Identification of mesenchymal collagenases? ? MT1-MMP-deficient mice temporarily survive despite a severe skeletal phenotype due to collagen indigestion. This led us to ask whether collagen remodeling ultimately is required for development and growth or if mammalian cells can modify their behavior and response to the surroundings when peri-cellular collagen remodeling in disrupted. In an effort to address this issue, we initiated a search for molecules that might possess collagenolytic properties and potentially compensate for the loss of MT1-MMP, and thus explain the survival of MT1-MMP deficient mice. We established by in situ hybridization that the molecular relative of MT1-MMP, MT3-MMP, is expressed abundantly in skeletal tissues in a pattern overlapping in part that of MT1-MMP. To test the hypothesis that MT3-MMP can compensate for the loss of collagenolytic activity, we generated mice with targeted disruption of MT3-MMP. These mice display a limited yet significant effect phenotype in adulthood, with diminished growth of the cranium and limbs compared to wildtype littermates. Mice deficient for both MT3-MMP and MT1-MMP, on the other hand, display profound developmental defects in the appendicular and craniofacial skeleton, and die immediately after birth. The observed phenotypic traits are consistent with loss of a second collagenolytic enzyme, and demonstrate that MT3-MMP is the molecule responsible for survival of MT1-MMP deficient mice. Consistent with this notion, MT3-MMP confers collagenolytic activity to collagenase-deficient MT1-MMP-null cells when expressed in these cells in vitro. Collectively these data identify MT3-MMP as a major mesenchymal collagenase in vivo, and demonstrate that collagen remodeling is an essential property for embryonic development.? ? Semaphorin 4D is a substrate of MT1-MMP? ? While collagen is the major structural substrate of MT1-MMP, additional non-extracellular matrix substrates such as MMP-2 and MMP-13 have also been suggested as targets of MT1-MMP activity. We have identified semaphorin 4D as a novel substrate of MT1-MMP. Semaphorin 4D is effectively liberated from its membrane association by MT1-MMP to facilitate interaction with the receptor, plexin B1. This pathway for shedding of semaphorin 4D demonstrates a novel paradigm for MT1-MMP-mediated cell signaling in blood vessel formation and growth, and also suggests a role for MT1-MMP in developmental and pathophysiological context beyond merely removing structural molecules blocking cell migration and cell proliferation? ? MT1-MMP and Cartilage Remodeling? ? The maintenance of a permanent hyaline cartilage is essential for 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 both permissive extracellular matrices, as well as inhibitor levels in the local environment tightly regulate cartilage dissolution. Interestingly, the expression of MT1-MMP completely rescues the 33% pre-weaning death observed in MT1-MMP deficient offspring. An analysis of transgene expression pattern reveals abundant levels of message in the cartilage as intended with the use of a type II collagen promoter. Moreover, immunoreactive MT1-MMP was detected in cartilage extracts and cultured chondrocytes. Contrary to expectations, 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 cranium and long bones of wild type mice do indeed express immunoreactive 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 an MT1-MMP deficient 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. To test the hypothesis that type II collagen expression facilitates expression of the MT1-MMP transgene in osteogenic cells and thus increases bone formation, we generated bone marrow stromal cell cultures and tested their osteogenic potential in vivo with or without collagen II-driven MT1-MMP expression. Confirming our initial observation in transgene expressing mice, cells carrying the transgene formed ectopic ossicles with more abundant bone than the null counterpart when implanted with and osteoconductive scaffold in nude mice. These data suggest that bone cells express type II collagen even after culture and that this expression is sufficient to drive MT1-MMP expression necessary for osteogenesis.? ? In summary our data demonstrate 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 accumulates in phagosomes, whereas the phagolysosomal compartment is devoid of collagen. Based on the literature describing phagocytic collagen degradation, we inferred 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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