Matrix Metalloproteinases: Remodeling of the Extracellular Matrix

我们以前已经确定，膜型金属蛋白酶（MT-MMP）是必不可少的骨骼发育的小鼠，其中胶原溶解活性是严重依赖于MT 1-MMP和MT3-MMP。 重要的是，与小鼠中MT 1-MMP缺失相关的性状是人类疾病温彻斯特综合征（OMIM # 259600）的显著表型，其现在被鉴定为MT 1-MMP基因座的纯合突变。 由于MT-MMP无效突变的多效性和MT-MMP在骨、骨相关组织和非骨组织中的广泛表达模式，我们已经产生并利用了条件性缺失突变小鼠品系。 随后，我们删除MT-MMP活性在结缔组织中的细胞成熟和发育阶段的特定方式分配细胞和组织特异性功能的MT-MMPs介导的细胞周蛋白水解。 此外，我们已经解决了MT-MMP活性在单核细胞/巨噬细胞/破骨细胞室中的作用，其中MT-MMP表达的意义知之甚少。 为了确定MT-MMP活性在周细胞样细胞（早期未定型间充质祖细胞）中的作用，我们消除了SM 22 α阳性细胞中的MT 1-MMP活性。 在该细胞亚群中MT 1-MMP的丢失使人联想到普遍的MT 1-MMP消融，包括侏儒症、猖獗的骨吸收减少骨形成、进行性消耗、纤维化和早期死亡。 这些观察结果表明，形成骨骼的细胞从血管周围细胞库中募集出来，并利用MT 1-MMP发挥其功能。 成骨命运的连续步骤是转录因子Sp 7/Osx的表达。 我们利用Osx-Cre表达小鼠消融早期定向骨祖细胞中的MT 1-MMP。 这导致明显的骨骼畸形，其次是猖獗的骨吸收，使人联想到无条件的MT 1-MMP缺乏，但严重程度低于SM 22 α特异性消融术。 该观察结果与MT 1-MMP切割和脱落RANKL的能力一致。 因此，蛋白水解与RANKL诱饵、骨保护素（OPG）一起作为破骨细胞募集的衰减因子。 接下来，将Osx阳性祖细胞中细胞周围蛋白水解的作用与骨钙素和胶原蛋白（Ocn，Col 1）阳性成熟成骨细胞的功能进行对比。 与Osx特异性缺失不同，Ocn和Col 1介导的MT 1-MMP消融导致大体正常的小鼠，然而其在成年期显示骨量减少。 因此，虽然祖细胞特异性缺失导致畸形和吸收，但成骨细胞特异性消融主要影响骨沉积，而不是吸收。 造血微环境对于适当的骨形成和重建是至关重要的，我们已经证明，缺乏MT 1-MMP活性的细胞不能支持出生后在异位骨/骨髓小骨模型中的骨髓形成。 我们认为MT-MMPs在造血干细胞的迁移和募集以及它们在出生后填充潜在造血环境的能力中起着关键作用。 因此，我们已经证明，MT 1-MMP缺陷小鼠的骨髓中保留的造血干细胞（HSC）是野生型同窝仔中观察到的两倍。 为了鉴定HSC保留细胞和负责动员HSC进入循环的细胞，我们证明了MT 1-MMP的Osx特异性损失减少了HSC的动员。 在骨骼稳态的背景下，HSC的重要后代之一是破骨细胞，其表达丰富水平的MT 1-MMP。 我们利用LysM-Cre小鼠特异性消融破骨细胞中的MT 1-MMP，与其他细胞特异性缺失不同，这导致骨含量增加。 该观察结果与破骨细胞中胶原溶解活性降低一致，并表明在矿化基质吸收之前蛋白水解去除软组织（始终存在于骨表面的薄层中）对破骨细胞功能至关重要。 MT-MMP活性在骨骼中特定的未矿化软骨的重塑中是必需的。 到目前为止，它仍然没有得到解决，如果这种重塑机制是软骨细胞或邻近软骨的结缔组织细胞的属性。 使用软骨特异性消融MT 1-MMP活性，我们已经确定软骨细胞是软骨重塑所必需的。 重要的是，邻近的细胞不能重塑这些未矿化的软骨，因此软骨细胞对于这种重塑过程至关重要。 我们进一步证明，MT 1-MMP在软骨中的组合损失和MT3-MMP的普遍损失显著加剧了软骨的破坏，并诱导严重的生长迟缓和侏儒症，以及极端的颅面畸形。

We have 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 and MT3-MMP. Importantly, the traits associated with loss of MT1-MMP in the mouse are a remarkable phenocopy of the human disease, Winchester syndrome (OMIM # 259600), which now is identified as a homozygous mutation of the MT1-MMP locus. Due to the pleiotropic nature of MT-MMP null-mutations and the widespread expression pattern of MT-MMP in bone, bone-associated tissues and non-bone tissues, we have generated and utilized a conditional deletion mutation mouse strain. We subsequently deleted MT-MMP activity in a progressive cell-maturity and developmental stage-specific fashion in connective tissues to assign the cell- and tissue-specific functions of pericellular proteolysis mediated by MT-MMPs. Additionally we have addressed the role of MT-MMP activity in the monocyte/macrophage/osteoclast compartment where the significance of MT-MMP expression is poorly understood. To establish the role of MT-MMP activity in pericyte-like cells, the early un-committed mesenchymal progenitor, we have ablated MT1-MMP activity in SM22alpha positive cells. 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 observations demonstrate that cells forming the skeleton are recruited out of the perivascular cell pool and utilize MT1-MMP to exert their function. A successive step towards osteogenic fate is expression of the transcription factor, Sp7/Osx. We utilized Osx-Cre expressing mice to ablate MT1-MMP in early committed osteoprogenitors. This leads to overt skeletal dysmorphism and secondarily, a rampant bone resorption reminiscent of unconditional MT1-MMP deficiency, yet less severe than that observed with SM22alpha-specific ablation. This observation is consistent with the ability of MT1-MMP to cleave and shed RANKL. Proteolysis thus works as an attenuation factor in osteoclast recruitment together with the RANKL decoy, osteoprotegerin (OPG). Next, the role of pericellular proteolysis in Osx positive progenitors was contrasted with the function of osteocalcin and collagen (Ocn, Col1) positive, mature osteoblasts. Unlike Osx-specific deletion, Ocn- and Col1- mediated ablation of MT1-MMP leads to grossly normal mice, which however display diminished bone mass in adulthood. Thus, while progenitor-specific deletion results in dysmorphism and resorption, osteoblast-specific ablation mainly affects bone apposition, but not resorption. The hematopoietic microenvironment is critical for proper bone formation and remodeling and we have demonstrated that cells devoid of MT1-MMP activity fail to support postnatal marrow formation in an ectopic bone/marrow ossicle model. We propose that MT-MMPs plays a pivotal role in the migration and recruitment of hematopoietic stem cells and their ability to populate potential hematopoietic environments postnatally. Accordingly, we have demonstrated that the bone marrow of MT1-MMP deficient mice retain twice as many hematopoietic stem cells (HSCs) as observed in wildtype littermates. In an effort to identify the HSC-retaining cell and the cell responsible for mobilization of HSCs into the circulation, we demonstrated that Osx-specific loss of MT1-MMP reduced mobilization of HSCs. In the context of skeletal homeostasis, one of the important descendants of HSCs are osteoclasts, which expresses abundant levels of MT1-MMP. We utilized LysM-Cre mice to specifically ablate MT1-MMP in osteoclasts, and unlike other cell specific-deletions, this leads to an increased bone content. This observation is consistent with diminished collagenolytic activity in osteoclasts and suggests that proteolytic removal of soft tissue (always present in a thin layer on bone surfaces) prior to mineralized matrix resorption is essential for osteoclast function. MT-MMP activity is required in remodeling of specific unmineralized cartilages in the skeleton. Until now, it has remained unresolved if this remodeling mechanism is a property of chondrocytes or connective tissue cells adjacent to the cartilage. Using cartilage specific ablation of MT1-MMP activity, we have established that chondrocytes are essential for the remodeling of the cartilages. Importantly, neighboring cells fail to remodel these unmineralized cartilages and chondrocytes are thus essential for this remodeling process. We furthermore demonstrate that the combined loss of MT1-MMP in cartilage and universal loss of MT3-MMP dramatically exacerbates the disruption of cartilage and induces a severe growth retardation and dwarfism, and extreme craniofacial dysmorphology.