The Matrix Metalloproteinase Unit in the Craniofacial and Skeletal Diseases Branch works towards understanding the biological roles of Matrix Metalloproteinases (MMPs) and their inhibitors in development, homeostasis and disease, with specific emphasis on the role of MMPs in the skeletal and peri-skeletal tissues. The MMP family is considered important in tissue remodeling and our studies using in vitro as well as mouse genetic approaches have demonstrated that MT1-MMP, a membrane-bound MMP, is essential for the timely removal of type I and type II collagen in non-mineralized matrices prior to mineralization events. We have maintained efforts to specifically identify substrates of the Membrane-Type MMPs, as well as their regulation in normal and disease processes.? ? Dissolution of Unmineralized Cartilages:? ? Bone is initially formed by two distinctly different processes leading to the establishment of a highly mineralized organic matrix of exceptional strength and flexibility. During endochondral bone formation, a cartilage template of embryonic origin undergoes a coordinated process of hypertrophy, mineralization, and partial removal by osteoclasts, leaving a scaffold on which bone cells deposit bone matrix. In contrast, intramembranous ossification proceeds by differentiation of bone cells and ensuing matrix deposition within a soft connective tissue to generate neurocranial bones and the cortices of most if not all bones.? ? Generation and analysis of the MT1-MMP deficient mouse revealed that even membranous bone invariably forms in association with a cartilage template. The properties separating membranous bone formation from endochondral bone formation is the mineralization state of the associated cartilage matrix. Endochondral bone formation takes place within a cartilage anlage whereas membranous bone formation takes place associated with, but physically removed from the cartilage. In the latter process, the cartilage is fully degraded as bone formation progresses, but importantly, this dissolution of cartilage happens without progression through classical hypertrophy, mineralization and osteoclast resorption. Instead, the cartilage is depleted of proteoglycans and subsequently the collagenous remnant is digested as resident chondrocytes increase MT1-MMP expression. This in turn, leads to a loss of cell-matrix contact and subsequently the chondrocytes undergo apoptotic demise. ? ? These novel observations moreover identify that cartilage is a template for bone almost without exception and that a substantial number of cartilages in the mouse maintain an unmineralized status, which render them resistant to osteoclast resorption. Furthermore, this mechanism has revealed that unmineralized cartilages maintain resistance to proteolysis in part through a continued proteoglycan synthesis or suppression of their degradation. The loss of proteoglycan enables chondrocytes to degrade their resident matrix by expression of MT1-MMP, which we have now demonstrated, endows chondrocytes with the ability to degrade collagen. ? ? MT1-MMP and Articular Cartilage Remodeling:? ? The maintenance of a permanent hyaline cartilage is essential to joint homeostasis, however, we observed in our analysis of MT1-MMP deficient animals that the interface between cartilage and bone is a site critically dependent on timely remodeling. We have shown that deficiency in this process significantly impairs growth and generates a severe arthropathy. This highlights the significance of MT1-MMP in cartilage degradation in the process of growth, but also suggests that MT1-MMP may be a significant proteolytic enzyme in pathological joint destruction. In an effort to understand this mechanism further we have directed MT1-MMP expression exclusively into chondrocytes using a type II collagen promoter/enhancer-driven MT1-MMP transgene. When this transgene is bred in to an MT1-MMP deficient background only collagen type II-expressing cells will express MT1-MMP and allow evaluation of MT1-MMP function in this cartilaginous tissues. Surprisingly, we observed none of the expected effects of cartilage degradation suggesting that cartilage dissolution is tightly regulated by both permissive extracellular matrices as well as inhibitor levels in the local environment. Interestingly, the expression of MT1-MMP completely rescues the 33% pre-weaning death observed in MT1-MMP deficient offspring. An analysis of the transgene expression pattern reveals abundant levels of message in the cartilage as intended with the use of a type II collagen promoter. In addition, the transgene is also expressed in bone. To rule out that this latter observation was caused by ectopic mis-expression of the transgene, we confirmed that bone lining cells in the head and long bones of wild type mice indeed express type II collagen. The observed expression of MT1-MMP in both bone and cartilage is therefore a natural consequence of type II collagen promoter activity in these tissues. Furthermore, mice carrying a type II collagen promoter-driven MT1-MMP transgene in a KO background display a significant gain in bone in both the cranium and the axial/appendicular skeleton. This was accompanied by a significant increase in both body weight and median lifespan. ? ? Based on these observations, we can state with certainty that the peri-natal and adolescent lethality in the MT1-MMP deficient mice is a consequence of insufficient expression solely in skeletal tissues and that the proper remodeling and development of the skeleton mediated by MT1-MMP in chondrocytes and bone cells is of utmost importance to viability in mice. Moreover, these findings have highlighted that type II collagen expression is not solely confined to chondrocytes, but is also abundant in at least a subset of bone cells at both the messenger RNA and protein levels. This leads us to suggest that at least a subset of bone cells have descended from a common type II collagen-expressing progenitor or stem cell, or have differentiated from a chondrocyte to a bone cell.? ? Extracellular and intracellular collagen Metabolism:? ? Coarse cross-banded intracellular collagen inclusions is a conspicuous finding associated with MT1-MMP deficiency. We have documented in detail that collagen is accumulating in the phagosomes whereas the phagolysosomal compartment is devoid of collagen. Based on the literature describing phagocytic collagen degradation, we infer that loss of pericellular collagenase activity is causing a switch in the degradative pathway that leads to an overload of phagocytic uptake. To test this hypothesis, we utilized mice deficient for uPARAP/endo180, which is required for integrin independent uptake of fibrillar collagen into the lysosomal compartment. Mice double deficient for MT1-MMP and uPARAP are viable, but uniformly die within the first nine days after birth. Our analysis of these animals demonstrate that they suffer from diminished bone formation in both the craniofacial and appendicular/axial skeleton over and above what is observed in MT1-MMP single deficient mice. At the cellular level, double deficiency for these two proteins specifically leads to loss of viability in chondrocytes and bone cells, explaining the severe delay in skeletal development that ultimately leads to premature death of the double deficient mice. Taken together, these observations highlight the important residual capacity of the cell for processing collagen by means of uPARAP mediated intracellular uptake in cases when the matrix load overwhelms the peri-cellular proteolysis.
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