Regulation of Epithelial Plasticity

上皮可塑性的调节

• 批准号: 9103387
• 负责人: DIANE L BARBER
• 金额: $ 6.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9103387
• 关键词: Adherens Junction; Adult; Affect; Animal Model; Bioinformatics; Biosensor; Breast Epithelial Cells; Carcinoma; Cell Differentiation process; Cell Lineage; Cell model; Cells; Cellular biology; Clinical; Data; Databases; Defect; Development; Disease; Drosophila genus; Embryonic Development; Environment; Epithelial; Epithelial Cells; Erinaceidae; Event; Extracellular Matrix Proteins; Fibrosis; Genetic; Homeostasis; Human Development; Image; Injury; Ions; Kidney; Life; Light; Liver; Lung; Malignant Neoplasms; Measures; Mediating; Mesenchymal; Mesoderm; Microfilaments; Molecular; Mus; Mutation; Neoplasm Metastasis; Neural Crest; Neural Crest Cell; Optics; Organ; Outcome; Ovarian Follicle; Ovary; Pathology; Pathway interactions; Pharmacologic Substance; Phenotype; Play; Positioning Attribute; Process; Proteins; Regenerative Medicine; Regulation; Resolution; Role; Signal Pathway; Signal Transduction; Staging; Stem cells; Techniques; Testing; Time; Transgenic Organisms; Work; Zebrafish; adult stem cell; base; cell behavior; cellular imaging; design; embryonic stem cell; gastrulation; human disease; in vivo; insight; migration; neoplastic cell; novel strategies; programs; protein structure; public health relevance; repaired; self-renewal; sensor; stem cell differentiation; stem cell fate; structural biology; therapeutic target; tissue repair; tissue/cell culture; transdifferentiation

 DESCRIPTION (provided by applicant): Our proposal is designed to determine how intracellular pH (pHi) dynamics regulates epithelial plasticity, with a focus on distinct types of ell differentiation. Transitions in the fate or morphological state of epithelial cells are central to metazoan development, homeostasis, and tissue repair. Our preliminary data indicate that increased pHi is necessary for three types of epithelial differentiation programs, the transdifferentiation of epithelial to mesenchymal cells (EMT), adult epithelial stem cell differentiation, and embryonic stem (ES) cell differentiation. EMT is necessary for normal development, contributes to organ repair after injury, including aberrant repair leading to fibrosis, and promotes cancer metastasis. The genetically distinct process of adult stem cell self-renewal and differentiation is a fundamental part of the program of adult homeostasis and tissue repair. The differentiation of ES cells mimics the process of lineage specification and expansion during embryonic development. Hence, resolving how these three types of epithelial differentiation are regulated has broad significance for both normal and pathological cell behaviors. Our data support testing the central hypothesis that increased pHi is necessary for different types of epithelial cell differentiation. In Aim 1 we will identify molecular mechanisms or pHi-regulated EMT based on our findings that increased pHi is necessary for EMT of lung and mammary epithelial cells. We will reveal stage-specific pHi dynamics and its regulation of actin filament remodeling and transcriptional events during EMT by using real-time imaging of genetically encoded biosensors with clonal cell models in 2D and 3D cultures as well as in vivo analysis of zebrafish neural crest development. We also will identify molecular mechanisms mediating pHi-dependent EMT by testing established and predicted pH-sensing proteins identified using an analytical combination of protein structures, cancer mutation databases, and a newly developed bioinformatics program that identifies titrating ionizable residues in proteins. In Aim 2 we will determine how pHi regulates differentiation of adult and embryonic stem cell lineages based on our findings that increased pHi is necessary for in vivo differentiation of the Drosophila ovarian follicle stem cell lineage, and for spontaneous differentiation of mouse ES cells. We will resolve how pHi dynamics regulates established cell lineage markers and the role that pH sensors play in Drosophila follicle cell and mouse ES cell differentiation, including investigating pHi-dependent hedgehog and wingless signaling, as suggested by our preliminary data. We bring to these studies new views on signaling mechanisms at the molecular level from our expertise in bridging structural and cell biology, quantitative live cell imaging, and Drosophia genetics. Although pHi is routinely measured in tissue culture cells, few studies have investigated pHi dynamics in vivo. Successful completion of these studies will provide substantial insight into a significant and unstudied mechanism for the regulation of epithelial plasticity, revealing new regulators for therapeutic targeting of disease-associated differentiatio programs.

 描述（由申请人提供）：我们的提案旨在确定细胞内 pH (pHi) 动态如何调节上皮可塑性，重点关注不同类型的细胞分化，上皮细胞的命运或形态状态的转变对于后生动物的发育至关重要。我们的初步数据表明，pHi 的增加对于三种类型的上皮分化程序（上皮细胞向间充质细胞的转分化）是必需的。 (EMT)、成体上皮干细胞分化和胚胎干细胞 (ES) 分化是正常发育所必需的，有助于损伤后的器官修复，包括导致纤维化的异常修复，并促进癌症转移的遗传独特过程。成体干细胞的自我更新和分化是成体稳态和组织修复程序的基本组成部分，ES 细胞的分化模仿胚胎发育过程中的谱系规范和扩展过程。三种类型的上皮细胞分化受到调节对于正常和病理细胞行为都具有广泛的意义，我们的数据支持检验这样的中心假设：pHi 的增加对于不同类型的上皮细胞分化是必要的。在目标 1 中，我们将确定 pHi 调节的分子机制。 EMT 基于我们的发现，即增加的 pHi 对于肺和乳腺上皮细胞的 EMT 是必要的，我们将通过使用 EMT 来揭示阶段特异性 pHi 动态及其对肌动蛋白丝重塑和转录事件的调节。我们还将通过测试已确定的和预测的 pH 传感蛋白来确定介导 pHi 依赖性 EMT 的分子机制。使用蛋白质结构、癌症突变数据库和新开发的生物信息学程序的分析组合来识别蛋白质中的滴定电离残基，在目标 2 中，我们将确定 pHi 如何调节。基于我们的发现，增加 pHi 对于果蝇卵巢滤泡干细胞谱系的体内分化和小鼠 ES 细胞的自发分化是必要的，我们将解决成体和胚胎干细胞谱系的分化问题，我们将解决 pHi 动力学如何调节已建立的细胞谱系标记。以及 pH 传感器在果蝇滤泡细胞和小鼠 ES 细胞分化中的作用，包括研究 pHi 依赖性刺猬蛋白和无翅信号传导，正如我们的初步数据所示，我们为这些研究带来了关于信号传导的新观点。我们在结构和细胞生物学、定量活细胞成像和果蝇遗传学方面的专业知识在分子水平上揭示了机制。 尽管 pHi 是在组织培养细胞中常规测量的，但很少有研究能够成功完成这些研究。对上皮可塑性调节的重要且未经研究的机制的深入了解，揭示了疾病相关分化程序治疗靶向的新调节剂。