Hippo suppression of NFkB controls pancreas morphogenesis and beta cell fate

Hippo 抑制 NFkB 控制胰腺形态发生和 β 细胞命运

• 批准号: 10016283
• 负责人: Ondine B Cleaver
• 金额: $ 40.5万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-11 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10016283
• 关键词: 3-Dimensional; Adhesions; Affect; American; Architecture; Autoimmune Diseases; B cell differentiation; Beta Cell; Binding; Bioinformatics; Cell Differentiation process; Cell Nucleus; Cell Polarity; Cell Proliferation; Cell model; Cells; Clinical Trials; Cultured Cells; Cytoplasm; Data; Defect; Development; Diabetes Mellitus; Embryo; Epithelial; Epithelial Cell Proliferation; Epithelial Cells; Epithelium; Evaluation; Failure; Gene Expression Profiling; Generations; Genes; Genetic; Goals; Homeostasis; In Vitro; Insulin; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Maintenance; Molecular; Morphogenesis; Mosaicism; Multipotent Stem Cells; Mus; Natural regeneration; Organ; Organ Size; Organoids; Pancreas; Pancreatic Bud; Pathway interactions; Pharmacology; Phosphotransferases; Population; Process; Regulation; Replacement Therapy; Role; Shapes; Signal Pathway; Signal Transduction; Testing; Therapeutic; Tissue Culture Techniques; Transcription Coactivator; Transgenic Mice; Transitional Cell; Vimentin; Work; beta cell replacement; diabetes mellitus therapy; epithelial to mesenchymal transition; improved; in vitro Model; in vivo; inhibitor/antagonist; innovation; insight; mouse model; mutant; novel; overexpression; pancreas development; pantetheinase; progenitor; regenerative therapy; stem cells; transcription factor; transcriptome sequencing; translational impact

More than 30 million Americans have pre- or existing diabetes. An innovative approach to treat diabetes is to generate functional beta cells, which originate from a multipotent progenitor population in the early pancreas bud. This approach is currently in clinical trials, but a recognized problem is suboptimal progenitor generation. While mechanisms regulating later pancreas development have been well defined, how pancreas progenitor specification is regulated remains unclear. This proposal will examine genetic pathways downstream of the Hippo signaling pathway using transgenic mouse models, as well as tissue culture techniques that include organ explants and pancreatic organoids. Bioinformatic approaches will be used to (1) determine if Hippo signaling regulates pancreatic progenitor specification, morphogenesis, or cell differentiation, and (2) determine crosstalk mechanisms between Yap1/Taz and NFκB that regulate progenitor specification and beta cell differentiation in the pancreas. We have found that deleting the Lats kinases dysregulates epithelial cell proliferation, leading to aberrant morphogenesis and cell differentiation, supporting the idea that pancreatic morphogenesis is closely tied to cell differentiation. Using transcriptional profiling, we have found that Yap1/Taz promote NFκB activator genes and here we will define the mechanisms involved. In this proposal, we propose the unique idea that Hippo pathway components act as a rheostat to control levels of NFκB activity during this process, sculpting the epithelial niche that generates beta cells. This niche is a specialized and transient epithelial plexus at the core of the embryonic pancreas. We hypothesize that Lats1/2 activity is required homeostatically to inactivate Yap1/Taz and thereby suppress aberrant NFκB signaling in the normal pancreas. Elevated NFκB activity in pancreatic epithelium results in dysregulated EMT initiation and loss of β-cell fate, due to disruption of the epithelial plexus niche. We hypothesize that elevated Yap1 activity stimulates NFκB signaling at least in part via the pantetheinase Vanin1 (Vnn1). Our aims will use in vivo and in vitro models to investigate these observations. Our hypotheses will be tested via: 1) an examination of how loss of Lats1 and Lats2 affects epithelial integrity and initiation of epithelial-to-mesenchymal transitions (EMTs); 2) an examination of whether overexpression of Yap1 mimics the Lats1/2 double deletion (1/2DKO) in single or clusters of epithelial cells, as well as an assessment of downstream targets known to be affected in 1/2DKO; and 3) an evaluation of the role of the NFkB pathway in pancreatic epithelial homeostasis and an examination of the role of Vnn1 in this process. We hope this work will enhance translational impact of downstream targets in pancreas progenitors and beta cells, with the goal of therapeutic beta cell replacement and regeneration to treat diabetes.

超过 3000 万美国人患有糖尿病或现有糖尿病。治疗糖尿病的一种创新方法是产生功能性β细胞，这些细胞源自早期胰腺芽中的多能祖细胞群。这种方法目前正在进行临床试验，但一个公认的问题是祖细胞生成不理想。虽然调节胰腺后期发育的机制已经明确，但胰腺祖细胞规格如何调节仍不清楚。该提案将使用转基因小鼠模型以及包括器官外植体和胰腺类器官在内的组织培养技术来检查河马信号通路下游的遗传通路。生物信息学方法将用于 (1) 确定 Hippo 信号传导是否调节胰腺祖细胞规格、形态发生或细胞分化，以及 (2) 确定 Yap1/Taz 和 NFκB 之间调节胰腺祖细胞规格和 β 细胞分化的串扰机制。我们发现，删除 Lats 激酶会导致上皮细胞增殖失调，导致形态发生和细胞分化异常，这支持了胰腺形态发生与细胞分化密切相关的观点。使用转录分析，我们发现 Yap1/Taz 促进 NFκB 激活基因，在这里我们将定义所涉及的机制。在此提案中，我们提出了一个独特的想法，即 Hippo 通路成分充当变阻器，在此过程中控制 NFκB 活性水平，塑造生成 β 细胞的上皮生态位。这个生态位是胚胎胰腺核心的特殊且短暂的上皮丛。我们假设 Lats1/2 活性需要体内稳态才能使 Yap1/Taz 失活，从而抑制正常胰腺中异常的 NFκB 信号传导。由于上皮神经丛生态位的破坏，胰腺上皮中 NFκB 活性升高导致 EMT 启动失调和 β 细胞命运丧失。我们假设 Yap1 活性升高至少部分通过泛酸酶 Vanin1 (Vnn1) 刺激 NFκB 信号传导。我们的目标将使用体内和体外模型来研究这些观察结果。我们的假设将通过以下方式进行检验：1）检查Lats1和Lats2的丢失如何影响上皮完整性和上皮间质转化（EMT）的启动； 2) 检查 Yap1 的过度表达是否模拟单个或上皮细胞簇中的 Lats1/2 双缺失 (1/2DKO)，以及评估已知在 1/2DKO 中受影响的下游靶点； 3) 评估 NFkB 通路在胰腺上皮稳态中的作用，并检查 Vnn1 在此过程中的作用。我们希望这项工作将增强胰腺祖细胞和β细胞下游靶标的转化影响，以实现治疗性β细胞替代和再生来治疗糖尿病。