Regulation of TGF-Beta Activity in the Lung by LTBP-4

描述（由申请人提供）：转化生长因子- β （TGF-ß）作为三方潜伏复合物的一部分从细胞中释放，该复合物除TGF-ß外，还包括潜伏期相关蛋白（LAP）和潜伏TGF-ß结合蛋白（LTBP）， LTBP与LAP二硫键结合。我们通过生成Ltbp4-/-逆转了LTBP-4缺失小鼠中受损的终末肺泡发育表型；Tgfb2-/-小鼠，从而降低TGF-ß水平。提示Ltbp4-/-动物肺隔缺损与TGF-ß2水平升高有关。我们提出LTBP-4主要作为弹性微纤维的组织者，多蛋白组合，其中包含纤维蛋白，纤维蛋白，弹性蛋白和ltbp，而不是作为潜在的TGF-ß的粘合剂。我们认为TGF-ß-介导的作用继发于基质异常。我们将用两个目标来检验这个假设。在Aim 1中，我们将产生LTBP-4中与LAP结合的两个半胱氨酸残基突变为丝氨酸的小鼠，使LTBP-4不能与TGF-ß结合。这些小鼠会产生Ltbp-4和TGF-ß，但不产生Ltbp-4-TGF-ß复合物。如果LTBP-4 -/-小鼠的肺泡化异常是由于缺乏LTBP-4的结构活性，这些新的突变动物应该具有正常的表型。相反，如果Ltbp-4-/-小鼠的肺缺陷与Ltbp-4结合的TGF-ß缺失有关，则突变动物会出现异常的气囊分隔。我们还将在LTBP-4的结构功能或TGF-ß水平归一化的条件下，使用LTBP-4 -/-细胞在体外验证我们的假设，并测量基质组织和活性TGF-ß水平。我们将通过加入表达WT LTBP-4或纯化LTBP-4蛋白的细胞来正常化LTBP-4的结构功能。通过对TGF-ß加入泛中和抗体，使TGF-ß水平正常化。在Aim 2中，我们将研究TGF-ß在肺部病理中的作用和来源。我们将通过产生Ltbp4-/-来表征TGF-ß对肺缺损的贡献；Tgfb1-/-小鼠并检测其表型。本实验的结果将建立Ltbp4-/-的肺表型是否正常化；Tgfb2-/-动物是由于总TGF-ß减少所致；即TGF-ß1与TGF-ß2之和，或者是针对TGF-ß2。我们还将通过使用潜在TGF-ß激活因子的特异性抑制剂或具有零突变的小鼠，确定培养细胞和/或LTBP-4缺乏的动物中潜在TGF-ß激活因子的性质。最后，我们将确定在缺乏LTBP- 4的情况下形成的过量活性TGF-ß是来自LTBP-1或LTBP-3与TGF-ß的复合物，还是来自未与LTBP结合的潜在TGF-ß。这些实验将产生重要的见解，如何潜伏TGF-ß是控制在肺和培养的肺细胞使用新的遗传和细胞方法。该结果可能提示TGF-ß在某些病理状态（如肺纤维化）中的正常化机制。

DESCRIPTION (provided by applicant): Transforming growth factor-beta (TGF-ß) is released from cells as part of a tripartite latent complex that includes, in addition to TGF-ß, the latency associated protein (LAP) and latent TGF-ß binding protein (LTBP), which is disulfide bonded to LAP. We have reversed the impaired terminal alveolar development phenotype observed in mice deficient in LTBP-4 by generating Ltbp4-/-;Tgfb2-/- mice and thereby lowering TGF-ß levels. This result suggests that the defect in lung septation in Ltbp4-/- animals is related to increased TGF-ß2 levels. We propose that LTBP-4 acts primarily as an organizer of elastic microfibrils, multi-protein assemblies, which contain fibrillins, fibulins, elastin, and LTBPs, and not as a binder of latent TGF-ß. In our view, the TGF-ß-mediated effects are secondary to abnormal matrix. We will test this hypothesis in two aims. In Aim 1, we will generate mice in which the two cysteine residues in LTBP-4 that bind to LAP are mutated to serines so that Ltbp-4 cannot bind to TGF-ß. These mice will produce Ltbp-4 and TGF-ß, but no Ltbp-4-TGF-ß complexes. If the lung alveolarization abnormality in Ltbp4-/- mice is due to the absence of the structural activity of LTBP-4, these new mutant animals should have a normal phenotype. Conversely, if the lung defect in Ltbp-4-/- mice relates to the loss of TGF-ß bound to Ltbp-4, the mutant animals will display abnormal air sac septation. We will also validate our hypothesis in vitro using Ltbp4-/- cells and measuring matrix organization and active TGF-ß levels under conditions in which either LTBP-4's structural function or TGF-ß levels are normalized. We will normalize the LTBP-4 structural function by adding either cells that express WT LTBP-4 or purified LTBP-4 protein. TGF-ß levels will be normalized by adding a pan-neutralizing antibody to TGF-ß. In Aim 2, we will examine the role and source of TGF-ß in the lung pathology. We will characterize the contribution of TGF-ß to the lung defect by producing Ltbp4-/-;Tgfb1-/- mice and examining their phenotypes. The results of this experiment will establish whether normalization of the lung phenotype in Ltbp4-/-;Tgfb2-/- animals is due to a decrease in total TGF-ß; i.e. the sum of TGF-ß1 and TGF-ß2, or is specific for TGF-ß2. We will also identify the nature of the activator of latent TGF-ß in cultured cells and/or animals deficient in LTBP-4 by using specific inhibitors of, or mice with null mutations for, latent TGF-ß activators. Finally, we will determine whether the excess active TGF-ß formed in the absence of LTBP- 4 derives from complexes of LTBP-1 or LTBP-3 with TGF-ß, or from latent TGF-ß not bound to an LTBP. These experiments will yield important insights as to how latent TGF-ß is controlled in the lung and by cultured lung cells using novel genetic and cellular approaches. The results may suggest mechanisms for normalizing TGF-ß in certain pathological states, such as lung fibrosis.