Cytoskeletal Regulation of Human Nucleus Pulposus Cell Phenotype

人髓核细胞表型的细胞骨架调节

基本信息

批准号：
9395650
负责人：
Bailey V Fearing
金额：
$ 5.92万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9395650
关键词：
Adolescent Adult Age Anabolism Back Pain Behavior Binding CRISPR interference Cell Differentiation process Cell Nucleus Cells Cellular Metabolic Process Cellular Morphology Collagen Complex Cues Cultured Cells Cytoskeletal Modeling Cytosol Data Disease Environment Exhibits Extracellular Matrix F-Actin Family member Fellowship Fibroblasts Focal Adhesions Future G Actin GTP-Binding Protein alpha Subunits, Gs Genes Genome Genome engineering Goals Grant Guide RNA Homologous Gene Human Hydrogels Image Immunohistochemistry Intervertebral disc structure Laminin Learning Manuscripts Measures Mechanics Mediating Mentors Metabolism Methods Morphology National Research Service Awards Nuclear Nuclear Translocation Operative Surgical Procedures Pathology Pathway interactions Phenotype Plasmids Preparation Production Protein Isoforms Proteins Proteoglycan Regenerative Medicine Regulation Reporter Research Research Methodology Research Personnel Role Serum Response Factor Signal Pathway Signal Transduction Signaling Molecule Stress Fibers Subfamily lentivirinae Techniques Testing Training Transcription Coactivator Transcriptional Coactivator with PDZ-Binding Motif Translations Vertebral column Viral Vector Work age related career cell type cellular engineering differential expression disc regeneration knock-down molecular marker myocardin novel nucleus pulposus physical property prevent repaired response senescence transcription factor transcriptome sequencing wasting

项目摘要

PROJECT SUMMARY/ABSTRACT Nucleus pulposus (NP) cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Age-related changes in these “physical cues” of matrix stiffness may be potent regulators of NP cell phenotype, and may contribute to transitions toward a senescent and fibroblastic NP cell with a limited capacity for repair. This project proposed in the NRSA Fellowship will study how “physical cues” of matrix stiffness can regulate NP cell phenotype and identify mechanisms that can preserve a biosynthetically active, juvenile NP cell phenotype. We have shown that NP cells forming multi-cell clusters on “soft” laminin-presenting substrates demonstrate biosynthetically active, juvenile NP phenotype with cytosolic localization of myocardin related transcription factor (MRTF-A), a co-activator of serum response factor (SRF) that promotes fibroblast-like behaviors in many cells. Additionally, it has been shown that other nuclear factors and signaling pathways may mediate mechanoresponses to matrix physical properties. In Specific Aim 1, we will determine if MRTF-A co-activation of SRF is necessary for promoting the fibroblastic differentiation of human NP cells, measured as decreased expression of NP-specific markers and reduced ECM biosynthesis, when cultured upon “stiff” substrates. Primary human NP cells will be transduced with SRE-luc or LifeAct GFP reporter plasmids, and then MRTF-A will be knocked down with CRISPRi/Cas9. Cells will be cultured on “stiff” PEG-laminin hydrogels and assessed for key NP-specific matrix synthesis and molecular markers, transcription factor and signaling molecule activity, and cytoskeletal organization. RNA-seq analysis will determine any downstream pathways implicated in MRTF-SRF signaling. In Specific Aim 2, we will evaluate if blocking YAP/TAZ activation of TEAD can promote NP-specific marker expression and elevated biosynthesis for adult human NP cells cultured on “stiff” substrates. Human NP cells will carry either TEAD-luc or LifeAct-GFP reporter plasmids and then transduced with lentivirus carrying YAP CRISPRi/Cas9 plasmid to disrupt YAP/TAZ signaling. These cells will be cultured on “stiff” laminin-presenting hydrogels (as well as “soft” for a control) and measured for the same NP-specific markers and RNA-seq analysis as in Specific Aim 1. The applicant will work with the Sponsor and mentoring team to learn new techniques in state-of-the-art genome engineering and RNA-seq methods as well as methods to quantify cytoskeletal organization that will advance both the applicant's career and the translation of cell phenotype manipulation for application to disc regeneration. The applicant will obtain broader training in research methodology, grant and manuscript preparation and responsible conduct in research that will prepare her for a career as an independent researcher.

项目摘要/摘要细胞核（NP）细胞居住在脱水并变成的柔软的胶状基质中随着年龄的增长，纤维化越来越多。这些与年龄相关的矩阵刚度的“物理线索”可能有效 NP细胞表型的调节剂，可能有助于向感觉和成纤维细胞NP细胞的过渡维修能力有限。 NRSA奖学金中提出的这个项目将研究“物理矩阵刚度的提示可以调节NP细胞表型，并确定可以保留一个的机制生物合成活性，青少年NP细胞表型。我们已经证明了形成多细胞簇的NP单元在“软”层粘连蛋白呈现底物上，表现出具有生物合成活性的幼年NP表型肌钙蛋白相关转录因子（MRTF-A）的胞质定位，血清反应的共激活剂因子（SRF）促进许多细胞中的成纤维细胞样行为。另外，已经证明了其他核因子和信号通路可能会介导机制提高到基质物理特性。在具体目的1，我们将确定MRTF-A共同激活SRF是否为促进人NP细胞的成纤维细胞分化，以NP特异性表达降低测量当在“刚性”底物上培养时，标记和减少ECM生物合成。原代人NP细胞将用SRE-LUC或LIFEACT GFP报告基因质粒转移，然后将MRTF-A击倒与CRISPRI/CAS9。细胞将在“硬” PEG - 氯胺酮水凝胶上培养，并评估关键NP特异性基质合成和分子标记，转录因子和信号分子活性以及细胞骨架组织。 RNA-seq分析将确定MRTF-SRF信号传导中实现的任何下游途径。在特定的目标2中，我们将评估阻塞YAP/TAZ激活TEAD是否可以促进NP特异性在“刚性”底物上培养的成年人类NP细胞的标记表达和升高的生物合成。人NP细胞将携带tead-luc或lifeAct-GFP报告基因质粒，然后翻译携带yap crispri/cas9质粒的慢病毒可破坏yap/taz信号传导。这些细胞将在 “刚性”呈粘着层粘连蛋白的水凝胶（以及对照的“软”），并针对相同的NP特异性测量标记和RNA-seq分析与特定目标1一样。申请人将与赞助商和心理团队合作，以最先进的方式学习新技术基因组工程和RNA-seq方法以及量化细胞骨架组织的方法推进应用程序的职业和细胞表型操纵的翻译以进行光盘再生。申请人将获得更广泛的研究方法，赠款和手稿的培训在研究中的准备和负责任的行为，这将为她做准备，使她成为独立的职业研究员。