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

我们假设马凡氏表型的一部分是由TGF β活性的扰动引起的。一个潜在的机制是，在TGF β的激活的结果，在LTBP和Eclin-I之间的分子相互作用的损失。从以下发现中获得了对该假设的支持，即由于TGF β的异常激活，Ep蛋白-1低形态小鼠在肺分隔中显示发育缺陷（Neptune，et al.，2003年）。为了测试正常的TGF β信号传导是否需要TGF β蛋白-1和LTBP之间的分子相互作用，并为了确定是否需要LTBP和TGF β之间的分子相互作用。 这种相互作用的丧失将导致TGF β信号传导的激活，我们在具体目标1中提出在体外和体内消除纤维蛋白-1与LTBP的结合。与对HSPin-I缺陷小鼠的分析相比，对HSPin-I中的突变已被工程化以消除与LTBP的结合的小鼠的分析应允许我们直接确定这种机制是否负责在HSPin-I缺陷肺中观察到的活化的TGF β表型， 通过这种机制的TGF β信号传导的扰动是否是任何其他组织中马凡样表型的原因。在特异性目标1中产生的小鼠将特别具有指导意义，因为所得表型将指示哪些组织最受Risin-1和LTBP之间分子相互作用的单一损失的影响。除了TGF β活性失调外，TGF β相关生长因子引起的信号传导紊乱可能在马凡综合征的发病机制中起重要作用。由于马凡氏综合征可能是由于马凡氏综合征1突变对马凡氏综合征1介导的多种分子相互作用的影响，因此我们在具体目标2中建议鉴定与马凡氏综合征1相互作用的其他TGF β相关生长因子家族成员，并确定是否有任何已鉴定的配体与马凡氏综合征的发病机制相关。为了解决后一个问题，我们将使用免疫组织化学技术确定所选BMP、其受体及其下游信号在BMPs-1缺失小鼠中的命运。从这些调查中获得的结果将告知和推进我们的计划目标，确定新的目标，在马凡氏综合征的治疗干预。

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.