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TGF-β的生物利用度是如何调节的，TGF-β在生理和病理过程中是重要的，如癌症、纤维化和自身免疫性疾病。TGF-β作为非活性复合物释放。该复合物由TGF-β、TGF-β前肽和第二基因产物、潜伏TGF-β结合蛋白（LTBP）组成。LTBP基因（Ltbp-1 L，3和4）的突变或亚型突变产生有限的，明显的异常，表明LTBP依赖性病理仅出现在组织中，其中多余的LTBP亚型不存在。然而，细胞和组织产生多种LTBP，表明LTBP具有非冗余功能。此外，Ltbp-3-/-或Ltbp-4-/-肺细胞具有仅通过表达缺失的LTBP逆转的细胞自主TGF-β依赖性表型，表明需要特异性LTBP同种型。为了解决这一明显的矛盾，我们假设LTBP亚型将潜伏性TGF-β定位于对个体激活机制特异的独特环境，并且这种特异性有助于潜伏性TGF-β激活和作用的多样性。我们将在体外和体内研究LTBP亚型的功能。目的1：从Ltbp缺失小鼠中分离肺细胞系，用Ltbp-1、-3、-4或嵌合Ltbp表达载体转染细胞系，以确定表型拯救所需的Ltbp结构。这种方法将阐明一个LTBP是否可以替代另一个，并将确定产生多样性的单个LTBP结构域。在目的2中，我们将分析Ltbp-1 L、3和4在肺中的表达模式，以测试无效表型是否反映Ltbp表达模式或另一参数，如基质定位或TGF-β活化。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Ltbp-1或Ltbp-4 cDNA敲入突变型Ltbp-3基因的小鼠。如通过组织（肺）表型监测的，“敲入”Ltbp替换缺失的Ltbp的能力将证明独特性和冗余性。这些实验将阐明LTBP在将潜伏的TGF-β引导至决定独特功能的不同细胞外位置中的作用。这些信息可能会产生关于TGF-β依赖性肺病理学的理解，并建议以组织特异性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.