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.
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