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: 8183734
• 负责人: DANIEL B RIFKIN
• 金额: $ 37.07万
• 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/8183734
• 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; human EBAF protein; 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-¿) 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. PUBLIC HEALTH RELEVANCE: This research focuses on the mechanisms controlling the action of a potent signaling molecule in the extra cellular environment of the lung. Abnormalities in this mechanism result in pathologies of lung development. We will explore the functions of molecules that bind to the signaling protein and how those interactions affect function. Our long-term goal is to utilize this information in developing new therapeutic strategies.

描述（由申请人提供）：转化生长因子-β（TGF-β）作为三重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终末肺泡发育表型。这一结果表明，Ltbp 4-/-动物的肺分隔缺陷与TGF-β 2水平增加有关。我们认为LTBP-4主要作为弹性微纤维的组织者，多蛋白组装体，其中含有原纤维蛋白，纤蛋白，弹性蛋白和LTBP，而不是作为潜在TGF-β的结合剂。在我们看来，TGF-β介导的效应是继发于异常基质。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-/-小鼠的肺缺陷与结合于Ltbp-4的TGF-β的丧失有关，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-/-细胞验证我们的假设，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。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添加泛中和抗体来正常化。在目的2中，我们将研究TGF-β在肺病理学中的作用和来源。我们将通过生产Ltbp 4-/-; Tgfb 1-/-小鼠并检查其表型来表征TGF-β对肺缺陷的贡献。本实验的结果将确定Ltbp 4-/-; Tgfb 2-/-动物中肺表型的正常化是否是由于总TGF-²的减少;即TGF-² 1和TGF-² 2的总和，或者是特异性的。针对TGF-² 2。我们还将通过使用潜在TGF-β激活剂的特异性抑制剂或具有无效突变的小鼠来鉴定培养细胞和/或LTBP-4缺陷动物中潜在TGF-β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-<$是否来自LTBP-1或LTBP-3与TGF-<$的复合物，或来自未与LTBP结合的潜在TGF-<$。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。这些结果可能提示在某些病理状态下，如肺纤维化，TGF-β正常化的机制。 公共卫生相关性：这项研究的重点是控制肺细胞外环境中有效信号分子作用的机制。这种机制的异常导致肺发育的病理学。我们将探索与信号蛋白结合的分子的功能以及这些相互作用如何影响功能。我们的长期目标是利用这些信息开发新的治疗策略。