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Background: Mori Radicis Cortex (MRC) is the root bark of the mulberry family as Morus alba L. In Korea, it is known as "Sangbaegpi". Although MRC has demonstrated anti-inflammatory and antioxidant effects, its specific mechanisms of action and impact on osteoporosis remain poorly understood., Methods: To investigate the antiosteoporosis effect of MRC, we examined the level of osteoclast differentiation inhibition in receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced-RAW 264.7 cells and animal models of ovariectomy (OVX) with MRC. Serum analysis in OVX animals was investigated by enzyme-linked immunosorbent assay (ELISA), and bone density analysis was confirmed by micro-computed tomography (micro-CT). The expression analysis of nuclear factor of activated T cells 1 (NFATc1) was confirmed by immunohistochemistry (IHC) in femur tissue. In addition, osteoclast differentiation inhibition was measured using tartrate-resistant acid phosphatase (TRAP). mRNA analysis was performed using reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression analysis was investigated by western blot., Results: Micro-CT analysis showed that MRC effectively inhibited bone loss in the OVX-induced rat model. MRC also inhibited the expression of alkaline phosphatase (ALP) and TRAP in serum. Histological analysis showed that MRC treatment increased bone density and IHC analysis showed that MRC significantly inhibited the expression of NFATc1. In RANKL-induced-RAW 264.7 cells, MRC significantly reduced TRAP activity and actin ring formation. In addition, MRC significantly inhibited the expression of NFATc1 and c-Fos, and suppressed the mRNA expression., Conclusion: Based on micro-CT, serum and histological analysis, MRC effectively inhibited bone loss in an OVX-induced rat model. In addition, MRC treatment suppressed the expression of osteoclast differentiation, fusion, and bone resorption markers through inhibition of NFATc1/c-Fos expression in RANKL-induced RAW 264.7 cells, ultimately resulting in a decrease in osteoclast activity. These results demonstrate that MRC is effective in preventing bone loss through inhibiting osteoclast differentiation and activity., Competing Interests: Conflicts of interest: The authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article., (Copyright © 2024, the Chinese Medical Association.)

Osteoclasts, derived from the monocyte/macrophage line of bone marrow hematopoietic stem cell progenitors, are the sole bone-resorbing cells of the body. Conventional osteoclast differentiation requires macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) signaling. Rheumatoid arthritis (RA) is the most prevalent systemic autoimmune disease and inflammatory arthritis characterized by bone destruction. Increased levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), in the serum and joints, cause excessive bone destruction. We have recently reported that stimulation of human peripheral blood monocytes with TNF-α and IL-6 induces the differentiation of osteoclasts with bone resorption activity. This review presents the functional differences between representative osteoclasts, conventional RANKL-induced osteoclasts, and recently identified proinflammatory cytokine (TNF-α and IL-6)-induced osteoclasts in RA patients. We believe novel pathological osteoclasts associated with RA will be identified, and new therapeutic strategies will be developed to target these osteoclasts and prevent the progression of bone destruction.

Osteoclasts are multinucleated bone-resorbing cells derived from the monocyte/macrophage lineage. The macrophage colony-stimulating factor/receptor activator of nuclear factor κB ligand (M-CSF/RANKL) signaling network governs the differentiation of precursor cells into fusion-competent mononucleated cells. Repetitive fusion of fusion-competent cells produces multinucleated osteoclasts. Osteoclasts are believed to die via apoptosis after bone resorption. However, recent studies have found that osteoclastogenesis in vivo proceeds by replacing the old nucleus of existing osteoclasts with a single newly differentiated mononucleated cell. Thus, the formation of new osteoclasts is minimal. Furthermore, the sizes of osteoclasts can change via cell fusion and fission in response to external conditions. On the other hand, osteoclastogenesis in vitro involves various levels of heterogeneity, including osteoclast precursors, mode of fusion, and properties of the differentiated osteoclasts. To better understand the origin of these heterogeneities and the plasticity of osteoclasts, we examine several processes of osteoclastogenesis in this review. Candidate mechanisms that create heterogeneity involve asymmetric cell division, osteoclast niche, self-organization, and mode of fusion and fission. Elucidation of the plasticity or fluctuation of the M-CSF/RANKL network should be an important topic for future researches., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Osteoclasts are multinucleated, specializes bone-resorbing cells that are derived from the monocyte/macrophage lineage. Excessive resorbing activities of osteoclasts are involved in destructive bone diseases. The detailed mechanism of acidification at the bone adhesion surface during the bone resorption process of osteoclasts remains to be defined. During glycolysis, pyruvate proceeds to the tricarboxylic cycle under aerobic conditions and pyruvate is converted to lactate via lactate dehydrogenase A (LDHA) under anaerobic conditions. However, tumor cells produce ATP during aerobic glycolysis and large amounts of pyruvate are converted to lactate and H + by LDHA. Lactate and H + are excreted outside the cell, whereby they are involved in invasion of tumor cells due to the pH drop around the cell. In this study, we focused on aerobic glycolysis and investigated the production of lactate by LDHA in osteoclasts. Expression of LDHA and monocarboxylate transporter 4 (MCT4) was upregulated during osteoclast differentiation. Intracellular and extracellular lactate levels increased with upregulation of LDHA and MCT4, respectively. FX11 (an LDHA inhibitor) inhibited osteoclast differentiation and suppressed the bone-resorbing activity of osteoclasts. We propose that inhibition of LDHA may represent a novel therapeutic strategy for controlling excessive bone resorption in osteoporosis and rheumatoid arthritis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

This review focuses on understanding the macroscopic and microscopic characteristics of bone tissue and reviews current knowledge of its physiology. It explores how these features intricately collaborate to maintain the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, which plays a pivotal role in shaping not only our physical framework but also overall health. In this work, a comprehensive exploration of microscopic and macroscopic features of bone tissue is presented.

Abstract: Cellular immune responses as well as generalized and periarticular bone loss are the key pathogenic features of rheumatoid arthritis (RA). Under the pathological conditions of RA, dysregulated inflammation and immune processes tightly interact with skeletal system, resulting in pathological bone damage via inhibition of bone formation or induction of bone resorption. Single-cell omics technologies are revolutionary tools in the field of modern biological research.They enable the display of the state and function of cells in various environments from a single-cell resolution, thus making it conducive to identify the dysregulated molecular mechanisms of bone destruction in RA as well as the discovery of potential therapeutic targets and biomarkers. Here, we summarize the latest findings of single-cell omics technologies in osteoimmunology research in RA. These results suggest that single-cell omics have made significant contributions to transcriptomics and dynamics of specific cells involved in bone remodeling, providing a new direction for our understanding of cellular heterogeneity in the study of osteoimmunology in RA., (Copyright © 2023 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

Osteoclasts are the cells responsible for the bone resorption process during bone remodeling. In a healthy situation, this process results from an equilibrium between new matrix formation by osteoblast and matrix resorption by osteoclast. Osteoporosis (OP) is a systemic bone disease characterized by a decreased bone mass density and alterations in bone microarchitecture, increasing fracture predisposition. Despite the variety of available therapies for OP management there is a growing gap in its treatment associated to the low patients' adherence owing to concerns related with long-term efficacy or safety. This makes the development of new and safe treatments necessary. Among the newly developed strategies, the use of synthetic and natural nanoparticles to modulate osteoclasts differentiation, activity, apoptosis or crosstalk with osteoblasts have arisen. Synthetic nanoparticles exert their therapeutic effect either by loading antiresorptive drugs or including molecules for osteoclasts gene regulation. Moreover, this control over osteoclasts can be improved by their targeting to bone extracellular matrix or osteoclast membranes. Furthermore, natural nanoparticles, also known as extracellular vesicles, have been identified to play a key role in bone homeostasis. Consequently, these systems have been widely studied to control osteoblasts and osteoclasts under variable environments. Additionally, the ability to bioengineer extracellular vesicles has allowed to obtain biomimetic systems with desirable characteristics as drug carriers for osteoclasts. The analyzed information reveals the possibility of modulating osteoclasts by different mechanisms through nanoparticles decreasing bone resorption. These findings suggest that controlling osteoclast activity using nanoparticles has the potential to improve osteoporosis management. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Nanotechnology Approaches to Biology > Nanoscale Systems in Biology., (© 2023 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.)

Osteoclasts are the only cells that can efficiently resorb bone. They do so by sealing themselves on to bone and removing the mineral and organic components. Osteoclasts are essential for bone homeostasis and are involved in the development of diseases associated with decreased bone mass, like osteoporosis, or abnormal bone turnover, like Paget's disease of bone. In addition, compromise of their development or resorbing machinery is pathogenic in multiple types of osteopetrosis. However, osteoclasts also have functions other than bone resorption. Like cells of the innate immune system, they are derived from myeloid precursors and retain multiple immune cell properties. In addition, there is now strong evidence that osteoclasts regulate osteoblasts through a process known as coupling, which coordinates rates of bone resorption and bone formation during bone remodeling. In this article we review the non-resorbing functions of osteoclasts and highlight their importance in health and disease., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation and bone erosion. The bones in the human body are constantly undergoing bone remodeling throughout their lives, which is the process of bone resorption by osteoclasts to damaged bone tissue and new bone formation by osteoblasts. Osteoblasts (OBs) are the main functional cells in bone formation, responsible for the synthesis, secretion and mineralization of the bone matrix. On the contrary, osteoclasts (OCs) mediate bone breakdown during natural bone turnover, but excessive breakdown occurs in RA. Under the condition of RA inflammation, many molecules, such as IL-1β, IL-6, TNF-α, IL-17 and hypoxia-inducible factor-1α (HIF-1α) are produced that could mediate bone loss. Studies have shown that cytokines mainly promote the formation of OCs and play a role in bone resorption by stimulating OBs to express receptor activator of NF-κB ligand (RANKL). JAK/STAT plays a crucial role in the process of bone destruction. And JAK/STAT pathway mediates the RANKL/receptor activator of NF-κB (RANK)/osteoprotegerin (OPG) axis. Tofacitinib, Baricitinib, Peficitinib and Filgotinib are now being used in patients with moderate to severe RA, as well as in patients with RA who have an inadequate response to methotrexate therapy and bone destruction. Currently, Tofacitiniband Baritinib areapprovedfor thetreatmentof moderate-to-severely active RA. JAK inhibitors have been reported to have better efficacy and lower adverse effects compared with methotrexate and adalimumab. In addition, two JAK inhibitors are currently in development: the JAK1 selective Upadacitinib, and the JAK3 selective inhibitor Decernotinib. In addition to the above JAK inhibitors, some small molecular compounds inhibit bone destruction by inhibiting the Phosphorylation of STAT3. In this paper, the research progress of bone destruction participated by JAK/ STAT in rheumatoid arthritis and therapeutic effect of JAK/STAT inhibitors were reviewed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Semen Cuscutae, called Tu-si-zi in Chinese, is a kind of dried mature seed in the Convolvulaceae family. It mainly distributes in China, Korea, Pakistan, Vietnam, India and Thailand. It is used as a kidney-tonifying drug for treatment of aging related diseases such as osteoporosis in traditional Chinese medicine. However, the exact mechanisms on bone resorption are poorly studied., Aim of the Study: The aim of this study was to investigate the potential effect of Semen Cuscutae on ovariectomy (OVX)-induced osteoporosis in mice and clarify the exact mechanisms by which Semen Cuscutae exert the anti-osteoporosis effect., Materials and Methods: Qualitative and quantitative analyses of Semen Cuscutae were performed by UPLC-Q-TOF-MS and HPLC-MS/MS, respectively. Changes in bone mineral density (BMD) induced by OVX in mice were measured by dual-energy X-ray absorptiometry and micro-computed tomography (μCT). Tartrate-resistant acid phosphatase (TRAP) staining as well as hematoxylin and eosin (HE) staining were used to observe bone microarchitectural changes. ELISA kits were used to assess the therapeutic effects of Semen Cuscutae on the serum levels of osteoprotegerin (OPG), tartrate-resistant acid phosphatase 5b (TRACP-5b), and receptor activator of nuclear factor-κB (RANKL). The effect of Semen Cuscutae on primary cell viability was assessed using CCK-8 and anti-tartrate phosphatase assays. TRAP staining and actin ring staining were used to observe the effect of Semen Cuscutae on osteoclast differentiation. Western blotting was used to measure the effects of Semen Cuscutae on expressions of NFATC1, c-Src kinase, and c-fos., Results: Results from UPLC-Q-TOF-MS showed that the main components of Semen Cuscutae were flavonoid compounds that included quercitrin, quercetin, hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercetin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, and astragalin. After the Semen Cuscutae extract was orally administered to OVX mice, bone density increased (P < 0.01) and bone microstructure was significantly improved (P < 0.01 or 0.05). Additionally, Semen Cuscutae exhibited a significant descending effect in the levels of serum TRACP-5b and RANKL, while there was a significant increase in OPG in the Semen Cuscutae group compared with the OVX group, especially at high doses. Moreover, we found that increasing of c-fos, c-Src kinase, and NFATC1 protein expressions were reversed by Semen Cuscutae in vitro and in vivo., Conclusions: Our results showed that Semen Cuscutae exhibited anti-osteoporosis effects through the c-fos/c-Src kinase/NFATC1 signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bone is a mineralized and elastic connective tissue that provides fundamental functions in the human body, including mechanical support to the muscles and joints, protection of vital organs and storage of minerals. Bone is a metabolically active organ that undergoes continuous remodeling processes to maintain its architecture, shape, and function throughout life. One of the most important medical discoveries of recent decades has been that the immune system is involved in bone remodeling. Indeed, chronic inflammation has been recognized as the most significant factor influencing bone homeostasis, causing a shift in the bone remodeling process toward pathological bone resorption. Bone osteolytic diseases typified by excessive bone resorption account for one of the greatest causes of disability worldwide, with significant economic and public health burdens. From this perspective, we discuss the recent findings and discoveries highlighting the cellular and molecular mechanisms that regulate this process in the bone microenvironment, in addition to the current therapeutic strategies for the treatment of osteolytic bone diseases.

Osteoporosis is a significant health problem, with skeletal fractures increasing morbidity and mortality. Excess glucocorticoids (GC) represents the leading cause of secondary osteoporosis. The first phase of glucocorticoid-induced osteoporosis is increased bone resorption. In this Chapter, in vitro studies of the direct glucocorticoid receptor (GR) mediated cellular effects of GC on osteoclasts to affect bone resorption and indirect effects on osteoblast lineage cells to increase the RANKL/OPG ratio and stimulate osteoclastogenesis and bone resorption are reviewed in detail, together with detailed descriptions of in vivo effects of GC in different portions of the skeleton in research animals and humans. Brief sections are devoted to contrasting functions of GC in osteonecrosis, vitamin D formation, in vitro and in vivo bone resorptive actions dependent on vitamin D receptor and vitamin D toxicity, as well as the molecular basis of GR action. Included are also more detailed assessments of the interactions of GC with the major calcium regulating hormones, 1,25(OH) 2 -vitamin D3 and parathyroid hormone, describing the in vitro increases in RANKL/OPG ratios, osteoclastogenesis and synergistic bone resorption that occurs when GC is combined with either 1,25(OH) 2 -vitamin D3 or parathyroid hormone. Additionally, a molecular basic for the synergistic interaction of GC with 1,25(OH) 2 -vitamin D3 is provided along with a suggested molecular basic for the interaction between GC and parathyroid hormone., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Receptor activator of nuclear factor kappa-B ligand (RANKL) is one of the key factors regulating the maturation of osteoclasts and an important target for osteoporosis treatment. A monoclonal antibody against RANKL showed effective therapeutic activity against osteoporosis by inhibiting bone resorption by osteoclasts. However, being an exogenous protein, its efficacy decreases after long-term use, and its discontinuation increases the risk of vertebral fractures. Here, we aimed to design an active immunotherapeutic agent to induce a T-cell dependent primary response. The agent, a mutant RANKL vaccine (mRv), was produced by cross-linking mutant RANKL, lacking the ability to stimulate osteoclast maturation, with the carrier protein keyhole limpet hemocyanin, a neo-antigen with a large molecular mass. Subcutaneous injection of mRv stimulated rats with ovariectomy-induced osteoporosis to produce high titers of anti-RANKL antibodies. The mutant RANKL vaccine decreased serum CTX-1 and BALP levels and inhibited the microstructural degeneration of trabecular bone in osteoporotic rats. mRv overcame immune system tolerance, stimulated rats to produce therapeutic antibodies, stabilized bone metabolism, and inhibited trabecular microstructural degeneration. These findings confirm the potential of the mutant RANKL vaccine to be developed into an effective preventive and therapeutic agent for osteoporosis., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: Parathyroid hormone (PTH) acts on bone to indirectly increase the number and activity of osteoclasts. Thus, PTH has a stimulatory effect on bone resorption and upregulates bone turnover. However, the responsiveness of bone to PTH varies widely among patients receiving dialysis. In fact, relative to the serum PTH level, the level of serum tartrate-resistant acid phosphatase-5b (TRACP-5b), a bone resorption marker derived from osteoclasts, varies as well. This study aimed to examine factors related to bone responsiveness to PTH in patients undergoing chronic hemodialysis (HD)., Methods: This study included patients receiving chronic HD in Kawasaki Municipal Tama Hospital (Kanagawa, Japan) and Yonaha Medical Clinic (Okinawa, Japan) and excluded patients who received HD for less than 6 months, those who received a combination of HD and peritoneal dialysis, and those who had cancer bone metastases or myeloma. The TRACP-5b/intact PTH (iPTH) ratio was created as an index of bone responsiveness to PTH, categorized into tertiles (low, medium, and high), and a cross-sectional study was conducted. P < 0.05 indicated statistically significant differences., Results: One hundred and six patients were analyzed. Age (P = 0.010), body mass index (BMI) (P = 0.003), use of calcium-sensing receptor (CaSR) agonists (P = 0.008), use of vitamin D receptor activators (VDRAs) (P = 0.012), plasma iPTH level (P < 0.001), serum 1,25(OH) 2 D level (P = 0.003), and serum TRACP-5b level (P < 0.001) were significantly different among the three categories. In the single linear regression analysis, age (P = 0.016), corrected serum calcium level (P = 0.007), and ln [1,25(OH) 2 D] (P = 0.044) showed a significant positive correlation with ln [TRACP-5b/iPTH], whereas BMI (P = 0.026), use of CaSR agonists (P = 0.001), use of VDRAs (P = 0.009), and serum phosphorus level (P = 0.018) showed a significant negative correlation. Upon conducting multiple linear regression analysis incorporating significant variables in the single linear regression analysis, a significant negative correlation was observed between the TRACP-5b/iPTH ratio and intravenous administration of a CaSR agonist (etelcalcetide) and/or a VDRA (calcitriol or maxacalcitol) in all the adjusted models., Conclusions: Bone responsiveness to PTH is negatively correlated with the intravenous administration of a CaSR agonist and/or a VDRA in patients undergoing chronic HD., (© 2021. The Author(s).)

Melatonin, a pineal gland hormone, has been suggested to treat postmenopausal osteoporosis due to its inhibitory effect on osteoclast differentiation. We previously reported that protein arginine methyltransferase 1 (PRMT1) was an important mediator of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the relationship between melatonin and PRMT1 in osteoclast differentiation and estrogen deficiency-induced osteoporosis is unclear. In this study, we investigated the inhibitory mechanisms of melatonin in vitro and in vivo by focusing on PRMT1. Melatonin treatment effectively blocked RANKL-induced osteoclastogenesis by inhibiting PRMT1 and asymmetric dimethylarginine (ADMA) expression. RANKL-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) and the phosphorylation of JNK were also suppressed by melatonin, and TRAF6 siRNA attenuated RANKL-induced p-JNK and PRMT1 production. Melatonin inhibited the transcriptional activity of NF-κB by interfering with the binding of PRMT1 and NF-κB subunit p65 in RANKL-treated bone marrow-derived macrophages. Our results also revealed that melatonin inhibits RANKL-induced PRMT1 expression through receptors-independent pathway. Thus, the anti-osteoclastogenic effect of melatonin was mediated by a cascade of inhibition of RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling in melatonin receptors-independent pathway. In vivo, ovariectomy caused significant decreases in bone mineral density, but melatonin treatment alleviated the ovariectomized (OVX)-induced bone loss by inhibiting bone resorption. Furthermore, the expression PRMT1 and TRAP mRNA was upregulated in OVX-femurs, but effectively suppressed by melatonin injection. These findings suggest that melatonin inhibited osteoclast differentiation and estrogen deficiency-induced osteoporosis by suppressing RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling cascades in melatonin receptors-independent pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Osteoporosis is one of the most common metabolic bone diseases among pre- and post-menopausal women. Despite numerous advances in the treatment of osteoporosis in recent years, the outcomes remain poor due to severe side effects. In this study, we investigated whether A-485, a highly selective catalytic p300/CBP inhibitor, could attenuate RANKL-induced osteoclast differentiation and explored the underlying molecular mechanisms. The protective role of A-485 in osteoporosis was verified using a mouse model of ovariectomy (OVX)-induced bone loss and micro-CT scanning. A-485 inhibited RANKL-induced osteoclast differentiation in vitro by reducing the number of tartrate-resistant acid phosphatase-positive osteoclasts without inducing significant cytotoxicity. In particular, A-485 dose-dependently disrupted F-actin ring formation and downregulated the expression of genes associated with osteoclast differentiation, such as CTSK, c-Fos, TRAF6, VATPs-d2, DC-STAMP, and NFATc1, in a time- and dose-dependent manner. Moreover, A-485 inhibited the RANKL-induced phosphorylation of MAPK pathways and attenuated OVX-induced bone loss in the mouse model while rescuing the loss of bone mineral density. Our in vitro and in vivo findings suggest for the first time that A-485 has the potential to prevent postmenopausal osteoporosis and could therefore be considered as a therapeutic molecule against osteoporosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Physical inactivity leads to losses of bone mass and strength in most mammalian species. In contrast, hibernating bears show no bone loss over the prolonged periods (4-6 months) of immobility during winter, which suggests that they have adaptive mechanisms to preserve bone mass. To identify transcriptional changes that underlie molecular mechanisms preventing disuse osteoporosis, we conducted a large-scale gene expression screening in the trabecular bone and bone marrow, comparing hibernating and summer active bears through sequencing of the transcriptome. Gene set enrichment analysis showed a coordinated down-regulation of genes involved in bone resorption, osteoclast differentiation and signaling, and apoptosis during hibernation. These findings are consistent with previous histological findings and likely contribute to the preservation of bone during the immobility of hibernation. In contrast, no significant enrichment indicating directional changes in gene expression was detected in the gene sets of bone formation and osteoblast signaling in hibernating bears. Additionally, we revealed significant and coordinated transcriptional induction of gene sets involved in aerobic energy production including fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, and mitochondrial metabolism. Mitochondrial oxidation was likely up-regulated by transcriptionally induced AMPK/PGC1α pathway, an upstream stimulator of mitochondrial function.

Osteoclasts, the multinucleated cells responsible for bone resorption, have an enormous destructive power which demands to be kept under tight control. Accordingly, the identification of molecular signals directing osteoclastogenesis and switching on their resorptive activity have received much attention. Mandatory factors were identified, but a very essential aspect of the control mechanism of osteoclastic resorption, i.e. its spatial control, remains poorly understood. Under physiological conditions, multinucleated osteoclasts are only detected on the bone surface, while their mono-nucleated precursors are only in the bone marrow. How are pre-osteoclasts targeted to the bone surface? How is their progressive differentiation coordinated with their approach to the bone surface sites to be resorbed, which is where they finally fuse? Here we review the information on the bone marrow distribution of differentiating pre-osteoclasts relative to the position of the mandatory factors for their differentiation as well as relative to physical entities that may affect their access to the remodelling sites. This info allows recognizing an "osteoclastogenesis route" through the bone marrow and leading to the coincident fusion/resorption site - but also points to what still remains to be clarified regarding this route and regarding the restriction of fusion at the resorption site. Finally, we discuss the mechanism responsible for the start of resorption and its spatial extension. This review underscores that fully understanding the control of bone resorption requires to consider it in both space and time - which demands taking into account the context of bone tissue., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bone remodeling is sophisticatedly regulated by two different cell types: bone-resorbing osteoclasts and bone-forming osteoblasts. Hochu-Ekki-To, a Japanese traditional herbal medicine, is commonly used for the treatment of chronic diseases or frailty after an illness; however, its effects on metabolic bone diseases such as osteoporosis are not well known. We herein report that daily oral Hochu-Ekki-To administration significantly inhibits osteoclast activation as well as the reduction in bone volume in ovariectomized mice. Our results suggest that supplementation with Hochu-Ekki-To might be beneficial for the prophylaxis and treatment of metabolic bone diseases associated with abnormal osteoclast activation., Competing Interests: Declaration of Competing Interest E. Hinoi is supported by a research fund from Tsumura & Co.. The other authors declare no potential conflicts of interest., (Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Osteoporosis is currently treated with drugs targeting the differentiation or viability osteoclasts, the cells responsible for physiological and pathological bone resorption. Nevertheless, osteoporosis drugs that target only osteoclast activity are expected to preserve bone formation by osteoblasts in contrast to current treatments. We report here the design, synthesis, and biological characterization of a series of novel N -arylsufonamides featuring a diazaspiro[4,4]nonane nucleus to target the guanine nucleotide exchange activity of DOCK5, which is essential for bone resorption by osteoclasts. These compounds can inhibit both mouse and human osteoclast activity. In particular, 4-chlorobenzyl-4-hydroxy-2-phenyl-1-thia-2,7-diazaspiro[4,4]nonane 1,1-dioxide (compound E197 ) prevented pathological bone loss in mice. Most interestingly, treatment with E197 did not affect osteoclast and osteoblast numbers and hence did not impair bone formation. E197 could represent a lead molecule to develop new antiosteoporotic drugs targeting the mechanism of osteoclast adhesion onto the bone.

Introduction: Prolonged use of anti-cancer treatments in breast and prostate tumors alters physiological bone turnover leading to adverse skeletal related events, such as osteoporosis, loss of bone mass, and increased risk of fractures. These complications known as cancer treatment-induced bone loss (CTIBL) should be managed with bone targeting agents such as the bisphosphonates and denosumab. The latter is a monoclonal antibody against the receptor activator of nuclear factor-kB ligand (RANKL) that suppresses osteoclasts function and survival increasing bone mass., Areas Covered: This review will focus on the mechanisms associated with bone loss induced by cancer treatments and the most recent evidence about the use of denosumab as preventive and therapeutic strategy to protect bone health. Moreover, we will discuss several key aspects regarding the clinical practical use of denosumab to optimize the management of CTLIB in breast and prostate cancer., Expert Opinion: Denosumab treatment strongly prevents cancer therapies-related skeletal issues in breast and prostate cancer with a good safety profile. Adjuvant six-monthly denosumab delays the time to first fracture onset in early stage breast cancer patients with normal or altered bone mineral density (BMD). Similarly, denosumab treatment is able to prevent fractures and BMD loss in nonmetastatic prostate cancer patients.

Dietary procyanidin has been shown to be an important bioactive component that regulates various pharmacological activities to maintain metabolic homeostasis. In particular, grape seed proanthocyanidin extract (GSPE) is a commercially available medicine for the treatment of venous and lymphatic dysfunction. This study aimed to investigate whether GSPE protects against lipopolysaccharide (LPS)-induced bone loss in vivo and the related mechanism of action in vitro. The administration of GSPE restored the inflammatory bone loss phenotype stimulated by acute systemic injection of LPS in vivo. GSPE strongly suppressed receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and bone resorption activity of mature osteoclasts by decreasing the RANKL-induced nuclear factor-κB transcription activity. GSPE mediates this effect through decreased phosphorylation and degradation of NF-κB inhibitor (IκB) by IκB kinaseβ, subsequently inhibiting proto-oncogene cellular Fos and nuclear factor of activated T cells. Additionally, GSPE promotes osteoclast proliferation by increasing the phosphorylation of components of the Akt and mitogen-activated protein kinase signaling pathways and it also inhibits apoptosis by decreasing the activity of caspase-8, caspase-9, and caspase-3, as corroborated by a decrease in the Terminal deoxynucleotidyl transferase dUTP nick end labeling -positive cells. Our study suggests a direct effect of GSPE on the proliferation, differentiation, and apoptosis of osteoclasts and reveals the mechanism responsible for the therapeutic potential of GSPE in osteoclast-associated bone metabolism disease.

Functional characterisation of cell-type-specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans -regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global ( Pik3cb -/- , Atp8b2 +/- , Igsf8 -/- , Eml1 -/- , Appl2 -/- , Deptor -/- ) and myeloid-specific Slc40a1 knockout mice displayed abnormal bone phenotypes. We report opposing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast multinucleation and bone mass., Competing Interests: MP, JK, JL, HP, KK, AT, PC, KP, MR, EP, JB, GW, JB No competing interests declared, (© 2020, Pereira et al.)

Background: Areca nut has anti-inflammatory, antiparasitic, antihypertensive, and antidepressant properties. The pathological hallmarks of inflammatory joint diseases are an increased number of osteoclasts and impaired differentiation of osteoblasts, which may disrupt the bone remodeling balance and eventually lead to bone loss., Purpose: The present study assessed the effects of arecoline, the main alkaloid found in areca nut, on osteoclast and osteoblast differentiation and function., Method: M-CSF/RANKL-stimulated murine bone marrow-derived macrophages (BMMs) were incubated with several concentrations of arecoline, and TRAP staining and pit formation were assessed to monitor osteoclast formation. Quantitative real-time RT-PCR and western blot analyses were used to analyze the expression of osteoclast-associated genes and signaling pathways. The effects of arecoline on bone were investigated in an in vivo mouse model of lipopolysaccharide (LPS)-induced trabecular bone loss after oral administration of arecoline. Alizarin red S staining and assays to measure ALP activity and the transcription level of osteoblast-related genes were used to evaluate the effects of arecoline on osteoblast differentiation and bone mineralization., Results: In a dose-dependent manner, arecoline at concentrations of 50-100 μM reduced both the development of TRAP-positive multinucleated osteoclasts and the formation of resorption pits in M-CSF/RANKL-stimulated BMMs. In M-CSF/RANKL-stimulated BMMs, arecoline also suppressed the expression and translocation of c-Fos and NFATcl, and osteoclast differentiated-related genes via interference with the AKT, MAPK, and NF-kB activation pathways. Femur bone loss and microcomputed tomography parameters were recovered by oral administration of arecoline in the mouse LPS-induced bone loss model. Lastly, arecoline increased ALP activity, bone mineralization, and the expression of osteoblast differentiation-related genes, such as ALP and Runx2, in MC3T3-E1 cells., Conclusion: Our data suggest that arecoline may attenuate or prevent bone loss by suppressing osteoclastogenesis and promoting osteoblastogenesis. These findings provide evidence supporting arecoline's use as a potential therapeutic agent in bone-loss disorders and diseases., Competing Interests: Declaration of Competing Interest The authors have declared no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbβ, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

Prostaglandins (PGs) are highly bioactive fatty acids. PGs, especially prostaglandin E 2 (PGE 2 ), are abundantly produced by cells of both the bone-forming (osteoblast) lineage and the bone-resorbing (osteoclast) lineage. The inducible cyclooxygenase, COX-2, is largely responsible for most PGE 2 production in bone, and once released, PGE 2 is rapidly degraded in vivo. COX-2 is induced by multiple agonists - hormones, growth factors, and proinflammatory factors - and the resulting PGE 2 may mediate, amplify, or, as we have recently shown for parathyroid hormone (PTH), inhibit responses to these agonists. In vitro, PGE 2 can directly stimulate osteoblast differentiation and, indirectly via stimulation of RANKL in osteoblastic cells, stimulate the differentiation of osteoclasts. The net balance of these two effects of PGE 2 in vivo on bone formation and bone resorption has been hard to predict and, as expected for such a widespread local factor, hard to study. Some of the complexity of PGE 2 actions on bone can be explained by the fact that there are four receptors for PGE 2 (EP1-4). Some of the major actions of PGE 2 in vitro occur via EP2 and EP4, both of which can stimulate cAMP signaling, but there are other distinct signaling pathways, important in other tissues, which have not yet been fully elucidated in bone cells. Giving PGE 2 or agonists of EP2 and EP4 to accelerate bone repair has been examined with positive results. Further studies to clarify the pathways of PGE 2 action in bone may allow us to identify new and more effective ways to deliver the therapeutic benefits of PGE 2 in skeletal disorders.

Osteoporosis is a condition where bone resorption exceeds bone formation leading to degeneration. With an aging population, the prevalence of osteoporosis is on the rise. Although advances in the field have made progress in targeting the mechanisms of the disease, the efficacy of current treatments remains limited and is complicated by unexpected side effects. Therefore, to overcome this treatment gap, new approaches are needed to identify and elucidate the cellular mechanisms mediating the pathogenesis of osteoporosis, which requires a strong understanding of bone biology. This chapter will focus on bone cells (osteoclasts, osteoblasts, and osteocytes) and their role in the bone turnover process in normal physiology and in pathology. With regard to osteoclast function, the regulators and underpinning signaling pathways leading to bone resorption will be discussed. Decreased osteoblastogenesis also contributes to bone deterioration with aging and osteoporosis; hence the factors and signaling pathways mediating osteoblast formation and function will be examined. Osteocytes are mature osteoblasts embedded in bone matrix and act as endocrine cells; their role in bone health and pathology will also be reviewed. In addition, this chapter will explore the emerging role of adipocytes in bone biology and the implications of increased bone marrow fat infiltration with aging on bone degeneration. In conclusion, a greater understanding of the pathogenesis of osteoporosis is of utmost importance in order to develop more effective treatments for osteoporosis and other bone diseases.

Inflammatory rheumatic diseases are often associated with secondary osteoporosis, as many inflammatory messengers can interfere with bone metabolism and adversely affect it. In addition to a decrease in densitometric bone density, remodeling occurs in the trabecular bone, which can lead to a disturbed microarchitecture and increase the risk of fracture.Central to this is the close integration of bone metabolism and the immune system. Proinflammatory cytokines play an important role not only in the inflammatory process, but also as mediators of bone resorption because they stimulate osteoclastogenesis and induce further signal transduction cascades with negative influence on the bone. The understanding gained in recent years of the underlying immunological processes has led to the development of new and targeted treatment approaches.

The balance between the osteoblasts and the osteoclasts is important for the maintenance of the skeleton of the human body. The osteoclasts absorb bone after differentiated into polymorphonuclear cells by the fusion of monocytes/macrophages. We have found that 6,7,4'-Trihydroxyflavone (THF), a compound from the heartwood of Dalbergia Odorifera inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, actin ring formation, and bone resorption in RAW 264.7 cells and bone marrow macrophage. THF significantly inhibited the c-Jun-N-terminal kinase signaling pathway without affecting extracellular signal-regulated kinase, p38, and AKT signaling. Moreover, THF inhibited the expression of c-Fos, nuclear factor-activated T cells cytoplasm 1, cathepsin K, and c-src by RANKL. We used a lipopolysaccharide (LPS)-induced bone loss model in mice. Consequently, bone volume per tissue volume, trabecular number's reduction was recovered in THF-treated mice, and trabecular separation's augmentation was also attenuated by THF administration. In summary, THF inhibits RANKL-induced osteoclast differentiation by MAPK signaling pathway and inhibits bone resorption by destroying the actin ring in mature osteoclasts. THF also prevented LPS-induced bone loss in a mice model. Thus, THF may be useful in the treatment of bone diseases associated with excessive osteoclast differentiation and bone resorption., (© 2019 John Wiley & Sons, Ltd.)

Objectives: This study aimed to investigate the role of JAK2-STAT3 (Janus kinase 2/signal transducer and activator of transcription 3) in periapical disease caused by Enterococcus faecalis, as well as the correlation between lipoteichoic acid (LTA) in E. faecalis and the activity of the JAK2-STAT3 signaling pathway and osteoclast formation., Materials and Methods: A rat model of periapical periodontitis induced by E. faecalis was established. Periapical bone resorption was confirmed by HE staining. The expression of JAK2, p-JAK2, STAT3, and p-STAT3 was assessed with immunohistochemical staining. Osteoclasts were observed through enzyme histochemical staining. LTA acted on mouse osteoclast precursor cells (RAW264.7 cells); a JAK2 inhibitor (AG490) was used to inhibit the JAK2-STAT3 pathway in RAW264.7 cells. The expression of proteins in the JAK2-STAT3 pathway and TRAP (tartrate resistant acid phosphatase) in RAW264.7 cells was also detected., Results: Rat periapical periodontitis was successfully established and bone resorption peaked at day 21. The expression of critical components in the JAK2-STAT3 pathway increased with the progression of inflammation. LTA promoted the differentiation of RAW264.7 cells into osteoclasts. NFATc1 was highly expressed and was inhibited by AG490., Conclusions: JAK2-STAT3 signaling pathway plays an important role in the process of periapical bone resorption and osteoclastogenesis., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Bone homeostasis requires a dynamic balance between osteogenesis and osteoclastogenesis, and osteolytic disorders are mainly attributed to aberrant osteoclastogenesis and bone resorption. Accumulating evidence has demonstrated that cyclin-dependent kinase 9 (CDK9) regulates some inflammatory diseases without affecting the cell cycle. Whether the specific inhibitor of CDK9, LDC000067 (abbreviated as LDC067), helps to prevent from osteolytic disorders has not been fully elucidated. Interestingly, this study demonstrated that LDC067 inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption in vitro, and suppressed the expression of osteoclast-related marker genes such as cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), dendrite cell-specific transmembrane protein (DC-STAMP), V-ATPase D2, calcitonin receptor (CTR) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). The bone protective effects of LDC067 can be partly explained by its suppression of nuclear factor-kappa B (NF-κB)-mediated NFATc1 activation via AKT signalling pathway. In keeping with the results obtained in vitro, inhibition of CDK9 with LDC067 was observed to delay subchondral osteolysis and substantially ameliorate LPS-induced osteolysis in murine calvaria. Collectively, these results highlight the positive effects of LDC067 in preventing osteolytic disorders and indicate that this CDK9 inhibitor may a promising therapeutic agent., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ovotransferrin, a member of the transferrin family, is the second main protein found in egg white. Ovotransferrin was reported to have antimicrobial, antioxidant, and immunomodulating activities. The aim of this work was to characterize the cellular and molecular functions of egg white ovotransferrin on osteoclasts differentiation and function. Osteoclasts were prepared from mouse macrophage RAW 264.7 cells stimulated with receptor activator of nuclear factor κB ligand (RANKL). Ovotransferrin inhibited osteoclasts differentiation and the calcium-phosphate resorptive ability via the suppression of RANKL-induced nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Ovotransferrin induced apoptosis of matured osteoclasts, accompanied by increased expression of Bcl-2-like protein 11 ( Bim ) and Bcl-2-assoicated death promoter ( Bad ), but decreased expression of B-cell lymphoma 2 ( Bcl-2 ) and B-cell lymphoma-extra-large ( Bcl-xl ). We established a novel role of egg white ovotransferrin as an inhibitor of osteoclastogenesis, which may be used for the prevention of osteoporosis.

Background: Many bone-related diseases such as osteoporosis and rheumatoid arthritis are commonly associated with the excessive activity of osteoclasts. Polyscias fruticosa has been used as traditional medicine for the treatment of ischemia and inflammation and also eaten as a salad. However, its effect on the bone related diseases has not been investigated yet., Purpose: This study aimed to investigate the effect of ethanol extract of P. fruticosa on RANKL-induced osteoclastogenesis in vitro and LPS-induced bone loss in mouse, and evaluate anti-osteoclastogenic activities of its major constituents., Methods: BMMs or RAW264.7 cells were treated with ethanol extract from P. fruticose leaves (EEPL), followed by an evaluation of cell viability, RANKL-induced osteoclast differentiation, actin-ring formation, and resorption pits activity. Effects of EEPL on RANKL-induced phosphorylation of MAPKs were evaluated by Western blotting. The expression levels of NFATc1 and c-Fos were evaluated by Western blotting or immunofluorescence assay. The expression levels of osteoclast-specific marker genes were evaluated by Western blotting and reverse transcription-qPCR analysis. A LPS-induced murine bone loss model was used to evaluate the protective effect of EEPL on inflammation-induced bone loss. HPLC analysis was performed to identify the major constituents of EEPL., Results: EEPL significantly inhibited RANKL-induced osteoclast differentiation by decreasing the number of osteoclasts, osteoclast actin-ring formation, and bone resorption. EEPL suppressed RANKL-induced phosphorylation of p38 and JNK MAPKs, as well as the expression of c-Fos and NFATc1. EEPL decreased the expression levels of osteoclast marker genes, including MMP-9, TRAP and CtsK. Mice treated with EEPL significantly protected the mice from LPS-induced osteoclast formation and bone destruction as indicated by micro-CT and histological analysis of femurs. We also identified 3-O-[β-d-glucopyranosyl-(1→4)-β-d-glucuronopyranosyl] oleanolic acid 28-O-β-d-glucopyranosyl ester (1) and quercitrin (3) as the active constituents in EEPL for inhibiting RANKL-induced osteoclast differentiation., Conclusion: The results showed that EEPL exerted anti-osteoclastogenic activity in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function, and suggested that EEPL could have beneficial applications for preventing or inhibiting osteoclast-mediated bone diseases., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

The purpose of this study was to determine the direct effects of phenobarbital (PB) on receptor activator of nuclear factor kappa-B ligand (RANKL) induced osteoclast differentiation and function in vitro and in vivo. Here, PB significantly inhibited osteoclast formation and bone resorption ability induced by RANKL in vitro. Meanwhile, intracellular signaling transduction analysis revealed PB specifically decreasing the phosphorylation level of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK), respectively. Besides, oral administration of PB at the dose of 60 mg/kg/day for 6 weeks led to improve the bone loss and to decrease the activity on both osteoblast and osteoclast. This suppression effect is more obvious in osteoblast-induced bone formation than that on osteoclast-induced bone resorption. Taken together, our findings demonstrated that PB down-regulate osteoclast differentiation and activity through modulation of NF-κB and MAPKs signaling pathway. The direct suppression effect on osteoclast can induce bone loss after long term oral administration. This bone loss is due to reducing bone turnover rate on both sides of bone formation and bone resorption., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bone is a highly dynamic organ that is continuously being remodeled by the reciprocal interactions between bone and immune cells. We have originally established an advanced imaging system for visualizing the in vivo behavior of osteoclasts and their precursors in the bone marrow cavity using two-photon microscopy. Using this system, we found that the blood-enriched lipid mediator, sphingosine-1-phosphate, controlled the migratory behavior of osteoclast precursors. We also developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo , and identified two distinct functional states of differentiated osteoclasts, "bone-resorptive" and "non-resorptive." Here, we summarize our studies on the dynamics and functions of bone and immune cells within the bone marrow. We further discuss how our intravital imaging techniques can be applied to evaluate the mechanisms of action of biological agents in inflammatory bone destruction. Our intravital imaging techniques would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would also be useful for evaluating novel therapies in animal models of bone-destroying diseases.

Bone loss is a non-motor symptom of Parkinson's disease (PD). It is unclear whether a patient's immobility or the endocrine changes in the body causes bone deterioration. To address this issue, we used an animal model of the disease where Swiss albino mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on day 1 and were left untreated for eight weeks. Behavioral phenotypes of PD, and striatal acetylcholinesterase and dopamine levels were measured. Cortical and trabecular bones were assessed by μ-CT and histology. Gene expression studies were done through quantitative real-time PCR. Effect of MPP+ and MPTP-treated mice serum on MC3T3E-1, SH-SY5Y, and primary osteoclast cells were also studied. Our results demonstrated that MPTP treatment leads to PD like symptoms. It shows a loss of trabecular bone mass and quality by decreasing osteoblast and increased osteoclast number and activity. This effect was accompanied by reduced osteogenic and elevated osteoclastogenic genes expression. While MPP+ had a cytotoxic effect on dopaminergic neurons, it did not affect bone cells. However, ex-vivo treatment of the serum from MPTP-treated mice decreased osteoblastogenesis and increased osteoclastogenesis in cell culture. In conclusion, our study suggests that MPTP-induced parkinsonian features in mice leads to trabecular bone loss by decreased bone formation and increased bone resorption due to changes in the serum circulating factors. This study characterizes the microarchitectural and cellular changes in the skeleton of a mouse model of PD that can be further utilized to investigate therapeutic avenues to treat bone loss in PD patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: This study was to determine the bone protective effects and underlying mechanisms of Osthole (OT) in ovariectomized (OVX) mice. We found that the inhibitory effects of OT on receptor activator of nuclear factor kappa-B ligand (RANKL)-activated osteoclastogenesis are responsible for its bone protective effects in OVX mice., Methods: Eight-week-old mice were ovariectomized and OT (10 mg/kg/d) was intraperitoneally administrated to OVX mice 7 days after the surgery and were sacrificed at the end of the 3 months. Osteoclasts were generated from primary bone marrow macrophages (BMMs) to investigate the inhibitory effects of OT. The activity of RANKL-activated signaling was simultaneously analyzed in vitro and in vivo using immunohistochemistry, Western blot, and PCR assays., Results: OT dose dependently inhibited RANKL-mediated osteoclastogenesis in BMM cultures. OT administration attenuated bone loss (mg Ha/cm: 894.68 ± 33.56 vs 748.08 ± 19.51, P < 0.05) in OVX mice. OT inhibits osteoclastogenesis (Oc.N/per view area: 72 ± 4.3 vs 0.8 ± 0.4, P < 0.05) and bone resorption activity (bone resorbed percentages %, 48.56 ± 7.25 vs 3.25 ± 1.37, P < 0.05) from BMMs. Mechanistically, OT inhibited the expressions of nuclear factor of activated T-cells c1 (NFATc1) and c-Fos. Moreover, OT suppressed the expression of RANKL-induced osteoclast marker genes, including matrix metalloproteinase 9 (MMP9), Cathepsin K (Ctsk), tartrate-resistant acid phosphatase (TRAP), and carbonic anhydrase II (Car2)., Conclusions: OT inhibits RANKL-mediated osteoclastogenesis and prevents bone loss in OVX mice. Our findings revealed that OT is a potential new drug for treating postmenopausal osteoporosis.

Osteoclasts (OCLs) are multinucleated phagocytes of monocytic origin responsible for physiological and pathological bone resorption including aging processes, chronic inflammation and cancer. Besides bone resorption, they are also involved in the modulation of immune responses and the regulation of hematopoietic niches. Accordingly, OCLs are the subject of an increasing number of studies. Due to their rarity and the difficulty to isolate them directly ex vivo , analyses on OCLs are usually performed on in vitro differentiated cells. In this state, however, OCLs represent a minority of differentiated cells. Since up to date a reliable purification procedure is still lacking for mature OCLs, all cells present in the culture are analyzed collectively to answer OCL-specific questions. With the development of in-depth transcriptomic and proteomic analyses, such global analyses on unsorted cells can induce severe bias effects in further results. In addition, for instance, analysis on OCL immune function requires working on purified OCLs to avoid contamination effects of monocytic precursors that may persist during the culture. This clearly highlights the need for a reliable OCL purification procedure. Here, we describe a novel and reliable method to sort OCLs based on cell multinucleation while preserving cell viability. Using this method, we successfully purified multinucleated murine cells. We showed that they expressed high levels of OCL markers and retained a high capacity of bone resorption, demonstrating that these are mature OCLs. The same approach was equally applied for the purification of human mature OCLs. Comparison of purified OCLs with mononucleated cells or unsorted cells revealed significant differences in the expression of OCL-specific markers at RNA and/or protein level. This exemplifies that substantially better outcomes for OCLs are achieved after the exclusion of mononucleated cells. Our results clearly demonstrate that the in here presented procedure for the analysis and sorting of pure OCLs represents a novel, robust and reliable method for the detailed examination of bona fide mature OCLs in a range that was previously impossible. Noteworthy, this procedure will open new perspectives into the biology of osteoclasts and osteoclast-related diseases.

Objectives: Bone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing CD115, Ly6C and CCR2) and non-classical monocytes (which are CD115 positive, but low in Ly6C and CCR2), in serving as precursors for OC in arthritis is still elusive., Methods: We investigated CCR2 -/- mice, which lack circulating classical monocytes, crossed into hTNFtg mice for the extent of joint damage. We analysed monocyte subsets in hTNFtg and K/BxN serum transfer arthritis by flow cytometry. We sorted monocyte subsets and analysed their potential to differentiate into OC and their transcriptional response in response to RANKL by RNA sequencing. With these data, we performed a gene ontology enrichment analysis and gene set enrichment analysis., Results: We show that in hTNFtg arthritis local bone erosion and OC generation are even enhanced in the absence of CCR2. We further show the numbers of non-classical monocytes in blood are elevated and are significantly correlated with histological signs of joint destruction. Sorted non-classical monocytes display an increased capacity to differentiate into OCs. This is associated with an increased expression of signal transduction components of RANK, most importantly TRAF6, leading to an increased responsiveness to RANKL., Conclusion: Therefore, non-classical monocytes are pivotal cells in arthritis tissue damage and a possible target for therapeutically intervention for the prevention of inflammatory joint damage., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.

Immunomodulatory drugs and monoclonal antibody-based immunotherapies have significantly improved the prognosis of the patients with multiple myeloma (MM) in the recent years. These new classes of reagents target malignant plasma cells (PCs) and further modulate the immune microenvironment, which prolongs anti-MM responses and may prevent tumor occurrence. Since MM remains an incurable cancer for most patients, there continues to be a need to identify new tumor target molecules and investigate alternative cellular approaches using gene therapeutic strategies and novel treatment mechanisms. Osteoclasts (OCs), as critical multi-nucleated large cells responsible for bone destruction in >80% MM patients, have become an attractive cellular target for the development of novel MM immunotherapies. In MM, OCs are induced and activated by malignant PCs in a reciprocal manner, leading to osteolytic bone disease commonly associated with this malignancy. Significantly, bidirectional interactions between OCs and MM cells create a positive feedback loop to promote MM cell progression, increase angiogenesis, and inhibit immune surveillance via both cell-cell contact and abnormal production of multiple cytokines/chemokines. Most recently, hyper-activated OCs have been associated with activation of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, which impairs T cell proliferation and cytotoxicity against MM cells. Importantly, therapeutic anti-CD38 monoclonal antibodies and checkpoint inhibitors can alleviate OC-induced immune suppression. Furthermore, a proliferation-inducing ligand, abundantly secreted by OCs and OC precursors, significantly upregulates PD-L1 expression on MM cells, in addition to directly promoting MM cell proliferation and survival. Coupled with increased PD-L1 expression in other immune-suppressive cells, i.e., myeloid-derived suppressor cells and tumor-associated macrophages, these results strongly suggest that OCs contribute to the immunosuppressive MM BM microenvironment. Based on these findings and ongoing osteoimmunology studies, therapeutic interventions targeting OC number and function are under development to diminish both MM bone disease and related immune suppression. In this review, we discuss the classical and novel roles of OCs in the patho-immunology of MM. We also describe novel therapeutic strategies simultaneously targeting OCs and MM interactions, including PD-1/PD-L1 axis, to overcome the immune-suppressive microenvironment and improve patient outcome.

Function studies of pectolinarigenin demonstrated that, as a natural product, it possesses the regulatory effects on transcription factors (TFs) such as: signal transducer and activator of transcription 3 (STAT3). Herein, we aimed to identify the regulatroy effects of pectolinarigenin on the osteoclastogenesis TFs such as: NFATc1 and c-Fos, and further identify the relevant up-stream signals activity. We initially found pectolinarigenin inhibited receptor activator of nuclear factor-kappa B ligand (RANKL) induced osteoclast formation during the bone marrow-derived macrophages (BMMs) cultures, suggesting that this natural product could act on osteoclast precursors by inhibiting the down signaling cascades of RANKL signaling. Moreover, mechanistical investigation showed pectolinarigenin inhibits RANKL-mediated osteoclastogenesis by attenuating the nuclear factor of activated T cells cytoplasmic 1 (NFATc-1) and c-Fos following the Akt and mitogen activated protein kinases (MAPKs) signaling costimulatory. These findings identify that pectolinarigenin may act as an anti-resorption agent by blocking osteoclast activation., (Copyright © 2018. Published by Elsevier B.V.)

Multiple myeloma (MM) is the second-most-common hematologic malignancy and the most frequent cancer to involve bone. MM bone disease (MMBD) has devastating consequences for patients, including dramatic bone loss, severe bone pain, and pathological fractures that markedly decrease the quality of life and impact survival of MM patients. MMBD results from excessive osteoclastic bone resorption and persistent suppressed osteoblastic bone formation, causing lytic lesions that do not heal, even when patients are in complete and prolonged remission. This review discusses the cellular and molecular mechanisms that regulate the uncoupling of bone remodeling in MM, the effects of MMBD on tumor growth, and potential therapeutic approaches that may prevent severe bone loss and repair damaged bone in MM patients., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)

Objectives: Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destruction in vivo has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted in vivo ., Methods: Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry., Results: Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation., Conclusions: Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Fusobacterium nucleatum is a Gram-negative anaerobic bacterium associated with periodontal disease. Some oral bacteria, like Porphyromonas gingivalis, evade the host immune response by inhibiting inflammation. On the other hand, F. nucleatum triggers inflammasome activation and release of danger-associated molecular patterns (DAMPs) in infected gingival epithelial cells., Methods: In this study, we characterized the pro-inflammatory response to F. nucleatum oral infection in BALB/c mice. Western blots and ELISA were used to measure cytokine and DAMP (HMGB1) levels in the oral cavity after infection. Histology and flow cytometry were used to observe recruitment of immune cells to infected tissue and pathology., Results: Our results show increased expression and production of pro-inflammatory cytokines during infection. Furthermore, we observe that F. nucleatum infection leads to recruitment of macrophages in different tissues of the oral cavity. Infection also contributes to osteoclast recruitment, which could be involved in the observed bone resorption., Conclusions: Overall, our findings suggest that F. nucleatum infection rapidly induces inflammation, release of DAMPs, and macrophage infiltration in gingival tissues and suggest that osteoclasts may drive bone resorption at early stages of the inflammatory process., (Copyright © 2018 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Objective: To study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function., Methods: Bone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area., Results: The flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS ( P <0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant ( P <0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group ( P <0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant ( P <0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant ( P <0.05)., Conclusion: LPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.

Rationale: Characterizing the regulation of bone-resorbing osteoclasts is central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis and periodontitis. 5-hydroxytryptamine (5-HT) has drawn considerable attention for its role in bone; however, it remains unknown whether the intracellular signaling of 5-HT receptors (5-HTRs) is linked to any of the regulatory mechanisms in osteoclasts. Herein, we report 5-HT 6 R to be a key regulatory receptor for osteoclastogenesis. Methods: In order to explore the critical role of 5-HT 6 R in bone-resorbing osteoclasts, in vitro experiments were performed using mouse whole bone marrow cells isolated from femora and tibiae and In vivo animal experiments were performed using 5-HT 6 R-deficient (5-HT 6 R KO-/- ) mice, bone resorption mice model, and osteoporosis mice model. Results: Compared to other 5HTRs, activation of 5-HT 6 R relatively increased TRAP (tartrate-resistant acid phosphatase) activity during osteoclastogenesis. 5-HT 6 R KO(-/-) mice and 5-HT 6 R KO(-/-) osteoclast lineages presented with an abnormal phenotype and impaired osteoclastogenesis and impaired osteoclastogenesis. Activation of 5-HT 6 R increased the number of TRAP-positive multinuclear osteoclasts, actin ring formation, and expression of early osteoclast markers with osteoclast lineage commitment. Intracellular 5-HT 6 R signaling was found to be linked to RhoA GTPase activation and was involved in the maturation of osteoclasts. This signaling pathway also showed enhanced bone destruction after lipopolysaccharide (LPS) administration in mice. Furthermore, inhibition of 5-HT 6 R-mediated RhoA GTPase signaling protected against ovariectomy(OVX)-induced bone loss in mice. Conclusion: Taken together, our findings place the 5-HT 6 R system in a new context of osteoclast lineages in both an in vitro and in vivo system, and also it may offer a novel molecular target for the treatment of bone diseases., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Head and neck squamous cell carcinoma (HNSCC) poses a significant challenge clinically where one of the mechanisms responsible for the invasion into facial bones occurs via the activation of osteoclasts. Copper has been demonstrated to play a key role in skeletal remodeling. However, the role of copper in cancer-associated bone destruction is thus far unknown. Lysyl oxidase (LOX) is a copper-dependent enzyme that promotes osteoclastogenesis. In the present study, we investigated the effects of copper on HNSCC with bone invasion by the copper chelator, ammonium tetrathiomolybdate (TM) in vitro and in vivo. We demonstrate that TM blocks the proliferation of HNSCC cells, inhibits LOX activation and decreases the expression of the receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts and osteocytes, subsequently suppressing bone destruction. These findings suggest that copper is a potential target for the treatment of HNSCCs associated with bone destruction.