Previous studies showed that inflammatory cytokines promote osteoclast formation, characterized by the function of bone resorption. However, it remains unclear whether inflammatory factors contribute to osteoporosis syndrome in postmenopausal women. Here, we found that postmenopausal women with osteoporosis (PO) had increased levels of TNF-α, compared with those without osteoporosis. TNF-α is highly correlated with the RANK and estrogen levels in PO patients. in vitro, TNF-α synergistically promotes RANKL-induced osteoclast formation by activation of NF-κB and PI3K/Akt signaling. Moreover, inhibition of PI3K/Akt totally blocked the synergistic effects of TNF-α on NF-κB activation as well as osteoclast formation. Together, these results demonstrate that TNF-α synergistically promotes RANKL-induced osteoclasts formation through activation of PI3K/Akt signaling, which ultimately contributes to osteoporosis syndrome in postmenopausal women. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  6. Cyanidin Chloride inhibits ovariectomy-induced osteoporosis by suppressing RANKL-mediated osteoclastogenesis and associated signaling pathways.

    Science.gov (United States)

    Cheng, Jianwen; Zhou, Lin; Liu, Qian; Tickner, Jennifer; Tan, Zhen; Li, Xiaofeng; Liu, Mei; Lin, Xixi; Wang, Tao; Pavlos, Nathan J; Zhao, Jinmin; Xu, Jiake

    2018-03-01

    Over-production and activation of osteoclasts is a common feature of osteolytic conditions such as osteoporosis, tumor-associated osteolysis, and inflammatory bone erosion. Cyanidin Chloride, a subclass of anthocyanin, displays antioxidant and anti-carcinogenesis properties, but its role in osteoclastic bone resorption and osteoporosis is not well understood. In this study, we showed that Cyanidin Chloride inhibits osteoclast formation, hydroxyapatite resorption, and receptor activator of NF-κB ligand (RANKL)-induced osteoclast marker gene expression; including ctr, ctsk, and trap. Further investigation revealed that Cyanidin Chloride inhibits RANKL-induced NF-κB activation, suppresses the degradation of IκB-α and attenuates the phosphorylation of extracellular signal-regulated kinases (ERK). In addition, Cyanidin Chloride abrogated RANKL-induced calcium oscillations, the activation of nuclear factor of activated T cells calcineurin-dependent 1 (NFATc1), and the expression of c-Fos. Further, we showed that Cyanidin Chloride protects against ovariectomy-induced bone loss in vivo. Together our findings suggest that Cyanidin Chloride is capable of inhibiting osteoclast formation, hydroxyapatite resorption and RANKL-induced signal pathways in vitro and OVX-induced bone loss in vivo, and thus might have therapeutic potential for osteolytic diseases. © 2017 Wiley Periodicals, Inc.

  7. TRAF6 Mediates Suppression of Osteoclastogenesis and Prevention of Ovariectomy-Induced Bone Loss by a Novel Prenylflavonoid.

    Science.gov (United States)

    Tan, Ee Min; Li, Lei; Indran, Inthrani Raja; Chew, Nicholas; Yong, Eu-Leong

    2017-04-01

    Given the limitations of current therapeutic options for postmenopausal osteoporosis, there is a need for alternatives with minimal adverse effects. In this study, we evaluated the effects of icaritin (ICT), a natural prenylflavonoid, on osteoclastogenesis both in vitro and in an ovariectomized (OVX) rat model and investigated its underlying molecular mechanism(s) of action. ICT inhibited osteoclast formation in two osteoclast precursor models, RAW 264.7 mouse monocyte cell line and human PBMC. ICT also inhibited sealing zone and resorption pit formation in a dose-dependent manner. Mechanistically, ICT inhibited RANKL-induced NF-κB and MAPK/AP-1 pathways to suppress gene expression of nuclear factor of activated T cells (NFAT)c1, the master transcription regulator of osteoclast differentiation. ICT, by inhibiting the TRAF6/c-Src/PI3K pathway, suppressed NADPH oxidase-1 activation to attenuate intracellular ROS production and downregulate calcineurin phosphatase activity. As a result, NFATc1 nuclear translocation and activity was suppressed. Crucially, ICT promoted proteasomal degradation of TRAF6, the critical adaptor protein that transduces RANKL/RANK signaling, and the inhibitory effect of ICT on osteoclastogenesis was reversed by the proteasomal inhibitor MG 132. ICT administration inhibited OVX-induced bone loss and resorption by suppressing osteoclast formation and activity. Consistent with cellular studies, ICT downregulated TRAF6 and NFATc1 protein expression in CD11b + /Gr-1 -/low osteoclast precursors isolated from OVX rats. Put together, we present novel findings that ICT, by downregulating TRAF6, coordinates inhibition of NF-κB, MAPK/AP-1, and ROS signaling pathways to reduce expression and activity of NFATc1. These results demonstrate the potential of ICT for treatment of postmenopausal osteoporosis and point to TRAF6 as a promising target for novel anti-osteoporotic drugs. © 2017 American Society for Bone and Mineral Research. © 2017 American

  8. A pterostilbene derivative suppresses osteoclastogenesis by regulating RANKL-mediated NFκB and MAPK signaling in RAW264.7 cells.

    Science.gov (United States)

    Nikhil, Kumar; Sharan, Shruti; Roy, Partha

    2015-12-01

    A dysfunctional osteoclast activity is often the cause of bone destructive diseases, such as osteoporosis, periodontitis, erosive arthritis, and cancer. The NFκB ligand (RANKL) has been identified as a major mediator of bone resorption. Agents that suppress RANKL signaling have the potential to inhibit bone resorption or osteoclastogenesis. The present study aimed to determine the effect of a pterostilbene derivative (PTERC-T) for suppressing RANKL or tumor cells-induced osteoclastogenesis in RAW264.7 murine macrophages. Cytotoxicity was measured by MTT assay and inhibitory effect on osteoclastogenesis was analyzed by counting the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and measuring the expression levels of the osteoclast-specific genes. The reactive oxygen species (ROS) generation was detected by FACS. Further, signaling pathways were analyzed by immunofluorescence and immunoblot analyses. PTERC-T suppressed the differentiation of monocytes to osteoclasts in a dose and time-dependent manner. The expression of osteoclast marker genes like TRAP, cathepsin K (CTSK), matrix metalloproteinase 9 (MMP9) and transcription factors c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were also diminished by PTERC-T. PTERC-T scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. Mechanistically, PTERC-T abrogated the phosphorylation of MAPKs (ERK and JNK) and inhibited RANKL-induced activation of NFκB by suppressing IκBα phosphorylation and preventing NFκB/p65 nuclear translocation. This study thus identifies PTERC-T as an inhibitor of osteoclast formation and provides evidence for its role in preventing osteoporosis and other bone related disorders. However, further studies are needed to establish its efficacy in vivo. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights

  9. Naringin abrogates osteoclastogenesis and bone resorption via the inhibition of RANKL-induced NF-κB and ERK activation.

    Science.gov (United States)

    Ang, Estabelle S M; Yang, Xiaohong; Chen, Honghui; Liu, Qian; Zheng, Ming H; Xu, Jiake

    2011-09-02

    Osteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid-induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK-mediated NF-κB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis. Copyright © 2011. Published by Elsevier B.V.

  10. Zebrafish scales respond differently to in vitro dynamic and static acceleration: analysis of interaction between osteoblasts and osteoclasts.

    Science.gov (United States)

    Kitamura, Kei-ichiro; Takahira, Koh; Inari, Masato; Satoh, Yusuke; Hayakawa, Kazuichi; Tabuchi, Yoshiaki; Ogai, Kazuhiro; Nishiuchi, Takumi; Kondo, Takashi; Mikuni-Takagaki, Yuko; Chen, Wenxi; Hattori, Atsuhiko; Suzuki, Nobuo

    2013-09-01

    Zebrafish scales consist of bone-forming osteoblasts, bone-resorbing osteoclasts, and calcified bone matrix. To elucidate the underlying molecular mechanism of the effects induced by dynamic and static acceleration, we investigated the scale osteoblast- and osteoclast-specific marker gene expression involving osteoblast-osteoclast communication molecules. Osteoblasts express RANKL, which binds to the osteoclast surface receptor, RANK, and stimulates bone resorption. OPG, on the other hand, is secreted by osteoblast as a decoy receptor for RANKL, prevents RANKL from binding to RANK and thus prevents bone resorption. Therefore, the RANK-RANKL-OPG pathway contributes to the regulation of osteoclastogenesis by osteoblasts. Semaphorin 4D, in contrast, is expressed on osteoclasts, and binding to its receptor Plexin-B1 on osteoblasts results in suppression of bone formation. In the present study, we found that both dynamic and static acceleration at 3.0×g decreased RANKL/OPG ratio and increased osteoblast-specific functional mRNA such as alkaline phosphatase, while static acceleration increased and dynamic acceleration decreased osteoclast-specific mRNA such as cathepsin K. Static acceleration increased semaphorin 4D mRNA expression, while dynamic acceleration had no effect. The results of the present study indicated that osteoclasts have predominant control over bone metabolism via semaphorin 4D expression induced by static acceleration at 3.0×g. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    OpenAIRE

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

    2013-01-01

    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-spe...

  12. Immune-suppressive activity of punicalagin via inhibition of NFAT activation

    International Nuclear Information System (INIS)

    Lee, Sang-Ik; Kim, Byoung-Soo; Kim, Kyoung-Shin; Lee, Samkeun; Shin, Kwang-Soo; Lim, Jong-Soon

    2008-01-01

    Since T cell activation is central to the development of autoimmune diseases, we screened a natural product library comprising 1400 samples of medicinal herbal extracts, to identify compounds that suppress T cell activity. Punicalagin (PCG) isolated from the fruit of Punica granatum was identified as a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated T cells (NFAT). PCG downregulated the mRNA and soluble protein expression of interleukin-2 from anti-CD3/anti-CD28-stimulated murine splenic CD4+ T cells and suppressed mixed leukocytes reaction (MLR) without exhibiting cytotoxicity to the cells. In vivo, the PCG treatment inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice and decreased CD3+ T cell infiltration of the inflamed tissue. These results suggest that PCG could be a potential candidate for the therapeutics of various immune pathologies

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

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Huixian [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Shi, Zhenqi [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Qiao, Ping [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Department of Pharmacology, Norman Bethune Medical College, Jilin University, Changchun, Jilin 130021 (China); Li, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); McCoy, Erin M. [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Mao, Ping [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Xu, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Feng, Xu [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Shunqing, E-mail: shqwang_cn@yahoo.com [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China)

    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

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

    Science.gov (United States)

    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

  15. Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts

    DEFF Research Database (Denmark)

    Sørensen, M G; Henriksen, K; Dziegiel, Morten Hanefeld

    2006-01-01

    + monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol...... inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did...

  16. The elementary fusion modalities of osteoclasts

    DEFF Research Database (Denmark)

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

    2015-01-01

    The last step of the osteoclast differentiation process is cell fusion. Most efforts to understand the fusion mechanism have focused on the identification of molecules involved in the fusion process. Surprisingly, the basic fusion modalities, which are well known for fusion of other cell types......, are not known for the osteoclast. Here we show that osteoclast fusion partners are characterized by differences in mobility, nuclearity, and differentiation level. Our demonstration was based on time-laps videos of human osteoclast preparations from three donors where 656 fusion events were analyzed. Fusions...... between a mobile and an immobile partner were most frequent (62%), while fusion between two mobile (26%) or two immobile partners (12%) was less frequent (p fusion partner contained more nuclei than the mobile one (p

  17. Covertly active and progressing neurochemical abnormalities in suppressed HIV infection.

    Science.gov (United States)

    Cysique, Lucette A; Jugé, Lauriane; Gates, Thomas; Tobia, Michael; Moffat, Kirsten; Brew, Bruce J; Rae, Caroline

    2018-01-01

    To assess whether HIV-related brain injury is progressive in persons with suppressed HIV infection. Seventy-three HIV+ virally suppressed men and 35 HIV- men, screened for psychiatric and alcohol/drug use disorders, underwent neuropsychological evaluation and proton magnetic resonance spectroscopy ( 1 H-MRS) at baseline and after and 23 ± 5 months. 1 H-MRS included brain regions known to be vulnerable to HIV and aging: frontal white matter (FWM), posterior cingulate cortex (PCC), and caudate area (CA). Major brain metabolites such as creatine (Cr: marker of cellular energy), N -acetyl aspartate (NAA: marker of neuronal integrity), choline (marker of cellular membrane turnover), glutamate/glutamine (excitatory/inhibitory neurotransmitter), and myo -Inositol (mI: marker of neuroinflammation) were calculated with reference to water signal. Neurocognitive decline was corrected for practice effect and baseline HIV-associated neurocognitive disorder (HAND) status. Across the study period, 44% had intact cognition, 42% stable HAND (including the single case that improved), 10% progressing HAND, and 4% incident HAND. When analyzing the neurochemical data per neurocognitive trajectories, we found decreasing PCC Cr in all subgroups compared with controls ( p < 0.002). In addition, relative to the HIV- group, stable HAND showed decreasing FWM Cr, incident HAND showed steep FWM Cr reduction, whereas progressing HAND had a sharply decreasing PCC NAA and reduced but stable CA NAA. When analyzing the neurochemical data at the group level (HIV+ vs HIV- groups), we found stable abnormal metabolite concentrations over the study period: decreased FWM and PCC Cr (both p < 0.001), decreased PCC NAA and CA NAA (both p < 0.05) and PCC mI increase ( p < 0.05). HIV duration and historical HAND had modest effects on metabolite changes. Our study reveals covertly active or progressing HIV-related brain injury in the majority of this virally suppressed cohort, reflecting ongoing

  18. Covertly active and progressing neurochemical abnormalities in suppressed HIV infection

    Science.gov (United States)

    Jugé, Lauriane; Gates, Thomas; Tobia, Michael; Moffat, Kirsten; Brew, Bruce J.; Rae, Caroline

    2018-01-01

    Objective To assess whether HIV-related brain injury is progressive in persons with suppressed HIV infection. Methods Seventy-three HIV+ virally suppressed men and 35 HIV− men, screened for psychiatric and alcohol/drug use disorders, underwent neuropsychological evaluation and proton magnetic resonance spectroscopy (1H-MRS) at baseline and after and 23 ± 5 months. 1H-MRS included brain regions known to be vulnerable to HIV and aging: frontal white matter (FWM), posterior cingulate cortex (PCC), and caudate area (CA). Major brain metabolites such as creatine (Cr: marker of cellular energy), N-acetyl aspartate (NAA: marker of neuronal integrity), choline (marker of cellular membrane turnover), glutamate/glutamine (excitatory/inhibitory neurotransmitter), and myo-Inositol (mI: marker of neuroinflammation) were calculated with reference to water signal. Neurocognitive decline was corrected for practice effect and baseline HIV-associated neurocognitive disorder (HAND) status. Results Across the study period, 44% had intact cognition, 42% stable HAND (including the single case that improved), 10% progressing HAND, and 4% incident HAND. When analyzing the neurochemical data per neurocognitive trajectories, we found decreasing PCC Cr in all subgroups compared with controls (p < 0.002). In addition, relative to the HIV− group, stable HAND showed decreasing FWM Cr, incident HAND showed steep FWM Cr reduction, whereas progressing HAND had a sharply decreasing PCC NAA and reduced but stable CA NAA. When analyzing the neurochemical data at the group level (HIV+ vs HIV− groups), we found stable abnormal metabolite concentrations over the study period: decreased FWM and PCC Cr (both p < 0.001), decreased PCC NAA and CA NAA (both p < 0.05) and PCC mI increase (p < 0.05). HIV duration and historical HAND had modest effects on metabolite changes. Conclusions Our study reveals covertly active or progressing HIV-related brain injury in the majority of this virally suppressed

  19. c-Fms signaling mediates neurofibromatosis Type-1 osteoclast gain-in-functions.

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    Yongzheng He

    Full Text Available Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1 patients. NF1 (Nf1 haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1(+/- mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21(Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF. Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1(+/ (- osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1(+/- osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1(+/- osteoporotic mice induced by ovariectomy (OVX and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1(+/--OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.

  20. A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

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    Amy B. Howell

    2011-03-01

    Full Text Available This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP staining, while the secretion of interleukin-8 (IL-8 and matrix metalloproteinases (MMPs was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

  1. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    International Nuclear Information System (INIS)

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

    2006-01-01

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity

  2. Cell-surface phosphatidylserine regulates osteoclast precursor fusion.

    Science.gov (United States)

    Verma, Santosh K; Leikina, Evgenia; Melikov, Kamran; Gebert, Claudia; Kram, Vardit; Young, Marian F; Uygur, Berna; Chernomordik, Leonid V

    2018-01-05

    Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in Caenorhabditis elegans , the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.

  3. Active Suppression of Narrowband Noise by Multiple Secondary Sources

    Directory of Open Access Journals (Sweden)

    Seokhoon Ryu

    2016-01-01

    Full Text Available This study presents theoretical and experimental investigation on the active suppression of narrowband noise with C1, C1.5, and C2 components by using multichannel secondary sources in a duct. The quality manipulation in the duct was controlled by changing quality factors which were incorporated into a multichannel FxLMS algorithm. The algorithm is extensively investigated in both theory and real-time control experiment. After analysing the primary and secondary paths of the duct system, an acoustic narrowband signal was chosen as a primary noise and the impulse responses were implemented as the secondary path models. Control results show that the quality factors in the algorithm that was implemented in a dSPACE 1104 provide a stable and excellent response compared to before control. It is obvious that the lower quality factor cancels the more primary noise as defined in the theory although the attenuation levels are not exactly and inversely proportional to the quality factor. The results in this study can be used for practical active sound quality control systems.

  4. Reversible suppression of sexual activity in tomcats with deslorelin implant.

    Science.gov (United States)

    Novotny, R; Cizek, P; Vitasek, R; Bartoskova, A; Prinosilova, P; Janosovska, M

    2012-09-01

    The aim of the study was to assess the efficacy of using a Gn-RH agonist implant (deslorelin, 4.7 mg, Suprelorin) to control sexual activity of male cats and reestablishment of sexual function after the implant removal 4 mo after placement. Using a control group (Group 1, n = 5), 22 domestic tomcats were given the implant subcutaneously in the region of the right shoulder blade and were then divided into two treatment groups. Animals in Group 2 (n = 14) were observed from the date of implant surgery and the observation lasted for 4 mo. In Group 3 (n = 8) all animals were monitored from the date of implant surgery. Then, after 4 mo, all implants were removed and the toms were observed for a further 4 mo. In all animals during their first visit and then in 1-mo intervals, changes in testosterone concentrations were assessed before (T0) and 4 h after (T4) human chorionic gonadotropin (HCG) administration and testis size was measured. In all tomcats, semen collection was performed, using an electroejaculator, in the course of the first visit and then in 2-mo intervals or at the end of observation. Total sperm count was determined in each semen sample. Two to four animals were castrated at weeks 4, 8, 12, 16, 20, 24, 28 and 32 and histologic assessment of the testes was performed. By evaluation of 200 cross sections of seminiferous tubules, the degree of spermatogenic suppression was assessed and animals in Groups 2 and 3 were assigned into groups according to most tubules with the most developed germ cell observed: G1, spermatocytes; G2, round spermatids; G3, elongating spermatids and G4, elongated spermatids. The mean area of Leydig-cell nuclei was calculated. In animals in Group 2, suppression after implant insertion was monitored. T4 concentrations, testis size, and total sperm count gradually decreased (P < 0.01; P < 0.01; and P < 0.05, respectively) within 4 mo after implantation. Histologic evaluation showed a high individual variation in the degree of

  5. Symmetrical retrograde actin flow in the actin fusion structure is involved in osteoclast fusion

    Directory of Open Access Journals (Sweden)

    Jiro Takito

    2017-07-01

    Full Text Available The aim of this study was to elucidate the role of the zipper-like structure (ZLS, a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3. Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

  6. The Suppression of Star Formation by Powerful Active Galactic Nuclei

    Science.gov (United States)

    Dwek, E.

    2012-01-01

    The old, red stars that constitute the bulges of galaxies, and the massive black holes at their centres, are the relics of a period in cosmic history when galaxies formed stars at remarkable rates and active galactic nuclei (AGN) shone brightly as a result of accretion onto black holes. It is widely suspected, but unproved, that the tight corre1ation between the mass of the black hole and the mas. of the stellar bulge results from the AGN quenching the surrounding star formation as it approaches its peak luminosity. X-rays trace emission from AGN unambiguously, whereas powerful star-forming ga1axies are usually dust-obscured and are brightest at infrared and submillimeter wavelengths. Here we report submillimetre and X-ray observations that show that rapid star formation was common in the host galaxies of AGN when the Universe was 2-6 billion years old, but that the most vigorous star formation is not observed around black holes above an X-ray luminosity of 10(exp 44) ergs per second. This suppression of star formation in the host galaxy of a powerful AGN is a key prediction of models in which the AGN drives an outflow, expe11ing the interstellar medium of its host and transforming the galaxy's properties in a brief period of cosmic time.

  7. The suppression of star formation by powerful active galactic nuclei.

    Science.gov (United States)

    Page, M J; Symeonidis, M; Vieira, J D; Altieri, B; Amblard, A; Arumugam, V; Aussel, H; Babbedge, T; Blain, A; Bock, J; Boselli, A; Buat, V; Castro-Rodríguez, N; Cava, A; Chanial, P; Clements, D L; Conley, A; Conversi, L; Cooray, A; Dowell, C D; Dubois, E N; Dunlop, J S; Dwek, E; Dye, S; Eales, S; Elbaz, D; Farrah, D; Fox, M; Franceschini, A; Gear, W; Glenn, J; Griffin, M; Halpern, M; Hatziminaoglou, E; Ibar, E; Isaak, K; Ivison, R J; Lagache, G; Levenson, L; Lu, N; Madden, S; Maffei, B; Mainetti, G; Marchetti, L; Nguyen, H T; O'Halloran, B; Oliver, S J; Omont, A; Panuzzo, P; Papageorgiou, A; Pearson, C P; Pérez-Fournon, I; Pohlen, M; Rawlings, J I; Rigopoulou, D; Riguccini, L; Rizzo, D; Rodighiero, G; Roseboom, I G; Rowan-Robinson, M; Sánchez Portal, M; Schulz, B; Scott, D; Seymour, N; Shupe, D L; Smith, A J; Stevens, J A; Trichas, M; Tugwell, K E; Vaccari, M; Valtchanov, I; Viero, M; Vigroux, L; Wang, L; Ward, R; Wright, G; Xu, C K; Zemcov, M

    2012-05-09

    The old, red stars that constitute the bulges of galaxies, and the massive black holes at their centres, are the relics of a period in cosmic history when galaxies formed stars at remarkable rates and active galactic nuclei (AGN) shone brightly as a result of accretion onto black holes. It is widely suspected, but unproved, that the tight correlation between the mass of the black hole and the mass of the stellar bulge results from the AGN quenching the surrounding star formation as it approaches its peak luminosity. X-rays trace emission from AGN unambiguously, whereas powerful star-forming galaxies are usually dust-obscured and are brightest at infrared and submillimetre wavelengths. Here we report submillimetre and X-ray observations that show that rapid star formation was common in the host galaxies of AGN when the Universe was 2-6 billion years old, but that the most vigorous star formation is not observed around black holes above an X-ray luminosity of 10(44) ergs per second. This suppression of star formation in the host galaxy of a powerful AGN is a key prediction of models in which the AGN drives an outflow, expelling the interstellar medium of its host and transforming the galaxy's properties in a brief period of cosmic time.

  8. Aesculin modulates bone metabolism by suppressing receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and transduction signals.

    Science.gov (United States)

    Zhao, Xiao-Li; Chen, Lin-Feng; Wang, Zhen

    2017-06-17

    Aesculin (AES), a coumarin compound derived from Aesculus hippocasanum L, is reported to exert protective role against inflammatory diseases, gastric disease and cancer. However, direct effect of AES in bone metabolism is deficient. In this study, we examined the effects of AES on osteoclast (OC) differentiation in receptor activator of NF-κB ligand (RANKL)-induced RAW264.7 cells. AES inhibits the OC differentiation in both dose- and time-dependent manner within non-toxic concentrations, as analyzed by Tartrate Resistant Acid Phosphatase (TRAP) staining. The actin ring formation manifesting OC function is also decreased by AES. Moreover, expressions of osteoclastogenesis related genes Trap, Atp6v0d2, Cathepsin K and Mmp-9 are decreased upon AES treatment. Mechanistically, AES attenuates the activation of MAPKs and NF-κB activity upon RANKL induction, thus leading to the reduction of Nfatc1 mRNA expression. Moreover, AES inhibits Rank expression, and RANK overexpression markedly decreases AES's effect on OC differentiation and NF-κB activity. Consistently, AES protects against bone mass loss in the ovariectomized and dexamethasone treated rat osteoporosis model. Taken together, our data demonstrate that AES can modulate bone metabolism by suppressing osteoclastogenesis and related transduction signals. AES therefore could be a promising agent for the treatment of osteoporosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Science.gov (United States)

    Pellegrini, Gretel G.; Morales, Cynthya C.; Wallace, Taylor C.; Plotkin, Lilian I.; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  10. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

    Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

  11. Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

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    Maria Arantzazu Sanchez-Fernandez

    Full Text Available BACKGROUND: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. METHODOLOGY/PRINCIPAL FINDING: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK, a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/-LacZ+/- adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

  12. Pu-erh Tea Extract Ameliorates Ovariectomy-Induced Osteoporosis in Rats and Suppresses Osteoclastogenesis In Vitro

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    Titi Liu

    2017-05-01

    Full Text Available Background and Objective: Tea drinking is associated with positive effects on bone health and may protect against osteoporosis, especially in elderly women. Pu-erh tea has many beneficial effects on human health; however, whether Pu-erh tea has anti-osteoporotic potential remains unclear. Thus, we investigated the effects of Pu-erh tea extract (PTE on ovariectomy-induced osteoporosis in rats and on osteoclastogenesis in vitro.Methods: Female Wistar rats were divided into six groups: the sham, model, and Xian-Ling-Gu-Bao capsule (XLGB groups, and the low-, medium-, and high-dose PTE groups. Ovariectomized (OVX rats were used as an animal model of osteoporosis. The animals were intragastrically administered distilled water, XLGB, or different concentrations of PTE for 13 weeks. Body weight, blood biochemical indicators, relative organ coefficients, femoral bone mineral density (BMD, bone biomechanical properties, and bone microarchitecture were examined and analyzed. Additionally, the in vitro effects of PTE on osteoclastic activities were investigated using the RAW 264.7 cell line as an osteoclast differentiation model. The effects of PTE on osteoclast differentiation and the expression of osteoclast-specific genes and proteins were determined.Results: PTE reduced OVX-induced body weight gain after 6 weeks of treatment, and the high-dose exerted a significant effect. High-dose PTE significantly ameliorated OVX-induced estradiol (E2 deficiency. PTE treatment maintained calcium and phosphorus homeostasis and improved other blood biochemical parameters to various degrees. In addition, PTE treatment improved organ coefficients of the femur, uterus, and vagina and improved femoral BMD and bone biomechanical properties. PTE treatment strikingly ameliorated bone microarchitecture. Moreover, in the in vitro studies, osteoclast differentiation using the differentiation cell model was significantly inhibited by PTE without cytotoxic effects. Additionally

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    Highlights: → Effect of compressive force on osteoblasts were examined. → Compressive force induced OPG expression and suppressed osteoclastogenesis. → 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 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-κB 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 IκBα, 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 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-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca 2+ pathway.

  14. Granulocyte colony-stimulating factor enhances bone tumor growth in mice in an osteoclast-dependent manner

    OpenAIRE

    Hirbe, Angela C.; Uluçkan, Özge; Morgan, Elizabeth A.; Eagleton, Mark C.; Prior, Julie L.; Piwnica-Worms, David; Trinkaus, Kathryn; Apicelli, Anthony; Weilbaecher, Katherine

    2007-01-01

    Inhibition of osteoclast (OC) activity has been associated with decreased tumor growth in bone in animal models. Increased recognition of factors that promote osteoclastic bone resorption in cancer patients led us to investigate whether increased OC activation could enhance tumor growth in bone. Granulocyte colony-stimulating factor (G-CSF) is used to treat chemotherapy-induced neutropenia, but is also associated with increased markers of OC activity and decreased bone mineral density (BMD). ...

  15. Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts

    Directory of Open Access Journals (Sweden)

    P. Narducci

    2010-03-01

    Full Text Available Bone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor κB (RANKL and its decoy receptor osteoprotegerin (OPG. Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption. Normally, one resorption cycle takes several hours as observed by monitoring actin ring formation and consequent disappearance in vitro. During these cyclic changes, the cytoskeleton undergoes remarkable dynamic rearrangement. Active cells show a continuous process of exocytosis that plays an essential role in transport of membrane components, soluble molecules and receptor-mediated ligands thus allowing them to communicate with the environment. The processes that govern intracellular transport and trafficking in mature osteoclasts are poorly known. The principal methodological problem that have made these studies difficult is a physiological culture of osteoclasts that permit observing the vesicle apparatus in conditions similar to the in vivo conditions. In the present study we have used a number of morphological approaches to characterize the composition, formation and the endocytic and biosynthetic pathways that play roles in dynamics of differentiation of mature bone resorbing cells using a tri-dimensional system of physiologic coculture.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jie Wang

    Full Text Available 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.

  18. Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-08-01

    Full Text Available Aconitum pseudo-laeve var. erectum (APE has been widely shown in herbal medicine to have a therapeutic effect on inflammatory conditions. However, there has been no evidence on whether the extract of APE is involved in the biological bone metabolism process, particularly osteoclast-mediated bone resorption. In this study, we confirmed that the administration of APE could restore normal skeletal conditions in a murine model of lipopolysaccharide (LPS-induced bone loss via a decrease in the receptor activator of nuclear factor kappa-B ligand (RANKL/osteoprotegerin (OPG ratio and osteoclast number. We then investigated the effect of APE on the RANKL-induced formation and function of osteoclasts to elucidate its underlying molecular mechanisms. APE suppressed the formation of tartrate-resistant acid phosphatase (TRAP-positive cells, as well as the bone-resorbing activity of mature osteoclasts. Furthermore, APE attenuated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1 and c-Fos without affecting any early signal pathway of osteoclastogenesis. Subsequently, APE significantly downregulated the expression of various genes exclusively expressed in osteoclasts. These results demonstrate that APE restores LPS-induced bone loss through a decrease of the serum RANKL/OPG ratio, and inhibits osteoclast differentiation and function, suggesting the promise of APE as a potential cure for various osteoclast-associated bone diseases.

  19. Cherubism Mice Also Deficient in c-Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts.

    Science.gov (United States)

    Kittaka, Mizuho; Mayahara, Kotoe; Mukai, Tomoyuki; Yoshimoto, Tetsuya; Yoshitaka, Teruhito; Gorski, Jeffrey P; Ueki, Yasuyoshi

    2018-01-01

    Currently, it is believed that osteoclasts positive for tartrate-resistant acid phosphatase (TRAP+) are the exclusive bone-resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2 KI/KI ) with a homozygous gain-of-function mutation in the SH3-domain binding protein 2 (SH3BP2) was shown to develop auto-inflammatory joint destruction. Here, we demonstrate that Sh3bp2 KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c-Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C-terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross-linked C-telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2 KI/KI mice deficient in c-Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c-Fos-deficient Sh3bp2 KI/KI mice. After activation of the NF-κB pathway, macrophage colony-stimulating factor (M-CSF)-dependent macrophages from c-Fos-deficient Sh3bp2 KI/KI mice expressed increased amounts of MMP14 compared with wild-type macrophages. Interestingly, receptor activator of NF-κB ligand (RANKL)-deficient Sh3bp2 KI/KI mice failed to show notable bone erosion, whereas c-Fos deletion did restore bone erosion to the RANKL-deficient Sh3bp2 KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss-of-function of c-Fos and gain-of-function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in

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

    Energy Technology Data Exchange (ETDEWEB)

    Hiruma, Yoshiharu, E-mail: hiruma.yoshiharu.hy@daiichisankyo.co.jp [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Hirai, Takehiro [Translational Medicine and Clinical Pharmacology Department, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan); Tsuda, Eisuke [Biological Research Laboratories, Daiichi Sankyo Co. Ltd., Tokyo 134-8630 (Japan)

    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.

  1. Intermedin suppresses pressure overload cardiac hypertrophy through activation of autophagy.

    Science.gov (United States)

    Chen, Huali; Wang, Xue; Tong, Mingming; Wu, Dan; Wu, Sisi; Chen, Jiaxiang; Wang, Xiaoxiao; Wang, Xulei; Kang, Yu; Tang, Hong; Tang, Chaoshu; Jiang, Wei

    2013-01-01

    Left ventricular hypertrophy is a maladaptive response to pressure overload and an important risk factor for heart failure. Intermedin (IMD), a multi-functional peptide, plays important roles in cardiovascular protection. In this study, we revealed an autophagy-dependent mechanism involved in IMD's protection against cardiac remodeling and cardiomyocyte death in heart hypertrophy. We observed that transverse aortic contraction (TAC) induction, Ang II or ISO exposure induced remarkable increase in the expression of endogenous IMD and its receptor components, CRLR, RAMP1 and RAMP3, in mouse hearts and H9c2 cell cultures, respectively. Furthermore, the heart size, heart weight/body weight ratios, cardiomyocyte size and apoptosis, interstitial collagen, hypertrophic markers including ANP and BNP expression were also significantly increased, which were effectively suppressed by IMD supplementation. In addition, IMD induced capillary angiogenesis and improved functions in hypertrophic hearts. We further observed that IMD induced strong autophagy in hypertrophic hearts and cultured cells, which was paralleling with the decrease in cardiomyocyte size and apoptosis. Furthermore, an autophagy inhibitor, 3-MA, was used to block the IMD-augmented autophagy level, and then the protection of IMD on cardiomyocyte hypertrophy and apoptosis was almost abrogated. We also observed that IMD supplementation stirred intracellular cAMP production, and augmented the ERK1/2 phosphorylation induced by Ang II/ISO exposure in H9c2 cells. In addition, we inhibited PI3K, PKA and MAPK/ERK1/2 signaling pathways by using wortamannin, H89 and PD98059, respectively, in H9c2 cells co-incubating with both IMD and Ang II or ISO, and observed that these inhibitors effectively reduced IMD-augmented autophagy level, but only H89 and PD98059 pre-incubation abrogated the anti-apoptotic action of IMD. These results indicate that the endogenous IMD and its receptor complexes are induced in hypertrophic

  2. Intermedin suppresses pressure overload cardiac hypertrophy through activation of autophagy.

    Directory of Open Access Journals (Sweden)

    Huali Chen

    Full Text Available Left ventricular hypertrophy is a maladaptive response to pressure overload and an important risk factor for heart failure. Intermedin (IMD, a multi-functional peptide, plays important roles in cardiovascular protection. In this study, we revealed an autophagy-dependent mechanism involved in IMD's protection against cardiac remodeling and cardiomyocyte death in heart hypertrophy. We observed that transverse aortic contraction (TAC induction, Ang II or ISO exposure induced remarkable increase in the expression of endogenous IMD and its receptor components, CRLR, RAMP1 and RAMP3, in mouse hearts and H9c2 cell cultures, respectively. Furthermore, the heart size, heart weight/body weight ratios, cardiomyocyte size and apoptosis, interstitial collagen, hypertrophic markers including ANP and BNP expression were also significantly increased, which were effectively suppressed by IMD supplementation. In addition, IMD induced capillary angiogenesis and improved functions in hypertrophic hearts. We further observed that IMD induced strong autophagy in hypertrophic hearts and cultured cells, which was paralleling with the decrease in cardiomyocyte size and apoptosis. Furthermore, an autophagy inhibitor, 3-MA, was used to block the IMD-augmented autophagy level, and then the protection of IMD on cardiomyocyte hypertrophy and apoptosis was almost abrogated. We also observed that IMD supplementation stirred intracellular cAMP production, and augmented the ERK1/2 phosphorylation induced by Ang II/ISO exposure in H9c2 cells. In addition, we inhibited PI3K, PKA and MAPK/ERK1/2 signaling pathways by using wortamannin, H89 and PD98059, respectively, in H9c2 cells co-incubating with both IMD and Ang II or ISO, and observed that these inhibitors effectively reduced IMD-augmented autophagy level, but only H89 and PD98059 pre-incubation abrogated the anti-apoptotic action of IMD. These results indicate that the endogenous IMD and its receptor complexes are induced in

  3. Alterations in osteoclast function and phenotype induced by different inhibitors of bone resorption--implications for osteoclast quality

    DEFF Research Database (Denmark)

    Neutzsky-Wulff, Anita V; Sørensen, Mette Guldmann; Kocijancic, Dino

    2010-01-01

    Normal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have secon...... secondary effects on bone remodeling, a process which is of importance for the pathogenesis of both osteoporosis and osteoarthritis. We treated human osteoclasts with different inhibitors and characterized their resulting function....

  4. C3 rho-inhibitor for targeted pharmacological manipulation of osteoclast-like cells.

    Directory of Open Access Journals (Sweden)

    Andrea Tautzenberger

    Full Text Available 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.

  5. Pantoprazole Decreases Cell Viability and Function of Human Osteoclasts In Vitro

    Directory of Open Access Journals (Sweden)

    Markus Prause

    2015-01-01

    Full Text Available Proton pump inhibitors (PPIs are commonly prescribed drugs that decrease stomach acidity and are thus often used to treat gastroesophageal reflux disease and as a preventative agent for the adverse effects of nonsteroidal anti-inflammatory drugs on the stomach mucosa. In recently published literature, an association between proton pump inhibitor administration and increased fracture risk has been stated. In order to reveal the underlying pathomechanisms of these observations, the effects of pantoprazole, a representative of the proton pump inhibitors, on human osteoclasts in vitro were evaluated in this study. Osteoclasts were stimulated with increasing concentrations of pantoprazole ranging from 0 μg/mL to 10 μg/mL over a period of seven days. Cell viability and tartrate-resistant acid phosphatase (TRAP activity assays were performed after 1 day, 3 days, and 7 days, respectively. Here, stimulated osteoclasts presented a significantly lower viability and TRAP activity than the negative controls. Osteoclast-specific gene expression was evaluated after seven days and revealed no significant differences between all samples. Overall, the bone degrading and resorptive function of osteoclasts is inhibited by the administration of proton pump inhibitors. While PPI-related fractures through “basic multicellular unit” dysfunction are unlikely, the underlying pathomechanism remains unknown.

  6. Characterization of two types of osteoclasts from human peripheral blood monocytes

    International Nuclear Information System (INIS)

    Yuasa, Kimitaka; Mori, Kouki; Ishikawa, Hitoshi; Sudo, Akihiro; Uchida, Atsumasa; Ito, Yasuhiko

    2007-01-01

    The two osteoclastogenesis pathways, receptor activator nuclear factor (NF)-κB ligand (RANKL)-mediated and fusion regulatory protein-1 (FRP-1)-mediated osteoclastogenesis, have recently been reported. There were significant differences in differentiation and activation mechanisms between the two pathways. When monocytes were cultured with FRP-1 without adding M-CSF, essential for the RANKL system, TRAP-positive polykaryocyte formation occurred. FRP-1-mediated osteoclasts formed larger pits on mineralized calcium phosphate plates than RANKL+M-CSF-mediated osteoclasts did. Lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were inclined to be single and isolated. However, osteoclasts induced by RANKL+M-CSF made many connected pits on dentin surfaces as if they crawled on there. Interestingly, FRP-1 osteoclastogenesis was enhanced by M-CSF/IL-1α, while chemotactic behavior to the dentin slices was not effected. There were differences in pH and concentration of HCO3- at culture endpoint and in adherent feature to dentin surfaces. Our findings indicate there are two types of osteoclasts with distinct properties

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Alterations in osteoclast function and phenotype induced by different inhibitors of bone resorption--implications for osteoclast quality

    DEFF Research Database (Denmark)

    Neutzsky-Wulff, Anita V; Sørensen, Mette Guldmann; Kocijancic, Dino

    2010-01-01

    Normal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have...

  9. Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner*

    Science.gov (United States)

    Chai, Ryan C.; Kouspou, Michelle M.; Lang, Benjamin J.; Nguyen, Chau H.; van der Kraan, A. Gabrielle J.; Vieusseux, Jessica L.; Lim, Reece C.; Gillespie, Matthew T.; Benjamin, Ivor J.; Quinn, Julian M. W.; Price, John T.

    2014-01-01

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss. PMID:24692538

  10. Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

    DEFF Research Database (Denmark)

    Søe, Kent; Andersen, Thomas Lykke; Hobolt-Pedersen, Anne-Sofie

    2011-01-01

    Generation of osteoclasts through fusion of mono-nucleated precursors is a key event of bone physiology and bone resorption is inefficient without osteoclast fusion. Several factors playing a critical role in the fusion process have already been recognized, but the factors involved in the actual...... fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer....... This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic...

  11. Expression of osteoblast and osteoclast regulatory genes in the bone marrow microenvironment in multiple myeloma

    DEFF Research Database (Denmark)

    Kristensen, Ida B; Christensen, Jacob Haaber; Lyng, Maria Bibi

    2014-01-01

    Multiple myeloma (MM) lytic bone disease (LBD) is caused by osteoclast activation and osteoblast inhibition. RANK/RANKL/OPG play central roles in osteoclast activation and Wnt inhibitor DKK1 in osteoblast inhibition. The role of other Wnt inhibitors is less clear. We evaluated gene expression...... of osteoclast regulators (RANK, RANKL, OPG, TRAIL, MIP1A), Wnt inhibitors (DKK1, SFRP2, SFRP3, sclerostin, WIF1) and osteoblast transcription factors (RUNX2, osterix) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the bone marrow (BM) microenvironment using snap-frozen BM biopsies...... radiographs and the bone resorption marker CTX-1. Protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Among Wnt inhibitors, only SFRP3 and DKK1 were significantly overexpressed in advanced LBD, correlating with protein levels. SFRP3 correlated with CTX-1. Our...

  12. Neuropeptide FF attenuates RANKL-induced differentiation of macrophage-like cells into osteoclast-like cells.

    Science.gov (United States)

    Sun, Yulong; Chen, Xiaohu; Chen, Zhihao; Ma, Xiaoli; Li, Dijie; Shang, Peng; Qian, Airong

    2015-02-01

    Neuropeptide FF (NPFF) has been implicated in many physiological processes but not osteoclastogenesis. We previously demonstrated that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. This study was designed to investigate the effect of NPFF on receptor activator of nuclear factor κB ligand (RANKL)-mediated differentiation of RAW264.7 cells into osteoclast-like cells. RAW264.7 cells were cultured in 96-stripwell plates or in Corning Osteo Assay Surface 96-well plates in the presence of various concentrations of NPFF with or without RANKL for 3 or 6 d. The differentiation of osteoclast-like cells was analyzed by tartrate-resistant acid phosphatase (TRAP) stain, TRAP activity and bone resorption capacity, respectively. The mRNA expression of NPFF2 receptor (NPFFR2) and osteoclast genes was evaluated by using real-time quantitative PCR which includes TRAP, RANK (receptor activator of NF-κB), Cathepsin K, MMP-9 (matrix metallopeptidase 9), Intβ3 (integrin β3) and NFATc1 (nuclear factor of activated T cells cytoplasmic 1). In addition, the influence of NPFF on the cell viability and NO release of RAW264.7 cells was measured by MTT assay and Griess method, respectively. NPFF dose-dependently inhibited RANKL-induced osteoclast-like cells differentiation including TRAP-positive cell formation, TRAP activity and bone resorption capacity. Moreover, NO release and osteoclast gene expression of osteoclast-like cells were downregulated by NPFF. In addition, NPFFR2 gene expression in osteoclast-like cells was augmented in response to NPFF treatment. Our findings showed that NPFF could attenuate osteoclast-like cells differentiation in an in vitro osteoclastogenesis model. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. The role of TGFβ receptor 1-smad3 signaling in regulating the osteoclastic mode affected by fluoride.

    Science.gov (United States)

    Yu, Haolan; Jiang, Ningning; Yu, XiuHua; Zhao, Zhitao; Zhang, Xiuyun; Xu, Hui

    2018-01-15

    Studies that have focused on the role TGFβ signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TβR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFβ receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFβ and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TβR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TβR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFβ signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It

  14. HMGB1 enhances immune suppression by facilitating the differentiation and suppressive activity of myeloid-derived suppressor cells.

    Science.gov (United States)

    Parker, Katherine H; Sinha, Pratima; Horn, Lucas A; Clements, Virginia K; Yang, Huan; Li, Jianhua; Tracey, Kevin J; Ostrand-Rosenberg, Suzanne

    2014-10-15

    Chronic inflammation often precedes malignant transformation and later drives tumor progression. Likewise, subversion of the immune system plays a role in tumor progression, with tumoral immune escape now well recognized as a crucial hallmark of cancer. Myeloid-derived suppressor cells (MDSC) are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. Thus, MDSCs may define an element of the pathogenic inflammatory processes that drives immune escape. The secreted alarmin HMGB1 is a proinflammatory partner, inducer, and chaperone for many proinflammatory molecules that MDSCs develop. Therefore, in this study, we examined HMGB1 as a potential regulator of MDSCs. In murine tumor systems, HMGB1 was ubiquitous in the tumor microenvironment, activating the NF-κB signal transduction pathway in MDSCs and regulating their quantity and quality. We found that HMGB1 promotes the development of MDSCs from bone marrow progenitor cells, contributing to their ability to suppress antigen-driven activation of CD4(+) and CD8(+) T cells. Furthermore, HMGB1 increased MDSC-mediated production of IL-10, enhanced crosstalk between MDSCs and macrophages, and facilitated the ability of MDSCs to downregulate expression of the T-cell homing receptor L-selectin. Overall, our results revealed a pivotal role for HMGB1 in the development and cancerous contributions of MDSCs. ©2014 American Association for Cancer Research.

  15. Identification of a novel compound that inhibits osteoclastogenesis by suppressing nucleoside transporters.

    Science.gov (United States)

    Katsuyama, Shun; Sugino, Kumi; Sasazawa, Yukiko; Nakano, Yoshihiko; Aono, Harumi; Morishita, Keisuke; Kawatani, Makoto; Umezawa, Kazuo; Osada, Hiroyuki; Simizu, Siro

    2016-04-01

    We screened small-molecule compounds that inhibit osteoclast differentiation to find new anti-osteoporosis agents and found that a novel compound, SUKU-1, suppressed osteoclastogenesis. We also synthesized 38 derivatives of SUKU-1 and discovered that nine of them had inhibitory effects on osteoclastogenesis and that SUKU-33 was the most potent inhibitor. Next, we investigated the mechanisms by which SUKU-33 suppressed osteoclast differentiation. By measuring the uptake of [(3) H]-uridine in cells, we found that SUKU-33 suppressed both equilibrative nucleoside transporters and concentrative nucleoside transporters. These results suggest that SUKU-33 inhibits osteoclast differentiation by suppressing nucleoside transporters. © 2016 Federation of European Biochemical Societies.

  16. Distinctive subdomains in the resorbing surface of osteoclasts.

    Directory of Open Access Journals (Sweden)

    Kinga A Szewczyk

    Full Text Available We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a 'sealing zone', encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but

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

    Science.gov (United States)

    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

    2017-11-01

    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.

  18. Acteoside suppresses RANKL-mediated osteoclastogenesis by inhibiting c-Fos induction and NF-κB pathway and attenuating ROS production.

    Directory of Open Access Journals (Sweden)

    Seung-Youp Lee

    Full Text Available Numerous studies have reported that inflammatory cytokines are important mediators for osteoclastogenesis, thereby causing excessive bone resorption and osteoporosis. Acteoside, the main active compound of Rehmannia glutinosa, which is used widely in traditional Oriental medicine, has anti-inflammatory and antioxidant potentials. In this study, we found that acteoside markedly inhibited osteoclast differentiation and formation from bone marrow macrophages (BMMs and RAW264.7 macrophages stimulated by the receptor activator of nuclear factor-kappaB (NF-κB ligand (RANKL. Acteoside pretreatment also prevented bone resorption by mature osteoclasts in a dose-dependent manner. Acteoside (10 µM attenuated RANKL-stimulated activation of p38 kinase, extracellular signal-regulated kinases, and c-Jun N-terminal kinase, and also suppressed NF-κB activation by inhibiting phosphorylation of the p65 subunit and the inhibitor κBα. In addition, RANKL-mediated increases in the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1 and in the production of tumor necrosis factor-α, interleukin (IL-1β, and IL-6 were apparently inhibited by acteoside pretreatment. Further, oral acteoside reduced ovariectomy-induced bone loss and inflammatory cytokine production to control levels. Our data suggest that acteoside inhibits osteoclast differentiation and maturation from osteoclastic precursors by suppressing RANKL-induced activation of mitogen-activated protein kinases and transcription factors such as NF-κB, c-Fos, and NFATc1. Collectively, these results suggest that acteoside may act as an anti-resorptive agent to reduce bone loss by blocking osteoclast activation.

  19. Time-lapse reveals that osteoclasts can move across the bone surface while resorbing

    DEFF Research Database (Denmark)

    Søe, Kent; Delaissé, Jean-Marie

    2017-01-01

    resulting in the formation of clusters of round pits. However, very importantly, we also demonstrate that more than half of the osteoclasts moved laterally, displacing their extracellular bone-resorbing compartment over the bone surface without disassembling and reconstructing it, thereby generating long......Bone erosion both demands that the osteoclast resorbs bone matrix and moves over the bone surface. It is widely accepted that these two activities alternate, because they are considered mutually exclusive since resorption is believed to involve an immobilizing seal to the bone surface. However......, clear real-time observations are still lacking. Herein, we used specific markers and time-lapse to monitor live the spatiotemporal generation of resorption events by osteoclasts cultured on bone slices. In accordance with the current view, we found alternating episodes of resorption and migration...

  20. Osteoclasts secrete non-bone derived signals that induce bone formation

    DEFF Research Database (Denmark)

    Karsdal, Morten A; Neutzsky-Wulff, Anita V; Dziegiel, Morten Hanefeld

    2008-01-01

    Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption......, in face of normal or even increased bone formation. This suggests that osteoclasts, not their resorptive activity, are important for sustaining bone formation. To investigate whether osteoclasts mediate control of bone formation by production of bone anabolic signals, we collected conditioned media (CM...

  1. Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression

    International Nuclear Information System (INIS)

    Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X.; Walterscheid, Jeffrey P.; Ullrich, Stephen E.

    2004-01-01

    Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependant manner. The release of biological response modifiers, particularly prostaglandin E 2 (PGE 2 ), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE 2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE 2 secretion. Jet fuel-induced PGE 2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin

  2. Active Suppression of Drilling System Vibrations For Deep Drilling

    Energy Technology Data Exchange (ETDEWEB)

    Raymond, David W.; Blankenship, Douglas A.; Buerger, Stephen; Mesh, Mikhail; Radigan, William Thomas; Su, Jiann-Cherng

    2015-10-01

    The dynamic stability of deep drillstrings is challenged by an inability to impart controllability with ever-changing conditions introduced by geology, depth, structural dynamic properties and operating conditions. A multi-organizational LDRD project team at Sandia National Laboratories successfully demonstrated advanced technologies for mitigating drillstring vibrations to improve the reliability of drilling systems used for construction of deep, high-value wells. Using computational modeling and dynamic substructuring techniques, the benefit of controllable actuators at discrete locations in the drillstring is determined. Prototype downhole tools were developed and evaluated in laboratory test fixtures simulating the structural dynamic response of a deep drillstring. A laboratory-based drilling applicability demonstration was conducted to demonstrate the benefit available from deployment of an autonomous, downhole tool with self-actuation capabilities in response to the dynamic response of the host drillstring. A concept is presented for a prototype drilling tool based upon the technical advances. The technology described herein is the subject of U.S. Patent Application No. 62219481, entitled "DRILLING SYSTEM VIBRATION SUPPRESSION SYSTEMS AND METHODS", filed September 16, 2015.

  3. Active vertical tail buffeting suppression based on macro fiber composites

    Science.gov (United States)

    Zou, Chengzhe; Li, Bin; Liang, Li; Wang, Wei

    2016-04-01

    Aerodynamic buffet is unsteady airflow exerting forces onto a surface, which can lead to premature fatigue damage of aircraft vertical tail structures, especially for aircrafts with twin vertical tails at high angles of attack. In this work, Macro Fiber Composite (MFC), which can provide strain actuation, was used as the actuator for the buffet-induced vibration control, and the positioning of the MFC patches was led by the strain energy distribution on the vertical tail. Positive Position Feedback (PPF) control algorithm has been widely used for its robustness and simplicity in practice, and consequently it was developed to suppress the buffet responses of first bending and torsional mode of vertical tail. However, its performance is usually attenuated by the phase contributions from non-collocated sensor/actuator configuration and plants. The phase lag between the input and output signals of the control system was identified experimentally, and the phase compensation was considered in the PPF control algorithm. The simulation results of the amplitude frequency of the closed-loop system showed that the buffet response was alleviated notably around the concerned bandwidth. Then the wind tunnel experiment was conducted to verify the effectiveness of MFC actuators and compensated PPF, and the Root Mean Square (RMS) of the acceleration response was reduced 43.4%, 28.4% and 39.5%, respectively, under three different buffeting conditions.

  4. Ethanol Extract of Atractylodes macrocephala Protects Bone Loss by Inhibiting Osteoclast Differentiation

    Directory of Open Access Journals (Sweden)

    Youn-Hwan Hwang

    2013-06-01

    Full Text Available The rhizome of Atractylodes macrocephala has been used mainly in Traditional Chinese Medicine for invigorating the functions of the stomach and spleen. In the present study, we investigated the inhibitory effect of the 70% ethanol extract of the rhizome of Atractylodes macrocephala (AMEE on osteoclast differentiation. We found that AMEE inhibits osteoclast differentiation from its precursors induced by receptor activator of nuclear factor-κB ligand (RANKL, an essential cytokine required for osteoclast differentiation. AMEE attenuated RANKL-induced activation of NF-κB signaling pathway, subsequently inhibiting the induction of osteoclastogenic transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1. Consistent with the in vitro results, administration of AMEE protected RANKL-induced bone loss in mice. We also identified atractylenolide I and II as active constituents contributing to the anti-osteoclastogenic effect of AMEE. Taken together, our results demonstrate that AMEE has a protective effect on bone loss via inhibiting osteoclast differentiation and suggest that AMEE may be useful in preventing and treating various bone diseases associated with excessive bone resorption.

  5. Inhibition of MDA-MB-231 breast cancer cell migration and invasion activity by andrographolide via suppression of nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

    Science.gov (United States)

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

    2015-02-01

    Breast cancer is one of the most common types of cancer worldwide. The majority of patients with cancer succumb to the disease as a result of distant metastases (for example, in the bones), which cause severe complications. Despite advancements in breast cancer treatment, chemotherapeutic outcomes remain far from satisfactory, prompting a search for effective natural agents with few side‑effects. Andrographolide (AP), a natural diterpenoid lactone isolated from Andrographis paniculata, inhibits cancer cell growth. The current study aimed to examine the effect of AP on breast cancer cell proliferation, survival and progression in vitro and also its inhibitory activity on breast cancer bone metastasis in vivo. To achieve this, CCK8, flow cytometry, migration, invasion, western blot, PCR and luciferase reporter assay analyses were performed in vitro as well as establishing intratibial xenograft model of breast cancer bone metastasis in vivo. The results demonstrated that AP inhibits the migration and invasion of the MBA‑MD‑231 aggressive breast cancer cell line at non‑lethal concentrations, in addition to suppressing proliferation and inducing apoptosis at high concentrations in vitro. In vivo, AP significantly inhibited the growth of tumors planted in bone and attenuated cancer‑induced osteolysis. Tartrate‑resistant acid phosphatase staining revealed osteoclast activation in tumor‑bearing mice and AP was observed to attenuate this activation. The anti‑tumor activity of AP in vitro and in vivo correlates with the downregulation of the nuclear factor κB signaling pathway and the inhibition of matrix metalloproteinase‑9 expression levels. These results indicate that AP may be an effective anti‑tumor agent for the treatment of breast cancer bone metastasis.

  6. Extinction cross-section suppression and active acoustic invisibility cloaking

    Science.gov (United States)

    Mitri, F. G.

    2017-10-01

    Invisibility in its canonical form requires rendering a zero extinction cross-section (or energy efficiency) from an active or a passive object. This work demonstrates the successful theoretical realization of this physical effect for an active cylindrically radiating acoustic body, undergoing periodic axisymmetric harmonic vibrations near a flat rigid boundary. Radiating, amplification and extinction cross-sections of the active source are defined. Assuming monopole and dipole modal oscillations of the circular source, conditions are found where the extinction energy efficiency factor of the active source vanishes, achieving total invisibility with minimal influence of the source size. It also takes positive or negative values, depending on its size and distance from the boundary. Moreover, the amplification energy efficiency factor is negative for the acoustically-active source. These effects also occur for higher-order modal oscillations of the active source. The results find potential applications in the development of acoustic cloaking devices and invisibility.

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

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-01-01

    Full Text Available 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.

  8. Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPα-interaction

    International Nuclear Information System (INIS)

    Lundberg, Pernilla; Koskinen, Cecilia; Baldock, Paul A.; Loethgren, Hanna; Stenberg, Asa; Lerner, Ulf H.; Oldenborg, Per-Arne

    2007-01-01

    Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPα) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPα-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPα strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP) + osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47 -/- mice were strongly reduced, and bones of CD47 -/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPα interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47 -/- mice

  9. Phloretin promotes osteoclast apoptosis in murine macrophages and inhibits estrogen deficiency-induced osteoporosis in mice.

    Science.gov (United States)

    Lee, Eun-Jung; Kim, Jung-Lye; Kim, Yun-Ho; Kang, Min-Kyung; Gong, Ju-Hyun; Kang, Young-Hee

    2014-09-15

    Bone-remodeling imbalance induced by increased osteoclast formation and bone resorption is known to cause skeletal diseases such as osteoporosis. The reduction of estrogen levels at menopause is one of the strongest risk factors developing postmenopausal osteoporosis. This study investigated osteoprotective effects of the dihydrochalcone phloretin found in apple tree leaves on bone loss in ovariectomized (OVX) C57BL/6 female mice as a model for postmenopausal osteoporosis. OVX demoted bone mineral density (BMD) of mouse femurs, reduced serum 17β-estradiol level and enhanced serum receptor activator of NF-κB ligand (RANKL)/osteoprotegerin ratio with uterine atrophy. Oral administration of 10 mg/kg phloretin to OVX mice for 8 weeks improved such effects, compared to sham-operated mice. Phloretin attenuated TRAP activity and cellular expression of β3 integrin and carbonic anhydrase II augmented in femoral bone tissues of OVX mice. This study further examined that osteogenic activity of phloretin in RANKL-differentiated Raw 264.7 macrophages into mature osteoclasts. Phloretin at 1-20 μM stimulated Smac expression and capase-3 activation concurrently with nuclear fragmentation of multi-nucleated osteoclasts, indicating that this compound promoted osteoclast apoptosis. Consistently, phloretin enhanced bcl-2 induction but diminished bax expression. Furthermore, phloretin activated ASK-1-diverged JNK and p38 MAPK signaling pathways in mature osteoclasts, whereas it dose-dependently inhibited the RANKL-stimulated activation of ERK. Therefore, phloretin manipulated ASK-1-MAPK signal transduction leading to transcription of apoptotic genes. Phloretin was effective in preventing estrogen deficiency-induced osteoclastogenic resorption. Copyright © 2014 Elsevier GmbH. All rights reserved.

  10. Helminth induced suppression of macrophage activation is correlated with inhibition of calcium channel activity.

    Directory of Open Access Journals (Sweden)

    Arun Chauhan

    Full Text Available Helminth parasites cause persistent infections in humans and yet many infected individuals are asymptomatic. Neurocysticercosis (NCC, a disease of the central nervous system (CNS caused by the cestode Taenia solium, has a long asymptomatic phase correlated with an absence of brain inflammation. However, the mechanisms of immune suppression remain poorly understood. Here we report that murine NCC displays a lack of cell surface maturation markers in infiltrating myeloid cells. Furthermore, soluble parasite ligands (PL failed to induce maturation of macrophages, and inhibited TLR-induced inflammatory cytokine production. Importantly, PL treatment abolished both LPS and thapsigargin-induced store operated Ca2+ entry (SOCE. Moreover, electrophysiological recordings demonstrated PL-mediated inhibition of LPS or Tg-induced currents that were TRPC1-dependent. Concomitantly STIM1-TRPC1 complex was also impaired that was essential for SOCE and sustained Ca2+ entry. Likewise loss of SOCE due to PL further inhibited NFkB activation. Overall, our results indicate that the negative regulation of agonist induced Ca2+ signaling pathway by parasite ligands may be a novel immune suppressive mechanism to block the initiation of the inflammatory response associated with helminth infections.

  11. The interpretation of mu suppression as an index of mirror neuron activity: past, present and future

    Science.gov (United States)

    2017-01-01

    Mu suppression studies have been widely used to infer the activity of the human mirror neuron system (MNS) in a number of processes, ranging from action understanding, language, empathy and the development of autism spectrum disorders (ASDs). Although mu suppression is enjoying a resurgence of interest, it has a long history. This review aimed to revisit mu's past, and examine its recent use to investigate MNS involvement in language, social processes and ASDs. Mu suppression studies have largely failed to produce robust evidence for the role of the MNS in these domains. Several key potential shortcomings with the use and interpretation of mu suppression, documented in the older literature and highlighted by more recent reports, are explored here. PMID:28405354

  12. REV-ERBs agonism suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss partially via FABP4 upregulation.

    Science.gov (United States)

    Song, Chao; Tan, Peng; Zhang, Zheng; Wu, Wei; Dong, Yonghui; Zhao, Liming; Liu, Huiyong; Guan, Hanfeng; Li, Feng

    2018-01-22

    REV-ERBs (REV-ERBα and REV-ERBβ) are transcription repressors and circadian regulators. Previous investigations have shown that REV-ERBs repress the expression of target genes, including MMP9 and CX3CR1, in macrophages. Because MMP9 and CX3CR1 reportedly participate in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, we inferred that REV-ERBs might play a role in osteoclastogenesis. In the present study, we found that the REV-ERBα level decreased significantly during RANKL-induced osteoclast differentiation from primary bone marrow-derived macrophages (BMMs). REV-ERBα knockdown by small interfering RNA in BMMs resulted in the enhanced formation of osteoclasts, whereas REV-ERBβ knockdown showed no effect on osteoclast differentiation. Moreover, the REV-ERB agonist SR9009 inhibited osteoclast differentiation and bone resorption. Intraperitoneal SR9009 administration prevented ovariectomy-induced bone loss; this effect was accompanied by decreased serum RANKL and C-terminal telopeptide of type I collagen levels and increased osteoprotegerin levels. Further investigation revealed that NF-κB and MAPK activation and nuclear factor of activated T cells, cytoplasmic 1, and c-fos expression were suppressed by SR9009. The level of reactive oxygen species was also decreased by SR9009, with NADPH oxidase subunits also being down-regulated. In addition, an expression microarray showed that FABP4, an intracellular lipid-binding protein, was up-regulated by REV-ERB agonism. BMS309403, an inhibitor of FABP4, partially prevented the suppression of osteoclastogenesis by SR9009 through stabilizing phosphorylation of p65. To summarize, our results proved that the REV-ERB agonism inhibited osteoclastogenesis partially via FABP4 up-regulation.-Song, C., Tan, P., Zhang, Z., Wu, W., Dong, Y., Zhao, L., Liu, H., Guan, H., Li, F. REV-ERBs agonism suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss partially via FABP4 upregulation.

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

    Science.gov (United States)

    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.

  14. Emergence of spatially heterogeneous burst suppression in a neural field model of electrocortical activity

    Directory of Open Access Journals (Sweden)

    Ingo eBojak

    2015-02-01

    Full Text Available Burst suppression in the electroencephalogram (EEG is a well described phenomenon that occurs during deep anaesthesia, as well as in a variety of congenital and acquired brain insults. Classically it is thought of as spatially synchronous, quasi-periodic bursts of high amplitude EEG separated by low amplitude activity. However, its characterisation as a ``global brain state'' has been challenged by recent results obtained with intracranial electrocortigraphy. Not only does it appear that burst suppression activity is highly asynchronous across cortex, but also that it may occur in isolated regions of circumscribed spatial extent. Here we outline a realistic neural field model for burst suppression by adding a slow process of synaptic resource depletion and recovery, which is able to reproduce qualitatively the empirically observed features during general anaesthesia at the whole cortex level. Simulations reveal heterogeneous bursting over the model cortex and complex spatiotemporal dynamics during simulated anaesthetic action, and provide forward predictions of neuroimaging signals for subsequent empirical comparisons and more detailed characterisation.Because burst suppression corresponds to a dynamical end-point of brain activity, theoretically accounting for its spatiotemporal emergence will vitally contribute to efforts aimed at clarifying whether a common physiological trajectory is induced by the actions of general anaesthetic agents. We have taken a first step in this direction by showing that a neural field model can qualitatively match recent experimental data that indicate spatial differentiation of burst suppression activity across cortex.

  15. Polyphenol derivatives inhibit human neutrophil activity by suppressing oxidative burst

    Czech Academy of Sciences Publication Activity Database

    Drábiková, K.; Perečko, T.; Nosáľ, R.; Harmatha, Juraj; Šmidrkal, J.; Jančinová, V.

    2012-01-01

    Roč. 5, Suppl.1 (2012), s. 31-31 ISSN 1337-6853. [Interdisciplinary Toxicological Conference & Advanced Toxicological Course /17./. 27.08.2012-31.08.2012, Stará Lesná] Institutional research plan: CEZ:AV0Z40550506 Keywords : polyphenol derivatives * neutrophil activity * pinosylvin Subject RIV: CC - Organic Chemistry

  16. Active power filter equivalent to an infinite feeder for harmonic suppression in radial distribution systems

    DEFF Research Database (Denmark)

    Shen, Hong; Yang, Lili; Sun, Xiaofeng

    2016-01-01

    This study proposes to install a novel active power filter (IF-APF) mimicking the character of an infinitely long transmission line at the end of a radial feeder. The proposed IF-APF eliminates the reflected wave and realises the background harmonic voltage suppression capability throughout...... the feeder by mimicking the characteristic of an infinite feeder. The proposed IF-APF shows a better harmonic damping performance than the traditional resistive active power filter (R-APF). Moreover, the proposed IF-APF achieves a better harmonic suppression performance than the traditional R-APF, especially...

  17. Dopa therapy and action impulsivity: subthreshold error activation and suppression in Parkinson's disease.

    Science.gov (United States)

    Fluchère, Frédérique; Deveaux, Manon; Burle, Borís; Vidal, Franck; van den Wildenberg, Wery P M; Witjas, Tatiana; Eusebio, Alexandre; Azulay, Jean-Philippe; Hasbroucq, Thierry

    2015-05-01

    Impulsive actions entail (1) capture of the motor system by an action impulse, which is an urge to act and (2) failed suppression of that impulse in order to prevent a response error. Several studies indicate that dopaminergic treatment can induce action impulsivity in patients diagnosed with Parkinson's disease (PD). Whether this effect is due to increased impulse expression or to decreased impulse suppression remains to be deciphered. We used a novel approach based on electromyographic (EMG) analyses to decipher the effects of the patient's usual dopaminergic therapy on the expression and suppression of subliminal erroneous impulses. To this end, we used a within-subject design and took advantage of the Simon task, that elicits prepotent response tendencies. The patients (N = 15) performed the task on their usual dopaminergic medication and after complete medication withdrawal (for at least 12 h). The correction rate that measures the ability to suppress subthreshold impulsive muscle activity was lower when the patients were on medication as compared to their off medication state (p < 0.05). The incorrect activation rate that measures the capture of the motor system by action impulses was unaffected by medication. Dopa therapy affected action impulsivity. Although medication did not influence the incidence of fast action impulses, it significantly reduced patients' ability to abort and suppress muscle activation related to the incorrect response alternative.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kaneuji, Takeshi [Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Takahashi, Tetsu [Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan); Oral Bioresearch Center, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580 (Japan)

    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

  19. Suppression of leukocyte inhibitory factor (LIF) production and [3H]thymidine incorporation by concanavalin A-activated mononuclear cells

    International Nuclear Information System (INIS)

    Lomnitzer, R.; Rabson, A.R.

    1979-01-01

    The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [ 3 H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production

  20. Commercial Honeybush (Cyclopia spp. Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages—An in vitro Study

    Directory of Open Access Journals (Sweden)

    Amcois Visagie

    2015-10-01

    Full Text Available 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.

  1. EBF2 regulates osteoblast-dependent differentiation of osteoclasts

    DEFF Research Database (Denmark)

    Kieslinger, Matthias; Folberth, Stephanie; Dobreva, Gergana

    2005-01-01

    of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences...

  2. Effects of osteoprotegerin from transfection of pcDNA3.1(+/chOPG on bioactivity of chicken osteoclasts

    Directory of Open Access Journals (Sweden)

    Yao Jing

    2011-03-01

    Full Text Available Abstract Background Osteoprotegerin (OPG has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG expressed in chicken embryo fibroblasts (CEFs on chicken osteoclast function in vitro. Methods The chOPG sequence containing the open reading frame (ORF was amplified from chicken embryo frontal bone and inserted into the pcDNA3.1 (+ vector. PcDNA3.1 (+/chOPG was transiently transfected into CEFs by lipofectamine 2000. Transcription of OPG mRNA and expression of chOPG recombinant protein were detected by reverse transcription polymerase chain reaction (RT-PCR and indirect immunofluorescence. The level of chOPG recombinant protein was detected by enzyme-linked immunosorbent assay. The suspension of osteoclasts was separated from chicken embryos and divided into three groups (control group, pcDNA3.1 (+ group and pcDNA3.1 (+/chOPG group. The percentage of osteoclast apoptosis was detected by flow cytometry. The tartrate-resistant acid phosphatase (TRAP secreted by osteoclasts was measured by the diazol method. The resorbing activity of osteoclasts was evaluated by the area of lacunae on bone flaps and the concentration of calcium in the supernatant liquid of osteoclasts. Results 48 h after transfection, the exogenous OPG gene transcription was detected by RT-PCR. After 72 h, the CEFs transfected from pcDNA3.1 (+/chOPG displayed green fluorescence and the concentration of chOPG protein was 15.78 ± 0.22 ng/mL. After chicken osteoclasts were cultured for 5 d in a medium containing supernatant from transfected CEFs, the percentage of osteoclast apoptosis was increased significantly, the concentration of TRAP, the area of lacunae on bone flaps and calcium concentration were decreased significantly in the

  3. Oral activated charcoal suppresses hyperphosphataemia in haemodialysis patients.

    Science.gov (United States)

    Wang, Zunsong; Cui, Meiyu; Tang, Lijun; Li, Wenbin; Wei, Yong; Zhu, Zhuxian; Jia, Xiaoyan; Kong, Xianglei; Xu, Dongmei

    2012-09-01

    Hyperphosphataemia is almost inevitable in end stage renal disease (ESRD) patients and is associated with increased morbidity and mortality. In this study we examined whether oral activated charcoal (oAC) reduces serum phosphate level in haemodialysis patients. This was an open-label, prospective, uncontrolled study. One hundred and thirty-five haemodialysis patients were included in this study, with cessation of treatment with any phosphate binders during a 2 week washout period. Patients with serum phosphate levels greater than 5.5 mg/dL during the washout period were included for treatment with oAC. oAC was started at a dose of 600 mg three times per day with meals and was administered for 24 weeks. oAC dose was titrated up during the 24 week period to achieve phosphate control (3.5-5.5 mg/dL). A second 2 week washout period followed the end of oAC treatment. In the 114 patients who successfully completed the trial, the mean dose of activated charcoal was 3190 ± 806 mg/day. oAC reduced mean phosphate levels to below 5.5 mg/dL, with mean decreases of 2.60 ± 0.11 mg/dL (P < 0.01) and 103 (90.4%) of the patients reached the phosphate target. After the second washout period the phosphate levels increased to 7.50 ± 1.03 mg/dL (P < 0.01). Serum intact parathyroid hormone (iPTH) levels declined from 338.75 ± 147.77 pg/mL to 276.51 ± 127.82 pg/mL (P < 0.05) during the study. oAC had no influence on serum prealbumin, total cholesterol, triglycerides, serum ferritin, haemoglobin or platelet levels and the levels of 1,25-dihydroxyvitamin D were stable during the study. In this open-label uncontrolled study, oAC effectively controls hyperphosphataemia and hyperparathyroidism in haemodialysis patients. The safety and efficacy of oAC needs to be assessed in a randomized controlled trial. © 2012 The Authors. Nephrology © 2012 Asian Pacific Society of Nephrology.

  4. Osteoclast Fusion is Based on Heterogeneity Between Fusion Partners

    DEFF Research Database (Denmark)

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

    2014-01-01

    Bone-resorbing osteoclasts are formed through fusion of mononucleated precursors. Their choice of partners during the fusion process remains unclear. We hypothesized that osteoclasts are selective in their choice of fusion partner and that this selectivity is based on heterogeneity among the cells...... with respect to their maturation stage and their expression and cellular organization of fusion factors. Support for this hypothesis was found from immunofluorescence staining of the osteoclast fusion factors CD47, dendritic cell-specific transmembrane protein (DC-STAMP), and syncytin-1. These stainings...... fusion steps was also suggested from experiments with a CD47 blocking antibody, which resulted in an inhibition of the fusion of small osteoclasts. Conversely, blocking of connexin 43 affected the fusion of larger osteoclasts with four or more nuclei. The suggestion that different fusion factors function...

  5. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yao Zhu

    2016-08-01

    Full Text Available Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL, one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2-regulated genes such as heme oxygenase-1 (HO-1 and NAD(PH dehydrogenase (quinone1 (NQO1. However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS and malondialdehyde (MDA, and improved the activities of superoxide dismutase (SOD and catalase (CAT, resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway.

  6. An adult osteopetrosis model in medaka reveals the importance of osteoclast function for bone remodeling in teleost fish.

    Science.gov (United States)

    To, Thuy Thanh; Witten, Paul Eckhard; Huysseune, Ann; Winkler, Christoph

    2015-12-01

    Osteoclasts play important roles during bone growth and in maintaining bone health and bone homeostasis. Dysfunction or lack of osteoclasts leads to increased bone mass and osteopetrosis phenotypes in mouse and human. Here we report a severe osteopetrosis-like phenotype in transgenic medaka fish, in which membrane bound EGFP (mEGFP) was expressed in osteoclasts under control of the cathepsin K promoter (ctsk:mEGFP). In contrast to reporter lines with GFP expression in the cytoplasm of osteoclasts, adult fish of the mEGFP line developed bone defects indicative for an osteoclast dysfunction. Activity of tartrate-resistant acid phosphatase (TRAP) was down-regulated and excess bone was observed in most parts of the skeleton. The osteopetrotic phenotype was particularly obvious at the neural and haemal arches that failed to increase their volume in growing fish. Excess bone caused severe constriction of the spinal cord and the ventral aorta. The continuation of tooth development and the failure to shed teeth resulted in severe hyperdontia. Interestingly, at the vertebral column vertebral body arches displayed a severe osteopetrosis, while vertebral centra had no or only a mild osteopetrotic phenotype. This confirms previous reports from cichlids that, different from the arches, allometric growth of fish vertebral centra initially does not depend on the action of osteoclasts. Independent developmental mechanism that shapes arches and vertebral centra can also lend support to the hypothesis that vertebral centra and arches function as independent developmental modules. Together, this medaka osteopetrosis model confirms the importance of proper osteoclast function during normal skeletal development in teleost fish that requires bone modeling and remodeling. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

    DEFF Research Database (Denmark)

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K

    2015-01-01

    an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme...... that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein...

  8. Suppression of Osteoclastogenesis by Melatonin: A Melatonin Receptor-Independent Action.

    Science.gov (United States)

    Kim, Hyung Joon; Kim, Ha Jin; Bae, Moon-Kyoung; Kim, Yong-Deok

    2017-05-26

    In vertebrates, melatonin is primarily secreted from the pineal gland but it affects various biological processes including the sleep-wake cycle, vasomotor control, immune system and bone homeostasis. Melatonin has been known to promote osteoblast differentiation and bone maturation, but a direct role of melatonin on osteoclast differentiation is still elusive. The present study investigated the effect of melatonin on the differentiation of macrophages to osteoclasts. The presence of melatonin significantly reduced receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and the siRNA-mediated knockdown of the melatonin receptor failed to overcome the anti-osteoclastogenic effect of melatonin. Although melatonin treatment did not affect the phosphorylation of extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), it markedly inhibited the activation of NF-κB and subsequent induction of nuclear factor of activated T cell cytoplasmic 1(NFATc1). Thus, our results suggest that melatonin could suppress osteoclast differentiation through downregulation of NF-κB pathway with concomitant decrease in the NFATc1 transcription factor induction. Furthermore, melatonin seems to have an anti-osteoclastogenic effect independent of plasma membrane melatonin receptors. In addition to previously reported properties of melatonin, our study proposes another aspect of melatonin and bone homeostasis.

  9. Pyrroloquinoline quinine inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos in mouse bone marrow cells and inhibits wear particle-induced osteolysis in mice.

    Directory of Open Access Journals (Sweden)

    Lingbo Kong

    Full Text Available The effects of pyrroloquinoline quinine (PQQ on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.

  10. Suppression of SOS-inducing activity of chemical mutagens by metabolites from microbial transformation of (-)-isolongifolene.

    Science.gov (United States)

    Sakata, Kazuki; Oda, Yoshimitsu; Miyazawa, Mitsuo

    2010-02-24

    In this study, biotransformation of (-)-isolongifolene (1) by Glomerella cingulata and suppressive effect on umuC gene expression by chemical mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and aflatoxin B(1) (AFB(1)) of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Initially, 1 was carried out the microbial transformation by G. cingulata. The result found that 1 was converted into (-)-isolongifolen-9-one (2), (-)-(2S)-13-hydroxy-isolongifolen-9-one (3), and (-)-(4R)-4-hydroxy-isolongifolen-9-one (4) by G. cingulata, and their conversion rates were 60, 25, and 15%, respectively. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB(1) in the umu test. Comound 2 showed gene expression by chemical mutagens furylfuramide and AFB(1) was suppressed 54 and 50% at <0.5 mM, respectively. Compound 2 is the most effective compound in this experiment.

  11. Saikosaponin a inhibits RANKL-induced osteoclastogenesis by suppressing NF-κB and MAPK pathways.

    Science.gov (United States)

    Zhou, Chi; Liu, Wengang; He, Wei; Wang, Haibin; Chen, Qunqun; Song, Houpan

    2015-03-01

    Inflammatory cytokines play an important role in osteoclastogenesis. Saikosaponin a (SSa) possesses anti-inflammatory activity. However, the role of SSa in osteoporosis is still unclear. Therefore, the objective of this study was to investigate the effects of SSa on receptor activator of the nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and signaling pathway by in vitro assay. In mouse bone marrow monocytes (BMMs), SSa suppressed RANKL plus macrophage colony-stimulating factor (M-CSF)-induced osteoclast differentiation in a dose-dependent manner. Moreover, SSa decreased osteoclastogenesis-related marker proteins expression, including NFATc1, c-fos and cathepsin K. At molecular levels, SSa inhibited RANKL-induced IκBα phosphorylation, p65 phosphorylation and NF-κB luciferase activity in RAW264.7 cells. And SSa also suppressed RANKL-induced p-38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) phosphorylation. Taken together, these findings suggest that SSa suppresses osteoclastogenesis through inhibiting RANKL-induced p-38, ERK, JNK and NF-κB activation. SSa is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. MicroRNA-214 suppresses gluconeogenesis by targeting activating transcriptional factor 4.

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-03-27

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. MicroRNA-214 Suppresses Gluconeogenesis by Targeting Activating Transcriptional Factor 4*

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-01-01

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. PMID:25657009

  14. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    Science.gov (United States)

    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

  15. Dual-specificity tyrosine phosphorylation-regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner.

    Science.gov (United States)

    Guterman-Ram, Gali; Pesic, Milena; Orenbuch, Ayelet; Czeiger, Tal; Aflalo, Anastasia; Levaot, Noam

    2018-01-01

    Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign-body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion-founder cells that fuse to fusion-follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells. © 2017 Wiley Periodicals, Inc.

  16. Lutein Enhances Bone Mass by Stimulating Bone Formation and Suppressing Bone Resorption in Growing Mice.

    Science.gov (United States)

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

    2017-01-01

    Lutein is a member of the xanthophyll family of carotenoids, which are known to prevent hypoxia-induced cell damage in the eye by removing free radicals. However, its role in other tissues is controversial, and the effects of lutein on bone tissues are unknown. To identify a possible role of lutein in bone tissues, we examined the effects of lutein on bone formation and bone resorption and on femoral bone mass in mice. Lutein enhanced the formation of mineralized bone nodules in cultures of osteoblasts. On the other hand, lutein clearly suppressed 1α, 25-dihydroxyvitamin D 3 -induced bone resorption as measured by pit formation in organ culture of mouse calvaria. In co-cultures of bone marrow cells and osteoblasts, lutein suppressed 1α, 25-dihydroxyvitamin D 3 -induced osteoclast formation. In cultures of bone marrow macrophages, lutein suppressed soluble RANKL, the receptor activator of nuclear factor-kappaB (NF-κB) ligand, induced osteoclast formation. When five-week-old male mice were orally administered lutein for 4 weeks, the femoral bone mass was clearly enhanced in cortical bone, as measured by bone mineral density in dual X-ray absorptiometry and micro computed tomography (µCT) analyses. The present study indicates that lutein enhances bone mass in growing mice by suppressing bone resorption and stimulating bone formation. Lutein may be a natural agent that promotes bone turnover and may be beneficial for bone health in humans.

  17. Suppression of superoxide anion generation catalyzed by xanthine oxidase with alkyl caffeates and the scavenging activity.

    Science.gov (United States)

    Masuoka, Noriyoshi; Kubo, Isao

    2016-01-01

    Alkyl caffeates are strong antioxidants and inhibitors of xanthine oxidase. However, it is unclear about the effect of caffeic acid and alkyl caffeates on superoxide anion (O2(-)) generation catalyzed by xanthine oxidase. Effects of caffeic acid and alkyl caffeates on the uric acid formation and O2(-) generation catalyzed by xanthine oxidase were analyzed. The scavenging activities of 1,1-diphenyl-2-picryhydrazyl (DPPH) radical and O2(-) generated with phenazine methosulfate (PMS) and NADH were examined. Caffeic acid derivatives equally suppressed O2(-) generation, and the suppression is stronger than inhibition of xanthine oxidase. Scavenging activity of O2(-) is low compared to the suppression of O2(-) generation. Suppression of O2(-) generation catalyzed by xanthine oxidase with caffeic acid derivatives was not due to enzyme inhibition or O2(-) scavenging but due to the reduction of xanthine oxidase molecules. Alkyl caffeates are effective inhibitors of uric acid and O2(-) catalyzed by xanthine oxidase as well as antioxidants for edible oil.

  18. Effects of carotenoids, immune activation and immune suppression on the intensity of chronic coccidiosis in greenfinches.

    Science.gov (United States)

    Sepp, Tuul; Karu, Ulvi; Sild, Elin; Männiste, Marju; Hõrak, Peeter

    2011-03-01

    Allocation trade-offs of carotenoids between their use in the immune system and production of integumentary colouration have been suggested as a proximate mechanism maintaining honesty of signal traits. We tested how dietary carotenoid supplementation, immune activation and immune suppression affect intensity of coccidian infection in captive greenfinches Carduelis chloris, a passerine with carotenoid-based plumage. Immune activation with phytohaemagglutinin (PHA) decreased body mass among birds not supplemented with lutein, while among the carotenoid-fed birds, PHA had no effect on mass dynamics. Immune suppression with dexamethasone (DEX) induced loss of body mass and reduced the swelling response to PHA. DEX and PHA increased the concentration of circulating heterophils. Lutein supplementation increased plasma carotenoid levels but had no effect on the swelling response induced by PHA. PHA and DEX treatments did not affect plasma carotenoids. Immune stimulation by PHA suppressed the infection, but only among carotenoid-supplemented birds. Priming of the immune system can thus aid in suppressing chronic infection but only when sufficient amount of carotenoids is available. Our experiment shows the importance of carotenoids in immune response, but also the complicated nature of this impact, which could be the reason for inconsistent results in studies investigating the immunomodulatory effects of carotenoids. The findings about involvement of carotenoids in modulation of an immune response against coccidiosis suggest that carotenoid-based ornaments may honestly signal individuals' ability to manage chronic infections. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Low-magnitude high-frequency vibration inhibits RANKL-induced osteoclast differentiation of RAW264.7 cells.

    Science.gov (United States)

    Wu, Song-Hui; Zhong, Zhao-Ming; Chen, Jian-Ting

    2012-01-01

    Osteoclasts are the key participants in regulation of bone mass. Low-magnitude high-frequency vibration (LMHFV) has been found to be anabolic to bone in vivo. This study aimed to investigate the effect of LMHFV on osteoclast differentiation in vitro. Murine monocyte cell line RAW264.7 cells in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) were treated with or without LMHFV at 45 Hz (0.3 g) for 15 min day(-1). Tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and actin ring formation were evaluated. Expression of the osteoclast-specific genes, such as cathepsin K, matrix metallopeptidase-9 (MMP-9) and TRAP, were analyzed using real time-PCR. c-Fos, an osteoclast-specific transcription factor, was determined using Western blot. We found that LMHFV significantly decreased the number of RANKL-induced TRAP-positive MNCs (P<0.01), and inhibited the actin ring formation. The mRNA expression of the cathepsin K, MMP-9 and TRAP were down-regulated by LMHFV intervention (all P<0.001). Furthermore, LMHFV also inhibited the expression of c-Fos protein in the RANKL-treated RAW264.7 cells (P<0.05). Our results suggest that LMHFV can inhibit the RANKL-induced osteoclast differentiation of RAW264.7 cells, which give some new insight into the anabolic effects of LMHFV on bone.

  20. P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis

    DEFF Research Database (Denmark)

    Wang, Ning; Agrawal, Ankita; Jørgensen, Niklas Rye

    2018-01-01

    Post-menopausal osteoporosis is a condition that affects millions worldwide and places a huge socio-economic burden on society. Previous research has shown an association of loss of function SNPs in the gene for the purinergic receptor P2X7R with low bone mineral density, increased rates of bone...... loss and vertebral fractures in post-menopausal women. In this study we use a mouse model of oestrogen deficiency-induced bone loss and the BALB/cJ P2X7R-/- to show that absence of the P2X7R resulted in increased bone loss. Osteoclast precursors were isolated from both BALB/cJ P2X7R-/- and BALB/cJ P2X7......R+/+ mice and then cultured in vitro to form mature resorbing osteoclasts. The BALB/cJ P2X7R-/- derived precursors generated slightly more osteoclasts but with a significant reduction in the amount of resorption per osteoclast. Furthermore, when using modified culture conditions osteoclast activity...

  1. The serotonin reuptake inhibitor citalopram suppresses activity in the neonatal rat barrel cortex in vivo.

    Science.gov (United States)

    Akhmetshina, Dinara; Zakharov, Andrei; Vinokurova, Daria; Nasretdinov, Azat; Valeeva, Guzel; Khazipov, Roustem

    2016-06-01

    Inhibition of serotonin uptake, which causes an increase in extracellular serotonin levels, disrupts the development of thalamocortical barrel maps in neonatal rodents. Previous in vitro studies have suggested that the disruptive effect of excessive serotonin on barrel map formation involves a depression at thalamocortical synapses. However, the effects of serotonin uptake inhibitors on the early thalamocortical activity patterns in the developing barrel cortex in vivo remain largely unknown. Here, using extracellular recordings of the local field potentials and multiple unit activity (MUA) we explored the effects of the selective serotonin reuptake inhibitor (SSRI) citalopram (10-20mg/kg, intraperitoneally) on sensory evoked activity in the barrel cortex of neonatal (postnatal days P2-5) rats in vivo. We show that administration of citalopram suppresses the amplitude and prolongs the delay of the sensory evoked potentials, reduces the power and frequency of the early gamma oscillations, and suppresses sensory evoked and spontaneous neuronal firing. In the adolescent P21-29 animals, citalopram affected neither sensory evoked nor spontaneous activity in barrel cortex. We suggest that suppression of the early thalamocortical activity patterns contributes to the disruption of the barrel map development caused by SSRIs and other conditions elevating extracellular serotonin levels. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Teramachi, Junpei [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto, Tokushima 770-8504 (Japan); Morimoto, Hiroyuki; Baba, Ryoko; Doi, Yoshiaki [Department of Anatomy, School of Medicine, University of Occupational and Environmental Health, Yahatanishi, Kitakyushu 807-8555 (Japan); Hirashima, Kanji [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto, Tokushima 770-8504 (Japan); Haneji, Tatsuji, E-mail: tat-hane@dent.tokushima-u.ac.jp [Department of Histology and Oral Histology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto, Tokushima 770-8504 (Japan)

    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

  3. Omega-3 free fatty acids suppress macrophage inflammasome activation by inhibiting NF-κB activation and enhancing autophagy.

    Directory of Open Access Journals (Sweden)

    Yolanda Williams-Bey

    Full Text Available The omega-3 (ω3 fatty acid docosahexaenoic acid (DHA can suppress inflammation, specifically IL-1β production through poorly understood molecular mechanisms. Here, we show that DHA reduces macrophage IL-1β production by limiting inflammasome activation. Exposure to DHA reduced IL-1β production by ligands that stimulate the NLRP3, AIM2, and NAIP5/NLRC4 inflammasomes. The inhibition required Free Fatty Acid Receptor (FFAR 4 (also known as GPR120, a G-protein coupled receptor (GPR known to bind DHA. The exposure of cells to DHA recruited the adapter protein β-arrestin1/2 to FFAR4, but not to a related lipid receptor. DHA treatment reduced the initial inflammasome priming step by suppressing the nuclear translocation of NF-κB. DHA also reduced IL-1β levels by enhancing autophagy in the cells. As a consequence macrophages derived from mice lacking the essential autophagy protein ATG7 were partially resistant to suppressive effects of DHA. Thus, DHA suppresses inflammasome activation by two distinct mechanisms, inhibiting the initial priming step and by augmenting autophagy, which limits inflammasome activity.

  4. Deep brain stimulation suppresses pallidal low frequency activity in patients with phasic dystonic movements.

    Science.gov (United States)

    Barow, Ewgenia; Neumann, Wolf-Julian; Brücke, Christof; Huebl, Julius; Horn, Andreas; Brown, Peter; Krauss, Joachim K; Schneider, Gerd-Helge; Kühn, Andrea A

    2014-11-01

    Deep brain stimulation of the globus pallidus internus alleviates involuntary movements in patients with dystonia. However, the mechanism is still not entirely understood. One hypothesis is that deep brain stimulation suppresses abnormally enhanced synchronized oscillatory activity within the motor cortico-basal ganglia network. Here, we explore deep brain stimulation-induced modulation of pathological low frequency (4-12 Hz) pallidal activity that has been described in local field potential recordings in patients with dystonia. Therefore, local field potentials were recorded from 16 hemispheres in 12 patients undergoing deep brain stimulation for severe dystonia using a specially designed amplifier allowing simultaneous high frequency stimulation at therapeutic parameter settings and local field potential recordings. For coherence analysis electroencephalographic activity (EEG) over motor areas and electromyographic activity (EMG) from affected neck muscles were recorded before and immediately after cessation of high frequency stimulation. High frequency stimulation led to a significant reduction of mean power in the 4-12 Hz band by 24.8 ± 7.0% in patients with predominantly phasic dystonia. A significant decrease of coherence between cortical EEG and pallidal local field potential activity in the 4-12 Hz range was revealed for the time period of 30 s after switching off high frequency stimulation. Coherence between EMG activity and pallidal activity was mainly found in patients with phasic dystonic movements where it was suppressed after high frequency stimulation. Our findings suggest that high frequency stimulation may suppress pathologically enhanced low frequency activity in patients with phasic dystonia. These dystonic features are the quickest to respond to high frequency stimulation and may thus directly relate to modulation of pathological basal ganglia activity, whereas improvement in tonic features may depend on long-term plastic changes within the

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

    OpenAIRE

    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

    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 ...

  6. Troglitazone inhibits endothelial cell proliferation through suppression of casein kinase 2 activity

    International Nuclear Information System (INIS)

    Lee, Kuy-Sook; Park, Jin-Hee; Lee, Seahyoung; Lim, Hyun-Joung; Jang, Yangsoo; Park, Hyun-Young

    2006-01-01

    Troglitazone, an agonist of peroxisome proliferator activated receptorγ (PPARγ), has been reported to inhibit endothelial cell proliferation by suppressing Akt activation. Recently, it has been also proposed that phosphatase and tensin homolog deleted from chromosome 10 (PTEN) plays an important role in such effect of troglitazone. However, the mechanism of how troglitazone regulates PTEN remains to be elucidated. We therefore investigated the effects of troglitazone on casein kinase 2 (CK2), which is known to negatively regulate PTEN activity. Troglitazone significantly inhibited serum-induced proliferation of HUVEC in a concentration dependent manner. Serum-induced Akt and its downstream signaling pathway activation was attenuated by troglitazone (10 μM) pretreatment. The phosphorylation of PTEN, which was directly related to Akt activation, was decreased with troglitazone pretreatment and was inversely proportional to CK2 activity. DRB, a CK2 inhibitor, also showed effects similar to that of troglitazone on Akt and its downstream signaling molecules. In conclusion, our results suggest that troglitazone inhibits proliferation of HUVECs through suppression of CK2 activity rendering PTEN to remain activated, and this effect of troglitazone in HUVECs seems to be PPARγ independent

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    Sundaram, Kumaran; Nishimura, Riko; Senn, Joseph; Youssef, Rimon F.; London, Steven D.; Reddy, Sakamuri V.

    2007-01-01

    Osteoclast differentiation is tightly regulated by receptor activator of NF-κB 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 in

  9. P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis.

    Science.gov (United States)

    Wang, Ning; Agrawal, Ankita; Jørgensen, Niklas Rye; Gartland, Alison

    2018-02-22

    Post-menopausal osteoporosis is a condition that affects millions worldwide and places a huge socio-economic burden on society. Previous research has shown an association of loss of function SNPs in the gene for the purinergic receptor P2X7R with low bone mineral density, increased rates of bone loss and vertebral fractures in post-menopausal women. In this study we use a mouse model of oestrogen deficiency-induced bone loss and the BALB/cJ P2X7R -/- to show that absence of the P2X7R resulted in increased bone loss. Osteoclast precursors were isolated from both BALB/cJ P2X7R -/- and BALB/cJ P2X7R +/+ mice and then cultured in vitro to form mature resorbing osteoclasts. The BALB/cJ P2X7R -/- derived precursors generated slightly more osteoclasts but with a significant reduction in the amount of resorption per osteoclast. Furthermore, when using modified culture conditions osteoclast activity was additionally increased in the absence of the P2X7R suggest that P2X7R may regulate the lifespan and activity of osteoclasts. Finally using mechanical loading as an anabolic stimulus for bone formation, we demonstrated that the increased oestrogen-deficient bone loss could be rescued, even in the absence of P2X7R. This study paves the way for clinical intervention for women with post-menopausal osteoporosis and P2XR7 loss of function polymorphisms.

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

    Directory of Open Access Journals (Sweden)

    Inês P. Perpétuo

    2017-01-01

    Full Text Available 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.

  11. Apparent suppression of MMP-9 activity by GD1a as determined by gelatin zymography.

    Science.gov (United States)

    Hu, Dan; Tan, Xuan; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2006-10-13

    Gelatin zymography is widely used to detect and evaluate matrix metalloproteinase-9 (MMP-9) activity. MMP-9 transcription was previously shown to be negatively regulated by ganglioside GD1a [D. Hu, Z. Man, T. Xuan, P. Wang, T. Takaku, S. Hyuga, X.S. Yao, T. Sato, S. Yamagata, T. Yamagata, Ganglioside GD1a regulation of matrix metalloproteinase-9 (MMP-9) expression in mouse FBJ cell Lines: GD1a suppression of MMP-9 expression stimulated by PI3K-Akt and p38 though not by the Erk signaling pathway, 2006, submitted for publication.]. Zymography of MMP-9 of FBJ-M5 cells preincubated with GD1a indicated a greater decrease in activity than expected from mRNA suppression. Incubation of conditioned medium containing MMP-9 with GD1a caused MMP-9 activity to decrease. Examination was thus made to confirm that MMP-9 activity is actually suppressed and/or MMP-9 protein undergoes degradation by GD1a. GD1a was found to have no effect on MMP-9 activity and Western blots indicated GD1a not to diminish MMP-9 during electrophoresis under reducing conditions. GD1a appeared to mediate the binding of a portion of MMP-9 with certain molecules, with consequently greater molecular mass on the gel, to cause decrease in the activity of MMP-9 at the site where it would normally appear. Caution should be used in doing gelatin zymography since molecules other than GD1a may similarly work, causing decrease in MMP-9 activity in zymography.

  12. Suppressed sympathetic outflow to skeletal muscle, muscle thermogenesis, and activity energy expenditure with calorie restriction.

    Science.gov (United States)

    Almundarij, Tariq I; Gavini, Chaitanya K; Novak, Colleen M

    2017-02-01

    During weight loss, adaptive thermogenesis occurs where energy expenditure (EE) is suppressed beyond that predicted for the smaller body size. Here, we investigated the contributions of resting and nonresting EE to the reduced total EE seen after 3 weeks of 50% calorie restriction (CR) in rats, focusing on activity-associated EE, muscle thermogenesis, and sympathetic outflow. Prolonged food restriction resulted in a 42% reduction in daily EE, through a 40% decrease in resting EE, and a 48% decline in nonresting EE These decreases in EE were significant even when the reductions in body weight and lean mass were taken into account. Along with a decreased caloric need for low-to-moderate-intensity treadmill activity with 50% CR, baseline and activity-related muscle thermogenesis were also suppressed, though the ability to increase muscle thermogenesis above baseline levels was not compromised. When sympathetic drive was measured by assessing norepinephrine turnover (NETO), 50% CR was found to decrease NETO in three of the four muscle groups examined, whereas elevated NETO was found in white adipose tissue of food-restricted rats. Central activation of melanocortin 4 receptors in the ventromedial hypothalamus stimulated this pathway, enhancing activity EE; this was not compromised by 50% CR These data suggest that suppressed activity EE contributes to adaptive thermogenesis during energy restriction. This may stem from decreased sympathetic drive to skeletal muscle, increasing locomotor efficiency and reducing skeletal muscle thermogenesis. The capacity to increase activity EE in response to central stimuli is retained, however, presenting a potential target for preventing weight regain. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  13. Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Sridhar

    2010-05-01

    Full Text Available Abstract Background Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene, a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established. Methods We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis. Results Resveratrol (100-150 μM exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G0/G1-S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway. Conclusions For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and

  14. Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways

    International Nuclear Information System (INIS)

    Vanamala, Jairam; Reddivari, Lavanya; Radhakrishnan, Sridhar; Tarver, Chris

    2010-01-01

    Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene), a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established. We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity) and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis. Resveratrol (100-150 μM) exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G 0 /G 1 -S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway. For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and activation of p53, suggesting its potential role as a

  15. The "love-hate" relationship between osteoclasts and bone matrix.

    Science.gov (United States)

    Rucci, Nadia; Teti, Anna

    2016-01-01

    Osteoclasts are unique cells that destroy the mineralized matrix of the skeleton. There is a "love-hate" relationship between the osteoclasts and the bone matrix, whereby the osteoclast is stimulated by the contact with the matrix but, at the same time, it disrupts the matrix, which, in turn, counteracts this disruption by some of its components. The balance between these concerted events brings about bone resorption to be controlled and to contribute to bone tissue integrity and skeletal health. The matrix components released by osteoclasts are also involved in the local regulation of other bone cells and in the systemic control of organismal homeostasis. Disruption of this regulatory loop causes bone diseases, which may end up with either reduced or increased bone mass, often associated with poor bone quality. Expanding the knowledge on osteoclast-to-matrix interaction could help to counteract these diseases and improve the human bone health. In this article, we will present evidence of the physical, molecular and regulatory relationships between the osteoclasts and the mineralized matrix, discussing the underlying mechanisms as well as their pathologic alterations and potential targeting. Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-08-04

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

  17. Active Vibration Suppression of a 3-DOF Flexible Parallel Manipulator Using Efficient Modal Control

    Directory of Open Access Journals (Sweden)

    Quan Zhang

    2014-01-01

    Full Text Available This paper addresses the dynamic modeling and efficient modal control of a planar parallel manipulator (PPM with three flexible linkages actuated by linear ultrasonic motors (LUSM. To achieve active vibration control, multiple lead zirconate titanate (PZT transducers are mounted on the flexible links as vibration sensors and actuators. Based on Lagrange’s equations, the dynamic model of the flexible links is derived with the dynamics of PZT actuators incorporated. Using the assumed mode method (AMM, the elastic motion of the flexible links are discretized under the assumptions of pinned-free boundary conditions, and the assumed mode shapes are validated through experimental modal test. Efficient modal control (EMC, in which the feedback forces in different modes are determined according to the vibration amplitude or energy of their own, is employed to control the PZT actuators to realize active vibration suppression. Modal filters are developed to extract the modal displacements and velocities from the vibration sensors. Numerical simulation and vibration control experiments are conducted to verify the proposed dynamic model and controller. The results show that the EMC method has the capability of suppressing multimode vibration simultaneously, and both the structural and residual vibrations of the flexible links are effectively suppressed using EMC approach.

  18. TET1 Suppresses Cancer Invasion by Activating the Tissue Inhibitors of Metalloproteinases

    Directory of Open Access Journals (Sweden)

    Chih-Hung Hsu

    2012-09-01

    Full Text Available Tumor suppressor gene silencing through cytosine methylation contributes to cancer formation. Whether DNA demethylation enzymes counteract this oncogenic effect is unknown. Here, we show that TET1, a dioxygenase involved in cytosine demethylation, is downregulated in prostate and breast cancer tissues. TET1 depletion facilitates cell invasion, tumor growth, and cancer metastasis in prostate xenograft models and correlates with poor survival rates in breast cancer patients. Consistently, enforced expression of TET1 reduces cell invasion and breast xenograft tumor formation. Mechanistically, TET1 suppresses cell invasion through its dioxygenase and DNA binding activities. Furthermore, TET1 maintains the expression of tissue inhibitors of metalloproteinase (TIMP family proteins 2 and 3 by inhibiting their DNA methylation. Concurrent low expression of TET1 and TIMP2 or TIMP3 correlates with advanced node status in clinical samples. Together, these results illustrate a mechanism by which TET1 suppresses tumor development and invasion partly through downregulation of critical gene methylation.

  19. USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

    Directory of Open Access Journals (Sweden)

    Zhenghong Lin

    2013-12-01

    Full Text Available The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

  20. Analysis of Harmonics Suppression by Active Damping Control on Multi Slim DC-link Drives

    DEFF Research Database (Denmark)

    Yang, Feng; Máthé, Lászlo; Lu, Kaiyuan

    2016-01-01

    Compared with conventional dc-link drive, slim dc-link drive is expected to achieve lower cost and longer life time. However, harmonics distortion problem may occur in such drive systems. This paper proposes to use an active damping control method to suppress the harmonic distortion...... with the benefit of low cost and also low loss. A new analysis method, based on the frequency domain impedance model, is presented to explore the mechanism of harmonics suppression. Also, a general method is presented to build the impedance model of a PMSM drive system using Field Oriented Control (FOC) method....... Some design issues, including power levels, current control bandwidth and harmonic interaction, are discussed when the drive system is fed by a weak grid. Case studies on a two-drive system composed by two slim dc-link drive units are provided to verify the proposed analysis method....

  1. Study on Active Suppression Control of Drivetrain Oscillations in an Electric Vehicle

    Science.gov (United States)

    Huang, Lei; Cui, Ying

    2017-07-01

    Due to the low damping in a central driven electric vehicle and lack of passive damping mechanisms as compared with a conventional vehicle, the vehicle may endure torsional vibrations which may deteriorates the vehicle’s drivability. Thus active damping control strategy is required to reduce the undesirable oscillations in an EV. In this paper, the origin of the vibration and the design of a damping control method to suppress such oscillations to improve the drivability of an EV are studied. The traction motor torque that is given by the vehicle controller is adjusted according to the acceleration rate of the motor speed to attenuate the resonant frequency. Simulations and experiments are performed to validate the system. The results show that the proposed control system can effectively suppress oscillations and hence improve drivability.

  2. Osteoimmunology and the influence of pro-inflammatory cytokines on osteoclasts

    Science.gov (United States)

    Zupan, Janja; Jeras, Matjaž; Marc, Janja

    2013-01-01

    Bone and immune system are functionally interconnected. Immune and bone cells derive from same progenitors in the bone marrow, they share a common microenvironment and are being influenced by similar mediators. The evidence on increased bone resorption associated with inappropriate activation of T cells such as during inflammation, is well established. However, the molecular mechanisms beyond this clinical observation have begun to be intensively studied with the advancement of osteoimmunology. Now days, we have firm evidence on the influence of numerous proinflammatory cytokines on bone cells, with the majority of data focused on osteoclasts, the bone resorbing cells. It has been shown that some proinflammatory cytokines could possess osteoclastogenic and/or anti-osteoclastogenic properties and can target osteoclasts directly or via receptor activator of nuclear factor κB (RANK)/RANK ligand(RANKL)/osteoprotegerin (OPG) system. Several studies have reported opposing data regarding (anti)osteoclastogenic properties of these cytokines. Therefore, the first part of this review is summarizing current evidence on the influence of pro-inflammatory cytokines on osteoclasts and thus on bone resorption. In the second part, the evidence on the role of pro-inflammatory cytokines in osteoporosis and osteoarthritis is reviewed to show that unravelling the mechanisms beyond such complex bone diseases, is almost impossible without considering skeletal and immune systems as an indivisible integrated system. PMID:23457765

  3. Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

    Science.gov (United States)

    Saito, Emi; Suzuki, Dai; Kurotaki, Daisuke; Mochizuki, Ayako; Manome, Yoko; Suzawa, Tetsuo; Toyoshima, Yoichi; Ichikawa, Takahiro; Funatsu, Takahiro; Inoue, Tomio; Takami, Masamichi; Tamura, Tomohiko; Inagaki, Katsunori; Kamijo, Ryutaro

    2017-06-01

    Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 -/- ) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 -/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+ ) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose

  4. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Fengbo [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Zhao, Bin; Zhang, Yang; Tian, Peng [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China); Li, Yanjun [Graduate School of Tianjin Medical University, No. 22, Qixiangtai Street, Heping District, Tianjin 300070 (China); Han, Zhe [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin TJ 300050 (China)

    2014-09-26

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

  5. Naringin prevents ovariectomy-induced osteoporosis and promotes osteoclasts apoptosis through the mitochondria-mediated apoptosis pathway

    International Nuclear Information System (INIS)

    Li, Fengbo; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhao, Bin; Zhang, Yang; Tian, Peng; Li, Yanjun; Han, Zhe

    2014-01-01

    Highlights: • Naringin possesses many pharmacological activities, promotes the proliferation of osteoblast. • Undecalcified histological obtain dynamic parameters of callus formation and remodeling. • Naringin regulate osteoclast apoptosis by mitochondrial pathway. - Abstract: Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats

  6. Chimaerin suppresses Rac1 activation at the apical membrane to maintain the cyst structure.

    Directory of Open Access Journals (Sweden)

    Shunsuke Yagi

    Full Text Available Epithelial organs are made of a well-polarized monolayer of epithelial cells, and their morphology is maintained strictly for their proper functions. Previously, we showed that Rac1 activation is suppressed at the apical membrane in the mature organoid, and that such spatially biased Rac1 activity is required for the polarity maintenance. Here we identify Chimaerin, a GTPase activating protein for Rac1, as a suppressor of Rac1 activity at the apical membrane. Depletion of Chimaerin causes over-activation of Rac1 at the apical membrane in the presence of hepatocyte growth factor (HGF, followed by luminal cell accumulation. Importantly, Chimaerin depletion did not inhibit extension formation at the basal membrane. These observations suggest that Chimaerin functions as the apical-specific Rac1 GAP to maintain epithelial morphology.

  7. Hybrid Active Filter with Variable Conductance for Harmonic Resonance Suppression in Industrial Power Systems

    DEFF Research Database (Denmark)

    Lee, Tzung-Lin; Wang, Yen-Ching; Li, Jian-Cheng

    2015-01-01

    Unintentional series and/or parallel resonances, due to the tuned passive filter and the line inductance, may result in severe harmonic distortion in the industrial power system. This paper presents a hybrid active filter to suppress harmonic resonance and reduce harmonic distortion as well......-tuned passive filter and an active filter in series connection, both dc voltage and kVA rating of the active filter are dramatically decreased compared with the pure shunt active filter. In real application, this feature is very attractive since the active power filter with fully power electronics is very....... The proposed hybrid filter is operated as variable harmonic conductance according to the voltage total harmonic distortion, so harmonic distortion can be reduced to an acceptable level in response to load change or parameter variation of power system. Since the hybrid filter is composed of a seventh...

  8. Endothelial cells suppress monocyte activation through secretion of extracellular vesicles containing antiinflammatory microRNAs.

    Science.gov (United States)

    Njock, Makon-Sébastien; Cheng, Henry S; Dang, Lan T; Nazari-Jahantigh, Maliheh; Lau, Andrew C; Boudreau, Emilie; Roufaiel, Mark; Cybulsky, Myron I; Schober, Andreas; Fish, Jason E

    2015-05-14

    The blood contains high concentrations of circulating extracellular vesicles (EVs), and their levels and contents are altered in several disease states, including cardiovascular disease. However, the function of circulating EVs, especially the microRNAs (miRNAs) that they contain, are poorly understood. We sought to determine the effect of secreted vesicles produced by quiescent endothelial cells (ECs) on monocyte inflammatory responses and to assess whether transfer of microRNAs occurs between these cells. We observed that monocytic cells cocultured (but not in contact) with ECs were refractory to inflammatory activation. Further characterization revealed that endothelium-derived EVs (EC-EVs) suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses. EVs isolated from mouse plasma also suppressed monocyte activation. Importantly, injection of EC-EVs in vivo repressed monocyte/macrophage activation, confirming our in vitro findings. We found that several antiinflammatory microRNAs were elevated in EC-EV-treated monocytes. In particular, miR-10a was transferred to monocytic cells from EC-EVs and could repress inflammatory signaling through the targeting of several components of the NF-κB pathway, including IRAK4. Our findings reveal that ECs secrete EVs that can modulate monocyte activation and suggest that altered EV secretion and/or microRNA content may affect vascular inflammation in the setting of cardiovascular disease. © 2015 by The American Society of Hematology.

  9. Principle and Control Design of Active Ground-Fault Arc Suppression Device for Full Compensation of Ground Current

    DEFF Research Database (Denmark)

    Wang, Wen; Zeng, Xiangjun; Yan, Lingjie

    2017-01-01

    . The commonly-used large-capacity reactive component may bring about overvoltage because of possible resonance with the distributed phase-to-ground capacitance. To solve these problems, an active ground-fault arc suppression device is presented. It employs a topology based on single-phase inverter to inject...... suppression without capacitive current detection. Its time-based feature also brings the benefit of fast response on ground-fault arc suppression. The principle of full current compensation is analyzed, together with the controller design method of the proposed device. Experiment on a prototype was carried......Traditional ground-fault arc suppression devices mainly deal with capacitive component of ground current and have weak effect on the active and harmonic ones, which limits the arc suppression performance. The capacitive current detection needed in them suffers from low accuracy and robustness...

  10. IL-6 alters osteocyte signaling toward osteoblasts but not osteoclasts.

    Science.gov (United States)

    Bakker, A D; Kulkarni, R N; Klein-Nulend, J; Lems, W F

    2014-04-01

    Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts.

  11. Fear conditioning suppresses large-conductance calcium-activated potassium channels in lateral amygdala neurons.

    Science.gov (United States)

    Sun, P; Zhang, Q; Zhang, Y; Wang, F; Wang, L; Yamamoto, R; Sugai, T; Kato, N

    2015-01-01

    It was previously shown that depression-like behavior is accompanied with suppression of the large-conductance calcium activated potassium (BK) channel in cingulate cortex pyramidal cells. To test whether BK channels are also involved in fear conditioning, we studied neuronal properties of amygdala principal cells in fear conditioned mice. After behavior, we made brain slices containing the amygdala, the structure critically relevant to fear memory. The resting membrane potential in lateral amygdala (LA) neurons obtained from fear conditioned mice (FC group) was more depolarized than in neurons from naïve controls. The frequencies of spikes evoked by current injections were higher in neurons from FC mice, demonstrating that excitability of LA neurons was elevated by fear conditioning. The depolarization in neurons from FC mice was shown to depend on BK channels by using the BK channel blocker charybdotoxin. Suppression of BK channels in LA neurons from the FC group was further confirmed on the basis of the spike width, since BK channels affect the descending phase of spikes. Spikes were broader in the FC group than those in the naïve control in a manner dependent on BK channels. Consistently, quantitative real-time PCR revealed a decreased expression of BK channel mRNA. The present findings suggest that emotional disorder manifested in the forms of fear conditioning is accompanied with BK channel suppression in the amygdala, the brain structure critical to this emotional disorder. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Osteoclastic finger arthrosis - a subtype of polyarthrosis of the hand; Osteoklastische Fingerarthrose - Subtyp der Handpolyarthrose

    Energy Technology Data Exchange (ETDEWEB)

    Dihlmann, W. [Radiologische Praxis, Hamburg-Barmbek (Germany); Dihlmann, A. [Berufsgenossenschaftliches Unfallkrankenhaus Hamburg (Germany)

    1998-02-01

    Aim: Description of a subtype of arthrosis deformans of the hand which is characterised as osteoclastic arthrosis. Patients and methods: Retrospective analysis of radiographs of the hands of 150 women and 100 men with radiological findings of arthrosis deformans. Results: 5% of women and 2% of men showed at least one digital joint with subchondral osteolysis of one or both articulating bones involving at least a third of the phalanx. This subchondral osteolysis far exceeds the cysts which are situated in the epiphyseal part of the articular region. It may develop within a year. Conclusion: Osteoclastic arthrosis of the finger is a subtype of polyarthrosis of the hand. Serial observations suggest that an osteoclast stimulating substance is produced by the cysts or arises directly from the synovial fluid; this enters the subchondral part of the bone through clefts which may or may not be visible radiologically and that this produces osteoclastic activity. The most important differential diagnoses are chronic tophacious gout and a benign tumor. (orig.) [Deutsch] Ziel: Beschreibung eines Subtyps der Arthrosis deformans an der Hand, der als osteoklastische Arthrose bezeichnet wird. Patienten und Methode: Retrospektive Analyse der Handroentgenaufnahmen von 150 Frauen und 100 Maennern mit Roentgenbefunden der Arthrosis deformans. Ergebnisse: 5% der Frauen und 2% der maennlichen Patienten des durchgesehenen Krankenguts zeigten an mindestens einem Fingergelenk eine Arthrose mit subchondralen Osteolysen an einem oder beiden artikulierenden Knochen, die mindestens ein Drittel der Phalanxlaenge erfasst hatten. Diese subchondralen Osteolysen gehen ueber die Groesse und Form der arthrotischen Geroellzysten, die lediglich im knoechernen (epiphysaeren) Gelenksockel sitzen, weit hinaus. Sie koennen innerhalb eines Jahres entstehen. Schlussfolgerung: Die osteoklastische Arthrose der Finger ist ein Subtyp der Handpolyarthrose. Nach Verlaufsbeobachtungen wird vermutet, dass eine

  13. IKKα Activation of NOTCH LinksTumorigenesis via FOXA2 Suppression

    Science.gov (United States)

    Liu, Mo; Lee, Dung-Fang; Chen, Chun-Te; Yen, Chia-Jui; Li, Long-Yuan; Lee, Hong-Jen; Chang, Chun-Ju; Chang, Wei-Chao; Hsu, Jung-Mao; Kuo, Hsu-Ping; Xia, Weiya; Wei, Yongkun; Chiu, Pei-Chun; Chou, Chao-Kai; Du, Yi; Dhar, Debanjan; Karin, Michael; Chen, Chung-Hsuan; Hung, Mien-Chie

    2011-01-01

    SUMMARY Pro-inflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/ S111, thereby suppressing FOXA2 transactivation activity, leading to decreased NUMB expression and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer. PMID:22196886

  14. The deacetylase Sirt6 activates the acetyltransferase GCN5 and suppresses hepatic gluconeogenesis.

    Science.gov (United States)

    Dominy, John E; Lee, Yoonjin; Jedrychowski, Mark P; Chim, Helen; Jurczak, Michael J; Camporez, Joao Paulo; Ruan, Hai-Bin; Feldman, Jessica; Pierce, Kerry; Mostoslavsky, Raul; Denu, John M; Clish, Clary B; Yang, Xiaoyong; Shulman, Gerald I; Gygi, Steven P; Puigserver, Pere

    2012-12-28

    Hepatic glucose production (HGP) maintains blood glucose levels during fasting but can also exacerbate diabetic hyperglycemia. HGP is dynamically controlled by a signaling/transcriptional network that regulates the expression/activity of gluconeogenic enzymes. A key mediator of gluconeogenic gene transcription is PGC-1α. PGC-1α's activation of gluconeogenic gene expression is dependent upon its acetylation state, which is controlled by the acetyltransferase GCN5 and the deacetylase Sirt1. Nevertheless, whether other chromatin modifiers-particularly other sirtuins-can modulate PGC-1α acetylation is currently unknown. Herein, we report that Sirt6 strongly controls PGC-1α acetylation. Surprisingly, Sirt6 induces PGC-1α acetylation and suppresses HGP. Sirt6 depletion decreases PGC-1α acetylation and promotes HGP. These acetylation effects are GCN5 dependent: Sirt6 interacts with and modifies GCN5, enhancing GCN5's activity. Lepr(db/db) mice, an obese/diabetic animal model, exhibit reduced Sirt6 levels; ectopic re-expression suppresses gluconeogenic genes and normalizes glycemia. Activation of hepatic Sirt6 may therefore be therapeutically useful for treating insulin-resistant diabetes. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Osteoclast abnormalities in fractured bone during bisphosphonate treatment for osteoporosis: a case report.

    Science.gov (United States)

    Vigorita, V J V; Silver, J S; Eisemon, E O E

    2012-07-01

    Bisphosphonates have been widely used in the treatment of an array of bone disorders. Recent complications have included unusual femoral fractures in patients who have received long term bisphosphonate treatment for osteoporosis. Although it has been shown that bisphosphonates are effective by blunting osteoclast resorption, there has been little morphologic description of the local tissue activity at the site of these unusual fractures. To evaluate for local changes to bone morphology at the fracture site in patients presenting with a bisphosphonate-related femur fracture, a sample of cortical bone was obtained at the site of a bisphosphonate fracture and was processed in a nondecalcified manner. The specimen was evaluated for potential cellular changes consistent with bisphosphonate treatment. Significant osteoclast abnormalities at the fracture site were found in a 69-year-old woman treated for 2 years with Fosamax substantiating that bone remodeling at this site is distinctly abnormal. Addressing the osteoclast dysfunction should be a focus of future therapeutic attention and intervention.

  16. Glucocorticoids: Dose-related effects on osteoclast formation and function via reactive oxygen species and autophagy.

    Science.gov (United States)

    Shi, Jun; Wang, Long; Zhang, Hongyang; Jie, Qiang; Li, Xiaojie; Shi, Qiyue; Huang, Qiang; Gao, Bo; Han, Yuehu; Guo, Kai; Liu, Jian; Yang, Liu; Luo, Zhuojing

    2015-10-01

    Whether glucocorticoids directly enhance or interrupt osteoclastogenesis is still a controversial subject. In this study, we ascertained the dose-dependent positive effects of glucocorticoids on osteoclastogenesis in vivo and in vitro as well as investigated the mechanism in vitro. As the dose of glucocorticoids increased, osteoclastogenesis was stimulated at 0.1 μM, a peak was achieved at 1 μM and a corresponding decrease occurred at 10 μM. Reactive oxygen species (ROS), which play a crucial role in osteoclastogenesis, and autophagy flux activity, a cellular recycling process, were consistently up-regulated along with the dose-dependent effects of the glucocorticoids on osteoclast formation and function. N-acetyl-cysteine (NAC), a ROS scavenger, abrogated the effects of the glucocorticoids on autophagy and osteoclastogenesis. Moreover, 3-methyladenine (3-MA), an autophagy inhibitor, interrupted osteoclastogenesis stimulation by the glucocorticoids. These results implied that with glucocorticoid administration, ROS and autophagy, as a downstream factor of ROS, played vital roles in osteoclast formation and function. 3-MA administration did not enhance ROS accumulation, so that autophagy had no effect on ROS induced by glucocorticoids. Our investigation demonstrated that glucocorticoids had dose-dependent positive effects on osteoclast formation and function via ROS and autophagy. These results provide support for ROS and autophagy as therapeutic targets in glucocorticoid-related bone loss diseases such as glucocorticoid-induced osteoporosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Substrate adhesion regulates sealing zone architecture and dynamics in cultured osteoclasts.

    Directory of Open Access Journals (Sweden)

    Fabian Anderegg

    Full Text Available The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ. The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data.

  18. Neutrophil-derived MRP-14 is up-regulated in infectious osteomyelitis and stimulates osteoclast generation.

    Science.gov (United States)

    Dapunt, Ulrike; Giese, Thomas; Maurer, Susanne; Stegmaier, Sabine; Prior, Birgit; Hänsch, G Maria; Gaida, Matthias M

    2015-10-01

    Bone infections of patients with joint replacement by endoprosthesis (so called "periprosthetic joint infection") pose a severe problem in the field of orthopedic surgery. The diagnosis is often difficult, and treatment is, in most cases, complicated and prolonged. Patients often require an implant exchange surgery, as the persistent infection and the accompanying inflammation lead to tissue damage with bone degradation and consequently, to a loosening of the implant. To gain insight into the local inflammatory process, expression of the proinflammatory cytokine MRP-14, a major content of neutrophils, and its link to subsequent bone degradation was evaluated. We found MRP-14 prominently expressed in the affected tissue of patients with implant-associated infection, in close association with the chemokine CXCL8 and a dense infiltrate of neutrophils and macrophages. In addition, the number of MRP-14-positive cells correlated with the presence of bone-resorbing osteoclasts. MRP-14 plasma concentrations were significantly higher in patients with implant-associated infection compared with patients with sterile inflammation or healthy individuals, advocating MRP-14 as a novel diagnostic marker. A further biologic activity of MRP-14 was detected: rMRP-14 directly induced the differentiation of monocytes to osteoclasts, thus linking the inflammatory response in implant infections with osteoclast generation, bone degradation, and implant loosening. © Society for Leukocyte Biology.

  19. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    Science.gov (United States)

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. PCAF Improves Glucose Homeostasis by Suppressing the Gluconeogenic Activity of PGC-1α

    Directory of Open Access Journals (Sweden)

    Cheng Sun

    2014-12-01

    Full Text Available PGC-1α plays a central role in hepatic gluconeogenesis and has been implicated in the onset of type 2 diabetes. Acetylation is an important posttranslational modification for regulating the transcriptional activity of PGC-1α. Here, we show that PCAF is a pivotal acetyltransferase for acetylating PGC-1α in both fasted and diabetic states. PCAF acetylates two lysine residues K328 and K450 in PGC-1α, which subsequently triggers its proteasomal degradation and suppresses its transcriptional activity. Adenoviral-mediated expression of PCAF in the obese mouse liver greatly represses gluconeogenic enzyme activation and glucose production and improves glucose homeostasis and insulin sensitivity. Moreover, liver-specific knockdown of PCAF stimulates PGC-1α activity, resulting in an increase in blood glucose and hepatic glucose output. Our results suggest that PCAF might be a potential pharmacological target for developing agents against metabolic disorders associated with hyperglycemia, such as obesity and diabetes.

  1. Osteoclastic differentiation and resorption is modulated by bioactive metal ions Co2+, Cu2+ and Cr3+ incorporated into calcium phosphate bone cements

    Science.gov (United States)

    Bernhardt, Anne; Schamel, Martha; Gbureck, Uwe; Gelinsky, Michael

    2017-01-01

    Biologically active metal ions in low doses have the potential to accelerate bone defect healing. For successful remodelling the interaction of bone graft materials with both bone-forming osteoblasts and bone resorbing osteoclasts is crucial. In the present study brushite forming calcium phosphate cements (CPC) were doped with Co2+, Cu2+ and Cr3+ and the influence of these materials on osteoclast differentiation and activity was examined. Human osteoclasts were differentiated from human peripheral blood mononuclear cells (PBMC) both on the surface and in indirect contact to the materials on dentin discs. Release of calcium, phosphate and bioactive metal ions was determined using ICP-MS both in the presence and absence of the cells. While Co2+ and Cu2+ showed a burst release, Cr3+ was released steadily at very low concentrations (below 1 μM) and both calcium and phosphate release of the cements was considerably changed in the Cr3+ modified samples. Direct cultivation of PBMC/osteoclasts on Co2+ cements showed lower attached cell number compared to the reference but high activity of osteoclast specific enzymes tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK) and significantly increased gene expression of vitronectin receptor. Indirect cultivation with diluted Co2+ cement extracts revealed highest resorbed area compared to all other modifications and the reference. Cu2+ cements had cytotoxic effect on PBMC/osteoclasts during direct cultivation, while indirect cultivation with diluted extracts from Cu2+ cements did not provoke cytotoxic effects but a strictly inhibited resorption. Cr3+ doped cements did not show cytotoxic effects at all. Gene expression and enzyme activity of CTSK was significantly increased in direct culture. Indirect cultivation with Cr3+ doped cements revealed significantly higher resorbed area compared to the reference. In conclusion Cr3+ doped calcium phosphate cements are an innovative cement

  2. Study of exteroceptive suppression of voluntary muscular activity in healthy volunteers and patients with paroxysmal neuropathic pain.

    Science.gov (United States)

    Gordeev, S A; Turbina, L G; Shtang, O M

    2014-07-01

    The exteroceptive suppression of voluntary electromyographic activity of the masseter and temporal muscles was studied in healthy volunteers and patients with paroxysmal neuropathic pain (trigeminal neuralgia). The latent period of the exteroceptive suppression was prolonged and the duration of its late fragment was shortened in the patients in comparison with normal subjects. A short exteroceptive suppression period in patients with trigeminal neuralgia reflected deficient activity of inhibitory interneurons of the reflector loop and excessive activity of the antinociceptive system of the brain stem, while prolongation of the latent period reflected prolongation of inhibitory interneurons activation. A direct correlation between the degree of changes in the exteroceptive suppression parameters and pain intensity, evaluated by the patients by the visual analog scale, was detected.

  3. Functional connectivity in raphé-pontomedullary circuits supports active suppression of breathing during hypocapnic apnea

    Science.gov (United States)

    Nuding, Sarah C.; Segers, Lauren S.; Iceman, Kimberly E.; O'Connor, Russell; Dean, Jay B.; Bolser, Donald C.; Baekey, David M.; Dick, Thomas E.; Shannon, Roger; Morris, Kendall F.

    2015-01-01

    Hyperventilation is a common feature of disordered breathing. Apnea ensues if CO2 drive is sufficiently reduced. We tested the hypothesis that medullary raphé, ventral respiratory column (VRC), and pontine neurons have functional connectivity and persistent or evoked activities appropriate for roles in the suppression of drive and rhythm during hyperventilation and apnea. Phrenic nerve activity, arterial blood pressure, end-tidal CO2, and other parameters were monitored in 10 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated cats. Multielectrode arrays recorded spiking activity of 649 neurons. Loss and return of rhythmic activity during passive hyperventilation to apnea were identified with the S-transform. Diverse fluctuating activity patterns were recorded in the raphé-pontomedullary respiratory network during the transition to hypocapnic apnea. The firing rates of 160 neurons increased during apnea; the rates of 241 others decreased or stopped. VRC inspiratory neurons were usually the last to cease firing or lose rhythmic activity during the transition to apnea. Mayer wave-related oscillations (0.04–0.1 Hz) in firing rate were also disrupted during apnea. Four-hundred neurons (62%) were elements of pairs with at least one hyperventilation-responsive neuron and a correlational signature of interaction identified by cross-correlation or gravitational clustering. Our results support a model with distinct groups of chemoresponsive raphé neurons contributing to hypocapnic apnea through parallel processes that incorporate disfacilitation and active inhibition of inspiratory motor drive by expiratory neurons. During apnea, carotid chemoreceptors can evoke rhythm reemergence and an inspiratory shift in the balance of reciprocal inhibition via suppression of ongoing tonic expiratory neuron activity. PMID:26203111

  4. Opiate-induced suppression of rat hypoglossal motoneuron activity and its reversal by ampakine therapy.

    Directory of Open Access Journals (Sweden)

    Amanda R Lorier

    2010-01-01

    Full Text Available Hypoglossal (XII motoneurons innervate tongue muscles and are vital for maintaining upper-airway patency during inspiration. Depression of XII nerve activity by opioid analgesics is a significant clinical problem, but underlying mechanisms are poorly understood. Currently there are no suitable pharmacological approaches to counter opiate-induced suppression of XII nerve activity while maintaining analgesia. Ampakines accentuate alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA receptor responses. The AMPA family of glutamate receptors mediate excitatory transmission to XII motoneurons. Therefore the objectives were to determine whether the depressant actions of mu-opioid receptor activation on inspiratory activity includes a direct inhibitory action at the inspiratory premotoneuron to XII motoneuron synapse, and to identify underlying mechanism(s. We then examined whether ampakines counteract opioid-induced depression of XII motoneuron activity.A medullary slice preparation from neonatal rat that produces inspiratory-related output in vitro was used. Measurements of inspiratory burst amplitude and frequency were made from XII nerve roots. Whole-cell patch recordings from XII motoneurons were used to measure membrane currents and synaptic events. Application of the mu-opioid receptor agonist, DAMGO, to the XII nucleus depressed the output of inspiratory XII motoneurons via presynaptic inhibition of excitatory glutamatergic transmission. Ampakines (CX614 and CX717 alleviated DAMGO-induced depression of XII MN activity through postsynaptic actions on XII motoneurons.The inspiratory-depressant actions of opioid analgesics include presynaptic inhibition of XII motoneuron output. Ampakines counteract mu-opioid receptor-mediated depression of XII motoneuron inspiratory activity. These results suggest that ampakines may be beneficial in countering opiate-induced suppression of XII motoneuron activity and resultant impairment of airway patency.

  5. [Observation of osteoclasts on the root surface during human deciduous teeth resorption].

    Science.gov (United States)

    Bao, Xiang-jun; Liang, Xing; Chen, Ming; Wang, Hang; Xie, Zhi-gang; Yang, Xiao-yu

    2004-08-01

    To observe osteoclasts on the resorbing surface of human deciduous teeth. After fixing the collected deciduous teeth, we prepared the tooth slices without decalcification, treated them with HE and TRAP dyestuff, and observed the osteoclasts under light and scanning electron microscope. There were large quantity of various forms of overlapping and huge osteoclasts with many nuclei and silk-like protuberances on the resorbing surface of deciduous teeth. The multinucleated osteoclasts align on the surface of coarse dentin. On the resorbing surface of human deciduous teeth there are large amount of osteoclasts which can be used as a source of studying human osteoclast.

  6. Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival

    DEFF Research Database (Denmark)

    Nielsen, Rasmus H; Karsdal, Morten A; Sørensen, Mette G

    2007-01-01

    osteoclast numbers, indicating increased life span of the osteoclasts. To investigate the role of the inorganic phase of bone with respect to osteoclast life span, we used the V-ATPase inhibitor bafilomycin and the calcium uptake antagonist ryanodine on human osteoclasts cultured on calcified and decalcified...... bone slices. Bafilomycin inhibited bone resorption and increased osteoclast survival on calcified but not decalcified bones. Ryanodine attenuated calcium uptake and thereby augmented osteoclast survival on calcified bones. In summary, we found that acidification leading to calcium release from bone...

  7. Scutellarin Suppresses NLRP3 Inflammasome Activation in Macrophages and Protects Mice against Bacterial Sepsis.

    Science.gov (United States)

    Liu, Yi; Jing, Yan-Yun; Zeng, Chen-Ying; Li, Chen-Guang; Xu, Li-Hui; Yan, Liang; Bai, Wen-Jing; Zha, Qing-Bing; Ouyang, Dong-Yun; He, Xian-Hui

    2017-01-01

    The NLRP3 inflammasome plays a critical role in mediating the innate immune defense against pathogenic infections, but aberrant activation of NLRP3 inflammasome has been linked to a variety of inflammatory diseases. Thus targeting the NLRP3 inflammasome represents a promising therapeutic for the treatment of such diseases. Scutellarin is a flavonoid isolated from Erigeron breviscapus (Vant.) Hand.-Mazz. and has been reported to exhibit potent anti-inflammatory activities, but the underlying mechanism is only partly understood. In this study, we aimed to investigate whether scutellarin could affect the activation of NLRP3 inflammasome in macrophages. The results showed that scutellarin dose-dependently reduced caspase-1 activation and decreased mature interleukin-1β (IL-1β) release in lipopolysaccharide (LPS)-primed macrophages upon ATP or nigericin stimulation, indicating that scutellarin inhibited NLRP3 inflammasome activation in macrophages. Consistent with this, scutellarin also suppressed pyroptotic cell death in LPS-primed macrophages treated with ATP or nigericin. ATP or nigericin-induced ASC speck formation and its oligomerization were blocked by scutellarin pre-treatment. Intriguingly, scutellarin augmented PKA-specific phosphorylation of NLRP3 in LPS-primed macrophages, which was completely blocked by selective PKA inhibitor H89, suggesting that PKA signaling had been involved in the action of scutellarin to suppress NLRP3 inflammasome activation. Supporting this, the inhibitory effect of scutellarin on NLRP3 inflammasome activation was completely counteracted by H89 or adenyl cyclase inhibitor MDL12330A. As NLRP3-dependent release of IL-1β has a critical role in sepsis, the in vivo activity of scutellarin was assayed in a mouse model of bacterial sepsis, which was established by intraperitoneally injection of a lethal dose of viable Escherichia coli . Oral administration of scutellarin significantly improved the survival of mice with bacterial sepsis

  8. Scutellarin Suppresses NLRP3 Inflammasome Activation in Macrophages and Protects Mice against Bacterial Sepsis

    Directory of Open Access Journals (Sweden)

    Yi Liu

    2018-01-01

    Full Text Available The NLRP3 inflammasome plays a critical role in mediating the innate immune defense against pathogenic infections, but aberrant activation of NLRP3 inflammasome has been linked to a variety of inflammatory diseases. Thus targeting the NLRP3 inflammasome represents a promising therapeutic for the treatment of such diseases. Scutellarin is a flavonoid isolated from Erigeron breviscapus (Vant. Hand.-Mazz. and has been reported to exhibit potent anti-inflammatory activities, but the underlying mechanism is only partly understood. In this study, we aimed to investigate whether scutellarin could affect the activation of NLRP3 inflammasome in macrophages. The results showed that scutellarin dose-dependently reduced caspase-1 activation and decreased mature interleukin-1β (IL-1β release in lipopolysaccharide (LPS-primed macrophages upon ATP or nigericin stimulation, indicating that scutellarin inhibited NLRP3 inflammasome activation in macrophages. Consistent with this, scutellarin also suppressed pyroptotic cell death in LPS-primed macrophages treated with ATP or nigericin. ATP or nigericin-induced ASC speck formation and its oligomerization were blocked by scutellarin pre-treatment. Intriguingly, scutellarin augmented PKA-specific phosphorylation of NLRP3 in LPS-primed macrophages, which was completely blocked by selective PKA inhibitor H89, suggesting that PKA signaling had been involved in the action of scutellarin to suppress NLRP3 inflammasome activation. Supporting this, the inhibitory effect of scutellarin on NLRP3 inflammasome activation was completely counteracted by H89 or adenyl cyclase inhibitor MDL12330A. As NLRP3-dependent release of IL-1β has a critical role in sepsis, the in vivo activity of scutellarin was assayed in a mouse model of bacterial sepsis, which was established by intraperitoneally injection of a lethal dose of viable Escherichia coli. Oral administration of scutellarin significantly improved the survival of mice with

  9. Activation of peroxisome proliferator-activated receptor-α (PPARα) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

    International Nuclear Information System (INIS)

    Kimura, Rino; Takahashi, Nobuyuki; Murota, Kaeko; Yamada, Yuko; Niiya, Saori; Kanzaki, Noriyuki; Murakami, Yoko; Moriyama, Tatsuya; Goto, Tsuyoshi; Kawada, Teruo

    2011-01-01

    Highlights: → PPARα activation increased mRNA expression levels of fatty acid oxidation-related genes in human intestinal epithelial Caco-2 cells. → PPARα activation also increased oxygen consumption rate and CO 2 production and decreased secretion of triglyceride and ApoB from Caco-2 cells. → Orally administration of bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and CO 2 production in small intestinal epithelial cells. → Treatment with bezafibrate decreased postprandial serum concentration of triglyceride after oral injection of olive oil in mice. → It suggested that intestinal lipid metabolism regulated by PPARα activation suppresses postprandial lipidemia. -- Abstract: Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO 2 and acid soluble metabolites in enterocytes. Moreover

  10. Multirate Flutter Suppression System Design for the Benchmark Active Controls Technology Wing. Part 2; Methodology Application Software Toolbox

    Science.gov (United States)

    Mason, Gregory S.; Berg, Martin C.; Mukhopadhyay, Vivek

    2002-01-01

    To study the effectiveness of various control system design methodologies, the NASA Langley Research Center initiated the Benchmark Active Controls Project. In this project, the various methodologies were applied to design a flutter suppression system for the Benchmark Active Controls Technology (BACT) Wing. This report describes the user's manual and software toolbox developed at the University of Washington to design a multirate flutter suppression control law for the BACT wing.

  11. Activation of the aryl hydrocarbon receptor suppresses sensitization in a mouse peanut allergy model.

    Science.gov (United States)

    Schulz, V J; Smit, J J; Willemsen, K J; Fiechter, D; Hassing, I; Bleumink, R; Boon, L; van den Berg, M; van Duursen, M B M; Pieters, R H H

    2011-10-01

    Food allergy is an increasing health problem in Western countries. Previously, it has been shown that the intensity of food allergic reactions can be regulated by regulatory T (T(reg)) cells. In addition, it has been shown that activation of the aryl hydrocarbon receptor (AhR) regulates T-cell responses by induction of T(reg) cells. Therefore, we hypothesized that activation of the AhR pathway can suppress development of food allergic responses through the induction of T(reg) cells. This was investigated by using a mouse model for peanut allergy. C3H/HeOuJ mice (AhR(b)(-2)) were sensitized to peanut by administering peanut extract (PE) by gavage in the presence of cholera toxin and were treated with the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.6, 1.7, 5, and 15 μg/kg body weight) on days 3 and 11 orally. The functional role of CD4(+)CD25(+)Foxp3(+) T(reg) cells was investigated by depleting these cells with anti-CD25 mAb during sensitization to PE. TCDD treatment dose dependently suppressed sensitization to peanut (PE-specific IgE, IgG1, and IgG2a and PE-induced IL-5, IL-10, and IL-13, respectively). The percentage, but not the number, of CD4(+)CD25(+)Foxp3(+) T(reg) cells dose dependently increased by AhR activation in both spleen and mesenteric lymph nodes. Depletion of CD4(+)CD25(+)Foxp3(+) T(reg) cells markedly reversed the suppressive effect of TCDD on PE-specific antibody levels and PE-induced IL-5, IL-10, and IL-13 cytokine production. Present data demonstrate for the first time that activation of the AhR by TCDD suppressed the development of Th2-mediated food allergic responses. A functional shift within the CD4(+) cell population toward CD4(+)CD25(+)Foxp3(+) T(reg) cells appeared to underlie this effect. This suggests that the AhR pathway might provide potential therapeutic targets to treat food allergic diseases.

  12. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage.

    OpenAIRE

    Friedman, A; Weiner, H L

    1994-01-01

    Oral tolerance was generated to hen egg white lysozyme in the mouse or to guinea pig myelin basic protein in the rat by a low-dose (1 mg) or a high-dose (5-20 mg) feeding regimen. High doses of antigen induced tolerance characterized by anergy with little or no active suppression and increased secretion of interleukin 4 (IL-4). Anergy was shown by an increase in frequency of IL-2-secreting cells following culture in recombinant IL-2. Low doses of antigen induced tolerance characterized by ant...

  13. Cholesterol synthesis inhibitor RO 48-8071 suppresses transcriptional activity of human estrogen and androgen receptor.

    Science.gov (United States)

    Mafuvadze, Benford; Liang, Yayun; Hyder, Salman M

    2014-10-01

    Breast cancer cells express enzymes that convert cholesterol, the synthetic precursor of steroid hormones, into estrogens and androgens, which then drive breast cancer cell proliferation. In the present study, we sought to determine whether oxidosqualene cyclase (OSC), an enzyme in the cholesterol biosynthetic pathway, may be targeted to suppress progression of breast cancer cells. In previous studies, we showed that the OSC inhibitor RO 48-8071 (RO) may be a ligand which could potentially be used to control the progression of estrogen receptor-α (ERα)-positive breast cancer cells. Herein, we showed, by real-time PCR analysis of mRNA from human breast cancer biopsies, no significant differences in OSC expression at various stages of disease, or between tumor and normal mammary cells. Since the growth of hormone-responsive tumors is ERα-dependent, we conducted experiments to determine whether RO affects ERα. Using mammalian cells engineered to express human ERα or ERβ protein, together with an ER-responsive luciferase promoter, we found that RO dose-dependently inhibited 17β-estradiol (E2)-induced ERα responsive luciferase activity (IC50 value, ~10 µM), under conditions that were non-toxic to the cells. RO was less effective against ERβ-induced luciferase activity. Androgen receptor (AR) mediated transcriptional activity was also reduced by RO. Notably, while ERα activity was reduced by atorvastatin, the HMG-CoA reductase inhibitor did not influence AR activity, showing that RO possesses broader antitumor properties. Treatment of human BT-474 breast cancer cells with RO reduced levels of estrogen-induced PR protein, confirming that RO blocks ERα activity in tumor cells. Our findings demonstrate that an important means by which RO suppresses hormone-dependent growth of breast cancer cells is through its ability to arrest the biological activity of ERα. This warrants further investigation of RO as a potential therapeutic agent for use against hormone

  14. Cellulase activity as a mechanism for suppression of phytophthora root rot in mulches.

    Science.gov (United States)

    Richter, Brantlee Spakes; Ivors, Kelly; Shi, Wei; Benson, D M

    2011-02-01

    Wood-based mulches are used in avocado production and are being tested on Fraser fir for reduction of Phytophthora root rot, caused by Phytophthora cinnamomi. Research with avocado has suggested a role of microbial cellulase enzymes in pathogen suppression through effects on the cellulosic cell walls of Phytophthora. This work was conducted to determine whether cellulase activity could account for disease suppression in mulch systems. A standard curve was developed to correlate cellulase activity in mulches with concentrations of a cellulase product. Based on this curve, cellulase activity in mulch samples was equivalent to a cellulase enzyme concentration of 25 U ml(-1) or greater of product. Sustained exposure of P. cinnamomi to cellulase at 10 to 50 U ml(-1) significantly reduced sporangia production, but biomass was only reduced with concentrations over 100 U ml(-1). In a lupine bioassay, cellulase was applied to infested soil at 100 or 1,000 U ml(-1) with three timings. Cellulase activity diminished by 47% between 1 and 15 days after application. Cellulase applied at 100 U ml(-1) 2 weeks before planting yielded activity of 20.08 μmol glucose equivalents per gram of soil water (GE g(-1) aq) at planting, a level equivalent to mulch samples. Cellulase activity at planting ranged from 3.35 to 48.67 μmol GE g(-1) aq, but no treatment significantly affected disease progress. Based on in vitro assays, cellulase activity in mulch was sufficient to impair sporangia production of P. cinnamomi, but not always sufficient to impact vegetative biomass.

  15. Magnetic suppression of turbulence and the star formation activity of molecular clouds

    Science.gov (United States)

    Zamora-Avilés, Manuel; Vázquez-Semadeni, Enrique; Körtgen, Bastian; Banerjee, Robi; Hartmann, Lee

    2018-03-01

    We present magnetohydrodynamic simulations aimed at studying the effect of the magnetic suppression of turbulence (generated through various instabilities during the formation of molecular clouds by converging) on the subsequent star formation (SF) activity. We study four magnetically supercritical models with magnetic field strengths B = 0, 1, 2, and 3 μG (corresponding to mass-to-flux ratios of ∞, 4.76, 2.38, and 1.59 times the critical value), with the magnetic field, initially being aligned with the flows. We find that, for increasing magnetic field strength, the clouds formed tend to be more massive, denser, less turbulent, and with higher SF activity. This causes the onset of SF activity in the non-magnetic or more weakly magnetized cases to be delayed by a few Myr in comparison to the more strongly magnetized cases. We attribute this behaviour to the suppression of the non-linear thin shell instability (NTSI) by the magnetic field, previously found by Heitsch and coworkers. This result is contrary to the standard notion that the magnetic field provides support to the clouds, thus reducing their star formation rate. However, our result is a completely non-linear one, and could not be foreseen from simple linear considerations.

  16. Folliculin contributes to VHL tumor suppressing activity in renal cancer through regulation of autophagy.

    Directory of Open Access Journals (Sweden)

    Prabhat Bastola

    Full Text Available Von Hippel-Lindau tumor suppressor (VHL is lost in the majority of clear cell renal cell carcinomas (ccRCC. Folliculin (FLCN is a tumor suppressor whose function is lost in Birt-Hogg-Dubé syndrome (BHD, a disorder characterized by renal cancer of multiple histological types including clear cell carcinoma, cutaneous fibrofolliculoma, and pneumothorax. Here we explored whether there is connection between VHL and FLCN in clear cell renal carcinoma cell lines and tumors. We demonstrate that VHL regulates expression of FLCN at the mRNA and protein levels in RCC cell lines, and that FLCN protein expression is decreased in human ccRCC tumors with VHL loss, as compared with matched normal kidney tissue. Knockdown of FLCN results in increased formation of tumors by RCC cells with wild-type VHL in orthotopic xenografts in nude mice, an indication that FLCN plays a role in the tumor-suppressing activity of VHL. Interestingly, FLCN, similarly to VHL, is necessary for the activity of LC3C-mediated autophagic program that we have previously characterized as contributing to the tumor suppressing activity of VHL. The results show the existence of functional crosstalk between two major tumor suppressors in renal cancer, VHL and FLCN, converging on regulation of autophagy.

  17. Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads.

    Science.gov (United States)

    Rando, R F; Ojwang, J; Elbaggari, A; Reyes, G R; Tinder, R; McGrath, M S; Hogan, M E

    1995-01-27

    An oligonucleotide (I100-15) composed of only deoxyguanosine and thymidine was able to inhibit human immunodeficiency virus type-1 (HIV-1) in culture assay systems. I100-15 did not block virus entry into cells but did reduce viral-specific transcripts. As assessed by NMR and polyacrylamide gel methods, I100-15 appears to form a structure in which two stacked guanosine tetrads are connected by three two-base long loops. Structure/activity experiments indicated that formation of intramolecular guanosine tetrads was necessary to achieve maximum antiviral activity. The single deoxyguanosine nucleotide present in each loop was found to be extremely important for the overall antiviral activity. The toxicity of I100-15 was determined to be well above the 50% effective dose (ED50) in culture which yielded a high therapeutic index (> 100). The addition of a cholesterol moiety to the 3' terminus of I100-15 (I100-23) reduced the ED50 value to less than 50 nM (from 0.12 microM for I100-15) and increased the duration of viral suppression to greater than 21 days (versus 7-10 days for I100-15) after removal of the drug from infected cell cultures. The favorable therapeutic index of such molecules coupled with the prolonged suppression of HIV-1, suggest that such compounds further warrant investigation as potential therapeutic agents.

  18. Structural requirements for the action of parathyroid hormone-related protein (PTHrP) on bone resorption by isolated osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Evely, R.S.; Bonomo, A.; Schneider, H.G.; Moseley, J.M.; Gallagher, J.; Martin, T.J. (St. Vincent' s Institute of Medical Research, Melbourne (Australia))

    1991-01-01

    Parathyroid hormone-related protein (PTHrP) plays a major role in the syndrome of humoral hypercalcemia of malignancy (HHM) by its actions on bone and kidney. In this study an isolated osteoclast bone resorption assay was used to investigate the actions of this peptide and the structure-activity relationships for its resorption effect. As with PTH, neither synthetic nor recombinant PTHrP preparations stimulated resorption within highly purified osteoclast populations. Resorption was stimulated only in the presence of contaminating osteoblasts or in cocultures with the osteoblast-like cell line UMR-106. In the presence of osteoblasts PTHrP-(1-34) and PTHrP-(1-84) stimulated bone resorption in a dose-dependent manner with a potency comparable to that of PTH-(1-34) on a molar basis. The biologic activity of the PTHrP was shown to reside in the first 34 amino acids, and within that region the structural requirements for promotion of osteoclastic resorption resembled closely those for promotion of cyclic AMP formation in osteoblast-like cells. Using emulsion autoradiography with iodinated PTHrP-(1-34) and PTHrP-(1-84) on mixed bone cell preparations from neonatal rats, specific binding was demonstrated only to osteoblasts, not to osteoclasts. These results clearly demonstrate that PTHrP is a potent stimulator of bone resorption and that these effects are, like those of PTH, mediated by initial actions upon cells of the osteoblast lineage.

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

    Science.gov (United States)

    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

    2015-03-31

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  1. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  2. Activated Charcoal Suppresses Breeding of the House Dust Mite, Dermatophagoides Pteronyssinus, in Culture

    Science.gov (United States)

    Nam, Hae-Seon; Lee, Sun-Hwa; Kim, Sung-Ho; Lee, Sang-Han; Crane, Julian

    2007-01-01

    House dust mite sensitized asthmatics are advised to practice allergen avoidance. Charcoal pillows are used in Korea with unsubstantiated claims regarding their efficacy in alleviating asthma symptoms. We tested the effects of activated charcoal on breeding of house dust mites in culture. Twenty live adult house dust mites (Dermatophagoides pteronyssinus) were inoculated, 10 replicates, on culture media containing 0%, 1%, 3%, 5%, 10%, and 20% activated charcoal and incubated at 25℃ and a relative humidity of 75%. After four weeks, the mean numbers of live house dust mites were 286, 176, 46, 16, 7, and 0 for the 0%, 1%, 3%, 5%, 10%, and 20% charcoal-containing culture media, respectively. Thus, activated charcoal suppresses breeding of house dust mites and offers a new promising method for house dust mite control. PMID:17449957

  3. Treadmill exercise ameliorates symptoms of Alzheimer disease through suppressing microglial activation-induced apoptosis in rats.

    Science.gov (United States)

    Baek, Seung-Soo; Kim, Sang-Hoon

    2016-12-01

    Alzheimer disease (AD) is a most common form of dementia and eventually causes impairments of learning ability and memory function. In the present study, we investigated the effects of treadmill exercise on the symptoms of AD focusing on the microglial activation-induced apoptosis. AD was made by bilateral intracerebroventricular injection of streptozotocin. The rats in the exercise groups were made to run on a treadmill once a day for 30 min during 4 weeks. The distance and latency in the Morris water maze task and the latency in the step-down avoidance task were increased in the AD rats, in contrast, treadmill exercise shortened these parameters. The numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive and caspase-3-positive cells in the hippocampal dentate gyrus were decreased in the AD rats, in contrast, treadmill exercise suppressed these numbers. Expressions of glial fibrillary acidic protein (GFAP) and cluster of differentiation molecule 11B (CD11b) in the hippocampal dentate gyrus were increased in the AD rats, in contrast, treadmill exercise suppressed GFAP and CD11b expressions. Bax expression was increased and Bcl-2 expression was decreased in the hippocampus of AD rats, in contrast, treadmill exercise decreased Bax expression and increased Bcl-2 expression. The present results demonstrated that treadmill exercise ameliorated AD-induced impairments of spatial learning ability and short-term memory through suppressing apoptosis. The antiapoptotic effect of treadmill exercise might be ascribed to the inhibitory effect of treadmill exercise on microglial activation.

  4. Andrographolide prevents human breast cancer-induced osteoclastic bone loss via attenuated RANKL signaling.

    Science.gov (United States)

    Zhai, Zanjing; Qu, Xinhua; Yan, Wei; Li, Haowei; Liu, Guangwang; Liu, Xuqiang; Tang, Tingting; Qin, An; Dai, Kerong

    2014-02-01

    Bone metastasis is a common and serious complication in advanced cancers such as breast cancer, prostate cancer, and multiple myeloma. Agents that prevent bone loss could be used to develop an alternative therapy for bone metastasis. RANKL, a member of the tumor necrosis factor superfamily, has been shown to play a significant role in cancer-associated bone loss. In this study, we examined the efficacy of the natural compound andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata, in reducing breast cancer-induced osteolysis. AP prevented human breast cancer-induced bone loss by suppressing RANKL-mediated and human breast cancer cell-induced osteoclast differentiation. Molecular analysis revealed that AP prevented osteoclast function by inhibiting RANKL-induced NF-κB and ERK signaling pathway in lower dose (20 μM), as well as inducing apoptosis at higher dose (40 μM). Thus, AP is a potent inhibitor of breast cancer-induced bone metastasis.

  5. TGF-β suppresses β-catenin-dependent tolerogenic activation program in dendritic cells.

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    Bryan Vander Lugt

    Full Text Available The mechanisms that underlie the critical dendritic cell (DC function in maintainance of peripheral immune tolerance are incompletely understood, although the β-catenin signaling pathway is critical for this role. The molecular details by which β-catenin signaling is regulated in DCs are unknown. Mechanical disruption of murine bone marrow-derived DC (BMDC clusters activates DCs while maintaining their tolerogenic potential and this activation is associated with β-catenin signaling, providing a useful model with which to explore tolerance-associated β-catenin signaling in DCs. In this report, we demonstrate novel molecular features of the signaling events that control DC activation in response to mechanical stimulation. Non-canonical β-catenin signaling is an essential component of this tolerogenic activation and is modulated by adhesion molecules, including integrins. This unique β-catenin-dependent signaling pathway is constitutively active at low levels, suggesting that mechanical stimulation is not necessarily required for induction of this unique activation program. We additionally find that the immunomodulatory cytokine TGF-β antagonizes β-catenin in DCs, thereby selectively suppressing signaling associated with tolerogenic DC activation while having no impact on LPS-induced, β-catenin-independent immunogenic activation. These findings provide new molecular insight into the regulation of a critical signaling pathway for DC function in peripheral immune tolerance.

  6. A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

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    Claude Jourdan Le Saux

    Full Text Available The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L, a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

  7. Osteoblast and osteoclast responses to A/B type carbonate-substituted hydroxyapatite ceramics for bone regeneration.

    Science.gov (United States)

    Germaini, Marie-Michèle; Detsch, Rainer; Grünewald, Alina; Magnaudeix, Amandine; Lalloue, Fabrice; Boccaccini, Aldo R; Champion, Eric

    2017-06-06

    The influence of carbonate substitution (4.4 wt%, mixed A/B type) in hydroxyapatite ceramics for bone remodeling scaffolds was investigated by separately analyzing the response of pre-osteoblasts and osteoclast-like cells. Carbonated hydroxyapatite (CHA) (Ca 9.5 (PO 4 ) 5.5 (CO 3 ) 0.5 (OH)(CO 3 ) 0.25 -CHA), mimicking the chemical composition of natural bone mineral, and pure hydroxyapatite (HA) (Ca 10 (PO 4 ) 6 (OH) 2 -HA) porous ceramics were processed to obtain a similar microstructure and surface physico-chemical properties (grain size, porosity ratio and pore size, surface roughness and zeta potential). The biological behavior was studied using MC3T3-E1 pre-osteoblastic and RAW 264.7 monocyte/macrophage cell lines. Chemical dissolution in the culture media and resorption lacunae produced by osteoclasts occur with both HA and CHA ceramics, but CHA exhibits much higher dissolution and greater bioresorption ability. CHA ceramics promoted a significantly higher level of pre-osteoblast proliferation. Osteoblastic differentiation, assessed by qRT-PCR of RUNX2 and COLIA2, and pre-osteoclastic proliferation and differentiation were not significantly different on CHA or HA ceramics but cell viability and metabolism were significantly greater on CHA ceramics. Thus, the activity of both osteoclast-like and osteoblastic cells was influenced by the carbonate substitution in the apatite structure. Furthermore, CHA showed a particularly interesting balance between biodegradation, by osteoclasts and chemical dissolution, and osteogenesis through osteoblasts' activity, to stimulate bone regeneration. It is hypothesized that this amount of 4.4 wt% carbonate substitution leads to an adapted concentration of calcium in the fluid surrounding the ceramic to stimulate the activity of cells. These results highlight the superior biological behavior of microporous 4.4 wt% A/B CHA ceramics that could beneficially replace the commonly used HA of biphasic calcium phosphates for future

  8. Suppression of adenosine-activated chloride transport by ethanol in airway epithelia.

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    Sammeta V Raju

    Full Text Available Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B adenosine receptor (A(2BAR, largely abolished the adenosine-stimulated chloride transport, suggesting that A(2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

  9. Drosera rotundifolia and Drosera tokaiensis suppress the activation of HMC-1 human mast cells.

    Science.gov (United States)

    Fukushima, Kenji; Nagai, Kanji; Hoshi, Yoshikazu; Masumoto, Saeko; Mikami, Ichiho; Takahashi, Yumiko; Oike, Hideaki; Kobori, Masuko

    2009-08-17

    Several Northern Hemisphere Drosera species have been used in the therapy of respiratory tract infections as the traditional medicine Droserae Herba. To determine the anti-inflammatory effects of Drosera species and to investigate a substitute material for Droserae Herba, we examined the effect of extracts of Drosera rotundifolia, Drosera tokaiensis and Drosera spatulata on activated T cell membrane (aTc-m)-induced inflammatory gene expression in HMC-1 human mast cells. Drosera rotundifolia, Drosera spatulata and Drosera tokaiensis were collected in Japan. Herbs were extracted with 80% EtOH, and subsequently applied to OASIS HLB column. HMC-1 cells were treated with each Drosera column-adsorbed fraction for 15min, and subsequently added to aTc-m and incubated for 16h. Inflammatory gene and protein expressions were determined by DNA microarray, RT-PCR and Western blotting. Drosera rotundifolia and Drosera tokaiensis fractions, but not the Drosera spatulata fraction, suppressed inflammatory gene expression induced by aTc-m in HMC-1 cells. Drosera rotundifolia and Drosera tokaiensis suppressed activation of HMC-1 cells induced by aTc-m. Since the Drosera tokaiensis fraction was more effective than the traditionally used Drosera rotundifolia, Drosera tokaiensis is a likely substitute as a source of Droserae Herba.

  10. Dormancy of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia.

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    Hiroko Endo

    Full Text Available A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death in vitro and a decreased number of dormant cells in vivo. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor-depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer.

  11. Rhodiola crenulata extract suppresses hepatic gluconeogenesis via activation of the AMPK pathway.

    Science.gov (United States)

    Lee, Shih-Yu; Lai, Feng-Yi; Shi, Li-Shian; Chou, Yu-Ching; Yen, I-Chuan; Chang, Tsu-Chung

    2015-04-15

    Rhodiola, a popular herb, has been used for treating high altitude sicknesses, depression, fatigue, and diabetes. However, the detailed mechanisms by which Rhodiola crenulata functions in the liver need further clarification. The current study was designed to examine the effects of Rhodiola crenulata root extract (RCE) on hepatic glucose production. Human hepatoma HepG2 cells were treated with RCE for 6 h. Glucose production, the expression level of p-AMPK, and the expression of key gluconeogenic genes were measured. The effects of RCE were also studied in Sprague-Dawley (SD) rats. The efficacy and underlying mechanism of RCE in the liver were examined. RCE significantly suppressed glucose production and gluconeogenic gene expression in HepG2 cells while activating the AMPK signaling pathway. Interestingly, RCE-suppressed hepatic gluconeogenesis was eliminated by an AMPK-specific inhibitor, but not by the PI3K/AKT-specific inhibitor. In addition, oral administration of RCE significantly increased phosphorylated AMPK levels and inhibited gluconeogenic gene expression in the rat liver. Furthermore, RCE treatment also decreased plasma glucose concentration in rats. We present in vitro and in vivo evidence that RCE might exert the glucose-lowering effect partly by inhibiting hepatic gluconeogenesis through activating the AMPK signaling pathway. These findings provide evidence that Rhodiola crenulata may be helpful for the management of type II diabetes. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9-tetrahydrocannabinol injection.

    Science.gov (United States)

    Newton, C A; Klein, T W; Friedman, H

    1994-01-01

    Resistance to infection with Legionella pneumophila is primarily dependent upon cell-mediated immunity rather than humoral immunity. Recent evidence suggests that activation of cell-mediated immunity depends on Th1 cells and activation of humoral immunity depends on Th2 cells. In this report, delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid of marijuana and an immunomodulator, suppressed development of secondary immunity to L. pneumophila, which correlated with a reduction in Th1 activity. BALB/c mice, infected with a primary sublethal dose of L. pneumophila, developed resistance to a larger challenge infection 3 to 4 weeks later. However, intravenous injection of THC (4 mg/kg of body weight) 1 day prior to primary infection resulted in increased mortality after the challenge infection. The level of anti-L. pneumophila antibodies in serum increased in both THC-treated and control mice; however, in the THC group IgG1 antibodies which are stimulated by Th2 cells were elevated while Th1-regulated, IgG2a antibodies were depressed. Furthermore, cultured splenocytes from THC-treated mice had less L. pneumophila-specific lymphoproliferation, indicating a deficiency in cell-mediated immunity. Normal mouse splenocytes treated in vitro with THC and pokeweed mitogen showed suppressed production of gamma interferon, a cytokine associated with Th1 cells, but increased production of interleukin 4, a cytokine produced by Th2 cells. Splenocytes from THC-treated mice, stimulated in vitro with either pokeweed mitogen or anti-CD3 antibodies, also produced less gamma interferon, indicating less Th1 activity in these mice. These results suggest that THC decreases the development of anti-L. pneumophila immunity by causing a change in the balance of Th1 and Th2 activities. PMID:8063421

  13. miR-191 suppresses angiogenesis by activation of NF-κB signaling.

    Science.gov (United States)

    Gu, Yuan; Ampofo, Emmanuel; Menger, Michael D; Laschke, Matthias W

    2017-08-01

    MicroRNAs (miRNAs) are powerful regulators of diverse biologic processes. However, the function of most miRNAs in angiogenesis remains elusive. In this study, we identified miR-191-5p (miR-191) as a potent inhibitor of blood vessel development. Transfection of human dermal microvascular endothelial cells with miR-191 mimic (miR-191m) inhibited their proliferation, migration, and tube formation. Moreover, vascular sprouting of miR-191m-transfected mouse aortic rings was significantly reduced when compared with controls. Transfection with miR-191 inhibitor (miR-191i) induced proangiogenic effects. The anti- and proangiogenic activities of miR-191m and -191i were further demonstrated in vivo Additional molecular biologic analyses revealed that miR-191m activates NF-κB signaling by up-regulating the mRNA expression of p65. miR-191 also increased the mRNA levels of the antiangiogenic factors p21 and tissue inhibitor of metalloproteinase-1 and reduced the expression of the proangiogenic factors eNOS and matrix metalloproteinase-1 and -9. Blockade of NF-κB activation with Bay 11-7082 reversed the antiangiogenic effects of miR-191m. These findings indicate that miR-191 effectively suppresses angiogenesis by activation of the NF-κB signaling pathway.-Gu, Y., Ampofo, E., Menger, M. D., Laschke, M. W. miR-191 suppresses angiogenesis by activation of NF-κB signaling. © FASEB.

  14. Osteoclast formation from peripheral blood of patients with bone-lytic diseases

    NARCIS (Netherlands)

    de Vries, T.J.; Everts, V.

    2009-01-01

    Recent literature indicates that osteoclast formation in vitro from peripheral blood of patients with diseases associated with bone loss such as rheumatoid arthritis, osteoporosis, periodontitis and bone metastatic cancer may occur spontaneously being independent of addition of osteoclast formation

  15. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    Science.gov (United States)

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

  16. Breast carcinoma with osteoclast-like giant cells

    DEFF Research Database (Denmark)

    Gjerdrum, L M; Lauridsen, M C; Sørensen, Flemming Brandt

    2001-01-01

    Primary carcinoma with osteoclast-like giant cells is a very rare tumour of the female breast. The clinical course, histological, immunohistochemical and ultrastructural features of 61 cases of invasive duct carcinoma with osteoclast-like multinucleated giant cells (OMGCs) are reviewed and a new...... in the literature have shown that 86% of patients with these tumours are still alive after 5 years. Histologically, these tumours are invasive ductal carcinomas with OMGCs next to the neoplastic glands and within their lumen. Signs of recent and past haemorrhage are ubiquitously present in the highly vascularized...

  17. Reverse feeding suppresses the activity of the GH axis in rats and induces a preobesogenic state.

    Science.gov (United States)

    Glad, Camilla A-M; Kitchen, Edward E J; Russ, Gemma C; Harris, Sophie M; Davies, Jeffrey S; Gevers, Evelien F; Gabrielsson, Britt G; Wells, Timothy

    2011-03-01

    Reversed feeding (RF) is known to disrupt hormone rhythmicity and metabolism. Although these effects may be mediated in part by phase inversion of glucocorticoid secretion, the precise mechanism is incompletely characterized. In this study, we demonstrate that acute nocturnal food deprivation in male rats suppressed the amplitude of spontaneous GH secretion during the dark phase by 62% (P inversion of core clock gene expression in liver, abdominal white adipose tissue (WAT) and skeletal muscle, without affecting their expression patterns in the suprachiasmatic nucleus. In addition, RF resulted in phase inversion of hepatic peroxisome proliferator-activated receptor γ2 mRNA expression, a 3- to 5-fold elevation in fatty acid synthase mRNA in WAT in both light- and dark-phase samples (P < 0.01) and an elevation in muscle uncoupling protein 3 mRNA expression at the beginning of the light phase (P < 0.01). Consumption of a high-fat diet increased inguinal (by 36%; P < 0.05) and retroperitoneal WAT weight (by 72%; P < 0.01) only in RF-maintained rats, doubling the efficiency of lipid accumulation (P < 0.05). Thus, RF not only desynchronizes central and peripheral circadian clocks, and suppresses nocturnal GH secretion, but induces a preobesogenic state.

  18. The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

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    Christoph Hemetsberger

    Full Text Available The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1 as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H₂O₂ strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

  19. H-reflex suppression and autonomic activation during lucid REM sleep: a case study.

    Science.gov (United States)

    Brylowski, A; Levitan, L; LaBerge, S

    1989-08-01

    A single subject, a proficient lucid dreamer experienced with signaling the onset of lucidity (reflective consciousness of dreaming) by means of voluntary eye movements, spent 4 nonconsecutive nights in the sleep laboratory. The subject reported becoming lucid and signaling in 8 of the 18 rapid-eye movement (REM) periods recorded. Ten lucid dream reports were verified by polygraphic examination of signals, providing a total of 12.5 min of signal-verified lucid REM. H-Reflex amplitude was recorded every 5 s, along with continuous recording of electroencephalogram, electrooculogram, electromyogram, electrocardiogram, finger pulse, and respiration. Significant findings included greater mean H-reflex suppression during lucid REM sleep than during nonlucid REM and correlations of H-reflex suppression with increased eye movement density, heart rate, and respiration rate. These results support previous studies reporting that lucid REM is not, as might be supposed, a state closer to awakening than ordinary, or nonlucid, REM; rather, lucid dreaming occurs during unequivocal REM sleep and is characteristically associated with phasic REM activation.

  20. Aryl Hydrocarbon Receptor Activation Suppresses EBF1 and PAX5 and Impairs Human B Lymphopoiesis.

    Science.gov (United States)

    Li, Jinpeng; Bhattacharya, Sudin; Zhou, Jiajun; Phadnis-Moghe, Ashwini S; Crawford, Robert B; Kaminski, Norbert E

    2017-11-15

    Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34 + hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo- p -dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo- p -dioxin-elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism. Copyright © 2017 by The American Association of Immunologists, Inc.

  1. Sodium salicylate suppresses GABAergic inhibitory activity in neurons of rodent dorsal raphe nucleus.

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    Yan Jin

    Full Text Available Sodium salicylate (NaSal, a tinnitus inducing agent, can activate serotonergic (5-HTergic neurons in the dorsal raphe nucleus (DRN and can increase serotonin (5-HT level in the inferior colliculus and the auditory cortex in rodents. To explore the underlying neural mechanisms, we first examined effects of NaSal on neuronal intrinsic properties and the inhibitory synaptic transmissions in DRN slices of rats by using whole-cell patch-clamp technique. We found that NaSal hyperpolarized the resting membrane potential, decreased the input resistance, and suppressed spontaneous and current-evoked firing in GABAergic neurons, but not in 5-HTergic neurons. In addition, NaSal reduced GABAergic spontaneous and miniature inhibitory postsynaptic currents in 5-HTergic neurons. We next examined whether the observed depression of GABAergic activity would cause an increase in the excitability of 5-HTergic neurons using optogenetic technique in DRN slices of the transgenic mouse with channelrhodopsin-2 expressed in GABAergic neurons. When the GABAergic inhibition was enhanced by optical stimulation to GABAergic neurons in mouse DRN, NaSal significantly depolarized the resting membrane potential, increased the input resistance and increased current-evoked firing of 5-HTergic neurons. However, NaSal would fail to increase the excitability of 5-HTergic neurons when the GABAergic synaptic transmission was blocked by picrotoxin, a GABA receptor antagonist. Our results indicate that NaSal suppresses the GABAergic activities to raise the excitability of local 5-HTergic neural circuits in the DRN, which may contribute to the elevated 5-HT level by NaSal in the brain.

  2. Sodium salicylate suppresses GABAergic inhibitory activity in neurons of rodent dorsal raphe nucleus.

    Science.gov (United States)

    Jin, Yan; Luo, Bin; Su, Yan-Yan; Wang, Xin-Xing; Chen, Liang; Wang, Ming; Wang, Wei-Wen; Chen, Lin

    2015-01-01

    Sodium salicylate (NaSal), a tinnitus inducing agent, can activate serotonergic (5-HTergic) neurons in the dorsal raphe nucleus (DRN) and can increase serotonin (5-HT) level in the inferior colliculus and the auditory cortex in rodents. To explore the underlying neural mechanisms, we first examined effects of NaSal on neuronal intrinsic properties and the inhibitory synaptic transmissions in DRN slices of rats by using whole-cell patch-clamp technique. We found that NaSal hyperpolarized the resting membrane potential, decreased the input resistance, and suppressed spontaneous and current-evoked firing in GABAergic neurons, but not in 5-HTergic neurons. In addition, NaSal reduced GABAergic spontaneous and miniature inhibitory postsynaptic currents in 5-HTergic neurons. We next examined whether the observed depression of GABAergic activity would cause an increase in the excitability of 5-HTergic neurons using optogenetic technique in DRN slices of the transgenic mouse with channelrhodopsin-2 expressed in GABAergic neurons. When the GABAergic inhibition was enhanced by optical stimulation to GABAergic neurons in mouse DRN, NaSal significantly depolarized the resting membrane potential, increased the input resistance and increased current-evoked firing of 5-HTergic neurons. However, NaSal would fail to increase the excitability of 5-HTergic neurons when the GABAergic synaptic transmission was blocked by picrotoxin, a GABA receptor antagonist. Our results indicate that NaSal suppresses the GABAergic activities to raise the excitability of local 5-HTergic neural circuits in the DRN, which may contribute to the elevated 5-HT level by NaSal in the brain.

  3. Targeted p53 activation by saRNA suppresses human bladder cancer cells growth and metastasis.

    Science.gov (United States)

    Wang, Chenghe; Ge, Qiangqiang; Zhang, Qingsong; Chen, Zhong; Hu, Jia; Li, Fan; Ye, Zhangqun

    2016-03-25

    Previous study showed that dsP53-285 has the capacity to induce tumor suppressor gene p53 expression by targeting promoter in non-human primates' cells. And it is well known that TP53 gene is frequently mutant or inactivated in human bladder cancer. Hereby, whether this small RNA can activate the expression of wild-type p53 and inhibit human bladder cancer cells remains to be elucidated. Oligonucleotide and lentivirus were used to overexpress dsP53-285 and dsControl. Real-time PCR and western blot were used to detect genes' mRNA and protein expression, respectively. Cell proliferation assay, colony formation, flow cytometry, transwell assay and wound healing assay were performed to determine the effects on bladder cancer cells proliferation and migration/invasion in vitro. Animal models were carried out to analyze the effects on cells growth and metastasis in vivo. Transfection of dsP53-285 into human bladder cancer cell lines T24 and EJ readily activate wild-type p53 expression by targeting promoter. Moreover, dsP53-285 exhibited robust capacity to inhibit cells proliferation and colony formation, induce cells G0/G1 arrest, suppress migration and invasion. Besides, the Cyclin-CDK genes (Cyclin D1 and CDK4/6) were down-regulated and the EMT-associated genes (E-cadherin, β-catenin, ZEB1 and Vimentin) were also expressed inversely after dsP53-285 treatment. In addition, dsP53-285 could also significantly suppress the growth of bladder cancer xenografts and metastasis in nude mice. Most importantly, the anti-tumor effects mediated by dsP53-285 were mainly achieved by manipulating wild-type p53 expression. Our findings indicate that the dsP53-285 can upregulate wild-type p53 expression in human bladder cancer cells through RNA activation, and suppresses cells proliferation and metastasis in vitro and in vivo.

  4. Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes

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    Baba Hideo

    2011-04-01

    Full Text Available Abstract Background Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1 is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro. Methods Primary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay. Results PAI-1 mRNA expression was up-regulated by 388% (P = 0.002 with dexamethasone, and down-regulated by 45% (P = 0.002 with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001 and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002. No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels. Conclusion The present study confirmed the suppressive

  5. Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption.

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    Zhong-Yuan Ren

    Full Text Available The cysteine protease cathepsin K (CatK, abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs to test their effects on osteoblasts and osteoclasts.Cell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices.Treatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10-100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed

  6. Osteoclasts Are Required for Hematopoietic Stem and Progenitor Cell Mobilization but Not for Stress Erythropoiesis in Plasmodium chabaudi adami Murine Malaria

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    Hugo Roméro

    2016-01-01

    Full Text Available The anemia and inflammation concurrent with blood stage malaria trigger stress haematopoiesis and erythropoiesis. The activity of osteoclasts seems required for the mobilization of hematopoietic stem and progenitor cells (HSPC from the bone marrow to the periphery. Knowing that BALB/c mice with acute Plasmodium chabaudi adami malaria have profound alterations in bone remodelling cells, we evaluated the extent to which osteoclasts influence their hematopoietic response to infection. For this, mice were treated with osteoclast inhibiting hormone calcitonin prior to parasite inoculation, and infection as well as hematological parameters was studied. In agreement with osteoclast-dependent HSPC mobilization, administration of calcitonin led to milder splenomegaly, reduced numbers of HSPC in the spleen, and their retention in the bone marrow. Although C-terminal telopeptide (CTX levels, indicative of bone resorption, were lower in calcitonin-treated infected mice, they remained comparable in naive and control infected mice. Calcitonin-treated infected mice conveniently responded to anemia but generated less numbers of splenic macrophages and suffered from exacerbated infection; interestingly, calcitonin also decreased the number of macrophages generated in vitro. Globally, our results indicate that although osteoclast-dependent HSC mobilization from bone marrow to spleen is triggered in murine blood stage malaria, this activity is not essential for stress erythropoiesis.

  7. Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation.

    Science.gov (United States)

    Fielding, Ceri A; Weekes, Michael P; Nobre, Luis V; Ruckova, Eva; Wilkie, Gavin S; Paulo, Joao A; Chang, Chiwen; Suárez, Nicolás M; Davies, James A; Antrobus, Robin; Stanton, Richard J; Aicheler, Rebecca J; Nichols, Hester; Vojtesek, Borek; Trowsdale, John; Davison, Andrew J; Gygi, Steven P; Tomasec, Peter; Lehner, Paul J; Wilkinson, Gavin W G

    2017-02-10

    The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.

  8. Inverse agonistic activity of antihistamines and suppression of histamine H1 receptor gene expression.

    Science.gov (United States)

    Mizuguchi, Hiroyuki; Ono, Shohei; Hattori, Masashi; Fukui, Hiroyuki

    2012-01-01

    Histamine H(1) receptor (H1R) expression influences the severity of allergy symptoms. We examined the effect of inverse agonists on H1R gene expression. Two inverse agonists (carebastine and mepyramine), but not the neutral antagonist oxatomide, decreased inositol phosphate accumulation. The inverse agonists also decreased H1R gene expression and down-regulated H1R mRNA below basal expression, while basal H1R mRNA expression was maintained after oxatomide treatment. These results suggest that inverse agonists more potently alleviate allergy symptoms by not only inhibiting stimulus-induced up-regulation of H1R gene expression but also by suppressing basal histamine signaling through their inverse agonistic activity.

  9. Sodium azide suppresses LPS-induced expression MCP-1 through regulating IκBζ and STAT1 activities in macrophages.

    Science.gov (United States)

    Park, Cho-Yi; Heo, Jae-Nyoung; Suk, Kyoungho; Lee, Won-Ha

    2017-05-01

    Sodium azide (NaN 3 ) is a chemical compound with multiple toxic effects on vascular and neuronal systems, causing hypotension and neurotoxicity, respectively. In order to test its effects on the immune system, human and mouse macrophage-like cell lines were treated with nontoxic doses of NaN 3 and the changes in LPS-induced inflammatory activation was measured. Interestingly, the LPS-induced expression of monocyte chemoattractant protein (MCP)-1 was suppressed by NaN 3 without affecting the expression of IL-8 and TNF-α. Further analysis of cellular signaling mediators involved in the expression of these cytokines revealed that NaN 3 suppressed the LPS-induced activation of signal transducers and activator of transcription (STAT)1 and inhibitor of κB (IκB) ς, which are involved in the LPS-induced expression of MCP-1, while the LPS-induced activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) was not affected. The LPS-induced expression of MCP-2 and CXCL10, which are also regulated by STAT1, was suppressed by NaN 3 . Similarly, the LPS-induced expression of IL-6, which is regulated by IκBζ, was suppressed by NaN 3 . These results demonstrate that NaN 3 selectively suppresses the LPS-induced expression of pro-inflammatory mediators through the suppression of STAT1 and IκBζ activation. These new findings about the activity of NaN 3 may contribute to the development of specific regulators of macrophage activity during acute and chronic inflammation. Copyright © 2017. Published by Elsevier Inc.

  10. Degradation of the organic phase of bone by osteoclasts

    DEFF Research Database (Denmark)

    Henriksen, Kim; Sørensen, Mette G; Nielsen, Rasmus H

    2006-01-01

    Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix...

  11. Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study

    DEFF Research Database (Denmark)

    Dizon, M A; Multhaupt, H A; Paskin, D L

    1996-01-01

    A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor....

  12. Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study.

    Science.gov (United States)

    Dizon, M A; Multhaupt, H A; Paskin, D L; Warhol, M J

    1996-03-01

    A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor.

  13. Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism

    Science.gov (United States)

    Chen, Xi; Xie, Chun; Fan, Xing-Xing; Jiang, Ze-Bo; Wong, Vincent Kam-Wai; Xu, Jia-Hui; Yao, Xiao-Jun; Liu, Liang; Leung, Elaine Lai-Han

    2017-01-01

    Drug resistance is becoming an obstacle in anti-cancer therapies. For target-based therapy of lung cancer, gefitinib, as the first generation of tyrosine kinase inhibitors (TKIs), demonstrated good initial response to the non-small cell lung cancer (NSCLC) patients whom harbors epidermal growth factor receptor (EGFR) mutation. However, within one year, additional EGFR mutation occurred, leading to eventual gefitinib-resistance. Therefore, it is urgently to discover novel effective small molecule inhibitors for those patients. Abnormal energy metabolism is accepted as new cancer hallmark. Recently, a metabolism rate-limiting enzyme 5’-adenosine menophosphate-activated protein kinase (AMPK) has become a promising anti-cancer target. In this study, we have identified a novel direct AMPK agonist, D561-0775 from a compound library by using molecular docking screening technique. We demonstrated that D561-0775 exhibited significant inhibitory effect on gefitinib-resistant NSCLC cell lines but less cytotoxicity on normal cells. Furthermore, D561-0775 demonstrated a remarkable in vitro AMPK enzyme activation effect. Taken together, D561-0775 showed potential anti-cancer activity via inducing apoptosis, cell cycle arrest, suppressing glycolysis and cholesterol synthesis after activation of AMPK in gefitinib-resistant H1975 cells. D561-0775 has provided a new chemical structure that could be developed as cancer drug for gefitinib-resistant NSCLC patients through inhibition lipid metabolism by directly targeting at AMPK directly. PMID:29221189

  14. Chicoric acid suppresses BAFF expression in B lymphocytes by inhibiting NF-κB activity.

    Science.gov (United States)

    Chen, Lingxi; Huang, Gang; Gao, Min; Shen, Xiaodong; Gong, Wei; Xu, Zhizhen; Zeng, Yijun; He, Fengtian

    2017-03-01

    B cell activating factor belonging to the TNF family (BAFF) plays a critical role in the pathogenesis of autoimmune diseases. The inhibition of BAFF expression is an emerging therapeutic approach for these disorders. Chicoric acid (CA), a bioactive phytochemical found in several widely used traditional medicinal plants, has significant anti-inflammatory activity and anti-arthritic effects. However, the role of CA in modulation of BAFF expression remains unknown. In this study, we demonstrated that CA reduced BAFF expression in human B lymphocyte cell lines and decreased the DNA-binding activity of nuclear factor-κB (NF-κB) in the BAFF promoter region. Furthermore, CA inhibited both the nuclear translocation of p65 (the subunit of NF-κB) and the phosphorylation of IκBα (inhibitor of NF-κB). These results suggest that CA suppresses BAFF expression by inhibiting NF-κB activity, and CA may serve as a novel therapeutic agent to down-regulate excessive BAFF expression in autoimmune diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. n-Butanol extracts of Panax notoginseng suppress LPS-induced MMP-2 expression in periodontal ligament fibroblasts and inhibit osteoclastogenesis by suppressing MAPK in LPS-activated RAW264.7 cells.

    Science.gov (United States)

    Jang, Young-Joo; Kim, Mee-Eun; Ko, Seon-Yle

    2011-11-01

    Periodontitis is a group of inflammatory diseases that affect connective tissue attachments and the supporting bone that surround the teeth. Osteoclasts are responsible for skeletal modeling and remodeling but may also destroy bone in several bone diseases, including osteoporosis and periodontitis. This study examined the anti-inflammatory effects of Panax notoginseng (PN) on periodontal ligament fibroblasts (PDLFs) and RAW264.7 cells under lipopolysaccharide (LPS) induced inflammatory conditions. The effects of PN on PDLFs were determined by measuring the cell viability and mRNA expression of tissue-destructive proteins. The effects of PN on osteoclasts were examined by measuring the following: (1) the cell viability, (2) the formation of Tartrate-resistant acid phosphatase (TRAP)(+) multinucleated cells, (3) MAPK signaling pathways, (4) mRNA expression of inflammatory-related proteins and (5) nitric oxide (NO) production. The n-butanol extracts of PN (bPN) increased the cell proliferation of the PDLFs and decreased the mRNA expression of matrix metalloproteinase (MMP)-2 in the PDLFs. bPN inhibited the formation of LPS-stimulated TRAP(+) multinucleated cells. bPN also inhibited the LPS-stimulated activation of JNK and ERK signaling, and inhibited the LPS-stimulated degradation of I(K)B in the RAW264.7 cells. In addition, bPN decreased the mRNA expression of MMP-9 and iNOS, which are involved in the range of pathophysiological processes, such as inflammation in the RAW264.7 cells. NO production was also decreased via the inhibition of iNOS. These findings suggest that bPN has therapeutic effects on bone-destructive processes, such as those that occur in periodontal diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis.

    Science.gov (United States)

    Ding, Hao; Jiang, Lei; Xu, Jing; Bai, Feng; Zhou, Yang; Yuan, Qi; Luo, Jing; Zen, Ke; Yang, Junwei

    2017-09-01

    Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy. Copyright © 2017 the American Physiological Society.

  17. Profiling of Sox4-dependent transcriptome in skin links tumour suppression and adult stem cell activation

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    Miguel Foronda

    2015-12-01

    Full Text Available Adult stem cells (ASCs reside in specific niches in a quiescent state in adult mammals. Upon specific cues they become activated and respond by self-renewing and differentiating into newly generated specialised cells that ensure appropriate tissue fitness. ASC quiescence also serves as a tumour suppression mechanism by hampering cellular transformation and expansion (White AC et al., 2014. Some genes restricted to early embryonic development and adult stem cell niches are often potent modulators of stem cell quiescence, and derailed expression of these is commonly associated to cancer (Vervoort SJ et al., 2013. Among them, it has been shown that recommissioned Sox4 expression facilitates proliferation, survival and migration of malignant cells. By generating a conditional Knockout mouse model in stratified epithelia (Sox4cKO mice, we demonstrated a delayed plucking-induced Anagen in the absence of Sox4. Skin global transcriptome analysis revealed a prominent defect in the induction of transcriptional networks that control hair follicle stem cell (HFSC activation such as those regulated by Wnt/Ctnnb1, Shh, Myc or Sox9, cell cycle and DNA damage response-associated pathways. Besides, Sox4cKO mice are resistant to skin carcinogenesis, thus linking Sox4 to both normal and pathological HFSC activation (Foronda M et al., 2014. Here we provide additional details on the analysis of Sox4-regulated transcriptome in Telogen and Anagen skin. The raw and processed microarray data is deposited in GEO under GSE58155.

  18. Organic amendments to avocado crops induce suppressiveness and influence the composition and activity of soil microbial communities.

    Science.gov (United States)

    Bonilla, Nuria; Vida, Carmen; Martínez-Alonso, Maira; Landa, Blanca B; Gaju, Nuria; Cazorla, Francisco M; de Vicente, Antonio

    2015-05-15

    One of the main avocado diseases in southern Spain is white root rot caused by the fungus Rosellinia necatrix Prill. The use of organic soil amendments to enhance the suppressiveness of natural soil is an inviting approach that has successfully controlled other soilborne pathogens. This study tested the suppressive capacity of different organic amendments against R. necatrix and analyzed their effects on soil microbial communities and enzymatic activities. Two-year-old avocado trees were grown in soil treated with composted organic amendments and then used for inoculation assays. All of the organic treatments reduced disease development in comparison to unamended control soil, especially yard waste (YW) and almond shells (AS). The YW had a strong effect on microbial communities in bulk soil and produced larger population levels and diversity, higher hydrolytic activity and strong changes in the bacterial community composition of bulk soil, suggesting a mechanism of general suppression. Amendment with AS induced more subtle changes in bacterial community composition and specific enzymatic activities, with the strongest effects observed in the rhizosphere. Even if the effect was not strong, the changes caused by AS in bulk soil microbiota were related to the direct inhibition of R. necatrix by this amendment, most likely being connected to specific populations able to recolonize conducive soil after pasteurization. All of the organic amendments assayed in this study were able to suppress white root rot, although their suppressiveness appears to be mediated differentially. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C

    Science.gov (United States)

    Cooper, D.; Pellis, N. R.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished. Activation marker expression was significantly reduced in RWV cultures. Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness. However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction. Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

  20. Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

    Directory of Open Access Journals (Sweden)

    Hanna Witwicka

    Full Text Available The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized

  1. Livers with constitutive mTORC1 activity resist steatosis independent of feedback suppression of Akt.

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    Heidi L Kenerson

    Full Text Available Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6-14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.

  2. [Stable isotopes of Mg2+ as activators of the suppressed ATP-generating function of mitochondria].

    Science.gov (United States)

    Kuznetsov, D A; Arkhantel'skiĭ, S E; Berdieva, A G; Markarian, A A; Khasigov, P Z; Gatagonova, T M; Ktsova, S A; Orlova, M A

    2005-01-01

    The ATP-generating activity of rat myocardial mitochondria and intramitochondrial creatine kinase was examined as a function of the isotopy of the incubation medium magnesium pool. The study was performed using in vitro systems prepared from the hearts of animals injected with 1-methylnicotine amide, which suppresses the NAD (NADP)-dependent reactions in vivo. It was shown that the presence of the 25Mg paramagnetic cations essential by compensates for the intramitochondrial ATP deficiency caused by the 1-methyl-nicotine amide-induced blockade of oxidative phosphorylation. This effect is hardey achievable in systems where the magnesium pool consists of isotopes with a zero nuclear spin (24Mg, 26Mg). The restoration of mitochondrial ATP synthesis involves the participation of creatine kinase since the activity of the latter does not depend on 1-methyl-nicotine amide. In this case, the high efficiency of this restaration seems to be a spin-selective phenomenon which requires predominantly 25Mg2+ cations. A possible meaning of the data for further studies on the mechanisms of enzymatic catalysis regulation is discussed.

  3. MST2 phosphorylation at serine 385 in mitosis inhibits its tumor suppressing activity.

    Science.gov (United States)

    Chen, Xingcheng; Chen, Yuanhong; Dong, Jixin

    2016-12-01

    Mammalian sterile 20-like kinase 1/2 (MST1/2) are core tumor suppressors in the Hippo signaling pathway. MST1/2 have been shown to regulate mitotic progression. Here, we report a novel mechanism for phospho-regulation of MST2 in mitosis and its biological significance in cancer. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates MST2 in vitro and in vivo at serine 385 during antimitotic drug-induced G2/M phase arrest. This phosphorylation occurs transiently during unperturbed mitosis. Mitotic phosphorylation of MST2 does not affect its kinase activity or Hippo-YAP signaling. We further showed that mitotic phosphorylation-deficient mutant MST2-S385A possesses higher activity in suppressing cell proliferation and anchorage-independent growth in vitro and tumorigenesis in vivo. Together, our findings reveal a novel layer of regulation for MST2 in mitosis and its role in tumorigenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

    Science.gov (United States)

    Kim, Sungjin; Alsaidan, Omar Awad; Goodwin, Octavia; Li, Qianjin; Sulejmani, Essilvo; Han, Zhen; Bai, Aiping; Albers, Thomas; Beharry, Zanna; Zheng, Y George; Norris, James S; Szulc, Zdzislaw M; Bielawska, Alicja; Lebedyeva, Iryna; Pegan, Scott D; Cai, Houjian

    2017-12-15

    Protein N -myristoylation enables localization to membranes and helps maintain protein conformation and function. N -myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N -myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. RAG-induced DNA lesions activate proapoptotic BIM to suppress lymphomagenesis in p53-deficient mice

    Science.gov (United States)

    Herold, Marco J.

    2016-01-01

    Neoplastic transformation is driven by oncogenic lesions that facilitate unrestrained cell expansion and resistance to antiproliferative signals. These oncogenic DNA lesions, acquired through errors in DNA replication, gene recombination, or extrinsically imposed damage, are thought to activate multiple tumor suppressive pathways, particularly apoptotic cell death. DNA damage induces apoptosis through well-described p53-mediated induction of PUMA and NOXA. However, loss of both these mediators (even together with defects in p53-mediated induction of cell cycle arrest and cell senescence) does not recapitulate the tumor susceptibility observed in p53−/− mice. Thus, potentially oncogenic DNA lesions are likely to also trigger apoptosis through additional, p53-independent processes. We found that loss of the BH3-only protein BIM accelerated lymphoma development in p53-deficient mice. This process was negated by concomitant loss of RAG1/2-mediated antigen receptor gene rearrangement. This demonstrates that BIM is critical for the induction of apoptosis caused by potentially oncogenic DNA lesions elicited by RAG1/2-induced gene rearrangement. Furthermore, this highlights the role of a BIM-mediated tumor suppressor pathway that acts in parallel to the p53 pathway and remains active even in the absence of wild-type p53 function, suggesting this may be exploited in the treatment of p53-deficient cancers. PMID:27621418

  6. Fem1b promotes ubiquitylation and suppresses transcriptional activity of Gli1.

    Science.gov (United States)

    Gilder, Andrew S; Chen, Yong-Bin; Jackson, Ramon J; Jiang, Jin; Maher, Joseph F

    2013-10-25

    The mammalian Fem1b gene encodes a homolog of FEM-1, a protein in the sex-determination pathway of the nematode Caenorhabditis elegans. Fem1b and FEM-1 proteins each contain a VHL-box motif that mediates their interaction with certain E3 ubiquitin ligase complexes. In C. elegans, FEM-1 negatively regulates the transcription factor TRA-1, and functions as an E3 ubiquitin ligase substrate recognition subunit to target TRA-1 for ubiquitylation. TRA-1 is homologous to the mammalian Gli1 protein, a transcription factor that mediates Hedgehog signaling as well as having Hedgehog-independent functions. Whether the interaction between nematode FEM-1 and TRA-1 proteins is conserved, between corresponding mammalian homologs, has not been reported. Herein, we show that Fem1b interacts with Gli1 within cells, and directly binds Gli1. Fem1b also promotes ubiquitylation of Gli1, suppresses transcriptional activation by Gli1, and attenuates an oncogenic Gli1 autoregulatory loop in cancer cells, all dependent on the VHL-box of Fem1b. These findings have implications for understanding the cellular functions of Fem1b, and the regulation of Gli1 oncoprotein activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Evodia alkaloids suppress gluconeogenesis and lipogenesis by activating the constitutive androstane receptor.

    Science.gov (United States)

    Yu, Lushan; Wang, Zhangting; Huang, Minmin; Li, Yingying; Zeng, Kui; Lei, Jinxiu; Hu, Haihong; Chen, Baian; Lu, Jing; Xie, Wen; Zeng, Su

    2016-09-01

    The constitutive androstane receptor (CAR) is a key sensor in xenobiotic detoxification and endobiotic metabolism. Increasing evidence suggests that CAR also plays a role in energy metabolism by suppressing the hepatic gluconeogenesis and lipogenesis. In this study, we investigated the effects of two evodia alkaloids, rutaecarpine (Rut) and evodiamine (Evo), on gluconeogenesis and lipogenesis through their activation of the human CAR (hCAR). We found that both Rut and Evo exhibited anti-lipogenic and anti-gluconeogenic effects in the hyperlipidemic HepG2 cells. Both compounds can potently activate hCAR, and treatment of cells with hCAR antagonists reversed the anti-lipogenic and anti-gluconeogenic effects of Rut and Evo. The anti-gluconeogenic effect of Rut and Evo was due to the CAR-mediated inhibition of the recruitment of forkhead box O1 (FoxO1) and hepatocyte nuclear factor 4α (HNF4α) onto the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene promoters. In vivo, we showed that treatment of mice with Rut improved glucose tolerance in a CAR-dependent manner. Our results suggest that the evodia alkaloids Rut and Evo may have a therapeutic potential for the treatment of hyperglycemia and type 2 diabetes. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity

    International Nuclear Information System (INIS)

    Pols, Thijs W.H.; Ottenhoff, Roelof; Vos, Mariska; Levels, Johannes H.M.; Quax, Paul H.A.; Meijers, Joost C.M.; Pannekoek, Hans; Groen, Albert K.; Vries, Carlie J.M. de

    2008-01-01

    NR4A nuclear receptors are induced in the liver upon fasting and regulate hepatic gluconeogenesis. Here, we studied the role of nuclear receptor Nur77 (NR4A1) in hepatic lipid metabolism. We generated mice expressing hepatic Nur77 using adenoviral vectors, and demonstrate that these mice exhibit a modulation of the plasma lipid profile and a reduction in hepatic triglyceride. Expression analysis of >25 key genes involved in lipid metabolism revealed that Nur77 inhibits SREBP1c expression. This results in decreased SREBP1c activity as is illustrated by reduced expression of its target genes stearoyl-coA desaturase-1, mitochondrial glycerol-3-phosphate acyltransferase, fatty acid synthase and the LDL receptor, and provides a mechanism for the physiological changes observed in response to Nur77. Expression of LXR target genes Abcg5 and Abcg8 is reduced by Nur77, and may suggest involvement of LXR in the inhibitory action of Nur77 on SREBP1c expression. Taken together, our study demonstrates that Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity

  9. Chronic recordings reveal tactile stimuli can suppress spontaneous activity of neurons in somatosensory cortex of awake and anesthetized primates.

    Science.gov (United States)

    Qi, Hui-Xin; Reed, Jamie L; Franca, Joao G; Jain, Neeraj; Kajikawa, Yoshinao; Kaas, Jon H

    2016-04-01

    In somatosensory cortex, tactile stimulation within the neuronal receptive field (RF) typically evokes a transient excitatory response with or without postexcitatory inhibition. Here, we describe neuronal responses in which stimulation on the hand is followed by suppression of the ongoing discharge. With the use of 16-channel microelectrode arrays implanted in the hand representation of primary somatosensory cortex of New World monkeys and prosimian galagos, we recorded neuronal responses from single units and neuron clusters. In 66% of our sample, neuron activity tended to display suppression of firing when regions of skin outside of the excitatory RF were stimulated. In a small proportion of neurons, single-site indentations suppressed firing without initial increases in response to any of the tested sites on the hand. Latencies of suppressive responses to skin indentation (usually 12-34 ms) were similar to excitatory response latencies. The duration of inhibition varied across neurons. Although most observations were from anesthetized animals, we also found similar neuron response properties in one awake galago. Notably, suppression of ongoing neuronal activity did not require conditioning stimuli or multi-site stimulation. The suppressive effects were generally seen following single-site skin indentations outside of the neuron's minimal RF and typically on different digits and palm pads, which have not often been studied in this context. Overall, the characteristics of widespread suppressive or inhibitory response properties with and without initial facilitative or excitatory responses add to the growing evidence that neurons in primary somatosensory cortex provide essential processing for integrating sensory stimulation from across the hand. Copyright © 2016 the American Physiological Society.

  10. Andrographolide Inhibits Ovariectomy-Induced Bone Loss via the Suppression of RANKL Signaling Pathways

    Science.gov (United States)

    Wang, Tao; Liu, Qian; Zhou, Lin; Yuan, Jin Bo; Lin, Xixi; Zeng, Rong; Liang, Xiaonan; Zhao, Jinmin; Xu, Jiake

    2015-01-01

    Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP), a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX) mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT). Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk), TRACP (Acp5), and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis. PMID:26593901

  11. Andrographolide Inhibits Ovariectomy-Induced Bone Loss via the Suppression of RANKL Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2015-11-01

    Full Text Available Osteoporosis is a debilitating skeletal disorder with an increased risk of low-energy fracture, which commonly occurs among postmenopausal women. Andrographolide (AP, a natural product isolated from Andrographis paniculata, has been found to have anti-inflammatory, anti-cancer, anti-asthmatic, and neuro-protective properties. However, its therapeutic effect on osteoporosis is unknown. In this study, an ovariectomy (OVX mouse model was used to evaluate the therapeutic effects of AP on post-menopausal osteoporosis by using micro-computed tomography (micro-CT. Bone marrow-derived osteoclast culture was used to examine the inhibitory effect of AP on osteoclastogenesis. Real time PCR was employed to examine the effect of AP on the expression of osteoclast marker genes. The activities of transcriptional factors NF-κB and NFATc1 were evaluated using a luciferase reporter assay, and the IκBα protein level was analyzed by Western blot. We found that OVX mice treated with AP have greater bone volume (BV/TV, trabecular thickness (Tb.Th, and trabecular number (Tb.N compared to vehicle-treated OVX mice. AP inhibited RANKL-induced osteoclastogenesis, the expression of osteoclast marker genes including cathepsin K (Ctsk, TRACP (Acp5, and NFATc1, as well as the transcriptional activities of NF-κB and NFATc1. In conclusion, our results suggest that AP inhibits estrogen deficiency-induced bone loss in mice via the suppression of RANKL-induced osteoclastogensis and NF-κB and NFATc1 activities and, thus, might have therapeutic potential for osteoporosis.

  12. Inhibitory effect of putranjivain A on allergic inflammation through suppression of mast cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hui-Hun; Park, Seung-Bin; Lee, Soyoung [CMRI, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kwon, Taeg Kyu [Department of Immunology, School of Medicine, Keimyung University, Daegu 704-701 (Korea, Republic of); Shin, Tae-Yong [College of Pharmacy, Woosuk University, Jeonju 565-701 (Korea, Republic of); Park, Pil-Hoon; Lee, Seung-Ho [College of Pharmacy, Youngnam University, Kyungsan 712-749 (Korea, Republic of); Kim, Sang-Hyun, E-mail: shkim72@knu.ac.kr [CMRI, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2014-02-01

    A great number of people are suffering from allergic inflammatory disease such as asthma, atopic dermatitis, and sinusitis. Therefore discovery of drugs for the treatment of these diseases is an important subject in human health. Putranjivain A (PJA), member of ellagitannin, is known to possess beneficial effects including anti-cancer and anti-viral activities. The aim of the present study was to elucidate whether PJA modulates the allergic inflammatory reaction and to study its possible mechanisms of action using mast cell-based in vitro and in vivo models. The study was performed in anaphylaxis mouse model and cultured mast cells. PJA inhibited the expression of pro-inflammatory cytokines in immunoglobulin E-stimulated mast cells. PJA reduced this expression by inhibiting nuclear factor (NF)-κB and nuclear factor of activated T cell. The oral administration of PJA reduced systemic and cutaneous anaphylaxis, the release of serum histamine, and the expression of the histamine H{sub 1} receptor. In addition, PJA attenuated the activation of mast cells. PJA inhibited the release of histamine from various types of mast cells by the suppression of intracellular calcium. The inhibitory activity of PJA on the allergic reaction was similar to that of disodium cromoglycate, a known anti-allergic drug. These results suggest that PJA can facilitate the prevention or treatment of allergic inflammatory diseases mediated by mast cells. - Highlights: • PJA reduced the degranulation of mast cells. • PJA inhibited the production of inflammatory cytokines. • The effect of PJA on allergic reaction was comparable to the DSCG. • PJA might be a candidate for the treatment of allergic inflammatory diseases.

  13. Antiplatelet Aggregation Activity of Walnut Hull Extract via Suppression of Reactive Oxygen Species Generation and Caspase Activation.

    Science.gov (United States)

    Meshkini, Azadeh; Tahmasbi, Masoumeh

    2017-06-01

    Walnut hull (wal hull) is an agricultural by-product that is widely used in traditional medicine for alleviating pain and treating skin diseases, however, recently it has gained much attention in modern pharmacology due to its antioxidant properties. The current study was aimed to determine the total phenolic, flavonoid, and tannin content of Persian wal hull extract and evaluate its biological effects on platelet function. Experimental data showed that acetone extract of wal hulls has a high content of polyphenolic compounds and antioxidant properties. The analytical study of crude extract by gas chromatography-mass spectrometry demonstrated different types of high- and low-molecular-weight compounds that are basically and biologically important. Moreover, an in vitro study revealed that wal hull extract at a concentration of 50 μg/mL inhibited thrombin-induced platelet aggregation and protein secretion by 50%, without any cytotoxic effects on platelets. The examined extract suppressed reactive oxygen species generation and also caspase activation in thrombin-stimulated platelets. Identically, N-acetylcysteine inhibited the increase of reactive oxygen species level induced by thrombin in platelets, and supported a link between cellular redox status and caspase activation in activated platelets. Presumably, the antiplatelet activity of wal hull extract is related to its polyphenolic compounds and their antioxidant properties. Therefore, wal hulls can be considered as a candidate for thrombotic disorders. Copyright © 2017. Published by Elsevier B.V.

  14. Antiplatelet Aggregation Activity of Walnut Hull Extract via Suppression of Reactive Oxygen Species Generation and Caspase Activation

    Directory of Open Access Journals (Sweden)

    Azadeh Meshkini

    2017-06-01

    Full Text Available Walnut hull (wal hull is an agricultural by-product that is widely used in traditional medicine for alleviating pain and treating skin diseases, however, recently it has gained much attention in modern pharmacology due to its antioxidant properties. The current study was aimed to determine the total phenolic, flavonoid, and tannin content of Persian wal hull extract and evaluate its biological effects on platelet function. Experimental data showed that acetone extract of wal hulls has a high content of polyphenolic compounds and antioxidant properties. The analytical study of crude extract by gas chromatography–mass spectrometry demonstrated different types of high- and low-molecular-weight compounds that are basically and biologically important. Moreover, an in vitro study revealed that wal hull extract at a concentration of 50 μg/mL inhibited thrombin-induced platelet aggregation and protein secretion by 50%, without any cytotoxic effects on platelets. The examined extract suppressed reactive oxygen species generation and also caspase activation in thrombin-stimulated platelets. Identically, N-acetylcysteine inhibited the increase of reactive oxygen species level induced by thrombin in platelets, and supported a link between cellular redox status and caspase activation in activated platelets. Presumably, the antiplatelet activity of wal hull extract is related to its polyphenolic compounds and their antioxidant properties. Therefore, wal hulls can be considered as a candidate for thrombotic disorders.

  15. Copper suppresses abscisic acid catabolism and catalase activity, and inhibits seed germination of rice.

    Science.gov (United States)

    Ye, Nenghui; Li, Haoxuan; Zhu, Guohui; Liu, Yinggao; Liu, Rui; Xu, Weifeng; Jing, Yu; Peng, Xinxiang; Zhang, Jianhua

    2014-11-01

    Although copper (Cu) is an essential micronutrient for plants, a slight excess of Cu in soil can be harmful to plants. Unfortunately, Cu contamination is a growing problem all over the world due to human activities, and poses a soil stress to plant development. As one of the most important biological processes, seed germination is sensitive to Cu stress. However, little is known about the mechanism of Cu-induced inhibition of seed germination. In the present study, we investigated the relationship between Cu and ABA which is the predominant regulator of seed germination. Cu at a concentration of 30 µM effectively inhibited germination of rice caryopsis. ABA content in germinating seeds under copper stress was also higher than that under control conditions. Quantitative real-time PCR (qRT-PCR) revealed that Cu treatment reduced the expression of OsABA8ox2, a key gene of ABA catabolism in rice seeds. In addition, both malondialdehyde (MDA) and H2O2 contents were increased by Cu stress in the germinating seeds. Antioxidant enzyme assays revealed that only catalase activity was reduced by excess Cu, which was consistent with the mRNA profile of OsCATa during seed germination under Cu stress. Together, our results demonstrate that suppression of ABA catabolism and catalase (CAT) activity by excess Cu leads to the inhibition of seed germination of rice. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Muramyl Dipeptide Enhances Lipopolysaccharide-Induced Osteoclast Formation and Bone Resorption through Increased RANKL Expression in Stromal Cells

    Directory of Open Access Journals (Sweden)

    Masahiko Ishida

    2015-01-01

    Full Text Available Lipopolysaccharide (LPS is bacterial cell wall component capable of inducing osteoclast formation and pathological bone resorption. Muramyl dipeptide (MDP, the minimal essential structural unit responsible for the immunological activity of peptidoglycans, is ubiquitously expressed by bacterium. In this study, we investigated the effect of MDP in LPS-induced osteoclast formation and bone resorption. LPS was administered with or without MDP into the supracalvariae of mice. The number of osteoclasts, the level of mRNA for cathepsin K and tartrate-resistant acid phosphatase (TRAP, the ratio of the bone destruction area, the level of tartrate-resistant acid phosphatase form 5b (TRACP 5b, and C-terminal telopeptides fragments of type I collagen as a marker of bone resorption in mice administrated both LPS and MDP were higher than those in mice administrated LPS or MDP alone. On the other hand, MDP had no effect on osteoclastogenesis in parathyroid hormone administrated mice. MDP enhanced LPS-induced receptor activator of NF-κB ligand (RANKL expression and Toll-like receptor 4 (TLR4 expression in vivo and in stromal cells in vitro. MDP also enhanced LPS-induced mitogen-activated protein kinase (MAPK signaling, including ERK, p38, and JNK, in stromal cells. These results suggest that MDP might play an important role in pathological bone resorption in bacterial infection diseases.

  17. Bulleyaconitine A prevents Ti particle-induced osteolysis via suppressing NF-κB signal pathway during osteoclastogenesis and osteoblastogenesis.

    Science.gov (United States)

    Zhang, Liwei; Feng, Mingxuan; Li, Zhiyan; Zhu, Min; Fan, Yongyong; Chu, Binxiang; Yuan, Chiting; Chen, Lihua; Lv, Haiyan; Hong, Zhenghua; Hong, Dun

    2018-02-01

    Balanced bone resorption and bone formation are vital for bone homeostasis. Excessive osteoclastic bone resorption in this process can cause a variety of bone disorders including osteoporosis, aseptic prosthetic loosening and tumor associated bone destruction. Bulleyaconitine A (BLA) is a natural compound that has been widely used for pain treatment but its role in osteolysis has not yet been investigated. In this study, we verified for the first time that BLA inhibited osteoclast formation, the mRNA expression of osteoclast-related genes and osteoclastic bone resorption by inhibiting NF-κB signal pathway and downstream NFATc1 expression. Meanwhile, BLA had a stimulatory effect in osteoblast differentiation and mineralization. Furthermore, BLA showed preventive effect in Ti particle-induced osteolysis model in vivo. Together, all our data demonstrated that BLA suppressed osteoclastogenesis and promoted osteoblastogenesis via suppressing NF-κB signal pathway and could be an alternative therapeutic choice against bone loss. © 2018 Wiley Periodicals, Inc.

  18. Cystatin E/M suppresses legumain activity and invasion of human melanoma

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    Fodstad Øystein

    2010-01-01

    Full Text Available Abstract Background High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied. Methods A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6, and migration and invasiveness were studied by a Matrigel invasion assay. Results Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD was predominant compared to the non-glycosylated form (14 kD. Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at l