ECM compliance and cell cycle control

描述（由申请人提供）：细胞外基质（ECM）控制大量的生理过程，包括分化、凋亡和增殖。此外，ECM组成和组织硬度的变化是纤维化、癌症和动脉粥样硬化等多种疾病的标志。在很大程度上，ECM通过结合和激活表面受体的整合素家族来调节细胞功能。整合素信号传导对G1期细胞周期进程的必要性现在已经得到了很好的证实，但是用于记录ECM/整合素效应的方法通常依赖于抑制细胞粘附、肌动蛋白聚合或Rho-Rho激酶信号传导，这些方法使用的是在刚性塑料或玻璃表面培养的细胞，这些细胞不能模拟生理组织的可变形性。由于细胞-ECM相互作用的一个标志是评估细胞外刚度的能力，因此ECM依从性可能是下游信号通路的重要决定因素。我们使用了可变形的ECM包被水凝胶，与细胞在体内遇到的生理顺应性相匹配，以确定ECM/整合素信号如何在生理上调节增殖。我们的初步数据表明，整合素依赖性细胞周期事件具有不同的顺应性阈值，乳腺和主动脉的组织顺应性特征通过对细胞周期蛋白D1的选择性作用发挥细胞周期抑制剂的作用。顺应性调节的信号通路涉及FAK，但与我们和其他人先前涉及的整合素依赖性诱导cyclin D1的信号通路不同。我们现在提出三个目标来确定ECM顺应性的生理相关变化如何调节细胞周期。目的1将使用ECM包被的水凝胶来确定ECM依从性和FAK调节mef和新分离的小鼠血管平滑肌细胞（VSMCs）中cyclin D1基因表达的信号机制。Aim 2将使用相同的实验系统来研究ECM依从性对cyclin D1功能和cdk4/6激活的新颖且意想不到的翻译后效应。然后，Aim 3将使用新获得的小鼠细丝血管损伤方法，在体内测试FAK对组织顺应性和VSMC增殖的作用。我们结合使用生物工程基质，组织弹性的生物物理测量，细胞和分子生物学，以及体内小鼠模型，为我们提供了一个强大的跨学科方法来确定ECM顺应性如何控制整合素信号传导到细胞周期。由于ECM在血管损伤部位重建，这些研究的结果也可能对理解组织顺应性变化如何影响动脉粥样硬化和再狭窄的VSMC增殖具有重要意义。公共卫生相关性：现代细胞生物学的主要限制之一是，细胞通常是在完全刚性的塑料表面上培养的，而不是在具有柔性的天然生物基质上培养的。底层基质的灵活性（其“顺应性”）对细胞结构、分化和增殖具有深远的影响，这提高了在传统培养中发现的一些信号传导、分化和增殖反应在体内可能不相关的可能性。我们最近采用了一种培养系统，使我们能够完全控制基质的顺应性，并使我们能够将培养细胞的顺应性与其天然组织的顺应性相匹配。利用这个系统，我们发现生理组织顺应性是血管平滑肌细胞细胞周期的负调节因子。我们现在提议确定这种效应背后的机制。由于生物学相关性的最终测试必须在活的生物体中进行，因此本应用程序还利用新获得的血管损伤小鼠模型来测试我们在顺应性培养中表征的信号事件的生物学相关性。除了在基础细胞生物学方面取得进展外，我们提出的工作具有很强的生物医学相关性，因为平滑肌细胞增殖和血管重塑是动脉粥样硬化和再狭窄（球囊血管成形术后平滑肌细胞增殖）的关键方面。因此，了解生理ECM顺应性如何抑制血管平滑肌细胞增殖，以及这种控制如何通过动脉的病理性硬化来克服，有可能成为生物医学的重大进展。

DESCRIPTION (provided by applicant): The extracellular matrix (ECM) controls a large number of physiological processes including differentiation, apoptosis, and proliferation. Moreover, changes in ECM composition and tissue stiffness are hallmarks of diseases as diverse as fibrosis, cancer, and atherosclerosis. In large part, the ECM regulates cellular function by binding to and activating the integrin family of surface receptors. The necessity of integrin signaling for G1 phase cell cycle progression is now well established, but the approaches that have been used to document ECM/integrin effects typically rely on inhibition of cell adhesion, actin polymerization, or Rho-Rho kinase signaling using cells cultured on rigid plastic or glass surfaces that do not model the deformability of physiological tissue. Since a hallmark of cell-ECM interactions is the ability to assess extracellular stiffness, ECM compliance may be an important determinant of downstream signaling pathways. We have used deformable ECM-coated hydrogels matched to the physiological compliance that cells encounter in vivo to determine how ECM/integrin signaling regulates proliferation physiologically. Our preliminary data show that integrin-dependent cell cycle events have distinct compliance thresholds, and that the tissue compliance characteristic of mammary glands and aortae acts as a cell cycle inhibitor through a selective effect on cyclin D1. The compliance-regulated signaling pathway involves FAK, but is distinguishable from the signaling pathways that we and others have previously implicated in integrin-dependent induction of cyclin D1. We now propose three aims to determine how physiologically relevant changes in ECM compliance regulate the cell cycle. Aim 1 will use ECM-coated hydrogels to determine the signaling mechanisms by which ECM compliance and FAK regulate cyclin D1 gene expression in MEFs and freshly isolated mouse vascular smooth muscle cells (VSMCs). Aim 2 will use the same experimental systems to study a novel and unexpected post- translational effect of ECM compliance on the function of cyclin D1 and activation of cdk4/6. Aim 3 will then test the roles of FAK on tissue compliance and VSMC proliferation in vivo, using newly acquired methodology for fine-wire vascular injury in the mouse. Our combined use of bioengineered substrata, biophysical measurements of tissue elasticity, cell and molecular biology, and in vivo mouse modeling provides us with a powerful interdisciplinary approach for determining how ECM compliance controls integrin signaling to the cell cycle. Since the ECM remodels at sites of vascular injury, the results from these studies may also have important implications for understanding how changes in tissue compliance affect VSMC proliferation in atherosclerosis and restenosis. PUBLIC HEALTH RELEVANCE: One of the main limitations of modern cell biology is that cells are usually cultured on a plastic surface which is completely rigid rather than its native biological substratum which is flexible. The flexibility of an underlying substratum (its "compliance") has profound effects on cellular architecture, differentiation, and proliferation, raising the possibility that some of the signaling, differentiation, and proliferative responses identified in traditional culture may not be relevant in vivo. We have recently adopted a culture system that gives us complete control of substratum compliance and allows us to match the compliance of cultured cells to the compliance of their native tissues. Using this system, we show that physiological tissue compliance is a negative regulator of the cell cycle in vascular smooth muscle cells. We now propose to determine the mechanism underlying this effect. Since the ultimate test of biological relevance must be made in a living organism, this application also exploit a newly acquired mouse model of vascular injury to test the biological relevance of the signaling events that we characterize in compliance-appropriate culture. In addition to the advance in basic cell biology, our proposed work has strong biomedical relevance because smooth muscle cell proliferation and vascular remodeling are critical aspects of both atherosclerosis and restenosis (smooth muscle cell proliferation after balloon angioplasty). Thus, understanding how physiological ECM compliance inhibits vascular smooth muscle cell proliferation, and how this control can be overcome by pathological stiffening of arteries, has the potential to be a significant biomedical advance.