钙振荡介导的calcineurin-NFAT信号通路在骨细胞力学信号转导中的作用研究

Bone can remodel its own structure to adapt to the external mechanical environment, and failure of normal bone adaptation is known to play a significant role in the etiology of metabolic bone diseases such as osteoporosis and osteopetrosis. Abundant evidence has substantiated that osteocytes play a significant role in bone mechanosensing. Our study for the first time demonstrates that osteocytes response to the mechanical stimuli via unique calcium oscillations with multiple robust calcium peaks, and osteocytes behave more active in calcium signaling than osteoblasts in response to mechanical signals. Our results provide direct evidence that osteocytes are qualified as the major "mechanical sensor" in bone. To date, however, how osteocytes decode and transduce the unique calcium oscillations and how calcium oscillations affect the cellular functions of osteocytes remain unknown. Several previous studies have shown that NFAT acts as a critical decoder for low-frequency calcium oscillations in cardiomyocytes and lymphocytes. Thus, the objective of the present study is to investigate the spatiotemporal characteristics of intracellular calcium and NFAT in osteocytes via real-time fluorescence imaging of calcium and NFAT molecules under mechanical loading. Moreover, the correlation of NFAT activities with the frequency and intensity of calcium oscillations will be also systematically analyzed. Then, the changes of osteocytic biological functions and secretions of cytokines related with bone remodeling will be also investigated after the inhibition of the NFAT signaling. The present study aims to decipher the intracellular decoding mechanism of osteocytes for the unique calcium oscillations, which contributes to uncovering the mechanism about how bone senses and transduces mechanical signals, and thus advancing our fundamental knowledge of bone adaptation and bone mechanotransduction.

骨能对外界应力环境发生适应性变化，骨适应能力的缺失是骨质疏松、骨硬化症等代谢骨病发生的重要原因。诸多研究揭示骨细胞（OCY）在骨力学信号响应中起重要作用，我们首次发现OCY是通过独特多尖峰钙振荡响应应力刺激的，且OCY应力响应敏感性远高于成骨细胞，该发现为揭示OCY是骨中最核心应力感受器提供了直接证据。但OCY是如何解码和转导这种独特钙振荡，钙振荡是如何影响OCY功能的，仍不得而知。NFAT被证实是心肌、淋巴细胞重要的低频钙振荡解码分子，本课题拟通过钙和NFAT实时荧光成像分析应力作用下OCY钙和NFAT时空变化规律，明确NFAT与钙振荡频率和强度的关系；随后分别阻断体外/体内OCY的NFAT信号，分析应力作用下OCY生物功能及其分泌的重要骨代谢调控因子的变化。本课题旨在明确OCY对力致钙振荡的解码机制，回答骨骼是如何响应和转导应力刺激这一重要问题，丰富对骨适应和骨力学信号转导机制的认识。

我们前期研究发现骨细胞在应力刺激下表现出独特的多尖峰钙振荡特性，但是骨细胞是如何解码和转导这种钙振荡，钙振荡是如何影响骨细胞功能的，仍是未知。本研究中，我们首先构建了程控式细胞流体剪切力系统，发现了NFAT拮抗剂（环孢霉素A）对MLO-Y4细胞进行干预后，剪切力诱发的骨细胞凋亡的降低、Wnt/β-catenin通路的激活和RANKL/OPG表达下调均被显著抑制；其次，我们分别对小鼠胫骨原位骨细胞和MLO-Y4细胞系中NFAT各亚型（NFATc1~4）进行了检测，我们发现体外/原位骨细胞中NFATc3的亚型表达最高；随后，我们通过构建HA-NFAT-GFP转染质粒协同rhod-2/AM钙荧光探针染色实现了钙和NFAT实时双通道荧光成像，以胞核NFAT（NFATNuc）与胞浆NFAT（NFATCyt）比值计算NFAT活性，我们发现正常的具有多尖峰钙振荡的骨细胞NFATNuc/NFATCyt呈现逐渐增加的趋势，而施加了ER或P2R阻断剂的细胞（钙尖峰数量显著降低）NFAT增加趋势不显著，证实了NFAT充当应力刺激下骨细胞钙振荡解码器的角色；随后，我们使用基因沉默技术阻断了骨细胞NFATc3表达，发现剪切力诱发的骨细胞凋亡率的降低被显著抑制，且剪切力诱发的Wnt3a、β-catenin与OPG上调以及SOST、RANKL下调同样被显著抑制，进一步证实了NFAT在骨细胞力学信号转导和力学生物学调控中同样发挥关键作用；最后，我们构建了新颖的活体小鼠长骨应力加载系统，通过尾静脉注射NFAT拮抗剂，发现了NFAT阻断后应力对于骨中骨细胞功能活性的调控被显著抑制，进一步证实了NFAT在骨细胞力学信号转导中起关键作用。本研究进一步丰富了骨细胞力学信号转导机制的认识，并有望为更加系统认识骨是如何响应和转导应力刺激这一重要科学问题提供有力依据。

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