Cytoskeletal Regulation of Human Nucleus Pulposus Cell Phenotype

项目总结/摘要 髓核（NP）细胞位于柔软的凝胶状基质中， 随着年龄的增长越来越纤维化。在这些基质硬度的“物理线索”中， 调节NP细胞表型，并可能有助于向衰老和成纤维细胞NP细胞的过渡 修复能力有限。NRSA奖学金中提出的这个项目将研究如何“物理” 基质硬度的“线索”可以调节NP细胞表型，并确定可以保持NP细胞表型的机制。 具有生物合成活性的幼年NP细胞表型。我们已经表明，形成多细胞簇的NP细胞 在"软"层粘连蛋白呈递底物上的细胞表现出生物合成活性，幼年NP表型， 血清反应共激活因子心肌蛋白相关转录因子（MRTF-A）的胞浆定位 在许多细胞中促进成纤维细胞样行为的SRF因子。此外，还表明， 核因子和信号通路可能介导基质物理性质的机械反应。在 具体目标1，我们将确定SRF的MRTF-A共激活是否是促进细胞增殖所必需的。 人NP细胞的成纤维细胞分化，测量为NP特异性 标记物和减少ECM生物合成。原代人NP细胞 将用SRE-luc或LifeAct GFP报告质粒转导，然后MRTF-A将被敲低 使用CRISPRi/Cas9。细胞将在"硬" PEG-层粘连蛋白水凝胶上培养，并评估关键的NP特异性免疫应答。 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 organization. RNA-seq分析将确定涉及MRTF-SRF信号传导的任何下游途径。 在具体目标2中，我们将评估阻断TEAD的雅普/TAZ活化是否可以促进NP特异性 标记物表达和提高的生物合成。 人NP细胞将携带TEAD-luc或LifeAct-GFP报告质粒，然后用TEAD-luc或LifeAct-GFP转导。 携带雅普CRISPRi/Cas9质粒的慢病毒以破坏雅普/TAZ信号传导。这些细胞将被培养在 "硬的"层粘连蛋白呈递水凝胶（以及"软的"对照），并测量相同的NP特异性凝胶。 标记和RNA-seq分析，如具体目标1中所述。 申请人将与赞助商和指导团队一起学习最先进的新技术 基因组工程和RNA-seq方法以及定量细胞骨架组织的方法， 促进申请人的职业生涯和细胞表型操作的翻译应用于光盘 再生申请人将获得更广泛的研究方法，赠款和手稿的培训 准备和负责任的研究行为，这将为她的职业生涯作为一个独立的 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.