The role of MT1-MMP proteolytic activity in osteogenesis

MT1-MMP 蛋白水解活性在成骨中的作用

• 批准号: 9386911
• 负责人: Paolo Mignatti
• 金额: $ 22.37万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9386911
• 关键词: 4-Hydroxy-Tamoxifen; Affect; Alpha Cell; Amino Acids; Apoptosis; Arthritis; Aspartic Acid; Biomechanics; Bone Development; Bone Growth; Bone Marrow; Bone Resorption; Catalytic Domain; Cell Proliferation; Cell membrane; Cell physiology; Cells; Charge; Collagen; Cytoplasmic Tail; Defect; Disease; Dwarfism; Embryo; Enterobacteria phage P1 Cre recombinase; Extracellular Matrix; Generations; Genetic; Genetic Models; Grant; Hajdu-Cheney Syndrome; Homeostasis; Human; In Vitro; Induced Mutation; Injectable; Joint structure of suture of skull; Knock-out; Knowledge; Lead; MAPK3 gene; MMP14 gene; Measures; Mediating; Mesenchymal Stem Cells; Mus; Mutant Strains Mice; Mutate; Mutation; Osteoblasts; Osteogenesis; Osteolysis; Osteopenia; Osteoporosis; Partner in relationship; Pathway interactions; Patients; Peptide Hydrolases; Phenotype; Play; Point Mutation; Property; Proteins; Proteolysis; Proto-Oncogene Proteins c-akt; Recombinants; Reporting; Role; Signal Pathway; Skeletal Development; Skeletal bone; Structure of fontanel of skull; Syndrome; Tarsal Bones; Techniques; Time; Tissues; Tyrosine; United States National Institutes of Health; Up-Regulation; WNT Signaling Pathway; bone; bone fragility; bone mass; carpus bone; cell motility; circulating biomarkers; design; embryonic stem cell; extracellular; homologous recombination; in vivo; innovation; insight; long bone; novel; osteogenic; postnatal; skeletal; skeletal maturation; tumor growth; vector

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.

摘要 我们建议研究膜型基质金属蛋白酶(MT1-1)的非蛋白降解功能的作用。 MMP1、MMP14)在体内调控骨形成中的作用。MT1-MMP1，一种细胞膜结合的蛋白水解酶 胞外催化部位和一个20个氨基酸(AA)的胞浆尾巴，降解多种细胞外基质(ECM) 并在出生后骨形成中起关键作用。MT1-MMP1(MT1-MM-/-)的遗传缺陷 小鼠造成严重的骨量减少、侏儒症、面颅骨畸形，并伴有颅缝未闭合 (Fontanelle)和全身性关节炎。在人类中，MT1-MMPs突变导致多中心骨溶解和关节炎 温彻斯特综合征，重述了MT1-MMP1-/-小鼠戏剧性的骨骼表型，显示 MT1-基质金属蛋白酶在人类出生后骨发育中的基础作用。因为MT1-MMPs在ECM中是必不可少的 重塑，人们普遍认为MT1-MMP1-/-小鼠的严重表型是由于胶原蛋白缺陷造成的 营业额。然而，大量证据表明，MT1-MMP胞浆尾含有多种氨基酸残基 以及通过非蛋白降解机制控制细胞内信号通路和细胞功能的基序。我们 通过MT1-MMP胞浆域(MT1-MMP点突变)产生了一只小鼠 Y573D)，并发现该非蛋白水解区在体内控制Wnt信号转导和骨稳态。在MT1中- MM-/-小鼠MT1-MMP的蛋白水解性和非蛋白水解性均被取消，但其表型效应 普遍归因于蛋白分解活性的缺乏。然而，由于MT1-基质金属蛋白酶是一种双功能蛋白，所以 要了解它在体内的作用，有必要研究它的蛋白分解活性，而不是它的非蛋白分解功能。 这可以通过突变MT1-MMP催化结构域中保守的Glu240(E240A)来实现，这种突变可以废除 在不影响MT1-基质金属蛋白酶非蛋白降解功能的前提下，具有较强的蛋白降解活性。因此，我们建议研究 MT1-MMPs在骨内稳态中的作用 条件表达MT1-MMPE240A小鼠的表型。我们将在中国培育一只转基因小鼠 蛋白水解性失活的MT1-MMPE240A的哪种表达可以以时间和组织特异性的方式诱导。我们 然后将分析条件MMP14 E240A、MT1-MM-/-和MT1-MMPY573D小鼠的骨表型 在骨骼发育的不同阶段，成骨细胞中分别诱导了相应的突变。成骨细胞和 这些小鼠的间充质干细胞也将被鉴定为Wnt信号和分化为 成骨血统。我们预计，缺乏MT1-MMPE240A蛋白水解酶活性的MT1-MMPE240A小鼠将表现出较温和的表现 骨质疏松症和侏儒症比MT1-MMP1-/-小鼠低，但骨表型与MT1-MMPY573D小鼠相反(轻度 骨质疏松症和侏儒症分别与骨量增加相比)。这一结果将阐明 MT1-MMPs的非蛋白水解性和非蛋白水解性功能 到目前为止还没有在体内检测到。从我们的研究中获得的知识最终可以为 设计高度创新的治疗骨质疏松症和其他骨脆性疾病的方法。