Matrix Metalloproteinases: Remodeling of the Extracellular Matrix

我们之前已经证实，膜型金属蛋白酶(MT-MMPs)对小鼠骨骼发育是必不可少的，其中胶原酶的活性关键依赖于MT1-MMPs和MT3-MMPs。重要的是，与小鼠MT1-MMPs丢失相关的特征是人类疾病温彻斯特综合征(OMIM#259600)的显著表型，该疾病现在被确认为Mt1-MMP座的纯合子突变。 鉴于MT-MMP零突变的多效性，以及MT-MMP在骨、骨相关组织和非骨组织中的广泛表达模式，我们培育并利用了一种条件缺失突变小鼠品系。随后，我们在结缔组织中以逐渐的细胞成熟和发育阶段特异性的方式删除了MT-MMPs的活性，以分配MT-MMPs介导的细胞和组织特异性的细胞内蛋白分解功能。此外，我们还讨论了MT-基质金属蛋白酶活性在单核/巨噬细胞/破骨细胞间的作用，在那里MT-基质金属蛋白酶表达的意义尚不清楚。 为了确定MT-基质金属蛋白酶活性在周细胞样细胞中的作用，我们去除了SM22pha阳性细胞中的金属硫蛋白1-基质金属蛋白酶活性。MT1-MMPs在这一亚群细胞中的丢失使人联想到普遍的MT1-MMPs消融，包括侏儒症、猖獗的骨吸收、骨形成减少、进行性消瘦、纤维化和早期死亡。这些观察表明，形成骨架的细胞是从血管周围细胞池中招募出来的，并利用MT1-MMP来发挥其功能。 通往成骨命运的一个连续步骤是转录因子SP7/OSX的表达。我们利用表达OSX-CRE的小鼠来去除早期定向成骨细胞中的MT1-MMPs。这会导致明显的骨骼畸形，其次是猖獗的骨吸收，这让人想起无条件的MT1-MMP缺乏症，但没有SM22pha特异性消融术观察到的那么严重。这与MT1-MMP裂解和脱落RANKL的能力是一致的。因此，蛋白分解与RANKL诱饵-骨保护素(OPG)一起，在破骨细胞招募中起到了衰减因素的作用。 接下来，将OSX阳性祖细胞的细胞周围蛋白分解作用与骨钙素和胶原(OCN，COL1)阳性的成熟成骨细胞的功能进行对比。与OSX特异性缺失不同，OCN和COL1介导的MT1-MMP消融导致小鼠大体正常，但成年后表现出骨量减少。因此，虽然祖细胞特异性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定位，但不影响吸收。 造血微环境对适当的骨形成和改建至关重要，我们已经证明，在异位骨/骨髓小骨模型中，缺乏MT1-MMP活性的细胞不能支持出生后的骨髓形成。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发挥着关键作用。因此，我们已经证明，MT1-MMP缺陷小鼠的骨髓中保留的造血干细胞(HSCs)是野生型小鼠的两倍。为了确定HSC保留细胞和负责动员HSCs进入循环的细胞，我们证明了OSX特异性MT1-MMPs的丢失减少了HSCs的动员。在骨骼动态平衡的背景下，HSCs的重要后代之一是破骨细胞，它表达丰富的MT1-MMPs。我们利用LysM-CRE小鼠特异性地去除破骨细胞中的MT1-MMP，与其他细胞特异性缺失不同，这会导致骨含量增加。这一观察结果与破骨细胞中胶原分解活性的减弱是一致的，并表明在矿化基质吸收之前对软组织(总是存在于骨表面的薄层中)进行蛋白质分解是破骨细胞功能所必需的。 MT-MMPs活性是骨骼中特定的未矿化软骨重塑所必需的。到目前为止，这种重塑机制是否是软骨细胞或邻近软骨的结缔组织细胞的特性仍未解决。通过软骨特异性消融MT1-基质金属蛋白酶活性，我们已经确定软骨细胞对于软骨的重塑是必不可少的。重要的是，邻近的细胞无法重塑这些未矿化的软骨，因此软骨细胞在重塑过程中是必不可少的。我们进一步证明，软骨中MT1-MMPs的丢失和MT3-MMPs的普遍丢失极大地加剧了软骨的破坏，并导致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.