[Show abstract][Hide abstract] ABSTRACT: 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.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 10/2008; Chapter 10:Unit 10.8. DOI:10.1002/0471143030.cb1008s40
[Show abstract][Hide abstract] ABSTRACT: Strains of B. gingivalis were shown to produce collagenolytic activity capable of dissolving reconstituted collagen (type I) fibrils and of cleaving the helical domain of types I. II and III collagens at 22° C. The catalytic activity was dependent on free thiol groups and on metal ions, as indicated by inhibition by thiol blocking reagents and metal chelators. The activity was associated with the bacterial cells and was not secreted to the medium. Under optimal conditions. 100 Units of collagenase per gram cell pellet (wet weight) were released by detergents such as Triton X-100 and SDS. Zymography of detergent extracts revealed that collagen-degrading strains, but not an inactive control strain (W), contained a discrete Mr 90 000 gelatin cleaving protease which may be identical to the collagenolytic enzyme. The initial attack on the helical domain of type I collagen occurred near the COOH-terminus. The a1 and a2 chains were cleaved at the same site, generating a major helical fragment consisting of three shortened (Mr 82 000) a-chains. Subsequent cleavages of this shortened collagen molecule resulted in generation of multiple fragments from the component a-chains in the Mr 60 000 to 6000 range. This cleavage pattern was clearly distinct from the characteristic 3/4–1/4 pattern produced by vertebrate collagenases. Type II and III collagens were also cleaved first near the COOH-terminus, generating fragments of similar size to those produced from type I collagen. In view of its ability to dissolve reconstituted collagen fibrils at 35°C and its ability to attack the helical domain of interstitial collagens in solution at 22°C, we suggest that this enzyme tentatively be classified as a true collagenase.
Journal of Periodontal Research 06/2006; 23(4):258 - 264. DOI:10.1111/j.1600-0765.1988.tb01369.x · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. Instead, we identify the membrane-anchored matrix metalloproteinase, MT1-MMP, as the key pericellular collagenolysin that controls the ability of VSMCs to degrade and infiltrate 3-D barriers of interstitial collagen, including the arterial wall. Furthermore, genetic deletion of the proteinase affords mice with a protected status against neointimal hyperplasia and lumen narrowing in vivo. These studies suggest that therapeutic interventions designed to target MT1-MMP could prove beneficial in a range of human vascular disease states associated with the destructive remodeling of the vessel wall extracellular matrix.
Journal of Experimental Medicine 10/2005; 202(5):663-71. DOI:10.1084/jem.20050607 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. In contrast, MT2-MMP was found exclusively in the mammary epithelium. Lactating gland expressed none of the examined MT-MMPs. Mammary gland tumors expressed MT1-MMP, MT2-MMP, and MT3-MMP while MT4-MMP was not expressed in any developmental or cancerous stage analyzed here. Our results suggest that MT1-MMP, MT2-MMP, and MT3-MMP may be involved in remodeling of both the normal and diseased mammary gland either directly or indirectly by activation of other MMPs.
[Show abstract][Hide abstract] ABSTRACT: 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. In the developing vasculature, mural cell recruitment is associated with the functionally undefined expression of the type I transmembrane proteinase, membrane-type 1 matrix metalloproteinase (MT1-MMP). In this paper, using VSMCs and tissues isolated from gene-targeted mice, we identify MT1-MMP as a PDGF-B-selective regulator of PDGFRbeta-dependent signal transduction and mural cell function. In VSMCs, catalytically active MT1-MMP associates with PDGFRbeta in membrane complexes that support the efficient induction of mitogenic signaling by PDGF-B in a matrix metalloproteinase inhibitor-sensitive fashion. In contrast, MT1-MMP-deficient VSMCs display PDGF-B-selective defects in chemotaxis and proliferation as well as ERK1/2 and Akt activation that can be rescued in tandem fashion following retroviral transduction with the wild-type protease. Consistent with these in vitro findings, MT1-MMP-deficient brain tissues display a marked reduction in mural cell density as well as abnormal vessel wall morphology similar to that reported in mice expressing PDGF-B or PDGFRbeta hypomorphic alleles. Together, these data identify MT1-MMP as a novel proteolytic modifier of PDGF-B/PDGFRbeta signal transduction that cooperatively regulates vessel wall architecture in vivo.
Genes & Development 05/2005; 19(8):979-91. DOI:10.1101/gad.1294605 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 < 0.01). MT1-MMP-/- airways and alveoli had an abnormal ultrastructural appearance, but epithelial cell differentiation markers were distributed similarly in both strains. There was no evidence of excess extracellular matrix deposition or inflammation at the time points examined. In contrast, by adulthood MMP-2-/- mice had normal alveolar size and structure, indicating normal alveolar development was not dependent on the ability of MT1-MMP to activate pro-MMP-2. These data indicate that MT1-MMP is required for normal lung development.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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., beta3 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.
The Journal of Cell Biology 12/2004; 167(4):757-67. DOI:10.1083/jcb.200405001 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Cell Biology 12/2004; 167(4):769-81. DOI:10.1083/jcb.200408028 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. These mice are viable and fertile and do not display any obvious abnormalities. However, Mmp19-null mice develop a diet-induced obesity due to adipocyte hypertrophy and exhibit decreased susceptibility to skin tumors induced by chemical carcinogens. Based on these results, we suggest that this enzyme plays an in vivo role in some of the tissue remodeling events associated with adipogenesis, as well as in pathological processes such as tumor progression.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Cell Biology 12/2003; 163(3):661-71. DOI:10.1083/jcb.200307061 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Cell 09/2003; 4(2):83-4. DOI:10.1016/S1535-6108(03)00196-X · 23.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Cell Biology 04/2003; 160(7):1009-15. DOI:10.1083/jcb.200211091 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 03/2003; Chapter 10:Unit 10.8. DOI:10.1002/0471143030.cb1008s17
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivobiological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although
mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype.
Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has
hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses.
Our findings demonstrate that enamelysin activity is essential for proper enamel development.
[Show abstract][Hide abstract] ABSTRACT: 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. The data showed that both eruption and root elongation were severely inhibited in animals lacking the enzyme. We also found, in periodontal ligament fibroblasts from MT1-MMP-deficient mice, a massive age-related accumulation (up to 60-fold over controls) of collagen fibril-containing phagosomes. Phagolysosomes, which represent the next downstream step in collagen fibril degradation by the lysosomal pathway, did not accumulate. These observations indicate that MT1-MMP plays a central role in periodontal remodeling. The stunted root growth and the failure to erupt indicate the important role of the enzyme in tooth development.
European Journal Of Oral Sciences 01/2003; 110(6):445-51. DOI:10.1034/j.1600-0722.2002.21384.x · 1.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Experimental Medicine 03/2002; 195(3):295-308. · 12.52 Impact Factor