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

LTBP-4 对肺中 TGF-β 活性的调节

• 批准号: 8665804
• 负责人: DANIEL B RIFKIN
• 金额: $ 34.09万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8665804
• 关键词: Affect; Air Sacs; Alveolar; Animals; Appearance; Binding; Binding Proteins; Cell Culture System; Cells; Complex; Cultured Cells; Cysteine; Defect; Development; Elastin; Environment; Extracellular Matrix; Fibrosis; Genes; Genetic; Goals; Growth Factor; Homeostasis; In Vitro; Inflammation; Lung; Measures; Mediating; Mediator of activation protein; Microfibrils; Mus; Mutate; Mutation; Nature; Outcome; Pathology; Pharmaceutical Preparations; Phenotype; Process; Production; Proteins; Regulation; Reporting; Research; Role; Secondary to; Serine; Signal Transduction; Signaling Molecule; Signaling Protein; Source; Structure; Sum; Testing; Transforming Growth Factor beta; Transforming Growth Factors; cell growth; disulfide bond; fibrillin; fibulin; inhibitor/antagonist; insight; latency-associated protein; lung development; mutant; neutralizing antibody; novel; novel therapeutic intervention; novel therapeutics; null mutation; protein complex; receptor; research study

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.

描述（由申请人提供）：转化生长因子-β（TGF-β）作为三联潜伏复合物的一部分从细胞中释放，该复合物除TGF-β外还包括潜伏相关蛋白（TGF-β）和与TGF-β二硫键结合的潜伏TGF-β结合蛋白（LTBP）。我们通过产生Ltbp 4-/-; Tgfb 2-/-小鼠，从而降低TGF-β水平，逆转了在LTBP-4缺陷小鼠中观察到的受损末端肺泡发育表型。该结果表明Ltbp 4-/-动物中肺分隔的缺陷与TGF-β 2水平的增加有关。我们提出LTBP-4主要作为弹性微纤维、多蛋白组装体（其含有原纤维蛋白、纤蛋白、弹性蛋白和LTBP）的组织者，而不是作为潜在TGF-β的结合剂。在我们看来，TGF-β介导的作用是继发于异常基质。我们将从两个方面来检验这一假设。在目标1中，我们将产生小鼠，其中LTBP-4中结合TGF-β的两个半胱氨酸残基突变为丝氨酸，使得Ltbp-4不能结合TGF-β。这些小鼠将产生Ltbp-4和TGF-β，但不产生Ltbp-4-TGF-β复合物。如果Ltbp 4-/-小鼠中的肺泡化异常是由于LTBP-4的结构活性的缺乏，则这些新的突变动物应该具有正常的表型。相反，如果Ltbp-4-/-小鼠中的肺缺陷涉及与Ltbp-4结合的TGF-β的丧失，则突变动物将显示异常的气囊分隔。我们还将在体外使用Ltbp-4-/-细胞并在LTBP-4的结构功能或TGF-β水平正常化的条件下测量基质组织和活性TGF-β水平来验证我们的假设。我们将通过添加表达WT LTBP-4或纯化的LTBP-4蛋白的细胞来使LTBP-4结构功能正常化。TGF-β水平将通过向TGF-β添加泛中和抗体来标准化。在目的2中，我们将研究TGF-β 1在肺病理中的作用和来源。我们将通过产生Ltbp 4-/-; Tgfb 1-/-小鼠并检查其表型来表征TGF-β对肺缺陷的贡献。该实验的结果将确定Ltbp 4-/-; Tgfb 2-/-动物中肺表型的正常化是否是由于总TGF-β 1（即TGF-β 1和TGF-β 2的总和）的减少所致，或者是TGF-β 2特异性的。我们还将通过使用潜伏TGF-β激活剂的特异性抑制剂或具有潜伏TGF-β激活剂无效突变的小鼠来鉴定培养细胞和/或LTBP-4缺陷动物中潜伏TGF-β激活剂的性质。最后，我们将确定在不存在LTBP- 4的情况下形成的过量活性TGF-β是否来自LTBP-1或LTBP-3与TGF-β的复合物，或来自未与LTBP结合的潜在TGF-β。这些实验将产生重要的见解，如何潜伏TGF-β是控制在肺和培养的肺细胞使用新的遗传和细胞的方法。这些结果可能提示了TGF-β在某些病理状态下（如肺纤维化）正常化的机制。