ECM compliance and cell cycle control

ECM 合规性和细胞周期控制

基本信息

批准号：
7737418
负责人：
Richard Assoian
金额：
$ 44.92万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-24 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7737418
关键词：
Abbreviations Acrylamides Actins Adhesions Adopted Adult Affect Aorta Apoptosis Architecture Arteries Atherosclerosis Attention Balloon Angioplasty Binding Biological Biological Assay Biological Testing Biomedical Engineering Blood Vessels CCND1 gene Cell Adhesion Inhibition Cell Culture Techniques Cell Cycle Cell Cycle Progression Cell Cycle Regulation Cell Nucleus Cell Proliferation Cell physiology Cells Cellular biology Characteristics Complement Cultured Cells Cyclin D1 Cyclin-Dependent Kinases Data Disease Elasticity Event Extracellular Matrix Extracellular Matrix Proteins Family Fibronectins Fibrosis Focal Adhesions G1 Phase Gene Expression Glass Growth Growth Factor Hallmark Cell Hydrogels Immunofluorescence Microscopy Immunohistochemistry Immunoprecipitation In Vitro Incubated Injury Integrins Knock-out Laboratories Life Ligand Binding Lysine Malignant Neoplasms Mammary Neoplasms Mammary gland Measurement Messenger RNA Methodology Mitogens Modeling Molecular and Cellular Biology Mus Null Lymphocytes Organism PTK2 gene Pathologic Processes Pathway interactions Pharmaceutical Preparations Phase Phosphorylation Physiological Physiological Processes Plastics Protein Binding Proteins Receptor Protein-Tyrosine Kinases Regulation Reverse Transcriptase Polymerase Chain Reaction Rho-associated kinase Role Signal Pathway Signal Transduction Site Smooth Muscle Myocytes Stimulation of Cell Proliferation Surface Suspension substance Suspensions System Testing Time Tissues Vascular remodeling Western Blotting Work bcl-1 Genes cell type chromatin immunoprecipitation extracellular femoral artery flexibility in vivo inhibitor/antagonist interdisciplinary approach intraperitoneal mouse model novel polymerization public health relevance receptor reconstitution response restenosis rho vascular smooth muscle cell proliferation

项目摘要

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.

描述（由申请人提供）：细胞外基质（ECM）控制大量生理过程，包括分化，凋亡和增殖。此外，ECM组成和组织刚度的变化是纤维化，癌症和动脉粥样硬化等疾病的标志。在很大程度上，ECM通过结合并激活表面受体的整合素家族来调节细胞功能。现在已经确定了整联蛋白信号传导用于G1相细胞周期进程的必要性，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，使用对刚性塑料或不能模拟生理组织不良能力的刚性塑料表面进行培养的细胞。由于细胞ECM相互作用的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。我们已经使用了可变形的ECM涂层水凝胶，该水凝胶与细胞在体内遇到的生理依从性相匹配，以确定ECM/整合素信号传导如何在生理上调节增殖。我们的初步数据表明，整联蛋白依赖性细胞周期事件具有不同的依从性阈值，并且通过对细胞周期蛋白D1的选择性作用，乳腺和主动脉的组织合规性特征是细胞周期抑制剂。合规性的信号通路涉及FAK，但与我们和其他人先前与整合素依赖性诱导细胞周期蛋白D1有关的信号传导途径可区分。现在，我们提出三个旨在确定ECM合规性中与生理相关的变化如何调节细胞周期的三个目标。 AIM 1将使用ECM涂层的水凝胶来确定ECM合规性和FAK调节Cyclin D1基因表达的信号传导机制，以及新鲜分离的小鼠血管平滑肌细胞（VSMC）。 AIM 2将使用相同的实验系统来研究ECM依从性对细胞周期蛋白D1功能和CDK4/6激活的新颖而意外的翻译后效应。然后，AIM 3将使用新获得的方法来测试FAK在体内在组织合规性和VSMC增殖中的作用，用于小鼠的细线血管损伤。我们对生物工程底层的联合使用，组织弹性，细胞和分子生物学的生物物理测量以及体内小鼠建模为我们提供了一种强大的跨学科方法，用于确定ECM合规性如何控制整合素信号对细胞周期。由于ECM在血管损伤部位进行了重塑，因此这些研究的结果可能对了解组织合规性的变化如何影响动脉粥样硬化和再狭窄中的VSMC增殖有重要意义。公共卫生相关性：现代细胞生物学的主要局限性之一是细胞通常在塑料表面上培养，该塑料表面完全是刚性的，而不是其天然生物基质的柔性。潜在的底层（其“合规性”）对细胞结构，分化和增殖具有深远的影响，从而提高了传统文化中确定的某些信号，分化和增殖反应的可能性可能与体内无关。我们最近采用了一种培养系统，该系统使我们完全控制了底层合规性，并使我们能够将培养细胞的依从性与其天然组织的依从性相匹配。使用该系统，我们表明生理组织依从性是血管平滑肌细胞中细胞周期的负调节剂。现在，我们建议确定此效果的基础机制。由于必须在活生物体中进行生物学相关性的最终测试，因此该应用还利用了新获得的血管损伤的小鼠模型来测试我们在适合合规性文化中表征的信号事件的生物学相关性。除了基本细胞生物学的进步外，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的关键方面（气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合规性如何抑制血管平滑肌细胞的增殖，以及如何通过病理僵硬来克服这种控制，这可能是显着的生物医学前进。