• YAKHAK HOEJI
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• v.53 no.4
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• pp.179-183
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• 2009

We explored the role of reactive oxygen species (ROS) in LPS-induced bone loss. LPS was shown to increase the concentration of ROS in osteoclast precursors. The antioxidant decreased osteoclast formation by LPS. Furthermore, the antioxidant decreased NFATc1 expression by LPS, suggesting that ROS mediates NFATc1 expression in the regulation of LPS-induced osteoclast formation. Finally, the antioxidant decreased LPS-induced RANKL mRNA expression in osteoblasts. Taken together, these data indicate that LPS mediates ROS to induce bone loss.

• International Journal of Oral Biology
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• v.30 no.2
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• pp.47-57
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• 2005

Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. Several results suggest that leptin is important mediators of bone metabolism. The present study was undertaken to determine the effects of leptin on anti-osteoclastogenesis using murine precursors cultured on Ca-P coated plates and on the production of osteoprotegerin (OPG) in osteoblastic cells. Additionally, this study examined the possible involvement of prostaglandin $E_2\;(PGE_2)$/protein kinase C (PKC)-mediated signals on the effect of leptin on anti-osteoclastogenesis to various culture systems of osteoclast precursors. Osteoclast generation was determined by counting tartrate-resistant acid phosphatase positive [TRAP (+)] multinucleated cells (MNCs). Osteoclastic activity was determined by measuring area of resorption pits formed by osteoclasts on Ca-P coated plate. The number of 1,25-dihydroxycholecalciferol $(1,25[OH]_2D_3)$- or $PGE_2$-induced TRAP (+) MNCs in the mouse bone marrow cell culture decreased significantly after treatment with leptin. The number of receptor activator of NF-kB ligand (RANKL)-induced TRAP (+) MNCs in M-CSF dependent bone marrow macrophage (MDBM) cell or RAW264.7 cell culture decreased significantly with leptin treatment. Indomethacin inhibited osteoclast generation induced by $1,25[OH]_2D_3$ and dexamethasone, however, no significant differences were found in the leptin treated group when compared to the corresponding indomethacin group. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited osteoclast generation induced by $1,25[OH]_2D_3$. The number of TRAP (+) MNCs decreased significantly with treatment by PMA at concentrations of 0.01 and $0.1{\mu}M$ in culture. Leptin inhibited PMA-mediated osteoclast generation. Isoquinoline-5-sulfonic 2-methyl-1-piperazide dihydrochloride (H7) had no effect on osteoclast generation induced by $1,25[OH]_2D_3$. Cell culture treatment with leptin resulted in no significant differences in osteoclast generation compared to the corresponding H7 group. Indomethacin showed no significant effect on TRAP (+) MNCs formation from the RAW264.7 cell line. PMA inhibited TRAP (+) MNCs formation induced by RANKL in the RAW264.7 cell culture. H7 had no effect on osteoclast generation from the RAW264.7 cell line. There was no difference compared with the corresponding control group after treatment with leptin. $1,25[OH]_2D_3$- or $PGE_2$-induced osteoclastic activity decreased significantly with leptin treatment at a concentration of 100 ng/ml in mouse bone marrow cell culture. Indomethacin, PMA, and H7 significantly inhibited osteoclastic activity induced by $1,25[OH]_2D_3$ in mouse bone marrow cell culture. No significant differences were found between the leptin treated group and the corresponding control group. The secretion of OPG, a substance known to inhibit osteoclast formation, was detected from the osteoblasts. Treatment by leptin resulted in significant increases in OPG secretion by osteoblastic cells. Taken these results, leptin may be an important regulatory cytokines within the bone marrow microenvironment.

• The Journal of Korean Obstetrics and Gynecology
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• v.25 no.2
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• pp.23-42
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• 2012

Objectives: This study was performed to evaluate the effect of Samkieumgamibang (SKG) on osteoporosis. Methods: The osteoclastogenesis and gene expression were determined in RANKL-stimulated RAW 264.7 cell. And, osteoblastogenesis was also determined in rat calvarial cell. Results: SKG decreased the number of TRAP positive cell in osteoclast. It also decreased the expression of Cathepsin K, MMP-9, TRAP, c-fos, NAFTc1 and JNK1 in osteoclast. SKG increased the expression of iNOS in RANKL-stimulated in osteoclast. Otherwise, SKG inhibited TRAP activity in osteoclast. SKG increased cell proliferation, ALP activity, bone martix protein, collagen and nodule in osteoblast. Conclusions: It is concluded that SKG might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression. And, SKG might increase the bone formation resulted from increase of osteoblast function.

• BMB Reports
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• v.51 no.5
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• pp.230-235
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• 2018

Bone resorption by multinucleated osteoclasts is a multistep process involving adhesion to the bone matrix, migration to resorption sites, and formation of sealing zones and ruffled borders. Macrophage colony-stimulating factor (M-CSF) and osteopontin (OPN) have been shown to be involved in the bone resorption process by respective activation of integrin ${\alpha}v{\beta}3$ via "inside-out" and "outside-in" signaling. In this study, we investigated the link between signal modulators known to M-CSF- and OPN-induced osteoclast adhesion and spreading. M-CSF- and OPN-induced osteoclast adhesion was achieved via activation of stepwise signals, including integrin ${\alpha}v{\beta}3$, $PLC{\gamma}$, $PKC{\delta}$, and Rac1. Osteoclast spreading induced by M-CSF and OPN was shown to be controlled via sequential activation, consistent with the osteoclast adhesion processes. In contrast to osteoclast adhesion, osteoclast spreading induced by M-CSF and OPN was blocked via activation of $PLC{\gamma}/PKC{\alpha}/RhoA$ signaling. The combined results indicate that osteoclast adhesion and spreading are selectively regulated via $PLC{\gamma}/PKC{\alpha}-PKC{\delta}/RhoA-Rac1$ signaling.

• YAKHAK HOEJI
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• v.54 no.6
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• pp.461-465
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• 2010

We investigated the role of L-type $Ca^{2+}$ channel in receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast formation. BayK 8644, a L-type $Ca^{2+}$ channel agonist, was shown to increase the RANKLinduced osteoclastogenesis and actin ring formation in mouse bone marrow-dereived macrophage (BMM) culture system. BayK 8644 stimulated RANKL-induced extracellular signal-regulated kinase (ERK) and p38 MAP kinase (MAPK) activation, which leads to increased nuclear factor of activated T cells (NFAT)c1 expression. Taken together, these data indicate that L-type $Ca^{2+}$ channel regulates osteoclast formation possibly through ERK- and p38-mediated NFATc1 expression.

• Journal of Periodontal and Implant Science
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• v.32 no.4
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• pp.721-731
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• 2002

Recently, soluble TNF receptor homolog osteoprotegerin(OPG) and its membrane-bound ligand osteoclast differentiation factor(ODF) were found to regulate osteoclast formation and function, and bone metabolism. It is now well established that ODF acts via RANK expressed on hematopoietic osteoclast precursor cells to facilitate their differentiation to osteoclasts, and OPG prevents the formation of osteoclasts by interfering the binding of ODF and RANK. Expression of OPG and ODF was believed to be closely related to the pathogenesis of bone resorption and destruction from osteoporosis, periodontal diseases, malignant bone tumor, and arthritis. The periodontal ligament fibroblasts (PDLF), located between the tooth and tooth socket, has been thought to play an important role in maintaining bone homeostasis of periodontal tissues. However, the exact mechanism by which bone formation and resorption are regulated by PDLF is not well understood. In this study we have prepared primary cultures of human PDLF from periodontium of malaligned tooth extracted due to orthodontic reason, and determined steady state or inflammatory signal-induced OPG and ODF expression using RT-PCR and western blot analysis. OPG and ODF mRNA and protein were expressed constitutively in the PDLF and these expression were slightly increased by osteotropic cytokine IL-1 ${\beta}$. Lipopolysaccharide-treated PDLF showed decrease in OPG mRNA and protein expression, and increase in ODF mRNA and protein expression. These results indicated that PDLF influence the osteoclastogenesis by OPG and ODF expression in the inflammatory situation as well as physiological condition, and thereby pathogenesis of periodontal alveolar bone destruction.

• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.141-148
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• 2022

Many bone diseases such as osteolysis, osteomyelitis, and septic arthritis are caused by gram-negative bacterial infection, and lipopolysaccharide (LPS), a bacterial product, plays an essential role in this process. Drugs that inhibit LPS-induced osteoclastogenesis are urgently needed to prevent bone destruction in infective bone diseases. Marein, a major bioactive compound of Coreopsis tinctoria, possesses anti-oxidative, anti-inflammatory, anti-hypertensive, anti-hyperlipidemic, and anti-diabetic effects. In this study, we measured the effect of marein on RAW264.7 cells by CCK-8 assay and used TRAP staining to determine osteoclastogenesis. The levels of osteoclast-related genes and NF-κB-related proteins were then analyzed by western blot, and the levels of pro-inflammatory cytokines were quantified by ELISA. Our results showed that marein inhibited LPS-induced osteoclast formation by osteoclast precursor RAW264.7 cells. The effect of marein was related to its inhibitory function on expressions of pro-inflammatory cytokines and osteoclast-related genes containing RANK, TRAF6, MMP-9, CK, and CAII. Additionally, marein leads to markedly inhibited NF-κB signaling pathway activation in LPS-induced RAW264.7 cells. Concurrently, when the NF-κB signaling pathway was inhibited, osteoclast formation and pro-inflammatory cytokine expression were decreased. Collectively, marein could inhibit LPS-induced osteoclast formation in RAW264.7 cells via regulating the NF-κB signaling pathway. Our data demonstrate that marein might be a potential drug for bacteria-induced bone destruction disease. Our findings provide new insights into LPS-induced bone disease.

• 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.

• The korean journal of orthodontics
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• v.25 no.6 s.53
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• pp.705-714
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• 1995

Due to the great deal of effort that has gone into the study of osteoclastic differentiation and activation over the last few decades, the mechanisms of these two events have been discovered gradually. Nitric oxide($NO^-$), which is produced from arginine by a nitric oxide synthase, opened up a new area of biological research. Recently, it has been reported that $NO^-$ is produced by osteoblasts stimulated by lipopolysaccharide and several other cytokines. In this study, the effect of sodium nitroprusside(SNP), a donor of nitric oxide($NO^-$), on osteoclast-like cell formation and on mature osteoclast function was examined. To determine the mechanism of the inhibitory effects of SNP decreased not only the basal $^{45}Ca$ release but also thee bone resorption induced by PTH and 1,25-dihydroxyvitamin $D_3\;(1,25[OH]_{2}D_3)$. The inhibitory effect of SNP on bone resorption induced by PTH appeared 2 dyas after treatment, whereas SNP effect on inhibiting bone resorption induced by $1,25[OH]_{2}D_3$ appeared at the thhird days. When chicken and rat osteeoclasts were cultured on dentin slices, treatment of $300{\mu}M$ SNP resulted in a significant decrease in dentin resorption by osteoclasts in terms of total resolution area and average individual area. We also examind the effect of SNP on formation of osteoclast-like cells that is TRAP-positive multinucleated cells from chicken and rat bone marrow cells in the presence or absence of $10^{-8}\;M\;1,25[OH]_{2}D_3$. The addition of $300{\mu}M$ SNP inhibiteed the formation of TRAP-positive multinucleated cells. The present data suggest that SNP, possibly as a $NO^-$ donor, inhibits the osteoclastic differentiation and osteoclastic activity.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.113-116
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• 2008

Cementum is the mineralized tissue of the tooth. It is similar to bone in several aspects but it differs from bone. Human bone marrow stromal cells (BMSC) and human cementum derived cells (HCDC) (10,000 $cells/cm^2$) were plated in 6 well plates as feeder cells. The next day, mouse bone marrow cells (1.5 million $cells/cm^2$) were added. One group of these plates were incubated in serum-free conditioned medium (SFCM) generated from BMSC or HCDC supplemented with 2% FBS, parathyroid hormone (PTH), 1, 25 dihydroxyvitamin $D_3$ (Vit. $D_3$) and dexamethasone, or plain medium with the same supplements. Another group of plates were cocultured with BMSC or HCDC in plain medium supplemented with 2% FBS, PTH, Vit. $D_3$ and dexamethasone. Plates grown without SFCM or coculture were used as controls. After 10 days, the cells were stained for tartrate-resistant acid phosphatase (TRAP). BMSC were found to support osteoclast formation under normal conditions. This was inhibited however by both SFCM generated from HCDC and also by coculture with HCDC. In addition, HCDC themselves did not support osteoclast formation under any conditions. Our results thus indicate that HCDC do not support osteoclast formation in vitro and that soluble factor (s) from HCDC may inhibit this process. In addition, we show that this inhibition also involves an active mechanism that is independent of osteoprotegerin, a feature that may distinguish cementoblasts from other cells present in periodontium.