ACTIVATION OF LATENT TGFB BY THE INTEGRIN ALPHA VB6

整合素 ALPHA VB6 激活潜在 TGFB

• 批准号: 6351591
• 负责人: John S Munger
• 金额: $ 40.64万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-07 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351591
• 关键词: genetically modified animals; integrins; laboratory mouse; protein binding; protein protein interaction; receptor expression; transforming growth factors

Transforming growth factor-beta1 (TGFbeta1) is a widely-expressed cytokine that has major effects on most cell types. TGFbeta1 is anti- inflammatory, pro-fibrotic, and casually linked to fibrotic diseases, e.g. pulmonary fibrosis. TGFbeta1 is secreted in an inactive complex (FTGFbeta1) with its pro-peptide dimer, which is called latency- associated (LAP). Activation of LTGFbeta1 is a key control point in TGFbeta1 biology, but is poorly understood. Only thrombospondin-1 (TSP1) has been shown previously to active PTGFbeta1 in normal animals. We found that LAP is a ligand for the epithelium-specific integrin alphavbeta6, and that cells expressing alphavbeta6 bind and activate latent TGFbeta1. This mechanism can explain the heretofore puzzling phenotype of beta6 integrin knock-out mice: inflammation in lung and skin, and protection from bleomycin-induced pulmonary fibrosis. Our results provide the first evidence that dysregulated TGFbeta1 activation causes fibrosis. Our goals are to understand quantitatively the interactions between LTGFbeta and alphavbeta6 that lead to activation, and to develop an animal model an animal model and knowledge to explore fully the biological role of a of avbeta6-mediated LTGFbeta1 activation. In Aim 1 we will analyze the activation mechanism by focusing on alphavbeta6- LTGFbeta1 interactions. We will make TGFbeta1-mull alphavbeta6- expressing cells to which specifically engineered forms of LTGFbeta1 will be added (either by transfection or as recombinant protein). In this system we will then determine the relative activatability of two major forms of LTGFbeta1 (the so-called small and large latent complexes), the relative effects of LTGFbeta1 concentration and alphavbeta6 expression levels of activation, the activatability of soluble and matrix-bound latent TGFbeta1, and the integrin: LTGFbeta1 stoichiometry required for activation. Also, we will assess the influence of integrin/LTGFbeta1 binding affinity on activation. The results will be incorporated into an activation model and related to activation in vivo. In Aim 2, we will create a mouse expressing a mutant form of LTGFbeta1 that cannot be activated by integrins (the RGD integrin binding sites in LAP will be mutated to RGE). The phenotypes of these mice and beta6 integrin null mice will be compared to confirm that the beta6 null phenotype is due specifically to loss of TGFbeta1 activation. To interpret the phenotype will test the ability of other RGD-binding integrins to activate LTGFbeta1. Finally, we will cross RGE-TGFbeta1 mice with TSP1 null mice to assess the summed effects of the two currently known TGFbeta1 activation mechanisms, namely alphavbeta6 and TSP1. The results of these aims will lead to better understanding of alphavbeta6-mediated TGFbeta1 activation in disease.

转化生长因子β 1（TGF β 1）是一种广泛表达的细胞因子，对大多数细胞类型具有重要作用。TGF β 1是抗炎的、促纤维化的，并且偶然地与纤维化疾病（例如肺纤维化）相关。TGF β 1与其前肽二聚体以非活性复合物（TGF β 1）的形式分泌，这被称为潜伏期相关（latency- associated，缩写为TGFbeta 1）。LTGFbeta 1的激活是TGF beta1生物学的关键控制点，但知之甚少。只有血小板反应蛋白-1（TSP 1）先前已被证明在正常动物中激活PTGF β 1。我们发现，TGF β 1是上皮特异性整合素α v β 6的配体，表达α v β 6的细胞结合并激活潜伏的TGF β 1。这一机制可以解释迄今为止令人困惑的β 6整合素基因敲除小鼠的表型：肺部和皮肤的炎症，以及对博莱霉素诱导的肺纤维化的保护。我们的研究结果提供了第一个证据表明，TGF β 1激活失调导致纤维化。我们的目标是定量地了解导致活化的LTGFbeta和alphavbeta 6之间的相互作用，并开发一种动物模型，充分探索alphavbeta 6介导的LTGFbeta 1活化的生物学作用。在目标1中，我们将通过关注alphavbeta 6-LTGFbeta 1相互作用来分析激活机制。我们将制备TGF β 1-穆尔α v β 6表达细胞，向其中加入特定工程形式的TGF β 1（通过转染或作为重组蛋白）。在这个系统中，我们将确定两种主要形式的LTGF β 1（所谓的小和大的潜在复合物）的相对活化性，LTGF β 1浓度和α v β 6表达水平的活化的相对影响，可溶性和基质结合的潜在TGF β 1的活化性，和整合素：活化所需的LTGF β 1的化学计量。此外，我们将评估整合素/LTGFbeta 1结合亲和力对激活的影响。结果将被纳入激活模型，并与体内激活。在目标2中，我们将创建表达不能被整联蛋白激活的LTGF β 1突变形式的小鼠（在LTG中的RGD整联蛋白结合位点将突变为RGE）。将比较这些小鼠和β 6整联蛋白缺失小鼠的表型，以确认β 6缺失表型是由于TGF β 1活化丧失所致。为了解释表型，将测试其他结合RGD的整合素激活LTGF β 1的能力。最后，我们将RGE-TGF β 1小鼠与TSP 1缺失小鼠杂交，以评估两种目前已知的TGF β 1激活机制（即α v β 6和TSP 1）的综合效应。这些目标的结果将导致更好地理解疾病中α v β 6介导的TGF β 1激活。