RESUMEN
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.
Asunto(s)
Metaloendopeptidasas/metabolismo , Invasividad Neoplásica , Neoplasias/enzimología , Neoplasias/patología , Animales , División Celular , Colágeno Tipo I/metabolismo , Fibrina/metabolismo , Metaloproteinasas de la Matriz Asociadas a la MembranaRESUMEN
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.
Asunto(s)
Arterias/metabolismo , Movimiento Celular/fisiología , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Miocitos del Músculo Liso/metabolismo , Enfermedades Vasculares/metabolismo , Animales , Apoptosis/fisiología , Arterias/ultraestructura , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Técnicas de Transferencia de Gen , Etiquetado Corte-Fin in Situ , Masculino , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Ratones Mutantes , Microscopía Electrónica , Miocitos del Músculo Liso/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Fibrina/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Metaloendopeptidasas/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Perros , Fibrinólisis , Fibroblastos/citología , Fibroblastos/metabolismo , Metaloproteinasa 14 de la Matriz , Metaloproteinasa 15 de la Matriz , Metaloproteinasa 16 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Metalotioneína 3 , Ratones , Ratones Noqueados , TransfecciónRESUMEN
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.
Asunto(s)
Apoptosis/genética , Huesos/embriología , Huesos/enzimología , Cartílago/enzimología , Condrocitos/enzimología , Metaloendopeptidasas/deficiencia , Animales , Remodelación Ósea/genética , Huesos/citología , Cartílago/citología , Linaje de la Célula/genética , Condrocitos/citología , Tejido Conectivo/embriología , Tejido Conectivo/enzimología , Regulación del Desarrollo de la Expresión Génica/genética , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Metamorfosis Biológica/genética , Ratones , Ratones Noqueados , Osteogénesis/genética , Cráneo/anomalías , Cráneo/enzimología , Cráneo/patologíaRESUMEN
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.
Asunto(s)
Vasos Sanguíneos/metabolismo , Colágeno Tipo I/metabolismo , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Metaloendopeptidasas/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Vasos Sanguíneos/citología , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Células Endoteliales/citología , Marcación de Gen , Humanos , Receptores de Hialuranos/metabolismo , Integrina beta3/metabolismo , Masculino , Metaloproteinasa 14 de la Matriz , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Modelos Biológicos , Fenotipo , Plasminógeno/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Movimiento Celular/genética , Matriz Extracelular/metabolismo , Metaloendopeptidasas/metabolismo , Neoplasias/enzimología , Animales , Células Cultivadas , Embrión de Pollo , Técnicas de Cocultivo , Colágeno/metabolismo , Colagenasas/metabolismo , Fibroblastos/citología , Fibroblastos/enzimología , Marcación de Gen , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 14 de la Matriz , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Invasividad Neoplásica , Neoplasias/genética , FenotipoRESUMEN
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.
Asunto(s)
Colágeno/metabolismo , Endocitosis , Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Colagenasas/metabolismo , Fibronectinas/metabolismo , Eliminación de Gen , Metaloproteinasa 13 de la Matriz , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Receptores de Superficie Celular/química , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores Mitogénicos/química , Receptores Mitogénicos/deficiencia , Receptores Mitogénicos/genética , Receptores Mitogénicos/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Transferrina/metabolismoRESUMEN
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.
Asunto(s)
Metaloendopeptidasas/deficiencia , Obesidad/etiología , Neoplasias Cutáneas/prevención & control , Adipocitos/enzimología , Adipocitos/patología , Animales , Secuencia de Bases , Carcinógenos/toxicidad , Tamaño de la Célula , ADN Complementario/genética , Dieta , Femenino , Marcación de Gen , Humanos , Técnicas In Vitro , Masculino , Metaloproteinasas de la Matriz Secretadas , Metaloendopeptidasas/genética , Metaloendopeptidasas/fisiología , Metilcolantreno/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica , Obesidad/enzimología , Obesidad/genética , Obesidad/patología , Fenotipo , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genéticaRESUMEN
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.
Asunto(s)
Bioquímica/métodos , Metaloproteinasas de la Matriz/metabolismo , Animales , Colágeno/metabolismo , Expresión Génica , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/genética , Ratas , Coloración y Etiquetado , Tendones/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , alfa-Macroglobulinas/metabolismoRESUMEN
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.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/genética , Glándulas Mamarias Animales/enzimología , Neoplasias Mamarias Animales/enzimología , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Envejecimiento/fisiología , Animales , Femenino , Salud , Lactancia/genética , Lactancia/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Metaloproteinasa 14 de la Matriz , Metaloproteinasa 15 de la Matriz , Metaloproteinasa 16 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Metalotioneína 3 , Ratones , EmbarazoRESUMEN
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.
Asunto(s)
Endotelio Vascular/citología , Metaloproteinasas de la Matriz/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/fisiología , Bromodesoxiuridina , Quimiotaxis/fisiología , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , TransfecciónRESUMEN
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.
Asunto(s)
Metaloendopeptidasas/deficiencia , Alveolos Pulmonares/embriología , Animales , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Alveolos Pulmonares/ultraestructuraRESUMEN
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.
Asunto(s)
Huesos/fisiología , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/fisiología , Osteocitos/enzimología , Osteocitos/fisiología , Animales , Colorantes Azulados , Colágeno Tipo I/metabolismo , Hibridación in Situ , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/ultraestructura , Ratones , Microscopía Electrónica de Transmisión , Osteocitos/ultraestructura , Tinción con Nitrato de Plata , Factores de TiempoRESUMEN
Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, including interstitial and basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. The enzymes are secreted or released in latent form and become activated in the pericellular environment by disruption of a Zn++-cysteine bond which blocks the reactivity of the active site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts, keratinocytes, endothelial cells, and macrophages) are capable of responding to growth factors and cytokines, as well as to products released from the microbial flora by induction of transcription of 1 or more MMP genes. Cytokines that are likely to regulate expression of MMP genes in periodontal tissues include IL-1, TNF-α, and TGF-α. In addition, triggered PMN leukocytes which express only 2 MMP (PMN-CL and Mr 92K GL) release these enzymes from specific granule storage sites in response to a number of stimuli. The evidence that MMP are involved in tissue destruction in human periodontal diseases is still indirect and circumstantial. Cells isolated from normal and inflamed gingiva are capable of expressing a wide complement of MMP in culture and several MMP can be detected in cells of human gingiva in vivo. In addition, PMN-CL and Mr 92K GL are readily detected in gingival crevicular fluid from gingivitis and Periodontitis patients. Osteoclastic bone resorption does not appear to directly involve MMP, but a body of evidence suggests that bone resorption is initiated by removal of the osteoid layer by osteoblasts by means of a collagenase-dependent process. J Periodontol 1993; 64:474-484.
RESUMEN
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.
Asunto(s)
Colagenasas/metabolismo , Metaloendopeptidasas/metabolismo , Animales , Huesos/patología , Huesos/fisiología , Membrana Celular/enzimología , Movimiento Celular , Tejido Conectivo/fisiología , Matriz Extracelular/metabolismo , Eliminación de Gen , Humanos , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/deficiencia , Invasividad NeoplásicaRESUMEN
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 vivo biological 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.
Asunto(s)
Amelogénesis Imperfecta/genética , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/fisiología , Amelogenina , Animales , Proteínas del Esmalte Dental/genética , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Exones , Vectores Genéticos , Genotipo , Heterocigoto , Intrones , Metaloproteinasa 20 de la Matriz , Ratones , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Modelos Genéticos , Diente Molar/fisiología , Diente Molar/ultraestructura , Mutación , Péptidos/química , Fenotipo , ARN/metabolismoRESUMEN
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.
Asunto(s)
Colágenos Fibrilares/metabolismo , Metaloendopeptidasas/fisiología , Ligamento Periodontal/enzimología , Erupción Dental/fisiología , Raíz del Diente/enzimología , Raíz del Diente/crecimiento & desarrollo , Animales , Fibroblastos/enzimología , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Ratones Mutantes , Diente Molar/enzimología , Diente Molar/crecimiento & desarrollo , Ligamento Periodontal/citología , Fagosomas/ultraestructuraRESUMEN
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.
Asunto(s)
Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/aislamiento & purificación , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidor Tisular de Metaloproteinasa-2/química , alfa-Macroglobulinas/química , Animales , Caseínas/química , Colágeno Tipo I/química , Cisteína/química , Precursores Enzimáticos/química , Precursores Enzimáticos/aislamiento & purificación , Colorantes Fluorescentes/química , Gelatina/química , Inhibidores de la Metaloproteinasa de la Matriz , Métodos , Ratas , Especificidad por SustratoRESUMEN
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. MMP-2 activation by the Mov-13 fibroblasts was rescued by transfection of a full-length alpha1(I) collagen cDNA, and to our surprise, also by transfection with an alpha1(I) collagen cDNA carrying a mutation at the C-proteinase cleavage, which almost abrogated fibrillogenesis. Although studies with ascorbate-cultured MT1-MMP-/- fibroblasts showed that MT1-MMP played a significant role in the collagen-induced MMP-2 activation, a residual MT1-MMP-independent activation of MMP-2 was seen which resembled the level of MMP-2 activation persisting when wild-type fibroblasts were cultured in the presence of both ascorbic acid and MMP inhibitors. We were also unable to block this residual activation with inhibitors specific for serinyl, aspartyl, or cysteinyl enzymes.
Asunto(s)
Ácido Ascórbico/farmacología , Colágeno/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloendopeptidasas/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Reactivos de Enlaces Cruzados , Activación Enzimática , Colágenos Fibrilares/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/fisiología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Piel/citología , Factores de TiempoRESUMEN
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.