Coordinated regulation of signaling events for insulin biosynthesis and secretion

项目描述（由申请人提供）：该项目的目标是破译控制胰岛素生物合成和分泌的分子信号机制，目前的重点是剖析Shp2酪氨酸磷酸酶在细胞内协调信号级联反应中的功能。虽然胰腺细胞衰竭是所有形式糖尿病的关键组成部分，但细胞功能障碍的分子基础尚不清楚。这主要是因为我们对细胞中介导葡萄糖和胰岛素信号的细胞质成分知之甚少。Shp2是一种细胞质酪氨酸磷酸酶，具有两个SH2结构域，参与信号通路的调节和协调。特别是，Shp2在体外已被证明可以促进胰岛素刺激的Erk激活，尽管Shp2在胰岛素信号传导中的功能的生理意义尚不清楚。在最近的研究中，我们成功地在小鼠中创建了一个条件Shp2敲除等位基因Shp2flox，这使我们能够在体内研究特定细胞类型或组织中的特定Shp2功能。我们已经在成熟细胞或Pdx1+胰腺前体细胞中产生了缺失Shp2的突变小鼠，并将对这些新型小鼠模型进行表征，以验证Shp2在调节胰岛素生物合成和分泌过程中协调和控制几种信号通路强度的工作假设。作为体内基因靶向方法的补充，我们还将使用siRNA介导的基因敲低技术来破译细胞中的分子信号机制。我们的具体目标是：1)确定Shp2在（-）细胞功能和葡萄糖稳态中的生理作用；2)解剖Shp2在（-）细胞中的作用的分子机制；3)研究Shp2在胰腺发育和（-）细胞再生中的功能。该实验的成功完成将填补我们在细胞胞质信号事件协调调节方面的知识空白，甚至可能导致在葡萄糖稳态和2型糖尿病发病机制中调节细胞功能的新范式。该项目的目标是了解细胞内控制胰岛素生物合成的信号机制，以及2型糖尿病（世界上最常见的代谢性疾病）细胞衰竭的分子基础。2型糖尿病的特征是胰腺细胞对葡萄糖的胰岛素释放缺陷和胰岛素对其靶组织的作用受损。细胞衰竭可能是由于细胞群扩张不足和/或细胞对葡萄糖反应不足引起的。现在，一个关键的问题是了解细胞感知葡萄糖和分泌胰岛素的分子信号机制。我们最初克隆了小鼠Shp2 （Syp）蛋白酪氨酸磷酸酶，该蛋白酪氨酸磷酸酶包含两个Src同源2 （SH2）结构域（Feng et al., Science 1993）。Shp2是一种细胞质酶，参与调节由生长因子、细胞因子和激素引发的信号事件。特别是，Shp2结合胰岛素受体底物（IRS）和grb2相关结合物（GAB）蛋白。一些研究小组已经在体外证明了Shp2在介导胰岛素刺激的Erk激活方面的积极作用。虽然这些研究推测了Shp2在胰岛素作用中的作用，但体内的生理证据尚未获得。为了确定Shp2在（-）细胞中的功能，我们产生了胰腺或（-）细胞中选择性删除Shp2的突变小鼠系。这些突变小鼠表现出糖耐量受损和胰岛素分泌和产生缺陷。我们的主要工作假设是，Shp2作为一个协调者，微调和整合胰岛素生物合成和分泌的多种信号。我们的初步实验数据表明，Shp2确实在促进胰岛素的生物合成和分泌中发挥关键作用，从而控制葡萄糖稳态。成功完成这三个目标的实验不仅将填补我们在(-细胞分子信号事件方面的知识空白，而且甚至可能刷新我们目前对(-细胞在葡萄糖刺激胰岛素分泌中的功能以及2型糖尿病病因的看法。

DESCRIPTION (provided by applicant): The goal of this project is to decipher molecular signaling mechanisms for control of insulin biosynthesis and secretion, and the immediate focus is on dissecting the function of Shp2 tyrosine phosphatase in orchestrating signaling cascades in ( cells. Although pancreatic ( cell failure is a critical component in all forms of diabetes, the molecular basis underlying ( cell dysfunction is poorly understood. This is mainly because that little is known for the cytoplasmic components mediating glucose and insulin signals in (-cells. Shp2 is a cytoplasmic tyrosine phosphatase with two SH2 domains that is implicated in regulation and coordination of signaling pathways. In particular, Shp2 has been shown to promote insulin-stimulated Erk activation in vitro, although the physiological significance of Shp2 function in insulin signaling is unclear. In recent studies, we have successfully created a conditional Shp2 knockout allele, Shp2flox, in mice, which allows us to investigate specific Shp2 functions in a specific cell type or tissue in vivo. We have generated mutant mice with Shp2 deleted in mature (-cells or in Pdx1+ pancreatic precursor cells, and will characterize these novel mouse models to test the working hypothesis that Shp2 acts to coordinate and control the strength of several signaling pathways in orchestrating insulin biosynthesis and secretion in (-cells. In complement with the gene targeting approach in vivo, we will also use siRNA- mediated gene knockdown technique to decipher the molecular signaling mechanisms in ( cells. Our specific aims are: 1) to determine the physiological role of Shp2 in (-cell function and glucose homeostasis; 2) to dissect the molecular mechanism for Shp2 action in (-cells; and 3) to investigate the Shp2 function in pancreatic development and (-cell regeneration. Successful completion of the proposed experiments will fill in a gap in our knowledge for coordinated regulation of cytoplasmic signaling events in (-cells, and may even lead to a new paradigm on regulation of (-cell functions in glucose homeostasis and also in pathogenesis of type 2 diabetes. PUBLIC HEALTH RELEVANCE The goal of this project is to understand the intracellular signaling mechanisms for control of insulin biosynthesis in ( cells and also the molecular basis for ( cell failure in Type 2 diabetes, the most common metabolic disease in the world. Type 2 diabetes is characterized by defective pancreatic ( cell insulin release in response to glucose and impaired insulin action on its target tissues. (-cell failure is likely caused by inadequate expansion of the (-cell mass and/or failure of the (-cells to respond to glucose. Now, a crucial issue is to understand the molecular signaling scheme for (-cell sensing of glucose and secretion of insulin. We originally cloned murine Shp2 (Syp) as a protein tyrosine phosphatase that contains two Src homology 2 (SH2) domains (Feng et al., Science 1993). Shp2 is a cytoplasmic enzyme and has been implicated in regulation of signaling events triggered by growth factors, cytokines and hormones. In particular, Shp2 binds the insulin receptor substrate (IRS) and Grb2-associated binder (GAB) proteins. Several groups have demonstrated a positive effect of Shp2 in mediating insulin-stimulated Erk activation in vitro. While these studies suggest a putative role of Shp2 in insulin action, the physiological evidence in vivo is yet to be obtained. To determine Shp2 function in (-cells, we have generated mutant mouse lines with Shp2 selectively deleted in the pancreas or (-cells. These mutant mice displayed impaired glucose tolerance and defective insulin secretion and production. Our central working hypothesis is that Shp2 acts as a coordinator to fine-tune and integrate multiple signals for insulin biosynthesis and secretion in (-cells. Our preliminary experimental data suggest that Shp2 indeed has a critical role in promoting insulin biosynthesis and secretion in (-cells for control of glucose homeostasis. Successful completion of the proposed experiments in these three Aims will not only fill in a gap in our knowledge in molecular signaling events in (- cells, but may even refresh our current view on (-cell functions in glucose-stimulated insulin secretion and also on the etiology of type 2 diabetes.