MAP Kinse in Islet Function

描述（由申请人提供）：糖尿病在美国和世界各地都是一个巨大的健康负担，无论是在生活质量下降还是在成本上。本研究旨在探讨控制胰腺p细胞功能的基本机制。更多的知识将有助于保存p细胞功能和开发安全有效的抗糖尿病疗法。MAP激酶（MAPKs） ERK1和ERK2参与了p细胞中整合营养和激素输入以维持胰岛素基因转录的信号转导机制。本研究的重点是阐明ERK1/2在胰腺p细胞正常生理和p细胞功能障碍中的功能。ERK1/2增强胰岛素基因转录必需因子的活性；抑制ERK1/2活性会损害胰岛素基因转录。ERK1/2还涉及多种p细胞功能受损的情况，包括高血糖和免疫抑制。糖尿病患者可能出现高浓度的循环葡萄糖，导致ERK1/2的长期过度激活和ERK1/2敏感转录因子复合物的形成，这与胰岛素基因转录减少有关；因此，ERK1/2也有助于降低p细胞在长期高血糖期间产生胰岛素的能力。我们已经证明，至少有六种调节胰岛素基因转录的转录因子是p细胞中ERK1/2的靶点，包括PDX-1、Beta2、MafA、NFAT和C/EBP-p。目的是确定ERK1/2增强转录的机制，确定ERK1/2抑制转录的机制，并确定ERK1/2与PEA-15 （II型糖尿病中过表达的蛋白质）相互作用对p细胞的影响。MafA和NFAT形成与胰岛素基因启动子活性增加相关的erk1 /2依赖性复合物。我们将研究ERK1/2如何调节它们的活性，并探索ERK1/2在辅助激活因子和其他蛋白质向转录因子复合物募集中的作用。在长期暴露于高糖的p细胞中，抑制ERK1/2导致编码应激诱导的C/EBP同源因子CHOP-10 （GADD153）的mRNA显著增加。我们将定义ERK1/2如何抑制CHOP-10 mRNA的表达并探索其在p细胞中的功能。p细胞暴露于高糖环境超过12小时会抑制胰岛素基因转录，部分原因是C/EBP-p的诱导。阻断ERK1/2会破坏dna结合的C/EBP-p复合物，从而抑制胰岛素基因转录。因此，我们将确定ERK1/2调控C/EBP-p的机制。PEA-15在2型糖尿病中过度表达，在小鼠中表达时引起高血糖，并在MIN6细胞中损害胰岛素释放。PEA-15直接与ERK1/2结合并阻止其在成纤维细胞中的核积累。我们将确定PEA-15的过表达是否会干扰p细胞中的ERK1/2功能，以及ERK1/2定位是否会影响其底物的功能。

DESCRIPTION (provided by applicant): Diabetes is a huge health burden in the United States and around the world, both in decreased quality of life and in cost. This proposal addresses basic mechanisms controlling the function of pancreatic p-cells. Greater knowledge will facilitate the preservation of p-cell function and the development of safe and effective anti-diabetic therapies. The MAP kinases (MAPKs) ERK1 and ERK2 participate in the signal transduction mechanisms that integrate nutrient and hormonal inputs to the maintenance of insulin gene transcription in p-cells. The focus of this proposal is the elucidation of the functions of ERK1/2 in the normal physiology of pancreatic p-cells and in p-cell dysfunction. ERK1/2 enhance the activity of factors essential for insulin gene transcription; inhibiting ERK1/2 activity impairs insulin gene transcription. ERK1/2 are also implicated in multiple conditions in which p-cell function is impaired, including hyperglycemia and immunosuppression. High concentrations of circulating glucose that can occur in diabetes cause prolonged hyperactivation of ERK1/2 and formation of ERK1/2-sensitive transcription factor complexes associated with decreased insulin gene transcription; thus, ERK1/2 also contribute to the reduced ability of p-cells to produce insulin during prolonged hyperglycemia. We have demonstrated that at least six transcription factors that regulate insulin gene transcription are ERK1/2 targets in p-cells including PDX-1, Beta2, MafA, NFAT, and C/EBP-p. The aims are to define mechanisms by which ERK1/2 enhance transcription, to define mechanisms by which ERK1/2 inhibit transcription, and to determine the impact on p-cells of interactions of ERK1/2 with PEA-15, a protein over-expressed in type II diabetes. MafA and NFAT form an ERK1/2-dependent complex associated with increased insulin gene promoter activity. We will examine how ERK1/2 regulate their activities and explore the role of ERK1/2 in recruitment of coactivators and other proteins to transcription factor complexes. Inhibition of ERK1/2 in p-cells chronically exposed to high glucose results in a marked increase in mRNA encoding the stress-induced C/EBP homologous factor CHOP-10 (GADD153). We will define how ERK1/2 inhibit expression of the CHOP-10 mRNA and explore its functions in p-cells. Exposure of p-cells to high glucose for more than 12 h inhibits insulin gene transcription in part due to induction of C/EBP-p. Blocking ERK1/2 disrupts a DNA-bound C/EBP-p complex that inhibits insulin gene transcription. Thus, we will determine mechanisms of C/EBP-p regulation by ERK1/2. PEA-15 is overexpressed in type 2 diabetes, causes hyperglycemia when expressed in mice, and impaired insulin release in MIN6 cells. PEA-15 binds directly to ERK1/2 and prevents their nuclear accumulation in fibroblasts. We will determine if overexpression of PEA-15 interferes with ERK1/2 function in p-cells and if ERK1/2 localization affects the functions of their substrates.