• Biomedical Science Letters
• /
• v.19 no.2
• /
• pp.164-167
• /
• 2013

Osteoclasts are originated from hemopoietic progenitors of the monocyte/macrophage lineage and resorb mineralized tissues. Elevated osteoclast numbers and activity result in bone disease such as osteoporosis, Paget's disease, and tumor osteolysis. In order to identify the genes that are involved in osteoclast differentiation, microarray was performed after treated with RANKL for 12 h and 24 h in osteoclast precursors. The genes that changed by RANKL treatment were grouped by biological process or molecular function. Among them, the number of genes involved in signal transduction and nucleic acid binding was 6065 and 3066, respectively. When analyzed the number of genes changed more than 1.5 fold in the cells treated with RANKL for 12 h or 24 h compared to when RANKL was not treated, 83 and 62 genes were up-regulated; 56 and 62 genes were downregulated, respectively. To verify the microarray results, real-time RT-PCR for Cxcl1 and Slfn1genes that have not been reported yet related to osteoclast differentiation, as well as Ccl2 gene associated with osteoclast differentiation were carried out. Both experiments showed a similar result of more than 1.5 fold induction of these genes by RANKL treatment. These results suggest the possibility that Cxcl1 and Slfn1 may associate with osteoclastogenesis and provide that microarray is a useful tool to analyze the profile of genes changed during osteoclast differentiation by RANKL. Moreover, this gene profile contributes to understand the regulatory mechanisms involved in osteoclast differentiation and the pathogenesis, thus developing therapeutics of bone diseases such as osteoporosis.

• BMB Reports
• /
• v.35 no.6
• /
• pp.609-614
• /
• 2002

The remodeling process of bone is accompanied by complex changes in the expression levels of various genes. Several approaches have been employed to detect differentially-expressed genes in regard to osteoclast differentiation. In order to identify the genes that are involved in osteoclast differentiation, we used a cDNA-array-nylon membrane. Among 1,200 genes that showed ameasurable signal, 19 genes were chosen for further study. Eleven genes were up-regulated; eight genes were down-regulated. TIS21 was one of the up-regulated genes which were highly expressed in mature osteoclasts. To verify the cDNA microarray results, we carried out RT-PCR and real-time RT-PCR for the TIS21 gene. The TIS21 mRNA level was higher in differentiated-osteoclasts when compared to undifferentiated bone-marrow macrophages. Furthermore, the treatment with $1\;{\mu}M$ of a TIS21 antisense oligonucleotide reduced the formation of osteoclasts from the bone-marrow-precursor cells by ~30%. These results provide evidence for the potential role of TIS21 in the differentiation of osteoclasts.

• Molecules and Cells
• /
• v.37 no.9
• /
• pp.685-690
• /
• 2014

Osteoclasts are large polykaryons that have the unique capacity to degrade bone and are generated by the differentiation of myeloid lineage progenitors. To identify the genes involved in osteoclast development, we performed microarray analysis, and we found that carboxypeptidase E (CPE), a prohormone processing enzyme, was highly upregulated in osteoclasts compared with their precursors, bone marrow-derived macrophages (BMMs). Here, we demonstrate a novel role for CPE in receptor activator of NF-${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation. The overexpression of CPE in BMMs increases the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts and the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are key regulators in osteoclastogenesis. Furthermore, employing CPE knockout mice, we show that CPE deficiency attenuates osteoclast formation. Together, our data suggest that CPE might be an important modulator of RANKL-induced osteoclast differentiation.

• Molecules and Cells
• /
• v.37 no.11
• /
• pp.827-832
• /
• 2014

The balance between bone formation by osteoblasts and destruction of mineralized bone matrix by osteoclasts is important for bone homeostasis. The increase of osteoclast differentiation by RANKL induces bone diseases such as osteoporosis. Recent studies have shown that insulin is one of main factors mediating the cross-talk between bone remodeling and energy metabolism. However, the systemic examination of insulin-induced differential gene expression profiles in osteoclasts has not been extensively studied. Here, we investigated the global effects of insulin on osteoclast precursors at the level of gene transcription by microarray analysis. The number of genes that were up-regulated by ${\geq}1.5$ fold after insulin treatment for 6 h, 12 h, or 24 h was 76, 73, and 39; and 96, 83, and 54 genes were down-regulated, respectively. The genes were classified by 20 biological processes or 24 molecular functions and the number of genes involved in 'development processes' and 'cell proliferation and differentiation' was 25 and 18, respectively, including Inhba, Socs, Plk3, Tnfsf4, and Plk1. The microarray results of these genes were verified by real-time RT-PCR analysis. We also compared the effects of insulin and RANKL on the expression of these genes. Most genes had a very similar pattern of expressions in insulin- and RANKL-treated cells. Interestingly, Tnfsf4 and Inhba genes were affected by insulin but not by RANKL. Taken together, these results suggest a potential role for insulin in osteoclast biology, thus contributing to the understanding of the pathogenesis and development of therapeutics for numerous bone and metabolic diseases.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.29 no.2
• /
• pp.278-284
• /
• 2002

To examine the global gene expression of osteoclastogenesis-related genes in RAW 264.7 and its differentiated OCLs through the use of Atlas Mouse cDNA Array 2.1 membranes printed with 1176 well-characterized mouse genes involved in biology. Both samples were screened in parallel using cDNA expression arrays. The array results were additionally validated using RT-PCR. The results of cDNA arrays showed that 6 genes were up-regulated >2.5-fold (PKC beta II. POMC, PTEN, etc) and 16 genes were down-regulated >2.5-fold (Osteopontin, Cyclin D1, Cathepsin C, PTMA, etc) in both samples at the mRNA level. RT-PCR analysis of PKC beta II of these differentially expressed genes gave result consistent with cDNA array findings. The result of osteoclastogenesis showed that the PKC beta II gene was overexpressed in OCLs compared with RAW264.7 cell line. Osteoclastogenesis-related genes are differentially expressed in RAW264.7 cell line and its differentiated OCLs. its gene overexpression correlates with osteoclast differentiation in RAW264.7 cell line.

• Molecules and Cells
• /
• v.40 no.10
• /
• pp.752-760
• /
• 2017

A2B adenosine receptor (A2BAR) is known to be the regulator of bone homeostasis, but its regulatory mechanisms in osteoclast formation are less well-defined. Here, we demonstrate the effect of A2BAR stimulation on osteoclast differentiation and activity by RANKL. A2BAR was expressed in bone marrow-derived monocyte/macrophage (BMM) and RANKL increased A2BAR expression during osteoclastogenesis. A2BAR stimulation with its specific agonist BAY 60-6583 was sufficient to inhibit the activation of ERK1/2, p38 MAP kinases and $NF-{\kappa}B$ by RANKL as well as it abrogated cell-cell fusion in the late stage of osteoclast differentiation. Stimulation of A2BAR suppressed the expression of osteoclast marker genes, such as c-Fos, TRAP, Cathepsin-K and NFATc1, induced by RANKL, and transcriptional activity of NFATc1 was also inhibited by stimulation of A2BAR. A2BAR stimulation caused a notable reduction in the expression of Atp6v0d2 and DC-STAMP related to cell-cell fusion of osteoclasts. Especially, a decrease in bone resorption activity through suppression of actin ring formation by A2BAR stimulation was observed. Taken together, these results suggest that A2BAR stimulation inhibits the activation of ERK1/2, p38 and $NF-{\kappa}B$ by RANKL, which suppresses the induction of osteoclast marker genes, thus contributing to the decrease in osteoclast cell-cell fusion and bone resorption activity.

• The Korean Journal of Physiology and Pharmacology
• /
• v.23 no.5
• /
• pp.411-417
• /
• 2019

Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclastassociated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.

• The Korea Journal of Herbology
• /
• v.38 no.5
• /
• pp.9-19
• /
• 2023

Objectives : Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density and increased risk of fractures. Bisphosphonates and selective estrogen receptors, which are bone resorption inhibitors that are currently widely used as osteoporosis treatments, show serious side effects when administered for a long time. Research on bone resorption inhibitors that complement the problems of existing treatments is needed. The purpose of this study was to investigate the effect of inhibiting osteoclast differentiation and activity on the tuberous root of Ampelopsis japonica (Thunb.) Makino (AM). Methods : After extracting AM using distilled water and ethanol, the inhibitory effects of the two solvents on osteoclast differentiation were compared using the RANKL-induced in vitro experimental model and the TRAP assay kit. The impact of AM on bone resorption was investigated through the pit formation assay, and its effect on F-actin formation was assessed through fluorescent staining. Additionally, protein and mRNA expression levels of osteoclast differentiation markers (NFATc1, c-Fos, TRAP and ATP6v0d2) and resorption markers (MMP-9, CTK, and CA2) were analyzed via western blot and RT-PCR. Results : AM treatment significantly decreased the number of TRAP-positive cells and pit formation area. Furthermore, AM suppressed both the protein and mRNA expression of NFATc1 and c-Fos, key transcription factors involved in osteoclast differentiation and it downregulated the expression of osteoclast-associated genes such as TRAP, CTK, MMP-9, CA2, and ATP6v0d2. Conclusions : These results suggest that AM can inhibit bone resorption and osteoclast differentiation, indicating its potential for use in the treatment and prevention of osteoporosis.

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

• International Journal of Oral Biology
• /
• v.38 no.1
• /
• pp.37-42
• /
• 2013

Receptor activator of NF-${\kappa}B$ ligand (RANKL) is an essential cytokine for osteoclast differentiation, activation and survival. T lymphocytes such as $T_{17}$ cells, a subset of T helper cells that produce IL-17, play an important role in rheumatoid arthritic bone resorption by producing inflammatory cytokines and RANKL. It has not yet been clearly elucidated how T cell activation induces RANKL expression. T cell receptor activation induces the activation of nuclear factor of activated T cell (NFAT) and expression of its target genes. In this study, we examined the role of NFAT in T cell activation-induced RANKL expression. EL-4, a murine T lymphocytic cell line, was used. When T cell activation was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin, RANKL expression increased in a time-dependent manner. In the presence of cyclosporin, an inhibitor of NFAT activation, this PMA/ionomycin-induced RANKL expression was blocked. Overexpression of either NFATc1 or NFATc3 induced RANKL expression. Chromatin immunoprecipitation results demonstrated that PMA/ionomycin treatment induced the binding of NFATc1 and NFATc3 to the mouse RANKL gene promoter. These results suggest that NFATc1 and NFATc3 mediates T cell receptor activation-induced RANKL expression in T lymphocytes.