Calcium/calmodulin-signaling supports TRPV4 activation in osteoclasts and regulates bone mass
Calcium/calmodulin-signaling supports TRPV4 activation in osteoclasts and regulates bone mass
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DOI：
10.1002/jbmr.1629
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发表时间：
2012-08-01
影响因子：
6.2
通讯作者：
Komori, Toshihisa
中科院分区：
文献类型：
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作者：
Masuyama, Ritsuko;Mizuno, Atsuko;Komori, Toshihisa
破骨细胞分化严重依赖于钙 (Ca2+) 信号传导。瞬时受体电位香草酸 4 (TRPV4) 在破骨细胞分化后期介导 Ca2+ 流入，从而调节 Ca2+ 信号传导。然而，TRPV4 活性的系统改变作用仍有待确定。为了阐明基于破骨细胞分化的TRPV4激活机制，通过TRPV4中的氨基酸取代R616Q和V620I产生TRPV4功能获得突变体，并将其引入Trpv4缺失小鼠的破骨细胞谱系中以产生Trpv4R616Q/V620I转基因小鼠。正如预期的那样，破骨细胞中 TRPV4 的激活增加了破骨细胞的数量及其吸收活性，从而导致骨质流失。在体外分析中，与缺乏 Trpv4 的破骨细胞相比，Trpv4R616Q/V620I 破骨细胞显示出激活的 Ca2+/钙调蛋白信号传导。此外，对缺乏钙调蛋白结合域的Trpv4R616Q/V620I小鼠的研究表明，TRPV4激活引起的骨质流失可通过Ca2+/钙调蛋白信号传导与TRPV4之间相互作用的丧失而消除。最后，通过蛋白质组分析研究了 TRPV4 与钙调蛋白结合域相互作用的调节剂。有趣的是，通过液相色谱串联质谱 (LC-MS/MS) 分析鉴定了非肌肉肌球蛋白 IIa，并通过与 TRPV4 免疫共沉淀后的免疫印迹证实了这一点。此外，肌球蛋白 IIa 基因沉默显着降低了 TRPV4 的激活，同时破骨细胞成熟受损。这些结果表明，TRPV4 激活可相互调节 Ca2+/钙调蛋白信号传导，其中涉及 TRPV4 与肌球蛋白 IIa 的关联，并促进充分的破骨细胞功能。 (C) 2012 年美国骨与矿物质研究学会。
Osteoclast differentiation is critically dependent on calcium (Ca2+) signaling. Transient receptor potential vanilloid 4 (TRPV4), mediates Ca2+ influx in the late stage of osteoclast differentiation and thereby regulates Ca2+ signaling. However, the system-modifying effect of TRPV4 activity remains to be determined. To elucidate the mechanisms underlying TRPV4 activation based on osteoclast differentiation, TRPV4 gain-of-function mutants were generated by the amino acid substitutions R616Q and V620I in TRPV4 and were introduced into osteoclast lineage in Trpv4 null mice to generate Trpv4R616Q/V620I transgenic mice. As expected, TRPV4 activation in osteoclasts increased the number of osteoclasts and their resorption activity, thereby resulting in bone loss. During in vitro analysis, Trpv4R616Q/V620I osteoclasts showed activated Ca2+/calmodulin signaling compared with osteoclasts lacking Trpv4. In addition, studies of Trpv4R616Q/V620I mice that lacked the calmodulin-binding domain indicated that bone loss due to TRPV4 activation was abrogated by loss of interactions between Ca2+/calmodulin signaling and TRPV4. Finally, modulators of TRPV4 interactions with the calmodulin-binding domain were investigated by proteomic analysis. Interestingly, nonmuscle myosin IIa was identified by liquid chromatographytandem mass spectroscopy (LC-MS/MS) analysis, which was confirmed by immunoblotting following coimmunoprecipitation with TRPV4. Furthermore, myosin IIa gene silencing significantly reduced TRPV4 activation concomitant with impaired osteoclast maturation. These results indicate that TRPV4 activation reciprocally regulates Ca2+/calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function. (C) 2012 American Society for Bone and Mineral Research.