ECM stiffness, mechanotransduction, and cell cycling

ECM 硬度、力转导和细胞循环

基本信息

批准号：
10210426
负责人：
Richard Assoian
金额：
$ 42.49万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10210426
关键词：
Abbreviations Address Adhesions Affect Age Area Arteries Atomic Force Microscopy BCAR1 gene Biological Biological Models Cardiovascular Diseases Cell Culture Techniques Cell Cycle Cell Cycle Regulation Cell Proliferation Cells Complement Connective Tissue Cues Cyclin D1 Cyclin-Dependent Kinases Cyclins Data Embryo Event Extracellular Matrix Extracellular Matrix Proteins Family Fibroblasts Fibronectins Fibrosis Fluorescent in Situ Hybridization Focal Adhesion Kinase 1 G1 Phase Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Histology Hydrogels Immunoprecipitation In Vitro Injury Integrins Lead Liver Fibrosis MALAT1 gene MYH11 gene Malignant Neoplasms Mechanics Mediating Molecular Molecular Analysis Mus Pathology Pathway Analysis Physiology Proliferating Protein-Lysine 6-Oxidase Proteins Pulmonary Fibrosis Regulation Repression Research S Phase Signal Pathway Signal Transduction Smooth Muscle Smooth Muscle Actin Staining Method Smooth Muscle Myocytes Specificity Surface Tamoxifen Testing Tissue Model Tissues Tunica Adventitia Untranslated RNA Vascular Smooth Muscle Western Blotting arterial stiffness base experimental study extracellular in vivo inhibitor/antagonist interest kinase inhibitor locked nucleic acid mechanotransduction mouse model myocardin novel paxillin prevent programs response rho transcription factor transcriptome sequencing vascular injury

项目摘要

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.

概括机械生物学——细胞和组织如何感知和响应机械影响——是一门快速发展的学科。对于理解生理学和纤维化相关病理学的重要性日益增长的领域包括癌症、肺纤维化和肝纤维化，尤其是心血管疾病。该应用程序研究如何细胞感知并响应细胞外基质 (ECM) 硬度中包含的机械信号。 ECM 中的机械信息通过整合素粘附和 Rho 家族 GTPases 传递，但是如何这些早期信号事件驱动细胞的命运和功能仍然知之甚少。解开这些连接是该领域的一个重大挑战。我们正在通过研究如何解决这一理解上的差距 ECM 硬度的变化被转化为控制细胞周期的信号事件。通过结合在可变形基质（水凝胶）上进行细胞培养的分子分析，我们最近表明，焦点粘附激酶 (FAK)、p130Cas 和 Rac 组成一个离散信号模块，充当正向信号通路硬度敏感的细胞周期蛋白 D1 表达和细胞周期进入 S 期的调节剂。但在非信号中转化细胞很少是线性和单向的：负调节通常补充正调节发出信号以严格控制命运。这些负面信号和途径通常没有被很好地理解，对于刚度调节的机械传导来说，情况确实如此。因此我们使用 RNASeq 寻找细胞限制刚度感应以防止过度刺激的方法。这项分析确定了长非编码 RNA，MALAT1，作为刚性依赖性细胞周期的新型负调节因子：MALAT1 刺激进入 S 期，但 ECM 僵硬会降低 MALAT1 的表达水平。奇怪的是，僵硬- 刺激的 Rac 活性介导细胞周期蛋白 D1 的诱导和 MALAT1 的抑制。我们现在提议检查 ECM 硬度、MALAT1 和细胞周期蛋白 D1 及其上游之间的关系激活剂。目标 1 将检查 MALAT1 对 G1 期细胞周期蛋白-cdks 的影响，评估之间的串扰 MALAT1 和细胞周期蛋白 D1，并确定 ECM 组成和整合素展示的变化如何影响 MALAT1、细胞周期蛋白 D1 和细胞周期的刚性依赖性调节。目标 2 着眼于细胞周期蛋白 D1 的上游， MALAT1 并将确定整合素粘附中的不同成分如何共享激活能力 Rac 可以差异调节 MALAT1。最后，目标 3 将通过以下方式测试我们的研究结果在体内的相关性：分析组织硬化和平滑肌细胞小鼠模型中的平滑肌细胞增殖血管损伤后增殖。