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小鼠的条件性缺失突变品系。 随后，我们删除MT-MMP活性的细胞和阶段特异性的方式来解释细胞和组织特异性功能的细胞周蛋白水解介导的MT-MMP。 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库，我们已经消融了SM 22阳性细胞中的MT-MMP活性。 在该细胞亚群中MT 1-MMP的损失导致颅骨和长骨中骨的总损失，其中骨小梁和皮质骨含量显著降低。 此外，明显的纤维化与骨含量减少有关。 总之，这些观察结果表明，参与骨骼形成的细胞在很大程度上是从血管周围细胞库中招募出来的，并利用MT 1-MMP发挥其功能。 为了进一步了解细胞周围蛋白水解在通过表达转录因子Sp 7/Osx而致力于成骨命运的细胞中的功能，在该细胞亚群中缺失MT 1-MMP。 这种缺陷导致明显的骨骼畸形，其次是猖獗的骨吸收，使人想起无条件的MT 1-MMP缺陷。 这些数据表明，Osx阳性细胞及其后代不仅影响骨形成，在普遍的MT 1-MMP缺乏症中观察到的疾病的大部分，而且还调节骨吸收的重要方面。 这一观察结果与MT 1-MMP切割和脱落RANKL的能力一致，RANKL是破骨细胞分化中的关键TNF超家族成员。 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蛋白水解的作用与骨钙素（OCN）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猖獗的骨丢失。 因此，虽然祖细胞介导的缺失导致畸形和吸收，成骨细胞特异性消融主要导致骨吸收。 从这一观察，我们推断，成熟的成骨细胞主要是通过蛋白水解调节吸收。 造血微环境对于适当的骨形成和重建是至关重要的，并且我们已经集中于MT-MMP活性在该组织隔室中的作用。 值得注意的是，缺乏MT-MMP活性的骨骼细胞不能支持出生后异位骨形成中的骨髓形成。 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HSC向循环募集的细胞，我们证明了MT 1-MMP的Osx特异性损失影响HSC向循环的动员。 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-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。

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.