Physiological role of MT1-MMP-mediated, proteolysis-independent signaling in vivo

描述(申请人提供)：膜型1基质金属蛋白酶(MT1-MMPs)是一种跨膜蛋白，具有细胞外催化、类血球蛋白(PEX)和铰链结构域，以及一条短的细胞质尾巴，参与了多种生理和病理过程。与其他MMPs不同的是，MT1-MMPs的基因缺陷会导致严重的骨骼发育缺陷，表现为出生后生长明显减慢，长骨变短，软骨血管形成缺陷和继发性骨化中心的形成延迟。此外，MT1-MMP缺陷小鼠在肺泡发育和成年血管生成方面表现出重要的缺陷，并在3周龄前死亡。据认为，这些小鼠的表型是由于缺乏MT1-MMP的蛋白分解活性所致。我们最近发表的大量研究表明，MT1-MMP通过一种不依赖于蛋白降解的机制，在体外和体内刺激细胞的增殖和迁移，介导细胞内信号的激活。MT1-MMP胞内信号的激活是由胞浆尾部的特定序列介导的，与MT1-MMP蛋白的降解活性无关。虽然这一新的和意想不到的发现并没有削弱MT1-MMP蛋白分解活性的公认重要性，但它强烈支持我们所确定的信号机制的一个非常重要的作用，并表明MT1-MMP零小鼠的表型至少部分原因是缺乏MT1-MMPs介导的信号。我们发现的信号机制是MT1-MMP一种新的、不依赖于蛋白降解的活性，它可能在生长板软骨细胞的增殖、次级骨化中心的形成、骨骼和牙槽骨的发育以及成人血管生成中发挥重要作用。因此，我们建议通过以下特定目的来研究MT1-MMP介导的细胞内信号的生理作用：建立并鉴定具有微小突变的转基因小鼠的表型，该突变可以取消MT1-MMP介导的细胞内信号。我们建议建立一株表达MT1-MMP胞浆尾突变的小鼠，在不影响MT1-MMP蛋白分解活性的情况下取消信号传递能力，并将它们的表型与wt和MT1-MMP零小鼠的表型进行比较。我们期望这种突变的MT1-MMPs的表达将导致一个表型，部分概括了MT1-MMP零小鼠的表型。因此，对我们的突变小鼠的表型分析将使我们了解MT1-MMPs介导的信号在发育、正常生理和病理中的作用。公共卫生相关性：我们发现了一种意想不到的、范式转换的机制，它控制着细胞在体外和体内的增殖和迁移。因此，我们建议在小鼠身上建立和表征一个遗传模型，以了解这种新的机制在发育、正常生理和病理中的作用。从我们的研究中获得的知识可以为了解骨骼和肺泡发育以及血管形成(血管生成)提供重要的见解。

DESCRIPTION (provided by applicant): Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with extracellular catalytic, hemopexin-like (PEX) and hinge domains, and a short cytoplasmic tail, has been implicated in a variety of physiological and pathological processes. Unlike the other MMPs, whose genetic deficiency has minor phenotypic effects, the genetic deficiency of MT1-MMP causes severe defects in skeletal development with marked deceleration of postnatal growth, shortening of long bones, defective vascularization of the cartilage and delayed formation of secondary ossification centers. In addition, MT1- MMP-deficient mice show important defects in alveolar development and adult angiogenesis, and die by 3 weeks of age. It has been proposed that the phenotype of these mice results from the lack of the proteolytic activity of MT1-MMP. Our recently published, extensive studies have shown that MT1-MMP mediates the activation of intracellular signaling by a proteolysis-independent mechanism that stimulates cell proliferation and migration in vitro and in vivo. MT1-MMP activation of intracellular signaling is mediated by a specific sequence of the cytoplasmic tail and independent of the MT1-MMP proteolytic activity. While this novel and unexpected finding does not diminish the well-established importance of the proteolytic activity of MT1-MMP, it strongly advocates a very important role for the signaling mechanism we identified, and indicates that the phenotype of MT1-MMP null mice can result at least in part from lack of MT1-MMP-mediated signaling. The signaling mechanism we discovered is a novel, proteolysis-independent activity of MT1-MMP that may have important roles in chondrocyte proliferation in the growth plates, formation of secondary ossification centers, skeletal and alveolar development, and in adult angiogenesis. Therefore, we propose to study the physiological role of MT1-MMP- mediated intracellular signaling by developing the following Specific Aim: To generate and characterize the phenotype of a transgenic mouse with a subtle mutation that abolishes MT1-MMP-mediated intracellular signaling. We propose to generate a strain of mice expressing a mutation in the MT1-MMP cytoplasmic tail that abolishes the signaling capacity without affecting the proteolytic activity of MT1-MMP, and to compare their phenotype to those of wt and MT1-MMP null mice. We expect that expression of this mutant MT1-MMP will result in a phenotype that partially recapitulates the phenotype of MT1-MMP null mice. Therefore, the analysis of the phenotype of our mutant mice will allow us to understand the role of MT1-MMP-mediated signaling in development, normal physiology and pathology. PUBLIC HEALTH RELEVANCE: We found an unexpected, paradigm-shifting mechanism that controls cell proliferation and migration in vitro and in vivo. Therefore, we propose to generate and characterize a genetic model in mice to understand the role of this novel mechanism in development, normal physiology and in pathology. The knowledge derived from our study can provide important insight into skeletal and pulmonary alveolar development and in blood vessel formation (angiogenesis).