CAVEOLIN-3 AND MUSCULAR DYSTROPHY

CAVEOLIN-3 和肌营养不良症

• 批准号: 6087881
• 负责人: MICHAEL P LISANTI
• 金额: $ 32.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6087881
• 关键词: 3T3 cells; antisense nucleic acid; biological signal transduction; caveolas; caveolins; cellular pathology; disease /disorder model; gene expression; gene mutation; gene targeting; genetically modified animals; laboratory mouse; molecular pathology; muscle cells; muscular dystrophy; myogenesis; tissue /cell culture

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.

该提案的长期目标是了解肌肉小窝和小窝蛋白-3 i）在正常肌肉发育中的作用;和ii）在肌肉营养不良的发病机制中。小窝是细胞表面的“小洞穴”。有人认为小窝是调节信号转导的信息中心。Caveolin-3是一种肌肉特异性Caveolin相关蛋白，是横纹肌细胞类型（心肌和骨骼肌）中Caveolae膜结构域的主要结构蛋白。最近，我们发现了一种新的常染色体显性形式的肢带型肌营养不良症（LGMD-1C）在人类，这是由于突变的编码序列的人小窝蛋白-3基因（3 p25）。该建议的目的是检验以下假设：小窝蛋白-3表达对于正常肌肉发育是重要的，并且小窝蛋白-3表达的变化（上调或下调）可导致肌营养不良表型。为了验证这一假设，我们将使用各种互补的体内方法，例如在培养细胞中使用caveolin-3反义和开发小鼠动物模型。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。我们将研究caveolin-3的LGMD-1C突变体在NIH 3 T3细胞中异源表达后的表型行为，并与野生型caveolin-3进行比较; 2）建立转基因小鼠模型，以超过野生型caveolin-3和caveolin-3的LGMD-1C突变体形式。我们将过度表达野生型和LGMD-1C突变形式的小窝蛋白-3作为转基因小鼠，并评估其对骨骼肌的影响。由于窖蛋白-3水平在杜氏肌营养不良症中上调，这些实验将帮助我们评估窖蛋白-3上调是否有助于这种疾病的发病机制;以及3）检查窖蛋白-3表达是否是正常肌肉发育所需的。使用反义方法，我们将废除小窝蛋白-3在C2 C12细胞，骨骼肌成肌细胞系，在文化中分化的表达。然后，我们将评估小窝蛋白-3下调对C2 C12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，我们将创建和表征缺乏小窝蛋白-3基因表达的“敲除”小鼠。这些研究将有助于了解肌细胞小窝在正常肌肉发育和肌营养不良中的作用。