Chiou Wen-Fei, Huang Yu-Ling, Liu Yen-Wenn
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan, ROC ; Department of Biotechnology, Hungkuang University, Taichung, Taiwan, ROC.
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.
PLoS One. 2014 Feb 18;9(2):e89159. doi: 10.1371/journal.pone.0089159. eCollection 2014.
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.
我们最近报道，口服由黑葡萄制备的富含(+)-葡萄素A的产品可显著改善去卵巢小鼠的骨质流失，且(+)-葡萄素A能够抑制RAW264.7细胞中核因子κB受体激活剂配体(RANKL)诱导的破骨细胞分化。在此，我们进一步阐明了(+)-葡萄素A靶向破骨细胞分化和活性的机制。使用凝胶酶谱法或基于荧光光谱的测定法测定破骨细胞特征性酶活性。通过蛋白质印迹或免疫沉淀分析信号分子的表达。结果显示，(+)-葡萄素A抑制RANKL诱导的多核细胞(MNCs)形成和骨吸收，同时伴随着β3整合素、破骨细胞刺激跨膜蛋白(OC-STAMP)、基质金属蛋白酶-9(MMP-9)和组织蛋白酶K蛋白表达的降低。(+)-葡萄素A还通过靶向后期功能下调MMP-9和组织蛋白酶K的蛋白水解活性。(+)-葡萄素A显著消除RANKL触发的核因子κB、活化蛋白-1(AP-1，c-Fos/c-Jun二聚体)的核转位以及活化T细胞核因子c1(NFATc1)的相关诱导和核积累。上游IκB降解以及细胞外信号调节激酶(ERK)和应激活化蛋白激酶(JNK)磷酸化也被显著抑制。用靶向肿瘤坏死因子受体相关因子6(TRAF6)的小干扰RNA(siRNA)转染可明显抑制RANKL诱导的MNCs形成和NFATc1诱导。有趣的是，RANKL触发TRAF6的多聚泛素化，且(+)-葡萄素A显著减弱相关的TRAF6-转化生长因子β激活激酶1(TAK1)复合物形成。此外，RANKL刺激可显著诱导c-src酪氨酸激酶(c-Src)与β3之间的相互作用。在RAW264.7细胞中，当(+)-葡萄素A与RANKL同时处理时，可显著减弱这种相互作用，但在用MNCs进行后期培养时，未能影响c-Src/β3复合物的形成。综上所述，(+)-葡萄素A可能通过中断RANKL诱导的TRAF6泛素化及相关下游信号通路来抑制骨吸收。此外，对MMP-9和组织蛋白酶K蛋白水解活性的负调控作用也可能有助于(+)-葡萄素A的抗吸收作用。
