ACTIVATION OF LATENT TGFB BY THE INTEGRIN ALPHA VB6

转化生长因子-β1(TGFbeta1)是一种广泛表达的细胞因子，对大多数细胞类型有重要影响。TGFbeta1是一种抗炎、促肝纤维化的药物，通常与肺纤维化等纤维化疾病有关。TGFbeta1与其前肽二聚体在非活性复合体(FTGFbeta1)中分泌，称为潜伏期相关(LAP)。LTGFbeta1的激活是TGFbeta1生物学中的一个关键控制点，但人们对此知之甚少。在正常动物中，只有凝血酶反应蛋白-1(TSP1)被证明具有PTGFbeta1的活性。我们发现LAP是上皮特异性整合素αvbeta6的配体，表达αvbeta6的细胞结合并激活潜伏的TGFbeta1。这一机制可以解释迄今为止令人困惑的Beta6整合素基因敲除小鼠的表型：肺和皮肤的炎症，以及对博莱霉素诱导的肺纤维化的保护。我们的结果首次提供了失调的TGFbeta1激活导致纤维化的证据。我们的目标是定量地了解LTGFbeta6和alphavbeta6之间导致激活的相互作用，并开发一种动物模型和知识来全面探索avbeta6介导的LTGFbeta1激活的生物学作用。在目标1中，我们将通过αvbeta6-LTGFbeta1相互作用来分析激活机制。我们将使TGFbeta1-Mullαvbeta6表达细胞，其中将添加特定工程形式的LTGFbeta1(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LTGFbeta1(所谓的小潜伏复合体和大潜伏复合体)的相对活性，激活的LTGFbeta1浓度和αvbeta6表达水平的相对影响，可溶性和基质结合的潜伏TGFbeta1的激活能力，以及激活所需的整合素：LTGFbeta1化学计量比。此外，我们还将评估整合素/LTGFbeta1结合亲和力对激活的影响。结果将被合并到激活模型中，并与体内激活相关。在目标2中，我们将创造一种表达LTGFbeta1突变形式的小鼠，该突变形式不能被整合素激活(LAP中的RGD整合素结合位点将突变为RGE)。这些小鼠和Beta6整合素缺失小鼠的表型将进行比较，以确认Beta6缺失表型是由于失去TGFbeta1的激活所致。解释表型将测试其他RGD结合整合素激活LTGFbeta1的能力。最后，我们将RGE-TGFbeta1小鼠与TSP1缺失的小鼠杂交，以评估目前已知的两种TGFbeta1激活机制，即αvbeta6和TSP1的总和效应。这些目标的结果将有助于更好地理解α-β6介导的TGFbeta1在疾病中的激活。

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.