细胞骨架蛋白介导Hippo-Yap信号调控对椎间盘髓核细胞形态及增殖活性的效应机制研究

Disc cell morphology and viability are spatiotemporally regulated during intervertebral disc (IVD) ageing and degeneration. To date, the molecular mechanism that underlies these dynamic cellular changes remains elusive. Hippo-YAP pathway is a newly discovered and evolutionally conserved signaling cascade that regulates cell proliferation and morphology alteration. In addition, evidence is mounting that a large number of Yap targets are modulated by cytoskeletal proteins, which sense and response to extracellular microenvironment and regulate the biological functions of the Hippo pathway. . Previously we have shown that Yap, the core protein of Hippo pathway, is constitutively expressed in disc cells, whereas the expression of cytoskeletal F-actin varies significant among different subgroup of nucelus pulpusos (NP) cells. However, more studies are needed to expand our understanding of the interactions between Hippo-Yap pathways and cytoskeletal dynamics, as well as their pathophysiological significance on NP cell morphology and viability. . To detect the changes of Hippo signals and cytoskeletal components during IVD ageing and degeneration, the transcriptional and protein level of F-actin, β-tubulin, vimentin, and Yap will be detected in rat NP with age-related or accelerated degeneration. The correlation between cytoskeletal signatures, Yap expression, and NP cells morphological features will be analyzed. . To investigate the regulatory mechanism of Hippo pathways by cytoskeletal dynamics, the notochordal-like NP cells and chondrocyte-like NP cell will be isolated from rat disc and cultured in monolayers. The changes in cellular morphology, cytoskeletal components, as well as the Yap expression level will be studied in different NP cells under varied adhesion and contact conditions. By up-regulating or down-regulating the expression of Mst and Lats, the feasibility of inhibit or activate Hippo pathways will be tested in the notochordal-like and the chondrocyte-like NP cell respectively. Subsequently, alterations in cell morphology, cell viability, proliferative capacity, as well as the transactivation of Yap targets such as β-catenin, GSK3β, and miR-29 will be evaluated and compared among different NP cells with enhanced or reduced Yap expression. . We hypothesize that the Hippo-Yap pathway, regulated by cytoskeletal dynamics, is critical to maintain disc cell viability and biological responsiveness to various culture conditions. Disturbance of Hippo signals might result in decreased proliferative capacity and viability, thus expending our understanding of the pathogenesis of early IVD degeneration.

细胞形态与活性在椎间盘不同区域和病理阶段差异显著。Hippo-Yap通路应答细胞骨架蛋白对胞外环境的感知，继而通过Yap下游靶点调控细胞增殖等诸多生物学行为。本研究首先在大鼠椎间盘自然衰老与加速退变过程中观察髓核细胞形态，骨架蛋白F-actin、β-tubulin、vimentin含量，YAP蛋白激活水平三者间的相关性与变化规律。继而培养软骨样髓核细胞和脊索样髓核细胞，在不同界面贴壁和接触环境下比较细胞形态、骨架蛋白成分、YAP激活等方面的差异；并在上游Mst、Lats环节强化或抑制Hippo信号，观察Hippo信号调控髓核细胞YAP激活及其下游增殖相关靶点β-catenin、GSK3β、miR-29的量效关系。旨在探明骨架蛋白介导Hippo信号调控并影响髓核细胞形态与活性的分子生物学机制，为从“Hippo-Yap信号调控异常→髓核细胞活性过早丢失”角度认知椎间盘退变的细胞学病因打开思路。

细胞骨架蛋白F-actin、β-tubulin、vimentin调节细胞形态同时可感知外界细胞间刺激，调节LATS或其上游MST1/2、 YAP和YAP蛋白磷酸化。我们提出并验证假说：髓核细胞内Hippo-YAP 信号调控能改变细胞形态、增殖等生物学行为，且细胞骨架蛋白介导该信号参与调控髓核细胞老化及椎间盘退变的发生发展。我们发现：.1.Hippo信号通路参与IDD的调节，其机制与IDD的年龄和损伤有关。对髓核中Hippo信号通路分析得知，YAP蛋白和基因的表达随年龄的增长而下降，而幼龄鼠的上游LATS1基因则增加，说明LATS1与YAP存在互相调控作用。损伤早期CTGF基因的表达与蛋白质的表达趋势不一致，提示存在一定的自我修复能力和不完全自我修复（转录后调节）；.2.细胞高密度条件下YAP被抑制（细胞拥挤状态），YAP表达降低，YAP定位从细胞核向细胞质中转移，反之低细胞密度条件下YAP被激活（细胞分散状态），YAP 由细胞质向细胞核聚集。与其他报道一致，细胞在高密度培养环境中，细胞核内YAP降低，表明生长抑制。.3.Hippo信号通路在衰老的NPCs中被激活，产生生长抑制信号。在衰老NPCs中，YAP主要集中在细胞质中，而在非衰老细胞中，YAP则主要分布细胞核；.4.细胞密度和ECM可调节Hippo信号通路和细胞骨架，β-tubulin和Vimentin 的主要功能是为细胞提供形状和弹性以抵抗机械力刺激。在低细胞密度下YAP 主要定位于细胞质，表明NPCs 的特异性和Hippo 信号通路复杂性；.5.YAP是LATS1和LATS2的直接靶分子，YAP活化时两者均增加，而奇怪的是在大鼠尾椎椎间盘自然IDD 进展过程中没有观察到LATS2 表达的明显变化，这表明LATS1是YAP的主要调节因子，LATS2作用微弱，但尚不清楚这种调节效应是否与物种有关；.6.AMOT130 可以结合并滞留YAP于细胞质中，并且可以不依赖于Hippo信号方式激活LATS进而抑制YAP活性，即可以通过两种不同的机制抑制YAP活性：1）骨架蛋白F-actin 与YAP 竞争性结合AMOT130调控YAP 活性；2）经典Hippo 信号途径对YAP磷酸化，磷酸化YAP和14-3-3σ结合滞留胞质内进一步降解。