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。 重要的是，与小鼠中MT 1-MMP丢失相关的性状是人类消失性骨病温彻斯特综合征（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 1-MMP缺陷的多效性和MT 1-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，我们删除了MT 1-MMP活性在一个渐进的细胞成熟和发育阶段特定的方式在结缔组织分配细胞和组织特异性功能的MT 1-MMP介导的细胞周蛋白水解。 此外，我们已经解决了MT 1-MMP活性在骨髓来源的单核细胞/巨噬细胞/破骨细胞隔室中的作用，其中MT 1-MMP表达的意义知之甚少。 为了确定MT 1-MMP活性在周细胞样细胞（其中一些已被证明是骨骼干细胞）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。 朝向成骨命运的连续步骤是转录因子Sp 7/Osterix（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成为生物学无活性形式的能力一致。 MT 1-MMP在这种能力中的活性与RANKL诱饵、骨保护素（OPG）一起抑制破骨细胞募集。 这与MT 1-MMP在表达1型胶原（Col 1a 1 -2.3kb）的成熟成骨细胞中的功能形成鲜明对比。 与Osx特异性缺失不同，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，成熟成骨细胞特异性消融主要影响骨沉积，但不影响吸收。 在骨骼稳态和骨转换的背景下，破骨细胞是一种重要的细胞类型，它表达大量的MT 1-MMP。 然而，MT 1-MMP在骨细胞活性中的作用还不清楚。 因此，我们利用LysM-Cre小鼠特异性地消融破骨细胞中的MT 1-MMP，与其他细胞特异性缺失不同，这导致了骨小梁含量的增加。 因此，破骨细胞特异性MT 1-MMP缺陷不是MT 1-MMP缺陷小鼠中观察到的骨侵蚀的原因。 MT 1-MMP-缺陷型破骨细胞以相同数量形成，显示与野生型破骨细胞无法区分的形态，并保留降解矿化基质的不受损害的能力。 然而，它们在降解纤维胶原（骨衬骨膜的主要成分）的能力上确实显示出几乎完全的缺陷。 这种结缔组织是破骨细胞和矿化骨之间的屏障，必须在矿物质溶解之前降解。 这些结果突出了中性蛋白酶活性在破骨细胞中的作用，并解释了该细胞亚群中MT 1-MMP丢失后骨含量的增加。 与我们的合作者一起，我们一直在为骨纤维异常增殖症（FD）的研究建立各种小鼠模型。 FD是一种致残性骨骼疾病，其特征在于正常骨和骨髓被矿化不足、无组织的骨和纤维化骨髓替代，缺乏造血，导致受影响骨的畸形和骨折。 该疾病是由编码刺激性G蛋白Gs的α亚基的基因的合子后突变（R201 C，R201 H）引起的。这种疾病在人类中缺乏遗传性被认为反映了种系传播的激活性GS-α突变的胚胎致死性，这种突变只能通过体细胞嵌合体存活。 产生了组成型表达Gs R201 C的多个小鼠系，并发现其产生了人FD的遗传的精确复制品。 在所有组织和鼠胚胎干细胞中稳健的转基因表达与骨骼组织的正常发育和骨骼细胞的分化相关。 与人类疾病一样，小鼠FD病变仅在出生后发生。 发现病变通过三个不同的阶段发展：1）由过量骨形成和正常再吸收定义的建模阶段; 2）过度重塑阶段;和3）纤维发育不良阶段，其在>1岁的小鼠中再现了人骨病理学的完整复制品。 因此，Gs-alpha突变足以导致FD，并且与小鼠的生殖系传播和正常胚胎发育相容。 这些新的鼠系构成了FD的第一个模型。

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. Importantly, the traits associated with loss of MT1-MMP in the mouse are a remarkable phenocopy 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 pleiotropic nature of MT1-MMP deficiency and the widespread expression pattern of MT1-MMP in bone, bone-associated tissues and non-bone tissues, we have generated and utilized a conditional deletion mutation mouse strain. We subsequently deleted MT1-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 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 skeletal stem cells), 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/Osterix (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 into a biologically inactive form. MT1-MMP activity in this capacity works to suppress osteoclast recruitment in conjunction the RANKL decoy, Osteoprotegerin (OPG). This was in stark contrast to the function of MT1-MMP in Type 1 Collagen (Col1a1-2.3kb)-expressing, mature osteoblasts. Unlike Osx-specific deletion, Col1-mediated ablation of MT1-MMP led to grossly normal mice, which however display diminished bone mass in adulthood. Thus, while progenitor-specific deletion results in dysmorphism and resorption, mature osteoblast-specific ablation mainly affects bone apposition, but not resorption. In the context of skeletal homeostasis and bone turnover, one of the essential cell types is an 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, and unlike other cell specific-deletions, this led to increased trabecular bone content. Osteoclast-specific MT1-MMP deficiency consequently is not the cause of the 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 the postnatal life. The lesions were found to develop via three distinct stages: 1) a modeling phase defined by excess bone formation and normal resorption; 2) an excessive remodeling phase; and 3) a fibrous dysplastic phase, which reproduced a complete replica of the human bone pathology in mice of age >1 year. Thus, Gs-alpha mutations are sufficient to cause FD, and are compatible with germline transmission and normal embryonic development in mice. These novel murine lines constitute the first model of FD.