Activation of latent TGF-beta by integrins

描述(申请人提供)：转化生长因子β以3种亚型存在，是一种多效性细胞因子，参与多种肺过程和疾病，如发展、肺纤维化、结核病、哮喘呼吸道重塑和癌症。转化生长因子β的生物利用度在细胞外空间受到严格的调控：转化生长因子β以潜伏期的形式分泌，在接头蛋白(LTBP)的作用下被隔离在基质结合部位，并且在通过转化生长因子β受体发出信号之前必须被激活。转化生长因子β潜伏期是与转化生长因子β前肽潜伏期相关肽(LAP)结合的结果，激活涉及从LAP释放转化生长因子β。 因为所有的TGFbeta信号都发生在TGFbeta激活之后，所以激活机制是关键的控制点。TGFbeta的激活机制已被提出(如凝血酶反应蛋白-1、蛋白水解酶和氧化剂)，但它们在体内的作用尚不清楚。我们发现，上皮特异性整合素αVbeta6在LAP内的RGD序列上与潜在的TGFbeta1结合。表达αVbeta6的细胞激活TGFbeta1，缺乏αVbeta6的小鼠不会发生博莱霉素诱导的肺纤维化。我们还发现αVbeta6可以激活TGFbeta3，而在肺上皮细胞中表达的另一种整合素(AlphaVbeta8)可以激活TGFbeta1/3。 这项工作的目的是确定整合素介导的TGFbeta1在体内的激活作用。为此，我们培育了TGFbeta1基因突变的敲门小鼠，该基因编码LAP前肽中的一个非功能性整合素结合部位。这些小鼠的表型(命名为TGFbeta1-RGE)再现了TGFbeta1-/-表型最突出的方面：严重的多器官炎症，导致3-4周大的死亡。这一重要的发现表明，整合素介导的TGFbeta1激活可能发生在大多数TGFbeta1效应的上游。这项工作的具体目的是(1)比较TGFbeta1-RGE小鼠和TGFbeta1-/-小鼠的特征，(2)检验体内唯一激活TGFbeta1的整合素是αVbeta6和αVbeta8的假设，以及(3)测试某些整合素KO小鼠(alphaV-/-和(beta8-/-)的表型是由于缺乏TGFbeta1和TGFbeta3的激活所致。这些研究将进一步阐明RGD结合整合素在转化生长因子β生物学中的作用，并为进一步研究其在肺部病理中的作用提供基础。

DESCRIPTION (provided by applicant): TGFbeta, which exists in 3 isoforms, is a pleiotropic cytokine involved in a variety of lung processes and diseases, e.g., development, pulmonary fibrosis, tuberculosis, asthmatic airway remodeling and cancer. TGFbeta bioavailability is tightly regulated in the extracellular space: TGFbeta is secreted in a latent form, which is sequestered in matrix binding sites by the action of an adapter protein (LTBP), and must be activated before it can signal via TGFbeta receptors. TGFbeta latency occurs as a result of binding to the TGFbeta propeptide Latency-Associated Peptide (LAP), and activation involves releasing TGFbeta from LAP. Because all TGFbeta signaling occurs subsequent to TGFbeta activation, the activation mechanisms are critical control points. Mechanisms of TGFbeta activation have been proposed (e.g., thrombospondin-1, proteases and oxidants), but their roles in vivo are unclear. We showed that an epithelium-specific integrin, alphaVbeta6, binds latent TGFbeta1 at an RGD sequence within LAP. Cells expressing alphaVbeta6 activate TGFbeta1, and mice lacking alphaVbeta6 do not develop bleomycin-induced pulmonary fibrosis. We have also shown that alphaVbeta6 can activate TGFbeta3, and that another integrin expressed in lung epithelium (alphaVbeta8) can activate TGFbeta1/3. The goal of this work is to define the role of integrin-mediated TGFbeta1 activation in vivo. To that end, we have generated knockin mice with a mutation in the TGFbeta1 gene that encodes a nonfunctional integrin-binding site in the LAP propeptide. The phenotype of these mice (designated TGFbeta1-RGE) reproduces the most prominent aspect of the TGFbeta1-/- phenotype: severe, multiorgan inflammation that causes death at 3-4 weeks of age. This important finding suggests the possibility that integrin-mediated TGFbeta1 activation occurs upstream of most TGFbeta1 effects. The specific aims of this work are to (1) characterize the TGFbeta1-RGE mice in comparison to TGFbeta1-/- mice, (2) test the hypothesis that the only TGFbeta1-activating integrins in vivo are alphaVbeta6 and alphaVbeta8, and (3) test the hypothesis that the phenotype of certain integrin KO mice (alphaV-/- and (beta8-/-) is due to combined lack of TGFbeta1 and TGFbeta3 activation. These studies will further delineate the role of RGD-binding integrins in TGFbeta biology and provide a basis for important studies of their role in lung pathology.