Isoform Specificities of the Latent TGF-beta Binding Proteins in Lung

描述(由申请人提供)：我们试图了解转化生长因子-β的生物利用度是如何调节的，转化生长因子-β在癌症、纤维化和自身免疫性疾病等生理和病理过程中是重要的。转化生长因子-β以非活性复合体的形式释放。该复合体由转化生长因子-β、转化生长因子-β前肽和第二基因产物--潜伏的转化生长因子-β结合蛋白(LTBP)组成。LTBP基因(LTBP-1L、3和4)的零突变或亚型突变会产生有限的、明显的异常，这表明LTBP依赖的病理只出现在缺乏冗余LTBP亚型的组织中。然而，细胞和组织产生多个LTBP，这表明LTBP具有非冗余功能。此外，肺细胞的LTBP-3-/-或LTBP-4-/-具有细胞自主的转化生长因子-β依赖表型，只有缺失的LTBP的表达才能逆转，这表明需要特定的LTBP亚型。为了解决这一明显的矛盾，我们假设LTBP亚型将潜在的转化生长因子-β定位于特定于个体激活机制的独特环境，并且这种特异性有助于潜在转化生长因子-β的激活和作用的多样性。我们将在体外和体内检测LTBP亚型的功能。在目标1中，我们将从LtBP基因缺失的小鼠中分离出肺细胞系，并将该细胞系与LtBP-1、-3、-4或嵌合的LtBP表达载体共转染，以确定表型挽救所需的LtBP结构。这种方法将澄清一个LTBP是否可以替代另一个LTBP，并将识别产生多样性的单个LTBP域。在目标2中，我们将分析LTBP-1、L、3和4在肺组织中的表达模式，以测试空表型是否反映了LTBP的表达模式或其他参数，如基质定位或转化生长因子-β的激活。作为唯一性或冗余性的进一步测试，我们将产生复合双Ltbp缺失小鼠，并确定是否出现新的表型或表型是相加的。最后，我们将产生小鼠，在小鼠中，LtBP-1或LtBP-4基因被敲击到突变的LtBP-3基因中。根据组织(肺)表型的监测，“敲门”的中位碱基取代缺失的中位碱基的能力将证明其独特性和冗余性。这些实验将阐明LTBP在引导潜在的转化生长因子-β到决定独特功能的不同细胞外位置方面的作用。这些信息可能会产生关于转化生长因子-β依赖的肺部病理的理解，并建议以组织特异性的方式控制这些不良事件的方法。

DESCRIPTION (provided by applicant): We seek to understand how the bioavailability of TGF-beta, which is important in physiological and pathological processes, such as cancer, fibrosis, and autoimmune diseases, is regulated. TGF-beta is released as an inactive complex. The complex consists of TGF-beta, the TGF-beta propeptide and a second gene product, the latent TGF-beta binding protein (LTBP). Null or hypomorphic mutations of the LTBP genes (Ltbp-1L, 3, and 4) produce limited, distinct abnormalities suggesting that LTBP-dependent pathologies appear only in tissues in which redundant LTBP isoforms are absent. However, cells and tissues produce multiple LTBPs suggesting LTBPs have non-redundant functions. Also, Ltbp-3-/- or Ltbp-4-/- lung cells have cell autonomous TGF-beta-dependent phenotypes reversed only by expression of the missing LTBP, indicating a requirement for specific LTBP isoforms. To address this apparent contradiction, we hypothesize that LTBP isoforms localize latent TGF-beta to unique environments specific for individual activation mechanisms and that this specificity contributes to the diversity of latent TGF-beta activation and action. We will examine LTBP isoform function in vitro and in vivo. In Aim 1, we will isolate lung cell lines from Ltbp null mice, transfect the cells lines with Ltbp-1, -3, -4 or chimeric Ltbp expression vectors to ascertain the Ltbp structural requirements for phenotype rescue. This approach will clarify whether one LTBP can substitute for another and will identify individual LTBP domains that generate diversity. In Aim 2, we will analyze the expression pattern of Ltbp-1 L, 3, and 4 in the lung to test whether null phenotypes reflect Ltbp expression patterns or another parameter, such as matrix localization or TGF-beta activation. As a further test of uniqueness or redundancy, we will generate compound double Ltbp null mice and determine if either novel phenotypes appear or the phenotypes are additive. Finally, we will generate mice in which the Ltbp-1 or Ltbp-4 cDNA is knocked into the mutant Ltbp-3 gene. The ability of the "knockin" Ltbp to replace the deleted Ltbp, as monitored by tissue (lung) phenotypes, will demonstrate uniqueness and redundancy. These experiments will clarify the role of the LTBPs in directing latent TGF-beta to distinct extracellular locations that determine unique functions. This information may yield understanding concerning TGF-beta- dependent lung pathologies and suggest ways to control these adverse events in a tissue-specific manner.