CAVEOLIN-3 AND MUSCULAR DYSTROPHY

这项建议的长期目标是了解肌小窝和小窝-3在正常肌肉发育中的作用；以及ii)在肌肉营养不良的发病机制中的作用。小窝是细胞表面的“小洞穴”。有人提出小窝作为“信息中心”来调节信号转导。小窝蛋白-3是一种肌肉特异性小窝蛋白相关蛋白，是横纹肌细胞(心脏和骨骼)小窝膜域的主要结构蛋白。最近，我们在人类中发现了一种新的常染色体显性遗传型肢体带状肌营养不良症(LGMD-1C)，其原因是人类小窝蛋白-3基因(3p25)的编码序列发生突变。这一建议的目的是验证一种假设，即小窝蛋白-3的表达对正常肌肉发育很重要，并且小窝蛋白-3表达的变化(上调或下调)可以导致肌营养不良表型。为了验证这一假说，我们将使用多种体内互补的方法，如在培养细胞中使用小窝蛋白-3反义抗体和发展小鼠动物模型。该项目的具体目标是：1)确定小窝蛋白-3突变在LGMD-1C发病机制中的作用。我们将检测小凹蛋白-3的LGMD-1C突变在NIH 3T3细胞中异源表达后的表型行为，并与野生型小凹蛋白-3进行比较；2)建立覆盖野生型小窝蛋白-3和LGMD-1C突变的小凹蛋白-3的转基因小鼠模型。我们将在小鼠中过量表达野生型和LGMD-1C突变形式的小窝蛋白-3作为转基因，并评估它们对骨骼肌的影响。由于小窝蛋白-3在Duchenne‘s肌营养不良症中表达上调，这些实验将帮助我们评估小窝蛋白-3的上调是否在该疾病的发病机制中起作用；以及3)检测小窝蛋白-3的表达是否是正常肌肉发育所必需的。使用反义方法，我们将取消小窝蛋白-3在C2C12细胞中的表达，C2C12细胞是一种在培养中分化的骨骼肌母细胞系。然后，我们将评估小窝蛋白-3下调对C2C12成肌细胞融合和肌管形成的影响。此外，通过一种有针对性的基因破坏方法，我们将创造并鉴定缺乏小窝蛋白-3基因表达的“敲除”小鼠。预计这些研究将有助于了解肌肉细胞小窝在正常肌肉发育和肌营养不良症中的作用。

The long-term objective of this proposal is to understand the role of muscle caveolae and caveolin-3 i) in normal muscle development; and ii) in the pathogenesis of muscle dystrophy. Caveolae are "little caves" at the surface of cells. It has been proposed that caveolae function as message centers" for regulating signal transduction. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Recently, we identified a novel autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). The aim of this proposal is to test the hypothesis that caveolin-3 expression is important for normal muscle development and that changes in caveolin-3 expression (either up-regulation or down-regulation) can result in muscular dystrophy phenotype. In order to test this hypothesis,, we will use a variety of complementary in vivo approaches, such as the use of caveolin-3 anti-senses in cultured cells and the development of mouse animal models. The specific aims of the project are: 1) To determine the role of caveolin-3 mutations in the pathogenesis of LGMD- 1C. We will examine the phenotypic behavior of LGMD-1C mutations of caveolin-3 after heterologous expression in NIH 3T3 cells, as compared with wild-type caveolin-3; 2) To develop transgenic mouse models that over wild-type caveolin-3 and LGMD-1C mutant forms of caveolin-3. We will over-express wild type and LGMD-1C mutant forms of caveolin- 3 as transgenes in mice and assess their effects on skeletal muscle. As caveolin-3 levels are up-regulated in Duchenne's muscular dystrophy, these experiments will help us evaluate if caveolin-3 up-regulation contributes to the pathogenesis of this diseases; and 3) To examine if caveolin-3 expression is required for normal muscle development. Using an anti-sense approach, we will abrogate caveolin-3 expression in C2C12 cells, a skeletal myoblast cell line that differentiates in culture. We will then assess the effects of caveolin-3 down-regulation on C2C12 myoblast fusion and myotube formation. In addition, through a targeted gene disruption approach, we will create and characterize "knock-out" mice that lack caveolin-3 gene expression. It is expected that these studies will contribute fundamen6tal knowledge toward understanding the role of muscle cell caveolae in normal muscle development and muscular dystrophy.