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(+)-Vitisin A inhibits osteoclast differentiation by preventing TRAF6 ubiquitination and TRAF6-TAK1 formation to suppress NFATc1 activation.

Chiou WF, Huang YL, Liu YW - PLoS ONE (2014)

Bottom Line: Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor β-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A.Furthermore, the interaction between c-src tyrosine kinase (c-Src) and β3 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/β3 complex formation when post-cultured with MNCs.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.

View Article: PubMed Central - PubMed

Affiliation: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, ROC ; Department of Biotechnology, Hungkuang University, Taichung, Taiwan, ROC.

ABSTRACT
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-κB 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 β3 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-κB, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream IκB 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 β-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and β3 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/β3 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.

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Effects of (+)-vitisin A (Vt-A) on RANKL-induced tartrate-resistance acid phosphatase (TRAP) activity and cell viability in RAW264.7 cells.(A) For osteoclast differentiation, RAW264.7 cells were stimulated with RANKL (100 ng/ml) and TRAP activity was assayed in cell lysates as described in methods. (B) The effect of Vt-A on cell viability was evaluated by MTT assay. Each value is the mean ±SEM of five independent experiments each performed in triplicate. *p<0.05 and **p<0.01, different from values after treatment with RANKL alone.
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pone-0089159-g002: Effects of (+)-vitisin A (Vt-A) on RANKL-induced tartrate-resistance acid phosphatase (TRAP) activity and cell viability in RAW264.7 cells.(A) For osteoclast differentiation, RAW264.7 cells were stimulated with RANKL (100 ng/ml) and TRAP activity was assayed in cell lysates as described in methods. (B) The effect of Vt-A on cell viability was evaluated by MTT assay. Each value is the mean ±SEM of five independent experiments each performed in triplicate. *p<0.05 and **p<0.01, different from values after treatment with RANKL alone.

Mentions: Signaling by RANKL is crucial for terminal differentiation of macrophages into osteoclasts. In undifferentiated cells (UND), there was no detectable TRAP staining observed, but RANKL produced numerous huge and TRAP-positive multinucleated cells (TRAP+ MNCs ≧ five nuclei). When (+)-vitisin A (5, 10 and 15 µM) was added together with RANKL, the number of TRAP+ MNCs was diminished concentration-dependently. Especially in the presence of 15 µM (+)-vitisin A, RANKL-induced fusion of MNCs was almost abolished (Figure 1). We also examined the inhibitory effect of (+)-vitisin A on TRAP activity. Figure 2A revealed that RANKL obviously fostered TRAP activity in RAW264.7 macrophages and this activity was dose-dependently declined in the presence of (+)-vitisin A. Cell viability was not affected by treating 1-20 µM (+)-vitisin A (Figure 2B), suggesting that ≦ 20 µM (+)-vitisin A was not cytotoxic.


(+)-Vitisin A inhibits osteoclast differentiation by preventing TRAF6 ubiquitination and TRAF6-TAK1 formation to suppress NFATc1 activation.

Chiou WF, Huang YL, Liu YW - PLoS ONE (2014)

Effects of (+)-vitisin A (Vt-A) on RANKL-induced tartrate-resistance acid phosphatase (TRAP) activity and cell viability in RAW264.7 cells.(A) For osteoclast differentiation, RAW264.7 cells were stimulated with RANKL (100 ng/ml) and TRAP activity was assayed in cell lysates as described in methods. (B) The effect of Vt-A on cell viability was evaluated by MTT assay. Each value is the mean ±SEM of five independent experiments each performed in triplicate. *p<0.05 and **p<0.01, different from values after treatment with RANKL alone.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928435&req=5

pone-0089159-g002: Effects of (+)-vitisin A (Vt-A) on RANKL-induced tartrate-resistance acid phosphatase (TRAP) activity and cell viability in RAW264.7 cells.(A) For osteoclast differentiation, RAW264.7 cells were stimulated with RANKL (100 ng/ml) and TRAP activity was assayed in cell lysates as described in methods. (B) The effect of Vt-A on cell viability was evaluated by MTT assay. Each value is the mean ±SEM of five independent experiments each performed in triplicate. *p<0.05 and **p<0.01, different from values after treatment with RANKL alone.
Mentions: Signaling by RANKL is crucial for terminal differentiation of macrophages into osteoclasts. In undifferentiated cells (UND), there was no detectable TRAP staining observed, but RANKL produced numerous huge and TRAP-positive multinucleated cells (TRAP+ MNCs ≧ five nuclei). When (+)-vitisin A (5, 10 and 15 µM) was added together with RANKL, the number of TRAP+ MNCs was diminished concentration-dependently. Especially in the presence of 15 µM (+)-vitisin A, RANKL-induced fusion of MNCs was almost abolished (Figure 1). We also examined the inhibitory effect of (+)-vitisin A on TRAP activity. Figure 2A revealed that RANKL obviously fostered TRAP activity in RAW264.7 macrophages and this activity was dose-dependently declined in the presence of (+)-vitisin A. Cell viability was not affected by treating 1-20 µM (+)-vitisin A (Figure 2B), suggesting that ≦ 20 µM (+)-vitisin A was not cytotoxic.

Bottom Line: Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor β-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A.Furthermore, the interaction between c-src tyrosine kinase (c-Src) and β3 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/β3 complex formation when post-cultured with MNCs.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.

View Article: PubMed Central - PubMed

Affiliation: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, ROC ; Department of Biotechnology, Hungkuang University, Taichung, Taiwan, ROC.

ABSTRACT
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-κB 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 β3 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-κB, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream IκB 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 β-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and β3 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/β3 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.

Show MeSH
Related in: MedlinePlus