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Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6.

ABSTRACT: Osteoclasts are poly-nuclear cells that resorb mineral components from old or damaged bone tissue. Primary mononuclear cells are activated by receptor activator of nuclear factor kappa-? ligand (RANKL) and differentiate into large multinucleated cells. Dysregulation of osteoclast differentiation can lead to pathological bone loss and destruction. Many studies have focused on the development of new molecules to regulate RANKL-mediated signaling. In this study, N-[2-(4-acetyl-1-piperazinyl)phenyl]-2-(2-chlorophenoxy) acetamide (PPOA-N-Ac-2-Cl) led to a significant decrease in the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells in a dose-dependent manner, without inducing significant cytotoxicity. PPOA-N-Ac-2-Cl affected the expression of osteoclast-specific marker genes, such as TRAF6, c-fos, DC-STAMP, NFATc1, MMP9, CtsK, and TRAP (Acp5), during RANKL-mediated osteoclastogenesis. Moreover, PPOA-N-Ac-2-Cl significantly attenuated the protein levels of CtsK, a critical protease involved in bone resorption. Accordingly, bone resorption activity and F-actin ring formation decreased in the presence of PPOA-N-Ac-2-Cl. In conclusion, this study shows that PPOA-N-Ac-2-Cl acts as an inhibitor of osteoclast differentiation and may serve as a potential candidate agent for the treatment of osteoclast-related bone diseases by virtue of attenuating bone resorption.

SUBMITTER: Chen Z

PROVIDER: S-EPMC6829416 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Inhibitory Effects of <i>N</i>-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6.

Chen Zhihao Z Cho Eunjin E Lee Jinkyung J Lee Sunwoo S Lee Tae-Hoon TH

International journal of molecular sciences 20191020 20

Osteoclasts are poly-nuclear cells that resorb mineral components from old or damaged bone tissue. Primary mononuclear cells are activated by receptor activator of nuclear factor kappa-Β ligand (RANKL) and differentiate into large multinucleated cells. Dysregulation of osteoclast differentiation can lead to pathological bone loss and destruction. Many studies have focused on the development of new molecules to regulate RANKL-mediated signaling. In this study, <i>N</i>-[2-(4-acetyl-1-piperazinyl) ...[more]

PMID: 31635168

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Project description:We recently reported that oral administration of a (+)-vitisin A-enriched product prepared from Vitis thunbergii obviously ameliorated bone loss in ovariectomized mice and (+)-vitisin A was able to inhibit receptor activator of NF-?B ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells. Here we further clarified the mechanism(s) by which (+)-vitisin A targets osteoclastic differentiation and activity. Osteoclast-characteristic enzyme activity was determined using gel zymography or spectroflurometric-based assay. Expression of signal molecules was analyzed via Western blot or immunoprecipitation. Results showed that (+)-vitisin A suppressed RANKL-induced multinuclear cells (MNCs) formation and bone resorption which was accompanied with reduction in ?3 integrin, osteoclast stimulatory transmembrane protein (OC-STAMP), matrix metalloproteinase-9 (MMP-9) and cathepsin K proteins expression. (+)-Vitisin A also down-regulated the proteolytic activities of MMP-9 and cathepsin K via targeting at the late stage function. (+)-Vitisin A prominently abrogated RANKL-triggered nuclear translocations of NF-?B, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream I?B degradation as well as ERK and JNK phosphorylation were also substantially repressed. Transfection with siRNA targeting tumor necrosis factor receptor associated factor 6 (TRAF6) clearly restrained RANKL-induced MNCs formation and NFATc1 induction. Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor ?-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and ?3 was markedly induced by RANKL stimulation. (+)-Vitisin A significantly attenuated this interaction when concomitant treated with RANKL in RAW264.7 cells, but failed to affect c-Src/?3 complex formation when post-cultured with MNCs. Taken together, (+)-vitisin A suppressed bone resorption possibly via interruption of RANKL-induced TRAF6 ubiquitination and associated downstream signaling pathways. Furthermore, action through negative regulation of the proteolytic activity of MMP-9 and cathepsin K might also contribute to the anti-resorption effect of (+)-vitisin A.

Project description:Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor in osteoclast differentiation by our previous study. However, the biological functions of Zscan10 have not been fully understood except its role in maintenance of genome stability and pluripotency in embryonic stem cells. Therefore, the purpose of this study is clarification of Zscan10 function in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established using CRISPR/Cas9 system and single cell sorting. Then, Control (Ctrl) and KO cells were differentiated into osteoclast by RANKL stimulation. As a result, TRAP activity and expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells as compared to Ctrl cells. These results suggested that Zscan10 might regulate transcription of the genes which negatively control osteoclastogenesis. To understand the transcriptomes controlled by Zscan10, RNA-seq was performed and the stringent analyses identified significantly down-regulated Haptoglobin (Hp) in KO cells. Additionally, Zscan10 binding sequence was located near the genomic region of Hp gene locus. ChIP against Zscan10 followed by qPCR for the region revealed that Zscan10 binds to the region located near Hp gene locus and transcript start site, suggesting that Zscan10 may regulate transcription of Hp. Next, to examine the effect of Hp under Zscan10 mediated osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). As a result, Hp treatment could suppress the elevated TRAP activity of KO cells without affecting cell viability. Furthermore, Hp KO mice exhibit decreased bone mass and increased osteoclast number, and Hp has been reported to be involved in suppression of osteoclastogenesis. Also, In hemolytic disease, Hp had been decreased and also bone density. These facts suggested that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Dataset Information

Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6.

Publications

Inhibitory Effects of <i>N</i>-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6.

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