探索“甜蜜”的力学信号——干细胞中蛋白质糖基化水平受胞外基质弹性影响的机制研究

Be a well-known phenomenon, substrate elasticity inducing stem cells differentiation has been intensely investigated during past several years. Despite these efforts, the mechanism of how substrate elasticity manipulates the cells function and the mechanosensing process remain largely unknown. Our group have been devoted to this topic for more than five years and have reported a new mechanosensing pathway, which is that soft substrate reduces the Bone Morphogenesis Protein (BMP) pathway via triggering the endocytosis of integrin and BMP, and subsequently induce the neurogenesis differentiation of mesenchymal stem cells (MSCs). During our research, we have found that the glycosylation of beta1 integrin and Wnt1 was significantly reduced by soft substrate when compared to the stiff one. As a key process of protein modification, the glycosylation of proteins is crucial to the proper folding, trafficking, positioning and degradation of proteins; thus contributes to may cellular functions, such as migration, differentiation and proliferation. Accordingly, the substrate elasticity inducing glycosylation variation may open a door to a better understanding of the mechanosensing mechanism of substrate elasticity. Based on our previous research, we proposed that soft substrate attenuates the focal adhesion assembly and the phosphorylation of FAK, hence induces the accumulation and transportation into the nucleus of p53. P53 then inhibits the expression of apt2c1 via promoting the expression of micro RNA532, and subsequently reduces the glycosylation levels of beta1 integrin and Wnt1 proteins. The present project aims to investigate the mechanism of substrate elasticity inducing variation of glycosylation. By successfully carrying out this project, we may expect a better understanding of the cellular mechanosensing mechanism of substrate elasticity.

胞外基质弹性诱导干细胞分化是一个广为人知的有趣话题，其背后的机理有许多未解之谜。本课题组致力于研究上述机理问题已逾5年，报道了一条新的力学信号转导通路：较软的胞外基质通过促进整合素内吞影响骨形态蛋白下游信号通路，促使间充质干细胞成神经系分化(2011，PNAS)。在前期工作中，我们首次发现胞外基质弹性能显著影响beta1整合素和Wnt1的糖基化水平。糖基化过程对蛋白质的正确折叠、运输、定位、酶解等起到关键作用。鉴于蛋白糖基化的重要作用，上述新现象为深入理解基质弹性调控细胞功能的机理打开了一个新的视角。分析已有实验数据，我们提出：较软基底通过削弱粘着斑合成，降低FAK磷酸化，使p53聚集、入核，启动微小RNA532的转录，导致atp2c1表达水平受抑制，降低了蛋白质糖基化水平。本项目将以间充质干细胞为研究对象，探索基质弹性调控蛋白糖基化水平的机理，以期深入理解胞外基质弹性调控细胞功能的奥秘。

本课题组在探索ECM弹性影响多种细胞功能的机理时发现：β1整合素和Wnt1a的N-连接糖基化水平在较软的ECM上发生了明显下调。蛋白质的糖基化是翻译后重要修饰过程之一，对于蛋白的正确折叠、运输、定位、酶解等起到关键性作用。糖基化包括N-连接的糖基化与O-连接的糖基化，二者均完成于高尔基体内。由于糖基化现象普遍存在于膜蛋白、分泌型蛋白、转录因子等蛋白中，蛋白质的糖基化过程对多种细胞的迁移、周期、分化等功能具有广泛的重要影响，并与多种病生理过程相关，因此在很多疾病的治疗和诊断中具有很好的应用前景。鉴于蛋白质糖基化的重要影响，我们认为糖基化水平的改变可能是ECM弹性对多种细胞功能产生广泛影响的原因之一。本课题以骨髓间充质干细胞、软骨细胞、乳腺癌细胞为研究模型，探索ECM弹性对多种细胞中蛋白质糖基化的影响及其机理。.第一，我们发现了细胞外基质弹性能够显著影响细胞糖基化水平；并找到了基质刚度通过miR-532/atp2c1影响整合素糖基化的信号通路。.第二，基质弹性影响细胞的糖基化水平是我们探索细胞弹性感知机理中的一部分。在本项目的资助下，我们也继续深入探索了细胞感知胞外基质弹性过程中力学信号是如何转化为生化信号的过程。研究发现整合素与DGEA形成的复合物的寿命随着复合物上的加载速率升高呈现出二相性特点，即：当加载速率高于5888N/s时，α2β1-DEGA复合物的寿命从2.36s跃升至90.3s。可以推论，在较软的基底上α2β1-DEGA复合物容易发生解离，触发整合素的内吞。从而据此提出了细胞通过感知整合素上的加载率感知胞外基质弹性的模型。.第三，我们发现并鉴定整合素与BMPR1A的结合位点，提出整合素与BMPR1A结合并携带其内吞从而影响BMP/Smad信号通路。.通过上述三部分研究结果，我们对细胞感知胞外基质弹性的原理有了更深入和全面的研究，其中在研究整合素与DGEA复合物寿命的过程中建立了云图分析法，纠正了以前AFM的数据分析中的错误，对这类研究方法的完善做出了贡献。

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