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1
Combs, Charlotte E., Karen Fuller, Hashethra Kumar, Anthony P. Albert, Grisha Pirianov, James McCormick, Ian C. Locke, Timothy J. Chambers und Kevin M. Lawrence. „Urocortin is a novel regulator of osteoclast differentiation and function through inhibition of a canonical transient receptor potential 1-like cation channel“. Journal of Endocrinology 212, Nr. 2 (14.11.2011): 187–97. http://dx.doi.org/10.1530/joe-11-0254.
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This study investigated the role of urocortin (UCN), a member of the corticotrophin-releasing factor (CRF) family of peptides, in osteoclast maturation and function. We found that 10−7 M UCN significantly (P<0.05) suppressed osteoclast differentiation from bone marrow precursor cells in culture and reduced the expression of several osteoclastic markers. Furthermore, UCN potently suppressed osteoclast bone resorption, by significantly inhibiting both the plan area of bone resorbed by osteoclasts and actin ring formation within osteoclasts at 10−9 M (P<0.05), with complete inhibition at 10−7 M (P<0.001). UCN also inhibited osteoclast motility (10−7 M) but had no effect on osteoclast survival. Osteoclasts expressed mRNA encoding both UCN and the CRF receptor 2β subtype. Pre-osteoclasts however, expressed CRF receptor 2β alone. Unstimulated osteoclasts contained constitutively active cation channel currents with a unitary conductance of 3–4 pS, which were inhibited by over 70% with UCN (10−7 M). Compounds that regulate calcium signalling and energy status of the cell, both crucial for osteoclast activity were investigated. The non-selective cation channel blockers, lanthanum (La3+) and gadolinium (Gd3+), inhibited actin ring formation in osteoclasts, whereas modulators of voltage-dependent Ca2+ channels and KATP channels had no effect. These findings show for the first time that UCN is a novel anti-resorptive molecule that acts through a direct effect on osteoclasts and their precursor cells.
2
Alatalo, Sari L., Jussi M. Halleen, Teuvo A. Hentunen, Jukka Mönkkönen und H. Kalervo Väänänen. „Rapid Screening Method for Osteoclast Differentiation in Vitro That Measures Tartrate-resistant Acid Phosphatase 5b Activity Secreted into the Culture Medium“. Clinical Chemistry 46, Nr. 11 (01.11.2000): 1751–54. http://dx.doi.org/10.1093/clinchem/46.11.1751.
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Abstract Background: Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay. Methods: We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3–7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium. Results: TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed (r = 0.94; P <0.0001; n = 120). Conclusions: The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.
3
Niida, Shumpei, Masato Kaku, Hitoshi Amano, Hisahiro Yoshida, Hiroshi Kataoka, Satomi Nishikawa, Kazuo Tanne, Norihiko Maeda, Shin-Ichi Nishikawa und Hiroaki Kodama. „Vascular Endothelial Growth Factor Can Substitute for Macrophage Colony-Stimulating Factor in the Support of Osteoclastic Bone Resorption“. Journal of Experimental Medicine 190, Nr. 2 (19.07.1999): 293–98. http://dx.doi.org/10.1084/jem.190.2.293.
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We demonstrated previously that a single injection of recombinant human macrophage colony-stimulating factor (rhM-CSF) is sufficient for osteoclast recruitment and survival in osteopetrotic (op/op) mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene. In this study, we show that a single injection of recombinant human vascular endothelial growth factor (rhVEGF) can similarly induce osteoclast recruitment in op/op mice. Osteoclasts predominantly expressed VEGF receptor 1 (VEGFR-1), and activity of recombinant human placenta growth factor 1 on osteoclast recruitment was comparable to that of rhVEGF, showing that the VEGF signal is mediated through VEGFR-1. The rhM-CSF–induced osteoclasts died after injections of VEGFR-1/Fc chimeric protein, and its effect was abrogated by concomitant injections of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF showed no significant difference in the bone-resorbing activity. op/op mice undergo an age-related resolution of osteopetrosis accompanied by an increase in osteoclast number. Most of the osteoclasts disappeared after injections of anti-VEGF antibody, demonstrating that endogenously produced VEGF is responsible for the appearance of osteoclasts in the mutant mice. In addition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclast differentiation. These results demonstrate that M-CSF and VEGF have overlapping functions in the support of osteoclastic bone resorption.
4
Fuller, K., und T. J. Chambers. „Localisation of mRNA for collagenase in osteocytic, bone surface and chondrocytic cells but not osteoclasts“. Journal of Cell Science 108, Nr. 6 (01.06.1995): 2221–30. http://dx.doi.org/10.1242/jcs.108.6.2221.
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Osteoclasts resorb the extracellular matrix of bone by secreting protons and enzymes into a circumpherentially sealed compartment between the osteoclast and the bone surface. Although the lysosomal cysteine proteinases play a major role in matrix degradation by osteoclasts, collagenase (matrix metalloproteinase-1, EC 3.4.24.7) is also required for osteoclastic bone resorption, and may be directly involved in collagen degradation in the hemivacuole. We assessed the effects of inhibitors of cysteine proteinases and collagenase on bone resorption by osteoclasts isolated from rodent bone. We found that while inhibition of cysteine proteinases strongly suppressed osteoclastic resorption, inhibitors of collagenase were without effect on the number, size, or demineralised fringe of excavations. We could find no evidence of expression of mRNA for collagenase in rat osteoclasts by in situ hybridisation, but found that it was expressed by chondrocytes, bone surface cells and osteocytes adjacent to osteoclasts. The distribution of these cells, and the correlation between increased collagenase production and increased stimulation of osteoclastic resorption in vitro by bone cells, suggests that these cells might be involved in the regulation of bone resorption in situ, and that collagenase production might play a role in this process.
5
Fuller, Karen, Brian Wong, Simon Fox, Yongwon Choi und Tim J. Chambers. „TRANCE Is Necessary and Sufficient for Osteoblast-mediated Activation of Bone Resorption in Osteoclasts“. Journal of Experimental Medicine 188, Nr. 5 (07.09.1998): 997–1001. http://dx.doi.org/10.1084/jem.188.5.997.
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TRANCE (tumor necrosis factor–related activation-induced cytokine) is a recently described member of the tumor necrosis factor superfamily that stimulates dendritic cell survival and has also been found to induce osteoclastic differentiation from hemopoietic precursors. However, its effects on mature osteoclasts have not been defined. It has long been recognized that stimulation of osteoclasts by agents such as parathyroid hormone (PTH) occurs through a hormonal interaction with osteoblastic cells, which are thereby induced to activate osteoclasts. To determine whether TRANCE accounts for this activity, we tested its effects on mature osteoclasts. TRANCE rapidly induced a dramatic change in osteoclast motility and spreading and inhibited apoptosis. In populations of osteoclasts that were unresponsive to PTH, TRANCE caused activation of bone resorption equivalent to that induced by PTH in the presence of osteoblastic cells. Moreover, osteoblast-mediated stimulation of bone resorption was abrogated by soluble TRANCE receptor and by the soluble decoy receptor osteoprotegerin (OPG), and stimulation of isolated osteoclasts by TRANCE was neutralized by OPG. Thus, TRANCE expression by osteoblasts appears to be both necessary and sufficient for hormone-mediated activation of mature osteoclasts, and TRANCE-R is likely to be a receptor for signal transduction for activation of the osteoclast and its survival.
6
Fuller, Karen, Chiho Murphy, Barrie Kirstein, Simon W. Fox und Timothy J. Chambers. „TNFα Potently Activates Osteoclasts, through a Direct Action Independent of and Strongly Synergistic with RANKL“. Endocrinology 143, Nr. 3 (01.03.2002): 1108–18. http://dx.doi.org/10.1210/endo.143.3.8701.
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Abstract TNFα is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFα promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFα promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats. TNFα was equipotent with RANKL for this action. Activation by TNFα was unaffected by blockade of RANKL by OPG, its soluble decoy receptor, suggesting that this was due to a direct action on osteoclasts. Bone resorption was similarly directly and potently stimulated, in a RANKL-independent manner in osteoclasts, whether these were formed in vitro or in vivo. Interestingly, TNFα promoted actin ring formation at concentrations an order of magnitude below those required for osteoclastic differentiation. Moreover, TNFα strongly synergized with RANKL, such that miniscule concentrations of TNFα were sufficient to substantially augment osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNFα suggests that the most sensitive osteolytic response of bone to TNFα is through activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism whereby increased osteolysis can be achieved without disturbance to the underlying pattern of osteoclastic localization.
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Yu, Anna Xiao-Dan, Jian Xiao, Shi-Zheng Zhao, Xiang-Peng Kong, Kenneth Kin-Leung Kwan, Brody Zhong-Yu Zheng, Kevin Qi-Yun Wu, Tina Ting-Xia Dong und Karl Wah-Keung Tsim. „Biological Evaluation and Transcriptomic Analysis of Corylin as an Inhibitor of Osteoclast Differentiation“. International Journal of Molecular Sciences 22, Nr. 7 (29.03.2021): 3540. http://dx.doi.org/10.3390/ijms22073540.
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Corylin, a flavonoid isolated from the fruit of Psoralea corylifolia, has an osteogenic effect on osteoblasts in vitro and bone micromass ex vivo. However, the effect and mechanism of corylin in regulating osteoclastogenesis remain unknown. By using murine bone marrow macrophages as the osteoclast precursor, corylin was found to inhibit the receptor activator of nuclear factor (NF) κB ligand (RANKL)-induced osteoclast differentiation via down-regulating osteoclastic marker genes. In parallel, F-actin formation and osteoclast migration were diminished in corylin-treated cultured osteoclasts, and subsequently the expressions of osteoclastic proteins were suppressed: the suppression of protein expression was further illustrated by transcriptomic analysis. Furthermore, corylin inhibited the nuclear translocation of p65, giving rise to a restraint in osteoclastic differentiation through the attenuation of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells c1 (NFATc1). There was no obvious change in apoptosis when the RANKL-induce osteoclasts were cultured in the presence of corylin. The finding supports the potential development of corylin as an osteoclast inhibitor against osteoporosis.
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Nakamura, I., M. F. Pilkington, P. T. Lakkakorpi, L. Lipfert, S. M. Sims, S. J. Dixon, G. A. Rodan und L. T. Duong. „Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone“. Journal of Cell Science 112, Nr. 22 (15.11.1999): 3985–93. http://dx.doi.org/10.1242/jcs.112.22.3985.
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The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50)= 0.1 nM) than those required to detach osteoclasts from bone (IC(50) approximately 1 microM); (2) echistatin (IC(50)= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50)= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50)= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).
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Kameda, Takashi, Hiroshi Mano, Tatsuhisa Yuasa, Yoshihisa Mori, Koshi Miyazawa, Miho Shiokawa, Yukiya Nakamaru et al. „Estrogen Inhibits Bone Resorption by Directly Inducing Apoptosis of the Bone-resorbing Osteoclasts“. Journal of Experimental Medicine 186, Nr. 4 (18.08.1997): 489–95. http://dx.doi.org/10.1084/jem.186.4.489.
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Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 β-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor– mediated mechanism.
10
Fuller, K., J. M. Owens und T. J. Chambers. „Macrophage inflammatory protein-1 alpha and IL-8 stimulate the motility but suppress the resorption of isolated rat osteoclasts.“ Journal of Immunology 154, Nr. 11 (01.06.1995): 6065–72. http://dx.doi.org/10.4049/jimmunol.154.11.6065.
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Abstract Cells of the osteoblastic lineage play a major role in the regulation of osteoclastic bone resorption. Recent studies have demonstrated production of chemokines by osteoblastic cells. Although these phagocyte-stimulating and proinflammatory cytokines act as chemoattractants and activators for other members of the hemopoietic lineage, their actions on osteoclasts have not been characterized. We found that macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-8 inhibited bone resorption by rat osteoclasts, primarily through reduction in the proportion of osteoclasts resorbing bone, a pattern of inhibition previously observed in response to macrophage CSF (M-CSF). MIP-2, RANTES, MIP-1 beta, and monocyte chemotactic protein-1 were without effect on resorption. MIP-1 alpha and IL-8, but not the other chemokines, also stimulated osteoclastic motility and increased the osteoclast spread area in a dose-dependent manner, over the same concentration range as that which inhibited bone resorption. In addition, MIP-1 alpha induced osteoclast orientation in a gradient of the chemokine, and stimulated osteoclast migration. We detected no effect of chemokines on osteoclast formation or survival. Our data suggest that chemokines can promote osteoclast orientation and migration, processes that might be involved in chemotaxis; it seems appropriate that resorptive functions should be suppressed during migration. Because chemokines are proinflammatory, their actions on osteoclasts might represent mechanisms by which bone resorption is modulated by the inflammatory process when this occurs in bone. However, given that chemokines are increasingly recognized to be multifunctional and that they are produced by cells of the osteoblastic lineage, they may also be components of the physiologic regulation of bone resorption.
11
Perkins, S. L., und S. J. Kling. „Local concentrations of macrophage colony-stimulating factor mediate osteoclastic differentiation“. American Journal of Physiology-Endocrinology and Metabolism 269, Nr. 6 (01.12.1995): E1024—E1030. http://dx.doi.org/10.1152/ajpendo.1995.269.6.e1024.
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Macrophage colony-stimulating factor (M-CSF) is essential for differentiation of osteoclasts and macrophages from a common bone marrow precursor. Using ST-2 stromal cell/murine bone marrow coculture, we studied the effects of increasing amounts of M-CSF on differentiation of macrophages and osteoclasts. Addition of exogenous M-CSF caused a dose-dependent 98% decrease in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a 2.5-fold increase in nonspecific esterase-staining macrophages. Similar decrease in osteoclastic functional activity, including 125I-labeled calcitonin binding and calcitonin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, were observed. Addition of exogenous M-CSF beyond 6 days in coculture had a decreasing ability to inhibit osteoclast formation, suggesting that M-CSF exerts its effects early in osteoclast differentiation, during the proposed proliferative phase of osteoclast formation. Similarly, early addition of neutralizing anti-M-CSF inhibited osteoclast formation, with diminishing effects beyond day 9. These results suggest that local high concentrations of M-CSF may influence the early determination of terminal differentiation into either macrophages or osteoclasts.
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Lerner, Ulf H. „New Molecules in the Tumor Necrosis Factor Ligand and Receptor Superfamilies with Importance for Physiological and Pathological Bone Resorption“. Critical Reviews in Oral Biology & Medicine 15, Nr. 2 (März 2004): 64–81. http://dx.doi.org/10.1177/154411130401500202.
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Osteoclasts are tissue-specific polykaryon bone-resorbing cells derived from the monocyte/macrophage hematopoietic lineage with specialized functions required for the adhesion of the cells to bone and the subsequent polarization of the cell membrane, secretion of acid to dissolve mineral crystals, and release of proteolytic enzymes to degrade the extracellular matrix proteins. Most pathological conditions in the skeleton lead to loss of bone due to excess osteoclastic bone resorption, including periodontal disease, rheumatoid arthritis, and osteoporosis. In rare cases, most of them genetic, patients with osteopetrosis exhibit sclerotic bone due either to a lack of osteoclasts or to non-functional osteoclasts. Mainly because of phenotypic findings in genetically manipulated mice or due to spontaneous mutations in humans, mice, and rats, several genes have been discovered as being crucial for osteoclast formation and activation. Recent breakthroughs in our understanding of osteoclast biology have revealed the critical roles in osteoclast differentiation played by RANKL, RANK, and OPG, three novel members of the tumor necrosis factor ligand and receptor superfamilies. The further study of these molecules and downstream signaling events are likely to provide a molecular basis for the development of new drugs for the treatment of diseases with excess or deficient osteoclastic bone resorption.
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Fuller, K., J. M. Owens, C. J. Jagger, A. Wilson, R. Moss und T. J. Chambers. „Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.“ Journal of Experimental Medicine 178, Nr. 5 (01.11.1993): 1733–44. http://dx.doi.org/10.1084/jem.178.5.1733.
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Macrophage colony-stimulating factor (M-CSF) is known to play an important role in osteoclast formation. However, its actions on mature cells have not been fully characterized. We now report that M-CSF dramatically stimulates osteoclastic motility and spreading; osteoclasts responded to a gradient of M-CSF with orientation, and random cell polarization occurred after isotropic exposure. M-CSF also supported the survival of osteoclasts by preventing apoptosis. Paradoxically, M-CSF inhibits bone resorption by isolated osteoclasts. We found that this was effected predominantly by reduction in the number of excavations. Thus, M-CSF showed a propensity to suppress resorption through a reduction in the proportion of cells that were resorbing bone. Our data suggest that apart from the established role of M-CSF in the provision of precursors for osteoclastic induction, a major role for M-CSF in bone resorption is to enhance osteoclastic survival, migration, and chemotaxis. It seems appropriate that during these processes resorptive functions should be suppressed. We suggest that M-CSF continues to modulate osteoclastic activity once osteoclasts are on resorptive sites, through regulation of the balance between resorption and migration, such that not only the quantity, but the spatial pattern of resorption can be controlled by adjacent M-CSF-secreting cells of osteoblastic lineage.
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Andersson, G. N., und S. C. Marks. „Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts.“ Journal of Histochemistry & Cytochemistry 37, Nr. 1 (Januar 1989): 115–17. http://dx.doi.org/10.1177/37.1.2461980.
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We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.
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Heinemann, Christiane, Josephine Adam, Benjamin Kruppke, Vera Hintze, Hans-Peter Wiesmann und Thomas Hanke. „How to Get Them off?—Assessment of Innovative Techniques for Generation and Detachment of Mature Osteoclasts for Biomaterial Resorption Studies“. International Journal of Molecular Sciences 22, Nr. 3 (29.01.2021): 1329. http://dx.doi.org/10.3390/ijms22031329.
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The fusion process of mononuclear monocytes into multinuclear osteoclasts in vitro is an essential process for the study of osteoclastic resorption of biomaterials. Thereby biomaterials offer many influencing factors such as sample shape, material composition, and surface topography, which can have a decisive influence on the fusion and thus the entire investigation. For the specific investigation of resorption, it can therefore be advantageous to skip the fusion on samples and use mature, predifferentiated osteoclasts directly. However, most conventional detachment methods (cell scraper, accutase), lead to a poor survival rate of osteoclasts or to a loss of function of the cells after their reseeding. In the present study different conventional and novel methods of detachment in combination with different culture surfaces were investigated to obtain optimal osteoclast differentiation, yield, and vitality rates without loss of function. The innovative method—using thermoresponsive surfaces for cultivation and detachment—was found to be best suited. This is in particular due to its ability to maintain osteoclast activity, as proven by TRAP 5b-, CTSK-activity and resorption pits on dentin discs and decellularized osteoblast-derived matrix plates. In conclusion, it is shown, that osteoclasts can be predifferentiated on cell culture dishes and transferred to a reference biomaterial under preservation of osteoclastic resorption activity, providing biomaterial researchers with a novel tool for material characterization.
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Møller, Anaïs M. J., Jean-Marie Delaissé, Jacob B. Olesen, Luisa M. Canto, Silvia R. Rogatto, Jonna S. Madsen und Kent Søe. „Fusion Potential of Human Osteoclasts In Vitro Reflects Age, Menopause, and In Vivo Bone Resorption Levels of Their Donors—A Possible Involvement of DC-STAMP“. International Journal of Molecular Sciences 21, Nr. 17 (02.09.2020): 6368. http://dx.doi.org/10.3390/ijms21176368.
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It is well established that multinucleation is central for osteoclastic bone resorption. However, our knowledge on the mechanisms regulating how many nuclei an osteoclast will have is limited. The objective of this study was to investigate donor-related variations in the fusion potential of in vitro-generated osteoclasts. Therefore, CD14+ monocytes were isolated from 49 healthy female donors. Donor demographics were compared to the in vivo bone biomarker levels and their monocytes’ ability to differentiate into osteoclasts, showing that: (1) C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels increase with age, (2) the number of nuclei per osteoclast in vitro increases with age, and (3) there is a positive correlation between the number of nuclei per osteoclast in vitro and CTX levels in vivo. Furthermore, the expression levels of the gene encoding dendritic cell-specific transmembrane protein (DCSTAMP) of osteoclasts in vitro correlated positively with the number of nuclei per osteoclast, CTX levels in vivo, and donor age. Our results furthermore suggest that these changes in gene expression may be mediated through age-related changes in DNA methylation levels. We conclude that both intrinsic factors and age-induced increase in fusion potential of osteoclasts could be contributing factors for the enhanced bone resorption in vivo, possibly caused by increased expression levels of DCSTAMP.
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Miyauchi, Yoshiteru, Ken Ninomiya, Hiroya Miyamoto, Akemi Sakamoto, Ryotaro Iwasaki, Hiroko Hoshi, Kana Miyamoto et al. „The Blimp1–Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis“. Journal of Experimental Medicine 207, Nr. 4 (05.04.2010): 751–62. http://dx.doi.org/10.1084/jem.20091957.
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Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte–induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets ‘osteoclastic’ molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1–Bcl6–osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.
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Moreaux, Jerome, Dirk Hose, Alboukadel Kassambara, Thierry Reme, Philippe Moine, Guilhem Requirand, Hartmut Goldschmidt und Bernard Klein. „Osteoclast-gene expression profiling reveals osteoclast-derived CCR2 chemokines promoting myeloma cell migration“. Blood 117, Nr. 4 (27.01.2011): 1280–90. http://dx.doi.org/10.1182/blood-2010-04-279760.
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Abstract Multiple myeloma is characterized by the clonal expansion of malignant plasma cells (multiple myeloma cells [MMCs]), in the bone marrow. Osteolytic bone lesions are detected in 80% of patients because of increased osteoclastic bone resorption and reduced osteoblastic bone formation. MMCs are found closely associated with sites of increased bone resorption. Osteoclasts strongly support MMC survival in vitro. To further elucidate the mechanisms involved in osteoclast/MMC interaction, we have identified 552 genes overexpressed in osteoclasts compared with other bone marrow cell subpopulations. Osteoclasts express specifically genes coding for 4 CCR2-targeting chemokines and genes coding for MMC growth factors. An anti-CCR2 monoclonal antibody blocked osteoclast chemoattractant activity for MMC, and CCR2 chemokines are also MMC growth factors, promoting mitogen-activated protein kinase activation in MMC. An anti-insulin growth factor-1 receptor monoclonal antibody completely blocked the osteoclast-induced survival of MMC suppressing both osteoclast and MMC survival. Specific a proliferation-inducing ligand or IL-6 inhibitors partially blocked osteoclast-induced MMC survival. These data may explain why newly diagnosed patients whose MMC express high levels of CCR2 present numerous bone lesions. This study displays additional mechanisms involved in osteoclast/MMC interaction and suggests using CCR2 and/or insulin growth factor-1 targeting strategies to block this interaction and prevent drug resistance.
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Cheng, Yin, Haixia Liu, Jing Li, Yujie Ma, Changheng Song, Yuhan Wang, Pei Li, Yanjing Chen und Zhiguo Zhang. „Evaluation of culture conditions for osteoclastogenesis in RAW264.7 cells“. PLOS ONE 17, Nr. 11 (17.11.2022): e0277871. http://dx.doi.org/10.1371/journal.pone.0277871.
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Osteoclasts are the only multinucleated cells in vivo responsible for bone resorption and are vital for regulating bone remodeling and maintaining bone mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation and biological molecular mechanism. However, protocols for inducing osteoclast formation in RAW264.7 cells vary considerably between laboratories, hindering the replication of results. Therefore, we tested the influence of culture conditions on osteoclast differentiation, including cell density and receptor activator of nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the morphology of osteoclasts. qPCR was used to detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), osteoclast transcription factors c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 gene expression. Concerning bone resorption activity, RANKL treatment increased bone resorption pits on bovine bone slices. Significantly higher levels of osteoclastogenesis were observed with RAW264.7-cell density of 2×104 cells/well in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no positive effect on osteoclastogenesis. RANKL treatment and cell density contribute to osteoclast formation, and the optimal conditions are beneficial when exploring osteoclast function and mechanism.
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Huang, WH, AT Lau, LL Daniels, H. Fujii, U. Seydel, DJ Wood, JM Papadimitriou und MH Zheng. „Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization“. Journal of Molecular Endocrinology 20, Nr. 2 (01.04.1998): 211–19. http://dx.doi.org/10.1677/jme.0.0200211.
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Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.
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Martin-Millan, Marta, Maria Almeida, Elena Ambrogini, Li Han, Haibo Zhao, Robert S. Weinstein, Robert L. Jilka, Charles A. O'Brien und Stavros C. Manolagas. „The Estrogen Receptor-α in Osteoclasts Mediates the Protective Effects of Estrogens on Cancellous But Not Cortical Bone“. Molecular Endocrinology 24, Nr. 2 (01.02.2010): 323–34. http://dx.doi.org/10.1210/me.2009-0354.
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Abstract Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)α from the monocyte/macrophage cell lineage in mice (ERαLysM−/−) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERαLysM−/− were resistant to the proapoptotic effect of 17β-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERα knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERα was as effective as 17β-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERα. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERα. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton.
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Hulley, Philippa A., und Helen J. Knowles. „A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups“. Cells 11, Nr. 24 (08.12.2022): 3973. http://dx.doi.org/10.3390/cells11243973.
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Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them prime therapeutic targets. Osteoclast research is limited by the heterogeneity of osteoclast populations generated in vitro, where the mixture of undifferentiated monocytes, binuclear pre-osteoclasts and multinucleated osteoclasts has by necessity been considered a single osteoclast population. This study describes the differentiation of primary human CD14+ monocyte-derived osteoclasts in 3D collagen gels. These osteoclasts remained small (>95% with ≤5 nuclei) but were viable and active; when released from the gel with collagenase, they fused rapidly when reseeded onto solid substrates and resorbed dentine for 2–3 weeks. 3D-generated osteoclasts expressed cell surface markers of osteoclast differentiation (e.g., CD9, RANK, OSCAR, CD63, CD51/61) which, with their small size, enabled live cell sorting of highly enriched viable subpopulations of human osteoclasts that retained full functional resorption capacity. Low-yield osteoclast preparations were strongly enriched to remove undifferentiated cells (e.g., 13.3% CD51/61+ to 84.2% CD51/61+), and subpopulations of CD9+CD51/61− early osteoclasts and CD9+CD51/61+ mature cells were distinguished. This novel approach allows the study of selected populations of differentiating osteoclasts in vitro and opens the door to in-depth transcriptomic and proteomic analysis of these cells, increasing our ability to study human osteoclast molecular mechanisms relevant to development, aging and disease.
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Dai, Jingjin, Rui Dong, Xinyun Han, Jianmei Li, Xiaoshan Gong, Yun Bai, Fei Kang et al. „Osteoclast-derived exosomal let-7a-5p targets Smad2 to promote the hypertrophic differentiation of chondrocytes“. American Journal of Physiology-Cell Physiology 319, Nr. 1 (01.07.2020): C21—C33. http://dx.doi.org/10.1152/ajpcell.00039.2020.
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The invasion of osteoclasts into the cartilage via blood vessels advances the process of endochondral ossification, and dysregulation of dynamic intercellular interactions results in skeletal dysplasias. Although the regulation of osteoclasts by growth plate chondrocytes has been reported in detail, the effect of osteoclasts on chondrocytes remains to be determined. In this study, ATDC5 cells and bone marrow mesenchymal stem cells were differentiated into chondrocytes and treated with conditioned medium obtained from bone marrow macrophages differentiated to osteoclast precursors and osteoclasts. Exosomes were inhibited in conditioned medium or isolated directly from osteoclasts to further determine whether osteoclast-derived exosomes play an important role in chondrocyte hypertrophy. Additionally, exosomal miRNAs were detected, and let-7a-5p was selected as an miRNA with significantly increased expression in osteoclast-derived exosomes. Experiments were performed to verify the potential target Smad2 and investigate how let-7a-5p affected chondrocytes. The results suggest that both osteoclast precursors and osteoclasts promote chondrocyte hypertrophy and that the promotive effect of osteoclasts is more significant than that of osteoclast precursors. Osteoclast-derived exosomes promote the hypertrophic differentiation of chondrocytes. Moreover, osteoclast-derived exosomal let-7a-5p inhibits Smad2 to decrease the transforming growth factor-β-induced inhibition of chondrocyte hypertrophy. Our research reveals the role of osteoclasts in the regulation of chondrocytes and provides insights into the highly coordinated intercellular process of endochondral ossification.
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Lee, Kyunghee, Incheol Seo, Mun Choi und Daewon Jeong. „Roles of Mitogen-Activated Protein Kinases in Osteoclast Biology“. International Journal of Molecular Sciences 19, Nr. 10 (01.10.2018): 3004. http://dx.doi.org/10.3390/ijms19103004.
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Bone undergoes continuous remodeling, which is homeostatically regulated by concerted communication between bone-forming osteoblasts and bone-degrading osteoclasts. Multinucleated giant osteoclasts are the only specialized cells that degrade or resorb the organic and inorganic bone components. They secrete proteases (e.g., cathepsin K) that degrade the organic collagenous matrix and establish localized acidosis at the bone-resorbing site through proton-pumping to facilitate the dissolution of inorganic mineral. Osteoporosis, the most common bone disease, is caused by excessive bone resorption, highlighting the crucial role of osteoclasts in intact bone remodeling. Signaling mediated by mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, has been recognized to be critical for normal osteoclast differentiation and activation. Various exogenous (e.g., toll-like receptor agonists) and endogenous (e.g., growth factors and inflammatory cytokines) stimuli contribute to determining whether MAPKs positively or negatively regulate osteoclast adhesion, migration, fusion and survival, and osteoclastic bone resorption. In this review, we delineate the unique roles of MAPKs in osteoclast metabolism and provide an overview of the upstream regulators that activate or inhibit MAPKs and their downstream targets. Furthermore, we discuss the current knowledge about the differential kinetics of ERK, JNK, and p38, and the crosstalk between MAPKs in osteoclast metabolism.
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Huang, W. H., L. L. Daniels, D. J. Wood, U. Seydel, J. M. Papadimitriou und M. H. Zheng. „VITAMIN D RECEPTOR mRNA IS EXPRESSED IN OSTEOCLAST-LIKE CELLS OF HUMAN GIANT CELL TUMOR OF BONE (OSTEOCLASTOMA)“. Journal of Musculoskeletal Research 03, Nr. 03 (September 1999): 201–7. http://dx.doi.org/10.1142/s021895779900021x.
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The conventional view of the effects of 1,25- (OH)2D3 on osteoclastogenesis and bone resorption are thought to be mediated by stromal cells or osteoblastic cell. Giant cell tumor of bone (GCT) is characterized by multinuclear giant cells (osteoclast-like cells) distributed among a mass of mononuclear cells. Because it is very difficult to obtain normal human osteoclasts, many investigators, including ourselves, have used GCT as a source of human osteoclasts to enhance the understanding of normal skeletal remodeling and especially the hormone regulation of osteoclast functions. In this report, expression of human vitamin D receptor (VDR) mRNA was examined in the various components of GCT using the fluorescence in situ hybridization (FISH) technique. The results showed that VDR gene transcripts were expressed in both multinuclear cells (osteoclast-like cells) and mononuclear macrophage-like cells but not in stromal-like tumor cells in two cases of GCT examined. Measurement of VDR gene transcripts in situ by confocal microscopy showed that the level of VDR mRNA in osteoclast-like giant cells is greater than that in macrophage-like cells (p < 0.05). These findings suggest that 1,25- (OH)2D3 mediated osteoclastic bone resorption or osteoclastogenesis could at least, in part, occur by direct action on osteoclasts/their precursor cells.
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Gwaltney, S. M., R. J. S. Galvin, K. B. Register, R. B. Rimler und M. R. Ackermann. „Effects of Pasteurella multocida Toxin on Porcine Bone Marrow Cell Differentiation into Osteoclasts and Osteoblasts“. Veterinary Pathology 34, Nr. 5 (September 1997): 421–30. http://dx.doi.org/10.1177/030098589703400506.
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The effect of Pasteurella multocida toxin (PMT) on porcine osteoclast and osteoblast differentiation was studied using in vitro cell culture systems. When grown in the presence of Vitamin D3, isolated porcine bone marrow cells formed multinucleated cells with features characteristic of osteoclasts. Exposure of bone marrow cells to Vitamin D3 and PMT during growth resulted in formation of increased numbers and earlier appearance of osteoclasts compared to controls. Ultrafiltered medium from PMT-treated cells likewise increased osteoclast numbers, suggesting that a soluble mediator may be involved in the action of PMT. When cell cultures were treated with fluorescein-labeled PMT, fluorescence was found within the cytoplasm of small, round cells that did not resemble either osteoclasts or osteoclastic precursor cells. Cultures of porcine bone marrow cells exposed to dexamethasone, ascorbic acid, and β-glycerophosphate developed into osteoblastic cells that formed multilayered, mineralized nodules. Exposure of osteoblastic cultures to low concentrations of PMT resulted in retarded cell growth, formation of decreased numbers of nodules, and minimal to no mineralization in the nodules; higher concentrations of PMT resulted in increased cellular debris and poor growth of cells, with no nodule formation. These findings suggest that PMT may induce turbinate atrophy in pigs by increasing osteoclast numbers and inhibiting osteoblastic bone formation. The effect of PMT on osteoclastic differentiation and growth may not be due to a direct effect on preosteoclastic cells, but rather due to alterations in the soluble mediator secretion by marrow stromal cells.
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Yagi, Mitsuru, Takeshi Miyamoto, Yumi Sawatani, Katsuya Iwamoto, Naobumi Hosogane, Nobuyuki Fujita, Kozo Morita et al. „DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells“. Journal of Experimental Medicine 202, Nr. 3 (01.08.2005): 345–51. http://dx.doi.org/10.1084/jem.20050645.
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Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Osteoclasts form large multinuclear giant cells by fusion of mononuclear osteoclasts. How cell fusion is mediated, however, is unclear. We identify the dendritic cell–specific transmembrane protein (DC-STAMP), a putative seven-transmembrane protein, by a DNA subtraction screen between multinuclear osteoclasts and mononuclear macrophages. DC-STAMP is highly expressed in osteoclasts but not in macrophages. DC-STAMP–deficient mice were generated, and osteoclast cell fusion was completely abrogated in homozygotes despite normal expression of osteoclast markers and cytoskeletal structure. As osteoclast multinucleation was restored by retroviral introduction of DC-STAMP, loss of cell fusion was directly attributable to a lack of DC-STAMP. Defects in osteoclast multinucleation reduce bone-resorbing activity, leading to osteopetrosis. Similar to osteoclasts, foreign body giant cell formation by macrophage cell fusion was also completely abrogated in DC-STAMP–deficient mice. We have thus identified an essential regulator of osteoclast and macrophage cell fusion, DC-STAMP, and an essential role of osteoclast multinucleation in bone homeostasis.
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Hatton, R., M. Stimpel und T. J. Chambers. „Angiotensin II is generated from angiotensin I by bone cells and stimulates osteoclastic bone resorption in vitro“. Journal of Endocrinology 152, Nr. 1 (Januar 1997): 5–10. http://dx.doi.org/10.1677/joe.0.1520005.
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Abstract During bone resorption, osteoclasts are closely associated with endothelial cells. The latter are able to produce several agents that regulate bone resorption. In view of the increasing evidence that angiotensin II, which can be generated by endothelial cells, has actions outside the traditional renin-angiotensin system, we tested the effect of angiotensin II on bone resorption. Angiotensin II showed no effect either on osteoclast formation or on bone resorption by isolated osteoclasts. However, in co-cultures of osteoclasts with calvarial or MC3T3-E1 osteoblastic cells, and in osteoclastic cultures co-cultured with other bone cells obtained by prolonged sedimentation, angiotensin II stimulated bone resorption to a similar degree to that observed with 1,25(OH)2 vitamin D3. Stimulation of resorption was noted at concentrations of 10−7 m and above. We found that angiotensin I also stimulated bone resorption in co-cultures of osteoclasts with osteoblastic cells, and that this action was inhibited by inhibitors of angiotensin-converting enzyme. These results identify angiotensin I and II as potent stimulators of osteoclastic bone resorption, and raise the possibility that bone might contain a tissue-renin-angiotensin system that might play a role in the regulation of bone resorption. Journal of Endocrinology (1997) 152, 5–10
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Na, Woojin, Eun-Jung Lee, Min-Kyung Kang, Yun-Ho Kim, Dong Yeon Kim, Hyeongjoo Oh, Soo-Il Kim, Su Yeon Oh und Young-Hee Kang. „Aesculetin Inhibits Osteoclastic Bone Resorption through Blocking Ruffled Border Formation and Lysosomal Trafficking“. International Journal of Molecular Sciences 21, Nr. 22 (13.11.2020): 8581. http://dx.doi.org/10.3390/ijms21228581.
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For the optimal resorption of mineralized bone matrix, osteoclasts require the generation of the ruffled border and acidic resorption lacuna through lysosomal trafficking and exocytosis. Coumarin-type aesculetin is a naturally occurring compound with anti-inflammatory and antibacterial effects. However, the direct effects of aesculetin on osteoclastogenesis remain to be elucidated. This study found that aesculetin inhibited osteoclast activation and bone resorption through blocking formation and exocytosis of lysosomes. Raw 264.7 cells were differentiated in the presence of 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL) and treated with 1–10 μM aesculetin. Differentiation, bone resorption, and lysosome biogenesis of osteoclasts were determined by tartrate-resistance acid phosphatase (TRAP) staining, bone resorption assay, Western blotting, immunocytochemical analysis, and LysoTracker staining. Aesculetin inhibited RANKL-induced formation of multinucleated osteoclasts with a reduction of TRAP activity. Micromolar aesculetin deterred the actin ring formation through inhibition of induction of αvβ3 integrin and Cdc42 but not cluster of differentiation 44 (CD44) in RANKL-exposed osteoclasts. Administering aesculetin to RANKL-exposed osteoclasts attenuated the induction of autophagy-related proteins, microtubule-associated protein light chain 3, and small GTPase Rab7, hampering the lysosomal trafficking onto ruffled border crucial for bone resorption. In addition, aesculetin curtailed cellular induction of Pleckstrin homology domain-containing protein family member 1 and lissencephaly-1 involved in lysosome positioning to microtubules involved in the lysosomal transport within mature osteoclasts. These results demonstrate that aesculetin retarded osteoclast differentiation and impaired lysosomal trafficking and exocytosis for the formation of the putative ruffled border. Therefore, aesculetin may be a potential osteoprotective agent targeting RANKL-induced osteoclastic born resorption for medicinal use.
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Roy, Michèle, und Sophie Roux. „Rab GTPases in Osteoclastic Bone Resorption and Autophagy“. International Journal of Molecular Sciences 21, Nr. 20 (16.10.2020): 7655. http://dx.doi.org/10.3390/ijms21207655.
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Small guanosine triphosphate hydrolases (GTPases) of the Rab family are involved in plasma membrane delivery, fusion events, and lysosomal and autophagic degradation pathways, thereby regulating signaling pathways and cell differentiation and function. Osteoclasts are bone-resorbing cells that maintain bone homeostasis. Polarized vesicular trafficking pathways result in the formation of the ruffled border, the osteoclast’s resorptive organelle, which also assists in transcytosis. Here, we reviewed the different roles of Rab GTPases in the endomembrane machinery of osteoclasts and in bone diseases caused by the dysfunction of these proteins, with a particular focus on autophagy and bone resorption. Understanding the molecular mechanisms underlying osteoclast-related bone disease development is critical for developing and improving therapies.
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Ewanchuk, Benjamin W., Corey R. Arnold, Dale R. Balce, Priyatha Premnath, Tanis L. Orsetti, Amy L. Warren, Alexandra Olsen, Roman J. Krawetz und Robin M. Yates. „A non-immunological role for γ-interferon–inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption“. Science Advances 7, Nr. 17 (April 2021): eabd3684. http://dx.doi.org/10.1126/sciadv.abd3684.
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The extracellular bone resorbing lacuna of the osteoclast shares many characteristics with the degradative lysosome of antigen-presenting cells. γ-Interferon–inducible lysosomal thiol reductase (GILT) enhances antigen processing within lysosomes through direct reduction of antigen disulfides and maintenance of cysteine protease activity. In this study, we found the osteoclastogenic cytokine RANKL drove expression of GILT in osteoclast precursors in a STAT1-dependent manner, resulting in high levels of GILT in mature osteoclasts, which could be further augmented by γ-interferon. GILT colocalized with the collagen-degrading cysteine protease, cathepsin K, suggesting a role for GILT inside the osteoclastic resorption lacuna. GILT-deficient osteoclasts had reduced bone-resorbing capacity, resulting in impaired bone turnover and an osteopetrotic phenotype in GILT-deficient mice. We demonstrated that GILT could directly reduce the noncollagenous bone matrix protein SPARC, and additionally, enhance collagen degradation by cathepsin K. Together, this work describes a previously unidentified, non-immunological role for GILT in osteoclast-mediated bone resorption.
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Kim, Tae-Ho, Chae Gyeong Jeong, Hyeong-U. Son, Man-Il Huh, Shin-Yoon Kim, Hong Kyun Kim und Sang-Han Lee. „Ethanolic Extract of Rubus coreanus Fruits Inhibits Bone Marrow-Derived Osteoclast Differentiation and Lipopolysaccharide-Induced Bone Loss“. Natural Product Communications 12, Nr. 12 (Dezember 2017): 1934578X1701201. http://dx.doi.org/10.1177/1934578x1701201228.
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The inhibition of osteoclast differentiation/bone resorption is a well-known therapeutic strategy for controlling pathological and postmenopausal bone loss. Natural products that specifically inhibit osteoclastogenesis could therefore be developed as antiresorptive drugs for the treatment of metabolic bone disorders characterized by excessive osteoclastic bone resorption. We therefore examined the effects of Rubus coreanus extract (eeRc) on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced differentiation of bone marrow macrophages (BMMs) into osteoclasts and pit formation in vitro. Additionally, the in vivo effects of the eeRc were observed in mice with lipopolysaccharide (LPS)-induced bone erosion. In this study, we found that the ethanolic extract of Rubus coreanus fruits considerably suppressed the RANKL-induced differentiation of primary BMMs into osteoclasts and bone-resorbing activity of mature osteoclasts. Oral administration of eeRc attenuated LPS-induced bone loss in vivo, as demonstrated by the reversal of LPS-induced reduction in bone volume per tissue volume, bone mineral density, and trabecular number to some extent in eeRc-treated mice. In addition, eeRc slightly decreased the serum levels of C-terminal telopeptide fragments of type I collagen, the collagen-breakdown product generated by osteoclasts. Collectively, our results indicate that eeRc has the potential to inhibit bone loss by blocking osteoclast differentiation and could therefore be a promising natural product for the prevention and/or treatment of inflammatory bone loss.
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Feng, Shengmei, Lianfu Deng, Wei Chen, Jianzhong Shao, Guoliang Xu und Yi-Ping Li. „Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts“. Biochemical Journal 417, Nr. 1 (12.12.2008): 195–203. http://dx.doi.org/10.1042/bj20081073.
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Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-κB (nuclear factor κB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3−/− osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.
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Chole, Richard A. „Osteoclasts in Chronic Otitis Media, Cholesteatoma, and Otosclerosis“. Annals of Otology, Rhinology & Laryngology 97, Nr. 6 (November 1988): 661–66. http://dx.doi.org/10.1177/000348948809700615.
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Bone resorption and remodeling are characteristic of chronic otitis media with and without cholesteatoma and otosclerosis. The consequences of this remodeling process may be hearing loss, repeated infection, vestibular disturbance, or intracranial complications. Evidence of osteoclastic bone resorption was found in surgical specimens of 11 of 24 cases of cholesteatoma, two of three cases of chronic otitis media, and three of ten cases of otosclerotic stapes; all three spongiotic lesions had osteoclasts. With careful serial sectioning these cells are almost always multinucleate and have the typical appearance of osteoclasts with ruffled borders. Some specimens had evidence of bone erosion in the absence of osteoclasts; this finding represents an inactive phase of the remodeling process. Since the osteoclast plays an important role in the resorption and remodeling of bone in these middle ear diseases, the source, physiology, and local control of these cells are of prime importance in investigating the pathophysiology of these diseases. At the present time, the local control of activation and recruitment of osteoclasts, as well as their chemotactic responses, is poorly understood.
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Athanasou, N. A., J. Quinn, A. Heryet und J. O. McGee. „Localization of platelet antigens and fibrinogen on osteoclasts“. Journal of Cell Science 89, Nr. 1 (01.01.1988): 115–22. http://dx.doi.org/10.1242/jcs.89.1.115.
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The antigenic phenotype of the human osteoclast, which is known to be derived from a circulating mononuclear precursor cell of haemopoietic origin, is controversial. Recent studies have shown that macrophage as well as megakaryocyte/platelet antigens are expressed by osteoclasts. In this study, we have sought to define, by immunohistochemistry, the nature and possible function of platelet antigens expressed by human osteoclasts in foetal and adult bone specimens. Monoclonal antibodies to platelet glycoprotein IIIa (gpIIIa) and CD9 antibodies stained osteoclasts in all bone specimens examined. Fibrinogen was also localized to the osteoclast membrane in foetal bone imprints. In addition, we found that CD9 and gpIIIa antibodies reacted weakly with monocytes in buffy coat smears. Antibodies to factor 8 and glycoproteins Ib and IIb/IIIa did not react with osteoclasts. These results show that osteoclasts, monocytes, macrophages, megakaryocytes and platelets possess common antigens and that fibrinogen is present on the surface of osteoclasts. By analogy with platelets, CD9 and gpIIIa may play a role in fibrinogen binding by osteoclasts. Possible mechanisms by which platelet antigens and fibrinogen binding could affect osteoclast function are proposed.
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Hayashi, Shin-Ichi, Toshiyuki Yamane, Akitomo Miyamoto, Hiroaki Hemmi, Hisashi Tagaya, Yasuko Tanio, Hidenobu Kanda, Hidetoshi Yamazaki und Takahiro Kunisada. „Commitment and differentiation of stem cells to the osteoclast lineage“. Biochemistry and Cell Biology 76, Nr. 6 (01.12.1998): 911–22. http://dx.doi.org/10.1139/o98-099.
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Osteoclasts are hematopoietic cells which play important roles in bone remodeling and resorption. They have phenotypic characteristics of the monocyte/macrophage lineages. In this review we first describe the phylogeny of osteoclasts. Osteoclast generation is closely linked to the presence of bone tissues. The formation of bone cavities in aquatic animals is underdeveloped, even though they have cells which have the potential to differentiate into osteoclasts. Next we describe recent advances in our understanding of osteoclastogenesis that have resulted from the identification of critical molecules and mutated genes of osteopetrotic mice. Reports that transcriptional factors PU.1 and c-Fos are essential for commitment and (or) differentiation into the osteoclast lineage and novel culture systems, which have clarified some characteristics of osteoclast precursors, are also described. We are now able to induce mature osteoclasts from hematopoietic stem cells and even from totipotent embryonic stem cells. Cell lines that differentiate into osteoclasts are also available. Using these culture systems and cell lines, the interactions of osteoclasts with osteoblastic stromal cells, which produce critical molecules for osteoclastogenesis, have been studied. Very recently, one of these critical molecules, osteoclast differentiation factor / osteoprotegerin-ligand, was cloned. The presence of this factor and macrophage-colony-stimulating factor is sufficient to induce osteoclast development in cultures inoculated only with an osteoclast precursor cell line. We review the present status and the remaining questions in osteoclast biology.Key words: osteoclast, stem cell, osteopetrosis, M-CSF, ODF/OPGL, hematopoiesis.
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Blair, H. C., S. L. Teitelbaum, L. E. Grosso, D. L. Lacey, H. L. Tan, D. W. McCourt und J. J. Jeffrey. „Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization“. Biochemical Journal 290, Nr. 3 (15.03.1993): 873–84. http://dx.doi.org/10.1042/bj2900873.
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Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.
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Wang, Qiang, Yi Xie, Quan-Sheng Du, Xiao-Jun Wu, Xu Feng, Lin Mei, Jay M. McDonald und Wen-Cheng Xiong. „Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin“. Journal of Cell Biology 160, Nr. 4 (10.02.2003): 565–75. http://dx.doi.org/10.1083/jcb.200207036.
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Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion–targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2–gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.
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Abdel Razik, Heba E., Miho Nakamura, Leire Bergara-Muguruza, Uruj Sarwar, Mohammad Hassan, Robert Horowitz und Ahmed El-Ghannam. „Osteoblast-Mediated Resorption of Porous Bioactive SCPC Granules Enhances Bone Regeneration in Human Extraction Sockets“. Solid State Phenomena 340 (23.12.2022): 107–12. http://dx.doi.org/10.4028/p-32eola.
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Bone graft materials are widely used in orthopedic and maxillofacial surgeries. The controlled resorbability of the graft material is essential for bone regeneration. Hydroxyapatite and biphasic calcium phosphate bone grafts have poor resorption and limited bone conductive effects. Histology analyses of bone biopsy from SCPC grafted human extraction sockets showed complete bone regeneration and graft resorption in absence of osteoclasts and macrophages. The hypothesis of the present study is that bioactive SCPC inhibits osteoclast’s activity due to the presence of resorbable silica phase in the material. Our objective is to analyze the effect of SCPC dissolution products on the resorption activity of osteoclasts. The conditioned medium was prepared by immersion of SCPC resorbable bioactive SCPC porous granules (Shefabone, Inc, USA) in cell culture medium at various ratios at 37°C for 3 days. The concentration of Si ions released from the SCPC granules into cell culture medium was measured using ICP-OES. Osteoclast precursors derived from human bone marrow were seeded on bone slices and cultured in the conditioned medium containing 10% FBS and osteoclast induction factors. Osteoclast differentiation and resorption were evaluated by TRAP staining and measurement of the volume of resorption pits on the bone slices. Mature multinuclear giant TRAP-positive osteoclasts were observed on the bone substrates after 14 days incubation in control medium containing osteoclast induction factors. In conditioned medium, the number of multinuclear TRAP-positive cells was significantly decreased as the concentration of SCPC dissolved silica increased. The dissolution of silica from SCPC into the culture medium correlates well with down regulation of osteoclast differentiation and the rapid bone regeneration in human bone defects.
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Fong, E. L. S., E. L. Prabha und T. Carney. „POS0348 DEVELOPING A WHOLE MOUNT FLUORESCENT OSTEOCLAST ACTIVITY ASSAY USING THE ELF97 PHOSPHATASE SUBSTRATE TO VISUALISE AND QUANTIFY IN SITU OSTEOCLAST ACTIVITY IN ZEBRAFISH (DANIO RERIO)“. Annals of the Rheumatic Diseases 81, Suppl 1 (23.05.2022): 427.3–428. http://dx.doi.org/10.1136/annrheumdis-2022-eular.5402.
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BackgroundOsteoporosis is a mineral bone disease arising from the predominance of osteoclastic bone resorption. Bisphosphonates which inhibit osteoclasts are commonly used in osteoporosis treatment, but are not without severe adverse effects like osteonecrosis of the jaw. The mechanisms behind the development of such phenomena is not well understood. Bone homeostasis is achieved through an intimate cross-talk between osteoclasts and osteoblasts. Thus, it is important to visualise activities of these cells simultaneously in situ. Currently, there are means to visualise osteoclast shape and numbers with tartrate-resistant alkaline phosphatase (TRAP) staining but no practical and accurate methods to quantify osteoclast activity in situ.ObjectivesThis investigation aims to establish the use of ELF97, a substrate of TRAP, to visualise and quantify osteoclast activity. This provides vital clues to mechanisms of various bone disorders. TRAP dephosphorylation of ELF97 results in a detectable fluorescent product at areas of osteoclast activity.MethodsOsteoclastic activity was initiated in zebrafish by inducing crush injuries in tail fin rays. Colocalisation of ELF97 fluorescence with osteoclast-specific DsRed in transgenic zebrafish, visualised under confocal microscopy, is used to further establish the specificity of ELF97 to sites of osteoclastic activity. Quantification is established by comparing fluorescence between wild type, osteoclast-deficient mutants and bisphosphonate-treated zebrafish. The utility of ELF97 will also be investigated in terms of the stability of the florescent product.ResultsThe investigation revealed that ELF97 and DsRed fluorescence were found commonly at crush sites with osteoclastic activity. Wild type zebrafish had greater fluorescence compared to osteoclast-deficient (p<0.0001) and bisphosphonate-treated zebrafish (p<0.0001) after 7 and 14 days post-crush, revealing that fluorescence from ELF97 corresponds to expected osteoclastic activity. Fluorescence of tail fins treated with ELF97 did not diminish over a period of 21 days of storage, demonstrating its stability.ConclusionELF97 is thus a useful means to visualise osteoclast activity, potentially crucial in more advanced investigations to understand bone disorders. It could be used in combination with other cellular markers in whole biological samples to study and experimentally manipulate bone remodelling.Disclosure of InterestsNone declared
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Cackowski, Frank C., Judith L. Anderson, Kenneth D. Patrene, Rushir J. Choksi, Steven D. Shapiro, Jolene J. Windle, Harry C. Blair und G. David Roodman. „Osteoclasts are important for bone angiogenesis“. Blood 115, Nr. 1 (07.01.2010): 140–49. http://dx.doi.org/10.1182/blood-2009-08-237628.
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Abstract Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-κB ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9−/− mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.
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Kolb, Alexus D., Jinlu Dai, Evan T. Keller und Karen M. Bussard. „‘Educated’ Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment“. Cancers 13, Nr. 2 (12.01.2021): 263. http://dx.doi.org/10.3390/cancers13020263.
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Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.
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Lee, B. S., L. S. Holliday, B. Ojikutu, I. Krits und S. L. Gluck. „Osteoclasts express the B2 isoform of vacuolar H(+)-ATPase intracellularly and on their plasma membranes“. American Journal of Physiology-Cell Physiology 270, Nr. 1 (01.01.1996): C382—C388. http://dx.doi.org/10.1152/ajpcell.1996.270.1.c382.
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Osteoclasts express high levels of vacuolar H(+)-ATPase (V-ATPase) in their ruffled membranes, driving the secretion of H+ required for normal bone resorption. Previous reports have suggested that the B subunit of the osteoclast V-ATPase differs from those expressed in kidney and other tissues. In this study, B subunit isoform-specific antibodies and cDNA probes were used to examine which B subunit isoform is expressed in osteoclasts and osteoclast-like cells. Immunoblotting and RNA hybridization analysis were used to demonstrate that cells from an osteoclast-rich mouse bone marrow culture model express the B2 but not the B1 subunit isoform. Immunocytochemical staining of murine osteoclasts generated in vitro and of native rat osteoclasts in bone sections showed that the B2 but not the B1 isoform was expressed at high levels and was polarized to the ruffled membrane. Human marrow cultures and monocyte-derived macrophages, used as models for osteoclasts, also expressed the B2 but not the B1 subunit isoform. These results indicate that V-ATPases containing the B2 subunit isoform mediate osteoclast bone resorption.
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Leightner, Amanda C., Carina Mello Guimaraes Meyers, Michael D. Evans, Kim C. Mansky, Rajaram Gopalakrishnan und Eric D. Jensen. „Regulation of Osteoclast Differentiation at Multiple Stages by Protein Kinase D Family Kinases“. International Journal of Molecular Sciences 21, Nr. 3 (05.02.2020): 1056. http://dx.doi.org/10.3390/ijms21031056.
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Balanced osteoclast and osteoblast activity is necessary for skeletal health, whereas unbalanced osteoclast activity causes bone loss in many skeletal conditions. A better understanding of pathways that regulate osteoclast differentiation and activity is necessary for the development of new therapies to better manage bone resorption. The roles of Protein Kinase D (PKD) family of serine/threonine kinases in osteoclasts have not been well characterized. In this study we use immunofluorescence analysis to reveal that PKD2 and PKD3, the isoforms expressed in osteoclasts, are found in the nucleus and cytoplasm, the mitotic spindle and midbody, and in association with the actin belt. We show that PKD inhibitors CRT0066101 and CID755673 inhibit several distinct aspects of osteoclast formation. Treating bone marrow macrophages with lower doses of the PKD inhibitors had little effect on M-CSF + RANKL-dependent induction into committed osteoclast precursors, but inhibited their motility and subsequent differentiation into multinucleated mature osteoclasts, whereas higher doses of the PKD inhibitors induced apoptosis of the preosteoclasts. Treating post-fusion multinucleated osteoclasts with the inhibitors disrupted the osteoclast actin belts and impaired their resorptive activity. In conclusion, these data implicate PKD kinases as positive regulators of osteoclasts, which are essential for multiple distinct processes throughout their formation and function.
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Robinson, Lisa J., Salvatore Mancarella, Irina L. Tourkova, John B. Barnett, Harry C. Blair und Jonathan Soboloff. „Critical Role for the Calcium-Release Activated Calcium Channel Orai1 In RANKL-Stimulated Osteoclast Formation From Monocytic Cells“. Blood 116, Nr. 21 (19.11.2010): 928. http://dx.doi.org/10.1182/blood.v116.21.928.928.
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Abstract Abstract 928 Calcium signals are major regulators of human osteoclast formation and function, and the molecular mechanisms underlying calcium effects are of interest as possible targets for pharmacologic regulation of bone resorption. IP3-receptor regulated release of calcium stores is linked to NFATc1 activation, which stimulates expression of key osteoclast genes in precursors, but the roles of other calcium channels in osteoclastogenesis are not clear. In particular, the identity of the channel(s) mediating extracellular calcium influx triggered by release of calcium stores remains uncertain. In lymphoid cells, a major mediator of this extracellular calcium influx is the Calcium-Release Activated Calcium (CRAC) channel consisting of Orai1, a plasma-membrane calcium channel, and the calcium-sensitive regulatory protein, STIM1. Calcium released from intracellular stores binds to a low affinity EF-hand in STIM1 causing a conformational change in STIM1 that permits binding to Orai1, aggregation in microscopically distinct puncta at the cell membrane, and opening of the Orai1 channel with consequent influx of extracellular calcium. Targeted deletion of Orai1 or Stim1 in mice results in severe immunodeficiency and early death; this has limited the assessment of Orai1 effects in other tissues. To evaluate the specific role of Orai1 in human osteoclasts, we used peripheral blood monocytes which form multinucleated osteoclasts capable of bone resorption when treated with CSF1 and RANKL. We confirmed Orai1 expression in human monocytes using Western blot and quantitative PCR assays, and found that the protein was down-regulated in mature osteoclasts. We used fura-2 to measure store-dependent and -independent changes in intracellular calcium during osteoclastic differentiation of monocytes over 10–14 days in RANKL and CSF1. RANKL-associated calcium oscillations were detected throughout differentiation, but calcium-release activated influx of extracellular calcium was markedly lower in the mature osteoclasts compared to precursors, paralleling their expression of Orai1. Human monocytes, transfected with Orai1-specific siRNA producing an 80% reduction in Orai1 protein compared to control siRNA treated cells, showed inhibition of store-regulated calcium influx during osteoclastogenesis. Furthermore, monocytes deficient in Orai1 showed impaired osteoclast formation; in particular, multinucleation resulting from osteoclast precursor cell fusion was markedly reduced, impairing bone resorption. Orai1 deficiency in T-cells inhibits activation of NFATc1, but this did not appear prominent in our cells: we found no significant inhibition of NFATc1 regulated gene expression in Orai1 siRNA-transfected cells compared to control siRNA-transfected cells, despite the marked difference in Orai1 protein. Other calcium channels may mediate calcium dependent NFATc1 activation in osteoclast precursors; alternatively, the low level of Orai1 protein remaining in Orai1 siRNA treated monocytes may be sufficient for NFATc1 activation. To define the effects of complete Orai1 deficiency, we examined osteoclast formation and in vivo skeletal development in mice with targeted deletion of the Orai1 gene (Gwack et al. Mol Cell Biol 28 (2008) 5209-22). Consistent with our in vitro results, multinucleated osteoclasts were nearly absent from Orai1-/- mice, but mononuclear cells expressing osteoclast markers such as TRAcP, were seen. Surprisingly, the knock-out mice did not show the osteopetrotic phenotype typical of osteoclast deficiency. Retention of fetal cartilage was seen, indicating defective osteoclastic function in Orai1-/- mice, but marked inhibition of bone formation was also present. Using micro-computed tomography we found significant reductions in both cortical ossification and trabecular bone formation in Orai1-/- mice. This raised the possibility of a previously unrecognized role for Orai1 in osteoblasts, or the osteoblast defect might simply reflect abnormalities of Orai1-/- osteoclasts and/or lymphocytes, since both cell types have regulatory effects on osteoblast formation and function. In summary, our studies identify a requirement for CRAC channel mediated calcium influx, and specifically the Orai1 channel, for normal formation and activity of human osteoclasts; these results are confirmed in an Orai1 knock-out mouse which also shows defective bone formation. Disclosures: No relevant conflicts of interest to declare.
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Klein-Nulend, J., M. A. van Duin, T. P. Green, V. Everts und T. J. de Vries. „The dual specific Src/Abl kinase inhibitor AZD0530 inhibits the formation and activity of human osteoclasts“. Journal of Clinical Oncology 25, Nr. 18_suppl (20.06.2007): 3602. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.3602.
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3602 Background: Gene knockout studies have demonstrated the critical importance of the non-receptor TK Src to osteoclast bone resorptive function. Deregulated Src TK activity is also reported as a hallmark of the invasive cancer cell. Bone metastatic cancer cells interact with and activate osteoclasts in a destructive cycle of bone degradation and stimulation of tumor cell growth. Therefore targeting Src activity would appear to be a rational therapeutic approach in treating metastatic bone disease. We have reported previously (AACR 2005) on the activity of the dual Src/Abl kinase inhibitor AZD0530 in inhibiting the bone resorptive activity of mature rabbit osteoclasts in a bone slice model. Here we tested the effect of AZD0530 in a human co-culture system, examining its activity on i) osteoclast formation by peripheral blood mononuclear cells (PBMCs) co-cultured with osteoblasts, and ii) osteoclastic bone resorption. Methods and Results: PBMC adhesion to osteoblasts and osteoblast morphology was not affected by AZD0530 (0.1–10 μM). However, AZD0530 inhibited the formation of multinucleated osteoclast-like cells dose dependently. PBMC-osteoblast co-cultures were then exposed to 1 μM AZD0530 for different time intervals. AZD0530 was most effective in inhibiting the formation of osteoclast-like cells when added at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial induction of osteoclast formation. Formation of actin rings, to which c-Src co-localizes, is a prerequisite for osteoclastic bone resorption. The effect of AZD0530 on formation of actin rings was analyzed using the co-culture system on cortical bone slices. AZD0530 prevented migration of osteoclast precursors to the bone surface, and the subsequent formation of actin rings. On withdrawal of the drug, this process was reversible. Conclusions: Our data suggest that Src activity is pivotal for the formation, migration and activity of osteoclasts. Data reported elsewhere suggest AZD0530 will also impact on tumor cells directly. AZD0530 is a promising new anti-cancer drug with potential to additionally treat metastatic bone disease through its inhibition of both osteoclast activity and tumor cell invasion into and within the bone micro-environment. No significant financial relationships to disclose.
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Chellaiah, M. A., N. Kizer, R. Biswas, U. Alvarez, J. Strauss-Schoenberger, L. Rifas, S. R. Rittling, D. T. Denhardt und K. A. Hruska. „Osteopontin Deficiency Produces Osteoclast Dysfunction Due to Reduced CD44 Surface Expression“. Molecular Biology of the Cell 14, Nr. 1 (Januar 2003): 173–89. http://dx.doi.org/10.1091/mbc.e02-06-0354.
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Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN−/− osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the αvβ3 integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.
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Frisch, Benjamin, John M. Ashton, Adam Olm-shipman, Lianping Xing, Craig T. Jordan und Laura Calvi. „Reciprocal Synergistic Interactions of Leukemic Cells with Osteoclast Progenitors in the Bone Microenvironment“. Blood 112, Nr. 11 (16.11.2008): 322. http://dx.doi.org/10.1182/blood.v112.11.322.322.
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Abstract The bone marrow provides an essential regulatory microenvironment for adult hematopoiesis, however the relationship between the bone marrow microenvironment and malignant hematopoiesis remains poorly understood. To investigate the interactions between leukemia and the bone marrow microenvironment we utilized a mouse model of blast-crisis chronic myelogenous leukemia (BC-CML), in which primitive normal murine hematopoietic cells are modified to leukemic cells by expressing the translocation products BCR/ABL and Nup98/HoxA9. The presence of each translocation was confirmed by their co-expression of Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP) respectively. Ten days after injection of GFP+/YFP+ leukemic cells into strain-matched immunocompetent, non-myeloablated recipient mice, 50% of the bone marrow was composed of leukemic cells as determined by flow cytometric analysis. Histologic analysis of the contralateral tibiae and femora demonstrated not only progressive replacement of the bone marrow by leukemic cells, but also a significant bone loss. Histomorphometric analysis confirmed 50% decreased trabecular bone volume in leukemic mice compared to control mice that were not injected with leukemic cells (bone volume/total volume (%): 12±2 vs 26±2 p=0.01). Interestingly, numerous multi-nucleated osteoclasts were observed in the bone marrow of leukemic mice and were localized adjacent to leukemic cells, suggesting that leukemic cells may affect osteoclastogenesis and result in massive bone loss. To test this hypothesis, we first measured the expression of known regulators of osteoclastogenesis, including RANKL, in our leukemic cells by quantitative RT-PCR analysis. Compared to GFP−/YFP− cells, GFP+/YFP+ cells have 3-fold increased expression of RANKL, a major osteoclastogenic cytokine. We then examined if leukemic cells can give rise to osteoclasts in the presence of RANKL and M-CSF in vitro and found that these cells were unable to differentiate into osteoclasts themselves. To determine if leukemic cells can induce osteoclastogenesis of normal osteoclast progenitors, we cocultured spleen-derived osteoclast precursors from wild-type mice with GFP+/YFP+ leukemic cells or GFP−/YFP− non-leukemic cells in osteoclastic differentiating media containing optimal concentrations of M-CSF and RANKL. As expected, there was abundant formation of mature osteoclasts, identified as TRAP+ multinucleated cells, in control cultures containing non-leukemic cells and osteoclast precursors. Leukemic cells significantly increased TRAP+ mono-nucleated osteoclast precursors (No. TRAP+ mononucleated cells/well: 34±3.3 vs 20±6.0 in non-leukemic cells, p=0.0136). Under this culture condition, we did not observe increased mature osteoclast formation by leukemic cells. Surprisingly, we found that osteoclast precursors strongly prolonged the survival of leukemia cells. In control cultures without a feeder layer of osteoclast precursors there were no viable leukemia cells present after 6 days in culture while in the co-culture system viable leukemia cells were still abundant after 6 days in culture, identifiable by their expression of GFP/YFP (No. GFP+/YFP+/high power field: 0 vs 142±6.4, p<0.01). In summary, in a murine model of BC-CML, there was a rapid loss of trabecular bone and an increase in the number of osteoclasts. Expression of osteoclast-regulating cytokines in leukemic cells favored osteoclastogenesis, however modified cells did not themselves give rise to osteoclasts, while increasing the population of normal immature osteoclasts. In turn, osteoclast progenitors prolonged survival of leukemic cells in vitro. Taken together, these data strongly suggest reciprocal synergistic interactions of leukemic cells with osteoclast progenitors in the bone microenvironment. These previously unrecognized interactions could be exploited to modify disease progression, providing a novel approach for leukemia treatment.
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Chen, Shi, Alexander Robling, Xiaohong Li, Jin Yuan, Janet Hock, David A. Ingram, D. Wade Clapp und Fengchun Yang. „Hyperactivation of p21ras and PI3-K Cooperate To Alter Murine and Human NF1 Haploinsufficient Osteoclast Functions.“ Blood 108, Nr. 11 (16.11.2006): 675. http://dx.doi.org/10.1182/blood.v108.11.675.675.
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Abstract Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase activating protein for Ras. Though nullizygous loss of NF1 is associated with the development of malignancies, haploinsufficient phenotypes are now being increasingly recognized to alter cell fates and functions in a number of tissues resulting in nonmalignant disease manifestations. Bone manifestations, including skeletal dysplasias, scoliosis, and osteoporosis occur in 30–60% of all NF1 patients and osteoporosis is an increasingly recognized health problem for women with NF1. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone resorbing cells of the skeleton. Using an established murine model of NF1 developed using homologous recombination, we found that Nf1+/− mice contain elevated numbers of multinucleated osteoclasts and osteoclast progenitors per femur in vivo. Both osteoclasts and osteoclast progenitors from Nf1+/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL, growth factors that are integral to osteoclast maturation and activation. M-CSF stimulated p21ras-GTP and Akt phosphorylation was elevated in Nf1+/− osteoclasts associated with gains in function in survival and proliferation. Bone resorption by osteoclasts is linked to the migration and adherence of these cells to a local bone surface. Purified populations of Nf1+/− osteoclasts were initially placed in the upper chamber of a transwell coated with vitronectin and haptotaxis to M-CSF was determined. Nf1+/− osteoclasts had a 2–3 fold increase in migration as compared to syngeneic wildtype cells. A similar increase in adhesion of Nf1+/− osteoclasts to the integrin avb3 was also observed. Following adhesion, osteoclasts form a specialized cell-extracellular matrix to initiate degradation of bone matrix by secreting proteinases. Nf1+/− osteoclasts had significantly increased bone resorption as measured by scoring the number and area of individual bone resorbing “pits” on dentine slices and by scoring the total area of resorption. These collective increases in osteoclast function were validated in vivo by the observation that serum TRAP5b activity, a sensitive measure of osteoclast lytic activity was 2.5 fold higher in Nf1+/− mice as compared to WT mice. Furthermore, we performed ovariectomy, an established model of osteoporosis associated with an increase in osteoclast function. In two independent experiments, we found that Nf1+/− mice had a significantly greater reduction in bone mineral density following ovariectomy as compared to syngeneic wildtype mice. We hypothesized that hyperactivation of class1A-PI3-K may contribute to these gains in cellular function. We found that intercrossing Nf1+/− and Class1A PI3-K deficient mice (p85a) restores elevated PI3-K activity, and Nf1+/− osteoclast functions to wildtype levels. Furthermore, in vitro differentiated osteoclasts from NF1 patients also display elevated Ras-PI3-K activity and increased lytic activity analogous to murine Nf1+/− osteoclasts. Collectively, we identify a novel cellular and biochemical NF1 haploinsufficient phenotype in osteoclasts that has potential implications in the pathogenesis of NF1 bone disease.
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Baek, Jong Min, Ju-Young Kim, Yoon-Hee Cheon, Sun-Hyang Park, Sung-Jun Ahn, Kwon-Ha Yoon, Jaemin Oh und Myeung Su Lee. „Dual Effect ofChrysanthemum indicumExtract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and ResorptionIn Vitro“. Evidence-Based Complementary and Alternative Medicine 2014 (2014): 1–13. http://dx.doi.org/10.1155/2014/176049.
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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 ofChrysanthemum indicumextract (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.