ECM stiffness, mechanotransduction, and cell cycling

ECM 硬度、力转导和细胞循环

• 批准号: 9978116
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978116
• 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

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.

摘要 机械生物学--细胞和组织如何感知和响应机械影响--是一种快速的 对生理学和纤维化相关病理的理解日益重要的领域 包括癌症、肺癌和肝纤维化，尤其是心血管疾病。这个应用程序研究如何 细胞感知并对细胞外基质(ECM)硬度中包含的机械信号做出反应。 细胞外基质中的机械信息是通过整合素粘连和Rho家族GTP酶传递的，但如何传递 这些早期的信号事件决定了细胞的命运和功能，目前仍知之甚少。解开这些 连通性是该领域的一大挑战。我们正在通过研究如何解决这一理解上的差距 细胞外基质硬度的变化被传导到控制细胞周期的信号事件中。通过组合 通过在可变形底物(水凝胶)上进行细胞培养的分子分析，我们最近发现 黏附蛋白激酶(FAK)、p130Cas和Rac组成一个独立的信号模块，其功能与 调节僵硬敏感的细胞周期蛋白D1的表达和细胞周期进入S期。但在非 转化的细胞很少是线性的和单向的：负调控通常与正调控互补 发出信号，提供对命运的严格控制。这些负面信号和途径通常没有被很好地理解， 这当然是刚度调节机械转导的情况。因此，我们使用RNAseq 寻找细胞可能限制僵硬感知以防止过度刺激的方法。这一分析确定了 长非编码RNA，MALAT1，作为一种新的僵硬依赖细胞周期负调控因子：MALAT1 刺激细胞进入S期，但细胞外基质僵硬降低MALAT1的表达水平。奇怪的是，僵硬- 受刺激的Rac活性既介导了细胞周期蛋白D1的诱导，又抑制了MALAT1的表达。我们现在 建议研究细胞外基质刚性、MALAT1和细胞周期蛋白D1及其上游之间的关系 激活剂。目标1将检测MALAT1对G1期细胞周期蛋白-CDKs的影响，评估 MALAT1和细胞周期蛋白D1，并确定细胞外基质组成和整合素显示的变化可能如何影响 MALAT1、细胞周期蛋白D1和细胞周期的刚性调节。AIM 2研究了细胞周期蛋白D1的上游 并将确定整合素黏附中的不同成分如何共享激活能力 Rac对MALAT1具有差异调节作用。最后，Aim 3将通过以下方式测试我们的研究结果在体内的相关性 分析小鼠组织僵硬和平滑肌细胞模型中的平滑肌细胞增殖 血管损伤后的增殖。