Cytoskeletal Regulation of Human Nucleus Pulposus Cell Phenotype

项目摘要/摘要 髓核(NP)细胞存在于一种柔软的凝胶基质中，这种基质脱水并成为 随着年龄的增长而变得越来越纤维化。与年龄相关的改变，这些“物理线索”的矩阵僵硬可能是强有力的 NP细胞表型的调节者，可能有助于向衰老和成纤维细胞的NP细胞过渡 维修能力有限。这项在NRSA奖学金上提出的项目将研究如何“物理 基质僵硬的“线索”可以调节NP细胞的表型，并识别可以保存 生物合成活性，幼年NP细胞表型。我们已经证明了NP细胞形成多个细胞团 在“柔软的”层粘连蛋白呈递底物上展示了具有生物合成活性的幼年NP表型 血清反应共同激活剂--心肌钙蛋白相关转录因子(MRTF-A)的胞浆定位 在许多细胞中促进成纤维细胞样行为的因子(SRF)。此外，已经表明，其他 核因子和信号通路可能介导对基质物理性质的机械反应。在……里面 具体目标1，我们将确定是否需要SRF的MRTF-A共激活来促进 人NP细胞的成纤维细胞分化，以NP特异性表达减少为指标 标记和减少细胞外基质的生物合成，当培养在“僵硬”的底物上时。原代人NP细胞 将用SRE-Luc或LifeAct GFP报告质粒转导，然后MRTF-A将被击倒 使用CRISPRi/Cas9。细胞将在“僵硬”的聚乙二醇层粘连蛋白水凝胶上培养，并评估关键的NP特异性 基质合成与分子标记、转录因子与信号分子活性、细胞骨架 组织。RNA-SEQ分析将确定与MRTF-SRF信号有关的任何下游通路。 在特定的目标2中，我们将评估阻断TEAD的YAP/TAZ激活是否能够促进NP特异性 培养在“僵硬”底物上的成人NP细胞的标志物表达和生物合成增加。 人NP细胞将携带Tead-Luc或LifeAct-GFP报告质粒，然后与 携带YAP CRISPRi/Cas9质粒的慢病毒干扰YAP/TAZ信号转导。这些细胞将被培养在 “硬的”层粘连蛋白呈现水凝胶(以及对照的“软”水凝胶)，并对相同的NP特异性进行测量 具体目标1中的标记和RNA-seq分析。 申请者将与赞助商和指导团队合作，学习最先进的新技术 基因组工程和rna-seq方法以及量化细胞骨架组织的方法 促进申请者的职业生涯和细胞表型操作的翻译以应用于圆盘 再生。申请者将获得更广泛的研究方法、补助金和手稿方面的培训 在研究方面的准备和负责任的行为，这将为她作为独立人士的职业生涯做好准备 研究员。

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.