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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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Effect of (+)-vitisin A (Vt-A) on pit-forming activity in RANKL stimulated RAW264.7 cells.(A) Cells cultured on bone slices were stimulated with RANKL for 8 days by concurrent addition of Vt-A. (B) Vt-A was added at 5 days after RANKL stimulation and differentiated for further 3 days. After removing the cells, the slices were stained with Mayer's haematoxylin to identify resorption pits. The resorption area was observed under a light microscope and analyzed by Image-Pro Plus™. Data represent the mean ± SEM of three to five independent experiments. *p<0.05 and **p <0.01, different from values after treatment with RANKL alone.
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pone-0089159-g003: Effect of (+)-vitisin A (Vt-A) on pit-forming activity in RANKL stimulated RAW264.7 cells.(A) Cells cultured on bone slices were stimulated with RANKL for 8 days by concurrent addition of Vt-A. (B) Vt-A was added at 5 days after RANKL stimulation and differentiated for further 3 days. After removing the cells, the slices were stained with Mayer's haematoxylin to identify resorption pits. The resorption area was observed under a light microscope and analyzed by Image-Pro Plus™. Data represent the mean ± SEM of three to five independent experiments. *p<0.05 and **p <0.01, different from values after treatment with RANKL alone.

Mentions: To determine if this suppression of TRAP+ MNCs formation affected the resorptive ability, RAW264.7 cells were cultured on bone slices, and then differentiated to mature osteoclasts by RANKL for 8 days in the absence or presence of (+)-vitisin A. When compared to the un-differentiated (UND) group, many resorption pits (discriminated from the gray background by an intensively dark stain) were formed on the bone slices after cultured with RANKL. As shown in Figure 3A, RANKL augmented the number and size of bone resorption areas in osteoclasts transformed from macrophages. (+)-Vitisin A strongly reduced the resorption area when added at an earlier stage of differentiation. It is surprising that (+)-vitisin A added at several days after differentiation still significantly mitigated bone resorption (Figure 3B). In particular, adding a higher concentration of (+)-vitisin A (15 µM) at late stage of differentiation also decreased the pit area, similar to treatment at earlier stage. This inhibition was not caused by cell death since (+)-vitisin A has no significant effect on mature osteoclast viability (data not shown).


(+)-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)

Effect of (+)-vitisin A (Vt-A) on pit-forming activity in RANKL stimulated RAW264.7 cells.(A) Cells cultured on bone slices were stimulated with RANKL for 8 days by concurrent addition of Vt-A. (B) Vt-A was added at 5 days after RANKL stimulation and differentiated for further 3 days. After removing the cells, the slices were stained with Mayer's haematoxylin to identify resorption pits. The resorption area was observed under a light microscope and analyzed by Image-Pro Plus™. Data represent the mean ± SEM of three to five independent experiments. *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-g003: Effect of (+)-vitisin A (Vt-A) on pit-forming activity in RANKL stimulated RAW264.7 cells.(A) Cells cultured on bone slices were stimulated with RANKL for 8 days by concurrent addition of Vt-A. (B) Vt-A was added at 5 days after RANKL stimulation and differentiated for further 3 days. After removing the cells, the slices were stained with Mayer's haematoxylin to identify resorption pits. The resorption area was observed under a light microscope and analyzed by Image-Pro Plus™. Data represent the mean ± SEM of three to five independent experiments. *p<0.05 and **p <0.01, different from values after treatment with RANKL alone.
Mentions: To determine if this suppression of TRAP+ MNCs formation affected the resorptive ability, RAW264.7 cells were cultured on bone slices, and then differentiated to mature osteoclasts by RANKL for 8 days in the absence or presence of (+)-vitisin A. When compared to the un-differentiated (UND) group, many resorption pits (discriminated from the gray background by an intensively dark stain) were formed on the bone slices after cultured with RANKL. As shown in Figure 3A, RANKL augmented the number and size of bone resorption areas in osteoclasts transformed from macrophages. (+)-Vitisin A strongly reduced the resorption area when added at an earlier stage of differentiation. It is surprising that (+)-vitisin A added at several days after differentiation still significantly mitigated bone resorption (Figure 3B). In particular, adding a higher concentration of (+)-vitisin A (15 µM) at late stage of differentiation also decreased the pit area, similar to treatment at earlier stage. This inhibition was not caused by cell death since (+)-vitisin A has no significant effect on mature osteoclast viability (data not shown).

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