• Molecules and Cells
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• v.37 no.8
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• pp.598-604
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• 2014

Fatty acids, important components of a normal diet, have been reported to play a role in bone metabolism. Osteoclasts are bone-resorbing cells that are responsible for many bone-destructive diseases such as osteoporosis. In this study, we investigated the impact of a medium-chain fatty acid, capric acid, on the osteoclast differentiation, function, and survival induced by receptor activator of NF-${\kappa}B$ ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Capric acid inhibited RANKL-mediated osteoclastogenesis in bone marrow-derived macrophages and suppressed RANKL-induced $I{\kappa}B{\alpha}$ phosphorylation, p65 nuclear translocation, and NF-${\kappa}B$ transcriptional activity. Capric acid further blocked the RANKL-stimulated activation of ERK without affecting JNK or p38. The induction of NFATc1 in response to RANKL was also attenuated by capric acid. In addition, capric acid abrogated M-CSF and RANKL-mediated cytoskeleton reorganization, which is crucial for the efficient bone resorption of osteoclasts. Capric acid also increased apoptosis in mature osteoclasts through the induction of Bim expression and the suppression of ERK activation by M-CSF. Together, our results reveal that capric acid has inhibitory effects on osteoclast development. We therefore suggest that capric acid may have potential therapeutic implications for the treatment of bone resorption-associated disorders.

• 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.

• Journal of Life Science
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• v.16 no.6
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• pp.1066-1070
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• 2006

Smooth muscle cell (SMC) proliferation is important in the pathogenesis of vascular proliferative disorders. Understanding of the molecular mechanism underlying SMC growth after arterial injury would have therapeutic implications. Here we report that receptor activator of $NF-{\kappa}B$ ligand (RANKL), a member of tumor necrosis factor (TNF) family, promotes the proliferation of SMC, leading to decreased expression of p21 and enhancement of SMC growth. ERK and p38 phosphorylation was enhanced after RANKL treatment in SMC. Inhibition of ERK/p38 MAPK activity by PD98059/SB203580 completely abolished RANKL-induced proliferation of SMC, indicating ERK and p38 MAPK are essential for RANKL-induced SMC proliferation. Taken together, our findings demonstrate that RANK-RANKL-ERK/p38 pathway is important for proliferation of SMC and that these molecules may be the new therapeutic targets for the prevention of vascular diseases.

• Korean Journal of Oriental Medicine
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• v.13 no.2 s.20
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• pp.157-164
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• 2007

An imbalance in bone remodeling that is caused by increased bone resorption over bone formation leads to most adult skeletal diseases including osteoporosis. Since the development of anti-resorptive agents from natural substances has recently gained more interest in the treatment of osteoporosis, we evaluated the effects of 222 natural compounds on receptor activator of $NF-{\kappa}B$ ligand (RANKL)-induced of tartrate-resistance acid phosphatase (TRAP) activity in RAW264.7 murine macrophage cell, and found that berberine chloride is one of compounds inhibiting RANKL-induced TRAP activity. Berberine chloride significantly inhibited the RANKL-induced TRAP activity and the formation of multinucleated osteoclasts in a dose-dependent manner. In addition, berberine chloride prevented the RANKL-induced mRNA expression of TRAP, matrix metalloproteinase 9 and c-Src, which have been known to be highly expressed in the process of osteoclastogenesis. Interestingly, berberine chloride prevented the RANKL-induced activation of extracellular signal-regulated kinase (ERK) which is one of mitogen-activated protein (MAP) kinases. In conclusion, berberine chloride could inhibit the osteoclastogenesis via preventing the activation of ERK/MAP kinase signaling pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.1
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• pp.31-36
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• 2012

The receptor activator of NF-${\kappa}B$ ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-${\kappa}B$ and other signal transduction pathways essential for osteoclastogenesis, such as $Ca^{2+}$ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate ($IP_3$) and $IP_3$-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of $IP_3$ and evaluated $IP_3$-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of $Ca^{2+}$ signaling proteins such as $IP_3$ receptors ($IP_3Rs$), plasma membrane $Ca^{2+}$ ATPase, and sarco/endoplasmic reticulum $Ca^{2+}$ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of $IP_3$ was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) ${\delta}$, a probe specifically detecting intracellular $IP_3$ levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)[ and of $IP_3Rs$ with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of $IP_3Rs$) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular $IP_3$ levels and the $IP_3$-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

• International Journal of Oral Biology
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• v.30 no.4
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• pp.143-148
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• 2005

Osteoclasts are multinucleated cells with bone resorbing activity and differentiated from hematopoietic cell lineages of monocyte/macrophages in the presence of receptor activator of NF-${\kappa}B$ ligand (RANKL) and M-CSF. However, the exact molecular mechanisms through which RANKL stimulates osteoclastogenesis remain to be elucidated. Here we report that activation of cAMP-response elementbinding protein (CREB) is not involved in osteoclastogenesis from osteoclast precursors in response to RANKL. RANKL induced CREB activation in osteoclast precursors. Using pharmacological inhibitors, we found that RANKL-induced CREB activation is dependent on p38 MAPK pathways. We also found that ectopic expressions of wild type and dominant negative forms of CREB in osteoclast precursors did not affect RANKL-induced osteoclast formation and bone resorbing activity. Furthermore, dominant negative forms of CREB did not alter the expression levels of osteoclast-specific marker genes. Taken together, these data suggest that CREB is dispensable for differentiation and resorbing activity of osteoclasts.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.2
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• pp.111-118
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• 2015

Osteoprotegerin (OPG), receptor activator of NF-${\kappa}B$ ligand (RANKL)/receptor activator of NF-${\kappa}B$ (RANK) axis, and TNF-related apoptosis-inducing ligand (TRAIL) participate in vascular calcification process including atherosclerosis, but their contributions under high glucose (HG) and phosphate (HP) condition for a long-term period (more than 2 weeks) have not been fully determined. In this study, we evaluated the effects of HG and HP levels over 2 or 4 weeks on the progression of vascular calcification in rat vascular smooth muscle cells (VSMCs). Calcium deposition in VSMCs was increased in medium containing HG (30 mmol/L D-glucose) with ${\beta}$-glycerophosphate (${\beta}$-GP, 12 mmol/L) after 2 weeks and increased further after 4 weeks. OPG mRNA and protein expressions were unchanged in HG group with or without ${\beta}$-GP after 2 weeks. However, after 4 weeks, OPG mRNA and protein expressions were significantly lower in HG group with ${\beta}$-GP. No significant expression changes were observed in RANKL, RANK, or TRAIL during the experiment. After 4 weeks of treatment in HG group containing ${\beta}$-GP and rhBMP-7, an inhibitor of vascular calcification, OPG expressions were maintained. Furthermore, mRNA expression of alkaline phosphatase (ALP), a marker of vascular mineralization, was lower in the presence of rhBMP-7. These results suggest that low OPG levels after long term HG and phosphate stimulation might reduce the binding of OPG to RANKL and TRAIL, and these changes could increase osteo-inductive VSMC differentiation, especially vascular mineralization reflected by increased ALP activity during vascular calcification.

• Molecules and Cells
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• v.37 no.10
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• pp.747-752
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• 2014

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-${\kappa}B$ ligand (RANKL) signaling has remained elusive. We now demonstrate that $PKC{\beta}$ acts as a positive regulator which inactivates glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, $PKC{\beta}$ expression is increased by RANKL. Pharmacological inhibition of $PKC{\beta}$ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-$3{\beta}$ was decreased by $PKC{\beta}$ inhibition. Likewise, down-regulation of $PKC{\beta}$ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-$3{\beta}$ phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the $PKC{\beta}$ pathway, leading to GSK-$3{\beta}$ inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for $PKC{\beta}$'s therapeutic targeting to treat inflammation-related bone diseases.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.3
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• pp.291-297
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• 2015

An oral environment is constantly exposed to environmental factors and microorganisms. The periodontal ligament (PDL) fibroblasts within this environment are subject to bacterial infection and allergic reaction. However, how these condition affect PDL fibroblasts has yet to be elucidated. PDL fibroblasts were isolated from healthy donors. We examined using reverse transcription-polymerase chain reaction and measuring the intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$). This study investigated the receptors activated by exogenous bacterial pathogens (Lipopolysaccharide and peptidoglycan) and allergens (German cockroach extract and house dust mite) as well as these pathogenic mediators-induced effects on the intracellular $Ca^{2+}$ signaling in human PDL fibroblasts. Moreover, we evaluated the expression of pro-inflammatory cytokines (interleukin (IL)-$1{\beta}$, IL-6, and IL-8) and bone remodeling mediators (receptor activator of NF-${\kappa}B$ ligand and osteoprotegerin) and intracellular $Ca^{2+}$-involved effect. Bacterial pathogens and allergic mediators induced increased expression of pro-inflammatory cytokines, and these results are dependent on intracellular $Ca^{2+}$. However, bacterial pathogens and allergic mediators did not lead to increased expression of bone remodeling mediators, except lipopolysaccharide-induced effect on receptor activator of NF-${\kappa}B$ ligand expression. These experiments provide evidence that a pathogens and allergens-induced increase in $[Ca^{2+}]_i$ affects the inflammatory response in human PDL fibroblasts.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.190-198
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• 2018

Periodontitis is an inflammatory disease caused by microbial lipopolysaccharide (LPS), destroying gingival tissues and alveolar bone in the periodontium. In the present study, we evaluated the anti-inflammatory and anti-osteoclastic effects of panduratin A, a chalcone compound isolated from Boesenbergia pandurata, in human gingival fibroblast-1 (HGF-1) and RAW 264.7 cells. Treatment of panduratin A to LPS-stimulated HGF-1 significantly reduced the expression of interleukin-$1{\beta}$ and nuclear factor-kappa B (NF-${\kappa}B$), subsequently leading to the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-8 compared with that in the LPS control ($^{**}p$ < 0.01). These anti-inflammatory responses were mediated by suppressing the mitogen-activated protein kinase (MAPK) signaling and activator protein-1 complex formation pathways. Moreover, receptor activator of NF-${\kappa}B$ ligand (RANKL)-stimulated RAW 264.7 cells treated with panduratin A showed significant inhibition of osteoclastic transcription factors such as nuclear factor of activated T-cells c1 and c-Fos as well as osteoclastic enzymes such as tartrate-resistant acid phosphatase and cathepsin K compared with those in the RANKL control ($^{**}p$ < 0.01). Similar to HGF-1, panduratin A suppressed osteoclastogenesis by controlling MAPK signaling pathways. Taken together, these results suggest that panduratin A could be a potential candidate for development as a natural anti-periodontitis agent.