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The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis of large collagen fragments. First, we show that collagen that has been pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native collagen by uPARAP/Endo180-positive cells. Second, we demonstrate that this preference is governed by the acquisition of a gelatin-like structure by the collagen, occurring upon collagenase-mediated cleavage under native conditions. Third, we demonstrate that the growth of uPARAP/Endo180-deficient fibroblasts on a native collagen matrix leads to substantial extracellular accumulation of well defined collagen fragments, whereas, wild-type fibroblasts possess the ability to direct an organized and complete degradation sequence comprising both the initial cleavage, the endocytic uptake, and the intracellular breakdown of collagen.

Collagen is the most abundant extracellular matrix protein in connective tissues of higher animals. The growth of connective tissues is intimately linked to the ability to model and remodel the collagen-rich matrices of the organism at critical points during development and growth to allow expansion and adaptation of tissue interfaces. The mammalian cranium is one such place where collagen remodeling is required for proper growth, and this review explores the consequences of abrogated collagen remodeling as they materialize in a mouse model deficient for the membrane type 1 matrix metalloproteinase., (2005 S. Karger AG, Basel)

Gene ablation in mice offers a powerful tool to assay in vivo the role of selected molecules. Numerous new mouse models of matrix metalloproteinases (MMP) deficiency have been developed in the past 5 years and have yielded a new understanding of the role of MMPs while also putting to rest assumptions based on data predating the days of mouse models. The phenotype of the MT1-MMP deficient mouse is one example which illustrates the sometimes rather surprising insights into extracellular matrix remodeling in development and growth that can be gained with mouse genetics. While MT1-MMP appears to play little or no role in embryonic development, loss of this enzyme results in progressive impairment of postnatal growth and development affecting both the skeleton and the soft connective tissues. The underlying pathologic mechanism is loss of an indispensable collagenolytic activity, which remains essentially uncompensated. Our findings demonstrate that growth and maintenance of the skeleton requires coordinated and simultaneous MT1-MMP-dependent remodeling of all soft tissue attachments (ligaments, tendons, joint capsules). We note that the phenotype of the MT1-MMP deficient mouse bears no resemblance to those of mice deficient in MMP-2 and tissue inhibitors of metallo-proteinase (TIMP)-2 all but dispelling the view that activation of MMP-2 by the MT1-MMP/TIMP-2/proMMP-2 axis plays a significant role in growth and development throughout life. It is of interest to note that loss of a single catabolic function such as selective collagen degradation mediated by MT1-MMP gives rise to profound impairment of a number of both anabolic and catabolic functions., (Copyright 2004 Wiley-Liss, Inc.)

Matrix metalloproteinase-14 is required for degradation of fibrillar collagen by mesenchymal cells. Here we show that keratinocytes use an alternative plasminogen and matrix metalloproteinase-13-dependent pathway for dissolution of collagen fibrils. Primary keratinocytes displayed an absolute requirement for serum to dissolve collagen. Dissolution of collagen was abolished in plasminogen-depleted serum and could be restored by the exogenous addition of plasminogen. Both plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase blocked collagen dissolution, demonstrating the requirement of both plasminogen activation and matrix metalloproteinase activity for degradation. Cell surface plasmin activity was critical for the degradation process as aprotinin, but not alpha(2)-antiplasmin, prevented collagen dissolution. Keratinocytes with single deficiencies in either urokinase or tissue plasminogen activator retained the ability to dissolve collagen. However, collagen fibril dissolution was abolished in keratinocytes with a combined deficiency in both urokinase and tissue plasminogen activator. Combined, but not single, urokinase and tissue plasminogen activator deficiency also completely blocked the activation of the fibrillar collagenase, matrix metalloproteinase-13, by keratinocytes. The activation of matrix metalloproteinase-13 in normal keratinocytes was prevented by plasminogen activator inhibitor-1 and aprotinin but not by tissue inhibitor of metalloproteinase-1 and -2, suggesting that plasmin activates matrix metalloproteinase-13 directly. We propose that plasminogen activation facilitates keratinocyte-mediated collagen breakdown via the direct activation of matrix metalloproteinase-13 and possibly other fibrillar collagenases.

A well-coordinated remodeling of uncalcified collagen matrices is a pre-requisite for bone development and homeostasis. Collagen turnover proceeds through different pathways, either involving extracellular reactions exclusively, or being dependent on endocytic processes. Extracellular collagen degradation requires the action of secreted or membrane attached collagenolytic proteases, whereas the alternative collagen degradation pathway proceeds intracellularly after receptor-mediated uptake and delivery to the lysosomes. In this study we have examined the functional interplay between the extracellular collagenase, MMP-2, and the endocytic collagen receptor, uPARAP, by generating mice with combined deficiency of both components. In both uPARAP-deficient and MMP-2-deficient adult mice the length of the tibia and femur was decreased, along with a reduced bone mineral density and trabecular bone quality. An additional decrease in bone length was observed when combining the two deficiencies, pointing to both components being important for the remodeling processes in long bone growth. In agreement with results found by others, a different effect of MMP-2 deficiency was observed in the distinct bone structures of the calvaria. These membranous bones were found to be thickened in MMP-2-deficient mice, an effect likely to be related to an accompanying defect in the canalicular system. Surprisingly, both of the latter defects in MMP-2-deficient mice were counteracted by concurrent uPARAP deficiency, demonstrating that the collagen receptor does not support the same matrix remodeling processes as the MMP in the growth of the skull. We conclude that both uPARAP and MMP-2 take part in matrix turnover processes important for bone growth. However, in some physiological situations, these two components do not support the same step in the growth process. [ABSTRACT FROM AUTHOR]

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation. [ABSTRACT FROM AUTHOR]

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype. [ABSTRACT FROM AUTHOR]

Collagen degradation is essential for cell migration, proliferation, and differentiation. Two key turnover pathways have been described for collagen: intracellular cathepsin-mediated degradation and pericellular collagenase-mediated degradation. However, the functional relationship between these two pathways is unclear and even controversial. Here we show that intracellular and pericellular collagen turnover pathways have complementary roles in vivo. Individual deficits in intracellular collagen degradation (urokinase plasminogen activator receptor-associated protein/Endo180 ablation) or pericellular collagen degradation (membrane type 1-matrix metalloproteinase ablation) were compatible with development and survival. Their combined deficits, however, synergized to cause postnatal death by severely impairing bone formation. Interestingly, this was mechanistically linked to the proliferative failure and poor survival of cartilage- and bone-forming cells within their collagen-rich microenvironment. These findings have important implications for the use of pharmacological inhibitors of collagenase activity to prevent connective tissue destruction in a variety of diseases. [ABSTRACT FROM AUTHOR]