Tead1 - A Regulator of Quiescence and Proliferation in Pancreatic Beta Cells

 描述(由申请人提供)： 糖尿病是导致退伍军人发病率和死亡率的主要原因之一。未能增加β细胞的增殖和功能性β细胞质量 与肥胖和衰老相关的胰岛素抵抗的代谢需求是退伍军人成人发病糖尿病的主要原因。因此，有必要更好地了解β-细胞增殖的调控，以便将其靶向用于治疗糖尿病。虽然Tead1是哺乳动物河马途径的关键转录效应因子，已被证明在许多组织中促进细胞增殖，但它在β细胞增殖中的作用是 未知。在初步研究中，我们证明了Tead1在小鼠和人的胰岛中都有强健的表达，并且在调节β细胞的增殖方面具有非冗余的作用。在增生性胰岛中，Tead1的共同激活子YAP和TAZ移位到细胞核，而β细胞特异性的Tead1缺失会导致糖尿病和葡萄糖耐量减低，继而导致体内葡萄糖刺激的胰岛素分泌的丧失。Tead1缺失的胰岛显示成熟的β细胞标志物的表达减少，而β细胞的增殖显著增加。这种β细胞增殖的增加甚至发生在老年小鼠的β细胞中，而对照组中这些细胞通常处于静止状态。对Tead1缺失的胰岛进行的整体转录组学分析显示，调控细胞周期的基因有显著的差异表达，关键的细胞周期抑制因子p16(Ink4a/CDKN2a)的表达显著降低。Tead1还直接与p16启动子结合，提示它可能是Tead1在β-细胞中的直接转录靶点，并介导Tead1对细胞周期的调控。此外，Tead1还占据了β细胞关键基因Pdx1、MafA和Nkx6.1的顺式启动子元件，而在Tead1缺失的胰岛中这些基因的表达降低，提示Tead1可能直接调节β细胞的功能。因此，我们假设Tead1调节静止期和增殖期之间的转换，并在维持成熟的β细胞表型时对β细胞功能质量施加关键控制。其总体目标是 通过使用体内小鼠模型和体外小鼠和人胰岛的遗传功能丧失和功能获得研究，描绘调控β细胞增殖的关键途径。我们将具体地说：1.检验Tead1是否调节成年β细胞的增殖和静止，并通过评估Tead1是否调节衰老相关的β细胞增殖的静止和下降来剖析潜在的机制。我们将验证我们的假设，即p16是Tead1介导β细胞中与年龄相关的增殖性衰老的直接靶点。2.检测β细胞功能是否需要Tead1，并通过评估Tead1缺陷的β细胞在Pdx1、MafA和Nkx6.1等调控基因中的变化，以及鉴定Tead1调控β细胞功能的直接转录靶点，确定其在β细胞中的分子靶点。我们还将测试去分化是否在β细胞功能丧失中发挥作用 团队1删除。我们将测试Tead1共激活因子YAP/TAZ及其共抑制因子Vgl14是否调节β细胞的增殖和功能。3.通过评估Tead1是否调节分离的人胰岛β细胞的增殖，以及这种调节作用在糖尿病中是否受损，来确定Tead1和哺乳动物河马途径在人类胰岛增殖和功能中的作用。总而言之，拟议的研究将关键地讨论Tead1如何调控β细胞的增殖 并发现β-细胞中调控细胞周期的新途径。我们预计，这项研究的结果将导致发现有针对性的治疗方法来调节β细胞的增殖，以保护在抗击糖尿病中恢复功能的β细胞群。随着退伍军人糖尿病发病率的显著上升，迫切需要开发新的疗法来扭转功能性β细胞质量的下降，拟议中的实验的成功完成将满足这一关键需求。

 DESCRIPTION (provided by applicant): Diabetes is one of the leading causes of morbidity and mortality in the veteran population. A failure to increase the β-cell proliferation and functional β-cell mass in response to increasing metabolic demand from insulin resistance associated with obesity and aging underlies most causes of adult onset diabetes in veterans. It is, therefore, imperative to better understand the regulation of β-cell proliferation, so that it may be targeted for therapeutic purposes to cure diabetes. While Tead1, a critical transcriptional effector of the mammalian Hippo pathway, has been shown to increase cellular proliferation in many tissues, its role in β-cell proliferation is unknown. In preliminary studies, we demonstrate that Tead1 is robustly expressed in mouse and human islets and has a non-redundant role in regulating β-cell proliferation. While Yap and Taz, co-activators of Tead1, translocate to the nucleus in hyperplastic islets, β-cell specific Tead1 deletion leads to diabetes and glucose intolerance, secondary to a loss of glucose stimulated insulin secretion in vivo. Tead1-null islets display a decrease in expression of mature β-cell markers and a surprising increase in β-cell proliferation. This increase in β-cell proliferation occurs even in β-cells of aged mice that are normally quiescent in controls. Global transcriptome analysis from Tead1-null islets reveals a significant differential expression of genes that regulate cell cycle with a significant decrease in expression of p16 (Ink4a/Cdkn2a), a critical cell cycle inhibitor. Tead1 also binds directly to the promoter of p16, suggesting it may e a direct transcriptional target of Tead1 in β- cells and mediates the Tead1 regulation of cell cycle. In addition, Tead1 also occupies the cis-promoter elements of critical β-cell genes, including Pdx1, MafA and Nkx6.1, while the expression of these genes is decreased in Tead1-null islets, suggesting that Tead1 may directly regulate β-cell function. Hence, we hypothesize that Tead1 regulates the switch between quiescence and proliferation and in maintaining the mature β-cell phenotype to exert critical control of β-cell functional mass. The broad goal is to delineate key pathways regulating β-cell proliferation through genetic loss- and gain-of-function studies using in vivo mouse models and ex vivo mouse and human islets. We will specifically 1. Test if Tead1 regulates adult β-cell proliferation and quiescence and dissect underlying mechanisms by assessing if Tead1 regulates aging-associated quiescence and decline in β-cell proliferation and dissect underlying mechanisms. We will test our hypothesis that p16 is a direct target of Tead1 mediating the age-associated proliferative senescence in β-cells. 2. Test if Tead1 is required for β-cell function and identify its molecular targets in β-cells by assessing changes in Tead1-deficient β-cells in genes regulating function, such as Pdx1, MafA and Nkx6.1, along with identification of direct transcriptional targets of Tead1 that regulate β-cell function. We will also test if de-differentiation plays a role in the loss of β-cell function with Tead1 deletion. We will test if Tead1 co-activators, Yap/Taz and its co-repressor, Vgll4, regulate β-cell proliferation and function. 3. Determine the role of Tead1 and the mammalian Hippo pathway in human islet proliferation and function by assessing if Tead1 regulates β-cell proliferation in isolated human islets and if this regulatory role is impaired in diabetes. Collectively the proposed studies will critically address how Tead1 regulates β-cell proliferation and identify novel pathways regulating cell cycle in β-cells. We envision that the results from ths study will lead to discovery of targeted therapies to modulate β-cell proliferation to preserve an restore functional β-cell mass in combating diabetes. With the significant rise in incidence of diabetes in veterans, there is an urgent need to develop novel therapies to reverse the decline in functional β-cell mass and successful completion of the proposed experiments will address this critical need.