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

RANKL-induced osteoclast differentiation is dependent on TRAF6.RAW264.7 cells were treated with RANKL in the absence or presence of TRAF6 siRNA or control siRNA as described in methods. TRAF6 expression was analyzed by western blotting before and after siRNA transfection. After incubation, cells were also subjected to analyze TRAP-positive multinuclear cells formation and bone resorption. Cell morphology was examined by light microscopy.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089159-g009: RANKL-induced osteoclast differentiation is dependent on TRAF6.RAW264.7 cells were treated with RANKL in the absence or presence of TRAF6 siRNA or control siRNA as described in methods. TRAF6 expression was analyzed by western blotting before and after siRNA transfection. After incubation, cells were also subjected to analyze TRAP-positive multinuclear cells formation and bone resorption. Cell morphology was examined by light microscopy.

Mentions: The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TRAF6 has recently been shown to be important in mediating this process. We therefore investigated whether the TRAF6 signaling pathway is essential for RANKL-induced osteoclast differentiation and bone resorption activity in RAW264.7 cells. As shown in Figure 9, the formation of MNCs (middle) and extensive resorption area (bottom) were noted in cells stimulated with RANKL, however, such differentiation and resoprtion activity were obviously repressed in the presence of siRNA that targeted TRAF6. These results demonstrated that TRAF6 is essential for RANKL-induced osteoclast differentiation and bone resorption activity in RAW264.7 macrophages, as in bone marrow-derived macrophages (BMMs).


(+)-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)

RANKL-induced osteoclast differentiation is dependent on TRAF6.RAW264.7 cells were treated with RANKL in the absence or presence of TRAF6 siRNA or control siRNA as described in methods. TRAF6 expression was analyzed by western blotting before and after siRNA transfection. After incubation, cells were also subjected to analyze TRAP-positive multinuclear cells formation and bone resorption. Cell morphology was examined by light microscopy.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089159-g009: RANKL-induced osteoclast differentiation is dependent on TRAF6.RAW264.7 cells were treated with RANKL in the absence or presence of TRAF6 siRNA or control siRNA as described in methods. TRAF6 expression was analyzed by western blotting before and after siRNA transfection. After incubation, cells were also subjected to analyze TRAP-positive multinuclear cells formation and bone resorption. Cell morphology was examined by light microscopy.
Mentions: The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TRAF6 has recently been shown to be important in mediating this process. We therefore investigated whether the TRAF6 signaling pathway is essential for RANKL-induced osteoclast differentiation and bone resorption activity in RAW264.7 cells. As shown in Figure 9, the formation of MNCs (middle) and extensive resorption area (bottom) were noted in cells stimulated with RANKL, however, such differentiation and resoprtion activity were obviously repressed in the presence of siRNA that targeted TRAF6. These results demonstrated that TRAF6 is essential for RANKL-induced osteoclast differentiation and bone resorption activity in RAW264.7 macrophages, as in bone marrow-derived macrophages (BMMs).

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