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

描述（由申请人提供）：膜型1基质金属蛋白酶（MT1-MMP）是一种跨膜蛋白酶，具有细胞外催化，血红素样（PEX）和铰链结构域，以及短的细胞质尾部，与多种生理和病理过程有关。与其他MMPs不同的是，MT1-MMP的遗传缺陷只会产生轻微的表型影响，而MT1-MMP的遗传缺陷会导致骨骼发育的严重缺陷，包括出生后生长明显减慢、长骨缩短、软骨血管化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人血管生成方面表现出重要缺陷，并在3周龄时死亡。有人提出，这些小鼠的表型是由于缺乏MT1-MMP的蛋白水解活性。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质水解的机制介导细胞内信号的激活，刺激细胞在体外和体内的增殖和迁移。MT1-MMP细胞内信号的激活是由细胞质尾部的特定序列介导的，与MT1-MMP蛋白水解活性无关。虽然这一意想不到的新发现并没有削弱MT1-MMP蛋白水解活性的重要性，但它强烈支持我们发现的信号传导机制的重要作用，并表明MT1-MMP缺失小鼠的表型至少部分是由于缺乏MT1-MMP介导的信号传导。我们发现的信号机制是一种新的、不依赖于蛋白水解的MT1-MMP活性，它可能在生长板的软骨细胞增殖、继发性骨化中心的形成、骨骼和肺泡发育以及成人血管生成中发挥重要作用。因此，我们提出通过以下具体目的来研究MT1-MMP介导的细胞内信号传导的生理作用：产生并表征具有消除MT1-MMP介导的细胞内信号传导的微妙突变的转基因小鼠的表型。我们建议培养一种表达MT1-MMP细胞质尾部突变的小鼠，该突变可以在不影响MT1-MMP蛋白水解活性的情况下消除信号传导能力，并将其表型与wt和MT1-MMP缺失小鼠进行比较。我们预计这种突变MT1-MMP的表达将导致部分重现MT1-MMP缺失小鼠的表型。因此，分析突变小鼠的表型将使我们能够了解mt1 - mmp介导的信号传导在发育、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、范式转移的机制，可以在体外和体内控制细胞增殖和迁移。因此，我们建议在小鼠中建立遗传模型并进行表征，以了解这种新机制在发育、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).