To define the role of SOX9 and Sox9+ cells in alveolar homeostasis and regeneration

定义 SOX9 和 Sox9 细胞在肺泡稳态和再生中的作用

• 批准号: 9377766
• 负责人: Purushothama Rao Tata
• 金额: $ 24.9万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9377766
• 关键词: Ablation; Address; Adult; Adult Respiratory Distress Syndrome; Alveolar; Alveolar Cell; Alveolus; Anatomy; Asthma; Behavior; Biochemical; Biological Assay; Biology; Breathing; Cause of Death; Cell Differentiation process; Cell physiology; Cells; Chromatin; Chronic Obstructive Airway Disease; Co-Immunoprecipitations; Data; Defect; Development; Diphtheria Toxin; Distal; Doctor of Philosophy; Ectopic Expression; Environment; Epigenetic Process; Epithelial Cells; Epithelium; Fostering; Foundations; Gases; Gene Expression; Gene Targeting; General Hospitals; Genes; Genetic; Genetic Transcription; Genomics; Grant; Hair follicle structure; Hamman-Rich syndrome; Heterogeneity; Inflammatory; Influenza; Injury; Institutes; Integrin alpha6beta4; Intestines; Lead; Liver; Luciferases; Lung; Lung Capacity; Lung diseases; Maintenance; Massachusetts; Mediator of activation protein; Mentors; Mentorship; Molecular; Molecular Biology; Multipotent Stem Cells; Mus; Natural regeneration; Pathogenesis; Phase; Phenotype; Physiological; Plasticizers; Population; Postdoctoral Fellow; Proteins; Pulmonary Emphysema; Pulmonary Fibrosis; Recurrence; Regenerative Medicine; Regulator Genes; Reporter; Research; Research Personnel; Respiratory physiology; Role; SOX9 protein; Signal Pathway; Skin; Stem cells; Structure of parenchyma of lung; System; Tamoxifen; Testing; Therapeutic; Thinness; Time; Toxin; Training Activity; Transcriptional Regulation; Transplantation; United States; Universities; WNT Signaling Pathway; alveolar homeostasis; base; beta catenin; career; career development; cell type; chromatin immunoprecipitation; design; experience; experimental study; in vivo regeneration; injury and repair; interest; loss of function; medical schools; member; mortality; mouse model; multipotent cell; novel; novel therapeutic intervention; pathogen; pollutant; post-doctoral training; progenitor; programs; regenerative; repaired; restoration; self-renewal; skill acquisition; skills; stem; stem cell biology; stem cell population; subcutaneous; surfactant; tool; transcription factor

 DESCRIPTION (provided by applicant): Lung epithelium is prone to frequent and recurrent injuries from inhaled pathogens and pollutants. Understanding the regenerative capacity and the role of resident alveolar stem and progenitor cells is therefore of considerable practical and therapeutic interest. Repair of damaged alveoli is crucial for restoration of normal lung function but the cellular and molecular mechanisms regulating repair are still not well understood. We have identified a novel lung stem/progenitor population in the distal alveoli that is marked by Sox9 and that these cells are anatomically and molecularly distinct from the known alveolar progenitor cells. We also find that Sox9 expressing cells contribute to the distal alveoli followin influenza injury and that the expression of SOX9 protein us increased following injury. Based on this preliminary data, we hypothesize that these novel Sox9+ cells are multipotent plastic progenitor cells of the alveoli that are essential for airway and alveolar injury repair. We also hypothesize that the transcription factor SOX9 is required to regulate injury repair by co-operating with key lung-specific transcription factor to regulate adult alveolar homeostasis and regeneration. To test these hypotheses, we propose the following specific aims. In the K99 phase of the proposal, we will 1) determine the role of a novel population of Sox9-expressing cells in alveolar homeostasis and in alveolar injury repair; 2) determine the effect of gain and loss of function of SOX9 on adult alveolar homeostasis and regeneration. In the ROO phase of this application, we will define the molecular mechanisms through which SOX9 regulates adult alveolar maintenance and regeneration. We will simultaneously use lineage tracing and cell ablation murine models in combination with influenza injury to study the contribution of Sox9+ cells to regenerated lung. We will also study the effects of Sox9 gain and loss of function using conditional ectopic expression and loss of function mice, respectively. Finally, we will identify Sox9 interacting proteins and Sox9 bound genomic loci using co-immunoprecipitation and Chromatin IP experiments, respectively, followed by functional analysis of target proteins and genes. We will also address the question whether activation of these novel cells and molecular mechanisms accelerates alveolar repair, making these cells therapeutically relevant. I am a postdoctoral fellow at the Center for Regenerative Medicine, Massachusetts General Hospital. From my masters, Ph.D. and current postdoctoral training I have gained substantial research experience in stem cell and molecular biology. I have gained substantial expertise in developing new mouse models of injury, identifying novel genes and novel genetic and epigenetic transcription regulatory elements. In recent years, I have developed novel tools for modulating gene expression specific to lung tissues and identified a remarkable cellular plasticity phenomenon in regenerating airway in which differentiated cells can become stem cells after injury. In the current proposal I seek support to study a novel putative stem cell population and the associated transcriptional and signaling pathways in the alveoli. I intend to gain both scientific and career development skills from my mentor Dr. Jayaraj Rajagopal and co-mentor Dr. David Scadden. Dr. Rajagopal has all the necessary stem cell and mouse genetics expertise as well as the experience with Sox proteins and Wnt signaling to guide my early research plan and I have already developed some of the expertise to handle the biochemical studies proposed in the R00 phase of this grant. I chose Dr. Rajagopal's lab since he is considered one of the rising stars in lung stem cell biology. But in the interests of having a breadth of mentorship, I wll take advantage of the expertise from of Dr. David Scadden, a renowned stem cell biologist and director of the Harvard Stem Cell Institute that himself has fostered the career of many independent PIs, Dr. Konrad Hochedlinger, an expert in transcriptional regulation and Sox-proteins, Dr. Wellington Cardoso, an expert in airway and lung specific signaling pathways, and Dr. Darrel Kotton, an expert in alveolar biology. The rich research environment and facilities at the Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard University, and Harvard Medical School and the expert guidance from my mentors and advisory members will help me to develop new skills to establish as an independent investigator. I will devote my 100% of my time to focused research program and to training activities including attendance and presentation at both scientific and career developmental activities.

 描述（由申请人提供）：肺上皮容易受到吸入病原体和污染物的频繁和反复损伤，因此了解驻留肺泡干细胞和祖细胞的作用具有相当大的实际和治疗意义。对于恢复正常肺功能至关重要，但调节修复的细胞和分子机制仍不清楚。我们在远端肺泡中发现了一种新的肺干/祖细胞群，其特征是。 Sox9 并且这些细胞在解剖学和分子上与已知的肺泡祖细胞不同，我们还发现表达 Sox9 的细胞有助于流感损伤后的远端肺泡，并且基于此初步数据，SOX9 蛋白的表达增加。我们发现这些新型 Sox9+ 细胞是肺泡的多能塑料祖细胞，对于气道和肺泡损伤修复至关重要。我们还发现转录因子 SOX9 是调节所必需的。通过与关键的肺特异性转录因子合作来调节成人肺泡稳态和再生来修复损伤 为了检验这些假设，我们提出以下具体目标 在该提案的 K99 阶段，我们将 1) 确定 a 的作用。肺泡稳态和肺泡损伤修复中表达 Sox9 的新细胞群；2) 确定 SOX9 功能的获得和丧失对成人肺泡稳态和再生的影响。在本申请的ROO阶段，我们将定义SOX9调节成人肺泡维持和再生的分子机制，我们将同时使用谱系追踪和细胞消融小鼠模型结合流感损伤来研究Sox9+细胞对再生肺的贡献。还将分别使用条件异位表达和功能丧失小鼠研究 Sox9 功能获得和丧失的影响。最后，我们将使用识别 Sox9 相互作用蛋白和 Sox9 结合基因组位点。我们还将分别进行免疫共沉淀和染色质 IP 实验，然后对目标蛋白和基因进行功能分析。我们还将解决这些新细胞和分子机制的激活是否加速肺泡修复的问题，从而使这些细胞具有治疗意义。在马萨诸塞州总医院再生医学中心，通过我的硕士、博士学位和目前的博士后培训，我在干细胞和分子生物学方面获得了丰富的专业知识，并在开发新的小鼠损伤模型方面获得了丰富的专业知识。新基因和近年来，我开发了调节肺组织特异性基因表达的新工具，并在当前的提议中发现了气道再生中显着的细胞可塑性现象，其中分化的细胞可以在损伤后变成干细胞。我寻求研究肺泡中新型假定干细胞群以及相关转录和信号通路的支持，我打算从我的导师 Jayaraj Rajagopal 博士和共同导师 David Scadden 博士那里获得科学和职业发展技能。 Rajagopal 拥有所有必要的干细胞和小鼠遗传学专业知识以及 Sox 蛋白和 Wnt 信号传导方面的经验来指导我的早期研究计划，我已经掌握了一些专业知识来处理本次资助的 R00 阶段提出的生化研究我选择了 Rajagopal 博士的实验室，因为他被认为是肺干细胞生物学领域的后起之秀之一，但为了获得广泛的指导，我将利用著名干细胞 David Scadden 博士的专业知识。哈佛干细胞研究所的生物学家和所长，他本人培养了许多独立 PI 的职业生涯，Konrad Hochedlinger 博士是转录调控和 Sox 蛋白方面的专家，Wellington Cardoso 博士是气道和肺部特异性信号通路方面的专家，以及肺泡生物学专家 Darrel Kotton 博士，麻省总医院、哈佛干细胞研究所、哈佛大学和哈佛医学院丰富的研究环境和设施以及我的导师和顾问成员的专家指导。将帮助我发展新技能，成为一名独立研究者，我将把 100% 的时间投入到重点研究项目和培训活动中，包括参加科学和职业发展活动并进行演讲。