MOLECULAR MECHANISMS CONTROLLING PROGRAMMED CELL DEATH

控制程序性细胞死亡的分子机制

• 批准号: 6417280
• 负责人: LAWRENCE M SCHWARTZ
• 金额: $ 4.74万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6417280
• 关键词: Drosophilidae; Manduca; antisense nucleic acid; apoptosis; cell differentiation; developmental genetics; focal adhesion kinase; gene mutation; gene targeting; genetically modified animals; immunocytochemistry; laboratory mouse; myoblasts; myogenesis; paxillin; regulatory gene; site directed mutagenesis; transcription factor

DESCRIPTION (adapted from investigator's abstract): During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal transduction molecules that determine if myoblasts should differentiate or die are largely unknown. In order to analyze the molecular mechanisms that mediate programmed cell death during development, we have cloned death-associated genes from the intersegmental muscles (ISMs) of the moth Manduca sexta. One of these genes encodes Death-Associated LIM-Only Protein (DALP). Forced expression of DALP in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian paralog Hic-5, blocks differentiation and induces apoptosis in mouse C2Ct2 myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die following removal of trophic support. Taken together, these data suggest DALP and Hic-5 act upstream of MyoD and function as phylogenetically-conserved "switches" to block muscle differentiation and induce death. Hic-5 shares high sequence identity with the focal adhesion protein paxillin. In the first aim of the proposal, they will test the hypothesis that Hic-5 acts in myoblasts to block focal adhesion kinase-dependent phosphorylation of paxillin. In the second aim, they use site-directed mutagenesis to identify essential motifs within Hic-5 and to generate possible dominant-negative regulators. In the third aim, they will test the hypothesis that blockade of the Hic-5 pathway enhances the survival of transplanted myoblasts in vivo. Lastly, they will target the expression of wild-type and dominant-negative (or antisense) Hic-5 to the muscle lineage of transgenic mice in order to determine its role in myogenesis. They hope to exploit Hic-5 in order to: 1) understand how myoblasts makes the decision to differentiate or die, and 2) develop strategies to enhance the utility of myoblast based transplantation strategies.

描述（改编自研究者的摘要）：在肌生成过程中， 营养因子可用性的减少表明大多数成肌细胞融合， 上调肌肉特异性基因的表达并形成肌管。那些 未能分化成肌管的细胞启动细胞凋亡并迅速 死。目前，决定成肌细胞是否存在的信号转导分子 应该分化还是死亡很大程度上是未知的。为了分析 在发育过程中介导程序性细胞死亡的分子机制，我们 从节间肌（ISM）中克隆了与死亡相关的基因 曼杜卡六角蛾。这些基因之一编码仅与死亡相关的 LIM 蛋白质（DALP）。 DALP在果蝇中的强制表达导致骨骼 肌肉萎缩。 DALP 或其哺乳动物旁系同源物 Hic-5 的异位表达， 阻断小鼠 C2Ct2 成肌细胞的分化并诱导细胞凋亡。两者都 这些影响可以通过与正常成肌细胞接触或通过异位成肌细胞来克服 肌肉特异性转录因子 MyoD 的表达。 Hic-5表达 在正常成肌细胞中被特异性且显着地诱导，这些成肌细胞随后死亡 去除营养支持。综合起来，这些数据表明 DALP 和 Hic-5 作用于 MyoD 的上游，并作为系统发育保守的“开关”发挥作用 阻断肌肉分化并诱导死亡。 Hic-5 共享高序列 与粘着斑蛋白桩蛋白的同一性。在第一个目标中 提案中，他们将测试 Hic-5 在成肌细胞中发挥作用以阻断这一假设 桩蛋白的粘着斑激酶依赖性磷酸化。在第二个目标中， 他们使用定点诱变来识别 Hic-5 中的基本基序 并产生可能的显性负调节因子。在第三个目标中，他们 将检验阻断 Hic-5 通路可增强 移植的成肌细胞在体内的存活率。最后，他们将瞄准 野生型和显性失活（或反义）Hic-5 的表达 转基因小鼠的肌肉谱系，以确定其在肌生成中的作用。 他们希望利用 Hic-5 来：1）了解成肌细胞如何产生 决定差异化或死亡，2）制定策略以增强 基于成肌细胞的移植策略的效用。