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

描述(申请人提供)：转化生长因子-β(转化生长因子-β)作为三方潜伏复合体的一部分从细胞中释放，除转化生长因子-β外，该复合体还包括潜伏期相关蛋白(LAP)和潜伏的转化生长因子-β结合蛋白(LTBP)，后者与LAP二硫键结合。我们已经逆转了在LTBP-4缺乏的小鼠中观察到的受损的终末肺泡发育表型，方法是产生Ltbp4-/-；Tgfb2-/-小鼠，从而降低转化生长因子-β水平。这一结果提示，Ltbp4-/-动物的肺间隔缺陷与转化生长因子-β2水平升高有关。我们认为LTBP-4主要作为弹性微纤维的组织者，多蛋白组合体，其中包含纤维蛋白、纤维蛋白、弹性蛋白和LTBP，而不是潜在的转化生长因子-β的结合剂。在我们看来，转化生长因子介导的效应是次要的异常基质。我们将在两个目标上检验这一假设。在目标1中，我们将产生小鼠，在小鼠中，LTBP-4中与LAP结合的两个半胱氨酸残基被突变为丝氨酸，从而使LtBP-4不能与转化生长因子结合。这些小鼠将产生LtBP-4和转化生长因子-？，但不会产生-4-转化生长因子？复合体。如果LTBP-4-/-小鼠的肺泡化异常是由于LTBP-4的结构活性缺失所致，那么这些新突变的动物应该具有正常的表型。相反，如果LtBP-4-/-小鼠的肺缺陷与其结合的转化生长因子的缺失有关，突变的动物将表现出异常的气囊分隔。我们还将在体外使用Ltbp4-/-细胞验证我们的假设，并在LTBP-4‘S结构功能或转化生长因子-β水平正常化的条件下测量基质组织和活性转化生长因子-β水平。我们将通过增加表达WT LTBP-4的细胞或纯化的LTBP-4蛋白来使LTBP-4的结构功能正常化。通过添加一种泛中和抗体来使转化生长因子-β水平正常化。在目标2中，我们将探讨转化生长因子在肺病理中的作用和来源。我们将通过制造Ltbp4-/-；Tgfb1-/-小鼠并检测它们的表型来表征转化生长因子-B对肺缺陷的贡献。这项实验的结果将确定在Ltbp4-/-；Tgfb2-/-动物中肺表型的正常化是由于总的转化生长因子β的减少；即转化生长因子1和转化生长因子2的总和，还是转化生长因子2的特异性。我们还将通过使用潜伏转化生长因子激活因子的特定抑制剂或突变为零的小鼠来鉴定培养细胞和/或缺乏LTBP-4的动物中潜伏转化生长因子激活因子的性质。最后，我们将确定在没有LTBP-4的情况下形成的多余的活性转化生长因子-β是来自于LTBP-1或LTBP-3与转化生长因子-β的络合物，还是来自潜在的未与LTBP结合的转化生长因子-β。这些实验将对如何利用新的遗传和细胞方法在肺和培养的肺细胞中控制潜伏的转化生长因子-？产生重要的见解。这一结果可能提示在某些病理状态下，如肺纤维化时，转化生长因子的正常化机制。

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.