Kenn Holmbeck

MT1-MMP-Deficient Mice Develop Dwarfism, Osteopenia, Arthritis, and Connective Tissue Disease due to Inadequate Collagen Turnover

MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP

As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from gene-targeted mice, a matrix metalloproteinase (MMP)–dependent activity is identified that drives invasion independently of plasminogen, the gelatinase A/TIMP-2 axis, gelatinase B, collagenase-3, collagenase-2, or stromelysin-1. In contrast, deleting or suppressing expression of the membrane-tethered MMP, MT1-MMP, in fibroblasts or tumor cells results in a loss of collagenolytic and invasive activity in vitro or in vivo. Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.

MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix.

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion

Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models.

Matrix Metalloproteinases (MMPs) Regulate Fibrin-invasive Activity via MT1-MMP–dependent and –independent Processes

Cross-linked fibrin is deposited in tissues surrounding wounds, inflammatory sites, or tumors and serves not only as a supporting substratum for trafficking cells, but also as a structural barrier to invasion. While the plasminogen activator-plasminogen axis provides cells with a powerful fibrinolytic system, plasminogen-deleted animals use alternate proteolytic processes that allow fibrin invasion to proceed normally. Using fibroblasts recovered from wild-type or gene-deleted mice, invasion of three-dimensional fibrin gels proceeded in a matrix metalloproteinase (MMP)-dependent fashion. Consistent with earlier studies supporting a singular role for the membrane-anchored MMP, MT1-MMP, in fibrin-invasive events, fibroblasts from MT1-MMP–null mice displayed an early defect in invasion. However, MT1-MMP–deleted fibroblasts circumvented this early deficiency and exhibited compensatory fibrin-invasive activity. The MT1-MMP–independent process was sensitive to MMP inhibitors that target membrane-anchored MMPs, and further studies identified MT2-MMP and MT3-MMP, but not MT4-MMP, as alternate pro-invasive factors. Given the widespread distribution of MT1-, 2-, and 3-MMP in normal and neoplastic cells, these data identify a subset of membrane-anchored MMPs that operate in an autonomous fashion to drive fibrin-invasive activity.

MT1-MMP: A tethered collagenase†

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.

The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.

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.

MT1-MMP Controls Tumor-induced Angiogenesis through the Release of Semaphorin 4D

The semaphorins are a family of proteins originally identified as regulators of axon growth that recently have been implicated in blood vessel development. The plexins are high affinity receptors for the semaphorins and are responsible for initiation of signaling upon ligation. Emerging evidence indicates that many human cancers overexpress Semaphorin 4D, which promotes neovascularization upon stimulating its receptor, Plexin-B1, on endothelial cells. However, to exert its pro-angiogenic functions, Semaphorin 4D must be processed and released from its membrane bound form to act in a paracrine manner on endothelial cells. Here we show that Semaphorin 4D is a novel target for the membrane-tethered collagenase membrane type 1-matrix metalloproteinase. We demonstrate that this metalloproteinase, which is not expressed in normal or immortal but non-tumorigenic epithelial cell lines, was present in several head and neck squamous cell carcinoma cell lines and was required for processing and release of Semaphorin 4D into its soluble form from these cells, thereby inducing endothelial cell chemotaxis in vitro and blood vessel growth in vivo. These results suggest that the proteolytic cleavage of Semaphorin 4D may provide a novel molecular mechanism by which membrane type 1-matrix metalloproteinase controls tumor-induced angiogenesis.

uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor–associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

MT1-MMP–dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)–dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of “ghost” cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.