Role of ER Stress in Beta Cell Apoptosis

描述(由申请人提供)：这项提案的总体目标是更好地了解导致1型胰岛素依赖型糖尿病(IDDM)α细胞凋亡的机制。虽然IDDM的自身免疫病因已经确定，但a细胞死亡信号机制还不完全清楚。内质网(ER)钙稳态破坏所致的内质网应激可导致MIN6细胞和小鼠胰岛细胞的凋亡。内质网应激与a细胞死亡之间的分子信号尚未解决。内质网应激在IDDM中的作用尚未明确。内质网应激诱导的细胞凋亡可能与半胱氨酸蛋白酶家族caspase的激活和转录因子CHOP(C/EBP同源蛋白，又称GADD153)基因表达增加有关。初步实验表明，肌内质网钙ATPase(SERCA)是内质网钙稳态的关键调节因子，在IDDM模型非肥胖糖尿病(NOD)小鼠的胰岛中受到损害。这提出了一种有趣的可能性，即糖尿病患者功能性a细胞质量的丧失与内质网应激有关。拟议的实验将：[1]确定内质网应激在a细胞中诱导的分子信号；[2]确定细胞因子是否诱导β细胞内质网应激；以及[3]确定内质网应激与NOD小鼠功能性a细胞质量损失之间的关系。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β细胞凋亡；[2]内质网应激诱导线粒体衍生的细胞死亡信号；[3]细胞毒性细胞因子扰乱β细胞内质网钙稳态，激活caspase并诱导CHOP表达；以及[4]NOD小鼠糖尿病的发展与内质网应激相关。将对MIN6细胞和小鼠胰岛细胞进行研究。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钙浓度梯度，测量caspase活性，以及应用激光捕获显微切割和实时聚合酶链式反应来监测胰岛中SERCA和CHOP的表达。拟议的研究将为调节β细胞活性的机制提供新的见解，并促进开发新的治疗策略，以保存和维持I型糖尿病患者的功能性β细胞群。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to provide better insight into mechanisms that cause a-cell apoptosis in Type 1, insulin-dependent diabetes mellitus (IDDM). Although the autoimmune etiology of IDDM is well established, a-cell death signaling mechanisms are incompletely understood. Endoplasmic reticulum (ER) stress induced by disruption of ER Ca2+ homeostasis causes apoptosis in MIN6 cells and in mouse islets of Langerhans. The molecular signals linking ER stress and a-cell death are unresolved. The role of ER stress in IDDM has not been defined. Apoptosis induced by ER stress may be related to activation of caspases, a family of cysteine proteases, and increased expression of the transcription factor CHOP (C/EBP homologous protein also known as GADD153) gene. Preliminary experiments suggest that sarcoendoplasmic reticulum calcium ATPase (SERCA), a key regulator of ER Ca2+ homeostasis, is impaired in islets from non-obese diabetic (NOD) mice, a model of IDDM. This raises an intriguing possibility that loss of functional a-cell mass in diabetes is related to ER stress. The proposed experiments will: [1] identify molecular signals induced by ER stress in a-cells; [2] determine whether cytokines induce beta-cell ER stress; and [3] define the relationship between ER stress and loss of functional a-cell mass in NOD mice. The following hypotheses will be tested: [1] ER stress activates beta-cell apoptosis via caspase- and CHOP-dependent pathways; [2] ER stress induces mitochondrially-derived cell death signals; [3] cytotoxic cytokines perturb beta-cell ER Ca2+ homeostasis, activate caspases and induce CHOP expression; and [4] development of diabetes in NOD mice is correlated with ER stress. MIN6 cells and mouse islet cells will be studied. Experimental procedures will include visualization of subcellular Ca2+ concentration gradients with genetically targeted Ca2+ biosensors, measurements of caspase activity, and application of Laser Capture Microdissection and real-time PCR to monitor SERCA and CHOP expression in islets. The proposed studies will provide new insights into mechanisms regulating beta-cell viability and facilitate development of novel therapeutic strategies to preserve and maintain functional beta-cell mass in patients with Type I diabetes.