Nkx2.2 in Pancreatic Development: The Role of the NK2-SD Domain

Nkx2.2 在胰腺发育中：NK2-SD 结构域的作用

• 批准号: 8403294
• 负责人: Joshua Adam Levine
• 金额: $ 2.62万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403294
• 关键词: Alanine; Amino Acids; Animals; Beta Cell; Blood Glucose; Cell Differentiation process; Cells; D Cells; DNA Binding; Development; Diabetes Mellitus; Ductal; Embryonic Development; Endocrine; Epithelial; Evolution; Family member; Functional disorder; Gene Expression; Generations; Genes; Gland; Glucagon; Gluconeogenesis; Hormonal; Human; In Vitro; Insulin; Investigation; Islet Cell; Islets of Langerhans; Knockout Mice; Knowledge; Laboratories; Maintenance; Mediating; Medical; Metabolic; Molecular; Mono-S; Mus; Mutant Strains Mice; Mutate; Mutation; Nkx-2.2 protein; Oral; Pancreas; Pancreatic Polypeptide; Pharmaceutical Preparations; Phenotype; Play; Point Mutation; Population; Production; Proteins; Protocols documentation; Regulation; Regulatory Pathway; Replacement Therapy; Role; Structure; TAC1 gene; Time; Transcriptional Regulation; Transplantation; blood glucose regulation; cell type; diabetic patient; embryonic stem cell; endocrine pancreas development; ghrelin; glycogenolysis; homeodomain; improved; in vivo; islet; pancreas development; postnatal; promoter; protein protein interaction; response; stem cell differentiation; stem cell technology; transcription factor

DESCRIPTION (provided by applicant): The endocrine pancreas, composed of the islets of Langerhans, is responsible for the maintenance of blood glucose homeostasis. Several cell types comprise the islets and include glucagon-producing . cells, insulin- producing ¿ cells, somatostatin-producing d cells, pancreatic polypeptide-producing PP cells, and ghrelin- producing e cells. Diabetes mellitus is associated with a loss of insulin-producing ¿ cells. Current medical treatment is focused on management of blood glucose levels through insulin replacement as well as oral medications that facilitate insulin release, inhibit gluconeogenesis, and inhibit glycogenolysis. However, in 2000, it was determined that islets can be successfully transplanted into diabetic patients using cadaveric islets. Although cell replacement therapies have been successful, the paucity of donor islets has limited its application and has led to the need to create islets in the laboratory using stem cell technologies. Current stem cell differentiation protocols have been adapted and improved in response to studies of normal mouse pancreas development, however complete ¿ cell differentiation has yet to be achieved. Thus, continued investigation into normal pancreas development will be of utmost importance for creating better in vitro ¿ cell differentiation protocols for the generation of transplantable cells. Nkx2.2 is a homeodomain transcription factor that is essential for the production of several islet cell types. Nkx2.2 null mice do not form ¿ cells and have reduced numbers of a and PP cells. Instead, the Nkx2.2 null islets are filled with ghrelin cells, the predominant mono-hormonal islet cell population that forms in the ES cell differentiation protocols. Therefore, knowledge of Nkx2.2 function and regulation will be important for the development of proper islet cell differentiation protocols. Studies from the Sussel lab have indicated that Nkx2.2 functions as an activator and a repressor at different developmental time points and in different cellular contexts. At this time, the functional structure of the Nkx2.2 protein is not well characterized, although three domains have been described, including the DNA binding homeodomain, the groucho co-repressor interaction domain (TN) and the NK2-specific domain (NK2-SD), which is highly conserved amongst family members and across species, but its function is unknown. Despite its high homology, the function of the NK2-SD domain is unknown. Characterization of the function of the NK2- SD domain during pancreas development will facilitate the development of optimized ¿ cell differentiation protocols. Specific aims: 1. To determine whether the highly conserved Nkx2.2 NK2-SD domain mediates Nkx2.2 function in vivo, I will investigate the phenotypic changes associated with Nkx2.2NK2-SD mutant mice during embryonic development and in postnatal animals. 2. To understand the mechanism by which the NK2-SD domain influences Nkx2.2 mediated transcriptional regulation, I will characterize the DNA binding and transcriptional activities of the NK2-SD domain on candidate Nkx2.2 target promoters.

描述（由申请方提供）：内分泌胰腺由胰岛组成，负责维持血糖稳态。几种细胞类型包括胰岛和胰高血糖素生产。细胞、胰岛素产生细胞、生长抑素产生细胞、胰多肽产生细胞和生长激素释放肽产生细胞。糖尿病与产生胰岛素的细胞的损失有关。目前的医学治疗集中于通过胰岛素替代以及促进胰岛素释放、抑制胰岛素生成和抑制糖原分解的口服药物来管理血糖水平。然而，在2000年，人们确定胰岛可以成功地移植到糖尿病患者使用尸体胰岛。虽然细胞替代疗法已经取得成功，但供体胰岛的缺乏限制了其应用，并导致需要在实验室中使用干细胞技术创造胰岛。目前的干细胞分化方案已经适应和改进，以响应正常小鼠胰腺发育的研究，然而尚未实现完全的细胞分化。因此，对正常胰腺发育的持续研究对于创建用于产生可移植细胞的更好的体外细胞分化方案至关重要。Nkx2.2是一种同源结构域转录因子，对几种胰岛细胞类型的产生至关重要。Nkx2.2基因敲除小鼠不形成α细胞，α和PP细胞数量减少。相反，Nkx2.2无效胰岛充满生长素释放肽细胞，其是在ES细胞分化方案中形成的主要单激素胰岛细胞群。因此，了解Nkx2.2的功能和调节对于开发适当的胰岛细胞分化方案将是重要的。Sussel实验室的研究表明，Nkx2.2在不同的发育时间点和不同的细胞环境中作为激活剂和抑制剂发挥作用。目前，Nkx2.2蛋白的功能结构尚未得到很好的表征，尽管已经描述了三个结构域，包括DNA结合同源结构域、groucho共阻遏物相互作用结构域（TN）和NK 2特异性结构域（NK 2-SD），其在家族成员和跨物种中高度保守，但其功能尚不清楚。尽管NK 2-SD结构域具有高度同源性，但其功能尚不清楚。NK 2- SD结构域在胰腺发育期间的功能的表征将促进优化的细胞分化方案的开发。具体目标：1.为了确定高度保守的Nkx2.2 NK 2-SD结构域是否在体内介导Nkx2.2功能，我将研究与Nkx2.2 NK 2-SD突变小鼠在胚胎发育过程中和出生后动物的表型变化。2.为了了解NK 2-SD结构域影响Nkx2.2介导的转录调控的机制，我将表征NK 2-SD结构域对候选Nkx2.2靶启动子的DNA结合和转录活性。