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，我们培育并利用了一株条件缺失突变株小鼠。随后，我们以细胞和阶段特定的方式删除了MT-MMPs的活性，以说明MT-MMPs介导的细胞和组织周围蛋白分解的特定功能。 为了确定MT-MMP活性在血管周围平滑肌/周细胞样细胞中的作用，这些细胞被认为是间充质祖细胞的潜在储存库，我们对SM22和SM22阳性细胞中的MT-MMP活性进行了去除。MT1-基质金属蛋白酶在这一亚群细胞中的丢失导致头盖骨和长骨中骨的大量丢失，在这些骨中，骨小梁和皮质骨的含量显著减少。此外，明显的纤维化与骨含量减少有关。综上所述，这些观察表明，在很大程度上参与骨骼形成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。 为了通过转录因子SP7/OSX的表达进一步了解成骨细胞中细胞周围蛋白分解的功能，MT1-MMPs在这一亚群细胞中被删除。这种缺陷导致明显的骨骼畸形，继而导致严重的骨吸收，使人联想到无条件的MT1-基质金属蛋白酶缺乏。这些数据表明，OSX阳性细胞及其后代不仅影响骨形成，而且还调节骨吸收的重要方面。骨形成是普遍存在的MT1-MMP缺乏症的主要组成部分。这一观察结果与MT1-MMPs切割和脱落RANKL的能力是一致的，RANKL是破骨细胞分化过程中的一个关键的肿瘤坏死因子超家族成员。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(OCN)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介导的MT1-MMP消融导致小鼠大体正常，但在成年后表现出严重的骨丢失。因此，祖细胞介导的缺失会导致畸形和吸收，而成骨细胞特异性消融则主要导致骨吸收。由此可见，成熟的成骨细胞主要通过蛋白分解来调节骨吸收。 造血微环境对适当的骨形成和改建至关重要，我们重点关注MT-MMP活性在这个组织间隔中的作用。值得注意的是，缺乏MT-MMP活性的骨骼细胞不能支持异位骨形成中的出生后骨髓形成。我们认为MT-MMPs在造血干细胞的迁移和募集以及它们在潜在的造血环境中的填充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HSC募集到循环的细胞，我们证明了OSX特异性MT1-MMPs的丢失影响HSC到循环的动员。 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，这种重建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得到解决。利用软骨特异性的MT-MMP活性消融，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，MT1-MMPs在软骨中的丢失和MT3-MMPs的普遍丢失共同加剧了软骨的破坏，并导致严重的生长迟缓和侏儒症。

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 strain of mice. We subsequently deleted MT-MMP activity in a cell and stage-specific fashion to account for the cell and tissue specific functions of pericellular proteolysis mediated by MT-MMPs. To establish the role of MT-MMP activity in perivascular smooth muscle/pericyte-like cells, which are considered a potential reservoir of mesenchymal progenitors, we have ablated MT-MMP activity in SM22α-positive cells. Loss of MT1-MMP in this subset of cells leads to gross loss of bone in the cranium and in long bones where the trabecular and cortical bone content is dramatically reduced. Additionally, conspicuous fibrosis is associated with the diminished bone content. Together, these observations indicate that cells engaging in the formation of the skeleton to a large degree are recruited out of the perivascular cell pool and utilize MT1-MMP to exert their function. To further understand the function of pericellular proteolysis in cells committed to osteogenic fate through expression of the transcription factor, Sp7/Osx, MT1-MMP was deleted in this subset of cells. This deficit leads to overt skeletal dysmorphism and secondarily, a rampant bone resorption reminiscent of unconditional MT1-MMP deficiency. These data demonstrate that Osx positive cells and their progeny not only affect bone formation, a large component of the disease observed in universal MT1-MMP deficiency, but also regulate significant aspects of bone resorption. This observation is consistent with the ability of MT1-MMP and to cleave and shed RANKL, a critical TNFα superfamily member in osteoclast differentiation. 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 (Ocn) positive, mature osteoblasts. Unlike Osx specific deletion, Ocn mediated ablation of MT1-MMP leads to grossly normal mice, which however display rampant bone loss in adulthood. Thus, while progenitor mediated deletion results in dysmorphism and resorption, osteoblast specific ablation leads to mainly bone resorption. From this observation we deduce that the mature osteogenic cell mainly serves to regulate resorption through proteolysis. The hematopoietic micro-environment is critical for proper bone formation and remodeling and we have focused on the role of MT-MMP activity in this tissue compartment. Notably, skeletal cells devoid of MT-MMP activity fail to support postnatal marrow formation in ectopic bone formation. We propose that MT-MMPs plays a pivotal role in the migration and recruitment of hematopoietic stem cells and their ability to populate a potential hematopoietic environment. 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 recruitment of HSCs to the circulation, we demonstrated that Osx-specific loss of MT1-MMP affect the mobilization of HSC to the circulation. MT-MMP activity is required in remodeling of specific unmineralized cartilages in the skeleton. Until now, it has remained unresolved if this remodeling mechanism was a property of chondrocytes or connective tissue cells adjacent to the cartilage. Using cartilage specific ablation of MT-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 induce a severe growth retardation and dwarfism.