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很重要，如癌症、纤维化和自身免疫性疾病。tgf -以非活性复合物的形式释放。该复合物由tgf - β、tgf - β前肽和第二个基因产物tgf - β结合蛋白（LTBP）组成。LTBP基因（LTBP- 1l、LTBP- 3和LTBP- 4）的零型或半形突变产生有限的、明显的异常，这表明LTBP依赖性病理只出现在缺乏冗余LTBP亚型的组织中。然而，细胞和组织产生多种ltbp，表明ltbp具有非冗余功能。此外，LTBP -3-/-或LTBP -4-/-肺细胞仅通过表达缺失的LTBP而逆转细胞自主tgf - β依赖性表型，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异构体将潜在的tgf - β定位到特定于个体激活机制的独特环境中，并且这种特异性有助于潜在tgf - β激活和作用的多样性。我们将在体内和体外检测LTBP异构体的功能。在Aim 1中，我们将从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Ltbp对表型拯救的结构要求。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Aim 2中，我们将分析Ltbp- 1l、3和4在肺中的表达模式，以测试空表型是否反映Ltbp表达模式或其他参数，如基质定位或tgf - β激活。作为独特性或冗余性的进一步测试，我们将产生复合双Ltbp缺失小鼠，并确定是否出现新表型或表型是加性的。最后，我们将产生Ltbp-1或Ltbp-4 cDNA敲入突变的Ltbp-3基因的小鼠。通过组织（肺）表型监测，“敲入”Ltbp取代缺失Ltbp的能力将证明其独特性和冗余性。这些实验将阐明ltbp在指导潜在tgf - β到决定独特功能的不同细胞外位置中的作用。这一信息可能有助于理解tgf -依赖性肺病理，并提出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.