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

描述(由申请人提供)：我们的长期目标是将细胞内钙([Ca]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的可检验假说提供了理由，即“肿瘤坏死因子-a诱导细胞内钙的失调，[钙]i，激活核因子-kB转录，进而导致b细胞的凋亡。”这项建议的重点是[Ca~(2+)]i失调，作为细胞因子诱导的NF-kB激活的中介，从而导致b细胞死亡。目的1：确定细胞因子诱导的[Ca~(2+)]i失衡引起的核因子-kB的激活。在RIN细胞、人类b细胞和人类胰岛中，我们将研究TNF-a诱导的(A)通过与Fura2进行比率荧光成像来测量[Ca~(2+)]i和[Ca~(2+)]i缓冲容量来调节[Ca~(2+)]i的失调，(B)通过使用FunctionEL ISA来降解IkBA亚单位，(C)通过免疫荧光方法将核因子-kB从胞浆移位到细胞核，以及(D)通过使用ELISA法测量核因子-kB的DNA结合活性来实现核因子-kB依赖的转录。目的2：筛选b细胞中肿瘤坏死因子-α调节基因的候选基因。从对照组和经肿瘤坏死因子-a处理的人类b细胞和RIN细胞中分离出的mrna水平，编码了一组被认为参与b细胞凋亡的六个由肿瘤坏死因子-a诱导的差异表达基因，将通过半定量多重聚合酶链式反应来测量。我们将展示这六个由肿瘤坏死因子-a诱导的差异表达基因如何对人类b细胞的b细胞功能做出贡献。我们将在加入胰岛素生长因子-1(IGF-1)的情况下，检测对照组和经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：“TNF-a诱导b细胞中p38和c-jun氨基末端MAPK的激活，从而导致b细胞死亡可被IGF1减弱”。目的3：确定线粒体诱导的半胱氨酸天冬氨酸氨基转移酶(Caspase)激活对b细胞死亡的影响。我们推测“肿瘤坏死因子-a诱导的Bax和Bcl2在b细胞线粒体中的共定位将导致线粒体细胞色素c的释放，当细胞色素c释放到胞浆中时，将激活caspase-3并导致b细胞死亡”。在RIN细胞和人类B细胞中，我们将研究TNF-a诱导的：(A)结合荧光共振能量转移(FRET)的荧光标记抗体的亚细胞定位的变化以及这两种蛋白之间的空间相互作用；(B)通过免疫荧光显微镜释放细胞色素c；(C)通过免疫荧光显微镜激活caspase 3；以及(D)细胞复制、凋亡率和胰岛素分泌的变化。这三个目标下的拟议研究结果应该为1型和2型糖尿病患者由于[钙]i失调而导致的b细胞死亡或存活中的细胞信号和下游效应因子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，为青少年和迟发性糖尿病设计有效的治疗策略和干预措施。 公共卫生相关性：糖尿病是一种复杂的不治之症，通过补充胰岛素进行治疗。我们的创新研究将提供对导致胰岛素分泌细胞死亡的分子和生理机制的见解，以及防止数百万美国人最终导致1型和2型糖尿病的β细胞死亡的新策略，以避免他们对每日注射胰岛素的依赖。

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.