• BMB Reports
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• v.50 no.3
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• pp.150-155
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• 2017

Non-small-cell lung cancer (NSCLC) is the third most common cancer that spreads to the bone, resulting in osteolytic lesions caused by hyperactivation of osteoclasts. Activating mutations in epidermal growth factor receptor-tyrosine kinase (EGF-TK) are frequently associated with NSCLC, and afatinib is a first-line therapeutic drug, irreversibly targeting EGF-TK. However, the effects of afatinib on osteoclast differentiation and activation as well as the underlying mechanism remain unclear. In this study, afatinib significantly suppressed receptor activator of nuclear factor ${\kappa}B$ (RANK) ligand (RANKL)-induced osteoclast formation in bone marrow macrophages (BMMs). Consistently, afatinib inhibited the expression of osteoclast marker genes, whereas, it upregulated the expression of negative modulator genes. The bone resorbing activity of osteoclasts was also abrogated by afatinib. In addition, afatinib significantly inhibited RANKL-mediated Akt/protein kinase B and c-Jun N-terminal kinase phosphorylation. These results suggest that afatinib substantially suppresses osteoclastogenesis by downregulating RANK signaling pathways, and thus may reduce osteolysis after bone metastasis.

• Molecules and Cells
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• v.40 no.5
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• pp.371-377
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• 2017

Despite the importance of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-RANK signaling mechanisms on osteoclast differentiation, little has been studied on how RANK expression is regulated or what regulates its expression during osteoclastogenesis. We show here that insulin signaling increases RANK expression, thus enhancing osteoclast differentiation by RANKL. Insulin stimulation induced RANK gene expression in time- and dose-dependent manners and insulin receptor shRNA completely abolished RANK expression induced by insulin in bone marrow-derived monocyte/macrophage cells (BMMs). Moreover, the addition of insulin in the presence of RANKL promoted RANK expression. The ability of insulin to regulate RANK expression depends on extracellular signal-regulated kinase 1/2 (ERK1/2) since only PD98059, an ERK1/2 inhibitor, specifically inhibited its expression by insulin. However, the RANK expression by RANKL was blocked by all three mitogen-activated protein (MAP) kinases inhibitors. The activation of RANK increased differentiation of BMMs into tartrate-resistant acid phosphatase-positive ($TRAP^+$) osteoclasts as well as the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of vacuolar ($H^+$) ATPase (v-ATPase) Vo domain (Atp6v0d2), genes critical for osteoclastic cell-cell fusion. Collectively, these results suggest that insulin induces RANK expression via ERK1/2, which contributes to the enhancement of osteoclast differentiation.

• International Journal of Oral Biology
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• v.40 no.1
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• pp.19-25
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• 2015

Osteocytes may function as mechanotransducers by regulating local osteoclastogenesis. Reduced availability of oxygen, i.e. hypoxia, could occur during disuse, bone development, and fracture. Receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) is an osteoblast/stromal cell derived essential factor for osteoclastogenesis. The hypoxia induced osteoclastogenesis via increased RANKL expression in osteoblasts was demonstrated. Hypoxic regulation of gene expression generally involves activation of the hypoxia-inducible factor (HIF) transcription pathway. In the present study, we investigated whether hypoxia regulates RANKL expression in murine osteocytes and HIF-$1{\alpha}$ mediates hypoxia-induced RANKL expression by transactivating RANKL promoter, to elucidate the role of osteocyte in osteoclastogenesis in the context of hypoxic condition. The expression levels of RANKL mRNA and protein, as well as hypoxia inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) protein, were significantly increased in hypoxic condition in MLO-Y4s. Constitutively active HIF-$1{\alpha}$ alone significantly increased the levels of RANKL expression in MLO-Y4s under normoxic conditions, whereas dominant negative HIF-$1{\alpha}$ blocked hypoxia-induced RANKL expression. To further explore to find if HIF-$1{\alpha}$ directly regulates RANKL transcription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity, whereas mutations of putative HIF-$1{\alpha}$ binding elements in RANKL promoter prevented this hypoxia-induced RANKL promoter activity in MLO-Y4s. These results suggest that HIF-$1{\alpha}$ mediates hypoxia-induced up-regulation of RANKL expression, and that in osteocytes of mechanically unloaded bone, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in osteocytes.

• BMB Reports
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• v.52 no.6
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• pp.409-414
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• 2019

Natural compounds isolated from medicinal herbs and plants have immense significance in maintaining bone health. Hydrolysable tannins have been shown to possess a variety of medicinal properties including antiviral, anticancer, and anti-osteoclastogenic activities. As a part of a study on the discovery of alternative agent against skeletal diseases, we isolated a hydrolysable tannin, 2-O-digalloyl-1,3,4,6-tetra-O-galloyl-${\beta}$-D-glucose (DTOGG), from Galla Rhois and examined the effect on osteoclast formation and function. We found that DTOGG significantly inhibited receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation by downregulating the expression of the key regulator in osteoclastogenesis as well as osteoclast-related genes. Analysis of RANKL/RANK signaling revealed that DTOGG impaired activation of $I{\kappa}B{\alpha}$ and p65 in the nuclear factor kappa-lightchain-enhancer of activated B cells (NF-${\kappa}B$) signaling pathway. Furthermore, DTOGG reduced bone resorbing activity of osteoclasts, compared to the vehicle-treated control. These results suggest that DTOGG could be a useful natural compound to manage osteoclast-mediated skeletal diseases.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.37-42
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• 2013

Receptor activator of NF-${\kappa}B$ ligand (RANKL) is an essential cytokine for osteoclast differentiation, activation and survival. T lymphocytes such as $T_{17}$ cells, a subset of T helper cells that produce IL-17, play an important role in rheumatoid arthritic bone resorption by producing inflammatory cytokines and RANKL. It has not yet been clearly elucidated how T cell activation induces RANKL expression. T cell receptor activation induces the activation of nuclear factor of activated T cell (NFAT) and expression of its target genes. In this study, we examined the role of NFAT in T cell activation-induced RANKL expression. EL-4, a murine T lymphocytic cell line, was used. When T cell activation was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin, RANKL expression increased in a time-dependent manner. In the presence of cyclosporin, an inhibitor of NFAT activation, this PMA/ionomycin-induced RANKL expression was blocked. Overexpression of either NFATc1 or NFATc3 induced RANKL expression. Chromatin immunoprecipitation results demonstrated that PMA/ionomycin treatment induced the binding of NFATc1 and NFATc3 to the mouse RANKL gene promoter. These results suggest that NFATc1 and NFATc3 mediates T cell receptor activation-induced RANKL expression in T lymphocytes.

• International Journal of Oral Biology
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• v.41 no.1
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• pp.9-15
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• 2016

Receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) is an osteoblast/stromal cell-derived essential factor for osteoclastogenesis. During endochondral bone formation, hypertrophic chondrocytes calcify cartilage matrix that is subsequently resorbed by osteoclasts in order to be replaced by new bone. Hypoxia-induced upregulation of RANKL expression has been previously demonstrated in an in vitro system using osteoblasts; however, the involved mechanism remains unclear in chondrocytes. In the present study, we investigated whether hypoxia regulates RANKL expression in ATDC5 cells, a murine chondrogenic cell line, and hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) mediates hypoxia-induced RANKL expression by transactivating the RANKL promoter. The expression levels of RANKL mRNA and protein, as well as HIF-$1{\alpha}$ protein, were significantly increased in ATDC5 cells under hypoxic condition. Constitutively active HIF-$1{\alpha}$ alone significantly increased the levels of RANKL expression under normoxic conditions, whereas dominant negative HIF-$1{\alpha}$ reduced hypoxia-induced RANKL expression. HIF-$1{\alpha}$ increased RANKL promoter reporter activity in a HIF-$1{\alpha}$ binding element-dependent manner in ATDC5 cells. Hypoxia-induced RANKL levels were much higher in differentiated ATDC5 cells, as compared to proliferating ATDC5 cells. These results suggested that under hypoxic conditions, HIF-$1{\alpha}$ mediates induction of RANKL expression in chondrocytes; in addition, hypoxia plays a role in osteoclastogenesis during endochondral bone formation, at least in part, through the induction of RANKL expression in hypertrophic chondrocytes.

• Journal of Veterinary Science
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• v.23 no.4
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• pp.47.1-47.11
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• 2022

Background: In lipopolysaccharide-induced RAW264.7 cells, Aster tataricus (AT) inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells and MAPKs pathways and critical pathways of osteoclast development and bone resorption. Objectives: This study examined how aster saponin A2 (AS-A2) isolated from AT affects the processes and function of osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264.7 cells and bone marrow macrophages (BMMs). Methods: The cell viability, tartrate-resistant acid phosphatase staining, pit formation assay, polymerase chain reaction, and western blot were carried out to determine the effects of AS-A2 on osteoclastogenesis. Results: In RAW264.7 and BMMs, AS-A2 decreased RANKL-initiated osteoclast differentiation in a concentration-dependent manner. In AS-A2-treated cells, the phosphorylation of ERK1/2, JNK, and p38 protein expression were reduced considerably compared to the control cells. In RAW264.7 cells, AS-A2 suppressed the RANKL-induced activation of osteoclast-related genes. During osteoclast differentiation, AS-A2 suppressed the transcriptional and translational expression of NFATc1 and c-Fos. AS-A2 inhibited osteoclast development, reducing the size of the bone resorption pit area. Conclusion: AS-A2 isolated from AT appears to be a viable therapeutic therapy for osteolytic illnesses, such as osteoporosis, Paget's disease, and osteogenesis imperfecta.

• The Korean Journal of Pain
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• v.30 no.2
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• pp.86-92
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• 2017

Osteoblasts, originating from mesenchymal cells, make the receptor activator of the nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in order to control differentiation of activated osteoclasts, originating from hematopoietic stem cells. When the RANKL binds to the RANK of the pre-osteoclasts or mature osteoclasts, bone resorption increases. On the contrary, when OPG binds to the RANK, bone resorption decreases. Denosumab (AMG 162), like OPG (a decoy receptor), binds to the RANKL, and reduces binding between the RANK and the RANKL resulting in inhibition of osteoclastogenesis and reduction of bone resorption. Bisphosphonates (BPs), which bind to the bone mineral and occupy the site of resorption performed by activated osteoclasts, are still the drugs of choice to prevent and treat osteoporosis. The merits of denosumab are reversibility targeting the RANKL, lack of adverse gastrointestinal events, improved adherence due to convenient biannual subcutaneous administration, and potential use with impaired renal function. The known adverse reactions are musculoskeletal pain, increased infections with adverse dermatologic reactions, osteonecrosis of the jaw, hypersensitivity reaction, and hypocalcemia. Treatment with 60 mg of denosumab reduces the bone resorption marker, serum type 1 C-telopeptide, by 3 days, with maximum reduction occurring by 1 month. The mean time to maximum denosumab concentration is 10 days with a mean half-life of 25.4 days. In conclusion, the convenient biannual subcutaneous administration of 60 mg of denosumab can be considered as a first-line treatment for osteoporosis in cases of low compliance with BPs due to gastrointestinal trouble and impaired renal function.

• Journal of Life Science
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• v.28 no.7
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• pp.874-883
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• 2018

Osteoporosis is a disease that increases the risk of fracture by decreasing the mass and strength of bone. It is caused by imbalance of osteoclast bone formation and osteoclast bone resorption. Bone formation by osteoblast is activated via bone morphogenetic proteins and runt-related transcription factor 2. $Wnt/{\beta}-catenin$ signaling and bone resorption by osteoclast are initiated by the binding of receptor activator of nuclear $factor-{\kappa}B$ ligand and receptor activator of nuclear $factor-{\kappa}B$. Menopausal women are at risk for many diseases due to hormonal imbalances, and osteoporosis is the most common metabolic disorder in 30% of postmenopausal women. When estrogen is deficient, bone resorption of osteoclasts is promoted, and the risk of osteoporosis especially increases in postmenopausal women. Hormone replacement therapy has been widely used to relieve or treat the symptoms of menopausal syndrome. However, long-term administration of hormone therapy has been associated with a high risk of side effects, such as breast cancer, ovarian cancer, and uterine cancer. Recently, phytochemicals have been actively studied as a phytoestrogen, which has an estrogen-like activity to cope with symptoms of menopausal syndrome. Therefore, in this review, we investigated the differentiation mechanism of osteoblast and osteoclast and the role of estrogen and phytoestrogen in bone metabolism in relation to previous studies.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.40 no.6
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• pp.291-296
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• 2014

Objectives: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphophonate therapy that has been reported in recent years. Osteoclastic inactivity by bisphosphonate is the known cause of BRONJ. Bone morphogenetic protein-2 (BMP-2) plays an important role in the development of bone. Recombinant human BMP-2 (rhBMP-2) is potentially useful as an activation factor for bone repair. We hypothesized that rhBMP-2 would enhance the osteoclast-osteoblast interaction related to bone remodeling. Materials and Methods: Human fetal osteoblast cells (hFOB 1.19) were treated with $100{\mu}M$ alendronate, and 100 ng/mL rhBMP-2 was added. Cells were incubated for a further 48 hours, and cell viability was measured using an MTT assay. Expression of the three cytokines from osteoblasts, receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF), were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results: Cell viability was decreased to $82.75%{\pm}1.00%$ by alendronate and then increased to $110.43%{\pm}1.35%$ after treatment with rhBMP-2 (P<0.05, respectively). OPG, RANKL, and M-CSF expression were all decreased by alendronate treatment. RANKL and M-CSF expression were increased, but OPG was not significantly affected by rhBMP-2. Conclusion: rhBMP2 does not affect OPG gene expression in hFOB, but it may increase RANKL and M-CSF gene expression.