The role of MT1-MMP proteolytic activity in osteogenesis

摘要 我们建议研究膜型1型基质金属蛋白酶（MT 1）的非蛋白水解功能的作用， MMP、MMP-14）在体内控制骨形成中的作用。MT 1-MMP是一种细胞膜结合蛋白酶， 细胞外催化位点和20个氨基酸（aa）的胞质尾，降解各种细胞外基质（ECM） 它是骨形成的重要组成部分，在出生后的骨形成中起着关键作用。MT 1-MM-/-基因缺陷的研究 小鼠引起严重骨质减少、侏儒症、颅面畸形伴颅缝闭合不全 （fontanelles）和全身性关节炎。在人类中，MT 1-MMP突变导致多中心骨质溶解和关节炎 疾病，温彻斯特综合征，它重演了戏剧性的骨骼表型的MT 1-MMP-/-小鼠，显示 MT 1-MMP在人类出生后骨发育中的基本作用。由于MT 1-MMP在ECM中是必需的， 由于MT 1-MMP-/-小鼠的严重表型是由胶原蛋白缺陷引起的， 周转然而，大量证据表明MT 1-MMP的胞质尾区含有多个氨基酸残基 以及通过蛋白水解非依赖性机制控制细胞内信号传导途径和细胞功能的基序。我们 产生了在MT 1-MMP胞质结构域（MT 1-MMP）中具有独特酪氨酸点突变的小鼠 Y 573 D），并发现该非蛋白水解结构域控制体内Wnt信号传导和骨稳态。在MT 1中- MM-/-小鼠MT 1-MMP的蛋白水解和非蛋白水解功能均被消除，但表型效应 通常仅归因于缺乏蛋白水解活性。然而，由于MT 1-MMP是一种双功能蛋白， 为了了解其在体内的作用，有必要独立于其非蛋白水解功能来研究其蛋白水解活性。 这可以通过突变MT 1-MMP催化结构域中的保守Glu 240（E240 A）来完成，该突变消除了MMP的活性。 蛋白水解活性而不影响MT 1-MMP的非蛋白水解功能。因此，我们建议研究 MT 1-MMP在骨稳态中的作用，具体目的如下： MT 1-MMP E240 A条件性表达小鼠的表型。我们将在2000年培育出一只转基因小鼠， 其蛋白水解失活的MT 1-MMP E240 A的表达可以以时间和组织特异性方式被诱导。我们 然后将分析条件MMP 14 E240 A、MT 1-MM-/-和MT 1-MMP Y 573 D小鼠的骨表型，其中 在骨骼发育的不同阶段在成骨细胞中诱导相应的突变。成骨细胞和 来自这些小鼠的间充质干细胞也将表征Wnt信号传导和分化为 成骨谱系。我们预期缺乏MT 1-MMP蛋白水解活性的MT 1-MMP E240 A小鼠将显示出更温和的 骨表型与MT 1-MMP Y 573 D小鼠相反（轻度）， 骨质减少和侏儒症与骨量增加）。这些结果将阐明 MT 1-MMP对骨稳态的蛋白水解依赖性和独立性功能，并揭示非蛋白水解功能 至今未在体内检测到。从我们的研究中获得的知识最终可以为 设计高度创新的方法治疗骨质疏松症和其他骨脆性疾病。

ABSTRACT We propose to investigate the role of non-proteolytic functions of membrane-type 1 matrix metalloproteinase (MT1- MMP, MMP-14) in the control of bone formation in vivo. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid (aa) cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components and plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP (MT1-MM-/-) in the mouse causes severe osteopenia, dwarfism, faciocranial dysmorphism with lack of closure of the cranial sutures (fontanelles), and generalized arthritis. In humans, mutation of MT1-MMP causes the multicentric osteolysis and arthritis disease, Winchester syndrome, which recapitulates the dramatic skeletal phenotype of the MT1-MMP-/- mouse, showing the fundamental role of MT1-MMP in postnatal bone development in humans. Because MT1-MMP is essential in ECM remodeling, it is universally accepted that the severe phenotype of MT1-MMP-/- mice results from defective collagen turnover. However, a large body of evidence shows that the cytoplasmic tail of MT1-MMP contains multiple aa residues and motifs that control intracellular signaling pathways and cell functions by proteolysis-independent mechanisms. We generated a mouse with a point mutation of the unique tyrosine in the MT1-MMP cytoplasmic domain (MT1-MMP Y573D), and found that this non-proteolytic domain controls Wnt signaling and bone homeostasis in vivo. In the MT1- MM-/- mouse both the proteolytic and non-proteolytic functions of MT1-MMP are abolished, but the phenotypic effects are universally ascribed only to the lack of proteolytic activity. However, because MT1-MMP is a bifunctional protein, to understand its roles in vivo it is necessary to study its proteolytic activity independently of its non-proteolytic functions. This can be done by mutating the conserved Glu240 (E240A) in the MT1-MMP catalytic domain, a mutation that abrogates the proteolytic activity without affecting the non-proteolytic functions of MT1-MMP. Therefore, we propose to study the roles of MT1-MMP in bone homeostasis by developing the following Specific Aim: To generate and characterize the phenotype of mice with conditional expression of MT1-MMP E240A. We will generate a genetically modified mouse in which expression of proteolytically inactive MT1-MMP E240A can be induced in a time- and tissue-specific manner. We will then analyze the bone phenotype of conditional MMP14 E240A, MT1-MM-/- and MT1-MMP Y573D mice in which the respective mutations are induced in osteoblasts at different stages of skeletal development. Osteoblasts and mesenchymal stem cells from these mice will also be characterized for Wnt signaling and differentiation into the osteogenic lineage. We expect that MT1-MMP E240A mice devoid of MT1-MMP proteolytic activity will show milder osteopenia and dwarfism than MT1-MMP-/- mice, but a bone phenotype opposite to that of MT1-MMP Y573D mice (mild osteopenia and dwarfism vs. increased bone mass, respectively). The results will elucidate the relative contribution of the proteolysis-dependent and independent functions of MT1-MMP to bone homeostasis, and unveil non-proteolytic functions thus far undetected in vivo. The knowledge derived from our study can ultimately provide important information for the design of highly innovative approaches for the treatment of osteoporosis and other bone fragility conditions.