• Title, Summary, Keyword: Receptor activator of nuclear factor kappa B ligand (RANKL)

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Bavachin counteracts receptor activator of nuclear factor-κB-induced osteoclastogenesis though the suppression of nuclear factor-κB signaling pathway in RAW264.7 cells

  • Kim, Bok-Hee;Cho, In-A;Kang, Kyeong-Rok;Lee, Sook-Young;Jung, Seo-Yun;Kim, Jae-Sung;Kim, Su-Gwan
    • Oral Biology Research
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    • v.42 no.3
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    • pp.130-139
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    • 2018
  • The aim of this study was to evaluate the biological effects and cellular signaling pathways associated with the anti-osteoclastogenesis effects of bavachin, a phytoestrogen, in the receptor activator of nuclear $factor-{\kappa}B$ ligand (RANKL)- treated RAW264.7 cells. The cell viability of RAW264.7 cells was not affected upon treatment with $5-20{\mu}M$ bavachin. Furthermore, osteoclastogenesis was suppressed by bavachin in a dose-dependent manner in RAW264.7 cells treated with RANKL. Tartrate-resistant acid phosphatase, matrix metalloproteinase-9, and cathepsin K, which are closely associated with osteoclastogenesis, were significantly downregulated by bavachin in the presence of RANKL. Additionally, bavachin decreased inflammatory molecules, such as nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 in RAW264.7 cells treated with RANKL. Bavachin suppressed the RANKL-induced phosphorylation of nuclear $factor-{\kappa}B$ and subsequently inhibited the translocation of nuclear $factor-{\kappa}B$ from the cytosol to the nucleus. Taken together, the obtained data suggest that bavachin may prevent the osteoclast-mediated bone destructive disorders.

Humanin suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via AMP-activated protein kinase activation

  • Kang, Namju;Kim, Ki Woo;Shin, Dong Min
    • The Korean Journal of Physiology and Pharmacology
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    • v.23 no.5
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    • pp.411-417
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    • 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 Effects of Cuscuta japonica Chois on Gene Expression in RANKL-induced RAW 264.7 Cell (도사자(菟絲子)가 RANKL 유도 파골세포(破骨細胞)에 미치는 영향)

  • Kim, Joon-Yeon;Hwang, Gwi-Seo
    • Journal of Society of Preventive Korean Medicine
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    • v.14 no.2
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    • pp.77-89
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    • 2010
  • Objectives : This study was performed to evaluate the effect of CJ(Cuscuta japonica Chois) on osteoclast differentiation and gene expression. Methods : The osteoclastogenesis and gene expression were determined in RANKL(receptor activator of nuclear factor kappa B ligand)-stimulated RAW 264.7. The results were summarized as followes. Results : CJ decreased the number of TRAP positive cell in RANKL-stimulated RAW264.7 cell. CJ decreased the expression of RANK(receptor activator of nuclear factor kappa B), $TNF{\alpha}$, and IL-6 in RANKL-stimulated RAW264.7 cell. CJ decreased the expression of iNOS and COX-2 in RANKL-stimulated RAW264.7 cell. CJ decreased the expression of Cathepsin K in RANKL-stimulated RAW264.7 cell. Conclusions : It is concluded that CJ might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression.

Current Understanding of RANK Signaling in Osteoclast Differentiation and Maturation

  • Park, Jin Hee;Lee, Na Kyung;Lee, Soo Young
    • Molecules and Cells
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    • v.40 no.10
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    • pp.706-713
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    • 2017
  • Osteoclasts are bone-resorbing cells that are derived from hematopoietic precursor cells and require macrophage-colony stimulating factor and receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) for their survival, proliferation, differentiation, and activation. The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts. This process depends on RANKL-RANK signaling, which is temporally regulated by various adaptor proteins and kinases. Here we summarize the current understanding of the mechanisms that regulate RANK signaling during osteoclastogenesis. In the early stage, RANK signaling is mediated by recruiting adaptor molecules such as tumor necrosis factor receptorassociated factor 6 (TRAF6), which leads to the activation of mitogen-activated protein kinases (MAPKs), and the transcription factors nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and activator protein-1 (AP-1). Activated NF-${\kappa}B$ induces the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is the key osteoclastogenesis regulator. In the intermediate stage of signaling, the co-stimulatory signal induces $Ca^{2+}$ oscillation via activated phospholipase $C{\gamma}2$ ($PLC{\gamma}2$) together with c-Fos/AP-1, wherein $Ca^{2+}$ signaling facilitates the robust production of NFATc1. In the late stage of osteoclastogenesis, NFATc1 translocates into the nucleus where it induces numerous osteoclast-specific target genes that are responsible for cell fusion and function.

Receptor activator of nuclear factor-κB ligand in T cells and dendritic cells communication

  • Nam, Sun-Young;Jeong, Hyun-Ja
    • CELLMED
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    • v.3 no.1
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    • pp.3.1-3.3
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    • 2013
  • The receptor activator of NF-${\kappa}B$ ligand (RANKL), a member of the tumor necrosis factor ligand family, has extensive functions beyond osteoclast development. RANKL is expressed in many immune cells such as osteoblasts, osteocytes, marrow stromal cells, activated T cells, synovial cells, keratinocytes, and mammary gland epithelial cells as well as in various tissues. The ligation of RANK by RANKL promotes dendritic cells (DCs) survival through prosurvival signals and the up-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-$x_L$ and plays a crucial role in DCs-mediated Th1 differentiation. Therefore, RANKL plays an important role in the regulation of DCs/T cells-mediated specific immunity. This review will briefly inform our current understanding of the role of RANKL signaling in T cells-DCs communication in the immune system.

NFATc1 and NFATc3 is Involved in the Expression of Receptor Activator of NF-${\kappa}B$ Ligand in Activated T Lymphocytes

  • Heo, Sun-Jae;Park, Hyun-Jung;Baek, Jeong-Hwa
    • International Journal of Oral Biology
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    • v.38 no.1
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    • pp.37-42
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    • 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.

Negative regulators in RANKL-induced osteoclastogenesis

  • Lee, Jun-Won;Kim, Kab-Sun;Kim, Nack-Sung
    • 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.

RANK Signaling Pathways and Key Molecules Inducing Osteoclast Differentiation

  • Lee, Na Kyung
    • 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.

Hypoxia Inducible Factor-$1{\alpha}$ Directly Induces the Expression of Receptor Activator of Nuclear Factor-${\kappa}B$ Ligand in MLO-Y4 Osteocytes

  • Baek, Kyunghwa;Park, Hyun-Jung;Baek, Jeong-Hwa
    • 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.

Soluble Expression and Purification of Receptor Activator of Nuclear Factor-Kappa B Ligand Using Escherichia coli

  • Park, Sol-Ji;Lee, Se-Hoon;Kim, Kwang-Jin;Kim, Sung-Gun;Kim, Hangun;Choe, Han;Lee, Sang Yeol;Yun, Jung-Mi;Cho, Jae Youl;Chun, Jiyeon;Choi, Kap Seong;Son, Young-Jin
    • 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.