Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic Ca2+ ([Ca2+]i), transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular Ca2+ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent Ca2+ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced [Ca2+]i increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.

Osteoclasts originated from hematopoietic stem cells are multi-nucleated cells that can resorb the bone matrix. Receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) signaling pathway is crucial for the differentiation and activation of osteoclasts. In this study, we investigated for the first time whether or not RANKL induced mitogen- and stress-activated kinase 1 (MSK1) phosphorylation at Ser 376. Activation of MSK1 was detected as soon as 5 min after RANKL stimulation and sparsely detected at 30 min after stimulation. RANKL-induced MSK1 phosphorylation occurred in a dose-dependent manner. MSK1 is known as a downstream signaling molecule of cAMP-dependent protein kinase (PKA). Treatment with the PKA inhibitor H89 significantly suppressed c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) induction upon RANKL stimulation. In addition, cAMP response element-binding protein (CREB) phosphorylation was extremely inhibited by H89 treatment. Mitogen-activated protein kinases (MAPKs) have been investigated for induction of MSK1 phosphorylation. Specific signaling pathway inhibitors for p38 and extracellular signal-regulated kinases (ERKs) significantly blocked RANKL-induced MSK1 activation. Finally, as a downstream effector of the p38-MSK1 pathway, c-Fos transcriptional activity was determined. RANKL-mediated elevation of c-Fos transcriptional activity was significantly suppressed by p38 inhibitor. Moreover, a dominant negative form of CREB suppressed activation of NFATc1. In conclusion, RANKL-stimulated MSK1 phosphorylation could play a role in induction of NFATc1 through CREB and c-Fos activation as a downstream molecule of p38, ERK MAPKs, and PKA. Our results support basic information for the development of osteoclast specific inhibitors.

Xylitol is a sugar alcohol with a variety of functions including bactericidal and anticariogenic effects. However, the cellular mechanisms underlying the role of xylitol in bone metabolism are not yet clarified. In our present study, we exploited the physiological role of xylitol on osteoclast dif-ferentiation in a co-culture system of osteoblastic and RAW 264.7 cells. Xylitol treatment of these co-cultures reduced the number of tartrate-resistant acid phosphatase (TRAP)- positive multinucleated cells induced by 10 nM 1α,25(OH)2 D3 in a dose‐dependent manner. A cell viability test revea-led no marked cellular damage by up to 100 mM of xylitol. Exposure of osteoblastic cells to xylitol decreased RANKL, but not OPG, mRNA expression in the presence of 10-8 M 1α,25(OH)2D3 in a dose‐dependent manner. Furthermore, bone resorption activity, assessed on bone slices in the co- culture system, was found to be dramatically decreased with increasing xylitol concentrations. RANKL and OPG proteins were assayed by ELISA and the soluble RANKL (sRANKL) concentration was decreased with an increased xylitol con-centration. In contrast, OPG was unaltered by any xylitol con-centration in this assay. These results indicate that xylitol inhibits 1α,25(OH)2D3-induced osteoclastogenesis by reducing the sRANKL/OPG expression ratio in osteoblastic cells.

This study examined the anti-osteoclastogenic effects of baicalin on receptor activator of NF-kB ligand (RANKL)- induced RAW264.7 cells. Baicalin is a flavonoid that is produced by Scutellaria baicalensis and is known to have multiple biological properties, including antibacterial, anti- inflammatory and analgesic effects. The effects of baicalin on osteoclasts were examined by measuring 1) cell via- bility; 2) the formation of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells; 3) RANK/RANKL signa- ling pathways and 4) mRNA levels of osteoclast-associated genes. Baicalin inhibited the formation of RANKL-stimu- lated TRAP (+) multinucleated cells and also suppressed the RANKL-stimulated activation of p-38, ERK, cSrc and AKT signaling. Baicalin also inhibited the RANKL-stimu- lated degradation of IĸB in RAW264.7 cells. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was found to be abrogated by this flavonoid. Baica- lin was further found to decrease the mRNA expression of osteoclast-associated genes, including carbonic anhydrase II, TRAP and cathepsin K in the RAW264.7 cells. Our data thus demonstrate that baicalin inhibits osteoclastogenesis by inhibiting the RANKL-induced activation of signaling molecules and transcription factors in osteoclast precursors.

The purpose of this study was to investigate the effect of cytokine-induced osteoclastogenesis on tooth movement related to orthodontic force. We evaluated the cytotoxicity as well as the expression of OPG and RANKL, which influence the homeostasis of bone metabolism. Titanium particles were applied to human periodontal ligament cells and subcultured fourth generation cells. The ALP assay and the MTT assay were used to assess changes in cytotoxicity. After 48 hours, cytotoxicity increased proportionally with the concentration of titanium. With 20 mg, the cytotoxicity was the lowest. R T-PCR was u sed for assessing m R NA l evels of O PG a nd R ANKL; after 96 hours, t he m R NAs of O PG a nd R ANKL increased steeply. A western blot analysis showed that with 20 mg of titanium, the protein expression of OPG increased linearly with time, especially a fter 96 hours, while t he p rotein e xpression o f RANKL d id n ot s how significant changes with titanium processing. Given the increase in OPG expression after the initial cytotoxicity, changes in cytotoxicity with titanium may be attributable to the antagonistic effect of OPG on cytotoxicity

The working mechanism of bisphosphonate on bone cells is unclear despite its powerful inhibitory activity on bone resorption. The differentiation and activation of osteoclasts are essential for bone resorption and are controlled by the stimulatory RANKL and inhibitory OPG molecules. Teeth exhibit a range of movement patterns during their eruption to establish their form and function, which inevitably accompanies peripheral bone resorption. Hence, the mandible, which contains the teeth during their eruption processes, is a good model for revealing the inhibitory mechanism of bisphosphonate upon bone resorption. In the present study, RANKL and OPG expression were examined immunohistochemically in the mandible of rats with developing teeth after alendronate administration (2.5 mg/kg). The preeruptive mandibular first molars at postnatal days 3 to 10 showed the developing stages from bell to crown. No morphological changes in tooth formation were observed after alendronate administration. The number of osteoclasts in the alveolar bone around the developing teeth decreased markedly at postnatal days 3, 7 and 10 compared with the control group. RANKL induced strong positive immunohistochemical reactions in the dental follicles and stromal cells around the mandibular first molar. In particular, many osteoclasts with strongly positive reactions to RANKL appeared above the developing mandibular first molars at postnatal days 3 and 10. Immunohistochemical reactions with RANKL after alendronate administration were weaker than the control groups. However, the immunohistochemical reactivity to OPG was stronger after alendronate administration, at postnatal days 3 and 10. These results suggest that alendronate may decrease bone resorption by regulating the RANKL/OPG pathway in the process of osteoclast formation, resulting in a delay in tooth eruption.

This study investigated whether orthodontic force influences the production of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) in vivo, both of which are affected by cortical activation. Mechanical force was applied to the maxillary premolars of orthodontic patients by fitting the transpalatal arch prior to cortical activation of the gingival tissue. Gingival crevicular fluid (GCF) samples were then collected from each patient using paper strips before and after 1, 3, 7 or 14 days of treatment. The OPG and RANKL levels in the GCF were determined by enzyme-linked immunosorbent assays. The levels of OPG were significantly increased after 1 day of fitting the appliance and decreased to basal levels at 3 days after fitting. In contrast, the RANKL levels were dramatically decreased at 1 day after fitting, but recovered to those of the untreated control at 3 days after the force application. The force-mediated changes in the OPG and RANKL levels of the GCF were unaffected by cortical activation during these experimental periods. Collectively, these results suggest that an acute and severe change between the OPG and RANKL levels plays an important role in stimulating the cellular responses required for alveolar bone remodeling by orthodontic treatment.

Receptor activator of nuclear factor xB ligand (RANKL) induces osteoclast formation from hematopoietic cells via up-regulation of positive regulators, including NF-xB, c-Fos, microphthalmia transcription factor (Mitf), PU.1, and nuclear factor of activated T cells (NFAT) c1. In addition to the positive regulation by these transcription factors, RANKL appears to regulate negative regulators such as MafB and inhibitors of differentiation (Ids). Ids and MafB are abundantly expressed in osteoclast precursors, bone marrowderived monocyte/macrophage lineage cells (BMMs). Expression levels of these genes are significantly reduced by RANKL during osteoclastogenesis. Overexpression of these genes in BMMs inhibits the formation of tartarate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts by down-regulation of NFATc1 and osteoclast-associated receptor (OSCAR), which are important for osteoclast differentiation. Furthermore, reduced expression of these genes enhances osteoclastogenesis and increases expression of NFATc1 and OSCAR. Taken together, RANKL induces osteoclastogenesis via up-regulation of positive regulators as well as down-regulation of negative regulators.

Bone remodeling is a process controlled by the action of two major bone cells; the bone forming osteoblast and the bone resorbing osteoclast. In the process of osteoclastogenesis, stromal cells and osteoblast produce RANKL, OPG, and M-CSF, which in turn regulate the osteoclastogenesis. During the bone resorption by activated osteoclasts, extracellular Ca²+/PO₄²- concentration and degraded organic materials goes up, providing the hypertonic microenvironment. In this study, we tested the effects of hypertonicity due to the degraded organic materials on osteoclastogenesis in co-culture system. It was examined the cellular response of osteoblastic cell in terms of osteoclastogenesis by applying the sucrose, and mannitol, as a substitute of degraded organic materials to co-culture system. Apart from the sucrose, mannitol, and NaCl was tested to be compared to the effect of organic osmotic particles. The addition of sucrose and mannitol (25, 50, 100, 150, or 200 mM) to co-culture medium inhibited the number of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells induced by 10 nM (). However, NaCl did exert harmful effect upon the cells in this co-culture system, which is attributed to DNA damage in high concentration of NaCl. To further investigate the mechanism by which hypertonicity inhibits -induced osteoclastogenesis, the mRNA expressions of receptor activator of nuclear factor (NF)-kB ligand (RANKL) and osteoprotegerin (OPG) were monitored by RT-PCR. In the presence of sucrose (50 mM), RANKL mRNA expression was decreased in a dose-dependent manner, while the change in OPG and M-CSF mRNA were not occurred in significantly. The RANKL mRNA expression was inhibited for 48 hours in the presence of sucrose (50 mM), but such a decrement recovered after 72 hours. However, there were no considerable changes in the expression of OPG and M-CSF mRNA. Conclusively, these findings strongly suggest that hypertonic stress down-regulates -induced osteoclastogenesis via RANKL signal pathway in osteoblastic cell, and may playa pivotal role as a regulator that modulates osteoclastogenesis.

Recently, we reported that high extracellular calcium increased receptor activator of nuclear factor- xB ligand (RANKL) expression via p44/42 mitogen-activated protein kinase (p44/42 MAPK) activation in mouse osteoblasts. However, the mechanism for p44/42 MAPK activation by high extracellular calcium is unclear. In this study, we examined the role of intracellular calcium increase in high extracellular calcium-induced RANKL induction and p44/42 MAPK activation. Primary cultured mouse calvarial osteoblasts were used. RANKL expression was highly induced by 10 mM calcium treatment. Ionomycin, a calcium ionophore, also increased RANKL expression and activated p44/42 MAPK. U0126, an inhibitor of MEK1/2, an upstream activator of p44/42 MAPK, blocked the RANKL induction by both high extracellular calcium and ionomycin. High extracellular calcium increased the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), one of the known upstream regulators of p44/42 MAPK activation. Bisindolylmaleimide, an inhibitor of protein kinase C, did not block RANKL induction and p44/42 MAPK activation induced by high extracellular calcium. 2-Aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, blocked the RANKL induction by high extracellular calcium. It also partially suppressed the activation of Pyk2 and p44/42 MAPK. Cyclosporin A, an inhibitor of calcineurin, also inhibited high calcium-induced RANKL expression in dose dependent manner. However, cyclosporin A did not affect the activation of Pyk2 and p44/42 MAPK by high extracellular calcium treatment. These results suggest that 1) the increase in intracellular calcium via IP3-mediated calcium release is necessary for RANKL induction by high extracellular calcium treatment, 2) Pyk2 activation, but not protein kinase C, following the increase in intracellular calcium might be involved in p44/42 MAPK activation, and 3) calcineurin-NFAT activation by the increase in intracellular calcium is involved in RANKL induction by high extracellular calcium treatment.

Background : Osteoclasts are differentiated from the monocytes/macrophages of hematopoietic cells, that excessive activities of bone-resorbing giant cells leads to pathological bone diseases such as osteoporosis (contained rheumatoid arthritis and autoimmune arthritis). Therefore, it is very important to suppress loss of bone mass by deactivation of osteoclast differentiation. In this context, we evaluated for the effects of black ginseng (BG) extract on TRAP activity, proliferation and differentiation in RANKL-induced osteoclastic RAW264.7 cells. Methods and Results : The aim of this study is to figure out the potential anti-osteoporosis effects and the underlying mechanism of BG extract in RANKL-induced osteoclastic RAW264.7 cells. The ginsenoside Rg3, Rg5, Rk1 and Rh4 content of BG was increased more than Red ginseng (RG). The extracts of BG markedly reduced the activity of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from osteoclastic RAW264.7 cells, without cytotoxicity. BG clearly inhibited RANKL-induced osteoclast differentiation by decreased calcitonin and TRAP (p < 0.01). Furthermore, ginseonside Rg5 and Rk1 significantly inhibited TRAP activity in formation of osteoclastic differentiation (p < 0.01). It is also found that Ginseonside Rg5 and Rk1 mixture more inhibits osteoclast differentiation activity. Conclusion : Our results suggest that Black ginseng extract has an anti-osteoporosis effects in bone disease when administered as a food supplement and has potential as a therapeutic agent for osteoporosis.

Background : This study aimed to determine the anti-osteoclastogenic effects of extracts from CK berry’s and identify the underlying mechanisms in vitro. Methods and Results : Reactive oxygen species (ROS) are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL) were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK) and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38, and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor, and integrin β3. Conclusion : In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB)-mediated c-Fos and NFATc1 signaling pathway.

Background : Capsaicin is an active component of chili peppers, which are plants belonging to the genus Capsicum. Research reported capsaicin has antioxidant and anti-inflamatory activity and osteoclast lineages are very susceptible to oxidative stress, as osteoclasts are produced by increased-generation of intracellular ROS and osteoclasts are activated by ROS. Therefore, our study was evaluated the influence of intracellular oxidative stress such as increased ROS level on RANKL-mediated osteoclast differenciation. Methods and Results : Capsaicin showed a good free radical scavenging activity at in-vitro antioxidant activity. The inhibitory effect of osteoclast differentiation on capsaicin was confirmed. Osteoclast differentiation from murine macrophage RAW 264.7 cells was induced by RANKL. The effect of capsaicin on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation was demonstrated using a tartrate-resistant acid phosphatase (TRAP) assay and TRAP staining. Capsaicin showed an inhibitory effect on TRAP activity. The TRAP staining showed that the number of TRAP positive osteoclasts was reduced in capsaicin-treated cells. Conclusion : Capsaicin revealed an inhibitory effect in osteoclast differentiation induced by RANKL. These results suggest that capsaicin may have a beneficial effect for the prevention or treatment of osteoclast caused bone diseases such as osteoporosis.

Background : Osteoclasts as multinucleated cells originate from hematopoietic monocyte/ macrophage precursor cell, shows the bone absorption through the commitment, differentiation, fusion, and bone resorption stages by regulation of M-CSF and RANKL. It has been reported a significant negative correlation between the increase of oxidative stress and the bone density, and when RANKL reaction to the osteoclasts precursor cells is mainly generated ROS is due to increased activity of NADPH oxidase1 (NOX1), and these ROS act as a factor which promotes osteoclasts differentiation. Thus, RANKL signaling process is important that excessive osteoclast formation and differentiation inhibited through the regulation of each step. Methods and Results : F3570 ethanol extract showed relatively high activity at in-vitro antioxidant activity. F3570 water extract inhibited ROS generation in RAW 264.7 cells stimulated with H2O2 and RANKL, even at low concentrations. The inhibitory effect of osteoclast differentiation on F3570 water extract was confirmed that shown through NF-κB pathway, MAPK pathway including ERK and JNK. F3570 ethanol extract is considered to be regulated by the p38 MAPK and the other signaling pathway. Also, F3570 both water and ethanol extract were significantly reduced gene expression such as TRAP, calcitonin receptors and integrin β3 of RANKL-induced mature osteoclast in the bone resorption stage. Conclusion : Through this study, F3570 extract revealed an outstanding inhibitory effect and signaling mechanisms in osteoclast differentiation induced by RANKL. These results suggest that F3570 is bone diseases associated with aging or osteoporosis caused by menopause in an aging society is expected to be a superior candidate for the treatment or the prevention