Cytokines dysregulate intracellular calcium and cause beta-cell death in diabetes

描述（由申请人提供）：我们的长期目标是表征细胞内钙（[Ca2+]i）稳态的变化作为细胞因子诱导的糖尿病b细胞功能障碍的中介。我们有充分和令人信服的证据为我们可测试的假设提供了基本原理，即“tnf -a诱导的细胞内钙[Ca2+]i失调，激活NF-kB转录，进而导致b细胞凋亡。”本提案的重点是[Ca2+]i失调作为细胞因子诱导的NF-kB激活的介质，导致b细胞死亡。三个具体目的是：目的1：确定细胞因子诱导的[Ca2+]i失调引起的NF-kB活化。在RIN细胞、人b细胞和人胰岛中，我们将研究tnf -a诱导的(a) [Ca2+]i的失调，通过使用Fura2的比例荧光成像测量[Ca2+]i和[Ca2+]i缓冲能力，(b)使用FunctionELISA检测IkBa亚基的降解，(c)通过免疫荧光检测NF-kB从细胞质到细胞核的易位，以及(d)通过使用ELISA检测NF-kB的DNA结合活性来检测NF-kB依赖性转录。目的2：筛选b细胞中TNF-a调控基因的候选基因。mRNA的水平，从对照和tnf -a处理的人b细胞和RIN细胞中分离出来，编码6组tnf -a诱导的差异表达基因，被认为参与b细胞凋亡，将通过半定量多重PCR来测量。我们将展示这六种tnf -a诱导的差异表达基因如何在人类b细胞中促进b细胞功能。我们将在添加胰岛素生长因子-1 （IGF-1）的情况下，测量对照和tnf -a处理的RIN细胞和人b细胞中磷酸化JNK和磷酸化p38的水平，以验证我们的假设，即“tnf -a诱导的b细胞中p38和c-JUN N-末端MAP激酶的激活导致b细胞死亡可以被IGF1减弱”。目的3：通过线粒体诱导的半胱天冬酶激活确定b细胞死亡的执行情况。我们假设“TNF-a诱导的b细胞线粒体中Bax与Bcl-2的共定位将导致线粒体细胞色素c的释放，当其释放到细胞质中时，将激活caspase-3并导致b细胞死亡”。在RIN细胞和人b细胞中，我们将研究tnf -a诱导的：(a) Bcl-2和Bax亚细胞定位的变化以及这两种蛋白之间的空间相互作用，使用荧光标记抗体结合荧光共振能量转移（FRET）方案；(b)免疫荧光显微镜观察细胞色素c的释放；(c)免疫荧光显微镜观察caspase 3的活化，(d)细胞复制、凋亡率和胰岛素分泌的变化。在三个AIMS下提出的研究结果应该为1型和2型糖尿病中由于[Ca2+]i失调而导致的b细胞死亡或存活的细胞信号和下游效应物的相互作用提供新的机制见解。我们的实验设计将确定关键的靶分子和细胞步骤，为设计有效的治疗策略和干预措施设计青少年和晚发性糖尿病。

DESCRIPTION (provided by applicant): Our long-term goal is to characterize the changes in intracellular calcium ([Ca2+]i) homeostasis as a mediator of cytokine-induced b-cell dysfunction in diabetes. We have sufficient and compelling evidence providing the rationale for our testable hypothesis that "TNF-a-induced dysregulation of intracellular calcium, [Ca2+]i, activates NF-kB transcription that in turn leads to b-cell apoptosis." The focus of this proposal is on [Ca2+]i dysregulation as a mediator of cytokine-induced NF-kB activation that leads to b-cell death. Three specific aims are: Aim 1: Determine the activation of NF-kB caused by cytokine-induced [Ca2+]i dysregulation. In RIN cells, human b-cells, and human islets, we will study TNF-a-induced (a) dysregulation of [Ca2+]i by measuring [Ca2+]i and [Ca2+]i buffering capacity using ratiometric fluorescence imaging with Fura2, (b) degradation of the IkBa subunit by using a FunctionELISA, (c) translocation of NF-kB from cytoplasm to nucleus by immunofluorescence, and (d) NF-kB-dependent transcription by measuring the DNA binding activity of NF-kB using ELISA. Aim 2: Characterization of candidate gene screen of TNF-a regulated genes in b-cells. The levels of mRNA, isolated from control and TNF-a-treated human b-cells and RIN cells, encoding a panel of six TNF-a-induced differentially expressed genes thought to participate in b-cell apoptosis will be measured by semiquantitative multiplex PCR. We will show how these six TNF-a-induced differentially expressed genes contribute to b-cell function in human b-cells. We will measure the levels of phosphorylated JNK and phosphorylated-p38 in control and TNF-a-treated RIN cells and human b-cells in presence of added insulin growth factor-1 (IGF-1) to test our hypothesis that "TNF-a-induced activation of p38 and c-JUN N- terminal MAP kinases in b-cells that leads to b-cells death could be attenuated by IGF1". Aim 3: Determine the execution of b-cell death by mitochondria-induced caspase activation. We hypothesize that "TNF-a- induced co-localization of Bax with Bcl-2 in mitochondria in b-cells will result in release of mitochondrial cytochrome c, which, when liberated to the cytosol, will activate caspase-3 and execute b-cell death". In RIN cells and human b-cells, we will study the TNF-a-induced: (a) changes in subcellular localizations of Bcl-2 and Bax and the spatial interaction between these two proteins by using fluorescent-labeled antibodies in conjunction with fluorescence resonance energy transfer (FRET) protocols; (b) release of cytochrome c by using immunofluorescence microscopy; (c) activation of caspase 3 by using immunofluorescence microscopy, and (d) changes in cell replication, apoptotic rates, and insulin secretion. The results of the proposed studies under the three AIMS should provide novel mechanistic insights into the cellular signals and interplay of downstream effectors in b-cell death or survival due to [Ca2+]i dysregulation in type 1 and type 2 diabetes. Our experimental design will identify critical target molecules and cellular steps for designing efficacious therapeutic strategies and interventions for juvenile and late onset diabetes. PUBLIC HEALTH RELEVANCE: Diabetes, a complex and incurable disease is managed by insulin supplementation. Our innovative studies will provide insights into molecular and physiological mechanisms that cause the death of insulin secreting pancreatic cells, and novel strategies to prevent beta cell death that culminates in diabetes type 1 and 2 in millions of Americans to avert their dependence on daily injections of insulin.