CAVEOLIN-3 AND MUSCULAR DYSTROPHY

这项建议的长期目标是了解肌肉小窝和小窝蛋白- 3i在正常肌肉发育中的作用；ii)肌肉萎缩的发病机制。小泡是细胞表面的“小洞”。有人提出，小泡在信号转导过程中起着“信息中心”的作用。小窝蛋白-3是一种肌肉特异性的小窝蛋白相关蛋白，是横纹肌细胞类型（心脏和骨骼）小窝膜结构域的主要结构蛋白。最近，我们在人类中发现了一种新的常染色体显性肢体带状肌营养不良（LGMD-1C），这是由于人类小洞蛋白-3基因（3p25）编码序列的突变引起的。本研究的目的是验证小窝蛋白-3表达对正常肌肉发育很重要的假设，以及小窝蛋白-3表达的变化（上调或下调）可导致肌肉营养不良表型。为了验证这一假设，我们将使用多种互补的体内方法，例如在培养细胞中使用caveolin-3反感官和开发小鼠动物模型。该项目的具体目的是：1)确定caveolin-3突变在LGMD- 1C发病机制中的作用。我们将研究在NIH 3T3细胞中异种表达caveolin-3后LGMD-1C突变的表型行为，并与野生型caveolin-3进行比较；2)建立野生型caveolin-3和LGMD-1C突变型caveolin-3的转基因小鼠模型。我们将在小鼠中过度表达野生型和LGMD-1C突变型的小窝蛋白- 3作为转基因，并评估它们对骨骼肌的影响。由于在杜氏肌营养不良症中，caveolin-3水平上调，这些实验将帮助我们评估caveolin-3的上调是否参与了该疾病的发病机制；3)检测caveolin-3的表达是否为正常肌肉发育所必需。使用反义方法，我们将消除C2C12细胞（一种在培养中分化的骨骼肌成细胞系）中caveolin-3的表达。我们将评估下调小室蛋白-3对C2C12成肌细胞融合和肌管形成的影响。此外，通过靶向基因破坏方法，我们将创建并表征缺乏caveolin-3基因表达的“敲除”小鼠。这些研究将为理解肌细胞小泡在正常肌肉发育和肌肉萎缩中的作用提供基础知识。

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.