ECM stiffness, mechanotransduction, and cell cycling

总结 机械生物学--细胞和组织如何感知和响应机械影响--是一个快速的 对生理学和纤维化相关病理学的理解越来越重要的日益增长的领域 包括癌症、肺和肝纤维化，尤其是心血管疾病。本应用程序研究如何 细胞感知并响应包含在细胞外基质（ECM）硬度内的机械信号。 ECM中的机械信息通过整合素粘附和Rho家族GTP酶传递，但如何传递呢？ 这些早期信号事件驱动细胞命运和功能仍然知之甚少。解开这些 连接是该领域的一项重大挑战。我们正在通过研究如何解决这一理解上的差距， ECM硬度的变化被转换成控制细胞周期的信号事件。通过组合 在可变形基质（水凝胶）上进行细胞培养的分子分析，我们最近表明， 粘附激酶（FAK）、p130 Cas和Rac组成了一个独立的信号传导模块， 刚性敏感性细胞周期蛋白D1表达和细胞周期进入S期的调节剂。但在非- 转化的细胞很少是线性和单向的：负调控通常补充正调控 发出信号以提供对命运的严格控制。这些负面信号和途径往往没有得到很好的理解， 这当然是刚度调节机械传导的情况。因此，我们使用RNASeq来 寻找细胞可能限制僵硬感的方法，以防止过度刺激。分析发现， 长链非编码RNA，MALAT 1，作为一种新的刚度依赖性细胞周期的负调控因子：MALAT 1 刺激进入S期，但ECM硬度降低MALAT 1的表达水平。奇怪的是，僵硬- 受刺激的Rac活性介导细胞周期蛋白D1的诱导和MALAT 1的阻遏。我们现在 建议检查ECM刚度，MALAT 1和细胞周期蛋白D1之间的关系， 活化剂。目标1将检查MALAT 1对G1相位周期-cdks的影响，评估 MALAT 1和细胞周期蛋白D1，并确定ECM组成和整合素显示的变化如何影响 MALAT 1、细胞周期蛋白D1和细胞周期的刚性依赖性调节。Aim 2位于细胞周期蛋白D1的上游， MALAT 1，并将确定整合素粘附中的不同组分如何共享激活 Rac可以差异调节MALAT 1。最后，目标3将通过以下方式测试我们的发现在体内的相关性： 分析组织硬化和平滑肌细胞增殖的小鼠模型中的平滑肌细胞增殖 血管损伤后的增殖。

SUMMARY Mechanobiology--how cells and tissues sense and respond to mechanical influences--is a rapidly growing field of increasing importance to the understanding of physiology and fibrosis-associated pathologies including cancer, lung and liver fibrosis, and especially cardiovascular disease. This application studies how cells sense and respond to mechanical cues contained within the stiffness of the extracellular matrix (ECM). Mechanical information in the ECM is relayed through integrin-adhesions and Rho family GTPases, but how these early signaling events drive cell fate and function remains poorly understood. Unraveling these connections is a major challenge in the field. We are addressing this gap in understanding by examining how changes in ECM stiffness are transduced into the signaling events that control cell cycling. By combining molecular analyses with cell culture on deformable substrata (hydrogels), we recently showed that focal adhesion kinase (FAK), p130Cas, and Rac comprise a discrete signaling module that functions as a positive regulator of stiffness-sensitive cyclin D1 expression and cell cycling into S phase. But signaling in non- transformed cells is rarely linear and uni-directional: negative regulation commonly complements positive signaling to provide tight control of fate. These negative signals and pathways are often not well understood, and this is certainly the case for stiffness-regulated mechanotransduction. We therefore used RNASeq to search for ways that cells might limit stiffness-sensing to prevent over-stimulation. This analysis identified the long noncoding RNA, MALAT1, as a novel negative regulator of stiffness-dependent cell cycling: MALAT1 stimulates entry into S phase, but ECM stiffness reduces the expression level of MALAT1. Curiously, stiffness- stimulated Rac activity mediates both the induction of cyclin D1 and the repression of MALAT1. We now propose to examine the relationships between ECM stiffness, MALAT1 and cyclin D1, and their upstream activators. Aim 1 will examine the impact of MALAT1 on the G1 phase cyclin-cdks, assess crosstalk between MALAT1 and cyclin D1, and determine how changes in ECM composition and integrin display may affect rigidity-dependent regulation of MALAT1, cyclin D1 and cell cycling. Aim 2 looks upstream of cyclin D1 and MALAT1 and will determine how distinct components in the integrin-adhesion that share an ability to activate Rac can differentially regulate MALAT1. Finally, Aim 3 will test the relevance of our findings in vivo by analyzing smooth muscle cell proliferation in a mouse model of tissue stiffening and smooth muscle cell proliferation after vascular injury.