Matrix metalloproteinases.

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods--cell-mediated dissolution of type-1 collagen fibrils, direct and reverse zymography, enzyme capture based on alpha2-macroglobulin and TIMP-1 and -2, and demonstration of cryptic thiol groups in metalloproteinase precursors--that are used to characterize the functions of matrix metalloproteinases and their inhibitors.

Matrix metalloproteinases.

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods-cell-mediated dissolution of type I collagen fibrils, direct and reverse zymography, enzyme capture based on a-2 macroglubulin and TIMP-1 and -2, and demonstration of crytic thiol groups in metalloproteinase precursors-that are used to characterize the functions of matrix metalloproteinases and their inhibitors.

MT1-matrix metalloproteinase directs arterial wall invasion and neointima formation by vascular smooth muscle cells.

During pathologic vessel remodeling, vascular smooth muscle cells (VSMCs) embedded within the collagen-rich matrix of the artery wall mobilize uncharacterized proteolytic systems to infiltrate the subendothelial space and generate neointimal lesions. Although the VSMC-derived serine proteinases, plasminogen activator and plasminogen, the cysteine proteinases, cathepsins L, S, and K, and the matrix metalloproteinases MMP-2 and MMP-9 have each been linked to pathologic matrix-remodeling states in vitro and in vivo, the role that these or other proteinases play in allowing VSMCs to negotiate the three-dimensional (3-D) cross-linked extracellular matrix of the arterial wall remains undefined. Herein, we demonstrate that VSMCs proteolytically remodel and invade collagenous barriers independently of plasmin, cathepsins L, S, or K, MMP-2, or MMP-9.

Expression pattern of four membrane-type matrix metalloproteinases in the normal and diseased mouse mammary gland.

Both mammary gland development and mammary carcinogenesis involve extensive remodeling of the mammary gland extracellular matrix. The expression of four membrane-type matrix metalloproteinases (MT-MMPs) with matrix remodeling potential in development and tumorigenesis was evaluated by in-situ hybridization on mouse mammary gland sections. MT1-MMP and MT3-MMP were found in the mammary stroma mainly around epithelial structures in both developing and mature mammary gland.

An MT1-MMP-PDGF receptor-beta axis regulates mural cell investment of the microvasculature.

Platelet-derived growth factor (PDGF)/PDGFRbeta-dependent investment of the vascular endothelium by mural cells (i.e., pericytes and vascular smooth muscle cells; VSMCs) is critical for normal vessel wall structure and function.

Membrane-type 1 matrix metalloproteinase is required for normal alveolar development.

Matrix metalloproteinases (MMPs) are expressed during lung development, but their role may be limited, as mice deficient in MMP-3, 7, 9, or 12 develop a normal adult lung. Because membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed in the developing lung epithelium, we examined the lung structure of MT1-MMP-deficient (-/-) mice. Branching morphogenesis was normal, but alveolar development was abnormal in the MT1-MMP-/- lungs with 40% less alveolar surface area at 1 month (P < 0.

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.

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.

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.

Diet-induced obesity and reduced skin cancer susceptibility in matrix metalloproteinase 19-deficient mice.

Matrix metalloproteinase 19 (MMP-19) is a member of the MMP family of endopeptidases that, in contrast to most MMPs, is widely expressed in human tissues under normal quiescent conditions. MMP-19 has been found to be associated with ovulation and angiogenic processes and is deregulated in diverse pathological conditions such as rheumatoid arthritis and cancer. To gain further insights into the in vivo functions of this protease, we have generated mutant mice deficient in Mmp19.

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.

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.

MT1-mmp: a collagenase essential for tumor cell invasive growth.

The manuscript discussed in this preview describes that reconstituted three-dimensional extracellular matrices such as fibrillar collagen and fibrin exert stringent territorial growth control on cells. The authors show that tumor cells are able to escape the matrix-enforced growth control effect (entrapment) by pericellular proteolysis mediated by MT1-MMP, a membrane bound matrix metalloproteinase capable of directly cleaving both type I collagen and fibrin but not by other, soluble matrix metalloprotinases. These data convincingly demonstrate one way that tumor cells orchestrate proteolysis to invade surrounding tissues.

Inhibition of molar eruption and root elongation in MT1-MMP-deficient mice.

To study whether eruption of teeth and root growth require remodeling of collagen in the peridental tissues, we studied molar development in mice deficient in MT1-MMP, an enzyme essential for remodeling of soft tissue-hard tissue interfaces. The lower jaws of deficient mice and their wildtype littermates were subjected to stereologic analysis. It was shown that in deficient animals, eruption and root elongation were severely inhibited, signifying a role of the enzyme in these developmental processes.

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.

On the role of MT1-MMP, a matrix metalloproteinase essential to collagen remodeling, in murine molar eruption and root growth.

Although the connective tissues of the periodontium are subject to a high turnover rate, no conclusive evidence has yet emerged that periodontal collagen turnover is essential for the eruption of teeth or for root elongation. These processes were studied in mice deficient in MT1-MMP, a membrane type matrix metalloproteinase essential for remodeling of soft tissue-hard tissue interfaces. Mandibular first molars of deficient mice and their wild-type littermates were subjected to stereological analysis in order to assess root length, eruption and the volume density of phagocytosed collagen in periodontal ligament fibroblasts.

Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype.

Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo.

Collagen dissolution by keratinocytes requires cell surface plasminogen activation and matrix metalloproteinase activity.

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.

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.

MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: regulation by fibrillar collagen.

Human skin fibroblasts were cultured long-term in the presence of ascorbic acid to allow formation of a three-dimensional collagen matrix, and the effects of this on activation of secreted matrix metalloproteinase-2 (MMP-2) were examined. Accumulation of collagen over time correlated with increased levels of both mature MMP-2 and cell-associated membrane type 1-MMP (MT1-MMP), and subsequently increased mRNA levels for MT1-MMP, providing temporal resolution of the "nontranscriptional" and "transcriptional" effects of collagen on MT-1MMP functionality. MMP-2 activation by these cultures was blocked by inhibitors of prolyl-4-hydroxylase, or when fibroblasts derived from the collagen alpha1(I) gene-deficient Mov-13 mouse were used.

A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondrial invasion and osteogenic differentiation during bone formation.

Tissue-remodeling processes are largely mediated by members of the matrix metalloproteinase (MMP) family of endopeptidases whose expression is strictly controlled both spatially and temporally. In this article, we have examined the molecular mechanisms that could contribute to modulate the expression of MMPs like collagenase-3 and MT1-MMP during bone formation. We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells.

The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis.

The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify intramembranously, and developing long bones undergoing endochondral ossification.

Using HSV-thymidine kinase for safety in an allogeneic salivary graft cell line.

Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland).

Gelatinase A (MMP-2) activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form.

The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation.

Monocyte membrane type 1-matrix metalloproteinase. Prostaglandin-dependent regulation and role in metalloproteinase-2 activation.

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs).