Real-Time Analyses of Apoptosis in Human Beta Cells

描述(由申请人提供)： 这项建议的总体目标是更好地洞察导致1型和2型糖尿病患者β细胞凋亡的机制。人类β细胞死亡的信号机制还不完全清楚，对人类β细胞凋亡的早期事件也知之甚少。内质网(ER)和线粒体在多种细胞的凋亡中起着重要的作用。细胞凋亡与半胱氨酸蛋白酶家族的激活有关，其中许多半胱氨酸蛋白酶起着细胞执行者的作用。内质网应激增加转录因子CHOP(C/EBP同源蛋白，也称为GADD153)基因的表达，并激活caspase家族的特定成员。在细胞凋亡过程中，线粒体膜通透性增加，线粒体释放蛋白质是细胞死亡的早期和必要步骤。内质网应激、线粒体功能和人类β细胞死亡之间的时间和因果关系尚不清楚。我们在MIN6细胞和小鼠胰岛上的初步实验表明，肌内质网钙-ATPase(SERCA)活性和内质网钙稳态的破坏所引起的内质网应激可导致细胞凋亡。拟议的实验将：[1]确定与内质网应激和线粒体功能障碍诱导的人β细胞凋亡相关的分子信号；[2]确定促炎细胞因子是否诱导人β细胞内质网应激和线粒体膜通透性的变化；以及[3]确定内质网应激和线粒体诱导的朗格汉斯胰岛细胞死亡信号之间的时间和因果关系。将检验以下假设：[1]内质网应激通过caspase和CHOP依赖的途径激活人β细胞的凋亡；[2]内质网应激诱导线粒体衍生的细胞死亡信号；以及[3]细胞毒性细胞因子扰乱β细胞内质网和线粒体钙稳态，诱导线粒体释放促凋亡蛋白(细胞色素c和Smac/Diablo)，激活半胱氨酸酶并诱导CHOP表达。人类的胰岛和β细胞将被研究。实验程序将包括用基因靶向的钙离子生物传感器显示亚细胞内钙浓度梯度，用荧光生物传感器实时测量caspase活性，细胞色素c和Smac/Diablo的释放，以及应用实时定量聚合酶链式反应来监测胰岛SERCA和CHOP的表达。拟议的研究将为调节人类β细胞活性的机制提供新的见解，并提供必要的信息，以促进我们对1型和2型糖尿病发病机制的理解。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to provide better insight into mechanisms that cause beta-cell apoptosis in Type 1 and Type 2 diabetes mellitus. Human beta-cell death signaling mechanisms are incompletely understood and very little is known about early events in human beta-cell apoptosis. The endoplasmic reticulum (ER) and mitochondria play essential roles in apoptosis in many types of cells. Apoptosis is associated with activation of caspases, a family of cysteine proteases, many of which function as cell executioners. ER stress increases expression of the transcription factor CHOP (C/EBP homologous protein also known as GADD153) gene and activates specific members of the caspase family. Mitochondria membrane permeability is increased during apoptosis and release of proteins from mitochondria is an early and necessary step in cell death. The temporal and causal interrelationships between ER stress, mitochondria function, and human beta-cell death are not understood. Our preliminary experiments in MIN6 cells and in mouse islets of Langerhans indicate that ER stress induced by disruption of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity and ER Ca2+ homeostasis causes apoptosis. The proposed experiments will: [1] identify molecular signals associated with apoptosis induced by ER stress and mitochondria dysfunction in human beta-cells; [2] determine whether proinflammatory cytokines induce human beta-cell ER stress and changes in mitochondrial membrane permeability; and [3] define the temporal and causal interrelationships between ER stress and mitochondria-induced cell death signaling in human islets of Langerhans. The following hypotheses will be tested: [1] ER stress activates human beta-cell apoptosis via caspase- and CHOP-dependent pathways; [2] ER stress induces mitochondrially-derived cell death signals; and [3] cytotoxic cytokines perturb beta-cell ER and mitochondrial Ca2+ homeostasis, induce release of pro-apototic proteins (cytochrome c and Smac/DIABLO) from mitochondria, activate caspases and induce CHOP expression. Human islets and beta-cells will be studied. Experimental procedures will include visualization of subcellular Ca2+ concentration gradients with genetically targeted Ca2+ biosensors, real-time measurements of caspase activity with fluorescent biosensors, cytochrome c and Smac/DIABLO release, and application of real-time quantitative PCR to monitor SERCA and CHOP expression in islets. The proposed studies will provide new insights into mechanisms regulating human beta-cell viability and information essential in advancing our understanding of the pathogenesis of both Type 1 and Type 2 diabetes mellitus.