• Molecules and Cells
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• v.38 no.3
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• pp.279-284
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• 2015

Obatoclax, a pan-Bcl2 inhibitor, shows antitumor activities in various solid malignancies. Bcl2-deficient mice have shown the importance of Bcl2 in osteoclasts, as the bone mass of the mice was increased by the induced apoptosis of osteoclasts. Despite the importance of Bcl2, the effects of obatoclax on the proliferation and differentiation of osteoclast precursors have not been studied extensively. Here, we describe the anti-proliferative effects of obatoclax on osteoclast precursors and its negative role on fusion of the cells. Stimulation with low doses of obatoclax significantly suppressed the proliferation of osteoclast precursors in a dose-dependent manner while the apoptosis was markedly increased. Its stimulation was sufficient to block the activation of ERK MAP kinase by RANKL. The same was true when PD98059, an ERK inhibitor, was administered to osteoclast precursors. The activation of JNK1/2 and p38 MAP kinase, necessary for osteoclast differentiation, by RANKL was not affected by obatoclax. Interestingly, whereas the number of TRAP-positive mononuclear cells was increased by both obatoclax and PD98059, fused, multinucleated cells larger than $100{\pm}m$ in diameter containing more than 20 nuclei were completely reduced. Consistently, obatoclax failed to regulate the expression of osteoclast marker genes, including c-Fos, TRAP, RANK and CtsK. Instead, the expression of DC-STAMP and Atp6v0d2, genes that regulate osteoclast fusion, by RANKL was significantly abrogated by both obatoclax and PD98059. Taken together, these results suggest that obatoclax down-regulates the proliferation and fusion of osteoclast precursors through the inhibition of the ERK1/2 MAP kinase pathway.

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

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.557-562
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• 2016

Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis are gram-negative bacteria frequently found in lesions from patients with periodontitis manifesting alveolar bone loss. Lipopolysaccharides are a major virulence factor of gram-negative bacteria. Bone resorption is known to be regulated by bacteria and their virulence factors. In the present study, we investigated the effects of A. actinomycetemcomitans and P. gingivalis on bone resorption. Heat-killed A. actinomycetemcomitans (HKAa) and heatkilled P. gingivalis (HKPg) induced bone loss in the femurs of mice after intraperitoneal administration. HKAa and HKPg augmented the differentiation of committed osteoclast precursors into osteoclasts, while they inhibited the differentiation of bone marrow-derived macrophages into osteoclasts. Concordant with the effects of the heat-killed whole cells, LPS purified from A. actinomycetemcomitans and P. gingivalis also augmented osteoclast differentiation from committed osteoclast precursors but attenuated it from bone marrow-derived macrophages. Taken together, these results suggest that the whole cells and lipopolysaccharides of A. actinomycetemcomitans and P. gingivalis induce the differentiation of committed osteoclast precursors into osteoclasts, potentially contributing to bone resorption in vivo.

• International Journal of Oral Biology
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• v.33 no.4
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• pp.179-186
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• 2008

Several lines of evidence suggest that osteocytes play a critical role in bone remodeling. Both healthy and apoptotic osteocytes can send signals to other bone surface cells such as osteoblasts, osteoclasts, osteoclast precursors, and bone lining cells through canalicular networks. Osteocytes responding to mechanical strain may also send signals to other cells. To determine the role for osteocytes an mechanical strain in bone remodeling, we examined the effects of fluid flow shear stress on osteoclast precursor cell and osteoblast proliferation and recruitment induced by osteocytes. In addition, the effects of fluid flow shear stress on osteocyte M-CSF, RANKL, and OPG mRNA expression were also examined. MLO-Y4 cells were used as an in vitro model for osteocytes, RAW 264.7 cells and MOCP-5 cells as osteoclast precursors, and 2T3 cells as osteoblasts. MLO-Y4 cells conditioned medium (Y4-CM) was collected after 24h culture. For fluid flow experiments, MLO-Y4 cells were exposed to 2h of pulsatile fluid flow (PFF) at 2, 4, 8, $16{\pm}0.6\;dynes/cm^2$ using the Flexcell $Streamer^{TM}$ system. For proliferation assays, MOCP-5, RAW 264.7, and 2T3 cells were cultured with control media or 10-100% Y4 CM. Cells were cultured for 3d, and then cells were counted. RAW 264.7 and 2T3 cell migration was assayed using transwells with control media or 10-100% Y4-CM. M-CSF, RANKL and OPG in MLO-Y4 mRNA expression was determined by semiquantitative RT-PCR. Y4-CM increased osteoclast precursor proliferation and migration, but decreased 2T3 cell proliferation and migration. CM from MLO-Y4 cells exposed to PFF caused decreased RAW 267.4 cell proliferation and migration and 2T3 migration compared to control Y4-CM. However, Y4-CM from cells exposed to PFF had no effect on 2T3 osteoblastic cell proliferation. PFF decreased RNAKL mRNA and increased OPG mRNA in MLO-Y4 cells compared to control(without PFF). PFF had no effect on M-CSF mRNA expression in MLO-Y4 cells. These results suggest that osteocytes can regulate bone remodeling by communication with osteoclast precursors and osteoblasts and that osteocytes can communicate mechanical signals to other cells.

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

• Molecules and Cells
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• v.43 no.1
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• pp.34-47
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• 2020

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbβ, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Rev-erbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo. Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

• Biomedical Science Letters
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• v.23 no.4
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• pp.295-302
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• 2017

Mononuclear osteoclast precursors derived from hematopoietic progenitors fuse together and then become multinucleated mature osteoclasts by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL). Especially, the binding of RANKL to its receptor RANK provides key signals for osteoclast differentiation and bone-resorbing function. RANK transduces intracellular signals by recruiting adaptor molecules such as TNFR-associated factors (TRAFs), which then activate mitogen activated protein kinases (MAPKs), Src/PI3K/Akt pathway, nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and finally amplify NFATc1 activation for the transcription and activation of osteoclast marker genes. This review will briefly describe RANKL-RANK signaling pathways and key molecules critical for osteoclast differentiation.

• 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 $IFN-{\beta}$.

• YAKHAK HOEJI
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• v.52 no.1
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• pp.43-47
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• 2008

To determine the regulatory roles of PDE4 inhibitor on LPS-induced osteoclastogenesis, we investigated the effect of a PDE4 inhibitor on osteoclast formation in the presence of LPS. A specific PDE4 inhibitor, rolipram, increased LPS-induced osteoclast formation in cocultures. To verify that whether rolipram acts indirectly on osteoblasts, we investigated the TRANCE and COX-2 mRNA expression levels in osteoblasts. Treatment of rolipram increased the expression of TRANCE and COX-2 mRNA in osteoblasts stimulated by LPS. On the contrary, rolipram did not augment the number of osteoclasts differentiated from bone marrow cells by LPS. In conclusion, the stimulation of LPS-induced osteoclast formation by the PDE4 inhibitor are attributable to its indirect effect on osteoblasts, not to their direct effect on bone marrow-derived osteoclast precursors.

• 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.