• International Journal of Oral Biology
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• v.49 no.3
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• pp.79-86
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• 2024

Periodontal disease (PD) is strongly linked to increased risk of oral squamous cell carcinoma (OSCC); however, the specific mechanism through which the development of PD and OSCC is simultaneously promoted remains unclear. This study explored the impact of periodontal pathogens on OSCC progression and the contribution of periodontal pathogen-stimulated OSCC to PD development. The expression of osteoclastogenesis-inducing factors was assessed using quantitative reverse transcription polymerase chain reaction analysis following stimulation of OSCC with lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis (Pg), a pathogen commonly responsible for PD. The cell counting kit-8 assay was used to determine the effects of Pg-LPS on OSCC proliferation and drug resistance to cisplatin and 5-fluorouracil. The effects of conditioned medium (CM) derived from Pg-LPS-stimulated OSCC on osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) staining on bone marrow-derived macrophages (BMMs). Pg-LPS administration in SCC-25 and YD-8 OSCC cell lines induced a significant increase in receptor activator of nuclear factor kappa-B ligand mRNA expression; however, it did not affect cell proliferation. Treatment with CM derived from Pg-LPS-stimulated SCC-25 or YD-8 cells markedly enhanced the formation of TRAP-positive multinucleated cells during osteoclast differentiation of BMMs. Altogether, these findings demonstrate that Pg-LPS-stimulated OSCC promoted osteoclastogenesis through a paracrine mechanism.

• The korean journal of orthodontics
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• v.35 no.4 s.111
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• pp.262-274
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• 2005

Tooth movement is a result of mutual physiologic responses between the periodontal ligament and alveolar bone stimulated by mechanical strain. The PDL cell and osteoblast are known to have an influence on bone formation by controlling collagen synthesis and alkaline phosphatase activation. Moreover. recent studies have shown that the PDL cell and osteoblast release osteoprotegerin (OPG) and the receptor activator of nuclear factor ぉ ligand (RANKL) to control the level of osteoclast differentiation and activation which in turn influences bone resorption. In this study. progressively increased, continuous tensional force was applied to PDL cells. The objective was to find out which kind of biochemical reactions occur after tensional force application and to illuminate the alveolar bone resorption and apposition mechanism. Continuous and progressively increased tensile force was applied to PDL cells cultured on a petriperm dish with a flexible membrane The amount of $PGE_2$ and ALP synthesis were measured after 1, 3, 0 and 12 hours of force application. Secondly RT-PCR analysis was carried out for OPG and RANKL which control osteoclast differentiation and MMP-1 -8, -9, -13 aud TIMP-1 which regulate the resolution of collagen and resorption of the osteoid layer According to the results. we concluded that progressively increased, concluded force application to human PDL cells reduces $PGE_2$ synthesis, and increases OPG mRNA expression.

• International Journal of Industrial Entomology and Biomaterials
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• v.48 no.1
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• pp.13-18
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• 2024

The rice field grasshopper, Oxya chinensis sinuosa (OC), has traditionally been utilized in Korea for various purposes; however, its potential benefits in the context of osteoporosis remain unclear. The results revealed that OC ethanol extract (OCE) significantly inhibited the formation and activity of tartrate-resistant acid phosphatase (TRAP)-positive cells in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated RAW264.7 cells. Furthermore, OCE, at concentrations ranging from 100 to 400 ㎍/mL, demonstrated a dose-dependent reduction in the protein expression of osteoclast-specific markers, including nuclear factor of activated T cell cytoplasmic 1, c-Src, and TRAP, when compared to RANKL stimulation alone. Additionally, OCE significantly inhibited RANKL-induced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) but not the activation of extracellular signal-regulated kinase. Collectively, these results indicate that OCE suppresses osteoclastogenesis by attenuating the phosphorylation of p38 MAPK and JNK. Consequently, these findings suggest that OCE holds promise for the prevention of osteoporosis.

• 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.50 no.9
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• pp.454-459
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• 2017

Tumor suppressor candidate 2 (Tusc2, also known as Fus1) regulates calcium signaling, and $Ca^{2+}$-dependent nuclear factor of activated T-cells (NFAT) and nuclear factor kappa B ($NF-{\kappa}B$) pathways, which play roles in osteoclast differentiation. However, the role of Tusc2 in osteoclasts remains unknown. Here, we report that Tusc2 positively regulates the differentiation of osteoclasts. Overexpression of Tusc2 in osteoclast precursor cells enhanced receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation. In contrast, small interfering RNA-mediated knockdown of Tusc2 strongly inhibited osteoclast differentiation. In addition, Tusc2 induced the activation of RANKL-mediated $NF-{\kappa}B$ and calcium/calmodulin-dependent kinase IV (CaMKIV)/cAMP-response element (CRE)-binding protein CREB signaling cascades. Taken together, these results suggest that Tusc2 acts as a positive regulator of RANKL-mediated osteoclast differentiation.

• Journal of Korean Medicine Rehabilitation
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• v.29 no.2
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• pp.135-147
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• 2019

Objectives This study was performed to evaluate the effect of Pyrola japonica extract (NJ) and its principal constituent, homoarbutin (HA) on osteoclast differentiation and gene expression and bone resorption. The osteoclastogenesis and gene expression were determined in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated RAW264.7 cell. Methods In order to evaluate the effect of HA extracted from NJ on bone resorption, osteoclasts were used to be differentiated and formed by stimulating RAW264.7 cells with RANKL. Tartarate-resistant acid phosphatase (TRAP) (+) polynuclear osteoclast formation ability was evaluated, and differentiation control genes including cathepsin K, matrix metalloproteinases-9 (MMP-9), and TRAP in osteoclast differentiation were analyzed by real-time polymerase chain reaction (PCR). Immunoblotting was performed to measure the effect of mitogen-activated protein kinase (MAPK) factors on bone resorption, and the effect of osteoclasts on osteoclast differentiation was measured. Results Both NJ and high concentration of HA blocked RANKL-stimulated differentiation from RAW264.7 cell to TRAP-positive multinucleated cells. NJ reduced RANKL-induced expression of TRAP, cathepsin K. Both NJ and high concentration of HA inhibited RANKL-mediated expression of MMP-9, nuclear factor of activated T-cells, cytoplasmic 1, and cellular Jun-fos. NJ suppressed RANKL-stimulated expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase, tumor necrosis factor-alpha, and levels of interleukins. Both NJ and HA decreased bone resorption in osteoclast-induced bone pit formation model. Conclusions These results suggest that NJ and HA blocked bone resorption by decreasing RANKL-mediated osteoclastogenesis through down-regulation of genes for osteoclast differentiation.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.1-5
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• 2007

Receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) induces osteoclast formation from hematopoietic cells via up-regulation of positive regulators, including $NF-{\kappa}B$, 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.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1270-1281
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• 2018

Periodontal disease is triggered by the host immune response to pathogens in the microbial biofilm. Worsening of periodontal disease destroys the tooth-supporting tissues and alveolar bone. As oral inflammation can induce systemic diseases in humans, it is important to prevent periodontal disease. In this study, we demonstrated that Curcuma xanthorrhiza supercritical extract (CXS) and its active compound, xanthorrhizol (XAN), exhibit anti-inflammatory effects on lipopolysaccharide (LPS)-treated human gingival fibroblast-1 cells and anti-osteoclastic effects on receptor activator of nuclear factor kappa B ligand (RANKL)-treated RAW264.7 cells. LPS-upregulated inflammatory factors, such as nuclear factor kappa B p65 and $interleukin-1{\beta}$, were prominently reduced by CXS and XAN. In addition, RANKL-induced osteoclastic factors, such as nuclear factor of activated T-cells c1, tartrate-resistant acid phosphatase, and cathepsin K, were decreased in the presence of CXS and XAN. CXS and XAN inhibited the mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathway. Collectively, these results provide evidence that CXS and XAN suppress LPS-induced inflammation and RANKL-induced osteoclastogenesis by suppressing the MAPK/AP-1 pathway.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.274-279
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• 2015

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-_D-1-thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

• Journal of Periodontal and Implant Science
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• v.43 no.5
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• pp.227-232
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• 2013

Purpose: The receptor activator of nuclear factor kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system plays a significant role in osteoclastogenesis, activation of osteoclasts, and regulation of bone resorption. This study aimed to evaluate the use of the salivary soluble RANKL (sRANKL)/OPG ratio as a diagnostic marker for periodontitis in nonsmokers. Methods: Twenty-five patients with chronic periodontitis and 25 individuals with a healthy periodontium were enrolled in this study. Samples containing 5 mL of unstimulated saliva were obtained from each subject. Salivary sRANKL and OPG concentrations were determined using a standard enzyme-linked immunosorbent assay. Statistical analysis was performed using SPSS ver. 18.0. Results: The levels of sRANKL and OPG were detectable in all of the samples. Positive relationships were found between the plaque index and clinical attachment level and both the salivary concentration of sRANKL and the salivary sRANKL/OPG ratio (P<0.05). The salivary concentration of sRANKL and the sRANKL/OPG ratio were significantly higher in the periodontitis group than in the healthy group (P=0.004 and P=0.001, respectively). In contrast, the OPG concentration showed no significant differences between the groups (P=0.455). Conclusions: These findings suggest that the salivary sRANKL/OPG ratio may be helpful in the screening and diagnosis of periodontitis. However, longitudinal studies with larger populations are needed to confirm these results.