title: (+)-Vitisin A inhibits osteoclast differentiation by preventing TRAF6 ubiquitination and TRAF6-TAK1 formation to suppress NFATc1 activation.
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.
authors.affiliation: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, ROC ; Department of Biotechnology, Hungkuang University, Taichung, Taiwan, ROC.
authors.forename: Wen-Fei
authors.initials: WF
authors.lastname: Chiou
authors.affiliation: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, ROC ; Department of Cosmetic Science, Chang Gung University of Science and Technology, Taoyuan, Taiwan, ROC.
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Huang
authors.affiliation: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, ROC.
authors.forename: Yen-Wenn
authors.initials: YW
authors.lastname: Liu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001572:Benzofurans / Q000494:pharmacology*; D015153:Blotting, Western; D056657:Cathepsin K / Q000037:antagonists & inhibitors; D002454:Cell Differentiation / Q000187:drug effects*; D047468:Immunoprecipitation; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D051379:Mice; D050778:NFATC Transcription Factors / Q000378:metabolism*; D010010:Osteoclasts / Q000187:drug effects / Q000502:physiology*; D010636:Phenols / Q000494:pharmacology*; D053245:RANK Ligand / Q000037:antagonists & inhibitors; D013050:Spectrometry, Fluorescence; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D001572:Benzofurans; D056657:Cathepsin K; D020930:MAP Kinase Kinase Kinases; D020780:Matrix Metalloproteinase 9; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D010636:Phenols; D053245:RANK Ligand; D048029:TNF Receptor-Associated Factor 6; D000099151:MAP Kinase Kinase Kinase 7; C494005:Nfatc1 protein, mouse; C527519:OC-STAMP protein, mouse; C505689:Tnfsf11 protein, mouse; C412469:vitisin A