MOLECULAR MECHANISMS CONTROLLING PROGRAMMED CELL DEATH

描述（改编自研究者摘要）：在肌肉发生期间， 营养因子可用性的降低发出信号通知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 不能分化成肌管的细胞启动凋亡并迅速 死的目前，决定成肌细胞 应该分化还是死亡在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 已经克隆了来自于猪的节间肌肉（ISM）的死亡相关基因， 天蛾这些基因之一编码死亡相关的LIM-仅 蛋白质（DALP）。DALP在果蝇中的强制表达导致骨骼肌的骨骼肌化。 肌肉萎缩DALP或其哺乳动物paraplasia Hic-5的异位表达， 阻断小鼠C2Ct 2成肌细胞的分化并诱导凋亡。两 这些作用可以通过与正常成肌细胞接触或通过异位成肌细胞的作用来克服。 肌肉特异性转录因子MyoD的表达。Hic-5表达 在正常的成肌细胞中特异性地显著诱导， 去除营养支持。综上所述，这些数据表明DALP和Hic-5 作用于MyoD的上游，并作为遗传上保守的“开关”发挥作用， 阻止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相同。在第一个目标中， 他们将测试Hic-5在成肌细胞中起作用以阻断 粘着斑激酶依赖的桩蛋白磷酸化。第二个目标， 他们使用定点突变来鉴定Hic-5中的基本基序 并产生可能的显性负调节子。第三个目标，他们 将检验阻断Hic-5通路可增强 移植成肌细胞在体内的存活。最后，他们将针对 表达野生型和显性阴性（或反义）Hic-5， 肌肉谱系的转基因小鼠，以确定其在肌肉发生中的作用。 他们希望利用Hic-5来：1）了解成肌细胞是如何使 决定差异化或死亡，2）制定战略，以提高 基于成肌细胞的移植策略的效用。

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.