PROJECT 2: MOLECULAR DISSECTION OF GROWTH FACTORS INVOLVED IN MFS (Lynn Y. Sakai,

我们假设马凡表型的一部分是由TGFβ活性的扰动引起的。一个潜在的机制是失去了纤维蛋白-1和LTBP之间的分子相互作用，导致了TGFβ的激活。支持这一假说的是，由于转化生长因子β的异常激活，纤维蛋白-1低形态小鼠在肺间隔中显示出发育缺陷(海王星等人，2003年)。为了测试纤维蛋白-1和LTBP之间的分子相互作用是否是正常的TGFbeta信号所必需的，并确定 失去这种相互作用将导致TGFβ信号的激活，我们在特定的目标1中提出了在体外和体内取消纤维蛋白Uin-1与LTBP的结合。与纤维蛋白-1缺乏小鼠的分析相比，对纤维蛋白-1突变被设计为取消与LTBP结合的小鼠的分析，应该允许我们直接确定这一机制是否与在纤维蛋白-1缺乏的肺中观察到的激活的TGFβ表型有关。 TGFbeta信号的扰动是否通过这一机制导致了任何其他组织中的马凡样表型。在特定目标1中产生的小鼠将特别有指导意义，因为由此产生的表型将表明哪些组织受到纤维蛋白-1和LTBP之间分子相互作用的单一丧失的影响最大。除了转化生长因子β活性异常外，转化生长因子相关生长因子对信号的干扰可能在马凡综合征的发病机制中发挥重要作用。由于马凡综合征可能是由于纤维蛋白-1基因突变对由纤维蛋白-1介导的多个分子相互作用的影响，因此我们在特定的目标2中建议确定其他与纤维蛋白-1相互作用的TGFβ相关生长因子家族成员，并确定是否有已识别的配体与马凡综合征的发病机制有关。为了解决后一个问题，我们将使用免疫组织化学技术确定选定的BMPs、它们的受体及其下游信号在纤维蛋白-1缺失小鼠中的命运。从这些研究中获得的结果将为我们确定马凡综合征治疗干预的新靶点提供信息并推进我们的规划目标。

We hypothesize that a portion of the Marfan phenotype is caused by perturbation of TGFbeta activity. A potential mechanism is that loss of the molecular interaction between fibrillin-1 and LTBP results in activation of TGFbeta. Support for this hypothesis was obtained from the finding that fibrillin-1 hypomorphic mice display developmental defects in lung septation due to abnormal activation of TGFbeta (Neptune, et al., 2003). In order to test whether the molecular interaction between fibrillin-1 and LTBP is required for normal TGFbeta signaling and to determine whether loss of this interaction will result in activation of TGFbeta signaling, we propose in Specific Aim 1 to abolish binding of fibriUin-1 to LTBP both in vitro and in vivo. Analyses of mice in which a mutation in fibrillin-1 has been engineered to abolish binding to LTBP, compared to analyses of fibrillin-1 deficient mice, should allow us to directly determine whether this mechanism is responsible for the activated TGFbeta phenotype observed in the fibrillin-1 deficient lung and whether perturbation of TGFbeta signaling through this mechanism is responsible for Marfan-like phenotypes in any other tissues. The mice generated in Specific Aim 1 will be especially instructive because resulting phenotypes will indicate which tissues are most affected by the singular loss of the molecular interaction between fibrillin-1 and LTBP. In addition to dysregulated TGFbeta activity, perturbations of signaling by TGFbeta -related growth factors may play important roles in the pathogenesis of the Marfan syndrome. Since it is possible that the Marfan syndrome results from the effects of mutations in fibrillin-1 on multiple molecular interactions mediated by fibrillin-1, we propose in Specific Aim 2 to identify other TGFbeta-related growth factor family members that interact with fibrillin-1 and to determine whether any of the identified ligands are relevant to pathogenesis of the Marfan syndrome. To address the latter issue, we will determine the fates of selected BMPs, their receptors, and their downstream signals in fibrillin-1 null mice using immunohistochemical techniques. Results obtained from these investigations will inform and advance our programmatic goal of identifying novel targets for therapeutic intervention in the Marfan syndrome.