Real-Time Analyses of Apoptosis in Human Beta Cells

人类 β 细胞凋亡的实时分析

• 批准号: 6916218
• 负责人: MICHAEL WILLIAM ROE
• 金额: $ 36.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916218
• 关键词: apoptosis; biosensor device; calcium flux; cell component structure /function; cellular pathology; confocal scanning microscopy; cysteine endopeptidases; cytokine; endoplasmic reticulum; enzyme activity; fluorescence resonance energy transfer; genetic regulation; human tissue; membrane permeability; mitochondria; pancreatic islet function; pancreatic islets; postmortem; stress; time resolved data

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.

描述（由申请人提供）： 该提案的总体目标是更好地了解导致1型和2型糖尿病β细胞凋亡的机制。人类β细胞死亡信号传导机制尚未完全了解，并且对人类β细胞凋亡的早期事件知之甚少。内质网（ER）和线粒体在许多类型的细胞凋亡中起重要作用。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作为细胞执行者发挥作用。ER应激增加转录因子CHOP（C/EBP同源蛋白，也称为GADD 153）基因的表达，并激活caspase家族的特定成员。线粒体膜通透性在细胞凋亡过程中增加，并且从线粒体释放蛋白质是细胞死亡的早期和必要步骤。ER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MIN 6细胞和小鼠胰岛中的初步实验表明，由肌内质网Ca ~（2+）-ATP酶（SERCA）活性和ER Ca ~（2+）稳态的破坏诱导的ER应激引起细胞凋亡。拟议的实验将：[1]鉴定与人β细胞中ER应激和线粒体功能障碍诱导的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ER应激和线粒体膜通透性的变化;[3]定义人胰岛中ER应激和胰岛β细胞诱导的细胞死亡信号传导之间的时间和因果关系。将检验以下假设：[1] ER应激通过半胱天冬酶和CHOP依赖性途径激活人β细胞凋亡; [2] ER应激诱导神经源性细胞死亡信号;和[3]细胞毒性细胞因子扰乱β细胞ER和线粒体Ca 2+稳态，诱导促凋亡蛋白（细胞色素c和Smac/DIABLO）从线粒体释放，激活半胱天冬酶并诱导CHOP表达。将研究人类胰岛和β细胞。实验程序将包括可视化的亚细胞Ca 2+浓度梯度与遗传靶向Ca 2+生物传感器，实时测量半胱天冬酶活性与荧光生物传感器，细胞色素c和Smac/DIABLO释放，和应用实时定量PCR监测SERCA和CHOP在胰岛中的表达。拟议的研究将为调节人类β细胞活力的机制提供新的见解，并为促进我们对1型和2型糖尿病发病机制的理解提供必要的信息。