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Sample records for carica inhibits osteoclast

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

    SciTech Connect

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

    2015-06-10

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

  2. TDAG8 activation inhibits osteoclastic bone resorption.

    PubMed

    Hikiji, Hisako; Endo, Daisuke; Horie, Kyoji; Harayama, Takeshi; Akahoshi, Noriyuki; Igarashi, Hidemitsu; Kihara, Yasuyuki; Yanagida, Keisuke; Takeda, Junji; Koji, Takehiko; Shimizu, Takao; Ishii, Satoshi

    2014-02-01

    Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis. PMID:24221084

  3. Stimulation by toll-like receptors inhibits osteoclast differentiation.

    PubMed

    Takami, Masamichi; Kim, Nacksung; Rho, Jaerang; Choi, Yongwon

    2002-08-01

    Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-kappa B activation and up-regulated TNF-alpha production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone. PMID:12133979

  4. Megakaryocyte-mediated inhibition of osteoclast development.

    PubMed

    Kacena, Melissa A; Nelson, Tracy; Clough, Mary E; Lee, Sun-Kyeong; Lorenzo, Joseph A; Gundberg, Caren M; Horowitz, Mark C

    2006-11-01

    A growing body of evidence indicates that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. We previously identified a novel regulatory pathway that controls bone formation, which is mediated by MK. In vivo megakaryocytosis resulted in massive bone formation. The co-culture of MK with osteoblasts (OB) resulted in increased OB proliferation in vitro, by a mechanism that required direct cell-to-cell contact. Here, we examined a second MK-mediated pathway that regulates osteoclast (OC) development. We have begun examining the unique inhibitory effect of MK on OC development. Spleen or bone marrow (BM) cells from C57BL/6 mice, as a source of OC precursors, were cultured with M-CSF and RANKL to induce OC development. MK were prepared by culturing fetal liver cells with thrombopoietin and separating cells into MK and non-MK populations. MK were titrated into spleen cell cultures and OC were identified as tartrate-resistant acid phosphatase-positive giant cells with >3 nuclei. There was a significant, P < 0.001, up to 10-fold reduction in OC formed when MK were added to the spleen cell cultures. We determined that 30% (vol:vol) MK conditioned media (CM) were able to completely block OC development from precursors, whereas 3% MK CM resulted in up to a 10-fold reduction in OC development, P < 0.001. These data indicate that a soluble factor(s) was responsible, at least in part, for the inhibition. We examined MK CM for known inhibitors of OC formation, using ELISAs. IL-4 was undetectable in MK CM, whereas IL-10 and IFN-gamma levels were similar in MK and non-MK CM. TGFbeta-1 levels were increased 2-fold in MK CM compared to control CM but were not responsible for the inhibition in OC development. Although, we found a significant increase in the levels of osteoprotegerin (OPG) in MK CM, antibody neutralization studies, MK derived from OPG-deficient mice, and tandem mass spectrophotometry, all confirm that OPG was not responsible for the MK

  5. Extract of acai-berry inhibits osteoclast differentiation and activity.

    PubMed

    Brito, C; Stavroullakis, A T; Ferreira, A C; Li, K; Oliveira, T; Nogueira-Filho, G; Prakki, A

    2016-08-01

    Osteoclastogenesis is the major cellular event responsible for bone loss and is triggered by inflammation. Acai-berry has proven anti-inflammatory effects. However, there is a lack of evidence for its effects on osteoclastogenesis. Thus, the aim of this study was to determine whether acai-berry extract (ABE) could inhibit osteoclastogenesis and osteoclast activity in vitro. The secretion of cytokines by osteoclasts has been also evaluated. RAW 264.7 cells were stimulated with RANKL (50ng/mL) and treated with various concentrations of ABE (25-100μg/mL) to verify: cell viability (MTT), total protein concentration (BCA), osteoclast differentiation and activity, and cytokine secretion. Cell viability and protein assays showed no toxicity to RAW cells for the tested ABE concentrations (p>0.05). ABE also showed a dose-dependent inhibition of osteoclastogenesis and osteoclast activity evaluated by tartrate-resistant acid phosphatase (TRAP) and hydroxylapatite resorption assay, respectively (p<0.05). ABE decreased the secretion of interleukin (IL)-1α, -6 and tumor necrosis factor alpha while increasing the secretion of IL-3, -4, -13 and interferon gamma when compared to the control group (p<0.05). Results of this study showed that acai-berry extract inhibits osteoclast differentiation and activity possibly due to the modulation of a vast number of cytokines produced by osteoclast precursor cells. PMID:27054700

  6. Orostachys japonicus Suppresses Osteoclast Differentiation by Inhibiting NFATc1 Expression.

    PubMed

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

    2015-01-01

    The herb Orostachys japonicus has been traditionally used to treat chronic diseases, such as hepatitis, hemorrhoids, and cancer, in Asia. In this study, we investigated the effect of Orostachys japonicus water extract (OJWE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone loss. We found that OJWE inhibited RANKL-induced osteoclast differentiation in a dose-dependent manner without affecting bone resorption in bone marrow-derived macrophage cells. Interestingly, OJWE significantly reduced serum levels of C-terminal telopeptide of type 1 collagen and tartrate-resistant acid phosphatase (TRAP) 5b, markers of bone resorption and osteoclast number, respectively, in an animal model of bone loss. Furthermore, OJWE suppressed the RANKL-induced up-regulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) expression, and activation of the p38 signaling pathway, but prevented the RANKL-mediated down-regulation of interferon regulatory factor-8 (IRF-8), which is known to be an anti-osteoclastogenic factor that represses NFATc1 expression. We also identified gallic acid and quercetin-3-O-β-D-glucoside as the OJWE components that inhibit RANKL-induced osteoclast differentiation. These results suggest that OJWE inhibits osteoclast differentiation by inhibiting RANKL-induced NFATc1 expression, which prevents osteoclast differentiation and bone loss. The present study elucidated a mechanism of action underlying the inhibitory effect of OJWE on osteoclast differentiation. Our findings suggest that O. japonicus has therapeutic potential for use in the treatment of bone diseases. PMID:26205967

  7. Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation

    PubMed Central

    Li, Defang; Liu, Jin; Guo, Baosheng; Liang, Chao; Dang, Lei; Lu, Cheng; He, Xiaojuan; Cheung, Hilda Yeuk-Siu; Xu, Liang; Lu, Changwei; He, Bing; Liu, Biao; Shaikh, Atik Badshah; Li, Fangfei; Wang, Luyao; Yang, Zhijun; Au, Doris Wai-Ting; Peng, Songlin; Zhang, Zongkang; Zhang, Bao-Ting; Pan, Xiaohua; Qian, Airong; Shang, Peng; Xiao, Lianbo; Jiang, Baohong; Wong, Chris Kong-Chu; Xu, Jiake; Bian, Zhaoxiang; Liang, Zicai; Guo, De-an; Zhu, Hailong; Tan, Weihong; Lu, Aiping; Zhang, Ge

    2016-01-01

    Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation. PMID:26947250

  8. Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts.

    PubMed

    Kanehisa, J

    1989-01-01

    The reversible calcitonin (CT)-induced inhibition of osteoclastic activity has been studied to clarify the mechanisms responsible for the so-called "escape phenomenon." Osteoclasts disaggregated from neonatal rabbits were cultured on glass coverslips or thin bovine bone slices. Resorption activity was evaluated by using time-lapse recording and scanning electron microscopy. Addition of CT to the cultures caused most osteoclasts on glass surfaces to be immotile and contracted. From 1.5 h onward, in cultures with CT, osteoclasts started to escape from CT-induced quiescence independently of other cells. CT also prevented osteoclasts on bone slices from excavating bone while concomitant cell immobility occurred. Inhibited osteoclasts were able to regain apparent bone-resorbing potency only after resumption of cytoplasmic immobility. The resumption of bone resorption could begin as early as 9.7 h after CT addition. The observations indicate that CT-induced inhibition of osteoclastic bone resorption is associated with inhibition of cytoplasmic motility and that the "escape" phenomenon reflects resumption of activity of osteoclasts that were previously inhibited by CT action rather than the resportive activity of newly formed osteoclasts. PMID:2765310

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

    PubMed Central

    Bendixen, Amy C.; Shevde, Nirupama K.; Dienger, Krista M.; Willson, Timothy M.; Funk, Colin D.; Pike, J. Wesley

    2001-01-01

    IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-κB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor γ1 (PPARγ1) ligands and can be blocked by the irreversible PPARγ antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARγ1, an interpretation strengthened by the observation that IL-4 can activate a PPARγ1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARγ1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARγ1 ligands. Our results reveal that PPARγ1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARγ1 ligands on bone loss in diabetic patients. PMID:11226258

  10. Eriodictyol Inhibits RANKL-Induced Osteoclast Formation and Function Via Inhibition of NFATc1 Activity.

    PubMed

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

    2016-09-01

    Receptor activator of nuclear factor kappa-B ligand (RANKL) induces differentiation and function of osteoclasts through triggering multiple signaling cascades, including NF-κB, MAPK, and Ca(2+) -dependent signals, which induce and activate critical transcription factor NFATc1. Targeting these signaling cascades may serve as an effective therapy against osteoclast-related diseases. Here, by screening a panel of natural plant extracts with known anti-inflammatory, anti-tumor, or anti-oxidant properties for possible anti-osteoclastogenic activities we identified Eriodictyol. This flavanone potently suppressed RANKL-induced osteoclastogenesis and bone resorption in a dose-dependent manner without detectable cytotoxicity, suppressing RANKL-induced NF-κB, MAPK, and Ca(2+) signaling pathways. Eriodictyol also strongly inhibited RANKL-induction of c-Fos levels (a critical component of AP-1 transcription factor required by osteoclasts) and subsequent activation of NFATc1, concomitant with reduced expression of osteoclast specific genes including cathepsin K (Ctsk), V-ATPase-d2 subunit, and tartrate resistant acid phosphatase (TRAcP/Acp5). Taken together, these data provide evidence that Eriodictyol could be useful for the prevention and treatment of osteolytic disorders associated with abnormally increased osteoclast formation and function. J. Cell. Physiol. 231: 1983-1993, 2016. © 2016 Wiley Periodicals, Inc. PMID:26754483

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

    SciTech Connect

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

    2014-01-10

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

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

    PubMed

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

    2016-07-01

    IL-37 is a newly defined member of the IL-1 cytokine family. It has been reported that IL-37 inhibited innate immunity and inflammatory responses in autoimmune diseases and tumors. IL-37 also inhibited Lipopolysaccharide (LPS)-induced immunological reaction. LPS is a bacterial cell wall component that is capable of inducing osteoclast formation and pathological bone resorption. However, there is no study to investigate the effect of IL-37 on LPS-induced osteoclast formation and bone resorption. The purpose of this study is to investigate the effect of IL-37 in LPS-induced osteoclast formation and bone resorption. LPS was administrated with or without IL-37 by subcutaneous injection on mice calvariae. The number of osteoclasts, the level of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA, the ratio of the bone resorption pits and the level of C-terminal telopeptide fragments of type I collagen cross-Links as a marker of bone resorption in mice administrated both LPS and IL-37 were lower than that in mice administrated LPS alone. Real-time RT-PCR was performed to analyze osteoclast related cytokines RANKL, TNF-α and IL-1β mRNA levels in vivo. RANKL, TNF-α and IL-1β mRNAs were increased in the LPS alone administrated mice as compared with PBS administrated groups. On the other hand, RANKL, TNF-α and IL-1β mRNAs were inhibited in the IL-37 and LPS administrated mice as compared with LPS alone administrated group. In vitro analysis, there was no effect of IL-37 in RANKL-induced osteoclast formation, TNF-α-induced osteoclast formation and cell viability from bone marrow macrophages as osteoclast precursor and LPS-induced RANKL expression from stromal cells. These results indicated that IL-37 inhibited LPS-induced osteoclast formation and bone resorption via inhibition of LPS-induced osteoclast related cytokines, but might not directly inhibit osteoclast formation on osteoclast precursor and RANKL expression on stromal cells. PMID:27154248

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-01

    Bone is a dynamic tissue that undergoes continuous remodeling coupled with the action of osteoblasts and osteoclasts. Osteoclast activity is elevated during osteoporosis and periodontitis resulting in excessive loss of trabecular and alveolar bone. Osteoclasts are formed in an inflammatory response to cytokine production receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL) and bacterial challenge lipopolysaccharide (LPS). Carvacrol, a monoterpenic phenol present in Origanum vulgare and Thymus vulgaris, is a natural compound with known medicinal properties. We investigated the effects of carvacrol on osteoclast formation induced by RANKL and LPS. Carvacrol suppressed RANKL-induced formation of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells in RAW264.7 macrophages and human CD14(+) monocytes. Furthermore, carvacrol inhibited LPS-induced osteoclast formation in RAW264.7 macrophages. Investigation of the underlying molecular mechanisms revealed that carvacrol downregulated RANKL-induced NF-κB activation in a dose-dependent manner. Furthermore, the suppression of NF-κB activation correlated with inhibition of inhibitor of kappaB (IκB) kinase (IKK) activation and attenuation of inhibitor of NF-κB (IκBa) degradation. Carvacrol potentiated apoptosis in mature osteoclasts by caspase-3 activation and DNA fragmentation. Moreover, carvacrol did not affect the viability of proliferating MC3T3-E1 osteoblast-like cells. Collectively, these results demonstrate that carvacrol mitigates osteoclastogenesis by impairing the NF-κB pathway and induction of apoptosis in mature osteoclasts. PMID:27170515

  15. The dynamin inhibitor dynasore inhibits bone resorption by rapidly disrupting actin rings of osteoclasts.

    PubMed

    Thirukonda, Gnanasagar J; Uehara, Shunsuke; Nakayama, Takahiro; Yamashita, Teruhito; Nakamura, Yukio; Mizoguchi, Toshihide; Takahashi, Naoyuki; Yagami, Kimitoshi; Udagawa, Nobuyuki; Kobayashi, Yasuhiro

    2016-07-01

    The cytoskeletal organization of osteoclasts is required for bone resorption. Binding of dynamin with guanosine triphosphate (GTP) was previously suggested to be required for the organization of the actin cytoskeleton. However, the role of the GTPase activity of dynamin in the organization of the actin cytoskeleton as well as in the bone-resorbing activity of osteoclasts remains unclear. This study investigated the effects of dynasore, an inhibitor of the GTPase activity of dynamin, on the bone-resorbing activity of and actin ring formation in mouse osteoclasts in vitro and in vivo. Dynasore inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation and rapidly disrupting actin rings in osteoclasts. A time-lapse image analysis showed that dynasore shrank actin rings in osteoclasts within 30 min. The intraperitoneal administration of dynasore inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced trabecular bone loss in mouse femurs. These in vitro and in vivo results suggest that the GTPase activity of dynamin is critical for the bone-resorbing activity of osteoclasts and that dynasore is a seed for the development of novel anti-resorbing agents. PMID:26063501

  16. Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.

    PubMed Central

    Kasten, T P; Collin-Osdoby, P; Patel, N; Osdoby, P; Krukowski, M; Misko, T P; Settle, S L; Currie, M G; Nickols, G A

    1994-01-01

    We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption. Images PMID:7513424

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:26704995

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

    PubMed

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

    2014-01-10

    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. PMID:24333429

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

    PubMed

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

    2015-12-10

    Osteoclasts are responsible for bone erosion in diseases such as osteoporosis and rheumatoid arthritis. In the present study, we investigate the effects of eriodictyol, a flavonoid found naturally in citrus fruits, on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation using mouse bone marrow macrophages (BMMs). Eriodictyol inhibited RANKL-induced osteoclast formation in a dose-dependent manner without cytotoxicity. In addition, eriodictyol suppressed bone resorption activity of differentiated osteoclasts. The inhibitory effect of eriodictyol was associated with impaired activation of multiple signaling events downstream of RANK, including extracellular signal-regulated kinase, p38, and c-Jun terminal kinase phosphorylation, followed by decreased nuclear factor of activated T cells (NFAT)c1 expression. Ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of eriodictyol, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Consistent with the in vitro anti-osteoclastogenic effect, eriodictyol suppressed lipopolysaccharide-induced osteoclast formation in the calvarial model and ovariectomy-induced bone loss in vivo. Taken together, our data demonstrate that eriodictyol is a new therapeutic agent with the potential to prevent bone destructive diseases by reducing both osteoclast differentiation and function. PMID:26450448

  20. Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption.

    PubMed

    Su, Nan; Li, Xiaogang; Tang, Yubin; Yang, Jing; Wen, Xuan; Guo, Jingyuan; Tang, Junzhou; Du, Xiaolan; Chen, Lin

    2016-09-01

    Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled-hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts derived from wild-type and Fgfr3-deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3-deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3-deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research. PMID:26990430

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

    PubMed

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

    2016-03-11

    The interplay between bone-forming osteoblasts and bone-resorbing osteoclasts is essential for balanced bone remodeling. In this study, we evaluate the ability of ethyl-2, 5-dihyrdoxybenzoate (E-2, 5-DHB) to affect both osteoblast and osteoclast differentiation for bone regeneration. Osteogenic differentiation of human mesenchymal stem cells (hMSCs) was quantified by measuring alkaline phosphatase (ALP) activity and calcium deposition. To evaluate osteoclast differentiation, we investigated the effect of E-2, 5-DHB on RANKL-activated osteoclastogenesis in RAW 264.7 cells. E-2, 5-DHB enhanced ALP activity and inhibited RAW 264.7 cell osteoclastogenesis in vitro. To assess the in vivo activity of E-2, 5-DHB, hMSCs were delivered subcutaneosuly alone or in combination with E-2, 5-DHB in an alginate gel into the backs of nude-mice. Histological and immunohistochemical evaluation showed significantly higher calcium deposition in the E-2, 5-DHB group. Osteocalcin (OCN) was highly expressed in cells implanted in the gels containing E-2, 5-DHB. Our results suggest that E-2, 5-DHB can effectively enhance osteoblast differentiation and inhibit osteoclast differentiation both in vitro and in vivo. Understanding the dual function of E-2, 5-DHB on osteoblast and osteoclast differentiation will aid in future development of E-2, 5-DHB as a material for bone tissue engineering. PMID:26869515

  2. Innovative approach for urease inhibition by Ficus carica extract-fabricated silver nanoparticles: An in vitro study.

    PubMed

    Borase, Hemant P; Salunkhe, Rahul B; Patil, Chandrashekhar D; Suryawanshi, Rahul K; Salunke, Bipinchandra K; Wagh, Nilesh D; Patil, Satish V

    2015-01-01

    In the present study, a rapid, low-cost, and ecofriendly method of stable silver nanoparticles (AgNPs) synthesis using leaves extract of Ficus carica (F. carica), a plant with diverse metabolic consortium, is reported for the first time. An absorption peak at 422 nm in UV-Vis spectroscopy, a spherical shape with an average size of 21 nm in transmission electron microscopy, and crystalline nature in X-ray powder diffraction studies were observed for the synthesized AgNPs. Fourier transform infrared analysis indicated that proteins of F. carica might have a vital role in AgNP synthesis and stabilization. AgNPs were found to inhibit urease, a key enzyme responsible for the survival and pathogenesis of the bacterium, Helicobacter pylori. Inhibition of urease by AgNPs was monitored spectrophotometrically by the evaluation of ammonia release. The urease inhibition potential of AgNPs can be explored in the treatment of H. pylori by preparing novel combinations of standard drugs with AgNPs- or AgNPs-encapsulated drug molecules. PMID:25560197

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

    SciTech Connect

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

    2013-11-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb+). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts and that survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abrogated by the addition of recombinant OA to cultures, which restored osteoclastogenesis to wild-type levels. Moreover, mix and match co-cultures demonstrated an induction of osteoclastogenesis in D2J osteoblasts co-cultured with osteoclasts of D2J or wild-type. Last, in functional osteo-assays, we show that bone resorption activity of D2J osteoclasts is dramatically reduced, and these osteoclasts present an abnormal ruffled border over the bone surface. Collectively, these data support a model whereby OA/Gpnmb acts as a negative regulator of osteoclast differentiation and survival but not function by inhibiting the ERK/AKT signaling pathways. PMID:25837253

  10. Dihydroartemisinin prevents breast cancer-induced osteolysis via inhibiting both breast caner cells and osteoclasts.

    PubMed

    Feng, Ming-Xuan; Hong, Jian-Xin; Wang, Qiang; Fan, Yong-Yong; Yuan, Chi-Ting; Lei, Xin-Huan; Zhu, Min; Qin, An; Chen, Hai-Xiao; Hong, Dun

    2016-01-01

    Bone is the most common site of distant relapse in breast cancer, leading to severe complications which dramatically affect the patients' quality of life. It is believed that the crosstalk between metastatic breast cancer cells and osteoclasts is critical for breast cancer-induced osteolysis. In this study, the effects of dihydroartemisinin (DHA) on osteoclast formation, bone resorption, osteoblast differentiation and mineralization were initially assessed in vitro, followed by further investigation in a titanium-particle-induced osteolysis model in vivo. Based on the proved inhibitory effect of DHA on osteolysis, DHA was further applied to MDA-MB-231 breast cancer-induced mouse osteolysis model, with the underlying molecular mechanisms further investigated. Here, we verified for the first time that DHA suppressed osteoclast differentiation, F-actin ring formation and bone resorption through suppressing AKT/SRC pathways, leading to the preventive effect of DHA on titanium-particle-induced osteolysis without affecting osteoblast function. More importantly, we demonstrated that DHA inhibited breast tumor-induced osteolysis through inhibiting the proliferation, migration and invasion of MDA-MB-231 cells via modulating AKT signaling pathway. In conclusion, DHA effectively inhibited osteoclastogenesis and prevented breast cancer-induced osteolysis. PMID:26743690

  11. Dihydroartemisinin prevents breast cancer-induced osteolysis via inhibiting both breast caner cells and osteoclasts

    PubMed Central

    Feng, Ming-Xuan; Hong, Jian-Xin; Wang, Qiang; Fan, Yong-Yong; Yuan, Chi-Ting; Lei, Xin-Huan; Zhu, Min; Qin, An; Chen, Hai-Xiao; Hong, Dun

    2016-01-01

    Bone is the most common site of distant relapse in breast cancer, leading to severe complications which dramatically affect the patients’ quality of life. It is believed that the crosstalk between metastatic breast cancer cells and osteoclasts is critical for breast cancer-induced osteolysis. In this study, the effects of dihydroartemisinin (DHA) on osteoclast formation, bone resorption, osteoblast differentiation and mineralization were initially assessed in vitro, followed by further investigation in a titanium-particle-induced osteolysis model in vivo. Based on the proved inhibitory effect of DHA on osteolysis, DHA was further applied to MDA-MB-231 breast cancer-induced mouse osteolysis model, with the underlying molecular mechanisms further investigated. Here, we verified for the first time that DHA suppressed osteoclast differentiation, F-actin ring formation and bone resorption through suppressing AKT/SRC pathways, leading to the preventive effect of DHA on titanium-particle-induced osteolysis without affecting osteoblast function. More importantly, we demonstrated that DHA inhibited breast tumor-induced osteolysis through inhibiting the proliferation, migration and invasion of MDA-MB-231 cells via modulating AKT signaling pathway. In conclusion, DHA effectively inhibited osteoclastogenesis and prevented breast cancer-induced osteolysis. PMID:26743690

  12. Acid extrusion is induced by osteoclast attachment to bone. Inhibition by alendronate and calcitonin.

    PubMed Central

    Zimolo, Z; Wesolowski, G; Rodan, G A

    1995-01-01

    Acid extrusion is essential for osteoclast (OC) activity. We examined Na+ and HCO3(-)-independent H+ extrusion in rat- and mouse OCs by measuring intracellular pH (pHi) changes, with the pHi indicator BCECF (biscarboxyethyl-5-(6) carboxyfluorescein) after H+ loading with an ammonium pulse. 90% of OCs attached to glass do not possess HCO3- and Na(+)-independent H(+)-extrusion (rate of pHi recovery = 0.043 +/- 0.007 (SEM) pH U/min, n = 26). In contrast, in OCs attached to bone, the pHi recovery rate is 0.228 +/- 0.011 pHi U/min, n = 25. OCs on bone also possess a NH(4+)-permeable pathway not seen on glass. The bone-induced H+ extrusion was inhibited by salmon calcitonin (10(-8) M, for 2 h), and was not present after pretreating the bone slices with the aminobisphosphonate alendronate (ALN). At ALN levels of 0.22 nmol/mm2 bone, H+ extrusion was virtually absent 12 h after cell seeding (0.004 +/- 0.002 pH U/min) and approximately 50% inhibition was observed at 0.022 pmol ALN/mm2 bone. The Na(+)-independent H+ extrusion was not inhibited by bafilomycin A1 (up to 10(-7) M), although a bafilomycin A1 (10(-8) M)-sensitive H+ pump was present in membrane vesicles isolated from these osteoclasts. These findings indicate that Na(+)-independent acid extrusion is stimulated by osteoclast attachment to bone and is virtually absent when bone is preincubated with ALN, or when osteoclasts are treated with salmon calcitonin. Images PMID:7593614

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

    SciTech Connect

    Choi, Bongkun; Kang, Soon-Suk; Kang, Sang-Wook; Min, Bon-Hong; Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min; Song, Youngsup; Yoon, Seung-Yong; Chang, Eun-Ju

    2014-07-18

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

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

    SciTech Connect

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

    2013-02-15

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

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

    SciTech Connect

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

    2009-02-20

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

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

    PubMed Central

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

    2013-01-01

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

  17. Inhibiting and stimulating effects of TGF-. beta. 1 on osteoclastic bone resorption in fetal mouse bone organ cultures

    SciTech Connect

    Dieudonne, S.C.; Foo, P.; van Zoelen, E.J.; Burger, E.H. )

    1991-05-01

    The effects of TGF-{beta} 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.

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

    PubMed Central

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

    2014-01-01

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

  19. Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models.

    PubMed

    Azuma, Y; Sato, H; Oue, Y; Okabe, K; Ohta, T; Tsuchimoto, M; Kiyoki, M

    1995-02-01

    Alendronate is an aminobisphosphonate that acts as a potent inhibitor of osteoclastic bone resorption. To understand the mechanism of action of alendronate in vivo, in this study we investigated the relationship between distribution of [14C]-alendronate in rat bone and its effects on bone resorption in vitro or in rat hypercalcemic models. A single IV dose of 0.05 approximately 1.25 mg/kg inhibited the increase in plasma calcium level induced by bovine PTH or 1 alpha(OH)D3. The minimal effective dose of pamidronate (1.25 mg/kg) and etidronate (over 31.25 mg/kg) were at least 5 times and 25 times, respectively, higher than the dose of alendronate in the rat hypercalcemic model prepared by 1 alpha(OH)D3. The relative potencies of compounds in the hypercalcemic rat models reflected those of inhibitory effects on bone resorption in vitro. We conducted the ivory-slice assay under two conditions: (a) addition of a given bisphosphonate after adherence of the osteoclasts; and (b) preincubation of the ivory slices with a given bisphosphonate. The inhibitory IC50 values of alendronate under condition (b) were similar to those under condition (a). To evaluate the interaction between osteoclasts and alendronate in bone, we investigated the localization of [14C]-alendronate in the tibia of growing rats (4-day-old rats). Alendronate did not distribute uniformly in the tibia. At 1 day after injection (0.05 mg SC), dense labeling was seen primarily under osteoclasts. We injected 0.05 mg/kg of [14C]-alendronate (single i.v.) into rats [14C]-alendronate was rapidly eliminated from plasma, and mainly distributed to the bone in rats. These data suggest that alendronate which distributed on bone surface mainly contributed to the antihypercalcemic action in vivo. PMID:7756053

  20. Osteoclast-derived microRNA-containing exosomes selectively inhibit osteoblast activity

    PubMed Central

    Sun, Weijia; Zhao, Chenyang; Li, Yuheng; Wang, Liang; Nie, Guangjun; Peng, Jiang; Wang, Aiyuan; Zhang, Pengfei; Tian, Weiming; Li, Qi; Song, Jinping; Wang, Cheng; Xu, Xiaolong; Tian, Yanhua; Zhao, Dingsheng; Xu, Zi; Zhong, Guohui; Han, Bingxing; Ling, Shukuan; Chang, Yan-Zhong; Li, Yingxian

    2016-01-01

    MicroRNAs have an important role in bone homeostasis. However, the detailed mechanism of microRNA-mediated intercellular communication between bone cells remains elusive. Here, we report that osteoclasts secrete microRNA-enriched exosomes, by which miR-214 is transferred into osteoblasts to inhibit their function. In a coculture system, inhibition of exosome formation and secretion prevented miR-214 transportation. Exosomes specifically recognized osteoblasts through the interaction between ephrinA2 and EphA2. In osteoclast-specific miR-214 transgenic mice, exosomes were secreted into the serum, and miR-214 and ephrinA2 levels were elevated. Therefore, these exosomes have an inhibitory role in osteoblast activity. miR-214 and ephrinA2 levels in serum exosomes from osteoporotic patients and mice were upregulated substantially. These exosomes may significantly inhibit osteoblast activity. Inhibition of exosome secretion via Rab27a small interfering RNA prevented ovariectomized-induced osteoblast dysfunction in vivo. Taken together, these findings suggest that exosome-mediated transfer of microRNA plays an important role in the regulation of osteoblast activity. Circulating miR-214 in exosomes not only represents a biomarker for bone loss but could selectively regulate osteoblast function. PMID:27462462

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

    PubMed

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

    2015-12-01

    Cytoskeletal changes in osteoclasts such as formation of actin ring is required for bone-resorbing activity. The tyrosine kinase Src is a key player in massive cytoskeletal change of osteoclasts, thereby in bone destruction. In order for Src to be activated, trafficking to the inner plasma membrane via myristoylation is of importance. A previous study reported that myristoleic acid derived from myristic acid, inhibited N-myristoyl-transferase, an essential enzyme for myristoylation process. This prompted us to investigate whether myristoleic acid could affect osteoclastogenesis. Indeed, we observed that myristoleic acid inhibited RANKL-induced osteoclast formation in vitro, especially, at later stages of differentiation. Myristoleic acid attenuated the tyrosine phosphorylation of c-Src and Pyk2, which associates with Src, by RANKL. When myristoleic acid was co-administered with soluble RANKL into mice, RANKL-induced bone loss was substantially prevented. Bone dissection clearly revealed that the number of multinucleated osteoclasts was significantly diminished by myristoleic acid. On the other hand, myristoleic acid treatment had little or no influence on early osteoclast differentiation markers, such as c-Fos and NFATc1, and proteins related to cytoskeletal rearrangement, including DC-STAMP, integrin αv and integrin β3 in vitro. Taken together, our data suggest that myristoleic acid is capable of blocking the formation of large multinucleated osteoclasts and bone resorption likely through suppressing activation of Src and Pyk2. PMID:26528796

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

    SciTech Connect

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

    2007-03-30

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

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

    SciTech Connect

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

    2015-05-01

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

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

    SciTech Connect

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

    2012-10-19

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  7. Drugs Which Inhibit Osteoclast Function Suppress Tumor Growth through Calcium Reduction in Bone

    PubMed Central

    Li, Xin; Liao, Jinhui; Park, Serk In; Koh, Amy J; Sadler, William D; Pienta, Kenneth J; Rosol, Thomas J; McCauley, Laurie K

    2011-01-01

    Prostate carcinoma frequently metastasizes to bone where the microenvironment facilitates its growth. Inhibition of bone resorption is effective in reducing tumor burden and bone destruction in prostate cancer. However, whether drugs that inhibit osteoclast function inhibit tumor growth independent of inhibition of bone resorption is unclear. Calcium is released during bone resorption and the calcium sensing receptor is an important regulator of cancer cell proliferation. The goal of this investigation was to elucidate the role of calcium released during bone resorption and to determine the impact of drugs which suppress bone resorption on tumor growth in bone. To compare tumor growth in a skeletal versus non-skeletal site, equal numbers of canine prostate cancer cells expressing luciferase (ACE-1luc) prostate cancer cells were inoculated into a simple collagen matrix, neonatal mouse vertebrae (vossicles), human de-proteinized bone, or a mineralized collagen matrix. Implants were placed subcutaneously into athymic mice. Luciferase activity was used to track tumor growth weekly and at one month tumors were dissected for histologic analysis. Luciferase activity and tumor size were greater in vossicles, de-proteinized bone and mineralized collagen matrix versus non-mineralized collagen implants. The human osteoblastic prostate carcinoma cell line C4-2b also grew better in a mineral rich environment with a greater proliferation of C4-2b cells reflected by Ki-67 staining. Zoledronic acid (ZA), a bisphosphonate, and recombinant OPG-Fc, a RANKL inhibitor, were administered to mice bearing vertebral implants (vossicles) containing ACE-1 osteoblastic prostate cancer cells. Vossicles or collagen matrices were seeded with ACE-1luc cells subcutaneously in athymic mice (2 vossicles, 2 collagen implants/mouse). Mice received ZA (5μg/mouse, twice/week), (OPG-Fc at 10mg/kg, 3 times/week) or vehicle, and luciferase activity was measured weekly. Histologic analysis of the tumors

  8. CGRP may regulate bone metabolism through stimulating osteoblast differentiation and inhibiting osteoclast formation.

    PubMed

    He, Haitao; Chai, Jianshen; Zhang, Shengfu; Ding, Linlin; Yan, Peng; Du, Wenjun; Yang, Zhenzhou

    2016-05-01

    Calcitonin-gene-related peptide (CGRP) is a neuropeptide, which is widely distributed throughout the central and peripheral nervous systems. Numerous mechanisms underlying the action of CGRP in osteoblast-associated cells have been suggested for bone growth and metabolism. The present study was designed to closely investigate the osteoblast‑ and osteoclast-associated mechanisms of the effect of CGRP administration on bone metabolism in primary osteoblasts. Primary osteoblasts were obtained from newborn rabbit calvaria and incubated with different concentrations of human CGRP (hCGRP), hCGRP and hCGRP (8‑37), or without treatment as a control. Intracellular calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) were detected following treatment, as well as the expression levels of osteoblast differentiation markers, including activating transcription factor‑4 (ATF4) and osteocalcin (OC), and receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG). The isolated primary osteoblasts were found to stain positively for ALP. hCGRP treatment had no significant effect on transient intracellular Ca2+ in the osteoblasts. Treatment of the osteoblasts with hCGRP led to elevations in the expression levels of cAMP, ATF4 and OPG, and downregulation in the expression of RANKL, in a dose‑dependent manner. These effects were markedly reversed by the addition of hCGRP (8‑37). The results of the present study demonstrated that CGRP administration not only stimulated osteoblast differentiation, as demonstrated by upregulated expression levels of ATF4 and OC in the hCGRP‑treated osteoblasts, but also inhibited OPG/RANKL‑regulated osteoclastogenesis. CGRP may act as a modulator of bone metabolism through osteoblast and osteoclast-associated mechanisms, which result in osteoblast formation with subsequent activation of bone formation. PMID:27035229

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2009-01-16

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

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

    PubMed Central

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

    2012-01-01

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

  15. Immature and Mature Megakaryocytes Enhance Osteoblast Proliferation and Inhibit Osteoclast Formation

    PubMed Central

    Ciovacco, Wendy A.; Cheng, Ying-Hua; Horowitz, Mark C.; Kacena, Melissa A.

    2011-01-01

    Recent data suggests that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC proliferation, that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs. PMID:20052670

  16. Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction*

    PubMed Central

    Kartner, Norbert; Yao, Yeqi; Li, Keying; Crasto, Gazelle J.; Datti, Alessandro; Manolson, Morris F.

    2010-01-01

    Vacuolar H+-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC50 of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis. PMID:20837476

  17. Knee loading inhibits osteoclast lineage in a mouse model of osteoarthritis

    PubMed Central

    Li, Xinle; Yang, Jing; Liu, Daquan; Li, Jie; Niu, Kaijun; Feng, Shiqing; Yokota, Hiroki; Zhang, Ping

    2016-01-01

    Osteoarthritis (OA) is a whole joint disorder that involves cartilage degradation and periarticular bone response. Changes of cartilage and subchondral bone are associated with development and activity of osteoclasts from subchondral bone. Knee loading promotes bone formation, but its effects on OA have not been well investigated. Here, we hypothesized that knee loading regulates subchondral bone remodeling by suppressing osteoclast development, and prevents degradation of cartilage through crosstalk of bone-cartilage in osteoarthritic mice. Surgery-induced mouse model of OA was used. Two weeks application of daily dynamic knee loading significantly reduced OARSI scores and CC/TAC (calcified cartilage to total articular cartilage), but increased SBP (subchondral bone plate) and B.Ar/T.Ar (trabecular bone area to total tissue area). Bone resorption of osteoclasts from subchondral bone and the differentiation of osteoclasts from bone marrow-derived cells were completely suppressed by knee loading. The osteoclast activity was positively correlated with OARSI scores and negatively correlated with SBP and B.Ar/T.Ar. Furthermore, knee loading exerted protective effects by suppressing osteoclastogenesis through Wnt signaling. Overall, osteoclast lineage is the hyper responsiveness of knee loading in osteoarthritic mice. Mechanical stimulation prevents OA-induced cartilage degeneration through crosstalk with subchondral bone. Knee loading might be a new potential therapy for osteoarthritis patients. PMID:27087498

  18. Knee loading inhibits osteoclast lineage in a mouse model of osteoarthritis.

    PubMed

    Li, Xinle; Yang, Jing; Liu, Daquan; Li, Jie; Niu, Kaijun; Feng, Shiqing; Yokota, Hiroki; Zhang, Ping

    2016-01-01

    Osteoarthritis (OA) is a whole joint disorder that involves cartilage degradation and periarticular bone response. Changes of cartilage and subchondral bone are associated with development and activity of osteoclasts from subchondral bone. Knee loading promotes bone formation, but its effects on OA have not been well investigated. Here, we hypothesized that knee loading regulates subchondral bone remodeling by suppressing osteoclast development, and prevents degradation of cartilage through crosstalk of bone-cartilage in osteoarthritic mice. Surgery-induced mouse model of OA was used. Two weeks application of daily dynamic knee loading significantly reduced OARSI scores and CC/TAC (calcified cartilage to total articular cartilage), but increased SBP (subchondral bone plate) and B.Ar/T.Ar (trabecular bone area to total tissue area). Bone resorption of osteoclasts from subchondral bone and the differentiation of osteoclasts from bone marrow-derived cells were completely suppressed by knee loading. The osteoclast activity was positively correlated with OARSI scores and negatively correlated with SBP and B.Ar/T.Ar. Furthermore, knee loading exerted protective effects by suppressing osteoclastogenesis through Wnt signaling. Overall, osteoclast lineage is the hyper responsiveness of knee loading in osteoarthritic mice. Mechanical stimulation prevents OA-induced cartilage degeneration through crosstalk with subchondral bone. Knee loading might be a new potential therapy for osteoarthritis patients. PMID:27087498

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

    PubMed Central

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N; Neutzsky-Wulff, Anita V; Geisler, Carsten; Karlsson, Lars; Söderström, Kalle

    2015-01-01

    Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions. PMID:25684021

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

    SciTech Connect

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

    2009-04-15

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

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

    PubMed

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

    2016-01-29

    Receptor activator of nuclear factor (NF)-κB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation, and survival. Salicortin is a phenolic glycoside that has been isolated from many plants such as Populus and Salix species, and has been shown to have anti-amnesic and anti-adipogenic effects. In this study, we investigated the effect of salicortin on RANKL-induced osteoclasts formation, bone resorption, and activation of osteoclast-related signaling pathways. Salicortin suppressed RANKL-induced osteoclastogenesis in bone marrow macrophage cultures in a dose-dependent manner, and inhibited osteoclastic bone resorption activity without any cytotoxicity. Salicortin inhibited RANKL-induced c-Jun N-terminal kinase and NF-κB activation, concomitant with retarded IκBα phosphorylation and inhibition of p65 nuclear translocation, leading to impaired transcription of nuclear factor of activated T cells c1 (NFATc1) and expression of osteoclastic-specific genes. Taken together, our findings demonstrate that salicortin inhibits NF-κB and NFATc1 activation, leading to attenuation of osteoclastogenesis and bone resorption. Thus, salicortin may be of interest in developments of treatment for osteoclast related diseases. PMID:26740180

  2. Inhibition of Platelet Aggregation by the Leaf Extract of Carica papaya During Dengue Infection: An In Vitro Study.

    PubMed

    Chinnappan, Shobia; Ramachandrappa, Vijayakumar Shettikothanuru; Tamilarasu, Kadhiravan; Krishnan, Uma Maheswari; Pillai, Agiesh Kumar Balakrishna; Rajendiran, Soundravally

    2016-04-01

    Dengue cases were reported to undergo platelet activation and thrombocytopenia by a poorly understood mechanism. Recent studies suggested that Carica papaya leaf extract could recover the platelet count in dengue cases. However, no studies have attempted to unravel the mechanism of the plant extract in platelet recovery. Since there are no available drugs to treat dengue and considering the significance of C. papaya in dengue treatment, the current study aimed to evaluate two research questions: First one is to study if the C. papaya leaf extract exerts its action directly on platelets and second one is to understand if the extract can specifically inhibit the platelet aggregation during dengue viral infection. Sixty subjects with dengue positive and 60 healthy subjects were recruited in the study. Platelet-rich plasma (PRP) and platelet-poor plasma were prepared from both the dengue-infected and healthy control blood samples. Effect of the leaf extract obtained from C. papaya leaves was assessed on plasma obtained as well as platelets collected from both healthy and dengue-infected individuals. Platelet aggregation was significantly reduced when leaf extract preincubated with dengue plasma was added into control PRP, whereas no change in aggregation when leaf extract incubated-control plasma was added into control PRP. Upon direct addition of C. papaya leaf extract, both dengue PRP and control PRP showed a significant reduction in platelet aggregation. Within the dengue group, PRP from severe and nonsevere cases showed a significant decrease in aggregation without any difference between them. From the study, it is evident that C. papaya leaf extract can directly act on platelet. The present study, the first of its kind, found that the leaf extract possesses a dengue-specific neutralizing effect on dengue viral-infected plasma that may exert a protective role on platelets. PMID:26910599

  3. Genistein Inhibits Osteoclastic Differentiation of RAW 264.7 Cells via Regulation of ROS Production and Scavenging

    PubMed Central

    Lee, Sang-Hyun; Kim, Jin-Kyoung; Jang, Hae-Dong

    2014-01-01

    Genistein, a phytoestrogen, has been demonstrated to have a bone-sparing and antiresorptive effect. Genistein can inhibit the osteoclast formation of receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW 264.7 cells by preventing the translocation of nuclear factor-κB (NF-κB), a redox-sensitive factor, to the nucleus. Therefore, the suppressive effect of genistein on the reactive oxygen species (ROS) level during osteoclast differentiation and the mechanism associated with the control of ROS levels by genistein were investigated. The cellular antioxidant capacity and inhibitory effect of genistein were confirmed. The translation and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1), as well as the disruption of the mitochondrial electron transport chain system were obviously suppressed by genistein in a dose-dependent manner. The induction of phase II antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and heme oxygenase-1 (HO-1), was enhanced by genistein. In addition, the translational induction of nuclear factor erythroid 2-related factor 2 (Nrf2) was notably increased by genistein. These results provide that the inhibitory effects of genistein on RANKL-stimulated osteoclast differentiation is likely to be attributed to the control of ROS generation through suppressing the translation and activation of Nox1 and the disruption of the mitochondrial electron transport chain system, as well as ROS scavenging through the Nrf2-mediated induction of phase II antioxidant enzymes, such as SOD1 and HO-1. PMID:24927148

  4. Characterization of vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase in osteoclasts

    SciTech Connect

    Yao, GuanFeng; Feng, HaoTian; Cai, YanLing; Qi, WeiLi; Kong, KangMei . E-mail: kangmeikong@21cn.com

    2007-06-15

    V-ATPase plays important roles in controlling the extra- and intra-cellular pH in eukaryotic cell, which is most crucial for cellular processes. V-ATPases are composed of a peripheral V{sub 1} domain responsible for ATP hydrolysis and integral V{sub 0} domain responsible for proton translocation. Osteoclasts are multinucleated cells responsible for bone resorption and relate to many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone loss. This review summarizes the structure and function of V-ATPase and its subunit, the role of V-ATPase subunits in osteoclast function, V-ATPase inhibitors for osteoclast function, and highlights the importance of V-ATPase as a potential prime target for anti-resorptive agents.

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

    PubMed Central

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

    2014-01-01

    Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of NF-κB ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and IκB, as well as IκB degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption. [BMB Reports 2014; 47(8): 451-456] PMID:24314143

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

    PubMed Central

    Visagie, Amcois; Kasonga, Abe; Deepak, Vishwa; Moosa, Shaakirah; Marais, Sumari; Kruger, Marlena C.; Coetzee, Magdalena

    2015-01-01

    Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone. PMID:26516894

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

    SciTech Connect

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

    2011-09-23

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

  8. Anthraquinone compounds from Morinda officinalis inhibit osteoclastic bone resorption in vitro.

    PubMed

    Bao, Leilei; Qin, Luping; Liu, Lei; Wu, Yanbin; Han, Ting; Xue, Liming; Zhang, Qiaoyan

    2011-11-15

    The root of Morinda officinalis has been claimed to have a protective effect against bone loss in sciatic neurectomized and ovariectomized osteoporotic rats, and this protective effect is supposed to be attributed to anthraquinone compounds in the plant. In the present study, we investigated the effects of three anthraquinones isolated from M. officinalis, including 1, 3, 8-trihydroxy-2-methoxy-anthraquinone (1), 2-hydroxy-1-methoxy-anthraquinone (2) and rubiadin (3) on bone resorption activity in vitro and the mechanism on osteoclasts derived from rat bone marrow cells. Compound 1, 2 and 3 decreased the formation of bone resorption pits, the number of multinucleated osteoclasts, and the activity of tartrate resistant acid phosphates (TRAP) and cathepsin K in the coculture system of osteoblasts and bone marrow cells in the presence of 1, 25-dihydroxyvitamine D(3) and dexamethasone. They also enhanced the apoptosis of osteoclasts induced from bone marrow cells with M-CSF and RANKL. In addition, Compound 1, 2 and 3 improved the ratio of mRNA and protein expression of OPG and RANKL in osteoblasts, interfered with the JNK and NF-κB signal pathway, and reduced the expression of calcitonin receptor (CTR) and carbonic anhydrase/II (CA II) in osteoclasts induced from bone marrow cells with M-CSF and RANKL. These findings indicate that the anthraquinone compounds from M. officinalis are potential inhibitors of bone resorption, and may also serve as evidence to explain the mechanism of the inhibitory effects of some other reported anthraquinones on bone loss. PMID:21945525

  9. WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling

    PubMed Central

    Chen, Cheng; Qin, Yi; Fang, Jian-ping; Ni, Xin-yan; Yao, Jian; Wang, Hai-ying; Ding, Kan

    2015-01-01

    Aim: WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice. Methods: RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg−1·d−1, po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured. Results: In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice. Conclusion: WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad

  10. Calcitonin controls bone formation by inhibiting the release of sphingosine 1-phosphate from osteoclasts

    PubMed Central

    Keller, Johannes; Catala-Lehnen, Philip; Huebner, Antje K.; Jeschke, Anke; Heckt, Timo; Lueth, Anja; Krause, Matthias; Koehne, Till; Albers, Joachim; Schulze, Jochen; Schilling, Sarah; Haberland, Michael; Denninger, Hannah; Neven, Mona; Hermans-Borgmeyer, Irm; Streichert, Thomas; Breer, Stefan; Barvencik, Florian; Levkau, Bodo; Rathkolb, Birgit; Wolf, Eckhard; Calzada-Wack, Julia; Neff, Frauke; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabě; Klutmann, Susanne; Tsourdi, Elena; Hofbauer, Lorenz C.; Kleuser, Burkhard; Chun, Jerold; Schinke, Thorsten; Amling, Michael

    2014-01-01

    The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signaling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the non-selective S1P receptor agonist FTY720 causes increased bone formation in wildtype, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo, and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts. PMID:25333900

  11. Calcitonin controls bone formation by inhibiting the release of sphingosine 1-phosphate from osteoclasts.

    PubMed

    Keller, Johannes; Catala-Lehnen, Philip; Huebner, Antje K; Jeschke, Anke; Heckt, Timo; Lueth, Anja; Krause, Matthias; Koehne, Till; Albers, Joachim; Schulze, Jochen; Schilling, Sarah; Haberland, Michael; Denninger, Hannah; Neven, Mona; Hermans-Borgmeyer, Irm; Streichert, Thomas; Breer, Stefan; Barvencik, Florian; Levkau, Bodo; Rathkolb, Birgit; Wolf, Eckhard; Calzada-Wack, Julia; Neff, Frauke; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabĕ; Klutmann, Susanne; Tsourdi, Elena; Hofbauer, Lorenz C; Kleuser, Burkhard; Chun, Jerold; Schinke, Thorsten; Amling, Michael

    2014-01-01

    The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts. PMID:25333900

  12. The effect of molecules in mother-of-pearl on the decrease in bone resorption through the inhibition of osteoclast cathepsin K.

    PubMed

    Duplat, Denis; Gallet, Marlène; Berland, Sophie; Marie, Arul; Dubost, Lionel; Rousseau, Marthe; Kamel, Saïd; Milet, Christian; Brazier, Michel; Lopez, Evelyne; Bédouet, Laurent

    2007-11-01

    This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial. PMID:17686515

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-09-23

    The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue. In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance osteoblast differentiation and to inhibit osteoclast differentiation simultaneously. PMID:21888898

  15. Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro.

    PubMed

    Yuan, Si-Yuan; Sheng, Tong; Liu, Lian-Qi; Zhang, Yun-Ling; Liu, Xue-Mei; Ma, Tao; Zheng, Hong; Yan, Yan; Ishimi, Yoshiko; Wang, Xin-Xiang

    2016-04-01

    The present study aimed at investigating the effects of Puerarin (PR), a major isoflavonoid isolated from the Chinese medicinal herb Puerariae radix, on bone metabolism and the underlying mechanism of action. The in vivo assay, female mice were ovariectomized (OVX), and the OVX mice were fed with a diet containing low, middle, and high doses of PR (2, 4, and 8 mg·d(-1), respectively) or 17β-estradiol (E2, 0.03 μg·d(-1)) for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight, compared with the control. The total femoral bone mineral density (BMD) was significantly reduced by OVX, which was reversed by intake of the diet with PR at any dose, especially at the low dose. In the in vitro assay, RAW264.7 cells were used for studying the direct effect of PR on the formation of osteoclasts. PR reduced the formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in the RAW 264.7 cells induced by receptor activator for nuclear factor-κB Ligand (RANKL). MC3T3-E1 cells were used for studying the effects of PR on the expression of osteoprotegerin (OPG) and RANKL mRNA expression in osteoblasts. The expression of OPG mRNA and RANKL mRNA was detected by RT-PCR on Days of 5, 7, 10, and 12 after PR exposure. PR time-dependently enhanced the expression of OPG mRNA and reduced the expression of RANKL mRNA in MC3T3-E1 cells. In conclusion, our results suggest that PR can effectively prevent bone loss in OVX mice without any hyperplastic effect on the uterus, and the antiosteoporosis activity of PR may be related to its effects on the formation of osteoclasts and the expression of RANKL OPG in osteoblasts. PMID:27114313

  16. Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice.

    PubMed

    Dankbar, Berno; Fennen, Michelle; Brunert, Daniela; Hayer, Silvia; Frank, Svetlana; Wehmeyer, Corinna; Beckmann, Denise; Paruzel, Peter; Bertrand, Jessica; Redlich, Kurt; Koers-Wunrau, Christina; Stratis, Athanasios; Korb-Pap, Adelheid; Pap, Thomas

    2015-09-01

    Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA. PMID:26236992

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

    PubMed Central

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

    2016-01-01

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

  18. Tatarinan O, a lignin-like compound from the roots of Acorus tatarinowii Schott inhibits osteoclast differentiation through suppressing the expression of c-Fos and NFATc1.

    PubMed

    Xu, Xiaohan; Liu, Ning; Wang, Yingjian; Pan, Lei-Chang; Wu, Donglin; Peng, Qisheng; Zhang, Maolin; Wang, Hong-Bing; Sun, Wan-Chun

    2016-05-01

    Osteoclasts (OC) are large multinucleated cells derived from monocyte/macrophage precursors. Suppressing osteoclastogenesis is considered as an effective therapeutic approach to erosive bone disease. The root of Acorus tatarinowii Schott, a well-known traditional Chinese medicine was used to treat rheumatosis and other inflammatory disease. However, the effects of tatarinan O (TO), one of the lignin-like compounds isolated from the roots of Acorus tatarinowii Schott during bone development are still unclear. In the present study, we explored the effect of TO on RANKL-induced osteoclastogenesis in vitro. TO was found to suppress osteoclast differentiation from RANKL-stimulated mouse bone marrow macrophages (BMMs) without significant cytotoxicity. TO also dose-dependently suppressed bone resorption activity of mature osteoclasts. Additionally, TO apparently inhibited the expression of osteoclastic marker genes, such as MMP-9, Cts K and TRAP. Furthermore, our results showed that TO decreased RANKL-induced expression of c-Fos and NFATc1 without influencing NF-κB activation and MAPK phosphorylation. Hence, for the first time we revealed that TO dose-dependently inhibited osteoclastogenesis from RANKL-stimulated mouse BMMs via decreasing the expression of NFATc1 and c-Fos. PMID:26971224

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

    PubMed Central

    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-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. PMID:24191169

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

    PubMed Central

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

    2016-01-01

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

  1. The Function of Naringin in Inducing Secretion of Osteoprotegerin and Inhibiting Formation of Osteoclasts

    PubMed Central

    Xu, Tong; Wang, Lu; Tao, You; Ji, Yan; Deng, Feng; Wu, Xiao-Hong

    2016-01-01

    Osteoporosis has become one of the most prevalent and costly diseases in the world. It is a metabolic disease characterized by reduction in bone mass due to an imbalance between bone formation and resorption. Osteoporosis causes fractures, prolongs bone healing, and impedes osseointegration of dental implants. Its pathological features include osteopenia, degradation of bone tissue microstructure, and increase of bone fragility. In traditional Chinese medicine, the herb Rhizoma Drynariae has been commonly used to treat osteoporosis and bone nonunion. However, the precise underlying mechanism is as yet unclear. Osteoprotegerin is a cytokine receptor shown to play an important role in osteoblast differentiation and bone formation. Hence, activators and ligands of osteoprotegerin are promising drug targets and have been the focus of studies on the development of therapeutics against osteoporosis. In the current study, we found that naringin could synergistically enhance the action of 1α,25-dihydroxyvitamin D3 in promoting the secretion of osteoprotegerin by osteoblasts in vitro. In addition, naringin can also influence the generation of osteoclasts and subsequently bone loss during organ culture. In conclusion, this study provides evidence that natural compounds such as naringin have the potential to be used as alternative medicines for the prevention and treatment of osteolysis. PMID:26884798

  2. The Function of Naringin in Inducing Secretion of Osteoprotegerin and Inhibiting Formation of Osteoclasts.

    PubMed

    Xu, Tong; Wang, Lu; Tao, You; Ji, Yan; Deng, Feng; Wu, Xiao-Hong

    2016-01-01

    Osteoporosis has become one of the most prevalent and costly diseases in the world. It is a metabolic disease characterized by reduction in bone mass due to an imbalance between bone formation and resorption. Osteoporosis causes fractures, prolongs bone healing, and impedes osseointegration of dental implants. Its pathological features include osteopenia, degradation of bone tissue microstructure, and increase of bone fragility. In traditional Chinese medicine, the herb Rhizoma Drynariae has been commonly used to treat osteoporosis and bone nonunion. However, the precise underlying mechanism is as yet unclear. Osteoprotegerin is a cytokine receptor shown to play an important role in osteoblast differentiation and bone formation. Hence, activators and ligands of osteoprotegerin are promising drug targets and have been the focus of studies on the development of therapeutics against osteoporosis. In the current study, we found that naringin could synergistically enhance the action of 1α,25-dihydroxyvitamin D3 in promoting the secretion of osteoprotegerin by osteoblasts in vitro. In addition, naringin can also influence the generation of osteoclasts and subsequently bone loss during organ culture. In conclusion, this study provides evidence that natural compounds such as naringin have the potential to be used as alternative medicines for the prevention and treatment of osteolysis. PMID:26884798

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2011-06-10

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

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

    PubMed Central

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

    2015-01-01

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

  6. Rhinacanthin C Inhibits Osteoclast Differentiation and Bone Resorption: Roles of TRAF6/TAK1/MAPKs/NF-κB/NFATc1 Signaling

    PubMed Central

    Tomomura, Mineko; Suzuki, Ryuichiro; Shirataki, Yoshiaki; Sakagami, Hiroshi; Tamura, Nobuaki; Tomomura, Akito

    2015-01-01

    Rhinacanthin C is a naphthoquinone ester with anti-inflammatory activity, found in Rhinacanthus nasutus (L) Kurz (Acanthaceae). We found that rhinacanthin C inhibited osteoclast differentiation stimulated by the receptor activator of nuclear factor-κB ligand (RANKL) in mouse bone marrow macrophage cultures, although the precise molecular mechanisms underlying this phenomenon are unclear. In this study, we investigated the inhibitory mechanisms of rhinacanthin C in osteoclastogenesis. Rhinacanthin C suppressed RANKL-induced nuclear factor of activated T cells c1 (NFATc1) expression. Phosphorylation of ERK, JNK, and NF-κB, but not p38, was inhibited by rhinacanthin C, which also inhibited RANKL-stimulated TRAF6-TAK1 complex formation. Thus, the anti-osteoclastogenic effect of rhinacanthin C is mediated by a cascade of inhibition of RANKL-induced TRAF6-TAK1 association followed by activation of MAPKs/NF-κB; this leads to suppression of c-Fos and NFATc1, which regulate transcription of genes associated with osteoclast differentiation. In vivo, rhinacanthin C also reduced RANKL-induced osteoclast formation and bone resorption in mouse calvaria. Rhinacanthin C also suppressed LPS-stimulated osteoclastogenesis and bone resorption in vitro and in vivo. Rhinacanthin C may provide a novel therapy for abnormal bone lysis that occurs during inflammatory bone resorption. PMID:26083531

  7. Inhibition of RANKL-induced osteoclast differentiation through the downregulation of c-Fos and NFATc1 by Eremochloa ophiuroides (centipedegrass) extract.

    PubMed

    Choi, Bo-Yun; Park, Chul-Hong; Na, Yun Hee; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-05-01

    Osteoclasts, derived from hematopoietic stem cells, are specialized macrophages and have a homeostatic role in skeletal modeling and remodeling with bone-forming osteoblasts. However, excessive osteoclast activity induces bone diseases, including osteoporosis, periodontitis and rheumatoid arthritis. Natural substances have received attention as therapeutic drugs in human diseases. In the current study, cells isolated from mouse bone marrow, and a mouse model, were used to determine the effect of centipedegrass extract (CGE) on osteoclasts. Multiple concentrations of CGE were administered to bone marrow cells for 24‑72 hours and, for the in vivo study, mice were treated with CGE for 8 days. The effects of CGE on transcription and translation of osteoclast-associated molecules were then determined using reverse transcription-polymerase chain reaction and immunoblotting, respectively. In the present study it was shown that CGE extracted from Eremochloa ophiuroides (centipedegrass) inhibited receptor activator of nuclear factor κ‑B ligand (RANKL)‑mediated osteoclast differentiation in bone marrow macrophages, without cytotoxicity, in a dose‑dependent manner. CGE decreased the expression levels of osteoclast‑specific genes, including matrix metalloproteinase‑9, osteoclast‑associated immunoglobulin‑like receptor and cathepsin K, however, CGE had no inhibitory effect on the expression levels of mitogen‑activated protein kinases, nuclear factor‑κB and Akt. Furthermore, the protein and RNA levels of RANKL‑induced c‑Fos and nuclear factor of activated T-cell cytoplasmic 1 were suppressed by CGE. These results indicated that CGE may serve as a useful drug in the prevention of bone loss. PMID:27035226

  8. In vitro antioxidant, collagenase inhibition, and in vivo anti-wrinkle effects of combined formulation containing Punica granatum, Ginkgo biloba, Ficus carica, and Morus alba fruits extract

    PubMed Central

    Ghimeray, Amal Kumar; Jung, Un Sun; Lee, Ha Youn; Kim, Young Hoon; Ryu, Eun Kyung; Chang, Moon Sik

    2015-01-01

    Background In phytotherapy, the therapeutic potential is based on the combined action of different herbal drugs. Our objective was to evaluate the antioxidant, anti-collagenase (in vitro), and anti-wrinkle (in vivo) effect of combined formulation containing Ginkgo biloba, Punica granatum, Ficus carica, and Morus alba fruits extract. Methods Antioxidant evaluation was based on the scavenging activity of free radicals (1,1-diphenyl-2-picrylhydrazyl, H2O2, and O2−) and the anti-collagenase activity was based on the reduction of collagenase enzyme in vitro. In an in vivo study, 21 female subjects were examined in a placebo-controlled trail. Facial wrinkle, especially the crow’s feet region of eyes, was treated with topical formulated 2% cream for 56 days and compared with the placebo. Results In the in vitro study, the combination of fruits extract showed a higher antioxidant activity which was comparable with the positive standard (ascorbic acid, butylated hydroxyanisole, and Trolox). The data also showed a dose-dependent inhibition of collagenase. In the in vivo study, treatment with 2% formulated cream for 56 days significantly reduced the percentage of wrinkle depth, length, and area with 11.5, 10.07, and 29.55, respectively. Conclusion The combined formulation of fruit extracts showed excellent antioxidative and anti-collagenase activity as well as a significant effect on anti-wrinkle activity on human skin. PMID:26203268

  9. Selective inhibition of TNFR1 reduces osteoclast numbers and is differentiated from anti-TNF in a LPS-driven model of inflammatory bone loss.

    PubMed

    Espirito Santo, A I; Ersek, A; Freidin, A; Feldmann, M; Stoop, A A; Horwood, N J

    2015-09-01

    The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening. PMID:26208457

  10. A possible new role for vitamin D-binding protein in osteoclast control: inhibition of extracellular Ca2+ sensing at low physiological concentrations.

    PubMed

    Adebanjo, O A; Moonga, B S; Haddad, J G; Huang, C L; Zaidi, M

    1998-08-28

    Upon removal of its sialic acid or galactose residue, vitamin D-binding protein (DBP) becomes a potent macrophage-activating factor, DBP-MAF. Here we document a new function of DBP-MAF and its parent molecule, DBP, in osteoclast control. We show that all DBPs potently inhibit extracellular Ca2+ (cation) sensing at low nanomolar concentrations with the following rank order of potency: native DBP = sialidase-treated DBP > beta-galactosidase-treated DBP. This attenuation remains unaffected despite co-incubation either with the native DBP ligand, 1,25-dihydroxyvitamin D3, or with an asialoglycoprotein receptor modulator, asialoorosomucoid. Taken together, the results suggest that circulating DBP may play a role in the systemic control of osteoclastic bone resorption, a hitherto unrecognized action of the protein. PMID:9731194

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

    PubMed

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

    2016-04-01

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

  12. Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells*

    PubMed Central

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

    2015-01-01

    Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption. PMID:25404736

  13. Chronic low dose tumor necrosis factor-α (TNF) suppresses early bone accrual in young mice by inhibiting osteoblasts without affecting osteoclasts.

    PubMed

    Gilbert, L C; Chen, H; Lu, X; Nanes, M S

    2013-09-01

    The inflammatory cytokine tumor necrosis factor-α (TNF-α) is known to cause bone resorption and inhibit bone formation in arthritis and aging but less is known about TNF effects in the young growing skeleton. While investigating the mechanism of bone loss in TNF transgenic mice, we identified an early TNF-sensitive period marked by suppression of osteoblasts and bone accrual as the sole mechanism of TNF action, without an effect on osteoclasts or bone resorption. TgTNF mice express low concentrations of hTNFα (≤5 pg/ml). Osteoblasts cultured from TgTNF mice express reduced levels of RUNX2, Osx, alkaline phosphatase, bone sialoprotein, and osteocalcin and have delayed formation of mineralized nodules. Early accrual of bone in TgTNF mice is suppressed until 6 weeks of age, after which the rate of bone accrual normalizes without catch up. Histomorphometry revealed that TgTNF mice fail to generate a transient surge in osteoblast number that is seen in wild type (WT) mice at 4 weeks. Osteoclasts, TRAP staining, erosive surfaces, serum CTx, and OPG/RANKL expression did not differ between young TgTNF and WT mice. Canonical Wnts and signaling through β-catenin were reduced in TgTNF mice at 4 weeks and partially recovered by 12 weeks, associated with reduced cytoplasm to nuclear transfer of β-catenin and Wnt regulated genes. TgTNF mice were crossed with BatGal Wnt reporter mice. Active Wnt signaling in tibial trabecular lining cells was reduced in TgTNF mice at 4 weeks compared to control littermates. Our results demonstrate that a low dose inflammatory stimulus is sufficient to inhibit the early surge in osteoblasts and optimal bone formation of young mice independent of changes in osteoclasts. TNF inhibition of the Wnt pathway contributes to the suppression of osteoblasts. PMID:23756233

  14. Microgravity induces pelvic bone loss through osteoclastic activity, osteocytic osteolysis, and osteoblastic cell cycle inhibition by CDKN1a/p21.

    PubMed

    Blaber, Elizabeth A; Dvorochkin, Natalya; Lee, Chialing; Alwood, Joshua S; Yousuf, Rukhsana; Pianetta, Piero; Globus, Ruth K; Burns, Brendan P; Almeida, Eduardo A C

    2013-01-01

    Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arrest during osteogenesis may also contribute to bone loss in space. To test this possibility, we exposed 16-week-old female C57BL/6J mice (n = 8) to microgravity for 15-days on the STS-131 space shuttle mission. Analysis of the pelvis by µCT shows decreases in bone volume fraction (BV/TV) of 6.29%, and bone thickness of 11.91%. TRAP-positive osteoclast-covered trabecular bone surfaces also increased in microgravity by 170% (p = 0.004), indicating osteoclastic bone degeneration. High-resolution X-ray nanoCT studies revealed signs of lacunar osteolysis, including increases in cross-sectional area (+17%, p = 0.022), perimeter (+14%, p = 0.008), and canalicular diameter (+6%, p = 0.037). Expression of matrix metalloproteinases (MMP) 1, 3, and 10 in bone, as measured by RT-qPCR, was also up-regulated in microgravity (+12.94, +2.98 and +16.85 fold respectively, p<0.01), with MMP10 localized to osteocytes, and consistent with induction of osteocytic osteolysis. Furthermore, expression of CDKN1a/p21 in bone increased 3.31 fold (p<0.01), and was localized to osteoblasts, possibly inhibiting the cell cycle during tissue regeneration as well as conferring apoptosis resistance to these cells. Finally the apoptosis inducer Trp53 was down-regulated by -1.54 fold (p<0.01), possibly associated with the quiescent survival-promoting function of CDKN1a/p21. In conclusion, our findings identify the pelvic and femoral region of the mouse skeleton as an active site of rapid bone

  15. Microgravity Induces Pelvic Bone Loss through Osteoclastic Activity, Osteocytic Osteolysis, and Osteoblastic Cell Cycle Inhibition by CDKN1a/p21

    PubMed Central

    Blaber, Elizabeth A.; Dvorochkin, Natalya; Lee, Chialing; Alwood, Joshua S.; Yousuf, Rukhsana; Pianetta, Piero; Globus, Ruth K.; Burns, Brendan P.; Almeida, Eduardo A. C.

    2013-01-01

    Bone is a dynamically remodeled tissue that requires gravity-mediated mechanical stimulation for maintenance of mineral content and structure. Homeostasis in bone occurs through a balance in the activities and signaling of osteoclasts, osteoblasts, and osteocytes, as well as proliferation and differentiation of their stem cell progenitors. Microgravity and unloading are known to cause osteoclast-mediated bone resorption; however, we hypothesize that osteocytic osteolysis, and cell cycle arrest during osteogenesis may also contribute to bone loss in space. To test this possibility, we exposed 16-week-old female C57BL/6J mice (n = 8) to microgravity for 15-days on the STS-131 space shuttle mission. Analysis of the pelvis by µCT shows decreases in bone volume fraction (BV/TV) of 6.29%, and bone thickness of 11.91%. TRAP-positive osteoclast-covered trabecular bone surfaces also increased in microgravity by 170% (p = 0.004), indicating osteoclastic bone degeneration. High-resolution X-ray nanoCT studies revealed signs of lacunar osteolysis, including increases in cross-sectional area (+17%, p = 0.022), perimeter (+14%, p = 0.008), and canalicular diameter (+6%, p = 0.037). Expression of matrix metalloproteinases (MMP) 1, 3, and 10 in bone, as measured by RT-qPCR, was also up-regulated in microgravity (+12.94, +2.98 and +16.85 fold respectively, p<0.01), with MMP10 localized to osteocytes, and consistent with induction of osteocytic osteolysis. Furthermore, expression of CDKN1a/p21 in bone increased 3.31 fold (p<0.01), and was localized to osteoblasts, possibly inhibiting the cell cycle during tissue regeneration as well as conferring apoptosis resistance to these cells. Finally the apoptosis inducer Trp53 was down-regulated by −1.54 fold (p<0.01), possibly associated with the quiescent survival-promoting function of CDKN1a/p21. In conclusion, our findings identify the pelvic and femoral region of the mouse skeleton as an active site of rapid bone

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

    PubMed Central

    Lee, Jiyeon

    2014-01-01

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

  17. Inhibition of TNF-α Reverses the Pathological Resorption Pit Profile of Osteoclasts from Patients with Acute Charcot Osteoarthropathy

    PubMed Central

    2015-01-01

    We hypothesised that tumour necrosis factor-α (TNF-α) may enhance receptor activator of nuclear factor-κβ ligand- (RANKL-) mediated osteoclastogenesis in acute Charcot osteoarthropathy. Peripheral blood monocytes were isolated from 10 acute Charcot patients, 8 diabetic patients, and 9 healthy control subjects and cultured in vitro on plastic and bone discs. Osteoclast formation and resorption were assessed after treatment with (1) macrophage-colony stimulating factor (M-CSF) and RANKL and (2) M-CSF, RANKL, and neutralising antibody to TNF-α (anti-TNF-α). Resorption was measured on the surface of bone discs by image analysis and under the surface using surface profilometry. Although osteoclast formation was similar in M-CSF + RANKL-treated cultures between the groups (p > 0.05), there was a significant increase in the area of resorption on the surface (p < 0.01) and under the surface (p < 0.01) in Charcot patients compared with diabetic patients and control subjects. The addition of anti-TNF-α resulted in a significant reduction in the area of resorption on the surface (p < 0.05) and under the surface (p < 0.05) only in Charcot patients as well as a normalisation of the aberrant erosion profile. We conclude that TNF-α modulates RANKL-mediated osteoclastic resorption in vitro in patients with acute Charcot osteoarthropathy. PMID:26137498

  18. Inhibition of TNF-α Reverses the Pathological Resorption Pit Profile of Osteoclasts from Patients with Acute Charcot Osteoarthropathy.

    PubMed

    Petrova, Nina L; Petrov, Peter K; Edmonds, Michael E; Shanahan, Catherine M

    2015-01-01

    We hypothesised that tumour necrosis factor-α (TNF-α) may enhance receptor activator of nuclear factor-κβ ligand- (RANKL-) mediated osteoclastogenesis in acute Charcot osteoarthropathy. Peripheral blood monocytes were isolated from 10 acute Charcot patients, 8 diabetic patients, and 9 healthy control subjects and cultured in vitro on plastic and bone discs. Osteoclast formation and resorption were assessed after treatment with (1) macrophage-colony stimulating factor (M-CSF) and RANKL and (2) M-CSF, RANKL, and neutralising antibody to TNF-α (anti-TNF-α). Resorption was measured on the surface of bone discs by image analysis and under the surface using surface profilometry. Although osteoclast formation was similar in M-CSF + RANKL-treated cultures between the groups (p > 0.05), there was a significant increase in the area of resorption on the surface (p < 0.01) and under the surface (p < 0.01) in Charcot patients compared with diabetic patients and control subjects. The addition of anti-TNF-α resulted in a significant reduction in the area of resorption on the surface (p < 0.05) and under the surface (p < 0.05) only in Charcot patients as well as a normalisation of the aberrant erosion profile. We conclude that TNF-α modulates RANKL-mediated osteoclastic resorption in vitro in patients with acute Charcot osteoarthropathy. PMID:26137498

  19. (+)-Vitisin A Inhibits Osteoclast Differentiation by Preventing TRAF6 Ubiquitination and TRAF6-TAK1 Formation to Suppress NFATc1 Activation

    PubMed Central

    Chiou, Wen-Fei; Huang, Yu-Ling; Liu, Yen-Wenn

    2014-01-01

    We recently reported that oral administration of a (+)-vitisin A-enriched product prepared from Vitis thunbergii obviously ameliorated bone loss in ovariectomized mice and (+)-vitisin A was able to inhibit receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells. Here we further clarified the mechanism(s) by which (+)-vitisin A targets osteoclastic differentiation and activity. Osteoclast-characteristic enzyme activity was determined using gel zymography or spectroflurometric-based assay. Expression of signal molecules was analyzed via Western blot or immunoprecipitation. Results showed that (+)-vitisin A suppressed RANKL-induced multinuclear cells (MNCs) formation and bone resorption which was accompanied with reduction in β3 integrin, osteoclast stimulatory transmembrane protein (OC-STAMP), matrix metalloproteinase-9 (MMP-9) and cathepsin K proteins expression. (+)-Vitisin A also down-regulated the proteolytic activities of MMP-9 and cathepsin K via targeting at the late stage function. (+)-Vitisin A prominently abrogated RANKL-triggered nuclear translocations of NF-κB, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream IκB degradation as well as ERK and JNK phosphorylation were also substantially repressed. Transfection with siRNA targeting tumor necrosis factor receptor associated factor 6 (TRAF6) clearly restrained RANKL-induced MNCs formation and NFATc1 induction. Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor β-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and β3 was markedly induced by RANKL stimulation. (+)-Vitisin A significantly attenuated this interaction when concomitant treated with RANKL in RAW264.7 cells, but failed to affect c-Src/β3 complex

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

    PubMed Central

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

    2015-01-01

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

  1. Inhibition of prostaglandin synthesis leads to a change in adherence of mouse osteoclasts from bone to periosteum.

    PubMed

    Marshall, M J; Holt, I; Davie, M W

    1996-09-01

    When mouse parietal bones were incubated for 1 day in medium containing indomethacin (Ind), the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP+OC) counted on the bone surface was drastically reduced. This reduction did not occur with calcitonin or if the endocranial membrane (periosteum) was removed prior to incubation with Ind. The aim of this work was to determine the mechanism involved. TRAP+OC were found to be increased on the endocranial membrane adjacent to the resorbing surface after Ind treatment, compared with cultures supplemented with parathyroid hormone (PTH) or prostaglandin E2 (PGE2). However, this increase accounted for only half of those lost from the bone surface. TRAP negative osteoclasts were also seen on the membrane and, to a lesser extent, on the bone. Increased TRAP specific activity could be extracted from the endocranial membranes of bones incubated with Ind compared with PGE2 controls. When bones that had been exposed to Ind were then cultured for 1 day in PGE2, an increase in TRAP+OC occurred. This increase was blocked by the removal of the endocranial membrane prior to incubation with PGE2. We conclude that when prostaglandin production ceases, TRAP+OC become less adherent to bone and more adherent to the endocranial membrane. Stimulators of bone resorption appear to reverse this process. PMID:8694899

  2. Pomegranate seed oil prevents bone loss in a mice model of osteoporosis, through osteoblastic stimulation, osteoclastic inhibition and decreased inflammatory status.

    PubMed

    Spilmont, Mélanie; Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Mercier, Sylvie; Miot-Noirault, Elisabeth; Pilet, Paul; Rios, Laurent; Wittrant, Yohann; Coxam, Véronique

    2013-11-01

    In the current context of longer life expectancy, the prevalence of osteoporosis is increasingly important. This is why development of new strategies of prevention is highly suitable. Pomegranate seed oil (PSO) and its major component, punicic acid (a conjugated linolenic acid), have potent anti-inflammatory and anti-oxidative properties both in vitro and in vivo, two processes strongly involved in osteoporosis establishment. In this study, we demonstrated that PSO consumption (5% of the diet) improved significantly bone mineral density (240.24±11.85 vs. 203.04±34.19 mg/cm(3)) and prevented trabecular microarchitecture impairment in ovariectomized (OVX) mice C57BL/6J, compared to OVX control animals. Those findings are associated with transcriptional changes in bone tissue, suggesting involvement of both osteoclastogenesis inhibition and osteoblastogenesis improvement. In addition, thanks to an ex vivo experiment, we provided evidence that serum from mice fed PSO (5% by gavage) had the ability to significantly down-regulate the expression of specific osteoclast differentiation markers and RANK-RANKL downstream signaling targets in osteoclast-like cells (RAW264.7) (RANK: negative 0.49-fold vs. control conditions). Moreover, in osteoblast-like cells (MC3T3-E1), it elicited significant increase in alkaline phosphatase activity (+159% at day 7), matrix mineralization (+271% on day 21) and transcriptional levels of major osteoblast lineage markers involving the Wnt/β-catenin signaling pathways. Our data also reveal that PSO inhibited pro-inflammatory factors expression while stimulating anti-inflammatory ones. These results demonstrate that PSO is highly relevant regarding osteoporosis. Indeed, it offers promising alternatives in the design of new strategies in nutritional management of age-related bone complications. PMID:23953990

  3. Antibacterial effects of Carica papaya fruit on common wound organisms.

    PubMed

    Dawkins, G; Hewitt, H; Wint, Y; Obiefuna, P C; Wint, B

    2003-12-01

    The purpose of the study was to investigate antibacterial activity of ripe and unripe Carica papaya on selected micro-organisms. Cultures of micro-organisms were routinely maintained in nutrient agar slants at 4 degrees C. Extracts of immature, mature and ripe Carica papaya fruit were obtained by separately grinding factions of the epicarp, endocarp and seeds and filtering them through gauze. Sensitivity tests were conducted by adding 0.06 ml of extract to agar wells (6 mm diameter) prepared from 20 ml agar seeded with 10(6) cells/ml suspension of one of the eight organisms per plate. The inoculated plates were allowed to equilibrate at 4 degrees C for 1 hour, incubated at 37 degrees C for 24 hours, and zones of inhibition measured in millimetres. Anti-bacterial activity was expressed in terms of the radius of zone of inhibition. Seed extracts from the fruit showed inhibition in the following order: B cereus > E coli > S faecalis > S aureus > P vulgaris > S flexneri. No significant difference was found in bacterial sensitivity between immature, mature and ripe fruits. No inhibition zone was produced by epicarp and endocarp extracts. Carica papaya seeds contain anti-bacterial activity that inhibits growth of gram-positive and gram-negative organisms. Observed activity was independent of stage of fruit maturity. Carica papaya has antibacterial effects that could be useful in treating chronic skin ulcers to promote healing. PMID:15040064

  4. Characterization of Regulatory Extracellular Vesicles from Osteoclasts.

    PubMed

    Huynh, N; VonMoss, L; Smith, D; Rahman, I; Felemban, M F; Zuo, J; Rody, W J; McHugh, K P; Holliday, L S

    2016-06-01

    Extracellular vesicles (EVs), which include exosomes and ectosomes/microvesicles, have emerged as important intercellular regulators. EVs can interact with surface receptors of target cells and can transport luminal components, including messenger RNAs (mRNAs), microRNAs, and enzymes, to the cytosol of the target cell. Here, we show that hematopoietic cells grown in culture shed exosome-like EVs as they differentiate from preosteoclasts into osteoclasts. These EVs were between 25 and 120 nm (mean, 40 nm) in diameter determined by transmission electron microscopy. The exosome-associated markers CD63 and EpCAM were enriched in the isolated EVs while markers of Golgi and endoplasmic reticulum were not detected. Treatment of isolated hematopoietic cells with EVs did not affect their receptor activator of nuclear factor κB-ligand (RANKL)-stimulated differentiation into osteoclasts. However, EVs from osteoclast precursors promoted 1,25-dihydroxyvitamin D3-dependent osteoclast formation in whole mouse marrow cultures, and EVs from osteoclast-enriched cultures inhibited osteoclastogenesis in the same cultures. These data suggested that osteoclast-derived EVs are paracrine regulators of osteoclastogenesis. EVs from mature osteoclasts contained receptor activator of nuclear factor κB (RANK). Immunogold labeling showed RANK was enriched in 1 in every 32 EVs isolated from osteoclast-enriched cultures. Depletion of RANK-rich EVs relieved the ability of osteoclast-derived EVs to inhibit osteoclast formation in 1,25-dihydroxyvitamin D3-stimulated marrow cultures. In summary, we show for the first time that EVs released by osteoclasts are novel regulators of osteoclastogenesis. Our data suggest that RANK in EVs may be mechanistically linked to the inhibition of osteoclast formation. RANK present in EVs may function by competitively inhibiting the stimulation of RANK on osteoclast surfaces by RANKL similar to osteoprotegerin. RANK-rich EVs may also take advantage of the RANK

  5. Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts.

    PubMed

    Pitari, Maria Rita; Rossi, Marco; Amodio, Nicola; Botta, Cirino; Morelli, Eugenio; Federico, Cinzia; Gullà, Annamaria; Caracciolo, Daniele; Di Martino, Maria Teresa; Arbitrio, Mariamena; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2015-09-29

    miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3'UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD. PMID:26160841

  6. Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts

    PubMed Central

    Pitari, Maria Rita; Rossi, Marco; Amodio, Nicola; Botta, Cirino; Morelli, Eugenio; Federico, Cinzia; Gullà, Annamaria; Caracciolo, Daniele; Di Martino, Maria Teresa; Arbitrio, Mariamena; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2015-01-01

    miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3′UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD. PMID:26160841

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

    PubMed Central

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

    2013-01-01

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

  8. Lysozyme synthesis in osteoclasts.

    PubMed

    Hilliard, T J; Meadows, G; Kahn, A J

    1990-12-01

    Osteoclasts may or may not be directly related to monocytes and macrophages, but it is well established that these cell types share a number of features in common. In the present study we sought to extend this comparison by assessing lysozyme synthesis in osteoclasts, an enzyme known to be produced and secreted in large amounts by monocytes and macrophages. Our data show that freshly isolated chicken osteoclasts and osteoclasts in situ contain an abundant amount of lysozyme and correspondingly high steady-state levels of the enzyme's messenger RNA. Marrow macrophages, at various stages of in vitro maturation, also possess lysozyme mRNA but in amounts approximately two to four times lower than osteoclasts. These observations reaffirm the monocyte-macrophage nature of the osteoclast but raise questions about the function of the lysozyme in this cell. At present, the role of the lysozyme in osteoclast activity remains unexplained. PMID:1706132

  9. 1α,25-Dihydroxyvitamin D3 inhibits the differentiation and bone resorption by osteoclasts generated from Wistar rat bone marrow-derived macrophages

    PubMed Central

    WANG, DONG; GU, JIAN-HONG; CHEN, YANG; ZHAO, HONG-YAN; LIU, WEI; SONG, RUI-LONG; BIAN, JIAN-CHUN; LIU, XUE-ZHONG; YUAN, YAN; LIU, ZONG-PING

    2015-01-01

    The steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] plays an important role in maintaining a balance in calcium and bone metabolism. To study the effects of 1α,25-(OH)2D3 on osteoclast (OC) formation and bone resorption, OC differentiation was induced in bone marrow-derived mononuclear cells from Wistar rats with the addition of macrophage colony stimulating factor and receptor activator for nuclear factor-κB ligand in vitro. Cells were then treated with 1α,25-(OH)2D3 at 10−9, 10−8 or 10−7 mol/l. OCs were identified using tartrate-resistant acid phosphatase staining and activity was monitored in the absorption lacunae by scanning electron microscopy. Expression levels of functional proteins associated with bone absorption, namely carbonic anhydrase II, cathepsin K and matrix metalloproteinase-9 were evaluated by western blot analysis. The results showed that 1α,25-(OH)2D3 inhibited the formation and activation of OCs in a dose-dependent manner and downregulated the expression levels of bone absorption-associated proteins. PMID:26622436

  10. Phosphatidylinositol 3-kinase association with the osteoclast cytoskeleton, and its involvement in osteoclast attachment and spreading.

    PubMed

    Lakkakorpi, P T; Wesolowski, G; Zimolo, Z; Rodan, G A; Rodan, S B

    1997-12-15

    Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts. PMID:9434625

  11. A new method for measuring osteoclast formation by electrical impedance.

    PubMed

    Emori, Haruka; Iwai, Shinichi; Ryu, Kakei; Amano, Hitoshi; Sambe, Takehiko; Kobayashi, Takahiro; Oguchi, Tatsunori; Ohura, Kiyoshi; Oguchi, Katsuji

    2015-06-01

    Osteoclasts are important target cells for osteoporosis treatment. Recently, a real-time cell analysis (RTCA) system was developed to observe cell morphology and adhesion; however, the use of RTCA to study osteoclastogenesis has not been reported. Here, we investigated whether osteoclast formation could be monitored in real-time using RTCA. The cell index determined via electrical impedance using RTCA, and the number of osteoclasts exhibited a significant positive correlation. RTCA was useful for determining the effect of (-)-epigallocatechin-3-gallate on the inhibition of bone resorption. We established a new method of measuring osteoclast formation in real-time using RTCA. PMID:26032840

  12. Zoledronate inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclast differentiation via suppression of expression of nuclear factor of activated T-cell c1 and carbonic anhydrase 2.

    PubMed

    Nakagawa, Takayuki; Ohta, Kouji; Kubozono, Kazumi; Ishida, Yoko; Naruse, Takako; Takechi, Masaaki; Kamata, Nobuyuki

    2015-04-01

    Bisphosphonates (BPs) are widely used in the prevention of skeletal-related events (SRE), including osteoporosis, skeletal metastases of malignant tumours, and multiple myeloma. Osteonecrosis of the jaw (ONJ) is frequently reported as a major adverse effect induced by BP treatment. The receptor activator of the nuclear factor kappa-B ligand (RANKL) inhibitor, denosumab, has recently been used to prevent SRE, but the frequency of ONJ induced by denosumab is similar to that by BPs. This finding suggests that the inhibition of RANKL-mediated osteoclastogenesis may have a close relationship with the occurrence of ONJ. We therefore investigated the expression status of RANKL-inducible genes in zoledronate-treated mouse osteoclast precursor cells. The molecular targets of zoledronate in the RANKL signal pathway and additional factors associated with osteoclastogenesis were analysed by genome-wide screening. Microarray analysis identified that among 31 genes on 44 entities of RANKL-inducible genes, the mRNA expression level of two genes, i.e., nuclear factor of activated T-cells c1 (NFATc1) and carbonic anhydrase 2 (CAII), was decreased in zoledronate-treated cells. Subsequent analyses verified that these two genes were significantly silenced by zoledronate treatment and that their expression was restored following inhibition of zoledronate action by geranylgeraniol. Zoledronate inhibited RANKL-induced osteoclast differentiation by suppression of NFATc1 and CAII gene expression. Our results suggest that these genes might be common targets for zoledronate and denosumab in the mechanism underlying RANKL-induced osteoclast differentiation. A clear understanding of the common molecular mechanisms of bone-remodelling agents is thus essential for prevention of ONJ. PMID:25601046

  13. Notch signaling promotes osteoclast maturation and resorptive activity.

    PubMed

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

    2015-11-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors. PMID:25914241

  14. Suppression of T cell-induced osteoclast formation

    SciTech Connect

    Karieb, Sahar; Fox, Simon W.

    2013-07-12

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

  15. Effects of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate on mouse osteoclasts

    SciTech Connect

    Marshall, M.J.; Wilson, A.S.; Davie, M.W. )

    1990-09-01

    A group of 5-day-old mice were injected intraperitoneally with (3-amino-1-hydroxypropylidine)-1,1-bisphosphonate (APD). Morphologic changes were observed in vitally stained osteoclasts on parietal bones 3 days later, and these were judged to be degenerative. At this time significantly increased numbers of nuclei per osteoclast and total numbers of osteoclast nuclei were observed. However, at 4 days after the injection of APD, the total numbers of osteoclasts were significantly reduced relative to controls. When parietal bones were maintained in culture, APD reduced osteoclast numbers and inhibited cell-mediated 45Ca2+ release. Exposure of bones to parathyroid hormone increased the number of osteoclasts counted 1 day later. This effect was not blocked by APD. Calcitonin prevented the reduction in osteoclast numbers due to APD in vitro. We conclude that APD has a direct effect on resorbing mouse osteoclasts.

  16. Calcitonin Induces Expression of the Inducible cAMP Early Repressor in Osteoclasts

    PubMed Central

    Yang, Maobin; Kream, Barbara E.

    2010-01-01

    The cAMP response element modulator gene (Crem) encodes a variety of transcriptional regulators including the inducible cAMP early repressor, ICER. We previously showed that Crem knockout mice, which are deficient in CREM and ICER factors, display slightly increased long bone mass and decreased osteoclast number. These data are consistent with the notion that Crem regulates bone mass in part through an effect on osteoclast formation and/or function. Since ICER is strongly induced by cAMP, we asked whether the calcium-regulating hormone calcitonin, which stimulates cAMP production and inhibits osteoclastic bone resorption, could induce ICER in osteoclasts. The monocytic cell line RAW264.7 was treated with receptor activator of NF-κB ligand (RANKL) to induce osteoclast formation. Calcitonin caused a time- and dose-dependent induction of ICER mRNA and an increase in ICER protein abundance in RANKL-treated RAW264.7 cells. Calcitonin also induced ICER mRNA and protein in osteoclasts derived from primary mouse bone marrow cell cultures. Calcitonin-treated osteoclasts showed immunoreactivity with an anti-CREM antibody. Calcitonin decreased the activity of wild type and Crem knockout osteoclasts in vitro, and this inhibitory effect was greater in Crem knockout osteoclasts. Furthermore, calcitonin decreased calcitonin receptor mRNA expression in wild type osteoclasts but not in Crem knockout osteoclasts. These data suggest that calcitonin induction of ICER in osteoclasts might regulate osteoclast activity. PMID:19016003

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. Molecular regulation of osteoclast activity.

    PubMed

    Bruzzaniti, Angela; Baron, Roland

    2006-06-01

    Osteoclasts are multinucleated cells derived from hematopoietic precursors that are primarily responsible for the degradation of mineralized bone during bone development, homeostasis and repair. In various skeletal disorders such as osteoporosis, hypercalcemia of malignancy, tumor metastases and Paget's disease, bone resorption by osteoclasts exceeds bone formation by osteoblasts leading to decreased bone mass, skeletal fragility and bone fracture. The overall rate of osteoclastic bone resorption is regulated either at the level of differentiation of osteoclasts from their monocytic/macrophage precursor pool or through the regulation of key functional proteins whose specific activities in the mature osteoclast control its attachment, migration and resorption. Thus, reducing osteoclast numbers and/or decreasing the bone resorbing activity of osteoclasts are two common therapeutic approaches for the treatment of hyper-resorptive skeletal diseases. In this review, several of the key functional players involved in the regulation of osteoclast activity will be discussed. PMID:16951988

  1. Role of osteoclasts in regulating hematopoietic stem and progenitor cells

    PubMed Central

    Miyamoto, Takeshi

    2013-01-01

    Bone marrow (BM) cavities are utilized for hematopoiesis and to maintain hematopoietic stem cells (HSCs). HSCs have the ability to self-renew as well as to differentiate into multiple different hematopoietic lineage cells. HSCs produce their daughter cells throughout the lifespan of individuals and thus, maintaining HSCs is crucial for individual life. BM cavities provide a specialized microenvironment termed “niche” to support HSCs. Niches are composed of various types of cells such as osteoblasts, endothelial cells and reticular cells. Osteoclasts are unique cells which resorb bones and are required for BM cavity formation. Loss of osteoclast function or differentiation results in inhibition of BM cavity formation, an osteopetrotic phenotype. Osteoclasts are also reportedly required for hematopoietic stem and progenitor cell (HSPC) mobilization to the periphery from BM cavities. Thus, lack of osteoclasts likely results in inhibition of HSC maintenance and HSPC mobilization. However, we found that osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization by using three independent osteoclast-less animal models. In this review, I will discuss the roles of osteoclasts in hematopoietic stem cell maintenance and mobilization. PMID:24147255

  2. Tumor-Induced Osteoclast miRNA Changes as Regulators and Biomarkers of Osteolytic Bone Metastasis

    PubMed Central

    Ell, Brian; Mercatali, Laura; Ibrahim, Toni; Campbell, Neil; Schwarzenbach, Heidi; Pantel, Klaus; Amadori, Dino; Kang, Yibin

    2013-01-01

    SUMMARY Understanding the mechanism by which tumor cells influence osteoclast differentiation is crucial for improving treatment of osteolytic metastasis. Here, we report broad microRNA (miRNA) expression changes in differentiating osteoclasts after exposure to tumor-conditioned media, in part through activation of NFκB signaling by soluble intracellular adhesion molecule (sICAM1) secreted from bone-metastatic cancer cells. Ectopic expression of multiple miRNAs down-regulated during osteoclastogenesis suppresses osteoclast differentiation by targeting important osteoclast genes. Intravenous delivery of these miRNAs in vivo inhibits osteoclast activity and reduces osteolytic bone metastasis. Importantly, serum levels of sICAM1 and two osteoclast miRNAs, miR-16 and miR-378, which are elevated in osteoclast differentiation, correlate with bone metastasis burden. These findings establish miRNAs as potential therapeutic targets and clinical biomarkers of bone metastasis. PMID:24135284

  3. Generation and culture of osteoclasts

    PubMed Central

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

    2014-01-01

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

  4. Smad 1/5 and Smad 4 Expression Are Important for Osteoclast Differentiation

    PubMed Central

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

    2015-01-01

    To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP’s effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity. PMID:25711193

  5. Signaling Pathways in Osteoclast Differentiation.

    PubMed

    Kim, Jung Ha; Kim, Nacksung

    2016-01-01

    Osteoclasts are multinucleated cells of hematopoietic origin that are responsible for the degradation of old bone matrix. Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). M-CSF and RANKL bind to their respective receptors c-Fms and RANK to stimulate osteoclast differentiation through regulation of delicate signaling systems. Here, we summarize the critical or essential signaling pathways for osteoclast differentiation including M-CSF-c-Fms signaling, RANKL-RANK signaling, and costimulatory signaling for RANK. PMID:26865996

  6. siRNA Knock-Down of RANK Signaling to Control Osteoclast-Mediated Bone Resorption

    PubMed Central

    Wang, Yuwei; Grainger, David W.

    2010-01-01

    Purpose To demonstrate the ability of small interfering (si)RNA targeting the cell receptor, RANK, to control osteoclast function in cultures of both primary and secondary osteoclasts and their precursor cells. Methods siRNA targeting RANK was transfected into both RAW264.7 and primary bone marrow cell cultures. RANK knock-down by siRNA and functional inhibition were assessed in both mature osteoclast and their precursor cell cultures. RANK mRNA message and protein expression after the transfections were analyzed by PCR and Western blot, respectively. Off-target effects were assessed. The inhibition of osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) assay, and subsequent bone resorption was determined by resorption pit assay. Results Both osteoclasts and osteoclast precursors can be targeted by siRNA in serum-containing media. Delivery of siRNA targeting RANK to both RAW 264.7 and primary bone marrow cell cultures produces short term repression of RANK expression without off-targeting effects, and significantly inhibits both osteoclast formation and bone resorption. Moreover, data support successful RANK knock-down by siRNA specifically in mature osteoclast cultures. Conclusions RANK is demonstrated to be an attractive target for siRNA control of osteoclast activity, with utility for development of new therapeutics for low bone mass pathologies or osteoporosis. PMID:20333451

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

    PubMed

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

    2015-06-26

    In an attempt to identify secretory products of osteoclasts that mediate the coupling of bone formation to resorption, we found that along with osteoclast differentiation, PDGF-A gene expression increase occurred first, by 12 h after stimulation of bone marrow macrophages with M-CSF and RANKL, and peaked at 36 h. This was next followed by a progressive increase in PDGF-B gene expression until a peak at 60 h, when mature osteoclasts formed. Isoform-specific ELISA of the conditioned medium collected every 24 h revealed that all three of the isoforms of PDGF-AA, AB and BB were secreted, in this temporal order as differentiation proceeded. Their secretion was enhanced when osteoclasts were activated by placing them on dentin slices. The secretion of all three isoforms was decreased in cathepsin K-deficient osteoclasts compared with wild-type osteoclasts. Pharmacological inhibition of cathepsin K with odanacatib also inhibited the secretion of all three isoforms, as was also the case with alendronate treatment. The secretion of sphingosine-1-phosphate, which increased during osteoclastogenesis, was reduced from cathepsin K-deficient osteoclasts, and was inhibited by treatment with odanacatib more profoundly than with alendronate. Thus, all three isoforms of PDGF, which are secreted at distinct differentiation stages of osteoclasts, appear to have distinct roles in the cell-cell communication that takes place in the microenvironment of bone remodeling, especially from the osteoclast lineage to mesenchymal cells and vascular cells, thereby stimulating osteogenesis and angiogenesis. PMID:25951977

  8. Implications of osteoblast-osteoclast interactions in the management of osteoporosis by antiresorptive agents denosumab and odanacatib.

    PubMed

    Sims, Natalie A; Ng, Kong Wah

    2014-03-01

    Antiresorptive agents, used in the treatment of osteoporosis, inhibit either osteoclast formation or function. However, with these approaches, osteoblast activity is also reduced because of the loss of osteoclast-derived coupling factors that serve to stimulate bone formation. This review discusses how osteoclast inhibition influences osteoblast function, comparing the actions of an inhibitor of osteoclast formation [anti-RANKL/Denosumab (DMAB)] with that of a specific inhibitor of osteoclastic cathepsin K activity [Odanacatib (ODN)]. Denosumab rapidly and profoundly, but reversibly, reduces bone formation. In contrast, preclinical studies and clinical trials of ODN showed that bone formation at some skeletal sites was preserved although resorption was reduced. This preservation of bone formation appears to be due to effects of coupling factors, secreted by osteoclasts and released from demineralized bone matrix. This indicates that bone resorptive activities of osteoclasts are separable from their coupling activities. PMID:24477416

  9. Effects of ionizing irradiation on formation and resorbing activity of osteoclasts in vitro

    SciTech Connect

    Scheven, B.A.; Burger, E.H.; Kawilarang-de Haas, E.W.; Wassenaar, A.M.; Nijweide, P.J.

    1985-07-01

    The effects of ionizing irradiation on the differentiation and activity of the osteoclast were investigated. Embryonic mouse metatarsal bones of different ages (14, 15, 16, 17 days) in which no osteoclasts had as yet been formed were irradiated with various x-ray doses and cultured until a marrow cavity became visible in the nonirradiated paired control bones. Bone growth and calcification were followed microscopically during culture. Irradiation caused a dose-dependent stunting of the longitudinal growth. Calcification was inhibited by high radiation doses (10 to 20 Gray (Gy), whereas a dose of 2.5 Gy stimulated the process in the early stages of long bone development. Histologic examination revealed complete inhibition of osteoclast formation in the 14- and 15-day-old bones after irradiation with 2.5 Gy or more. The number of osteoclasts in cultured older bones (16 days) was significantly reduced by irradiation, but osteoclast formation could not be completely prevented even by high dosages. Irradiation of explanted bone rudiments which were in a stage 1 day prior to the appearance of osteoclasts in vivo (17 days) did not significantly influence the formation of osteoclasts. Autoradiographic experiments using young bones showed that differentiation of osteoclast precursors into multinucleated osteoclasts is preceded by one or more divisions of the precursors in the periosteum. Furthermore, it was established from continuous /sup 3/H-thymidine-labeling experiments that in older bones (16 days) a part of the osteoclast nuclei originated from postmitotic osteoclast precursors. Irradiation mainly inhibited the appearance of labeled osteoclast nuclei in these bones.

  10. How the osteoclast degrades bone.

    PubMed

    Blair, H C

    1998-10-01

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

  11. Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

    PubMed

    Rieman, D J; McClung, H A; Dodds, R A; Hwang, S M; Holmes, M W; James, I E; Drake, F H; Gowen, M

    2001-03-01

    Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae. PMID:11248658

  12. The number of tartrate-resistant acid phosphatase-positive osteoclasts on neonatal mouse parietal bones is decreased when prostaglandin synthesis is inhibited and increased in response to prostaglandin E2, parathyroid hormone, and 1,25 dihydroxyvitamin D3.

    PubMed

    Marshall, M J; Holt, I; Davie, M W

    1995-03-01

    The culture of parietal bones from 4-day old mice in indomethacin (Ind) for 1 day caused a large reduction in the number of tartrate-resistant acid phosphatase positive osteoclasts (TRAP + OC) relative to both control bones and to freshly isolated bones. This reduction did not occur if prostaglandin E2 (PGE2) was present. When 5-bromo-2'-deoxyuridine (BDU) was injected into 4-day old mice, newly formed TRAP + OC nuclei became labeled 1 day later; these bones were then cultured with Ind for 1 day. TRAP + OC and newly labeled TRAP+OC nuclei were commensurately decreased in number. This suggests an active down-regulation rather than merely the inhibition of new TRAP+OC formation. Incubation of bones with Ind and either PGE2, parathyroid hormone, or 1,25 dihydroxyvitamin D3 for 6 hours following a 1-day preincubation in Ind, resulted in an increase in TRAP + OC compared with Ind alone. Using BDU labeling in vitro and in vivo, we show that this increase in number of TRAP+OC is not the result of cell proliferation, but rather differentiation of postmitotic precursors. PMID:7538445

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

    PubMed

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

    2016-06-01

    Osteoclasts are bone-degrading cells that are formed through fusion of their monocytic precursors. Three distinct subsets of monocytes have been identified in human peripheral blood: classical, intermediate, and non-classical monocytes. They are known to play different roles in physiology and pathology, but their capacity to differentiate into osteoclasts and whether inflammatory cytokines influence this differentiation is unknown. We hypothesized that classical, intermediate, and non-classical monocytes generate functionally different osteoclasts and that they respond in different ways to the inflammatory cytokine interleukin-17A (IL-17A). To investigate this, the different monocyte subsets were isolated from human peripheral blood and osteoclastogenesis was induced with the cytokines M-CSF and RANKL, with or without IL-17A. We found that all subsets are able to differentiate into osteoclasts in vitro, and that both osteoclastogenesis and subsequent bone resorption was distinctly affected by IL-17A. Osteoclastogenesis and bone resorption by osteoclasts derived from classical monocytes remained unaffected by IL-17A, while osteoclast formation from intermediate monocytes was inhibited by the cytokine. Surprisingly, bone resorption by osteoclasts derived from intermediate monocytes remained at similar levels as control cultures, indicating an increased bone resorbing activity by these osteoclasts. Limited numbers of osteoclasts were formed from non-classical monocytes on bone and no bone resorption was detected, which suggest that these cells belong to a cell lineage different from the osteoclast. By providing more insight into osteoclast formation from human blood monocytes, this study contributes to the possible targeting of specific osteoclast precursors as a therapeutic approach for diseases associated with inflammatory bone loss. J. Cell. Physiol. 231: 1249-1260, 2016. © 2015 Wiley Periodicals, Inc. PMID:26491867

  14. In vitro antimicrobial activity of four Ficus carica latex fractions against resistant human pathogens (antimicrobial activity of Ficus carica latex).

    PubMed

    Aref, Houda Lazreg; Salah, Karima Bel Hadj; Chaumont, Jean Pierre; Fekih, Abdelwaheb; Aouni, Mahjoub; Said, Khaled

    2010-01-01

    Methanolic, hexanoïc, chloroformic and ethyl acetate extracts of Ficus carica latex were investigated for their in vitro antimicrobial proprieties against five bacteria species and seven strains of fungi. The green fruit latex was collected from Chott Mariam Souse, Middle East coast of Tunisia. The antimicrobial activity of the extracts was evaluated and based respectively on the inhibition zone using the disc-diffusion assay, minimal inhibition concentration (MIC) for bacterial testing and the method by calculating inhibition percentage (I%) for fungi-inhibiting activities. The methanolic extract had no effect against bacteria except for Proteus mirabilis while the ethyl acetate extract had inhibition effect on the multiplication of five bacteria species (Enterococcus fecalis, Citobacter freundei, Pseudomonas aeruginosa, Echerchia coli and Proteus mirabilis). For the opportunist pathogenic yeasts, ethyl acetate and chlorophormic fractions showed a very strong inhibition (100%); methanolic fraction had a total inhibition against Candida albicans (100%) at a concentration of 500 microg/ml and a negative effect against Cryptococcus neoformans. Microsporum canis was strongly inhibited with methanolic extract (75%) and totally with ethyl acetate extract at a concentration of 750 microg/ml. Hexanoïc extract showed medium results. PMID:20067867

  15. Resolvin E1 regulates osteoclast fusion via DC-STAMP and NFATc1

    PubMed Central

    Zhu, Min; Van Dyke, Thomas E.; Gyurko, Robert

    2013-01-01

    Interactions between the immune and skeletal systems in inflammatory bone diseases are well appreciated, but the underlying molecular mechanisms that coordinate the resolution phase of inflammation and bone turnover have not been unveiled. Here we investigated the direct actions of the proresolution mediator resolvin E1 (RvE1) on bone-marrow-cell-derived osteoclasts in an in vitro murine model of osteoclast maturation and inflammatory bone resorption. Investigation of the actions of RvE1 treatment on the specific stages of osteoclast maturation revealed that RvE1 targeted late stages of osteoclast maturation to decrease osteoclast formation by 32.8%. Time-lapse vital microscopy and migration assays confirmed that membrane fusion of osteoclast precursors was inhibited. The osteoclast fusion protein DC-STAMP was specifically targeted by RvE1 receptor binding and was down-regulated by 65.4%. RvE1 did not affect the induction of the essential osteoclast transcription factor nuclear factor of activated T cells c1 (NFATc1) or its nuclear translocation; however, NFATc1 binding to the DC-STAMP promoter was significantly inhibited by 60.9% with RvE1 treatment as shown in electrophoresis mobility shift assay. Our findings suggest that proresolution mediators act directly on osteoclasts, in addition to down-regulation of inflammation, providing a novel mechanism for modulating osteoclast signaling in osteolytic inflammatory disease.—Zhu, M., Van Dyke, T. E., Gyurko, R. Resolvin E1 regulates osteoclast fusion via DC-STAMP and NFATc1. PMID:23629863

  16. Polycan suppresses osteoclast differentiation and titanium particle-induced osteolysis in mice.

    PubMed

    Lee, Young-Eun; Park, Kwang-Soo; Park, Eui-Kyun; Im, Sang-Uk; Choi, Youn-Hee; Song, Keun-Bae

    2016-08-01

    Particle-induced osteolysis is a major issue, and it is most likely the result of enhanced osteoclast activation in the pathogenesis of various skeletal diseases. This study investigated whether the inhibitory effect that Polycan has on osteoclast differentiation can be used to treat osteolysis induced by titanium (Ti) particles. To this end, the effects of Polycan were examined in terms of the cytotoxicity, osteoclast differentiation, cytokine expression, and Ti-induced calvarial osteolysis. Polycan had no significant cytotoxic effects on bone marrow macrophages (BMMs) but instead increased BMM proliferation. High levels of interleukin (IL)-6, IL-12, and macrophage colony-stimulating factor (M-CSF) were expressed in BMM cells in the presence of Polycan, suggesting that Polycan drives the differentiation of BMMs into M1 macrophages. Polycan significantly inhibited osteoclast differentiation induced by M-CSF and the receptor activator of nuclear factor kappa-B ligand (RANKL). The expression levels of the osteoclast marker genes significantly decreased, and Polycan induced and maintained the expression of IL-12, which suppressed osteoclast differentiation. In contrast, the RANKL signaling pathway was not inhibited by Polycan. An in vivo calvarial osteolysis model revealed that Polycan significantly decreased the osteoclast numbers and suppressed osteolysis. Our results suggest that the natural compound Polycan is a good candidate for therapeutic intervention against enhanced osteoclast differentiation and Ti particle-induced osteolysis. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1170-1175, 2016. PMID:26097144

  17. [Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro].

    PubMed

    Ming, Lei-Guo; Wang, Ming-Gang; Chen, Ke-Ming; Zhou, Jian; Han, Gui-Qiu; Zhu, Rui-Qing

    2012-02-01

    This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption. PMID:22512027

  18. Bafilomycin A1 Attenuates Osteoclast Acidification and Formation, Accompanied by Increased Levels of SQSTM1/p62 Protein.

    PubMed

    Zhu, Sipin; Rea, Sarah L; Cheng, Taksum; Feng, Hao Tian; Walsh, John P; Ratajczak, Thomas; Tickner, Jennifer; Pavlos, Nathan; Xu, Hua-Zi; Xu, Jiake

    2016-06-01

    Vacuolar proton pump H(+)-adenosine triphosphatases (V-ATPases) play an important role in osteoclast function. Further understanding of the cellular and molecular mechanisms of V-ATPase inhibition is vital for the development of anti-resorptive drugs specifically targeting osteoclast V-ATPases. In this study, we observed that bafilomycin A1, a naturally-occurring inhibitor of V-ATPases, increased the protein level of SQSTM1/p62, a known negative regulator of osteoclast formation. Consistently, we found that bafilomycin A1 diminishes the intracellular accumulation of the acidotropic probe lysotracker in osteoclast-like cells; indicative of reduced acidification. Further, bafilomycin A1 inhibits osteoclast formation with attenuation of cell fusion and multi-nucleation of osteoclast-like cells during osteoclast differentiation. Taken together, these data indicate that bafilomycin A1 attenuates osteoclast differentiation in part via increased levels of SQSTM1/p62 protein, providing further mechanistic insight into the effect of V-ATPase inhibition in osteoclasts. PMID:27043248

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

    PubMed Central

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

    2013-01-01

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

  20. Osteoclast recruitment in mice is stimulated by (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate.

    PubMed

    Marshall, M J; Holt, I; Davie, M W

    1993-01-01

    Though some evidence suggests that bisphosphonates (BPs) act directly on osteoclasts to inhibit bone resorption, other evidence suggests that they inhibit the development of the osteoclast. We found an increase in osteoclast recruitment in 2-day-old mice given (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD). A threefold increase in 5-bromo-2'-deoxyuridine (BrdU)-labeled osteoclast nuclei was observed on mouse parietal bones 3 days after APD injection. This suggests that inhibition of osteoclast development is not an action of APD in mice of this age. The mechanism of the increased recruitment was investigated. As osteoclast progenitors were not detected on parietal bones in vitro, we looked for an increase in circulating monocytes to account for the recruitment. No such increase was found, but when 51Cr-labeled bone marrow was injected intraperitoneally into mice given APD there was an increase in accumulation of 51Cr in calvaria and in femur and tibia over controls. This increase did not occur when 51Cr-labeled erythrocytes or free 51Cr was injected. We conclude that APD causes increased recruitment of osteoclast precursors by increasing the avidity of bone for hematopoietically derived cells. PMID:8453501

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

    PubMed

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

    2015-01-01

    Increased bone fracture is one of the health risk factors in patients with bone loss related disorders such as osteoporosis and breast cancer metastasis to bone. Over activity of osteoclasts leads to uncoupling of bone remodeling favoring bone loss over bone formation. Receptor activator of nuclear factor-κβ ligand (RANKL) triggers the differentiation pathway leading to multinucleated osteoclast formation. Modulation of RANKL or its downstream signaling pathways involved in osteoclast formation is of significant interest in the development of anti-resorptive agents. In this study, the effects of piperine, an alkaloid present in Piper nigrum L. on osteoclast formation was investigated. Piperine inhibited tartrate-resistant acid phosphatase-positive multinucleated osteoclast formation in murine RAW264.7 macrophages and human CD14+ monocytes induced by RANKL and breast cancer cells. Piperine attenuated the p38-mitogen activated protein kinase pathway activation, while the extracellular-signal-regulated kinase, c-Jun N-terminal kinase, or NF-κβ pathways downstream of RANKL remained unaffected. Concomitantly, expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), the key transcription factors involved in osteoclastogenesis were remarkably inhibited by piperine. Furthermore, piperine disrupted the actin ring structure and bone resorption, a characteristic hallmark of osteoclasts. Collectively, these results suggested that piperine inhibited osteoclast differentiation by suppressing the p38/NFATc1/c-Fos signaling axis.. PMID:26627060

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  4. Ficus carica L.: Metabolic and biological screening.

    PubMed

    Oliveira, Andreia P; Valentão, Patrícia; Pereira, José A; Silva, Branca M; Tavares, Fernando; Andrade, Paula B

    2009-11-01

    Ficus carica L. is one of the earliest cultivated fruit trees. In this work, metabolite profiling was performed on the leaves, pulps and peels of two Portuguese white varieties of F. carica (Pingo de Mel and Branca Tradicional). Phenolics and organic acids profiles were determined by HPLC/DAD and HPLC/UV, respectively. All samples presented a similar phenolic profile composed by 3-O- and 5-O-caffeoylquinic acids, ferulic acid, quercetin-3-O-glucoside, quercetin-3-O-rutinoside, psoralen and bergapten. 3-O-Caffeoylquinic acid and quercetin-3-O-glucoside are described for the first time in this species. Leaves' organic acids profile presented oxalic, citric, malic, quinic, shikimic and fumaric acids, while in pulps and peels quinic acid was absent. The antioxidant potential of the different plant parts was checked. All materials exhibited activity against DPPH and nitric oxide radicals in a concentration-dependent way. However, only the leaves presented capacity to scavenge superoxide radical. Leaves were always the most effective part, which seems to be related with phenolics compounds. Additionally, acetylcholinesterase inhibitory capacity was evaluated, but no effect was observed. Antimicrobial potential was also assessed against several bacterial species, although no activity was noticed. This is the first study comparing the chemical composition and biological potential of F. carica pulps, peels and leaves. PMID:19747518

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

    SciTech Connect

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

    2014-07-04

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

  6. Osteoclasts: more than ‘bone eaters’

    PubMed Central

    Charles, Julia F.; Aliprantis, Antonios O.

    2014-01-01

    As the only cells definitively shown to degrade bone, osteoclasts are key mediators of skeletal diseases including osteoporosis. Bone forming osteoblasts, and hematopoietic and immune system cells, each influence osteoclast formation and function, but the reciprocal impact of osteoclasts on these cells is less well appreciated. Here, we highlight functions osteoclasts perform beyond bone resorption. First, we consider how osteoclast signals may contribute to bone formation by osteoblasts and the pathology of bone lesions, such as fibrous dysplasia and giant cell tumors. Second, we review the interaction of osteoclasts with the hematopoietic system, including the stem cell niche and adaptive immune cells. Connections between osteoclasts and other cells in the bone microenvironment are discussed within a clinically relevant framework. PMID:25008556

  7. Formation of osteoclast-like cells is suppressed by low frequency, low intensity electric fields.

    PubMed

    Rubin, J; McLeod, K J; Titus, L; Nanes, M S; Catherwood, B D; Rubin, C T

    1996-01-01

    With use of a solenoid to generate uniform time-varying electric fields, the effect of extremely low frequency electric fields on osteoclast-like cell formation stimulated by 1,25(OH)2D3 was studied in primary murine marrow culture. Recruitment of osteoclast-like cells was assessed by counting multinuclear, tartrate-resistant acid phosphatase positive cells on day 8 of culture. A solenoid was used to impose uniform time-varying electric fields on cells; sham exposures were performed with an identical solenoid with a null net electric field. During the experiments, both solenoids heated interiorly to approximately 1.5 degrees C above ambient incubator temperature. As a result of the heating, cultures in the sham solenoid formed more osteoclast-like cells than those on the incubator shelf (132 +/- 12%). For this reason, cells exposed to the sham solenoid were used for comparison with cultures exposed to the active coil. Marrow cells were plated at 1.4 x 10(6)/cm2 in square chamber dishes and exposed to 60 Hz electric fields at 9.6 muV/cm from days 1 to 8. Field exposure inhibited osteoclast-like cell recruitment by 17 +/- 3% as compared with sham exposure (p < 0.0001). Several variables, including initial cell plating density, addition of prostaglandin E2 to enhance osteoclast-like cell recruitment, and field parameters, were also assessed. In this secondary series, extremely low frequency fields inhibited osteoclast-like cell formation by 24 +/- 4% (p < 0.0001), with their inhibitory effect consistent throughout all variations in protocol. These experiments demonstrate that extremely low intensity, low frequency sinusoidal electric fields suppress the formation of osteoclast-like cells in marrow culture. The in vitro results support in vivo findings that demonstrate that electric fields inhibit the onset of osteopenia and the progression of osteonecrosis; this suggests that extremely low frequency fields may inhibit osteoclast recruitment in vivo. PMID:8618169

  8. Synchronized Cell Cycle Arrest Promotes Osteoclast Differentiation.

    PubMed

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

    2016-01-01

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

  9. Synchronized Cell Cycle Arrest Promotes Osteoclast Differentiation

    PubMed Central

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

    2016-01-01

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

  10. Effects of IL-23 and IL-27 on osteoblasts and osteoclasts: inhibitory effects on osteoclast differentiation.

    PubMed

    Kamiya, Sadahiro; Nakamura, Chika; Fukawa, Takeshi; Ono, Katsuhiro; Ohwaki, Toshiyuki; Yoshimoto, Takayuki; Wada, Seiki

    2007-01-01

    Interleukin (IL)-23 and IL-27 are IL-6/IL-12 family members that play a role in the regulation of T helper 1 cell differentiation. Cytokines are known to be involved in the bone remodeling process, although the effects of IL-23 and IL-27 have not been clarified. In this study, we examined the possible roles of these cytokines on osteoblast phenotypes and osteoclastogenesis. We found that IL-27 induced signal transducers and activators of transcription 3 activation in osteoblasts. However, neither IL-23 nor IL-27 showed any significant effects on alkaline phosphatase activity, receptor activator of nuclear factor kappaB ligand (RANKL) expression, mRNA expression such as alkaline phosphatase type I procollagen, or the proliferation of osteoblasts. Osteoclastogenesis from bone marrow cells induced by soluble RANKL was partially inhibited by IL-23 and IL-27 with reduced multinucleated cell numbers, but these interleukins did not affect the proliferation of osteoclast progenitor cells. These results indicate that IL-23 and IL-27 could partly modify cell fusion or the survival of multinucleated osteoclasts. On the other hand, partially purified T cells, which are activated by 2 microg/ml anti-CD3 antibody, completely inhibited osteoclastogenesis by M-CSF/RANKL. On using T cells activated with 0.2 microg/ml anti-CD3 antibody, in which osteoclastogenesis was partially inhibited, the interleukins had additive effects for inhibiting osteoclastogenesis. Although the consequences of phosphorylated signals in osteoblasts have not been identified, IL-23 and IL-27, partly and indirectly through activated T cells, inhibited osteoclastogenesis, indicating that these interleukins may protect against bone destructive autoimmune disorders. PMID:17704992

  11. Antibacterial substance from Carica papaya fruit extract.

    PubMed

    Emeruwa, A C

    1982-01-01

    Ripe and unripe Carica papaya fruits (epicarp, endocarp, seeds and leaves) were extracted separately and purified. All the extracts except that of leaves produced very significant antibacterial activity on Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Shigella flexneri. The MIC of the substance was small (0.2-0.3 mg/ml) for gram-positive bacteria and large (1.5-4 mg/ml) for gram-negative bacteria. The substance was bactericidal and showed properties of a protein. Other proteins previously found in C. papaya did not show antibacterial activity. PMID:7097295

  12. Differences in osteoclast formation between proximal and distal tibial osteoporosis in rats with adjuvant arthritis: inhibitory effects of bisphosphonates on osteoclasts.

    PubMed

    Shu, Goukei; Yamamoto, Kaname; Nagashima, Masakazu

    2006-01-01

    Patients with rheumatoid arthritis commonly suffer both systemic and periarticular osteoporosis. Bisphosphonates (BPs) are inhibitors of bone resorption, and several derivatives have been developed for treatment of enhanced bone resorption. We aimed to characterize osteoclast formation in two different sites, the proximal tibial and distal tibial areas, in rats with adjuvant arthritis, and to investigate the impact of amino or non-amino types of bisphosphonate. Adjuvant arthritis was initiated in rats while administering daily injections of either etidronate, a non-amino BP, or alendronate, an amino BP, for 3 weeks. On the day following the last injection, bone mineral density (BMD) was measured in the proximal tibia to assess systemic osteoporosis and in the distal tibia for periarticular osteoporosis using dual-energy X-ray absorptiometry. Subsequently, bone marrow cells from either end of the tibia were collected and incubated for 7 days before staining and counting tartrate-resistant acid phosphatase positive cells. In the rats with adjuvant arthritis, BMD of either end of the tibia was lower than in normal rats. Although etidronate prevented bone mineral loss at both ends, distal loss was significantly less than proximal. In contrast, alendronate significantly inhibited mineral loss primarily in the proximal area. Large osteoclasts, defined as having five or more nuclei, formed preferentially in the proximal tibia, while small osteoclasts with fewer than four nuclei were found mainly distally. The suppressive effect of alendronate was greater on the large osteoclasts, while etidronate had a greater effect on the small osteoclasts. These results show that the size and multinuclearity of osteoclasts and the number of osteoclasts formed are different in the distal and proximal areas of the tibia, and that alendronate and etidronate may suppress different types of osteoclasts as discriminated by the number of nuclei. PMID:17164994

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

    PubMed

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

    2012-12-01

    Accumulating evidences suggest that Herba epimedii has the potential benefits against osteoporosis. However, previous studies were focused on the crude extract, total flavonoids (TF) and icariin (ICA), and the detailed molecular mechanisms of action and structure-activity relationship (SAR) remain unclear. Herein we aimed to systematically investigate the effects of Herba epimedii flavonoids (HEF) on the activity of osteoclasts, and explore the potential SAR. Both ICA and baohuoside-1 (BS) significantly inhibited the proliferation of RAW 264.7 cells (IC(50) 25 μM and 67 μM, respectively). Treatment of ICA resulted in G2/M arrest and apoptosis in RAW 264.7 cells as early as 12 h. Besides, HEF remarkably suppressed vitamin D-induced differentiation of osteoclasts in rabbit bone marrow cells and the bone resorption of rabbit mature osteoclasts in vitro. It is notable that the inhibitory effect of 100 μM ICA and BS on osteoclast formation is almost 90%; and the inhibition rate on bone resorption is 50% and 80%, respectively. Besides, RANKL-induced osteoclast formation from RAW 264.7 cells and the expression of TRAP, CA II, CTSK and MMP-9 was significantly reduced by the treatment of 25 μM HEF and 17β-estradiol (ES), and the inhibitory strength increases in the order TF < ES < ICA < BS, which was blocked by ICI182780 suggesting that the regulation of osteoclast activity might be ER dependent. Furthermore, the free hydroxyl group at C-7 of BS played an important role in the SAR for anti-osteoclast action. To conclude, HEF could regulate the formation and activity of osteoclasts by inhibiting the proliferation and differentiation, inducing apoptosis and cell cycle arrest and suppressing bone resorption of osteoclasts. Changes in osteoclast activity are probably mediated predominantly by interaction with nuclear estrogen receptors and via mitochondrial pathway. HEF, especially BS, has great potential for the prevention and treatment of osteoporosis. PMID:22796380

  14. Bone resorption by isolated human osteoclasts in vitro: effects of calcitonin.

    PubMed

    Murrills, R J; Shane, E; Lindsay, R; Dempster, D W

    1989-04-01

    Human osteoclasts were isolated from 12- to 17-week-old fetal tissue and from transiliac crest bone biopsies for an in vitro study of their biology. A hypodermic needle was used to flush either the fetal long bones or the trabeculae of the iliac crest bone biopsy with tissue culture medium and the resulting cell suspension sedimented briefly either onto the surface of plastic tissue culture dishes, for time-lapse microcinematography, or onto slices of devitalized bovine cortical bone for quantitative assay of bone resorption. The osteoclasts were motile, tartrate-resistant acid phosphatase positive and capable of excavating pits in slices of devitalized bovine cortical bone. Human calcitonin, at doses of 1 ng/ml and 1 microgram/ml, caused a 70% inhibition of bone resorption by human fetal osteoclasts over a 24 h period but had no apparent effect on the morphology or motility of either fetal or adult osteoclasts. PMID:2728929

  15. Jaw bone marrow-derived osteoclast precursors internalize more bisphosphonate than long-bone marrow precursors.

    PubMed

    Vermeer, Jenny A F; Jansen, Ineke D C; Marthi, Matangi; Coxon, Fraser P; McKenna, Charles E; Sun, Shuting; de Vries, Teun J; Everts, Vincent

    2013-11-01

    Bisphosphonates (BPs) are widely used in the treatment of several bone diseases, such as osteoporosis and cancers that have metastasized to bone, by virtue of their ability to inhibit osteoclastic bone resorption. Previously, it was shown that osteoclasts present at different bone sites have different characteristics. We hypothesized that BPs could have distinct effects on different populations of osteoclasts and their precursors, for example as a result of a different capacity to endocytose the drugs. To investigate this, bone marrow cells were isolated from jaw and long bone from mice and the cells were primed to differentiate into osteoclasts with the cytokines M-CSF and RANKL. Before fusion occurred, cells were incubated with fluorescein-risedronate (FAM-RIS) for 4 or 24h and uptake was determined by flow cytometry. We found that cultures obtained from the jaw internalized 1.7 to 2.5 times more FAM-RIS than long-bone cultures, both after 4 and 24h, and accordingly jaw osteoclasts were more susceptible to inhibition of prenylation of Rap1a after treatment with BPs for 24h. Surprisingly, differences in BP uptake did not differentially affect osteoclastogenesis. This suggests that jaw osteoclast precursors are less sensitive to bisphosphonates after internalization. This was supported by the finding that gene expression of the anti-apoptotic genes Bcl-2 and Bcl-xL was higher in jaw cells than long bone cells, suggesting that the jaw cells might be more resistant to BP-induced apoptosis. Our findings suggest that bisphosphonates have distinct effects on both populations of osteoclast precursors and support previous findings that osteoclasts and precursors are bone-site specific. This study may help to provide more insights into bone-site-specific responses to bisphosphonates. PMID:23962725

  16. Antiulcerogenic activity of Carica papaya seed in rats.

    PubMed

    Pinto, Lorraine Aparecida; Cordeiro, Kátia Wolff; Carrasco, Viviane; Carollo, Carlos Alexandre; Cardoso, Cláudia Andréa Lima; Argadoña, Eliana Janet Sanjinez; Freitas, Karine de Cássia

    2015-03-01

    The purpose of the present study was to evaluate the gastroprotective and healing effects of the methanolic extract of the seed of the papaya Carica papaya L. (MECP) in rats. Models of acute gastric ulcer induction by ethanol and indomethacin and of chronic ulcer by acetic acid were used. The gastric juice and mucus parameters were evaluated using the pylorus ligation model, and the involvement of sulfhydryl compounds (GSH) and nitric oxide in the gastroprotective effect was analyzed using the ethanol model. The toxicity was assessed through toxicity tests. No signs of toxicity were observed when the rats received a single dose of 2000 mg/kg of extract. The MECP in doses of 125, 250, and 500 mg/kg significantly reduced the gastric lesion with 56, 76, and 82 % inhibition, respectively, and a dose of 30 mg/kg lansoprazole showed 79 % inhibition in the ethanol model. MECP (125, 250, 500 mg/kg) and cimetidine (200 mg/kg) reduced the gastric lesion in the indomethacin model, with 62, 67, 81, and 85 % inhibition, respectively. The MECP (500 mg/kg) and cimetidine (200 mg/kg) treatments showed a reduction in ulcerative symptoms induced by acetic acid by 84 and 73 %, respectively. The antiulcerogenic activity seems to involve GSH because the inhibition dropped from 72 to 13 % in the presence of a GSH inhibitor. Moreover, the MECP showed systemic action, increasing the mucus production and decreasing gastric acidity. Treatments with MECP induce gastroprotection without signs of toxicity. This effect seems to involve sulfhydryl compounds, increased mucus, and reduced gastric acidity. PMID:25418890

  17. Fibrillin-1 directly regulates osteoclast formation and function by a dual mechanism

    PubMed Central

    Tiedemann, Kerstin; Boraschi-Diaz, Iris; Rajakumar, Irina; Kaur, Jasvir; Roughley, Peter; Reinhardt, Dieter P.; Komarova, Svetlana V.

    2016-01-01

    Summary Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca2+ signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function. PMID:24039232

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

    SciTech Connect

    Hattersley, G.; Chambers, T.J. )

    1991-02-01

    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.

  19. Decreased ferroportin promotes myeloma cell growth and osteoclast differentiation.

    PubMed

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

    2015-06-01

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

  20. Distinct osteoclast precursors in the bone marrow and extramedullary organs characterized by responsiveness to Toll-like receptor ligands and TNF-alpha.

    PubMed

    Hayashi, Shin-Ichi; Yamada, Takayuki; Tsuneto, Motokazu; Yamane, Toshiyuki; Takahashi, Masayuki; Shultz, Leonard D; Yamazaki, Hidetoshi

    2003-11-15

    Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues. PMID:14607912

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

    SciTech Connect

    Hiruma, Yoshiharu; Hirai, Takehiro; Tsuda, Eisuke

    2011-06-10

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

  2. Osteoclast TGF-β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

    PubMed Central

    Weivoda, Megan M; Ruan, Ming; Pederson, Larry; Hachfeld, Christine; Davey, Rachel A; Zajac, Jeffrey D; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclastspecific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. PMID:26108893

  3. A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomal vacuolar ATPase

    PubMed Central

    Niikura, Kazuaki; Takano, Mikiko; Sawada, Masae

    2004-01-01

    Vacuolar ATPase (V-ATPase) has been proposed as a drug target in lytic bone diseases. Studies of bafilomycin derivatives suggest that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is selective inhibition of osteoclast V-ATPase. Previous efforts to develop therapeutic inhibitors of osteoclast V-ATPase have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, we tried to find novel potent and specific V-ATPase inhibitors, which have new structural features and inhibition selectivity, from random screening using osteoclast microsomes. Finally, a novel V-ATPase inhibitor, FR167356, was obtained through chemical modification of a parental hit compound. FR167356 inhibited not only H+ transport activity of osteoclast V-ATPase but also H+ extrusion from cytoplasm of osteoclasts, which depends on the V-ATPase activity. As expected, FR167356 remarkably inhibited bone resorption in vitro. FR167356 also showed inhibitory effects on other V-ATPases, renal brush border V-ATPase, macrophage microsome V-ATPase and lysosomal V-ATPase. However, FR167356 was approximately seven-fold less potent in inhibiting lysosomal V-ATPase compared to osteoclast V-ATPase. Moreover, LDL metabolism in cells, which depends on acidification of lysosome, was blocked merely at higher concentration than bone resorption, suggesting that FR167356 inhibits V-ATPase of osteoclast ruffled border membrane still more selectively than lysosome at the cellular level. These results from the experiments seem to indicate that osteoclast V-ATPase may be different from lysosomal V-ATPase in respect of their structure. FR167356 had a novel chemical structural feature as well as inhibitory characteristics distinctly different from any previously known V-ATPase inhibitor family. Therefore, FR167356 is thought to be a useful tool for estimating the essential characteristics of V-ATPase inhibitors for drug development. PMID:15148249

  4. The Great Beauty of the osteoclast.

    PubMed

    Cappariello, Alfredo; Maurizi, Antonio; Veeriah, Vimal; Teti, Anna

    2014-09-15

    Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an "orchestrator" implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the '00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting "clastokines" that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast. PMID:24976175

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

    SciTech Connect

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

    2014-05-02

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

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

    PubMed

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

    2016-03-01

    Osteoclasts are the only cells capable of breaking down bone matrix, and excessive activation of osteoclasts is responsible for bone-destructive diseases. In this study, we investigated the effects of decursin from extract of Angelica gigas root on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast formation using mouse bone marrow-derived macrophages (BMMs). Decursin inhibited RANKL-induced osteoclast formation without cytotoxicity. In particular, decursin maintains the characteristics of macrophages by blocking osteoclast differentiation by RANKL. Furthermore, the RANKL-stimulated bone resorption was diminished by decursin. Mechanistically, decursin blocked the RANKL-triggered ERK mitogen-activated protein kinases (MAPK) phosphorylation, which results in suppression of c-Fos and the nuclear factor of activated T cells (NFATc1) expression. In accordance with the in vitro study, decursin reduced lipopolysaccharide (LPS)- or ovariectomy (OVX)-induced bone loss in vivo. Therefore, decursin exerted an inhibitory effect on osteoclast formation and bone loss in vitro and in vivo. Decursin could be useful for the treatment of bone diseases associated with excessive bone resorption. PMID:26825541

  7. LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption.

    PubMed

    Luo, Jian; Yang, Zhengfeng; Ma, Yu; Yue, Zhiying; Lin, Hongyu; Qu, Guojun; Huang, Jinping; Dai, Wentao; Li, Chenghai; Zheng, Chunbing; Xu, Leqin; Chen, Huaqing; Wang, Jiqiu; Li, Dali; Siwko, Stefan; Penninger, Josef M; Ning, Guang; Xiao, Jianru; Liu, Mingyao

    2016-05-01

    Tumor necrosis factor (TNF) superfamily member 11 (TNFSF11, also known as RANKL) regulates multiple physiological or pathological functions, including osteoclast differentiation and osteoporosis. TNFRSF11A (also called RANK) is considered to be the sole receptor for RANKL. Herein we report that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL. LGR4 competes with RANK to bind RANKL and suppresses canonical RANK signaling during osteoclast differentiation. RANKL binding to LGR4 activates the Gαq and GSK3-β signaling pathway, an action that suppresses the expression and activity of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (NFATC1) during osteoclastogenesis. Both whole-body (Lgr4(-/-)) and monocyte conditional knockout mice of Lgr4 (Lgr4 CKO) exhibit osteoclast hyperactivation (including elevation of osteoclast number, surface area, and size) and increased bone erosion. The soluble LGR4 extracellular domain (ECD) binds RANKL and inhibits osteoclast differentiation in vivo. Moreover, LGR4-ECD therapeutically abrogated RANKL-induced bone loss in three mouse models of osteoporosis. Therefore, LGR4 acts as a second RANKL receptor that negatively regulates osteoclast differentiation and bone resorption. PMID:27064449

  8. The in vitro osteoclastic degradation of nacre.

    PubMed

    Duplat, D; Chabadel, A; Gallet, M; Berland, S; Bédouet, L; Rousseau, M; Kamel, S; Milet, C; Jurdic, P; Brazier, M; Lopez, E

    2007-04-01

    Osteoclast activity was studied on nacre, the mother of pearl (MOP) in order to assess the plasticity of bone resorbing cells and their capacity to adapt to a biomineralized material with a different organic and mineral composition from that of its natural substrate, bone. Pure MOP, a natural biomineralized CaCO(3) material, was obtained from Pinctada oyster shell. When implanted in the living system, nacre has proven to be a sustainable bone grafting material although a limited surface degradation process. Osteoclast stem cells and mature osteoclasts were cultured on MOP substrate and osteoclast precursor cells were shown to differentiate into osteoclasts capable of resorbing nacre substrate. However, analysis of the organization of the cytoskeleton showed that both a sealing zone and a podosome structure were observed on the nacre substrate. Moreover, MOP resorption efficiency was consistently found to be lower than that of bone and appeared to be a limited process. PMID:17258312

  9. The Roles of Acidosis in Osteoclast Biology

    PubMed Central

    Yuan, Feng-Lai; Xu, Ming-Hui; Li, Xia; Xinlong, He; Fang, Wei; Dong, Jian

    2016-01-01

    The adverse effect of acidosis on the skeletal system has been recognized for almost a century. Although the underlying mechanism has not been fully elucidated, it appears that acidosis acts as a general stimulator of osteoclasts derived from bone marrow precursors cells and enhances osteoclastic resorption. Prior work suggests that acidosis plays a significant role in osteoclasts formation and activation via up-regulating various genes responsible for its adhesion, migration, survival and bone matrix degradation. Understanding the role of acidosis in osteoclast biology may lead to development of novel therapeutic approaches for the treatment of diseases related to low bone mass. In this review, we aim to discuss the recent investigations into the effects of acidosis in osteoclast biology and the acid-sensing molecular mechanism. PMID:27445831

  10. The origin of the non-recombining region of sex chromosomes in Carica and Vasconcellea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carica and Vasconcellea are two closely related sister genera in the family Caricaceae, and were once classified as two sections under Carica. After the section Vasconcellea with 21 species was reinstated as a separate genus based on molecular marker data, papaya became the sole species in Carica. S...

  11. Anticancer activity of Carica papaya: a review.

    PubMed

    Nguyen, Thao T T; Shaw, Paul N; Parat, Marie-Odile; Hewavitharana, Amitha K

    2013-01-01

    Carica papaya is widely cultivated in tropical and subtropical countries and is used as food as well as traditional medicine to treat a range of diseases. Increasing anecdotal reports of its effects in cancer treatment and prevention, with many successful cases, have warranted that these pharmacological properties be scientifically validated. A bibliographic search was conducted using the key words "papaya", "anticancer", and "antitumor" along with cross-referencing. No clinical or animal cancer studies were identified and only seven in vitro cell-culture-based studies were reported; these indicate that C. papaya extracts may alter the growth of several types of cancer cell lines. However, many studies focused on specific compounds in papaya and reported bioactivity including anticancer effects. This review summarizes the results of extract-based or specific compound-based investigations and emphasizes the aspects that warrant future research to explore the bioactives in C. papaya for their anticancer activities. PMID:23212988

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-01-01

    Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression. PMID:26759696

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

    PubMed Central

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

    2016-01-01

    Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression. PMID:26759696

  15. Suppressive effects of Anoectochilus formosanus extract on osteoclast formation in vitro and bone resorption in vivo.

    PubMed

    Masuda, Kikuko; Ikeuchi, Mayumi; Koyama, Tomoyuki; Yamaguchi, Kohji; Woo, Je-Tae; Nishimura, Tomio; Yazawa, Kazunaga

    2008-01-01

    Anoectochilus formosanus, a plant native to Taiwan, is used as a folk medicine. It was found that oral administration of A. formosanus extract (AFE) (500 mg/kg) for 4 weeks suppressed bone weight loss and trabecular bone loss in ovariectomized mice, an experimental model of osteoporosis. Although AFE at 12.5 and 25 mug/ml inhibited osteoclast formation in co-culture of osteoblasts and bone marrow cells, AFE did not inhibit the formation of osteoclast progenitor cells and preosteoclast cells in bone marrow cells and RAW264 cells. However, AFE (at 12.5 and 25 microg/ml) decreased RANKL expression. These results suggested that AFE might suppress the bone loss caused by estrogen deficiency through suppression of RANKL expression required for osteoclast formation. PMID:18301967

  16. Mechanically loaded myotubes affect osteoclast formation.

    PubMed

    Juffer, Petra; Jaspers, Richard T; Klein-Nulend, Jenneke; Bakker, Astrid D

    2014-03-01

    In response to mechanical loading skeletal muscle produces numerous growth factors and cytokines that enter the circulation. We hypothesized that myotubes produce soluble factors that affect osteoclast formation and aimed to identify which osteoclastogenesis-modulating factors are differentially produced by mechanically stimulated myotubes. C2C12 myotubes were subjected to mechanical loading by cyclic strain for 1 h, and postincubated with or without cyclic strain for 24 h. The effect of cyclic strain on gene expression in myotubes was determined by PCR. Conditioned medium (CM) was collected from cultures of unloaded and loaded myotubes and from MLO-Y4 osteocytes. CM was added to mouse bone marrow cells containing osteoclast precursors, and after 6 days osteoclasts were counted. Compared to unconditioned medium, CM from unloaded osteocytes increased osteoclast formation, while CM from unloaded myotubes decreased osteoclast formation. Cyclic strain strongly enhanced IL-6 expression in myotubes. CM from cyclically strained myotubes increased osteoclast formation compared to CM from unloaded myotubes, but this effect did not occur in the presence of an IL-6 antibody. In conclusion, mechanically loaded myotubes secrete soluble factors, among others IL-6, which affect osteoclast formation. These results suggest that muscle could potentially affect bone homeostasis in vivo via production of growth factors and/or cytokines. PMID:24264813

  17. Microcracks and osteoclast resorption activity in vitro.

    PubMed

    Rumpler, Monika; Würger, Tanja; Roschger, Paul; Zwettler, Elisabeth; Peterlik, Herwig; Fratzl, Peter; Klaushofer, Klaus

    2012-03-01

    During bone remodeling osteoclasts resorb bone, thus removing material, e.g., damaged by microcracks, which arises as a result of physiological loading and could reduce bone strength. Such a process needs targeted bone resorption exactly at damaged sites. Osteocytic signaling plays a key role in this process, but it is not excluded that osteoclasts per se may possess toposensitivity to recognize and resorb damaged bone since it has been shown that resorption spaces are associated with microcracks. To address this question, we used an in vitro setup of a pure osteoclast culture and mineralized substrates with artificially introduced microcracks and microscratches. Histomorphometric analyses and statistical evaluation clearly showed that these defects had no effect on osteoclast resorption behavior. Osteoclasts did not resorb along microcracks, even when resorption started right beside these damages. Furthermore, quantification of resorption on three different mineralized substrates, cortical bone, bleached bone (bone after partial removal of the organic matrix), and dentin, revealed lowest resorption on bone, significantly higher resorption on bleached bone, and highest resorption on dentin. The difference between native and bleached bone may be interpreted as an inhibitory impact of the organic matrix. However, the collagen-based matrix could not be the responsible part as resorption was highest on dentin, which contains collagen. It seems that osteocytic proteins, stored in bone but not present in dentin, affect osteoclastic action. This demonstrates that osteoclasts per se do not possess a toposensitivity to remove microcracks but may be influenced by components of the organic bone matrix. PMID:22271249

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

    PubMed

    Gu, Dong Ryun; Hwang, Jin-Ki; Erkhembaatar, Munkhsoyol; Kwon, Kang-Beom; Kim, Min Seuk; Lee, Young-Rae; Lee, Seoung Hoon

    2013-01-01

    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- κ B 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 Ca(2+)-oscillation via the inactivation of PLC γ 2, 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. PMID:24174976

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

    PubMed Central

    Gu, Dong Ryun; Hwang, Jin-Ki; Erkhembaatar, Munkhsoyol; Kwon, Kang-Beom; Lee, Young-Rae; Lee, Seoung Hoon

    2013-01-01

    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. PMID:24174976

  20. Antifungal Activity in Ethanolic Extracts of Carica papaya L. cv. Maradol Leaves and Seeds.

    PubMed

    Chávez-Quintal, Pedro; González-Flores, Tania; Rodríguez-Buenfil, Ingrid; Gallegos-Tintoré, Santiago

    2011-01-01

    Bioactive compounds from vegetal sources are a potential source of natural antifungic. An ethanol extraction was used to obtain bioactive compounds from Carica papaya L. cv. Maradol leaves and seeds of discarded ripe and unripe fruit. Both, extraction time and the papaya tissue flour:organic solvent ratio significantly affected yield, with the longest time and highest flour:solvent ratio producing the highest yield. The effect of time on extraction efficiency was confirmed by qualitative identification of the compounds present in the lowest and highest yield extracts. Analysis of the leaf extract with phytochemical tests showed the presence of alkaloids, flavonoids and terpenes. Antifungal effectiveness was determined by challenging the extracts (LE, SRE, SUE) from the best extraction treatment against three phytopathogenic fungi: Rhizopus stolonifer, Fusarium spp. and Colletotrichum gloeosporioides. The leaf extract exhibited the broadest action spectrum. The MIC(50) for the leaf extract was 0.625 mg ml(-1) for Fusarium spp. and >10 mg ml(-1) for C. gloeosporioides, both equal to approximately 20% mycelial growth inhibition. Ethanolic extracts from Carica papaya L. cv. Maradol leaves are a potential source of secondary metabolites with antifungal properties. PMID:22282629

  1. Osteoprotegerin exposure at different stages of osteoclastogenesis differentially affects osteoclast formation and function.

    PubMed

    Zhao, Hongyan; Gu, Jianhong; Dai, Nannan; Gao, Qian; Wang, Dong; Song, Ruilong; Liu, Wei; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Liu, Zongping

    2016-08-01

    This study aimed to investigate the effects of osteoprotegerin (OPG), a decoy receptor for receptor activator for nuclear factor κB ligand (RANKL), during the various stages of osteoclast differentiation, and additionally investigate its effects on osteoclast adhesion and activity. RAW264.7 murine monocytic cells were incubated with macrophage colony-stimulating factor and RANKL for 1, 3, 5, or 7 days, followed by an additional 24-h incubation in the presence or absence of OPG (80 ng/mL). We examined osteoclast differentiation and adhesion capacity using the tartrate-resistant acid phosphatase (TRAP) assay and immunofluorescence microscopy, and additionally examined cell growth in real time using the xCELLigence system. Furthermore, the expression levels of TRAP, RANK, integrin β3, matrix metalloproteinase 9, cathepsin K, carbonic anhydrase II, and vesicular-type H(+)-ATPase A1 were examined using western blotting. OPG exposure on day 1 enhanced the osteoclast growth curve as well as adhesion, and increased RANK and integrin β3 expression. In contrast, exposure to OPG at later time points (days 3-7) inhibited osteoclast differentiation, adhesion structure formation, and protease expression. In conclusion, the biological effects of OPG exposure at the various stages of osteoclast differentiation were varied, and included the enhanced adhesion and survival of preosteoclasts, the block of differentiation from the early to the terminal stages of osteoclastogenesis, and suppression of mature osteoclast activation following OPG exposure during the terminal differentiation stage, suggesting that the effects of OPG exposure differ based on the stage of differentiation. PMID:26044733

  2. Malignant Melanoma With Osteoclast-Like Differentiation.

    PubMed

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

    2015-09-01

    Osteoclast-like giant cells are frequently encountered in nonskeletal malignancies; however, the evidence to date suggests that they represent a tissue response to the lesion rather than neoplastic differentiation. We describe a case of metastatic melanoma demonstrating osteoclast-like differentiation in the lung. The lung nodule was diagnosed as a metastatic melanoma by histological features and confirmed by immunohistochemistry. Resection specimen showed numerous multinucleated giant cells exhibiting osteoclast-like morphology dispersed throughout the lesion. Both the neoplastic melanocytes and giant cells were reactive for HMB-45, Melan-A, and S100. In addition, the multinucleated neoplastic giant cells were also reactive for the monocyte/macrophage lineage markers CD68 and CD163, and alkaline phosphatase, an enzyme present in normal osteoclasts. The neoplastic melanocytes and the multinucleated neoplastic giant cells were also reactive for microphthalmia-associated transcription factor, a protein required for the development of both melanocytes and osteoclasts. Collectively, a co-expression of monocyte/macrophage markers along with melanocytic markers and alkaline phosphatase in the multinucleated neoplastic giant cells in metastatic melanoma suggest that malignant melanocytes are capable of differentiating into osteoclast-like cells and consequently aid invasion into various structures and eliciting the aggressive behavior. PMID:26113663

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

    PubMed

    Ying, Hua; Qin, An; Cheng, Tak S; Pavlos, Nathan J; Rea, Sarah; Dai, Kerong; Zheng, Ming H

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. Regulation of cytochrome c oxidase activity by c-Src in osteoclasts

    PubMed Central

    Miyazaki, Tsuyoshi; Neff, Lynn; Tanaka, Sakae; Horne, William C.; Baron, Roland

    2003-01-01

    The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane–associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria. PMID:12615910

  6. Hif1α is required for osteoclast activation and bone loss in male osteoporosis.

    PubMed

    Tando, Toshimi; Sato, Yuiko; Miyamoto, Kana; Morita, Mayu; Kobayashi, Tami; Funayama, Atsushi; Kanaji, Arihiko; Hao, Wu; Watanabe, Ryuichi; Oike, Takatsugu; Nakamura, Masaya; Matsumoto, Morio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2016-02-01

    The number of osteoporosis patients is increasing not only in women but in men. Male osteoporosis occurs due to aging or androgen depletion therapies, leading to fractures. However, molecular mechanisms underlying male osteoporosis remain unidentified. Here, we show that hypoxia inducible factor 1 alpha (Hif1α) is required for development of testosterone deficiency-induced male osteoporosis. We found that in mice Hif1α protein accumulates in osteoclasts following orchidectomy (ORX) in vivo. In vitro, Hif1α protein accumulated in osteoclasts cultured in hypoxic conditions, but Hif1α protein rather than mRNA levels were suppressed by testosterone treatment, even in hypoxia. Administration of a Hif1α inhibitor to ORX mice abrogated testosterone deficiency-induced osteoclast activation and bone loss but did not alter osteoclast activities or bone phenotypes in sham-operated, testosterone-sufficient animals. We conclude that Hif1α protein accumulation due to testosterone-deficiency promotes development of male osteoporosis. Thus Hif1α protein could be targeted to inhibit pathologically-activated osteoclasts under testosterone-deficient conditions to treat male osteoporosis patients. PMID:26792721

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

    PubMed

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

    2016-09-01

    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. PMID:27416754

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

    SciTech Connect

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

    2015-05-29

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

  9. Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol.

    PubMed

    Ishizuka, Kyoko; Hirukawa, Koji; Nakamura, Hiroshi; Togari, Akifumi

    2005-04-29

    The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo. PMID:15814197

  10. TRAFD1 (FLN29) Interacts with Plekhm1 and Regulates Osteoclast Acidification and Resorption

    PubMed Central

    Witwicka, Hanna; Jia, Hong; Kutikov, Artem; Reyes-Gutierrez, Pablo; Li, Xiangdong; Odgren, Paul R.

    2015-01-01

    Plekhm1 is a large, multi-modular, adapter protein implicated in osteoclast vesicle trafficking and bone resorption. In patients, inactivating mutations cause osteopetrosis, and gain-of-function mutations cause osteopenia. Investigations of potential Plekhm1 interaction partners by mass spectrometry identified TRAFD1 (FLN29), a protein previously shown to suppress toll-like receptor signaling in monocytes/macrophages, thereby dampening inflammatory responses to innate immunity. We mapped the binding domains to the TRAFD1 zinc finger (aa 37-60), and to the region of Plekhm1 between its second pleckstrin homology domain and its C1 domain (aa 784-986). RANKL slightly increased TRAFD1 levels, particularly in primary osteoclasts, and the co-localization of TRAFD1 with Plekhm1 also increased with RANKL treatment. Stable knockdown of TRAFD1 in RAW 264.7 cells inhibited resorption activity proportionally to the degree of knockdown, and inhibited acidification. The lack of acidification occurred despite the presence of osteoclast acidification factors including carbonic anhydrase II, a3-V-ATPase, and the ClC7 chloride channel. Secretion of TRAP and cathepsin K were also markedly inhibited in knockdown cells. Truncated Plekhm1 in ia/ia osteopetrotic rat cells prevented vesicle localization of Plekhm1 and TRAFD1. We conclude that TRAFD1, in association with Plekhm1/Rab7-positive late endosomes-early lysosomes, has a previously unknown role in vesicle trafficking, acidification, and resorption in osteoclasts. PMID:25992615

  11. Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

    NASA Technical Reports Server (NTRS)

    Miyauchi, A.; Hruska, K. A.; Greenfield, E. M.; Duncan, R.; Alvarez, J.; Barattolo, R.; Colucci, S.; Zambonin-Zallone, A.; Teitelbaum, S. L.; Teti, A.

    1990-01-01

    The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

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

    PubMed

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

    2016-09-01

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

  13. Post-irradiation identification of papaya ( Carica papaya L.) fruit

    NASA Astrophysics Data System (ADS)

    Chatterjee, Suchandra; Variyar, Prasad S.; Sharma, Arun

    2012-03-01

    Impact of radiation processing on the volatile essential oil profile of papaya ( Carica papaya) was investigated. Gamma-radiation processing resulted in the appearance of a new peak in the GLC profile that was identified as phenol. The observed dose dependent increase in phenol content suggested possible use of this compound as a marker for radiation processed papaya.

  14. Ecophysiology of papaya Carica papaya L.: a review

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Papaya (Carica papaya L.) is a principal horticultural crop of tropical and subtropical regions. Knowledge of how papaya responds to environmental factors provides a scientific basis to develop management strategies to optimize fruit yield and quality. A better understanding of genotypic responses t...

  15. In-vivo imaging of the fracture healing in medaka revealed two types of osteoclasts before and after the callus formation by osteoblasts.

    PubMed

    Takeyama, Kazuhiro; Chatani, Masahiro; Takano, Yoshiro; Kudo, Akira

    2014-10-15

    The fracture healing research, which has been performed in mammalian models not only for clinical application but also for bone metabolism, revealed that generally osteoblasts are induced to enter the fracture site before the induction of osteoclasts for bone remodeling. However, it remains unknown how and where osteoclasts and osteoblasts are induced, because it is difficult to observe osteoclasts and osteoblasts in a living animal. To answer these questions, we developed a new fracture healing model by using medaka. We fractured one side of lepidotrichia in a caudal fin ray without injuring the other soft tissues including blood vessels. Using the transgenic medaka in which osteoclasts and osteoblasts were visualized by GFP and DsRed, respectively, we found that two different types of functional osteoclasts were induced before and after osteoblast callus formation. The early-induced osteoclasts resorbed the bone fragments and the late-induced osteoclasts remodeled the callus. Both types of osteoclasts were induced near the surface on the blood vessels, while osteoblasts migrated from adjacent fin ray. Transmission electron microscopy revealed that no significant ruffled border and clear zone were observed in early-induced osteoclasts, whereas the late-induced osteoclasts had clear zones but did not have the typical ruffled border. In the remodeling of the callus, the expression of cox2 mRNA was up-regulated at the fracture site around vessels, and the inhibition of Cox2 impaired the induction of the late-induced osteoclasts, resulting in abnormal fracture healing. Finally, our developed medaka fracture healing model brings a new insight into the molecular mechanism for controlling cellular behaviors during the fracture healing. PMID:25131195

  16. Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium.

    PubMed

    Lakkakorpi, P T; Lehenkari, P P; Rautiala, T J; Väänänen, H K

    1996-09-01

    The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca2+]e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca2+]i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca2+]i increased when [Ca2+]e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca2+]e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca2+]e (up to 20 mM [Ca2+]e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca2+]e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca2+]e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca2+]i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca2+]i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C. PMID:8816921

  17. Bovine parathyroid hormone enhances osteoclast bone resorption by modulating V-ATPase through PTH1R

    PubMed Central

    LIU, SHUANGXIN; ZHU, WEIPING; LI, SIJIA; MA, JIANCHAO; ZHANG, HUITAO; LI, ZHONGHE; ZHANG, LI; ZHANG, BIN; LI, ZHUO; LIANG, XINLING; SHI, WEI

    2016-01-01

    The vacuolar-type H+ adenosine triphosphatase (V-ATPase) plays an important role in cellular acidification and bone resorption by osteoclasts. However, the direct effect of bovine parathyroid hormone (bPTH) on V-ATPase has not yet been elucidated. The aim of the present study was to assess the effects of bPTH on V-ATPase and osteoclasts. Osteoclasts from bone marrow (BM)-derived monocytes of C57BL/6 mice were cultured with or without bPTH. The mRNA and protein expression levels of the V-ATPase a3-subunit and d2-subunit (by RT-qPCR and western blot analysis), V-ATPase activity (using the V type ATPase Activity Assay kit) and the bone resorption function of osteoclasts (by bone resorption assay) were examined following treatment with various concentrations of bPTH (0.1, 1.0, 10 and 100 ng/ml) alone or with bPTH and its inhibitor, bafilomycin A1. Furthermore, the expression of parathyroid hormone (PTH) receptors in osteoclasts was also detected. The results revealed that the mRNA and protein expression levels of V-ATPase a3-subunit and d2-subunit increased in a dose-dependent manner, paralleling the level of bPTH present. In addition, an increase in the concentration of bPTH was accompanied by the increased resorption capability of osteoclasts, whereas bone resorption was inhibited in the presence of bafilomycin A1. In addition, we confirmed the existence of parathyroid hormone 1 receptor (PTH1R) in osteoclasts using three different methods (RT-qPCR, western blot analysis and immunofluorescence staining). We found that bPTH enhanced the bone resorption capability of osteoclasts by modulating the expression of V-ATPase subunits, intracellular acidification and V-ATPase activity. Thus, we propose that PTH has a direct effect on osteoblasts and osteoclasts, and that this effect is mediated through PTH1R, thus contributing to bone remodeling. PMID:26647715

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

    PubMed Central

    ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I.; Davison, Noel L.; de Vries, Teun J.; Everts, Vincent

    2015-01-01

    cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K. PMID:26426806

  19. Osteoclasts in the interface with electrospun hydroxyapatite.

    PubMed

    Pasuri, Jenni; Holopainen, Jani; Kokkonen, Hanna; Persson, Maria; Kauppinen, Kyösti; Lehenkari, Petri; Santala, Eero; Ritala, Mikko; Tuukkanen, Juha

    2015-11-01

    Electrospinning is a method to produce lightweight, resorbable and bioinspired scaffolds for tissue engineering. Here we investigated the influence of electrospun hydroxyapatite fibers (HA) on macrophages and osteoclasts. A mouse macrophage cell line (RAW 264.7) and human bone marrow derived primary osteoclasts (hOC) were cultured with electrospun HA fibers embedded in Matrigel. Cell morphology and the secretion of pro-inflammatory cytokines (IL-6 and TNF-α) were analyzed using macrophages. Both fluorescent microscopy and scanning electron microscopy indicated that the cell morphology differed on the various materials (HA fibers on Matrigel, pure Matrigel and a glass control). Control macrophages were activated with bacterial lipopolysaccharide (LPS) but electrospun HA did not provoke an inflammatory response. Cytokine secretion detected with enzyme-linked immunosorbent assay (ELISA) also supported this observation. LPS, but not HA fibers, stimulated TNF-α and IL-6 secretion by macrophages at the 2 day time point. After 4 days in culture there was an increasing trend in cytokine secretion in the HA fiber samples. Human bone marrow myeloid precursor cells were able to fuse and differentiate on the fibrous mineral scaffold to form functional multinuclear osteoclasts that were able to resorb the HA nanofibers. This indicates that osteoclasts do not necessarily need a continuous bone surface but osteoclast ruffled border membranes can form a resorption interface with a fibrous mineral scaffold. PMID:26342323

  20. Mechanisms of osteoclast-dependent bone formation

    PubMed Central

    Teti, Anna

    2013-01-01

    Should we believe that osteoclasts are only involved in bone resorption? What about their contribution to bone formation? In this article I will review evidence that bone formation can be regulated by osteoclasts. Why is this? Likely because in the physiologic condition of bone remodeling, bone resorption and formation are balanced, and there is no better way to control this equilibrium than through a concerted action between the two cell types. Although the influence of osteoblasts on osteoclastic bone resorption is well documented and consolidated over time, what osteoclasts do to regulate osteoblast activity is still matter of intense investigation. The original hypothesis that all is in the osteoblast-seeking factors stored in the bone matrix, released and activated during bone resorption, is now being challenged by several studies, suggesting that osteoclasts are also capable of producing ‘clastokines' that regulate osteoblast performance. Indeed, several of them have been demonstrated to orchestrate osteoclast–osteoblast activities. However, we are probably still at the dawn of a new era, and future work will tell us whether any of these clastokines can be exploited to stimulate bone formation and rebalance bone remodeling in skeletal diseases. PMID:24422142

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-21

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

  3. Carica papaya (Paw-Paw) unripe fruit may be beneficial in ulcer.

    PubMed

    Ezike, A C; Akah, P A; Okoli, C O; Ezeuchenne, N A; Ezeugwu, S

    2009-12-01

    The anti-ulcer potentials of aqueous (AE) and methanol (ME) extracts of whole unripe Carica papaya fruit were evaluated using ethanol- and indomethacin-induced gastric ulcer models in rats. The effect of the extracts on small intestinal propulsion was also investigated. The extracts significantly reduced the ulcer index in both experimental models (P < .05) compared to the control group. ME showed a better protection against indomethacin-induced ulcers, whereas AE was more effective against ethanol-induced gastric ulcers. The extracts also significantly (P < .05) inhibited intestinal motility, with ME showing greater activity. Oral administration of AE and ME up to 5,000 mg/kg did not produce lethality or signs of acute toxicity in mice after 24 hours. The extracts of unripe C. papaya contain terpenoids, alkaloids, flavonoids, carbohydrates, glycosides, saponins, and steroids. The cytoprotective and antimotility properties of the extracts may account for the anti-ulcer property of the unripe fruit. PMID:20041780

  4. 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

    PubMed Central

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

    2015-01-01

    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. PMID:26536233

  5. Methanolic Extract of Ficus carica Linn. Leaves Exerts Antiangiogenesis Effects Based on the Rat Air Pouch Model of Inflammation

    PubMed Central

    Eteraf-Oskouei, Tahereh; Allahyari, Saeideh; Akbarzadeh-Atashkhosrow, Arezu; Delazar, Abbas; Pashaii, Mahdiyeh; Gan, Siew Hua; Najafi, Moslem

    2015-01-01

    The antiangiogenesis effect of Ficus carica leaves extract in an air pouch model of inflammation was investigated in rat. Inflammation was induced by injection of carrageenan into pouches. After antioxidant capacity and total phenolic content (TPC) investigations, the extract was administered at 5, 25, and 50 mg/pouch, and then the volume of exudates, the cell number, TNFα, PGE2, and VEGF levels were measured. Angiogenesis of granulation tissues was determined by measuring hemoglobin content. Based on the DPPH assay, the extract had significant antioxidant activity with TPC of 11.70 mg GAE/100 g dry sample. In addition, leukocyte accumulation and volume of exudate were significantly inhibited by the extract. Moreover, it significantly decreased the production of TNFα, PGE2, and VEGF, while angiogenesis was significantly inhibited by all administered doses. Interestingly, attenuation of angiogenesis and inflammatory parameters (except leukocyte accumulation) by the extract was similar to that shown by diclofenac. The extract has anti-inflammatory effects and ameliorated cell influx and exudation to the site of the inflammatory response which may be related to the local inhibition of TNFα, PGE2, and VEGF levels as similarly shown by diclofenac. The antiangiogenesis and anti-VEGF effects of Ficus carica may be correlated with its significant antioxidant potentials. PMID:25977699

  6. Methanolic Extract of Ficus carica Linn. Leaves Exerts Antiangiogenesis Effects Based on the Rat Air Pouch Model of Inflammation.

    PubMed

    Eteraf-Oskouei, Tahereh; Allahyari, Saeideh; Akbarzadeh-Atashkhosrow, Arezu; Delazar, Abbas; Pashaii, Mahdiyeh; Gan, Siew Hua; Najafi, Moslem

    2015-01-01

    The antiangiogenesis effect of Ficus carica leaves extract in an air pouch model of inflammation was investigated in rat. Inflammation was induced by injection of carrageenan into pouches. After antioxidant capacity and total phenolic content (TPC) investigations, the extract was administered at 5, 25, and 50 mg/pouch, and then the volume of exudates, the cell number, TNFα, PGE2, and VEGF levels were measured. Angiogenesis of granulation tissues was determined by measuring hemoglobin content. Based on the DPPH assay, the extract had significant antioxidant activity with TPC of 11.70 mg GAE/100 g dry sample. In addition, leukocyte accumulation and volume of exudate were significantly inhibited by the extract. Moreover, it significantly decreased the production of TNFα, PGE2, and VEGF, while angiogenesis was significantly inhibited by all administered doses. Interestingly, attenuation of angiogenesis and inflammatory parameters (except leukocyte accumulation) by the extract was similar to that shown by diclofenac. The extract has anti-inflammatory effects and ameliorated cell influx and exudation to the site of the inflammatory response which may be related to the local inhibition of TNFα, PGE2, and VEGF levels as similarly shown by diclofenac. The antiangiogenesis and anti-VEGF effects of Ficus carica may be correlated with its significant antioxidant potentials. PMID:25977699

  7. How are osteoclasts induced to resorb bone?

    PubMed

    Chambers, T J; Fuller, K

    2011-12-01

    Although much is known about how osteoclasts are formed, we know little about how they are activated, or how they recognize bone as the substrate appropriate for resorption. Bone mineral is considered to be essential to this recognition process, but a "mineral receptor" has never been identified. Recently, we found that resorptive behavior, as judged by the formation of ruffled borders and actin rings, occurs on ordinary tissue culture substrates if they are first coated with vitronectin. Similarly, vitronectin-coated substrates induce osteoclasts to secrete tartrate-resistant acid phosphatase and to form podosome belts, and to make resorption trails in the protein that coat the substrate. The same applies to bone mineral, which only induces resorptive behavior if coated with vitronectin. In contrast, fibronectin has none of these effects, despite inducing adhesion and spreading. It appears that osteoclasts recognize bone as the substrate appropriate for resorption through the high affinity of vitronectin-receptor ligands for bone mineral. PMID:22172032

  8. Comparison of the ability of chondroitin sulfate derived from bovine, fish and pigs to suppress human osteoclast activity in vitro.

    PubMed

    Cantley, M D; Rainsford, K D; Haynes, D R

    2013-12-01

    Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 μg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed. PMID:23644893

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-02-01

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

  11. Biphasic influence of PGE2 on the resorption activity of osteoclast-like cells derived from human peripheral blood monocytes and mouse RAW264.7 cells.

    PubMed

    Lutter, Anne-Helen; Hempel, Ute; Anderer, Ursula; Dieter, Peter

    2016-08-01

    Osteoclasts are large bone-resorbing cells of hematopoietic origin. Their main function is to dissolve the inorganic component hydroxyapatite and to degrade the organic bone matrix. Prostaglandin E2 (PGE2) indirectly affects osteoclasts by stimulating osteoblasts to release factors that influence osteoclast activity. The direct effect of PGE2 on osteoclasts is still controversial. To study the influence of PGE2 on osteoclast activity, human peripheral blood monocytes (hPBMC) and mouse RAW264.7 cells were cultured on osteoblast-derived extracellular matrix. hPBMC and RAW264.7 cells were differentiated by the addition of macrophage colony-stimulation factor and receptor activator of NFκB ligand and treated with PGE2 before and after differentiation induction. The pit area, an indicator of resorption activity, and the activity of tartrate-resistant acid phosphatase were dose-dependently inhibited when PGE2 was present ab initio, whereas the resorption activity remained unchanged when the cells were exposed to PGE2 from day 4 of culture. These results lead to the conclusion that PGE2 treatment inhibits only the differentiation of precursor osteoclasts whereas differentiated osteoclasts are not affected. PMID:27499447

  12. Osteoclast function and bone-resorbing activity: An overview.

    PubMed

    Soysa, Niroshani Surangika; Alles, Neil

    2016-07-29

    Bone resorption is an important cellular function in skeletal development and remodeling of the adult skeleton. Most of the pathological bone disease conditions like osteoporosis reflect increased osteoclast activity; hence, increased bone resorption. Researchers have unraveled most of the intracellular mechanisms responsible for osteoclast bone-resorbing activity in last few decades. Therefore, understanding the fundamentals of osteoclast-induced bone resorption and the cytokines and other substances that modulate the osteoclast activity unequivocally provide insights into the development of drugs to ameliorate pathological bone diseases with enhanced bone resorption. The aim of this review is to examine the literature on osteoclast function and bone-resorbing activity. PMID:27157135

  13. Nano-topography sensing by osteoclasts

    PubMed Central

    Geblinger, Dafna; Addadi, Lia; Geiger, Benjamin

    2010-01-01

    Bone resorption by osteoclasts depends on the assembly of a specialized, actin-rich adhesive ‘sealing zone’ that delimits the area designed for degradation. In this study, we show that the level of roughness of the underlying adhesive surface has a profound effect on the formation and stability of the sealing zone and the associated F-actin. As our primary model substrate, we use ‘smooth’ and ‘rough’ calcite crystals with average topography values of 12 nm and 530 nm, respectively. We show that the smooth surfaces induce the formation of small and unstable actin rings with a typical lifespan of ~8 minutes, whereas the sealing zones formed on the rough calcite surfaces are considerably larger, and remain stable for more than 6 hours. It was further observed that steps or sub-micrometer cracks on the smooth surface stimulate local ring formation, raising the possibility that similar imperfections on bone surfaces may stimulate local osteoclast resorptive activity. The mechanisms whereby the physical properties of the substrate influence osteoclast behavior and their involvement in osteoclast function are discussed. PMID:20375065

  14. Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone.

    PubMed

    Wan, Qilong; Schoenmaker, Ton; Jansen, Ineke D C; Bian, Zhuan; de Vries, Teun J; Everts, Vincent

    2016-05-01

    Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of

  15. Ficus carica L. (Moraceae): Phytochemistry, Traditional Uses and Biological Activities.

    PubMed

    Mawa, Shukranul; Husain, Khairana; Jantan, Ibrahim

    2013-01-01

    This paper describes the botanical features of Ficus carica L. (Moraceae), its wide variety of chemical constituents, its use in traditional medicine as remedies for many health problems, and its biological activities. The plant has been used traditionally to treat various ailments such as gastric problems, inflammation, and cancer. Phytochemical studies on the leaves and fruits of the plant have shown that they are rich in phenolics, organic acids, and volatile compounds. However, there is little information on the phytochemicals present in the stem and root. Reports on the biological activities of the plant are mainly on its crude extracts which have been proven to possess many biological activities. Some of the most interesting therapeutic effects include anticancer, hepatoprotective, hypoglycemic, hypolipidemic, and antimicrobial activities. Thus, studies related to identification of the bioactive compounds and correlating them to their biological activities are very useful for further research to explore the potential of F. carica as a source of therapeutic agents. PMID:24159359

  16. Ficus carica L. (Moraceae): Phytochemistry, Traditional Uses and Biological Activities

    PubMed Central

    Mawa, Shukranul; Husain, Khairana; Jantan, Ibrahim

    2013-01-01

    This paper describes the botanical features of Ficus carica L. (Moraceae), its wide variety of chemical constituents, its use in traditional medicine as remedies for many health problems, and its biological activities. The plant has been used traditionally to treat various ailments such as gastric problems, inflammation, and cancer. Phytochemical studies on the leaves and fruits of the plant have shown that they are rich in phenolics, organic acids, and volatile compounds. However, there is little information on the phytochemicals present in the stem and root. Reports on the biological activities of the plant are mainly on its crude extracts which have been proven to possess many biological activities. Some of the most interesting therapeutic effects include anticancer, hepatoprotective, hypoglycemic, hypolipidemic, and antimicrobial activities. Thus, studies related to identification of the bioactive compounds and correlating them to their biological activities are very useful for further research to explore the potential of F. carica as a source of therapeutic agents. PMID:24159359

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

    PubMed Central

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

    2015-01-01

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

  18. The role of microRNAs in osteoclasts and osteoporosis.

    PubMed

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

    2014-01-01

    Osteoclasts are the exclusive cells of bone resorption. Abnormally activating osteoclasts can lead to low bone mineral density, which will cause osteopenia, osteoporosis, and other bone disorders. To date, the mechanism of how osteoclast precursors differentiate into mature osteoclasts remains elusive. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in numerous cellular processes, including cell differentiation and apoptosis, by post-transcriptional regulation of genes. Recently, a number of studies have revealed that miRNAs participate in bone homeostasis, including osteoclastic bone resorption, which sheds light on the mechanisms underlying osteoclast differentiation. In this review, we highlight the miRNAs involved in regulating osteoclast differentiation and bone resorption, and their roles in osteoporosis. PMID:25692234

  19. The role of MicroRNAs in Osteoclasts and Osteoporosis

    PubMed Central

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

    2014-01-01

    Osteoclasts are the exclusive cells of bone resorption. Abnormally activating osteoclasts can lead to low bone mineral density, which will cause osteopenia, osteoporosis, and other bone disorders. To date, the mechanism of how osteoclast precursors differentiate into mature osteoclasts remains elusive. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in numerous cellular processes, including cell differentiation and apoptosis, by post-transcriptional regulation of genes. Recently, a number of studies have revealed that miRNAs participate in bone homeostasis, including osteoclastic bone resorption, which sheds light on the mechanisms underlying osteoclast differentiation. In this review, we highlight the miRNAs involved in regulating osteoclast differentiation and bone resorption, and their roles in osteoporosis. PMID:25692234

  20. The Multifaceted Osteoclast; Far and Beyond Bone Resorption.

    PubMed

    Drissi, Hicham; Sanjay, Archana

    2016-08-01

    The accepted function of the bone resorbing cell, osteoclast, has been linked to bone remodeling and pathological osteolysis. Emerging evidence points to novel functions of osteoclasts in controlling bone formation and angiogenesis. Thus, while the concept of a "clastokine" with the potential to regulate osteogenesis during remodeling did not come as a surprise, new evidence provided unique insight into the mechanisms underlying osteoclastic control of bone formation. The question still remains as to whether osteoclast precursors or a unique trap positive mononuclear cell, can govern any aspect of bone formation. The novel paradigm eloquently proposed by leaders in the field brings together the concept of clastokines and osteoclast precursor-mediated bone formation, potentially though enhanced angiogenesis. These fascinating advances in osteoclast biology have motivated this short review, in which we discuss these new roles of osteoclasts. J. Cell. Biochem. 117: 1753-1756, 2016. © 2016 Wiley Periodicals, Inc. PMID:27019318

  1. Osteoclastic giant cell tumor of the pancreas☆

    PubMed Central

    Temesgen, Wudneh M.; Wachtel, Mitchell; Dissanaike, Sharmila

    2014-01-01

    INTRODUCTION Pancreatic giant cell tumors are rare, with an incidence of less than 1% of all pancreatic tumors. Osteoclastic giant cell tumor (OGCT) of the pancreas is one of the three types of PGCT, which are now classified as undifferentiated carcinoma with osteoclast-like giant cells. PRESENTATION OF CASE The patient is a 57 year old woman who presented with a 3 week history of epigastric pain and a palpable abdominal mass. Imaging studies revealed an 18 cm × 15 cm soft tissue mass with cystic components which involved the pancreas, stomach and spleen. Exploratory laparotomy with distal pancreatectomy, partial gastrectomy and splenectomy was performed. Histology revealed undifferentiated pancreatic carcinoma with osteoclast-like giant cells with production of osteoid and glandular elements. DISCUSSION OGCT of the pancreas resembles benign-appearing giant cell tumors of bone, and contain osteoclastic-like multinucleated cells and mononuclear cells. OGCTs display a less aggressive course with slow metastasis and lymph node spread compared to pancreatic adenocarcinoma. Due to the rarity of the cancer, there is a lack of prospective studies on treatment options. Surgical en-bloc resection is currently considered first line treatment. The role of adjuvant therapy with radiotherapy or chemotherapy has not been established. CONCLUSION Pancreatic giant cell tumors are rare pancreatic neoplasms with unique clinical and pathological characteristics. Osteoclastic giant cell tumors are the most favorable sub-type. Surgical en bloc resection is the first line treatment. Long-term follow-up of patients with these tumors is essential to compile a body of literature to help guide treatment. PMID:24631915

  2. Integrin subunit beta3 plays a crucial role in the movement of osteoclasts from the periosteum to the bone surface.

    PubMed

    Holt, I; Marshall, M J

    1998-04-01

    We have shown that, when mouse parietal bones were incubated in culture medium containing indomethacin, the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP + OCs) on the bone surface was drastically reduced (down-regulation), and the number on the periosteal membrane adjacent to the resorbing surface was increased. Subsequent incubation of bones with prostaglandin E2 (PGE2) rapidly reversed these changes (up-regulation). In the work reported here, the osteoclast-associated integrin subunit beta3 was stained by immunohistochemistry. The beta3-positive osteoclast (beta3 + OC) population on freshly isolated bone was comprised of about 67% TRAP + OCs and 33% TRAP OCs. Like TRAP + OCs, beta3 + OCs were reduced in number on the surface of bones incubated with indomethacin, but, in contrast to the TRAP + OCs, beta3 + OCs were not seen on the periosteal membrane. Following up-regulation of TRAP + OCs with PGE2, large numbers of beta3 + OCs appeared on the bone surface and, again, were not seen on the periosteal membrane. Echistatin, a peptide that binds to the alphavbeta3 integrin on osteoclasts, was found to inhibit the up-regulation of TRAP + OCs in a dose-dependent manner but had no effect on the down-regulation of TRAP + OCs. Similarly, echistatin inhibited the upregulation of beta3 + OCs on the bone surface, and, under these conditions, beta3 + OCs were observed on the periosteal membrane. The addition of anti-beta3 antibody also inhibited the up-regulation of TRAP + OCs in response to PGE2. The association of beta3 protein expression with the up-regulated osteoclast and the inhibition of up-regulation by echistatin and by anti-beta3 antibody provide strong evidence that beta3 plays an essential role in the movement of osteoclasts from the membrane to the bone. PMID:9491775

  3. Calcium sensing and cell signaling processes in the local regulation of osteoclastic bone resorption.

    PubMed

    Zaidi, Mone; Moonga, Baljit S; Huang, Christopher L H

    2004-02-01

    The skeletal matrix in terrestrial vertebrates undergoes continual cycles of removal and replacement in the processes of bone growth, repair and remodeling. The osteoclast is uniquely important in bone resorption and thus is implicated in the pathogenesis of clinically important bone and joint diseases. Activated osteoclasts form a resorptive hemivacuole with the bone surface into which they release both acid and osteoclastic lysosomal hydrolases. This article reviews cell physiological studies of the local mechanisms that regulate the resorptive process. These used in vitro methods for the isolation, culture and direct study of the properties of neonatal rat osteoclasts. They demonstrated that both local microvascular agents and products of the bone resorptive process such as ambient Ca2+ could complement longer-range systemic regulatory mechanisms such as those that might be exerted through calcitonin (CT). Thus elevated extracellular [Ca2+], or applications of surrogate divalent cation agonists for Ca2+, inhibited bone resorptive activity and produced parallel increases in cytosolic [Ca2+], cell retraction and longer-term inhibition of enzyme release in isolated rat osteoclasts. These changes showed specificity, inactivation, and voltage-dependent properties that implicated a cell surface Ca2+ receptor (CaR) sensitive to millimolar extracellular [Ca2+]. Pharmacological, biophysical and immunochemical evidence implicated a ryanodine-receptor (RyR) type II isoform in this process and localized it to a unique, surface membrane site, with an outward-facing channel-forming domain. Such a surface RyR might function either directly or indirectly in the process of extracellular [Ca2+] sensing and in turn be modulated by cyclic adenosine diphosphate ribose (cADPr) produced by the ADP-ribosyl cyclase, CD38. The review finishes by speculating about possible detailed models for these transduction events and their possible interactions with other systemic mechanisms involved

  4. Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

    PubMed Central

    Tanaka, S; Takahashi, N; Udagawa, N; Tamura, T; Akatsu, T; Stanley, E R; Kurokawa, T; Suda, T

    1993-01-01

    The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts. Images PMID:8423223

  5. Effects of cyclic tension stress on the apoptosis of osteoclasts in vitro

    PubMed Central

    LI, FENGBO; SUN, XIAOLEI; ZHAO, BIN; MA, JIANXIONG; ZHANG, YANG; LI, SHUANG; LI, YANJUN; MA, XINLONG

    2015-01-01

    The aim of the present study was to investigate the effect of cyclic tension stress on osteoclast apoptosis in vitro using murine RAW264.7 cells treated with receptor activator of nuclear factor-κB. Using the EF3200 mechanical testing instrument with BioDynamic bioreactor system, cultured osteoclasts which were seeded in a silicone rubber membrane load carrier, were loaded with periodic cyclic stretch microstrain. The induced osteoclasts were subjected to 0, 5, 10 and 15% stretch microstrain for 1 h daily for three days. The number of tartrate-resistant acid phosphatase-positive osteoclasts and the resorption area were assessed. Osteoclast apoptosis was detected by the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide binding assay. The mRNA expression of Bcl-2, Bax, caspase-3 and cytochrome c was detected following force loading using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Compared with the cells under no cyclic tension stress, the number of osteoclasts and the resorption area were increased in the cells under 10 and 15% stretch microstrain. The Annexin V binding assay showed that the early apoptosis rate of the 5, 10 and 15% stretch microstrain groups was decreased compared with that of the control group. RT-qPCR results showed that the Bcl-2/Bax ratio was significantly increased in the cells subjected to 5, 10 and 15% stretch microstrain compared with that in the control cells (P<0.05), while the expression of cytochrome c in the 10 and 15% stretch microstrain groups was decreased significantly (P<0.05). No significant difference was observed between the cytochrome c expression of the 5% stretch microstrain group and that of the control group (P>0.05). The expression of caspase-3 in the 5, 10 and 15% stretch microstrain groups was decreased significantly compared with that in the control group (P<0.05). These data suggest that cyclic tension stress can inhibit apoptosis in osteoclasts, possibly by increasing

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    2008-12-19

    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.

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

    PubMed

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

    2016-02-12

    Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation. PMID:26670608

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

    SciTech Connect

    Lee, Sang-Hyun; Jang, Hae-Dong

    2015-02-15

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

  10. The Inhibitory Effects of Forsythia Koreana Extracts on the Metastatic Ability of Breast Cancer Cells and Bone Resorption by Osteoclasts

    PubMed Central

    Kim, Yu Li; Lee, Sun Kyoung; Park, Kwang-Kyun; Chung, Won-Yoon

    2016-01-01

    Background: Breast cancer is the most common malignant disease in women. The patients with advanced breast cancer develop metastasis to bone. Bone metastasis and skeletal-related events by breast cancer are frequently associated with the invasiveness of breast cancer cells and osteoclasts-mediated bone resorption. Forsythia koreana is used in oriental traditional medicine to treat asthma, atopy, and allergic diseases. The aim of this study was to evaluate the inhibitory effects of F. koreana extracts on the invasion of breast cancer cells and bone resorption by osteoclasts. Methods: Cell viability was measured by an MTT assay and the migration and invasion of MDA-MB-231 cells were detected by a Boyden chamber assay. The formation of osteoclasts and pit was detected using tartrate-resistant acid phosphatase staining and calcium phosphate-coated plates, respectively. The activities of matrix metalloproteinases (MMPs) and cathepsin K were evaluated by gelatin zymography and a cathepsin K detection kit. Results: The fruit and leaf extracts of F. koreana significantly inhibited the invasion of MDA-MB-231 cells at noncytotoxic concentrations. The fruit extract of F. koreana reduced the transforming growth factor β1-induced migration, invasion and MMPs activities of MDA-MB-231 cells. In addition, the fruit, branch, and leaf extracts of F. koreana also inhibited the receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and osteoclast-mediated bone-resorbing activity by reducing the activities of MMPs and cathepsin K. Conclusions: The extracts of F. koreana may possess the potential to inhibit the breast cancer-induced bone destruction through blocking invasion of breast cancer cells, osteoclastogenesis, and the activity of mature osteoclasts. PMID:27390737

  11. Dihydroartemisinin, an Anti-Malaria Drug, Suppresses Estrogen Deficiency-Induced Osteoporosis, Osteoclast Formation, and RANKL-Induced Signaling Pathways.

    PubMed

    Zhou, Lin; Liu, Qian; Yang, Mingli; Wang, Tao; Yao, Jun; Cheng, Jianwen; Yuan, Jinbo; Lin, Xixi; Zhao, Jinmin; Tickner, Jennifer; Xu, Jiake

    2016-05-01

    Osteoporosis is an osteolytic disease that features enhanced osteoclast formation and bone resorption. Identification of agents that can inhibit osteoclast formation and function is important for the treatment of osteoporosis. Dihydroartemisinin is a natural compound used to treat malaria but its role in osteoporosis is not known. Here, we found that dihydroartemisinin can suppress RANKL-induced osteoclastogenesis and bone resorption in a dose-dependent manner. Dihydroartemisinin inhibited the expression of osteoclast marker genes such as cathepsin K, calcitonin receptor, and tartrate-resistant acid phosphatase (TRAcP). Furthermore, dihydroartemisinin inhibited RANKL-induced NF-κB and NFAT activity. In addition, using an in vivo ovariectomized mouse model, we show that dihydroartemisinin is able to reverse the bone loss caused by ovariectomy. Together, this study shows that dihydroartemisinin attenuates bone loss in ovariectomized mice through inhibiting RANKL-induced osteoclast formation and function. This indicates that dihydroartemisinin, the first physiology or medicine nobel prize discovery of China, is a potential treatment option against osteolytic bone disease. © 2015 American Society for Bone and Mineral Research. PMID:26684711

  12. THE BISPHOSPHONATE ZOLEDRONIC ACID DECREASES TUMOR GROWTH IN BONE IN MICE WITH DEFECTIVE OSTEOCLASTS*

    PubMed Central

    Hirbe, Angela C.; Roelofs, Anke J.; Floyd, Desiree H.; Deng, Hongju; Becker, Stephanie N.; Lanigan, Lisa G.; Apicelli, Anthony J.; Xu, Zhiqiang; Prior, Julie L.; Eagleton, Mark C.; Piwnica-Worms, David; Rogers, Michael J.; Weilbaecher, Katherine

    2009-01-01

    Bisphosphonates (BPs), bone targeted drugs that disrupt osteoclast function, are routinely used to treat complications of bone metastasis. Studies in preclinical models of cancer have shown that BPs reduce skeletal tumor burden and increase survival. Similarly, we observed in the present study that administration of the Nitrogen-containing BP (N-BP), zoledronic acid (ZA) to osteolytic tumor-bearing Tax+ mice beginning at 6 months of age led to resolution of radiographic skeletal lesions. N-BPs inhibit farnesyl diphosphate (FPP) synthase, thereby inhibiting protein prenylation and causing cellular toxicity. We found that ZA decreased Tax+ tumor and B16 melanoma viability and caused the accumulation of unprenylated Rap1a proteins in vitro. However, it is presently unclear whether N-BPs exert anti-tumor effects in bone independent of inhibition of osteoclast (OC) function in vivo. Therefore, we evaluated the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src-/- mice, which have non-functioning OCs. OC-defective mice treated with ZA demonstrated a significant 88% decrease in tumor growth in bone compared to vehicle-treated OC-defective mice. These data support an osteoclast-independent role for N-BP therapy in bone metastasis. PMID:19442620

  13. CCL2 and CCR2 are Essential for the Formation of Osteoclasts and Foreign Body Giant Cells.

    PubMed

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

    2016-02-01

    Osteoclasts are multinucleated cells responsible for bone resorption. They are derived from the fusion of cells in the monocyte/macrophage lineage. Monocytes and macrophages can also fuse to form foreign body giant cells (FBGC). Foreign body giant cells are observed at the interface between a host and a foreign body such as implants during a foreign body reaction. Macrophages are attracted to the site of bone resorption and foreign body reactions by different cytokines. Chemokine (C-C) ligand-2 (CCL2) is an important chemotactic factor and binds to a receptor CCR2. In this study we investigated the importance of CCL2 and the receptor CCR2 in the formation of osteoclasts and FBGC. CCL2 mRNA was more highly expressed in giant cell culture than macrophages, being 9-fold and 16-fold more abundant in osteoclasts and FBGC respectively. Significantly fewer osteoclasts and FBGC were cultured from the bone marrow of CCL2 and CCR2 knockout mice, when compared to wild type. Not only were the number of giant cells reduced but there was a significant reduction in the number of nuclei and the size of these cells in the cultures of CCL2 and CCR2 knockout mice. Formation of osteoclasts and FBGC were recovered in cultures by addition of exogenous CCL2 to the media containing marrow cells from CCL2-/- mice. We conclude that CCL2 and its receptor CCR2 are important for the formation of osteoclasts and FBGC and absence of these genes causes inhibition of osteoclast and FBGC formation. PMID:26205994

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

    SciTech Connect

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

    2012-01-06

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

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

    PubMed Central

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

    2016-01-01

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

  16. Two nematicidal furocoumarins from Ficus carica L. leaves and their physiological effects on pine wood nematode (Bursaphelenchus xylophilus).

    PubMed

    Guo, Qunqun; Du, Guicai; He, Hongwei; Xu, Hongkai; Guo, Daosen; Li, Ronggui

    2016-09-01

    The ethanol extract of the Ficus carica L. leaves was tested to show strong nematicidal activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, causing 90.93% corrected mortality within 72 h at 1.0 mg/mL. From the ethyl acetate soluble fraction of the F. carica L. leaves extract, the main nematicidal constituents were obtained by bioassay-guided isolation and identified as linear furocoumarins bergapten (1) and psoralen (2) by mass and NMR spectral data analysis. Bergapten and psoralen had significant nematicidal activity against PWN with the LC50 values of 97.08 aKSnd 115.03  μ g/mL within 72 h, respectively. The two furocoumarins could inhibit the activities of amylase, cellulase and acetylcholinesterase (AchE) from PWN. The morphologies of PWNs changed much after they were treated by bergapten and psoralen. The physiological effects of bergapten and psoralen on PWN might provide helpful clues to elucidate their nematicidal mechanisms. PMID:26479900

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

    PubMed

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

    2012-04-01

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

  18. Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts*

    PubMed Central

    Nagase, Yuichi; Iwasawa, Mitsuyasu; Akiyama, Toru; Kadono, Yuho; Nakamura, Masaki; Oshima, Yasushi; Yasui, Tetsuro; Matsumoto, Takumi; Hirose, Jun; Nakamura, Hiroaki; Miyamoto, Takeshi; Bouillet, Philippe; Nakamura, Kozo; Tanaka, Sakae

    2009-01-01

    The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2−/− mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2−/− mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2−/− mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2−/−Bim+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2+/−Bim+/− littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2−/− mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts. PMID:19846553

  19. Osteoclast differentiation and function in aquaglyceroporin AQP9 null mice

    PubMed Central

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

    2008-01-01

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

  20. Supplementation of broccoli or Bifidobacterium longum-fermented broccoli suppresses serum lipid peroxidation and osteoclast differentiation on alveolar bone surface in rats fed a high-cholesterol diet.

    PubMed

    Tomofuji, Takaaki; Ekuni, Daisuke; Azuma, Tetsuji; Irie, Koichiro; Endo, Yasumasa; Yamamoto, Tatsuo; Ishikado, Atsushi; Sato, Takehiko; Harada, Kayo; Suido, Hirohisa; Morita, Manabu

    2012-04-01

    High-cholesterol diet enhances osteoclastic activity on alveolar bone by increasing serum lipid peroxidation. We hypothesized that supplementation with dietary antioxidants, such as found in broccoli and its fermented products, might suppress increases in serum lipid peroxidation, contributing to the inhibition of osteoclastic activity after high-cholesterol diet intake. The purpose of the present study was to investigate the effects of broccoli and fermented broccoli consumption on serum lipid peroxidation and osteoclast differentiation in alveolar bone of rats fed a high-cholesterol diet. In this 12-week study, rats were divided into 4 groups (n = 6/group): a control group (fed regular diet) and 3 experimental groups (fed a high-cholesterol [1% wt/wt] diet, or a high-cholesterol diet supplemented with either broccoli powder [5% wt/wt] or Bifidobacterium longum-fermented broccoli powder [5% wt/wt]). Serum hexanoyl-lysine (HEL) levels were measured as a parameter of lipid peroxidation. The number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in alveolar bone was enumerated to evaluate osteoclast differentiation. When compared with regular diet, the high-cholesterol diet increased serum HEL levels and resulted in a higher number of TRAP-positive osteoclasts at 12 weeks. The high-cholesterol diet supplemented with broccoli or B. longum-fermented broccoli showed lower levels of serum HEL and fewer TRAP-positive osteoclasts than the high-cholesterol diet at 12 weeks. In conclusion, consumption of broccoli, or its fermented product, inhibited the effects of a high-cholesterol diet on osteoclast differentiation in rat alveolar bone by suppressing serum lipid peroxidation. PMID:22575044

  1. Nicotine Affects Bone Resorption and Suppresses the Expression of Cathepsin K, MMP-9 and Vacuolar-Type H+-ATPase d2 and Actin Organization in Osteoclasts

    PubMed Central

    Tanaka, Hideki; Tanabe, Natsuko; Kawato, Takayuki; Nakai, Kumiko; Kariya, Taro; Matsumoto, Sakurako; Zhao, Ning; Motohashi, Masafumi; Maeno, Masao

    2013-01-01

    Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization. PMID

  2. Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

    PubMed

    Benucci, Ilaria; Esti, Marco; Liburdi, Katia

    2015-01-01

    The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level. PMID:25376439

  3. Antiosteoporotic activity of anthraquinones from Morinda officinalis on osteoblasts and osteoclasts.

    PubMed

    Wu, Yan-Bin; Zheng, Cheng-Jian; Qin, Lu-Ping; Sun, Lian-Na; Han, Ting; Jiao, Lei; Zhang, Qiao-Yan; Wu, Jin-Zhong

    2009-01-01

    Bioactivity-guided fractionation led to the successful isolation of antiosteoporotic components, i.e. physicion (1), rubiadin-1-methyl ether (2), 2-hydroxy-1-methoxy- anthraquinone (3), 1,2-dihydroxy-3-methylanthraquinone (4), 1,3,8-trihydroxy-2-methoxy- anthraquinone (5), 2-hydroxymethyl-3-hydroxyanthraquinone (6), 2-methoxyanthraquinone (7) and scopoletin (8) from an ethanolic extract of the roots of Morinda officinalis. Compounds 4-8 are isolated for the first time from M. officinalis. Among them, compounds 2 and 3 promoted osteoblast proliferation, while compounds 4, 5 increased osteoblast ALP activity. All of the isolated compounds inhibited osteoclast TRAP activity and bone resorption, and the inhibitory effects on osteoclastic bone resorption of compounds 1 and 5 were stronger than that of other compounds. Taken together, antiosteoporotic activity of M. officinalis and its anthraquinones suggest therapeutic potential against osteoporosis. PMID:19169204

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

    PubMed Central

    Athanasou, N A; Quinn, J

    1990-01-01

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

  5. Dengue fever treatment with Carica papaya leaves extracts

    PubMed Central

    Ahmad, Nisar; Fazal, Hina; Ayaz, Muhammad; Abbasi, Bilal Haider; Mohammad, Ijaz; Fazal, Lubna

    2011-01-01

    The main objective of the current study is to investigate the potential of Carica papaya leaves extracts against Dengue fever in 45 year old patient bitten by carrier mosquitoes. For the treatment of Dengue fever the extract was prepared in water. 25 mL of aqueous extract of C. papaya leaves was administered to patient infected with Dengue fever twice daily i.e. morning and evening for five consecutive days. Before the extract administration the blood samples from patient were analyzed. Platelets count (PLT), White Blood Cells (WBC) and Neutrophils (NEUT) decreased from 176×103/µL, 8.10×103/µL, 84.0% to 55×103/µL, 3.7×103/µL and 46.0%. Subsequently, the blood samples were rechecked after the administration of leaves extract. It was observed that the PLT count increased from 55×103/µL to 168×103/µL, WBC from 3.7×103/µL to 7.7×103/µL and NEUT from 46.0% to 78.3%. From the patient feelings and blood reports it showed that Carica papaya leaves aqueous extract exhibited potential activity against Dengue fever. Furthermore, the different parts of this valuable specie can be further used as a strong natural candidate against viral diseases. PMID:23569787

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

    PubMed Central

    ARAÚJO, Adriele Silveira; FERNANDES, Alline Birra Nolasco; MACIEL, José Vinicius Bolognesi; NETTO, Juliana de Noronha Santos; BOLOGNESE, Ana Maria

    2015-01-01

    Orthodontic tooth movement (OTM) is a dynamic process of bone modeling involving osteoclast-driven resorption on the compression side. Consequently, to estimate the influence of various situations on tooth movement, experimental studies need to analyze this cell. Objectives The aim of this study was to test and validate a new method for evaluating osteoclastic activity stimulated by mechanical loading based on the fractal analysis of the periodontal ligament (PDL)-bone interface. Material and Methods The mandibular right first molars of 14 rabbits were tipped mesially by a coil spring exerting a constant force of 85 cN. To evaluate the actual influence of osteoclasts on fractal dimension of bone surface, alendronate (3 mg/Kg) was injected weekly in seven of those rabbits. After 21 days, the animals were killed and their jaws were processed for histological evaluation. Osteoclast counts and fractal analysis (by the box counting method) of the PDL-bone interface were performed in histological sections of the right and left sides of the mandible. Results An increase in the number of osteoclasts and in fractal dimension after OTM only happened when alendronate was not administered. Strong correlation was found between the number of osteoclasts and fractal dimension. Conclusions Our results suggest that osteoclastic activity leads to an increase in bone surface irregularity, which can be quantified by its fractal dimension. This makes fractal analysis by the box counting method a potential tool for the assessment of osteoclastic activity on bone surfaces in microscopic examination. PMID:25760264

  7. Reprint of: The Great Beauty of the osteoclast.

    PubMed

    Cappariello, Alfredo; Maurizi, Antonio; Veeriah, Vimal; Teti, Anna

    2014-11-01

    Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an "orchestrator" implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the '00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting "clastokines" that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast. PMID:25282390

  8. Regulation of osteoclast structure and function by FAK family kinases

    PubMed Central

    Ray, Brianne J.; Thomas, Keena; Huang, Cynthia S.; Gutknecht, Michael F.; Botchwey, Edward A.; Bouton, Amy H.

    2012-01-01

    Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAKΔmyeloid), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAKΔmyeloid mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells. PMID:22941736

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

    SciTech Connect

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

    2009-10-21

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

  10. RNA synthesis in isolated rat osteoclasts: inhibitory effect of calcitonin.

    PubMed

    Zheng, M H; Papadimitriou, J M; Nicholson, G C

    1991-01-01

    The metabolism of RNA has not been studied in the osteoclast (OC) because these bone-resorbing cells are only available in small numbers and cultures are always contaminated with other cells. Using two single-cell assay techniques, tritiated uridine (3H-UdR) autoradiography and gallocyanin quantitative cytophotometry, we have examined RNA synthesis in OCs isolated from neonatal rats. Oligo-nuclear OCs showed greater nuclear uptake of 3H-UdR than cells with many nuclei, and the variance of nuclear labeling within polykarya was greater in the latter, possibly because they contain nuclei of various ages. Salmon calcitonin (sCT) was a potent (ED50 approximately 5 x 10(-12) M) and rapid (40% reduction in 2 h, 75% reduction in 6 h) inhibitor of 3H-UdR uptake, and also reduced cytochemical total cellular RNA by 22% within 4 h. Forskolin (10(-5) M) inhibited nuclear uptake of 3H-UdR, suggesting that the sCT response may be mediated by cyclic AMP. Following a short (30 min) exposure to sCT, there was a progressive decline in labeling, followed by complete recovery by 4.5 h, a response possibly related to the phenomenon of calcitonin-induced persistent activation of adenylate cyclase. Inhibition of OC RNA synthesis may be an important component of its anti-resorptive action. PMID:1723609