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Journal articles on the topic 'Osteoclast inhibition'
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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, and 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, no. 2 (November 14, 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
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, no. 4 (August 18, 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.
3
Zavrski, Ivana, Monica Hecht, Holger Krebbel, Claudia Fleissner, Maren Mieth, Martin Kaiser, Ulrike Heider, et al. "Bortezomib Inhibits Human Osteoclastogenesis." Blood 108, no. 11 (November 16, 2006): 1395. http://dx.doi.org/10.1182/blood.v108.11.1395.1395.
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Abstract Enhanced osteoclastogenesis in cancer-induced bone disease may be caused by intercellular interactions between tumor cells and cells of the bone marrow microenvironment. In multiple myeloma, overexpression of RANKL in the bone marrow microenvironment may lead to the activation of TRAF-signaling and in consequence to increased NF-κB and AP-1 transcriptional activities in osteoclastic lineage cells. This results in enhanced osteoclast differentiation, activation and increased bone resorption. In this study, we have examined the effects of two NF-κB inhibitors towards their inhibitory potency on human osteoclastogenesis: proteasome inhibitor bortezomib and selective IKK inhibitor PS-1145. CD14+ osteoclast precursors from peripheral blood were stimulated with RANKL and M-CSF up to four weeks. Using MTT- and TUNEL-assays, cytotoxicity levels of each drug were determined on the differentiation stage day +1 (early osteoclast precursors), day +8 (preosteoclasts) and day +21 (osteoclasts). To evaluate the effects of both drugs considering osteoclast differentiation and -function, 2 sub-apoptotic doses of bortezomib, one sub-apoptotic and one low-apoptotic dose of PS-1145 were used. As revealed by the microscopic quantification of mature osteoclasts (TRAP-positive and multi-nucleated cells), the osteoclast differentiation was diminished by both drugs, whereas the effects were dose- and time-dependent. The microscopic quantification of resorption lacunae on dentine pits revealed that the resorptional activity was reduced by 65% for 0.1 nM bortezomib (p=0.007), by 79% for 1 nM bortezomib (p<0.0005), by 60% for 1 μM PS-1145 (p=0.023) and by 91% for 10 μM PS-1145 (p<0.0005). As shown by immunoblotting and by ELISA-based methods, the subcellular mechanisms of action involved in inhibition of early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase (MAPK) pathways, whereas the advanced differentiation and activation occurred in course of inhibition of AP-1 and NF-κB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor (VEGF) production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutical principle of cancer-induced lytic bone disease.
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Lentzsch, Suzanne, Gulsum Anderson, Noriyoshi Kurihara, Tadashi Honjo, Judith Anderson, Markus Y. Mapara, David Stirling, and David Roodman. "Thalidomide Derivative CC-4047 Inhibits Osteoclast Formation by down Regulation of PU.1." Blood 106, no. 11 (November 16, 2005): 629. http://dx.doi.org/10.1182/blood.v106.11.629.629.
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Abstract CC-4047 (Actimid) is an immunomodulatory analog of thalidomide that has stronger anti-myeloma and anti-angiogenic activity than thalidomide, but its effects on human osteoclast lineage are unknown. Early osteoclast progenitors are of hematopoietic origin and progressively differentiate into mature bone resorbing multinucleated osteoclasts. We investigated the effects of CC-4047 and thalidomide on human osteoclastogenesis, using in vitro receptor activator of NFκ-B ligand/M-CSF stimulated culture system of bone marrow cells. Three weeks of treatment of primary bone marrow cultures with 100 μM CC-4047 decreased osteoclast formation accompanied by complete inhibition of bone resorption. Interestingly, osteoclast formation was also inhibited when cultures were treated with CC-4047 only for the first week (90% inhibition). In contrast, inhibitory effect was greatly diminished when the drug was given for only the last week (25% inhibition), indicating that inhibition of osteoclast formation is an early event. The inhibitory effect of CC-4047 on osteoclastogenesis was not induced by cell death, but by a shift of lineage commitment to granulocyte-CFU at the expense of GM-CFU that are osteoclast progenitors. Further studies revealed that this shift is mediated through down regulation of the transcription factor PU.1, which is critical for early osteoclast formation. In contrast to CC-4047, thalidomide was a significantly less potent inhibitor of osteoclast formation and bone resorption. These results provide the first evidence that CC-4047 blocks osteoclast differentiation at the early phase of osteoclastogenesis. Therefore, CC-4047 might be a valuable drug targeting both the tumor and osteoclastic activity in patients with multiple myeloma and potentially other diseases associated with the development of osteolytic lesions.
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Klein-Nulend, J., M. A. van Duin, T. P. Green, V. Everts, and 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, no. 18_suppl (June 20, 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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Bouyer, Patrice, Hiroaki Sakai, Takashi Itokawa, Tsutomu Kawano, Christiaan M. Fulton, Walter F. Boron, and Karl L. Insogna. "Colony-Stimulating Factor-1 Increases Osteoclast Intracellular pH and Promotes Survival via the Electroneutral Na/HCO3 Cotransporter NBCn1." Endocrinology 148, no. 2 (February 1, 2007): 831–40. http://dx.doi.org/10.1210/en.2006-0547.
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Colony-stimulating factor-1 (CSF-1) promotes the survival of osteoclasts, short-lived cells that resorb bone. Although a rise in intracellular pH (pHi) has been linked to inhibition of apoptosis, the effect of CSF-1 on pHi in osteoclasts has not been reported. The present study shows that, in the absence of CO2/HCO3−, CSF-1 causes little change in osteoclast pHi. In contrast, exposing these cells to CSF-1 in the presence of CO2/HCO3− causes a rapid and sustained cellular alkalinization. The CSF-1-induced rise in pHi is not blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, an inhibitor of HCO3− transporters but is abolished by removing extracellular sodium. This inhibition profile is similar to that of the electroneutral Na/HCO3 cotransporter NBCn1. By RT-PCR, NBCn1 transcripts are present in both osteoclasts and osteoclast-like cells (OCLs), and by immunoblotting, the protein is present in OCLs. Moreover, CSF-1 promotes osteoclast survival in the presence of CO2/HCO3− buffer but not in its absence. Preventing the activation of NBCn1 markedly attenuates the ability of CSF-1 to 1) block activation of caspase-8 and 2) prolong osteoclast survival. Inhibiting caspase-3 or caspase-8 in OCLs prolongs osteoclast survival to the same extent as does CSF-1. This study provides the first evidence that osteoclasts express a CSF-1-regulated Na/HCO3 cotransporter, which may play a role in cell survival.
7
Brooks, Kalia, C. Ireland, Beeton, and Rushton. "Direct Inhibition of Osteoclast Formation and Activity by the Vitamin E Isomer gamma-Tocotrienol." International Journal for Vitamin and Nutrition Research 81, no. 6 (November 1, 2011): 358–67. http://dx.doi.org/10.1024/0300-9831/a000087.
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Vitamin E homologues, specifically tocotrienols, have been shown to have favorable effects on bone. They possess properties that are indicative of anti-resorptive activity, suggesting the potential for vitamin E in preventing bone loss. To investigate the anti-resorptive activity of the various vitamin E homologues, we cultured human osteoclasts from blood-derived CD14+ cells on collagen, dentin, and calcium phosphate substrates, with some samples supplemented with vitamin E homologues in their cell culture medium. These were compared to the clinically used bisphosphonate, pamidronate. Compounds were either added at the start of culture to study effects on osteoclast formation, or at the start of osteoclastic resorption to determine their effects on activity. The alpha- and gamma-tocotrienol isomers inhibited osteoclast formation without consequent reduction in total cell number. Only gamma-tocotrienol inhibited osteoclast activity without toxicity. Gamma-tocotrienol was the most potent inhibitor of both osteoclast formation and activity and requires further investigation into its anti-resorptive effects on bone.
8
Anderson, Gülsüm, Margarete Gries, Noriyoshi Kurihara, Tadashi Honjo, Judy Anderson, Vera Donnenberg, Albert Donnenberg, et al. "Thalidomide derivative CC-4047 inhibits osteoclast formation by down-regulation of PU.1." Blood 107, no. 8 (April 15, 2006): 3098–105. http://dx.doi.org/10.1182/blood-2005-08-3450.
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Abstract CC-4047, an immunomodulatory analog of thalidomide, inhibits multiple myeloma with unknown effects on the human osteoclast lineage. Early osteoclast progenitors are of hematopoietic origin and differentiate into mature bone resorbing multinucleated osteoclasts. We investigated the effects of CC-4047 and thalidomide on human osteoclastogenesis, using in vitro receptor activator of NFκ-B ligand/macrophage colony-stimulating factor–stimulated bone marrow cell cultures. Treating bone marrow cultures with CC-4047 for 3 weeks decreased osteoclast formation accompanied by complete inhibition of bone resorption. The inhibitory effect was similar when cultures were treated for 3 weeks or for only the first week (90% inhibition), indicating that CC-4047 inhibits early stages of osteoclast formation. Inhibition of osteoclastogenesis by CC-4047 was mediated by a shift of lineage commitment to granulocyte colony-forming units at the expense of granulocyte-macrophage colony-forming units. Further studies revealed that this shift in lineage commitment was mediated through down-regulation of PU.1. Treatment with thalidomide resulted in significantly less potent inhibition of osteoclast formation and bone resorption. These results provide evidence that CC-4047 blocks osteoclast differentiation during early phases of osteoclastogenesis. Therefore, CC-4047 might be a valuable drug for targeting both tumors and osteoclastic activity in patients with multiple myeloma and other diseases associated with osteolytic lesions.
9
Kim, Hyo Jeong, and Youngkyun Lee. "Endogenous Collagenases Regulate Osteoclast Fusion." Biomolecules 10, no. 5 (May 1, 2020): 705. http://dx.doi.org/10.3390/biom10050705.
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The precise regulation of osteoclast differentiation and function is crucial for the maintenance of healthy bone. Despite several reports of collagenase expression in bone tissues, the precise isoform expression as well as the role in osteoclasts are still unclear. In the present report, the expression of matrix metalloprotease (MMP)8 and MMP13 was confirmed in mouse bone marrow macrophage osteoclast precursors. The mRNA and protein expressions of both collagenases were significantly reduced by receptor activator of nuclear factor κB ligand (RANKL) stimulation. Notably, either inhibition of MMP expression by siRNA or treatment of cells with collagenase inhibitor Ro 32-3555 significantly augmented osteoclast fusion and resorption activity without affecting the osteoclast number. The inhibition of collagenase by Ro 32-3555 increased the expression of osteoclast fusion genes, Atp6v0d2 and Dcstamp, without affecting nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) protein expression. The enhanced osteoclast fusion by collagenase inhibition appears to be mediated through an extracellular signal regulated kinase (ERK)-dependent pathway. Collectively, these data provide novel information on the regulation of osteoclast fusion process.
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Masarachia, Patricia, Michiko Yamamoto, Chih-Tai Leu, Gideon Rodan, and Le Duong. "Histomorphometric Evidence for Echistatin Inhibition of Bone Resorption in Mice with Secondary Hyperparathyroidism." Endocrinology 139, no. 3 (March 1, 1998): 1401–10. http://dx.doi.org/10.1210/endo.139.3.5828.
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Echistatin, an RGD-containing peptide, was shown to inhibit the acute calcemic response to exogenous PTH or PTH-related protein (PTH-rP) in thyroparathyroidectomized rats, suggesting that echistatin inhibits bone resorption. In this study: 1) we present histological evidence for echistatin inhibition of bone resorption in mice with secondary hyperparathyroidism, and show that 2) echistatin binds to osteoclasts in vivo, 3) increases osteoclast number, and 4) does not detectably alter osteoclast morphology. Infusion of echistatin (30μ g/kg·min) for 3 days prevented the 2.6-fold increase in tibial cancellous bone turnover and the 36% loss in bone volume, produced by a low calcium diet. At the light microscopy level, echistatin immunolocalized to osteoclasts and megakaryocytes. Echistatin treatment increased osteoclast-covered bone surface by about 50%. At the ultrastructural level, these osteoclasts appeared normal, and the fraction of cells containing ruffled borders and clear zones was similar to controls. Echistatin was found on the basolateral membrane and in intracellular vesicles of actively resorbing osteoclasts. Weak labeling was found in the ruffled border, and no immunoreactivity was detected at the clear zone/bone surface interface. These findings provide histological evidence for echistatin binding to osteoclasts and for inhibition of bone resorption in vivo, through reduced osteoclast efficacy, without apparent changes in osteoclast morphology.
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Carron, CP, DM Meyer, VW Engleman, JG Rico, PG Ruminski, RL Ornberg, WF Westlin, and GA Nickols. "Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone." Journal of Endocrinology 165, no. 3 (June 1, 2000): 587–98. http://dx.doi.org/10.1677/joe.0.1650587.
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Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.
12
Zaidi, Mone. "Modularity of osteoclast behaviour and “mode-specific” inhibition of osteoclast function." Bioscience Reports 10, no. 6 (December 1, 1990): 547–56. http://dx.doi.org/10.1007/bf01116615.
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This study is part of an attempt to understand the role of specific cellular activities in the bone resorptive process. Experiments were performed whereby known pharmacological agents were used to inhibit individual modes of osteoclastic activity, such as motility and secretion. The effects of such treatments on bone resorption were assessed by quantitative scanning electron microscopy. The compounds included colchicine, which was used to inhibit osteoclast motility; molybdate ions which were used to selectively inhibit the catalytic activity of secreted acid phosphatase, and omeprazole which was employed to inhibit the secretion of hydrogen ions. All compounds inhibited osteoclastic bone resorption, but singularly affected defined modes of activity. These findings suggest that each mode of osteoclastic activity is essential for the bone resorptive process, and that “mode-specific” inhibition may provide a means whereby excessive activity of the osteoclast can be regulated in disease.
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Holliday, L. S., A. D. Dean, R. H. Lin, J. E. Greenwald, and S. L. Gluck. "Low NO concentrations inhibit osteoclast formation in mouse marrow cultures by cGMP-dependent mechanism." American Journal of Physiology-Renal Physiology 272, no. 3 (March 1, 1997): F283—F291. http://dx.doi.org/10.1152/ajprenal.1997.272.3.f283.
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High concentrations of nitric oxide (NO) inhibit bone resorption by mature osteoclasts. We examined the effects of low NO concentrations on osteoclast formation in mouse bone marrow cultures. The NO releasers sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine inhibited the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (a marker for osteoclasts) when administered during the last 3 days of 6-day cultures (differentiation stage) but not during the first 3 days (proliferation stage). SNP (1 microM) completely inhibited pit formation on dentine wafers when added to cultures during osteoclast formation, but 100 microM SNP was required to inhibit pitting by mature osteoclasts. Conversely, the NO synthase inhibitors aminoguanidine and nitro-L-arginine methyl ester both increased osteoclast formation. Inhibition of osteoclast formation by NO likely was guanosine 3',5'-cyclic monophosphate (cGMP) dependent, as SNP increased cGMP in marrow cultures, and 1 mM 8-bromo-cGMP or dibutyryl-cGMP reduced osteoclast formation when administered during the differentiation stage. The cGMP-specific type V phosphodiesterase inhibitor, zaprinast (M & B 22948) also inhibited osteoclast formation (half-maximal inhibitory constant, 100 microM) only when added during the differentiation stage. We conclude that the differentiation stage of osteoclast formation is inhibited by increases in cGMP levels elicited by NO.
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Boissy, Patrice, Thomas Lund, Thomas L. Andersen, Torben Plesner, and Jean-Marie Delaisse. "Bortezomib Transiently Inhibits Osteoclast Resorptive Activity in Cell Culture Conditions Mimicking In Vivo Intermittent Treatment." Blood 108, no. 11 (November 16, 2006): 3508. http://dx.doi.org/10.1182/blood.v108.11.3508.3508.
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Abstract Multiple myeloma (MM) leads to high risk for bone pain and fractures. MM-induced bone disease is due to acute degradation of bone matrix by osteoclasts, and absence of repair by bone forming osteoblasts. It is currently treated with bisphosphonates, highly effective bone resorption inhibitors, which do not promote but rather inhibit bone formation and may cause renal damage and osteonecrosis of the jaw. Thus, it is important to reconsider the management of MM bone disease in long-term treatment. Recent preclinical studies reported that the proteasome inhibitor Bortezomib (V) used for the treatment of MM patients can stimulate bone formation, and that in MM patients treated with V, serum levels of bone formation markers are increased. The present study aims at investigating if V may inhibit osteoclast activity. Methods: Osteoclasts were differentiated from pure populations of blood derived CD14-positive monocytes cultured with M-CSF and RANKL for 6–7 days, and treated continuously with V at various concentrations. As prolonged inhibition of proteasome activity has been reported to be toxic for any cell type, and in vivo pharmacodynamic studies have shown V to be eliminated from the vascular compartment as soon as 30min after intravenous injection, displaying maximal inhibitory activity of the proteasome within 24 hours subsiding rapidly thereafter, V was also given intermittently, to mimick the in vivo situation. Osteoclast differentiation and activity were assessed by measuring Tartrate-Resistant Acid Phosphatase (TRACP) activity in the medium. Cell viability was determined with Celltiter Blue measuring metabolic activity. To extend our observations to the clinical situation, serum levels of CTX-I, a bone resorption marker, were measured during the 3 days following therapeutic V administration in a single patient. Results: A continuous treatment of cultures with V at 4 nM and higher concentrations proved to be highly toxic for differentiating osteoclasts but also monocytes. A 3-hour-pulse treatment with V followed by a 3-day culture in the absence of V, was not toxic neither to monocytes nor to osteoclasts, even at a concentration as high as 100 nM. This 3-hour pulse was however highly toxic for myeloma cells. Interestingly, a 3-hour pulse with 25 nM V induced a 50% inhibition of the resorptive activity of osteoclasts, as assessed by culturing them for 3 days on bone slices and measuring the formation of resorption pits. The release of TRACP in the medium was inhibited to a similar extent within the first 24 hours post-pulse, but tended to return to the control level during the next 2 days. This 3-hour pulse with 25 nM V inhibited strongly RANKL-induced translocation of NF-KB in the osteoclast nuclei, an event dependent on proteasome function and critical for osteoclastic activity. Serum CTX-I levels decreased during the first 48 hours after each V injection (n = 3), and tended to increase again after 72 hours suggesting a partial recovery of osteoclast activity between each administration. Conclusions: Our results suggest that Bortezomib temporarily inhibits osteoclast activity in vitro and in vivo. This effect is linked to RANKL-induced translocation of NF-KB in the osteoclast nuclei and proteasome function. Since recent reports suggested that formation of new bone requires at least a transient activity of osteoclasts transient inhibition of osteoclasts could be an advantage compared to the more persistent inhibition of osteoclast activity by bisphosphonate.
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Arnett, T. R., R. Lindsay, J. M. Kilb, B. S. Moonga, M. Spowage, and D. W. Dempster. "Selective toxic effects of tamoxifen on osteoclasts: comparison with the effects of oestrogen." Journal of Endocrinology 149, no. 3 (June 1996): 503–8. http://dx.doi.org/10.1677/joe.0.1490503.
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Abstract We investigated the actions of the trans- and cis-isomers of tamoxifen on the function of neonatal rat osteoclasts in vitro. Both compounds inhibited resorption pit formation by osteoclast-containing mixed bone cell cultures incubated for 24 h on cortical bone slices. Cell counts revealed that the inhibition was closely related to a cytotoxic effect, to which osteoclasts appeared particularly sensitive. Partial inhibition of resorption was seen in the presence of 2 μm trans-tamoxifen, whereas complete abolition of resorption and osteoclast viability occurred with 10 μm trans-tamoxifen; survival of mononuclear cells was unimpaired at either concentration. Cis-tamoxifen appeared to be slightly more toxic, with significant inhibitions of osteoclast viability and thus resorption pit formation at a concentration of 2 μm, and also of mononuclear cell numbers at 10 μm. Time-lapse video observations indicated that osteoclast death occurred rapidly (within 2–3 h) following exposure to 10 μm of either trans-tamoxifen or cis-tamoxifen. The morphological appearance of the dying cells was consistent with apoptosis. These results may help to explain the anti-resorptive action of tamoxifen seen in vivo in rats and humans. In contrast, oestradiol-17β consistently exerted no significant effects on resorption pit formation by rat osteoclasts over 24 h, even at grossly supraphysiological concentrations (up to 10 μm). Journal of Endocrinology (1996) 149, 503–508
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Nakamura, I., M. F. Pilkington, P. T. Lakkakorpi, L. Lipfert, S. M. Sims, S. J. Dixon, G. A. Rodan, and L. T. Duong. "Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone." Journal of Cell Science 112, no. 22 (November 15, 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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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, no. 1 (July 1, 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.
18
Seidlitz, Eric P., Mohit K. Sharma, and Gurmit Singh. "Extracellular glutamate alters mature osteoclast and osteoblast functions." Canadian Journal of Physiology and Pharmacology 88, no. 9 (September 2010): 929–36. http://dx.doi.org/10.1139/y10-070.
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Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.
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Kim, Jung Ha, Kabsun Kim, Inyoung Kim, Semun Seong, Hyun Kook, Kyung Keun Kim, Jeong-Tae Koh, and Nacksung Kim. "Bifunctional Role of CrkL during Bone Remodeling." International Journal of Molecular Sciences 22, no. 13 (June 29, 2021): 7007. http://dx.doi.org/10.3390/ijms22137007.
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Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals.
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Pennanen, Paula, Roope A. Kallionpää, Sirkku Peltonen, Liisa Nissinen, Veli-Matti Kähäri, Eetu Heervä, and Juha Peltonen. "Signaling pathways in human osteoclasts differentiation: ERK1/2 as a key player." Molecular Biology Reports 48, no. 2 (January 24, 2021): 1243–54. http://dx.doi.org/10.1007/s11033-020-06128-5.
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AbstractLittle is known about the signaling pathways involved in the differentiation of human osteoclasts. The present study evaluated the roles of the Ras/PI3K/Akt/mTOR, Ras/Raf/MEK1/2/ERK1/2, calcium-PKC, and p38 signaling pathways in human osteoclast differentiation. Mononuclear cells were isolated from the peripheral blood of control persons and patients with neurofibromatosis 1 (NF1), and the cells were differentiated into osteoclasts in the presence of signaling pathway inhibitors. Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase 5B. Inhibition of most signaling pathways with chemical inhibitors decreased the number of human osteoclasts and disrupted F-actin ring formation, while the inhibition of p38 resulted in an increased number of osteoclasts, which is a finding contradictory to previous murine studies. However, the p38 inhibition did not increase the bone resorption capacity of the cells. Ras-inhibitor FTS increased osteoclastogenesis in samples from control persons, but an inhibitory effect was observed in NF1 samples. Inhibition of MEK, PI3K, and mTOR reduced markedly the number of NF1-deficient osteoclasts, but no effect was observed in control samples. Western blot analyses showed that the changes in the phosphorylation of ERK1/2 correlated with the number of osteoclasts. Our results highlight the fact that osteoclastogenesis is regulated by multiple interacting signaling pathways and emphasize that murine and human findings related to osteoclastogenesis are not necessarily equivalent.
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Robinson, Lisa J., Salvatore Mancarella, Irina L. Tourkova, John B. Barnett, Harry C. Blair, and Jonathan Soboloff. "Critical Role for the Calcium-Release Activated Calcium Channel Orai1 In RANKL-Stimulated Osteoclast Formation From Monocytic Cells." Blood 116, no. 21 (November 19, 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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Moreno, Jose L., Michele Kaczmarek, Achsah D. Keegan, and Mehrdad Tondravi. "IL-4 suppresses osteoclast development and mature osteoclast function by a STAT6-dependent mechanism: irreversible inhibition of the differentiation program activated by RANKL." Blood 102, no. 3 (August 1, 2003): 1078–86. http://dx.doi.org/10.1182/blood-2002-11-3437.
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Abstract Numerous reports have described the effects of interleukin-4 (IL-4) on bone biology. Previous studies, performed using complex coculture systems, demonstrated the effects of IL-4 on osteoblasts and osteoclasts. To directly test the effect of IL-4 on osteoclasts, we took advantage of a simplified system using recombinant receptor activator of nuclear factor κB ligand (RANKL) as the osteoclast differentiation factor. We analyzed the ability of IL-4 to directly regulate osteoclast differentiation and mature osteoclast function. We found that IL-4 inhibited the differentiation of osteoclasts from bone marrow precursors in an irreversible manner and also inhibited the resorptive capacity of mature osteoclasts. In the presence of IL-4, we detected the appearance of tartrate-resistant acid phosphatase (TRAP)–negative multinucleated giant (MNG) cells. Both IL-4 effects were dependent on signal transducer and activator of transcription 6 (STAT6). We found that IL-4 suppresses RANK mRNA expression in the developing precursor cells. When RANK was ectopically expressed under the cytomegalovirus (CMV) promoter in RAW264.7 macrophages, IL-4 treatment did not inhibit osteoclast development. Furthermore, when osteoclastogenesis was induced independently of RANKL by using tumor necrosis factor-α (TNF-α), IL-4 inhibited osteoclast differentiation through a STAT6-dependent mechanism. These results suggest that IL-4 regulates osteoclast development by regulating gene expression, including RANK. We propose that IL-4 irreversibly regulates the lineage commitment of precursor cells by regulating gene expression, resulting in the suppression of osteoclast development and the generation of MNG cells as an alternative pathway of differentiation.
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Lin, Shang Chien, Sheng Yu Kao, Nien Tzu Keng, and Wei Chung Liu. "Resveratrol Inhibition Osteoclastogenesis Induced by RANKL through Decrease Preosteoclast Formation." Advanced Materials Research 647 (January 2013): 124–28. http://dx.doi.org/10.4028/www.scientific.net/amr.647.124.
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It has been demonstrated that resveratrol can inhibit osteoclastogenesis induced by receptor activator of nuclear factor kappaB ligand (RANKL) in several cell models. However, the mechanism has not yet been completely clarified. In this study, we investigated the effects of resveratrol on osteoclasts differentiation induced by RANKL. Preosteoclast or osteoclast derived from the murine monocytic cell line RAW264.7 treated with RANKL. In RAW264.7 cells culture, data indicated that resveratrol at non-toxic concentrations dose-dependently inhibited the formation of osteoclasts and the activation of tartrate-resistant acid phosphatase (TRAP). Using flow cytometry assay, the results indicated that the percentage of preosteoclast differentiation was decreased by resveratrol, whereas the apoptosis rate of preosteoclasts was not changed. Our results suggest that resveratrol might inhibit the differentiation of RAW264.7 cells into osteoclasts and decrease osteoclast activation possibly via suppressing monocytes to differentiate preosteoclasts.
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Shanmugarajan, Srinivasan, Craig C. Beeson, and Sakamuri V. Reddy. "Osteoclast Inhibitory Peptide-1 Binding to the FcγRIIB Inhibits Osteoclast Differentiation." Endocrinology 151, no. 9 (July 7, 2010): 4389–99. http://dx.doi.org/10.1210/en.2010-0244.
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Osteoclast inhibitory peptide-1 (OIP) is an autocrine/paracrine inhibitor of osteoclast differentiation, and mice that overexpress OIP-1 in osteoclast lineage cells develop an osteopetrosis bone phenotype. In this study, we show that OIP-1 binding to the Fcγ receptor IIB (FcγRIIB) inhibits osteoclast differentiation. Confocal microscopy revealed colocalization of OIP-1 with FcγRIIB in osteoclasts, and we observed that OIP-1 carboxy-terminal GPI-linked peptide forms a 1:1 complex with recombinant FcγRIIB protein with an affinity binding of a dissociation constant of approximately 4 μm. Immunoreceptor tyrosine-based activation motif (ITAM)-bearing adapter proteins (FcRγ and DNAX-activating protein of molecular mass 12 kDa) are critical for osteoclast development, and OIP-1 transgenic mouse-derived preosteoclast cells demonstrated suppression (6-fold) of ITAM phosphorylation of FcRγ but not DNAX-activating protein of molecular mass 12 kDa. Interestingly, these preosteoclast cells demonstrated increased levels (4-fold) of immunoreceptor tyrosine-based inhibitory motif phosphorylation of FcγRIIB and Src homology 2-domain-containing proteins tyrosine phosphatase 1 activation. Further, OIP-1 mouse-derived preosteoclasts cells demonstrated inhibition of spleen tyrosine kinase activation (4.5-fold), compared with wild-type mice. These results suggest that cross-regulation of immunoreceptor tyrosine-based inhibitory motif and ITAM bearing Fc receptors may play a role in OIP-1 suppression of spleen tyrosine kinase activation and inhibition of osteoclast differentiation. Thus, OIP-1 is an important physiologic regulator of osteoclast development and may have therapeutic utility for bone diseases with high bone turnover.
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Odes-Barth, Shlomit, Marina Khanin, Karin Linnewiel-Hermoni, Yifat Miller, Karina Abramov, Joseph Levy, and Yoav Sharoni. "Inhibition of Osteoclast Differentiation by Carotenoid Derivatives through Inhibition of the NF-κB Pathway." Antioxidants 9, no. 11 (November 23, 2020): 1167. http://dx.doi.org/10.3390/antiox9111167.
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The bone protective effects of carotenoids have been demonstrated in several studies, and the inhibition of RANKL-induced osteoclast differentiation by lycopene has also been demonstrated. We previously reported that carotenoid oxidation products are the active mediators in the activation of the transcription factor Nrf2 and the inhibition of the NF-κB transcription system by carotenoids. Here, we demonstrate that lycopene oxidation products are more potent than intact lycopene in inhibiting osteoclast differentiation. We analyzed the structure–activity relationship of a series of dialdehyde carotenoid derivatives (diapocarotene-dials) in inhibiting osteoclastogenesis. We found that the degree of inhibition depends on the electron density of the carbon atom that determines the reactivity of the conjugated double bond in reactions such as Michael addition to thiol groups in proteins. Moreover, the carotenoid derivatives attenuated the NF-κB signal through inhibition of IκB phosphorylation and NF-κB translocation to the nucleus. In addition, we show a synergistic inhibition of osteoclast differentiation by combinations of an active carotenoid derivative with the polyphenols curcumin and carnosic acid with combination index (CI) values < 1. Our findings suggest that carotenoid derivatives inhibit osteoclast differentiation, partially by inhibiting the NF-κB pathway. In addition, carotenoid derivatives can synergistically inhibit osteoclast differentiation with curcumin and carnosic acid.
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Crockett, Julie C., Norbert Schütze, Denise Tosh, Susanne Jatzke, Angela Duthie, Franz Jakob, and Michael J. Rogers. "The Matricellular Protein CYR61 Inhibits Osteoclastogenesis by a Mechanism Independent of αvβ3 and αvβ5." Endocrinology 148, no. 12 (December 1, 2007): 5761–68. http://dx.doi.org/10.1210/en.2007-0473.
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Cysteine-rich protein 61 (CYR61/CCN1) belongs to the family of CCN matricellular proteins. Most of the known effects of CCN proteins appear to be due to binding to extracellular growth factors or integrins, including αvβ3 and αvβ5. Although CYR61 can stimulate osteoblast differentiation, until now the effect of CYR61 on osteoclasts was unknown. We demonstrate that recombinant human CYR61 inhibits the formation of multinucleated, αvβ3-positive, or tartrate-resistant acid phosphatase-positive human, mouse, and rabbit osteoclasts in vitro. CYR61 markedly reduced the expression of the osteoclast phenotypic markers tartrate-resistant acid phosphatase, matrix metalloproteinase-9, calcitonin receptor, and cathepsin K. However, CYR61 did not affect the formation of multinucleated osteoclasts when added to osteoclast precursors prior to fusion or affect the number or resorptive activity of osteoclasts cultured on dentine discs, indicating that CYR61 affects early osteoclast precursors but not mature osteoclasts. CYR61 did not affect receptor activator of nuclear factor-κB (RANK) ligand-induced phosphorylation of p38 or ERK1/2 in human macrophages and did not affect RANK ligand-induced activation of nuclear factor-κB, indicating that CYR61 does not appear to inhibit osteoclastogenesis by affecting RANK signaling. Furthermore, a mutant form of CYR61 defective in binding to αvβ3 also inhibited osteoclastogenesis, and CYR61 inhibited osteoclastogenesis similarly in cultures of mouse wild-type or β5−/− macrophages. Thus, CYR61 does not appear to inhibit osteoclast formation by interacting with αvβ3 or αvβ5. These observations demonstrate that CYR61 is a hitherto unrecognized inhibitor of osteoclast formation, although the exact mechanism of inhibition remains to be determined. Given that CYR61 also stimulates osteoblasts, CYR61 could represent an important bifunctional local regulator of bone remodeling.
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Kim, Mi, Won Kim, Jae-Eun Byun, Jung Choi, Suk Yoon, Inpyo Choi, and Haiyoung Jung. "Inhibition of Osteoclastogenesis by Thioredoxin-Interacting Protein-Derived Peptide (TN13)." Journal of Clinical Medicine 8, no. 4 (March 29, 2019): 431. http://dx.doi.org/10.3390/jcm8040431.
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Overactivated osteoclasts lead to many bone diseases, including osteoporosis and rheumatoid arthritis. The p38 MAPK (p38) is an essential regulator of the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and bone loss. We previously reported TAT conjugated thioredoxin-interacting protein-derived peptide (TAT-TN13) as an inhibitor of p38 in hematopoietic stem cells (HSCs). Here, we examined the role of TAT-TN13 in the differentiation and function of osteoclasts. TAT-TN13 significantly suppressed RANKL-mediated differentiation of RAW 264.7 cells and bone marrow macrophages (BMMs) into osteoclasts. TAT-TN13 also inhibited the RANKL-induced activation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), leading to the decreased expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP) and Cathepsin K. Additionally, TAT-TN13 treatment protected bone loss in ovariectomized (OVX) mice. Taken together, these results suggest that TAT-TN13 inhibits osteoclast differentiation by regulating the p38 and NF-κB signaling pathway; thus, it may be a useful agent for preventing or treating osteoporosis.
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Lentzsch, Suzanne, Gulsum Anderson, Cuiling Li, Chandra P. Belani, Markus Y. Mapara, and David Roodman. "Combination of Proteasome Inhibitor PS 341 (Velcade®) with Histone Acetylase Inhibitor (HDAC) PXD 101 Shows Superior Anti-Myeloma Activity and Inhibits Osteoclastogenesis." Blood 106, no. 11 (November 16, 2005): 2488. http://dx.doi.org/10.1182/blood.v106.11.2488.2488.
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Abstract Background: PS 341 has demonstrated activity in multiple myeloma (MM), and PXD 101 is a new HDAC inhibitor, currently being evaluated in Phase II clinical trials for MM. In addition to the anti-myeloma activity of PS 341 and PXD 101, HDAC inhibitors are strong inhibitors of osteoclast formation. Therefore, the combination of the two agents, targeting both the tumor and the development of osteolytic lesions should lead to an optimal treatment strategy for MM. Methods: MM cell lines (MM.1S, OPM 2, RPMI 8226) were exposed to serial dilutions of PS 341 (1–1000 nM) and PXD 101 (10–1000 nM) for 48 hours. Thymidine uptake was measured to assess inhibition of proliferation. To evaluate the effects of PS 341 and PXD 101 on the formation of osteoclasts from mononuclear hematopoietic precursors, we used a human bone marrow culture system from healthy donors and MM patients to generate osteoclasts using in vitro RANKL/M-CSF stimulation. Results: Dose-dependent inhibition of proliferation was achieved in all MM cell lines after a 48-hour treatment with PS 341 and PXD 101. For further experiments we used PS 341 at a fixed low concentration (10 nM), which showed no significant inhibition of proliferation of MM cell lines. PXD 101 was used at 100 nM and demonstrated median inhibition of 46% (range 0–48%). The combination of PS 341 with PXD 101 induced synergism with inhibition of proliferation of median 73% (range 44–73%). Although 100 nM PXD 101 and 50 nM PS 341 used as single agents were required to inhibit the proliferation of MM cells, combination of 25 nM PXD 101 and 1 nM PS 341 was sufficient to inhibit OCL formation completely, indicating that the drug combination results also in a highly synergistic inhibition of osteoclast formation. Conclusions: These studies provide the framework for clinical evaluation of PS 341 combined with PXD 101 in patients with MM and supports the hypothesis that the drug combination targets both the tumor and osteoclastic activity. (Drugs provided by NIH/NCI; Support: UO1 CA099168)
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Smieszek, Agnieszka, Klaudia Marcinkowska, Ariadna Pielok, Mateusz Sikora, Lukas Valihrach, and Krzysztof Marycz. "The Role of miR-21 in Osteoblasts–Osteoclasts Coupling In Vitro." Cells 9, no. 2 (February 19, 2020): 479. http://dx.doi.org/10.3390/cells9020479.
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MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor κB ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts’ differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-κB ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.
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Feng, Rentian, Xiangao Huang, Les Coulton, Hendrik De Raeve, Maurizio DiLiberto, Scott Ely, Guozhi Xiao, et al. "Targeting CDK4/CDK6 Impairs Osteoclast Progenitor Pool Expansion and Blocks Osteolytic Lesion Development in Multiple Myeloma." Blood 114, no. 22 (November 20, 2009): 298. http://dx.doi.org/10.1182/blood.v114.22.298.298.
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Abstract Abstract 298 Background: Multiple myeloma (MM) is characterized by increased osteoclast activity resulting in bone destruction and development of lytic lesions. PD0332991 is a selective small molecule inhibitor of cyclin-dependent kinase (CDK)4 and CDK6 with oral bioavailability. Recently we demonstrated that inhibition of CDK4/CDK6 by PD0332991 effectively controls MM tumor expansion in animal models and sensitizes MM for cytotoxic killing (Baughn et al, Cancer Res. 2006; Menu et al, Cancer Res. 2008; Huang et al, unpublished). Currently clinical phase I/II trials are ongoing to test the efficacy of the combination of PD0332991 and bortezomib. In vivo data further indicate that PD0332991 preferentially targets tumor cells and rapidly cycling bone marrow cells. This led us to investigate the possibility that PD0332991 may also inhibit osteoclastogenesis via restricting progenitor cell expansion and MM-induced bone destruction. PD0332991 significantly (p<0.01) decreased the number of lytic lesions by 81%, in addition to reducing tumor burden in the bone marrow of immunocompetent 5T2MM murine model. In a dose-dependent manner, PD0332991 inhibited osteoclastogenesis and the fusion of osteoclasts in human (IC50 <50 nM) marrow cultures in vitro. Importantly, treatment with PD0332991 for the first week, but not the second or third week, was sufficient to inhibit osteoclast formation. These data suggest that PD0332991 acts preferentially on the early stage of OCL development. This was confirmed by a reduction of osteoclast precursor colonies (CFU-M, CFU-GM) under PD0332991 treatment, due to inhibition of DNA synthesis and diminished expansion of the osteoclast progenitor pool. The basis for the inhibition of osteoclast precursor proliferation was G1 cell cycle arrest following inhibition of CDK4/CDK6-specific phosphorylation of Rb by PD0332991, but not cell death, as evidenced by the intact cell morphology and absence of caspase activation. The combination of PD0332991 and bortezomib synergistically abrogated human osteoclast formation. Further, our in vivo and in vitro data showed that PD0332991 has no effects on osteoblastogenesis or genes inducing osteoblast development including Bsp, Ocn, and Runx2. Conclusions: Collectively, our data suggest that by inducing G1 arrest in osteoclast precursors and inhibiting the osteoclast progenitor pool expansion, PD0332991 is a powerful and selective treatment for MM-induced osteolytic bone lesions. We propose that targeting CDK4/CD6 with PD0332291 in combination therapy is a promising therapeutic strategy to both suppress tumor expansion and improve bone integrity in MM. Disclosures: Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.
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Tenshin, Hirofumi, Jumpei Teramachi, Asuka Oda, Ryota Amachi, Masahiro Hiasa, Ariunzaya Bat-Erdene, Keiichiro Watanabe, et al. "TAK1 inhibition subverts the osteoclastogenic action of TRAIL while potentiating its antimyeloma effects." Blood Advances 1, no. 24 (October 26, 2017): 2124–37. http://dx.doi.org/10.1182/bloodadvances.2017008813.
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Key Points TRAIL enhances receptor activator of NF-κB ligand–induced osteoclastogenesis and c-FLIP upregulation without osteoclast apoptosis induction. TAK1 inhibition triggers TRAIL-induced apoptosis in osteoclasts, while potentiating TRAIL-induced myeloma cell death.
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Fuller, K., and T. J. Chambers. "Localisation of mRNA for collagenase in osteocytic, bone surface and chondrocytic cells but not osteoclasts." Journal of Cell Science 108, no. 6 (June 1, 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.
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Hwang, Youn-Hwan, Taesoo Kim, Rajeong Kim, and Hyunil Ha. "The Natural Product 6-Gingerol Inhibits Inflammation-Associated Osteoclast Differentiation via Reduction of Prostaglandin E2 Levels." International Journal of Molecular Sciences 19, no. 7 (July 16, 2018): 2068. http://dx.doi.org/10.3390/ijms19072068.
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The natural product 6-gingerol, a major bioactive component of the rhizome of ginger (Zingiber officinale), is known to have several beneficial effects on health, including anti-inflammatory activity. The present study aimed to investigate the effects of 6-gingerol on osteoclast differentiation associated with inflammation. 6-Gingerol inhibited osteoclast differentiation in co-cultures of osteoblasts and osteoclast precursor cells in response to the pro-inflammatory cytokine, interleukin (IL)-1. However, it did not affect osteoclast precursor differentiation into osteoclasts induced by the receptor activator of nuclear factor-κB ligand (RANKL), a key cytokine causing osteoclast differentiation. 6-Gingerol inhibited IL-1-induced RANKL expression in osteoblasts, and the addition of RANKL to the co-cultures overcame 6-gingerol-mediated inhibition of osteoclast differentiation. It also suppressed IL-1-induced prostaglandin E2 (PGE2) production in osteoblasts, and the addition of exogenous PGE2 reversed 6-gingerol-mediated inhibition of IL-induced RANKL expression in osteoblasts and osteoclast differentiation in the co-cultures. We found that 6-gingerol reduced PGE2 levels by suppressing enzymatic activities of cyclooxygenase and PGE synthase, which cooperatively catalyze the conversion of arachidonic acid to PGE2. Our findings demonstrate that 6-gingerol inhibits IL-1-induced osteoclast differentiation via suppression of RANKL expression in osteoblasts though reduction of PGE2 levels, suggesting its potential use in treating inflammatory bone destruction associated with excessive PGE2 production.
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Baek, Jong Min, Ju-Young Kim, Yoon-Hee Cheon, Sun-Hyang Park, Sung-Jun Ahn, Kwon-Ha Yoon, Jaemin Oh, and 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.
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Knowles. "The Adenosine A2B Receptor Drives Osteoclast-Mediated Bone Resorption in Hypoxic Microenvironments." Cells 8, no. 6 (June 21, 2019): 624. http://dx.doi.org/10.3390/cells8060624.
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Osteoclast-mediated bone destruction is amplified in the hypoxic synovial microenvironment of rheumatoid arthritis (RA). This increased bone resorption is driven by the hypoxia-inducible transcription factor HIF. We identified hypoxic induction of the HIF-regulated adenosine A2B receptor in primary human osteoclasts (mRNA, 3.8-fold increase, p < 0.01) and sought to identify the role(s) of purinergic signaling via this receptor in the bone resorption process. Primary human osteoclasts were differentiated from CD14+ monocytes and exposed to hypoxia (2% O2) and A2B receptor inhibitors (MRS1754, PSB603). The hypoxic increase in bone resorption was prevented by the inhibition of the A2B receptor, at least partly by the attenuation of glycolytic and mitochondrial metabolism via inhibition of HIF. A2B receptor inhibition also reduced osteoclastogenesis in hypoxia by inhibiting early cell fusion (day 3–4, p < 0.05). The A2B receptor is only functional in hypoxic or inflammatory environments when the extracellular concentrations of adenosine (1.6-fold increase, p < 0.05) are sufficient to activate the receptor. Inhibition of the A2B receptor under normoxic conditions therefore did not affect any parameter tested. Reciprocal positive regulation of HIF and the A2B receptor in a hypoxic microenvironment thus enhances glycolytic and mitochondrial metabolism in osteoclasts to drive increased bone resorption. A2B receptor inhibition could potentially prevent the pathological osteolysis associated with hypoxic diseases such as rheumatoid arthritis.
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Kuramoto, Kazuya, Ayumi Kodama, Junko Homan, Yohei Nakaya, Toshio Kitamura, and Haruna Naito. "Dual Inhibition of the STAT3 and Src Pathways by NS-018, a JAK2 and Src-Family Kinase Inhibitor, Reduces Myeloma Cell Proliferation and Adhesion and Suppresses Osteoclast Formation." Blood 118, no. 21 (November 18, 2011): 2900. http://dx.doi.org/10.1182/blood.v118.21.2900.2900.
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Abstract Abstract 2900 Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells in the bone marrow, and it finally leads to osteolytic bone destruction and impaired hematopoiesis. The pathophysiology of MM is closely linked with the bone marrow microenvironment, which consists of various cell components including bone marrow stromal cells (BMSCs) and bone marrow endothelial cells as well as osteoclasts and osteoblasts. Interaction between MM cells and BMSCs through cell-adhesion molecules confers drug resistance to myeloma cells and stimulates the release of cytokines such as interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) from BMSCs. IL-6 is a major cytokine which enhances cell proliferation and promotes the survival of MM cells by downstream signaling through Janus kinase (JAK) and signal transducer and activator of transcription (STAT). RANKL triggers osteoclast differentiation and activation leading to bone resorption, lytic bone lesion, and osteopenia. The Src family kinases (SFKs), c-Src and Fyn, mediate signaling by cell-adhesion molecules and the RANKL receptor, and they play important roles in cell adhesion and osteoclast differentiation. NS-018 is a potent and selective dual JAK2/SFKs inhibitor which is under phase1/2 clinical development for the treatment of myelofibrosis. A previous study has shown that NS-018 inhibits JAK2, c-Src and Fyn kinases with IC50 values of 0.72, 6.0 and 7.3 nmol/L, respectively. NS-018 also inhibits JAK2 and SFKs in cells as evidenced by its antiproliferative effect against Ba/F3 cells expressing constitutively activated JAK2 and NIH3T3 cells transformed by v-Src. In the present study, the ability of NS-018 to inhibit JAK2/STAT3 signaling was examined in IL-6-responsive human MM cell lines such as U266, RPMI 8226, and PCM6. NS-018 suppressed IL-6-induced phosphorylation of STAT3 in a dose-dependent manner at concentrations greater than 100 nmol/L. In addition, NS-018 inhibited the IL-6-enhanced the proliferation of PCM6 cells at concentrations similar to those required to inhibit STAT3 phosphorylation. To assess whether SFKs inhibition by NS-018 could contribute to an improvement in MM pathology, we next investigated the effect of NS-018 on the adhesion of myeloma cells to cell-adhesion molecules and on osteoclast formation. NS-018 (100 nmol/L) inhibited the adhesion of RPMI 8226 cells to collagen type 1 and VCAM-1 by about 40%. NS-018 also suppressed RANKL-induced differentiation of human osteoclast precursor cells to mature osteoclasts. The numbers of TRAP-positive multinucleated osteoclasts were reduced to about one-half at 100 nmol/L NS-018 and none were observed at 1000 nmol/L. NS-018 similarly suppressed the differentiation of murine RAW264.7 cells to mature osteoclasts. The suppression of cell adhesion and osteoclast formation by NS-018 could both be mediated by c-Src and/or Fyn inhibition, because both inhibitory effects were observed with a typical SFKs inhibitor but not with a typical JAK2 inhibitor. In conclusion, NS-018 reduced IL-6-enhanced myeloma cell proliferation through inhibition of the JAK2/STAT3 signaling pathway and suppressed cell adhesion and osteoclast formation through inhibition of c-Src and/or Fyn. These results suggest that NS-018 has a dual mechanism of action in MM by simultaneously blocking the JAK2/STAT3 and SFKs pathways. Treatment with NS-018 is a potential new therapeutic option to improve the complex pathological condition of patients with MM. Disclosures: No relevant conflicts of interest to declare.
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Chen, Shi, Jincheng Yan, Yingze Zhang, Yan Li, Xiaohong Li, Jin Yuan, Selina Eskwick, et al. "Rac1, but Not Rac2, Mediates Osteoclast Gain-of-Function Induced by Nf1 Haploinsufficiency." Blood 110, no. 11 (November 16, 2007): 237. http://dx.doi.org/10.1182/blood.v110.11.237.237.
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Abstract Neurofibromatosis type I (NF1) is a congenital disorder resulting from loss-of-function of the tumor suppressor gene, NF1. 50% of NF1 patients have osseous manifestations, including short stature, scoliosis, and reduced bone mineral density. Osteoclasts are hematopoietic stem cell-derived cells that function to resorb bone. We recently reported that osteoclasts derived from NF1 patients and Nf1 heterozygous (Nf1+/−) mice have elevated migration, adhesion, and bone resorption and our studies indicate that the gain-of-function of the Nf1+/− osteoclasts is, at least in part, caused by hyperactivation of macrophage colony-stimulating factor (M-CSF)-stimulated Ras, phosphoinositol-3-kinase (PI3K), and Erk. Rho GTPases function downstream of Ras and PI3K and act as binary switches, cycling between an inactive (GDP-bound) and active (GTP-bound) state, to regulate osteoclast actin ring formation, bone resorption, and development of filamentous actin structures associated with migration and adhesion. We hypothesized that hyperactivation of Rac1, Rac2, or both Rac1 and Rac2 contribute to the increased osteoclast function observed in Nf1+/− mice and NF1 patients. To examine this hypothesis, we intercrossed Nf1+/− mice with conditional Rac1flox/floxMxcre+ mice or with Rac2−/− mice to generate WT, Nf1+/−, Rac1−/−, Nf1+/−;Rac1−/−, Rac2−/−, and Nf1+/−;Rac2−/− mice. Genetic disruption of Rac1, but not of Rac2, restored the increased colony forming unit-macrophage (CFU-M), tatrate resistant acid phosphate+ (TRAP+) CFU-M, osteoclast migration, and bone resorption observed in Nf1+/− cultures. Osteoclast bone resorbing capacity is dependent on the organization of the actin cytoskeleton into a large f-actin-rich structure referred to as the sealing zone. The podosome belt evolves into the sealing zone in actively resorbing osteoclasts. A significantly higher level of belt formation, seen in mature osteoclasts, was observed in Nf1+/− cultures as compared to WT. Upon genetic deletion of Rac1, the Nf1+/−;Rac1−/− osteoclasts demonstrated belt formation at a similar level to that of WT osteoclasts. These data indicate that Rac1 plays an essential role in functional f-actin organization and suggest that inhibition of Rac1 in the setting of Nf1 haploinsufficiency is able to normalize osteoclast hyperactivity by correcting the cytoskeletal organization of f-actin-based structures. Mechanistically, Rac1 deficiency normalized M-CSF-stimulated phospho-Erk and phospho-Akt and pharmacologic inhibition of MEK and PI3K using PD98059 or Ly294002, respectively, normalized Nf1+/− osteoclast development and maturation. The critical role of Rac1, but not of Rac2, in osteoclast function is significant as it suggests that the Rac GTPases contribute non-redundant functions in various myeloid cell types and imply that blocking Rac1 function, while sparing that of Rac2, may provide a level of specificity to therapeutics for skeletal diseases. Collectively, these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1 and imply that Rac1 may be a rational therapeutic target for dysplastic and erosive bone diseases.
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Vallet, Soni a., Nileshwari Vaghela, Mariateresa Fulciniti, Petter Veiby, Teru Hideshima, Samantha Pozzi, Loredana Santo, et al. "CCR1 Inhibition Targets Osteoclast-Myeloma Adhesion and Exerts in Vivo Anti-Osteoclast Activity." Blood 112, no. 11 (November 16, 2008): 2757. http://dx.doi.org/10.1182/blood.v112.11.2757.2757.
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Abstract Among the components of the tumor microenvironment, osteoclasts (OC) and bone marrow stromal cells (BMSC) play a key role in stimulating multiple myeloma (MM) cell proliferation via cell-to-cell contact and cytokine secretion. In a vicious loop, MM cells promote OC development and impair osteoblasts (OB) differentiation shaping the microenvironment to support tumor growth and chemoresistance. These effects translate in osteolytic lesions that negatively affect patient quality of life and survival. CCL3 is involved in the pathogenesis of MM bone disease. Indeed, high bone marrow plasma levels of CCL3 correlate with osteolytic lesions in MM and CCL3 has a pro-osteoclastogenic effect. We have previously shown that inhibition of CCL3 receptor, CCR1, results in impaired osteoclastogenesis and blocks the proliferative advantage conferred by OCs to MM cells in vitro. (Vallet et al, Blood 2007) Here, we extend our studies and demonstrate in an in-vivo SCID-hu mouse model the anti-OC effects of MLN3897, a small molecule CCR1 inhibitor (Millennium Pharmaceuticals, Cambridge). CB17 SCID mice bearing a human fetal bone implant engrafted with INA6 MM cells were treated orally with MLN3897 for a total of 49 doses. After 4 weeks of treatment the bones were harvested and stained for TRAP activity and hematoxylin-eosin. The number of OC/400x field was significantly reduced in the treated group (2.7 vs 1.9, p<0.05), thus confirming in vivo the anti-osteoclastogenic effect of CCR1 inhibition. Although a 2-fold inhibition of tumor growth in the treated group was noted, this difference did not reach statistical significance. Our previous data suggested that CCR1 inhibition impaired OC-MM cell adhesion with subsequent reduced MM cell proliferation. Here, we observed a specific effect of CCR1 inhibition on MM cell/OC adhesion rather than BMSCs and fibronectin. Interestingly, these effects translated in anti-proliferative effects of the CCR1 inhibitor in OC-MM cells coculture with no effects in BMSCs-MM coculture, suggesting a specific inhibitory effect on the OC compartment. Ongoing studies to characterize the underlying mechanism of CCR1 inhbition on OC-MM cell adhesion will be presented. Combination approaches to study whether forced OB differentiation by the proteasome inhibitor, bortezomib, will improve BMSC sensitivity to CCR1 inhibition are being studied. These data confirm the specific in vivo anti-OC effects of CCR1 inhibition and will highlight the role of novel combinations by using anti-OC agents like MLN3897 and anabolic drugs like Bortezomib.
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Kim, Tae-Ho, Chae Gyeong Jeong, Hyeong-U. Son, Man-Il Huh, Shin-Yoon Kim, Hong Kyun Kim, and Sang-Han Lee. "Ethanolic Extract of Rubus coreanus Fruits Inhibits Bone Marrow-Derived Osteoclast Differentiation and Lipopolysaccharide-Induced Bone Loss." Natural Product Communications 12, no. 12 (December 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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Choi, Mun Hwan, Kyunghee Lee, Mi Yeong Kim, Hong-In Shin, and Daewon Jeong. "Pisidium coreanum Inhibits Multinucleated Osteoclast Formation and Prevents Estrogen-Deficient Osteoporosis." International Journal of Molecular Sciences 20, no. 23 (December 2, 2019): 6076. http://dx.doi.org/10.3390/ijms20236076.
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Mollusks have served as important sources of human food and medicine for a long time. Raw Pisidium coreanum, a freshwater bivalve of the phylum Mollusca, is used in traditional therapies in parts of Asia. However, the therapeutic effects of Pisidium coreanum on bone diseases are not known. We investigated the functional roles of Pisidium coreanum in osteoporotic bone diseases. Pisidium coreanum inhibited the differentiation of bone marrow-derived monocytic cells into mature osteoclasts in vitro. The ovariectomized mice that received oral administration of Pisidium coreanum showed improvements in both trabecular and cortical bones. This preventive activity of Pisidium coreanum against bone loss was due to limited osteoclast maturation with reduced osteoclast surface extent in trabecular bone tissue. The formation of large multinucleated osteoclasts in vitro was significantly decreased in response to Pisidium coreanum, consistent with the reduced expression levels of osteoclast markers and fusion-related genes, such as NFATc1, p65, integrin αvβ3, DC-STAMP, OC-STAMP, Atp6v0d2, FAK, CD44, and MFR. These data suggest that Pisidium coreanum inhibits osteoclast differentiation by negatively regulating the fusion of mononuclear osteoclast precursors. Thus, our data demonstrate the ability of Pisidium coreanum to effectively prevent estrogen-deficient osteoporosis through inhibition of multinucleated osteoclast formation.
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Khadeer, Mohammed A., Zhihui Tang, Harriet S. Tenenhouse, Maribeth V. Eiden, Heini Murer, Natividad Hernando, Edward J. Weinman, Meenakshi A. Chellaiah, and Anandarup Gupta. "Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions." American Journal of Physiology-Cell Physiology 284, no. 6 (June 1, 2003): C1633—C1644. http://dx.doi.org/10.1152/ajpcell.00580.2002.
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We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Picotransporters in the osteoclast plasma membrane provide Pifor ATP synthesis and that the osteoclast may utilize part of the Pireleased from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Picotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Picotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Picotransporters in the osteoclast.
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Nguyen, John, Semaj Kelly, Ryan Wood, Brian Heubel, and Anja Nohe. "A Synthetic Peptide, CK2.3, Inhibits RANKL-Induced Osteoclastogenesis through BMPRIa and ERK Signaling Pathway." Journal of Developmental Biology 8, no. 3 (July 9, 2020): 12. http://dx.doi.org/10.3390/jdb8030012.
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The skeletal system plays an important role in the development and maturation process. Through the bone remodeling process, 10% of the skeletal system is renewed every year. Osteoblasts and osteoclasts are two major bone cells that are involved in the development of the skeletal system, and their activity is kept in balance. An imbalance between their activities can lead to diseases such as osteoporosis that are characterized by significant bone loss due to the overactivity of bone-resorbing osteoclasts. Our laboratory has developed a novel peptide, CK2.3, which works as both an anabolic and anti-resorptive agent to induce bone formation and prevent bone loss. We previously reported that CK2.3 mediated mineralization and osteoblast development through the SMAD, ERK, and AKT signaling pathways. In this study, we demonstrated the mechanism by which CK2.3 inhibits osteoclast development. We showed that the inhibition of MEK by the U0126 inhibitor rescued the osteoclast development of RAW264.7 induced by RANKL in a co-culture system with CK2.3. We observed that CK2.3 induced ERK activation and BMPRIa expression on Day 1 after stimulation with CK2.3. While CK2.3 was previously reported to induce the SMAD signaling pathway in osteoblast development, we did not observe any changes in SMAD activation in osteoclast development with CK2.3 stimulation. Understanding the mechanism by which CK2.3 inhibits osteoclast development will allow CK2.3 to be developed as a new treatment for osteoporosis.
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Feng, Rentian, Ana B. Oton, Kenneth Patrene, Gulsum Anderson, Markus Y. Mapara, Chandra Belani, David Roodman, and Suzanne Lentzsch. "Combination of the Proteasome Inhibitor Bortezomib and a Histone Deacetylase Inhibitor PXD101 Results in Synergistic Inhibition of Osteoclastogenesis and Significantly Stronger Inhibition of Multiple Myeloma Growth In Vitro and In Vivo." Blood 108, no. 11 (November 16, 2006): 507. http://dx.doi.org/10.1182/blood.v108.11.507.507.
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Abstract Bortezomib, a proteasome inhibitor, has been known to have in vitro and in vivo activity against multiple myeloma (MM). PXD101, a novel, low molecular weight histone deacetylase (HDAC) inhibitor recently has been recognized to induce growth arrest and apoptosis in MM cells. We hypothesized that these two agents, with different molecular targets, can be combined with a high probability of additive or synergistic effects without exerting cross-resistance. In this study, we investigated the activity of the combination of PXD101 and bortezomib against osteoclast generation and MM cell growth. To evaluate the effects of bortezomib and PXD101 on the formation of osteoclasts from mononuclear hematopoietic precursors, we generated osteoclasts cultured from human bone marrow cells from healthy donors and MM patients using in vitro RANKL/M-CSF stimulation. Proliferation of MM cell lines treated with PXD101, bortezomib, or their combination was determined by measuring 3H-Thymidine incorporation. Annexin V-FITC/PI staining was used to determine the degree of apoptosis, and trypan blue was used to assess cell viability. We utilized a xenograft murine model to test the in vivo synergistic activity of the two agents against MM tumor formation. We found that even though either drug alone significantly inhibited osteoclast formation at concentrations of PXD101 at 25 nM or bortezomib at 1 nM, the combination resulted in synergistic inhibition in osteoclast assays. Treatment of MM cells lines (MM.1S, RPMI-8226, OPM2) with serial dilutions (10–100 nM) of the combination also led to significantly stronger inhibition of proliferation, apoptosis and cell death than single drug treatment. In addition, the combination demonstrated synergistic induction of cell death in primary human CD138+ myeloma cells isolated from patient samples. In vivo, the combination of PXD101 and bortezomib resulted in significantly higher xenograft tumor inhibition compared to either drug treatment alone and was not more toxic. The current study provides the rationale for clinical evaluation of this novel PXD101-bortezomib combination for patients with MM. The data generated here support the hypothesis that simultaneous targeting of different proliferative and anti-apoptotic pathways both in tumor cells and activated osteoclasts avoids cross-resistance and constitutes a dynamic approach to treat MM.
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Karkache, Ismael Y., Jeyaram R. Damodaran, David H. H. Molstad, Kim C. Mansky, and Elizabeth W. Bradley. "Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice." International Journal of Molecular Sciences 22, no. 18 (September 8, 2021): 9702. http://dx.doi.org/10.3390/ijms22189702.
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Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1−/− cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.
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Wani, Mohan R., Karen Fuller, Nack Sung Kim, Yongwon Choi, and Tim Chambers. "Prostaglandin E2 Cooperates with TRANCE in Osteoclast Induction from Hemopoietic Precursors: Synergistic Activation of Differentiation, Cell Spreading, and Fusion." Endocrinology 140, no. 4 (April 1, 1999): 1927–35. http://dx.doi.org/10.1210/endo.140.4.6647.
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Abstract It was recently found that osteoblastic cells express TRANCE (tumor necrosis factor-related activation-induced cytokine), a newly identified member of the tumor necrosis factor superfamily, and that expression was increased by calciotropic hormones. Furthermore, soluble recombinant TRANCE induces osteoclast formation and resorption in stroma-free populations of hemopoietic precursor cells. However, overexpression of the decoy receptor osteoprotegerin in vivo shows that there are substantial differences in the sensitivity of different sites to resorption-inhibition, suggesting that either alternative ligands exist or the sensitivity of osteoclasts to TRANCE can be modified by cofactors. We therefore tested the possibility that cofactors might enhance osteoclast formation by TRANCE. We found that the number of tartrate-resistant acid phosphatase-positive and calcitonin receptor-positive cells was increased by a factor of 10 by the presence of PGE2 in the absence of stromal cells. Moreover, although the tartrate-resistant acid phosphatase-positive cells that formed in TRANCE alone were typically mononuclear and poorly spread, the addition of PGE2 induced the formation of large, well spread multinuclear cells. There was an increase in bone resorption that corresponded with the increase in osteoclast number. PGE2 did not synergize with TRANCE for resorption-stimulation in mature cells. 8-Bromo-cAMP showed a similar syngergistic effect on osteoclastic differentiation. Thus, PGE2 appears to stimulate bone resorption through a direct effect on hemopoietic precursors, primarily through a synergistic effect on the ability of TRANCE to induce osteoclastic differentiation.
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Filipović, M., A. Šućur, D. Flegar, Z. Jajić, M. Ikić Matijašević, N. Lukač, N. Kovačić, et al. "POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 226.3–226. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2601.
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Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.
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Lymperi, Stefania, Adel Ersek, Francesca Ferraro, Francesco Dazzi, and Nicole J. Horwood. "Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo." Blood 117, no. 5 (February 3, 2011): 1540–49. http://dx.doi.org/10.1182/blood-2010-05-282855.
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Abstract Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin−cKit+Sca1+ Flk2− (LKS Flk2−) and long-term culture–initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.
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Maurer, Thomas, Gerald Zimmermann, Susanne Maurer, Sabine Stegmaier, Christof Wagner, and G. Maria Hänsch. "Inhibition of Osteoclast Generation: A Novel Function of the Bone Morphogenetic Protein 7/Osteogenic Protein 1." Mediators of Inflammation 2012 (2012): 1–9. http://dx.doi.org/10.1155/2012/171209.
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Monocytes have the potential to differentiate to either macrophages, dendritic cells, or to osteoclasts. The microenvironment, particularly cytokines, directs the monocyte differentiation. Receptors of NFκB (RANK) ligand, tumor necrosis factor (TNF)α, or interleukin- (IL-) 8 have be identified as inducers of osteoclastogenesis, whereas others, such as IL-10 or transforming growth factor (TGF)ßinhibit osteoclast generation or induce differentiation towards a dendritic cell type. We now describe that bone morphogenetic protein (BMP) 7/osteogenic protein- (OP-) 1 inhibited the differentiation of human CD14+ monocytes to osteoclasts. In the presence of BMP7/OP-1 the transcription factors c-Fos and NFATc1, though upregulated and translocated to the nucleus in response to either RANKL or IL-8, did not persist. In parallel, MafB, a transcription factor expressed by monocytes and required for differentiation to macrophages but inhibiting osteoclast generation, was preserved. Because both persistence of NFATc1 and downregulation of MafB are crucial for osteoclastogenesis, we conclude that BMP7/OP-1 inhibits the generation of osteoclasts by interfering with signalling pathways.
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Acharya, Chirag, Mike Y. Zhong, Yolanda Calle, Stephen Schey, Michelle Chen, Daniel Tannenbaum, Michaela R. Reagan, et al. "CRM1 Inhibition Abrogates Osteoclast Formation and Bone Resorption Via Inhibition of RANKL-Induced NFκB While Sparing Osteoblastogenesis: Further Therapeutic Implication in Multiple Myeloma." Blood 120, no. 21 (November 16, 2012): 1835. http://dx.doi.org/10.1182/blood.v120.21.1835.1835.
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Abstract Abstract 1835 Blocking CRM1 by novel selective inhibitors of nuclear export (SINE: KPT-185, KPT-251, KPT-276, and KPT-330) induced potent MM cell apoptosis in vitro and in vivo (Abstract #46829). In addition, these compounds inhibited NFkB p65 DNA-binding activity in MM cells. Here, we investigated whether SINEs have effects on bone and whether this is mediated only through anti-MM activity, or additional effects directly on osteoclasts (OC) are in play. We asked whether SINEs could prevent RANKL/M-CSF-induced osteoclastogenesis via blockade of NFkB activation. Mature OC (TRAP+ multinucleated cell) were derived from the CD138-negative cell fraction from MM patient samples (n=4) stimulated with RANKL/M-CSF for 3 weeks, in the presence or absence of KPT-185. KPT-185 significantly blocked formation of TRAP+ multinucleated OC in a dose-dependent manner, further confirmed by reduction of the selective osteoclastic marker TRAP5b in cell culture supernatant. NFkB p65 activity was induced in nuclear extracts of CD14+ OC precursor cells following RANKL stimulation for 30 min. Importantly, KPT-185 and KPT-330 blocked such induction in a dose-dependent manner. When KPT-185 was added 2 weeks following OC differentiation by RANKL/M-CSF, the effects of KPT-185 on osteoclast culture were not as prominent as when drug was added from the onset. Immunofluorescence staining to examine the actin cytoskeleton in OC cultures performed on glass cover slips further confirmed that actin belt formation in mature OCs is required for bone resorption activity. In the presence of KPT-185 or KPT-330, such critical structure was significantly decreased, consistent with diminished mature OC number and reduced TRAP5b. Pit formation assays on dentine slices clearly showed that KPT-185 and KPT-330, as low as 10 nM, inhibited % erosion area when compared with control group (p<0.005). In RANKL-activated preosteoclasts, both compounds further blocked expression levels of NFATC1, the key osteoclast differentiation transcription factor, as well as fusion-related (Atp6v0d2 and DC-STAMP) and adhesion (integrin αv and integrin β3) molecules. We also assessed the effect of SINE on osteogenesis derived from mesenchymal stem cells of normal healthy donors (n=3). Neither KPT-276 nor KPT-330 blocked calcium deposition, an indicator of bone formation in in vitro culture. Moreover, KPT-185 did not alter INA6 MM cell-inhibited calcium deposition of osteoblasts. Thus, SINEs specifically blocked osteoclast formation and bone resorption activity without significantly impacting osteogenesis. This is the first study to demonstrate a novel role of CRM1 regulating osteoclast formation at least in part by blocking NFkB activity triggered in osteoclast precursor cells by RANKL stimulation. Decreased NFkB p65 activity essential for osteoclast differentiation and fusion was associated with suppressed bone resorption. The potent MM cytotoxicity and prolonged host survival (p=0.0004) demonstrated in our disseminated SCID mouse model of human MM (Abstract#46829), coupled with these bone effects, provide the framework for clinical trials targeting CRM1 with SINEs to simultaneously inhibit both tumor progression and bone destruction in MM. Disclosures: Ghobrial: Millennium pharmaceuticals Inc.: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.
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Ghayor, Chafik, Rita M. Correro, Katrin Lange, Lindsay S. Karfeld-Sulzer, Klaus W. Grätz, and Franz E. Weber. "Inhibition of Osteoclast Differentiation and Bone Resorption by N-Methylpyrrolidone." Journal of Biological Chemistry 286, no. 27 (May 25, 2011): 24458–66. http://dx.doi.org/10.1074/jbc.m111.223297.
Full textAbstract:
Regulation of RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption.