Matrix Metalloproteinases: Remodeling of the Extracellular Matrix

基质金属蛋白酶：细胞外基质的重塑

• 批准号: 9155519
• 负责人: PAMELA G ROBEY
• 金额: $ 113.77万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9155519
• 关键词: Ablation; Address; Adipocytes; Adult; Affect; Alleles; Area; Attenuated; Binding; Biology; Bone Marrow; Bone Resorption; Cell Separation; Cells; Cleaved cell; Connective Tissue; Data; Defect; Deformity; Deletion Mutation; Development; Disease; Disease model; Drosophila genus; Dwarfism; Dysplasia; Embryo; Environment; Extracellular Matrix; Family; Fibrillar Collagen; Fibrosis; Fracture; GTP-Binding Proteins; Genes; Gs alpha mutations; Hematopoiesis; Homeostasis; Homologous Gene; Human; Inherited; Laboratories; Lesion; Life; Ligands; MMP14 gene; Maintenance; Marrow; Matrix Metalloproteinases; Mature Bone; Mediating; Membrane; Mesenchymal Stem Cells; Metalloproteases; Minerals; Morphology; Mosaicism; Mouse Strains; Mus; Mutate; Mutation; Online Mendelian Inheritance In Man; Osteitis Fibrosa Disseminata; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Pathological fracture; Pattern; Peptide Hydrolases; Perception; Pericytes; Periosteum; Phenotype; Play; Population; Process; Proteolysis; Recruitment Activity; Regulation; Reporter; Resources; Role; Signal Transduction; Skeletal Development; Skeleton; Staging; Stem cells; Syndrome; Tissue Differentiation; Tissues; Transplantation; Vanishing bone disease; Vascular Cell Adhesion Molecule-1; beta-Galactosidase; bone; bone cell; bone erosion; bone mass; bone turnover; cell type; embryonic stem cell; human disease; in vivo; inhibitor/antagonist; interest; lipid biosynthesis; macrophage; membrane-type matrix metalloproteinase; monocyte; mouse model; notch protein; novel; paracrine; postnatal; progenitor; promoter; skeletal; skeletal disorder; skeletal tissue; stem; substantia spongiosa; trait; transgene expression; wasting

We previously established that membrane-type metalloproteinases (MT-MMPs) are essential for skeletal development in the mouse, where collagenolytic activity is critically dependent on MT1-MMP. Importantly, the traits associated with loss of MT1-MMP in the mouse are a remarkably identical to those of the human vanishing bone disease, Winchester syndrome (OMIM # 259600), which now is identified as a homozygous mutation of the MT1-MMP locus. Due to the wide-spread expression pattern of MT1-MMP in non-skeletal tissues, and in the skeleton and associated tissues, we have generated and utilized a conditional deletion mutation mouse strain (MT1-MMP f/f). We subsequently deleted MT1-MMP activity in a progressive cell-maturity and developmental stage-specific fashion to assign the cell- and tissue-specific functions of pericellular proteolysis mediated by MT1-MMP. Additionally we have addressed the role of MT1-MMP activity in the bone marrow-derived monocyte/macrophage/osteoclast compartment, where the significance of MT1-MMP expression is poorly understood. To establish the role of MT1-MMP activity in pericyte-like cells (some of which have been proven to be SSCs), we have ablated MT1-MMP activity in SM22alpha positive cells (MT1-MMP f/f;SM22alpha-Cre). Loss of MT1-MMP in this subset of cells is reminiscent of universal MT1-MMP ablation including dwarfism, rampant bone resorption, diminished bone formation, progressive wasting, fibrosis and early demise. These studies demonstrate that cells forming the skeleton are recruited out of the perivascular cell pool and utilize MT1-MMP to exert their function. Using a SM22alpha-driven beta-galactosidase reporter, we have demonstrated that SM22alpha-positive cells expessing either VCAM-1 or PDGFRbeta isolated by cells cell sorting before development of the skeleton can be expanded ex vivo and give rise to bone upon in vivo transplantation. In contrast, cells with similar marker profiles in the absence of MT1-MMP fail to support bone formation, thus underscoring the importance of proteolysis in the skeletal stem/progenitor population. We have subsequently contrasted the role of pericellular proteolysis in SM22alpha-positive progenitors with the function of MT1-MMP in Osteocalcin (Ocn)-expressing, mature osteoblasts (MT1-MMP f/f;Ocn-Cre). Unlike SM22-specific deletion, Ocn-mediated ablation of MT1-MMP leads to grossly normal mice, which however display diminished bone mass and spontaneous fractures in adulthood. Thus, while progenitor-specific deletion results in dysmorphism and resorption, osteoblast-specific ablation mainly affects bone apposition, but not resorption. Additionally, Ocn-mediated ablation of MT1-MMP causes a prominent increase in marrow adipocyte expansion the default terminal differentiation state of SSCs. We hypothesized that MT1-MMP-mediated proteolysis in mature osteoblasts controls the fate of uncommitted progenitors by juxtacrine or paracrine regulation of adipogenic fate. Analysis of marrow-ablated bone from mice with Ocn-specific MT1-MMP deletion demonstrates a dramatic increase in the 56kDa delta-like (drosophila homolog), DLK1 levels. We have demonstrated that DLK1 is a novel substrate of MT1-MMP; i.e., membrane-bound DLK1 is efficiently cleaved by MT1-MMP to a soluble ligand presumably required for suppression of adipogenesis. The true function of DLK1 leading to this suppression is, however, poorly understood. The partial homology of DLK1 to canonical Notch ligands of the delta-like and Jagged family has earned DLK1 the presumptive role of a non-canonical Notch ligand. We have demonstrated that soluble, but not immobilized DLK1 efficiently attenuates Dll4-mediated Notch signaling, and as such suggests that DLK1 is a competitive inhibitor in canonical Notch signaling. Consistent with this hypothesis, the Notch responsive genes, Hes1 and Hey1, are also elevated in the absence of MT1-MMP pointing to disruption of Notch signaling regulation in the absence of protease-mediated DLK1 shedding. These results demonstrate the important (and previously unrecognized0 role of the mature bone cell as a regulator of progenitor fate via protease-mediated DLK1 shedding. In the context of skeletal homeostasis and bone turnover, one of the essential cell types is the osteoclast, which expresses abundant levels of MT1-MMP. However, the role that MT1-MMP plays in osteoclastic activity is not well understood. For that reason, we utilized LysM-Cre mice to specifically ablate MT1-MMP in osteoclasts (MT1-MMP f/f;LysM-Cre), and unlike other cell specific-deletions, this led to increased trabecular bone content. Consequently, osteoclast-specific MT1-MMP deficiency is not the cause of the dramatic bone erosion observed in MT1-MMP deficient mice. MT1-MMP-deficient osteoclasts form in equivalent numbers, display a morphology indistinguishable from wild-type osteoclasts and retain an uncompromised ability to degrade mineralized matrix. They do, however, display a near complete defect in ability to degrade fibrillar collagen, the major component of bone-lining periostea. This connective tissue is a barrier between the osteoclast and the mineralized bone that must be degraded prior to mineral dissolution. These results highlight the function of neutral proteinase activity in osteoclasts and explain the increase in bone content following loss of MT1-MMP in this cell subset. With our collaborators, we have been generating various mouse models for the study of fibrous dysplasia of bone (FD). FD is a crippling skeletal disease characterized by replacement of normal bone and marrow with hypomineralized, unorganized bone and a fibrotic marrow, devoid of hematopoiesis, leading to deformity and fracture of the affected bones. The disease is caused by post-zygotic mutations (R201C, R201H) of the gene encoding the alpha subunit of the stimulatory G protein, Gs. Lack of inheritance of the disease in humans is thought to reflect embryonic lethality of germline-transmitted activating Gs-alpha mutations, which would only survive through somatic mosaicism. Multiple lines of mice that express GsαR201C constitutively were generated and were found to develop an inherited, exact replica of human FD. Robust transgene expression in all tissues and murine embryonic stem cells was associated with normal development of skeletal tissues and differentiation of skeletal cells. As in the human diseases, FD lesions in mice developed only in postnatal life. One remaining question not addressed by the model disease induced by ubiquitous expression of the R201C mutation is which cell subset drives the disease process. To resolve this issue, R201C was expressed in mice under the control of the osteoblast specific Col1α1 2.3kb promoter. This forced expression of the mutated allele, however does not replicate the fibrous dysplasia phenotype, but rather induces a high bone mass phenotype with osteopetrotic bone. These data demonstrate that fibrous dysplasia, contrary to common perception, is not a disease of the osteoblast, but originates in upstream skeletal progenitors of osteoblasts, presumably in the bone marrow stroma.

我们先前确定膜型金属蛋白酶（MT-MMP）对于小鼠的骨骼发育至关重要，在小鼠中，胶原式活性严重取决于MT1-MMP。重要的是，与小鼠中MT1-MMP丢失相关的特征与人类消失的骨骼疾病，温彻斯特综合征（OMIM＃259600）的特征完全相同，该综合征（OMIM＃259600）现在被识别为MT1-MMP基因座的纯合突变。 由于非骨骼组织中MT1-MMP的广泛表达模式，以及在骨骼和相关组织中，我们已经产生并利用了条件缺失突变小鼠菌株（MT1-MMP F/F）。随后，我们以进行性细胞成熟度和​​发育阶段特异性的方式删除了MT1-MMP活性，以分配由MT1-MMP介导的细胞细胞蛋白水解的细胞和组织特异性功能。此外，我们已经解决了MT1-MMP活性在骨髓衍生的单核细胞/巨噬细胞/破骨细胞室中的作用，其中了解MT1-MMP表达的意义很少。 为了确定MT1-MMP活性在周细胞样细胞中的作用（其中一些已被证明为SSC），我们在SM22Alpha阳性细胞（MT1-MMP F/F; SM222Alpha-cre）中具有消融的MT1-MMP活性。在这一细胞子集中，MT1-MMP的丧失让人联想到通用MT1-MMP消融，包括矮人，骨骼猖ramp的骨吸收，骨形成减少，进行性浪费，纤维化和早期灭亡。这些研究表明，形成骨骼的细胞是从血管周细胞库中募集的，并利用MT1-MMP发挥其功能。使用SM22Alpha驱动的β-半乳糖苷酶报告基因，我们证明了SM22Alpha阳性细胞可以在骨骼开发之前通过细胞分类分离的VCAM-1或PDGFRBETA，可以扩展骨架，并在体内扩展，并在体内引起骨骼的骨骼。 相反，在没有MT1-MMP的情况下，具有相似标记物谱的细胞无法支撑骨形成，因此强调了骨骼茎/祖细胞种群中蛋白水解的重要性。 随后，我们将周围蛋白水解在SM22Alpha阳性祖细胞中的作用与MT1-MMP在骨钙素（OCN）表达，成熟的成熟成骨细胞（MT1-MMP F/F; OCN-CRE）中的作用。 与SM22特异性缺失不同，OCN介导的MT1-MMP消融导致严重正常的小鼠，但是成年期的骨骼质量和自发性骨折显示出降低。 因此，尽管特定于祖细胞的缺失会导致畸形和吸收，但成骨细胞特异性消融主要影响骨骼的骨骼，但不影响吸收。 此外，OCN介导的MT1-MMP的消融导致骨髓脂肪细胞扩展显着增加SSC的默认末端分化状态。 我们假设成熟成骨细胞中的MT1-MMP介导的蛋白水解控制着掺杂脂肪命运的近二氨酸或旁分泌调节的祖细胞的命运。 对具有OCN特异性MT1-MMP缺失的小鼠的骨髓释放的骨骼的分析表明，DLK1水平的56kDa Delta样（果蝇同源物）急剧增加。我们已经证明了DLK1是MT1-MMP的新型底物。即，膜结合的DLK1被MT1-MMP有效地裂解至可能需要抑制脂肪形成所需的可溶性配体。然而，导致这种抑制的DLK1的真实功能知之甚少。 DLK1的部分同源于类似三角洲和锯齿状家族的规范缺口配体获得了DLK1的dlk1，这是非典型档位配体的推定作用。 我们已经证明了可溶性但未固定的DLK1有效地减弱DLL4介导的Notch信号传导，因此表明DLK1是规范缺口信号传导中的竞争性抑制剂。 与该假设一致，在没有MT1-MMP指向的情况下，Notch响应基因HES1和HEY1在没有蛋白酶介导的DLK1脱落的情况下也会升高。这些结果表明，通过蛋白酶介导的DLK1脱落，成熟的骨细胞作为祖细胞命运的调节剂的重要（且以前未被认可的作用。 在骨骼稳态和骨转换的背景下，必需的细胞类型之一是破骨细胞，它表达了大量的MT1-MMP水平。然而，MT1-MMP在整骨活性中起着作用，尚不清楚。因此，我们利用Lysm-Cre小鼠在破骨细胞（MT1-MMP f/f; lysm-cre）中特异性烧蚀了MT1-MMP，与其他细胞特异性缺失不同，这导致小径骨含量增加。因此，破骨细胞特异性的MT1-MMP缺乏并不是MT1-MMP缺乏小鼠中观察到的巨大骨侵蚀的原因。 MT1-MMP缺陷的破骨细胞形成等效数，表现出与野生型破骨细胞不能区分的形态，并保留降低矿物化基质的毫不妥协的能力。但是，它们确实显示出降解纤维化胶原蛋白的能力，这是骨衬里骨膜的主要成分。该结缔组织是破骨细胞和矿物化骨之间的障碍，在矿物质溶解之前必须降解。这些结果突出了整骨细胞中中性蛋白酶活性的功能，并解释了该细胞子集中MT1-MMP丢失后骨含量的增加。 与我们的合作者一起，我们一直在生成各种小鼠模型来研究骨骼纤维发育不良（FD）。 FD是一种残酷的骨骼疾病，其特征是替换正常的骨骼和骨髓，用降压骨，无组织的骨骼和一个缺乏造血的纤维骨髓，导致受影响骨骼的畸形和断裂。该疾病是由编码刺激G蛋白Alpha亚基的基因GS的基因的杂结结突变（R201C，R201H）引起的。人们认为，这种疾病的遗传缺乏反映了种系传播激活GS-α突变的胚胎致死性，这只能通过体细胞镶嵌物生存。组成性地产生了多种表达GSαR201c的小鼠，并被发现会发展出人类FD的遗传，精确的复制品。在所有组织和鼠类胚胎干细胞中的稳健转基因表达与骨骼组织的正常发育和骨骼细胞的分化有关。与人类疾病一样，小鼠的FD病变仅在产后生活中发展。 R201c突变无处不在的模型疾病未解决的一个剩下的问题是哪个细胞子集驱动疾病过程。 为了解决此问题，在成骨细胞特异性COL1α12.3KB启动子的控制下，R201c在小鼠中表达。 然而，这种强迫的突变等位基因的表达不会复制纤维发育不良表型，而是诱导具有骨质骨骨的高骨质量表型。 这些数据表明，与共同感知相反的纤维发育不良不是成骨细胞的疾病，而是起源于成骨细胞的上游骨骼祖细胞，大概是在骨髓基质中。