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: 8761275
• 负责人: DANIEL B RIFKIN
• 金额: $ 34.45万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-11-27 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8761275
• 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-¿) 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.

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。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（即TGF-β 1和TGF-β 2的总和）的减少，或者是TGF-β 2特异性的。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。