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（由应用程序提供）：转化生长因子-Beta（TGF- - ）是从细胞中释放的，作为三方潜在复合物的一部分，除TGF-®外，还包括潜伏期相关蛋白（LAP）和潜在的TGF-ood结合蛋白（LTBP），该蛋白（LTBP）二硫键粘合到LAP。我们通过生成LTBP4 - / - ; TGFB2 - / - 小鼠，从而逆转了在LTBP-4缺乏的小鼠中观察到的末端肺泡发育表型的受损受损，从而降低了TGF-¿水平。该结果表明，LTBP4 - / - 动物中肺分离的缺陷与TGF-€2水平升高有关。我们提出，LTBP-4主要是弹性微纤维，多蛋白质组件的组织者，其中包含纤维蛋白，斐bulin蛋白，弹性蛋白，弹性蛋白和LTBP，而不是作为潜在TGF- - 的粘合剂。在我们看来，TGF-介导的效应是继发于异常基质的。我们将以两个目的检验这一假设。在AIM 1中，我们将生成小鼠，其中两个半胱氨酸在LTBP-4中保留的结合与LAP结合的小鼠被突变为丝氨酸，从而使LTBP-4不能与TGF- - 结合。这些小鼠将产生LTBP-4和TGF- - ，但没有LTBP-4-TGF-€。如果LTBP4 - / - 小鼠中的肺肺泡绝对是由于没有LTBP-4的结构活性引起的，则这些新的突变动物应具有正常的表型。相反，如果LTBP-4 - / - 小鼠中的肺缺损与与LTBP-4结合的TGF-4的损失有关，则突变动物将显示出异常的空气SAC分离。我们还将使用LTBP4 - / - 细胞在体外验证我们的假设，并在LTBP-4的结构函数或TGF- - 水平归一化的条件下测量矩阵组织和主动TGF-？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结构功能归一化。 TGF- - 将通过将泛和中和抗体添加到TGF- - 将其归一化。在AIM 2中，我们将研究TGF-在肺病理中的作用和来源。我们将通过产生LTBP4 - / - ; TGFB1 - / - 小鼠并检查其表型来表征TGF- - 对肺部缺陷的贡献。该实验的结果将确定LTBP4 - / - ; TGFB2 - / - 动物中肺表型的归一化是由于总TGF- - （即TGF-€1和TGF- - 2和TGF- - 2的总和）所致，还是针对TGF-®进行了特异性，我们还将通过TGF-4的本质和/criede的本质。潜在的TGF- - 活化剂的特异性抑制剂或无效突变的小鼠。最后，我们将确定在没有LTBP-4的情况下形成的过量活性TGF- - 源自LTBP-1或LTBP-3与TGF-3的复合物，或者来自LETENT TGF- - 不与LTBP结合。这些实验将对肺中潜在的TGF- - 如何通过使用新型遗传和细胞方法在肺中以及培养的肺部细胞中控制潜在的TGF- - 产生重要的见解。结果可能表明在某些病理状态（例如肺纤维化）中使TGF- - 标准化的机制。