• Molecules and Cells
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• v.39 no.10
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• pp.734-741
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• 2016

Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had $MHCII^{low}F4/80^{high}$ as well as $CD11c^+CD11b^{high}CD80^-CD64^+MerTK^+$ phenotypes. In contrast, GM-BMDCs had $MHCII^{high}F4/80^{low}$ and $CD11c^{high}CD8{\alpha}^-CD11b^+CD80^+CD64^-MerTK^{low}$ phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing $TNF{\alpha}$, $IL-1{\beta}$, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells.

• BMB Reports
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• v.47 no.8
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• pp.451-456
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• 2014

Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of $NF-{\kappa}B$ ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and $I{\kappa}B$, as well as $I{\kappa}B$ degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption.

• Journal of Korean society of Dental Hygiene
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• v.23 no.4
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• pp.219-226
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• 2023

Objectives: This study aimed to investigate the effects of rhubarb extract on osteoclast differentiation in bone marrow-derived macrophages (BMMs). Osteoclasts are vital for bone resorption and remodeling. Osteoclast dysregulation can contribute to various bone-related disorders that directly affect oral health. Rhubarb, a medicinal plant with anti-inflammatory properties, has been shown to modulate bone metabolism. Methods: BMMs were isolated from the femurs and tibias of 5-week-old C57BL/6 mice and cultured in the presence of mouse macrophage colony-stimulating factor (M-CSF) for 3 days. Subsequently, BMMs were treated with M-CSF and receptor activator of nuclear factor-κB ligand (RANKL) to induce osteoclast differentiation. Results: Rhubarb extract effectively suppressed osteoclast differentiation in BMMs. Furthermore, rhubarb extract inhibited the mRNA expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), which are essential for osteoclastogenesis. Moreover, it inhibited the RANKL-induced expression of nuclear factor of activated T cell c1 (NFATc1), a crucial transcription factor in osteoclast differentiation. Conclusions: These results suggest that rhubarb extract promotes oral health by inhibiting osteoclastogenesis in BMMs. Thus, rhubarb extract shows promise as a therapeutic agent for bone-related disorders that directly affect oral health, particularly those associated with abnormal osteoclast activity. Further research and exploration of the underlying mechanisms are warranted to fully understand their potential clinical applications.

• YAKHAK HOEJI
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• v.53 no.2
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• pp.78-82
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• 2009

We explored the effects of dexamethasone on osteoclast precursors using BMMs. Dexamethasone inhibited the proliferation of BMMs. Furthermore, it stimulated the osteoclast formation via NFATc1 activation in the presence of RANKL. Since dexamethasone targeted the early stage of osteoclast formation, we investigated its effect on mRNA expression of GR and $IFN-{\beta}$. Whereas dexamethasone had no effects on GR expression in the presence of RANKL, it reduced the expression of $IFN-{\beta}$, suggesting that dexamethasone increased RANKL-induced osteoclast formation by modulating $IFN-{\beta}$.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.5
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• pp.807-813
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• 2010

Impairment of balance between bone-resorbing osteoclasts and bone-forming osteoblasts result in bone disease. Especially, increased osteoclast formation and activity are responsible for bone diseases such as osteoporosis, rheumatoid arthritis, periodontal disease. Natural metabolites of plants have recently received much attention as an alternative tools for the development of novel therapeutic strategy. The aim of this study was to search the natural products to inhibit osteoclast differentiation and was to evaluate of its mechanism. Water extract of Gastrodia elata Blune significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages (BMMs) in a dose dependent manner. However, water extract of Gastrodia elata Blune did not affect cytotoxicity when compared with control. The mRNA expression of c-Fos, NFATc1, and TRAP induced by RANKL was inhibited by water extract of Gastrodia elata Blune treatment. Also, water extract of Gastrodia elata Blune inhibited the protein expression of c-Fos and NFATc1 expression in BMMs treated with RANKL. Water extract of Gastrodia elata Blune suppressed the phosphorylation of p38 induced by RANKL. In general, RANKL considerably inhibited the expression level of Id2 and MafB known as negative regulators of osteoclastogenesis, but RANKL did not inhibit Id2 and MafB expression in BMMs when it was co-treated with Gastrodia elata Blune. Taken together, these results suggest that Gastrodia elata Blune may be a useful drug in the treatment of bone-related disease.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.4
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• pp.891-895
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• 2008

Natural substances have recently received much attention as therapeutic drugs to prevent many diseases in humans because they avoid the many side effects of treatment with chemical compounds. We examined the effect of water extract of deer antler in RANKL-induced osteoclast differentiation. The effects of water extract of deer antler in osteoclast differentiation were determined by culture of bone marrow macrophages (BMMs). The mRNA expression levels of c-Fos, NFATc1, TRAP, and GAPDH in BMMs were analyzed by RT-PCR. Cell lysates were obtained from the treated cells, the expression levels of c-Fos and NFATc1 were determined by western blotting with antibodies for c-Fos and NFATc1. Water extract of deer antler greatly inhibited RANKL-mediated osteoclast differentiation in osteoclast precursors without cytotoxicity. Water extract of deer antler inhibited the expression of c-Fos and NFATc1 in BMMs treated with RANKL. Our findings suggest that water extract of deer antler inhibited osteoclast differentiation by suppressing c-Fos and NFATc1 expression in response to RANKL. These results demonstrate that water extract of deer antler may be a useful the treatment of bone-related disease such as osteoporosis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.4
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• pp.860-865
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• 2009

Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage. The differentiation of osteoclasts are regulated by osteoblastic cells expressed RANKL, which is the most critical molecule for osteoclast differentiation. In this study, we found that water extracts of cuscuta inhibited RANKL-mediated osteoclast differentiation by direct action on bone marrow macrophages (BMMs) without cytotoxicity. In BMMs, water extracts of cuscuta inhibited the mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR. Also, the protein expression of c-Fos and NFATc1 was inhibited by water extracts of cuscuta treatement. Water extracts of cuscuta inhibited the phosphorylation of p38, ERK, and JNK in BMMs treated with RANKL. However, water extracts of cuscuta did not inhibit RANKL-induced I-${\kappa}B$ activation. Water extract of cuscuta failed to inhibit bone resorption by osteoclasts cultured on hydroxyapatite plates. These results suggest that cuscuta may be a promising drug for use against bone disorders such as osteoporosis and rheumatoid arthritis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.1
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• pp.65-70
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• 2011

Osteoclasts play a critical role in bone-related diseases such as osteoporosis and rheumatoid arthritis by resorbing the bone. Recently, natural products from plants have been extensively studied as therapeutic drugs to treat and prevent various diseases. Here, we examined the effects of rhizoma arisaematis on ostoclast differentiation and bone resorption. We showed that rhizoma arisaematis significantly suppressed receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation in bone marrow-derived macrophages (BMMs) in a dose dependent manner but have little or no effect on the cytotoxicity of BMMs and RAW264.7 cells. We found that rhizoma arisaematis iarrow-ed the RANKL-induced c-Fos and nuclear factor of activated T cells (NFAT)c1, which is a master regulator of osteoclast differentiation. Furthermore, rhizoma arisaematis suppressed the mRNA expression of tartrate resistant-acid phosphatase and cathepsin K iaduced by RANKL in BMMs. in y chanistic studies, rhizoma arisaematis considerably iarrow-ed I-${\kappa}B$ degradation, which is a negative regulator of NF-${\kappa}B$, but iaduced the phosphderlation of p-38, ERK, and JNK.MMlso, we found that rhizoma arisaematis significantly iarrow-ed osteoclastic bone resorption. Taken tarether, our results suggest that rhizoma arisaematis suppresses osteoclast differentiation through down-regulatd the mRANKL-induced c-Fos and NFATc1 expression and iarrow-s bone resorption.

• The Journal of Advanced Prosthodontics
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• v.6 no.3
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• pp.157-164
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• 2014

PURPOSE. This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source. MATERIALS AND METHODS. 40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs). RESULTS. MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM. CONCLUSION. Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.

• YAKHAK HOEJI
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• v.59 no.5
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• pp.207-214
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• 2015

P2Y receptors, a type of P2 receptor family, are G-protein coupled receptors and 8 subtypes have been characterized ($P2Y_1$, $P2Y_2$, $P2Y_4$, $P2Y_6$, $P2Y_{11-14}$). Recently, several studies have shed light on the role of P2Y receptors in bone biology. Among them, little is known on the role of $P2Y_6$ receptor on osteoclast differentiation. Thus, we investigated the role of $P2Y_6$ receptor on osteoclastogenesis using $P2Y_6$ receptor selective antagonist, MRS 2578. When osteoblasts and bone marrow cells were co-cultured in the presence of $VitD_3$ and $PGE_2$, $P2Y_6$ antagonist increased the formation of TRAP positive osteoclasts. To elucidate the target cells of $P2Y_6$ antagonist, we first checked the effect of MRS 2578 on osteoblasts. Treatment of MRS 2578 did not affect OPG : RANKL mRNA ratio in osteoblasts. Next, we checked the effects of $P2Y_6$ antagonist on osteoclast precursors using mouse bone marrow macrophages (BMMs). Addition of MRS 2578 increased the number of osteoclasts in RANKL-treated BMMs. Although $P2Y_6$ antagonist had no effect on RANKL-induced NFATc1, c-Fos and MafB expression levels, it significantly stimulated RANKL-induced Blimp1 mRNA expression in BMMs. Taken together, these data indicate that $P2Y_6$ antagonist increases osteoclast formation by upregulation of Blimp1 expression.