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-¿）作为三方潜伏复合物的一部分从细胞中释放，该复合物除了TGF-¿外，还包括潜伏期相关蛋白（LAP）和潜伏TGF-¿结合蛋白（LTBP），后者与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-¿水平。本结果提示Ltbp4-/-动物肺隔缺损与TGF-¿2水平升高有关。我们认为LTBP-4主要作为弹性微原纤维的组织者，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TGF-¿的粘合剂。我们认为TGF-¿-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-¿结合。这些小鼠会产生Ltbp-4和TGF-¿，但不会产生Ltbp-4-TGF-¿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-¿缺失有关，则突变动物将表现出异常的气囊分隔。我们还将在体外使用LTBP-4 -/-细胞验证我们的假设，并在LTBP-4的结构功能或TGF-¿水平归一化的条件下测量基质组织和活性TGF-¿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。通过向TGF-¿中加入泛中和抗体，TGF-¿水平将正常化。在Aim 2中，我们将探讨TGF-¿在肺部病理中的作用和来源。我们将通过产生Ltbp4-/-来表征TGF-¿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；动物Tgfb2-/-是由于总TGF-¿减少所致；即TGF-¿1与TGF-¿2之和，或仅针对TGF-¿2。我们还将通过使用潜在TGF-¿激活剂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-¿激活剂的性质。最后，我们将确定在缺乏LTBP- 4的情况下形成的过量活性TGF-¿是来自LTBP-1或LTBP-3与TGF-¿的复合物，还是来自未与LTBP结合的潜在TGF-¿。这些实验将产生重要的见解，关于潜伏TGF-¿是如何在肺和培养的肺细胞中使用新的遗传和细胞方法控制的。该结果可能提示TGF-¿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-¿) is released from cells as part of a tripartite latent complex that includes, in addition to TGF-¿, the latency associated protein (LAP) and latent TGF-¿ 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-¿ levels. This result suggests that the defect in lung septation in Ltbp4-/- animals is related to increased TGF-¿2 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-¿. In our view, the TGF-¿-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-¿. These mice will produce Ltbp-4 and TGF-¿, but no Ltbp-4-TGF-¿ 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-¿ 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-¿ levels under conditions in which either LTBP-4's structural function or TGF-¿ 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-¿ levels will be normalized by adding a pan-neutralizing antibody to TGF-¿. In Aim 2, we will examine the role and source of TGF- ¿ in the lung pathology. We will characterize the contribution of TGF-¿ 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-¿; i.e. the sum of TGF-¿1 and TGF-¿2, or is specific for TGF-¿2. We will also identify the nature of the activator of latent TGF-¿ in cultured cells and/or animals deficient in LTBP-4 by using specific inhibitors of, or mice with null mutations for, latent TGF-¿ activators. Finally, we will determine whether the excess active TGF-¿ formed in the absence of LTBP- 4 derives from complexes of LTBP-1 or LTBP-3 with TGF-¿, or from latent TGF-¿ not bound to an LTBP. These experiments will yield important insights as to how latent TGF-¿ is controlled in the lung and by cultured lung cells using novel genetic and cellular approaches. The results may suggest mechanisms for normalizing TGF-¿ in certain pathological states, such as lung fibrosis.