Limits...
(+)-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.

Show MeSH

Related in: MedlinePlus

Enzyme activities of MMP-9 and cathepsin K were repressed by subsequent addition of (+)-vitisin A (Vt-A).MMP-9 activities (A) were analyzed using gelatin zymographic assays and cathepsin K activity (B) was measured by using QFRET Technology as described in methods. Data represent the mean ± SEM of four independent experiments.*p<0.05, **p<0.01 and ***p< 0.001, different from values after stimulated with RANKL alone.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3928435&req=5

pone-0089159-g005: Enzyme activities of MMP-9 and cathepsin K were repressed by subsequent addition of (+)-vitisin A (Vt-A).MMP-9 activities (A) were analyzed using gelatin zymographic assays and cathepsin K activity (B) was measured by using QFRET Technology as described in methods. Data represent the mean ± SEM of four independent experiments.*p<0.05, **p<0.01 and ***p< 0.001, different from values after stimulated with RANKL alone.

Mentions: It is known that β3 integrin plays a role in regulation of cell migration and maintenance of the sealing zone required for effective osteoclastic bone resorption [30], [31]. After RANKL stimulation for 5 days, the protein expression of β3 integrin was significantly up-regulated (Figure 4). However, the induction of β3 integrin was attenuated by addition of (+)-vitisin A concentration-dependently. OC-STAMP protein expression was increased fivefold in RANKL-stimulated cells. In the presence of (+)-vitisin A, the induction of OC-STAMP protein was reduced by at least 86% by 5 µM (+)-vitisin A and almost completely abolished by 10 µM (+)-vitisin A treatment. MMP-9 is responsible for the bone resorption mediated by mature osteoclasts [20], [32]. Compared with un-differentiated cells, MMP-9 protein expression increased approximately threefold in cells stimulated with RANKL. The addition of (+)-vitisin A decreased the induction of MMP-9 by RANKL. As shown in Figure 4, 5 µM of (+)-vitisin A had slight effect on MMP-9 expression, with up to 95% reduction after 15 µM (+)-vitisin A treatment. On the other hand, osteoclast-specific cathepsin K is elevated during maturation. Cathepsin K is secreted in a sealed zone beneath the ruffled border of the osteoclast and plays a pivotal role in bone resorption [33]. Results shown in Figure 4 also demonstrate that RANKL enhanced cathepsin K expression in RAW264.7 macrophages and such responsiveness was significantly attenuated by (+)-vitisin A.


(+)-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)

Enzyme activities of MMP-9 and cathepsin K were repressed by subsequent addition of (+)-vitisin A (Vt-A).MMP-9 activities (A) were analyzed using gelatin zymographic assays and cathepsin K activity (B) was measured by using QFRET Technology as described in methods. Data represent the mean ± SEM of four independent experiments.*p<0.05, **p<0.01 and ***p< 0.001, different from values after stimulated with RANKL alone.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089159-g005: Enzyme activities of MMP-9 and cathepsin K were repressed by subsequent addition of (+)-vitisin A (Vt-A).MMP-9 activities (A) were analyzed using gelatin zymographic assays and cathepsin K activity (B) was measured by using QFRET Technology as described in methods. Data represent the mean ± SEM of four independent experiments.*p<0.05, **p<0.01 and ***p< 0.001, different from values after stimulated with RANKL alone.
Mentions: It is known that β3 integrin plays a role in regulation of cell migration and maintenance of the sealing zone required for effective osteoclastic bone resorption [30], [31]. After RANKL stimulation for 5 days, the protein expression of β3 integrin was significantly up-regulated (Figure 4). However, the induction of β3 integrin was attenuated by addition of (+)-vitisin A concentration-dependently. OC-STAMP protein expression was increased fivefold in RANKL-stimulated cells. In the presence of (+)-vitisin A, the induction of OC-STAMP protein was reduced by at least 86% by 5 µM (+)-vitisin A and almost completely abolished by 10 µM (+)-vitisin A treatment. MMP-9 is responsible for the bone resorption mediated by mature osteoclasts [20], [32]. Compared with un-differentiated cells, MMP-9 protein expression increased approximately threefold in cells stimulated with RANKL. The addition of (+)-vitisin A decreased the induction of MMP-9 by RANKL. As shown in Figure 4, 5 µM of (+)-vitisin A had slight effect on MMP-9 expression, with up to 95% reduction after 15 µM (+)-vitisin A treatment. On the other hand, osteoclast-specific cathepsin K is elevated during maturation. Cathepsin K is secreted in a sealed zone beneath the ruffled border of the osteoclast and plays a pivotal role in bone resorption [33]. Results shown in Figure 4 also demonstrate that RANKL enhanced cathepsin K expression in RAW264.7 macrophages and such responsiveness was significantly attenuated by (+)-vitisin A.

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