Ca2+/Calmodulin/NFATc1信号通路在双膦酸盐诱发破骨细胞生成抑制中作用及机制研究

Bisphosphonates (BPs) are widely used for treatment of many bone diseases involving excessive bone resorption and they act mainly through inhibiting the formation and function of osteoclasts. However,the molecular mechanisms of BPs remains far less understood and this contricts their further clinical application and development of new drugs for treatment of above diseases..Ca2+/calmodulin/NFATc1(neclear factor of activated T cells c 1) signaling pathway is essential for differentiation and formation of osteoclast, while its function in BPs-induced inhibition of osteoclastogenesis remains unknown. Recently, we found alendronate (ALN), a third generation of BPs, could significantly downregulate gene expression of NFATc1 and inhibit osteoclastogenesis of RAW264.7 cells. The above effects of ALN was partly inverted by Gd3+, an antagonist of Ca2+ channel for control of Ca2+ influx. based on above results, we hypothesize that Ca2+/calmodulin/NFATc1 signaling pathway may play a key role in the intracellular action of ALN. To prove the hypothesis: ①The influences of ALN on [Ca2+]i, oscillation of Ca2+, activation of NFATc1, as well as gene expression of calmodulin, calcineurin, CaMKII and CaMKIV, are to be studied. The relationships among the molecules are also to be explored. ② Protein activity analysis and protein-binding experiment are to be performed to verify the activity and interaction of the molecules. ③ RNA interference and gene recombinant technique are also used to verify the function of these molecules on ALN-induced inhibition of osteoclastogenesis. The results of this study will broaden our knowledge on molecular mechanisms of BPs and benefit the development of new, more potent, osteoclast-targeting drugs for treatment of diseases invovling excessive bone resorption.

双膦酸盐（BPs）通过抑制破骨细胞（OC）治疗骨过度吸收性疾病，作用机制远未弄清，制约了临床应用及新药开发。Ca2+/calmodulin/NFATc1信号通路对OC生成至关重要；但是否参与BPs细胞效应尚不清楚。我们前期研究发现，阿伦膦酸(ALN)使OC[Ca2+]i显著上升，NFATc1表达下调，并抑制OC生成；Ca2+通道拮抗剂则逆转了上述效应；提示Ca2+/calmodulin/NFATc1通路可能参与其中。为了证实该假设，本课题拟：①进一步对BPs作用下OC[Ca2+]i及Ca2+波动、calmodulin、NFATc1等基因表达进行检测，明确信号分子间联动关系；②应用酶活性分析及蛋白结合试验，评价信号蛋白活性及相互作用；③采用RNA干扰、基因重组技术证实上述分子在BPs信号传导中的关键作用。以进一步阐明BPs分子机制；为开发新的OC靶向药物，治疗骨过度吸收疾病提供理论依据。

项目背景：双膦酸盐（BPs）通过抑制破骨细胞（OC）治疗骨过度吸收性疾病，作用机制远未弄清；Ca2+/calmodulin/NFATc1 信号通路对OC 生成至关重要，但其是否参与BPs对OC生成的抑制尚不清楚。.主要研究内容：为此，本课题研究探讨了：（1）双膦酸盐对OC生成、OC分化中NFATc1及下游TRAP、Cathepsin K、TRPV5、(p)NF-κB p50、(p)c-Jun、integrin αvβ3、Syk、Atp6v0d2等基因表达的影响；（2）NFATc1基因过表达对唑唻膦酸诱发的破骨细胞抑制的挽救效应；（3）唑唻膦酸对OC胞内Ca2+波动及与NFATc1 基因表达联动关系的影响；（4）唑唻膦酸对Ca2+信号分子Calmodulin、Calcineurin、CaMKII/IV基因表达及Calmodulin与CaMKII蛋白结合的影响；（4）体内唑唻膦酸促进骨质疏松时自体骨移植内种植体骨结合及高频低幅振动、掺锶透钙磷石涂层促进种植体骨结合的效果。.重要结果及关键数据：双膦酸盐可通过抑制OC 胞内NFATc1及下游TRAP、Cathepsin K、TRPV5、(p)NF-κB p50、(p)c-Jun、integrin αvβ3、Syk、Atp6v0d2等基因表达抑制OC生成；双膦酸盐的上述作用是通过影响Ca2+波动，进而抑制Ca2+下游Calmodulin、Calcineurin、CaMKII/IV等基因表达及Calmodulin与CaMKII蛋白结合发挥作用的；体内应用唑唻膦酸、高频低幅振动、掺锶透钙磷石涂层等抑制OC的手段均可有效促进种植体骨结合。.科学意义：本课题研究并证实了Ca2+/calmodulin/NFATc1信号轴是双膦酸盐抑制破骨细胞生成的重要信号通路，从而为双膦酸盐作用机制研究及新药开发提供了新的思路。