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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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TRAF6 is poly-ubiquitinated after RANKL stimulation and RANKL-induced expression of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A) RAW264.7 cells were stimulated with RANKL in the absence or presence of TRAF6 siRNA (the results represented are two separate experiments) or control siRNA. After incubation, cell lyses were immunoprecipitated with anti-TRAF6 antibody. Bound proteins were further immunoblotted with anti-ubiquitin or anti-β-actin as described in methods. (B) RAW264.7 cells were stimulated with RANKL in the presence or absence of TRAF6 siRNA or control siRNA. After stimulation, cells lysates were immunoblotted with anti-NFATc1 or anti-β-actin antibodies. Results are expressed as the mean ± SEM for each group from three to four separate experiments. **p<0.01, different from values after treatment with RANKL alone.
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pone-0089159-g010: TRAF6 is poly-ubiquitinated after RANKL stimulation and RANKL-induced expression of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A) RAW264.7 cells were stimulated with RANKL in the absence or presence of TRAF6 siRNA (the results represented are two separate experiments) or control siRNA. After incubation, cell lyses were immunoprecipitated with anti-TRAF6 antibody. Bound proteins were further immunoblotted with anti-ubiquitin or anti-β-actin as described in methods. (B) RAW264.7 cells were stimulated with RANKL in the presence or absence of TRAF6 siRNA or control siRNA. After stimulation, cells lysates were immunoblotted with anti-NFATc1 or anti-β-actin antibodies. Results are expressed as the mean ± SEM for each group from three to four separate experiments. **p<0.01, different from values after treatment with RANKL alone.

Mentions: TRAF6 is a master signaling molecule controlling multiple downstream pathways induced by RANKL. Ubiquitination of TRAF6 plays an important role in its signaling function, including the regulation of osteoclastogenesis [5], [6], [40]. The finding that (+)-vitisin A down-regulated both RANK signaling and osteoclastogenesis prompted us to examine whether this compound altered ubiquitination of TRAF6. As shown in Figure 10A, poly-ubiquitinated TRAF6 was detected in RANKL-stimulated RAW264.7 cells. The ubiquitination is TRAF6-dependent since less ubiquitinated TRAF6 was accumulated in siTRAF6-transfected cells. Moreover, silencing of TRAF6 by siRNA substantially inhibited RANKL-induced induction of NFATc1 in RAW264.7 macrophages (Figure 10B). These results clearly demonstrate that RANKL-induced ubiquitination of TRAF6 is involved in NFATc1 activation and osteoclastogenesis. We next examined the effect of (+)-vitisin A on the accumulation of ubiquitinated TRAF6. As shown in Figure 11A, the expression of TRAF6 was up-regulated when stimulated with RANKL in RAW264.7 macrophages. Significantly, RANKL-mediated TRAF6 ubiquitination was severely impaired in (+)-vitisin A treated cells (Figure 11A). In contrast, (+)-vitisin A failed to affect the total level of TRAF6.


(+)-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)

TRAF6 is poly-ubiquitinated after RANKL stimulation and RANKL-induced expression of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A) RAW264.7 cells were stimulated with RANKL in the absence or presence of TRAF6 siRNA (the results represented are two separate experiments) or control siRNA. After incubation, cell lyses were immunoprecipitated with anti-TRAF6 antibody. Bound proteins were further immunoblotted with anti-ubiquitin or anti-β-actin as described in methods. (B) RAW264.7 cells were stimulated with RANKL in the presence or absence of TRAF6 siRNA or control siRNA. After stimulation, cells lysates were immunoblotted with anti-NFATc1 or anti-β-actin antibodies. Results are expressed as the mean ± SEM for each group from three to four separate experiments. **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-g010: TRAF6 is poly-ubiquitinated after RANKL stimulation and RANKL-induced expression of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A) RAW264.7 cells were stimulated with RANKL in the absence or presence of TRAF6 siRNA (the results represented are two separate experiments) or control siRNA. After incubation, cell lyses were immunoprecipitated with anti-TRAF6 antibody. Bound proteins were further immunoblotted with anti-ubiquitin or anti-β-actin as described in methods. (B) RAW264.7 cells were stimulated with RANKL in the presence or absence of TRAF6 siRNA or control siRNA. After stimulation, cells lysates were immunoblotted with anti-NFATc1 or anti-β-actin antibodies. Results are expressed as the mean ± SEM for each group from three to four separate experiments. **p<0.01, different from values after treatment with RANKL alone.
Mentions: TRAF6 is a master signaling molecule controlling multiple downstream pathways induced by RANKL. Ubiquitination of TRAF6 plays an important role in its signaling function, including the regulation of osteoclastogenesis [5], [6], [40]. The finding that (+)-vitisin A down-regulated both RANK signaling and osteoclastogenesis prompted us to examine whether this compound altered ubiquitination of TRAF6. As shown in Figure 10A, poly-ubiquitinated TRAF6 was detected in RANKL-stimulated RAW264.7 cells. The ubiquitination is TRAF6-dependent since less ubiquitinated TRAF6 was accumulated in siTRAF6-transfected cells. Moreover, silencing of TRAF6 by siRNA substantially inhibited RANKL-induced induction of NFATc1 in RAW264.7 macrophages (Figure 10B). These results clearly demonstrate that RANKL-induced ubiquitination of TRAF6 is involved in NFATc1 activation and osteoclastogenesis. We next examined the effect of (+)-vitisin A on the accumulation of ubiquitinated TRAF6. As shown in Figure 11A, the expression of TRAF6 was up-regulated when stimulated with RANKL in RAW264.7 macrophages. Significantly, RANKL-mediated TRAF6 ubiquitination was severely impaired in (+)-vitisin A treated cells (Figure 11A). In contrast, (+)-vitisin A failed to affect the total level of TRAF6.

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