模拟微重力通过cAMP-PKA通路介导初级纤毛消失抑制骨形成

The sense and response of cells to microgravity is one of the important research areas of space biology. As a raised structure on the surface of a cell, primary cilium has been identified as a sensor to convey multiple mechanical and biochemical signals since it contains a variety of receptors, ion channels and transducers. In our previous work, it was found that the inhibition of mineralization and differentiation induced by simulated microgravity in ostoblasts were accompanied by disappearance of the primary cilium. In this project, rat calvarial osteoblasts treated with a three-dimentional random positioning machine and rats treated with hindlimb suspension will be used to simulate microgravity. The molecular mechanisms of bone loss induced by microgravity will be elucidated through observing the cAMP/PKA signaling pathway, the subcellular remodeling of cytosleleton and primary cilium by confocal microscope z-stake, FRET-based cAMP biosensor and RNA interference techniques. The activation of cAMP/PKA by the cilliary adenylate cyclases in microgavity condition, the phosphorylation of microtubule binding proteins, the depolymerization of microtubules connected to the roots of primary cilium and the resulting in disappearance of primary cilium and decrease of osteogenic formation will be clarified. The aim of this project is to promote the researh of bone metabolism in space environment, and to lay a foundation for the searching of effective targets to prevent the bone loss induced by microgravity.

细胞对微重力的感知和响应是空间生物学的重要研究方向之一。初级纤毛作为细胞表面的单根凸起结构，内部含有大量的受体、离子通道和信号分子，在物理和化学信号的感受和转导中有重要作用。我们前期的研究发现：模拟微重力抑制成骨细胞成熟矿化的过程伴随着初级纤毛的消失。本项目以三维回旋仪处理大鼠颅骨成骨细胞并用大鼠后肢悬浮法模拟微重力，通过激光共聚焦显微镜z-stake、FRET生物传感器及RNA干扰等技术，围绕cAMP/PKA信号通路、细胞骨架和初级纤毛的亚细胞重构这两个关键点，阐明微重力通过纤毛内腺苷酸环化酶使cAMP聚集并激活PKA，激活的PKA进入细胞质引起微管结合蛋白磷酸化，使得连接于初级纤毛根部的微管解聚，初级纤毛萎缩并逐渐消失，成骨细胞成骨能力显著降低。目的在于通过对微重力导致的骨流失过程和分子机制进行解析，推进空间骨代谢研究的发展，并为寻找防治空间微重力导致骨流失的有效作用靶点奠定基础。

初级纤毛能够响应流体剪切力、电磁场及化学分子等胞外信号的变化并激活定位在它上面的钙离子通路、Wnt及cAMP等信号通路促进成骨细胞分化。项目基于之前已经证实并报道的模拟微重力引起成骨细胞初级纤毛逐渐缩短甚至消失的现象，以三维回旋仪处理大鼠颅骨成骨细胞并用大鼠尾吊法模拟微重力，通过免疫荧光、激光共聚焦显微镜及RNA干扰等技术，研究了模拟微重力条件下cAMP-PKA通路介导的细胞微管解聚与初级纤毛缩短和骨形成抑制之间的关系，发现模拟微重力通过激活初级纤毛内的腺苷酸环化酶sAC促使cAMP聚集，进而激活PKA引起微管弯曲程度下降、线条状结构减少，呈疏松状分布，同时纤毛根部微管数量减少并呈现明显解聚状态；而用腺苷酸环化酶抑制剂或PKA抑制剂阻断cAMP/PKA信号通路时，能够缓解模拟微重力引起的细胞微管解聚和初级纤毛消失。利用微管稳定剂Doctaxel Trihydrate (DOC)处理成骨细胞，可以缓解模拟微重力引起的碱性磷酸酶活性下降，骨形成相关因子RUNX-2、BMP-2和OSX的基因和蛋白水平降低及钙化结节减少。这些结果阐明了模拟微重力通过诱导成骨细胞纤毛内cAMP/PKA激活，引起细胞微管疏松、纤毛根部微管解聚，进而导致初级纤毛萎缩甚至消失，使成骨细胞骨形成能力显著降低的过程和分子机制，并从动物水平得到了验证。项目结果为寻找防治空间微重力导致骨流失的有效靶点提供了新思路。