RESUMO
AIM: This review aims to summarise our understanding of the destructive role of acid environment and metalloproteinases in dentin caries progression using a review process. METHOD: The acids resulting from consumption of sugars by acidogenic and aciduric bacteria can cause demineralisation of the tooth surface, but are not able to cause caries-like lesions. The appearance of such lesions requires the activation of enzymatic proteolysis in an acidic environment for degradation of the dentin organic matrix, leading to cavity formation. Bacterial collagenases have long been considered responsible for organic matrix destruction; host cell-derived matrix metalloproteinases (MMPs) have recently been considered to be involved in the dentinal matrix destruction of carious lesions. DISCUSSION AND CONCLUSION: MMPs are initially synthesised as inactive zymogens to be activated in acid environment of dentinal fluid during the carious process, resulting in destruction of the collagenous matrix. The role of acid environment on enamel and dentin demineralisation and the role of salivary and dentinal MMPs in dentin progression of caries has encouraged general dentists to include the monitoring of oral environment not only by control of bacterial oral flora in caries treatment protocol, but mainly by inhibition of dentinal and salivary MMPs through the use of toothpaste and/or mouthwash containing specific active agents.
Assuntos
Cárie Dentária/enzimologia , Dentina/enzimologia , Metaloproteinases da Matriz/fisiologia , Ácidos , Bactérias/enzimologia , Colagenases/fisiologia , Cárie Dentária/fisiopatologia , Progressão da Doença , Ativação Enzimática , Humanos , Inibidores de Metaloproteinases de Matriz/uso terapêuticoRESUMO
Bacterial collagenases are metalloproteinases involved in the degradation of the extracellular matrices of animal cells, due to their ability to digest native collagen. These enzymes are important virulence factors in a variety of pathogenic bacteria. Nonetheless, there is a lack of scientific consensus for a proper and well-defined classification of these enzymes and a vast controversy regarding the correct identification of collagenases. Clostridial collagenases were the first ones to be identified and characterized and are the reference enzymes for comparison of newly discovered collagenolytic enzymes. In this review we present the most recent data regarding bacterial collagenases and overview the functional and structural diversity of bacterial collagenases. An overall picture of the molecular diversity and distribution of these proteins in nature will also be given. Particular aspects of the different proteolytic activities will be contextualized within relevant areas of application, mainly biotechnological processes and therapeutic uses. At last, we will present a new classification guide for bacterial collagenases that will allow the correct and straightforward classification of these enzymes.
Assuntos
Bactérias/enzimologia , Colagenases/fisiologia , Animais , Bactérias/classificação , Bactérias/genética , Técnicas de Cultura de Células , Colágeno/química , Colágeno/genética , Colágeno/metabolismo , Colagenases/química , Colagenases/classificação , Colagenases/uso terapêutico , Cosméticos , Tecnologia de Alimentos , Gelatinases/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , ProteóliseRESUMO
Dentin organic matrix, with type I collagen as the main component, is exposed after demineralization in dentinal caries, erosion or acidic conditioning during adhesive composite restorative treatment. This exposed matrix is prone to slow hydrolytic degradation by host collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins. Here we review the recent findings demonstrating that inhibition of salivary or dentin endogenous collagenolytic enzymes may provide preventive means against progression of caries or erosion, just as they have been shown to retain the integrity and improve the longevity of resin composite filling bonding to dentin. This paper also presents the case that the organic matrix in caries-affected dentin may not be preserved as intact as previously considered. In partially demineralized dentin, MMPs and cysteine cathepsins with the ability to cleave off the terminal non-helical ends of collagen molecules (telopeptides) may lead to the gradual loss of intramolecular gap areas. This would seriously compromise the matrix ability for intrafibrillar remineralization, which is considered essential in restoring the dentin's mechanical properties. More detailed data of the enzymes responsible and their detailed function in dentin-destructive conditions may not only help to find new and better preventive means, but better preservation of demineralized dentin collagenous matrix may also facilitate true biological remineralization for the better restoration of tooth structural and mechanical integrity and mechanical properties.
Assuntos
Cárie Dentária/enzimologia , Dentina/enzimologia , Metaloproteinases da Matriz/fisiologia , Catepsinas/fisiologia , Colagenases/fisiologia , Cisteína Proteases/fisiologia , Colagem Dentária , Cárie Dentária/prevenção & controle , Dentina/efeitos dos fármacos , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Remineralização Dentária/métodosAssuntos
Colagenases/fisiologia , Rejuvenescimento/fisiologia , Envelhecimento da Pele/fisiologia , Colágeno/efeitos da radiação , Fármacos Dermatológicos/uso terapêutico , Humanos , Metaloproteases/efeitos da radiação , Proteínas Tirosina Fosfatases/efeitos da radiação , Espécies Reativas de Oxigênio/efeitos da radiação , Receptores Proteína Tirosina Quinases/efeitos da radiação , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Raios UltravioletaRESUMO
En la enfermedad periodontal, la acumulación de bacterias gramnegativas, genera una respuesta inmunoinflamatoria que es modulada por el mecanismo de defensa del paciente. El tratamiento de modulación del huésped (TMH), ha sido incorporado como una opción farmacológica para el control de la enfermedad periodontal. El objetivo de la revisión fue investigar los efectos de los inhibidores de la colagenasa tisular y de los analgésicos antiinflamatorios no esteroides (AINES) como agentes moduladores de la enfermedad periodontal. A tal fin, se realizó una búsqueda de estudios de casos, controles y revisiones, empleando las bases de datos Medline-PubMed, LILACS y Dialnet. Se encontró que los resultados de las terapias de modulación del huésped tienen como blanco los mediadores proinflamatorios y enzimas destructivas que degradan el colágeno y destruyen tejido óseo, equilibrando y aumentando las acciones antiinflamatorias y protectivas. Los fármacos usados en el TMH regulan los procesos destructivos de la respuesta inmunoinflamatoria en presencia de placa dental, sobre todo en pacientes susceptibles.
Assuntos
Humanos , Anti-Inflamatórios não Esteroides/imunologia , Autoimunidade/fisiologia , Doenças Periodontais/imunologia , Doenças Periodontais/tratamento farmacológico , Estudos de Casos e Controles , Colagenases/fisiologia , Bases de Dados BibliográficasRESUMO
The core of an atheromatous plaque contains lipids, macrophages, and cellular debris, typically covered by a fibrous cap that separates the thrombogenic core from the blood. Rupture of the fibrous cap causes most fatal myocardial infarctions. Interstitial collagen confers tensile strength on the cap, as it does in skin and tendons. In 1994, Peter Libby and colleagues demonstrated overexpression of collagenolytic enzymes in atheromatous plaques and implicated MMPs in the destabilization of these lesions.
Assuntos
Aterosclerose/enzimologia , Colagenases/fisiologia , Placa Aterosclerótica/enzimologia , Animais , Aterosclerose/complicações , Humanos , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/etiologia , Placa Aterosclerótica/complicaçõesRESUMO
Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.
Assuntos
Linfangiogênese/genética , Vasos Linfáticos/embriologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Colágeno Tipo I/metabolismo , Colagenases/genética , Colagenases/metabolismo , Colagenases/fisiologia , Embrião não Mamífero , Líquido Extracelular/enzimologia , Líquido Extracelular/metabolismo , Feminino , Humanos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/fisiologia , Masculino , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peixe-ZebraRESUMO
Efficient deposition of type I collagen is fundamental to healing after myocardial infarction. Whether there is also a role for cleavage of type I collagen in infarct healing is unknown. To test this, we undertook coronary artery occlusion in mice with a targeted mutation (Col1a1(r/r)) that yields collagenase-resistant type I collagen. Eleven days after infarction, Col1a1(r/r) mice had a lower mean arterial pressure and peak left ventricular systolic pressure, reduced ventricular systolic function, and worse diastolic function, compared with wild-type littermates. Infarcted Col1a1(r/r) mice also had greater 30-day mortality, larger left ventricular lumens, and thinner infarct walls. Interestingly, the collagen fibril content within infarcts of mutant mice was not increased. However, circular polarization microscopy revealed impaired collagen fibril organization and mechanical testing indicated a predisposition to scar microdisruption. Three-dimensional lattices of collagenase-resistant fibrils underwent cell-mediated contraction, but the fibrils did not organize into birefringent collagen bundles. In addition, time-lapse microscopy revealed that, although cells migrated smoothly on wild-type collagen fibrils, crawling and repositioning on collagenase-resistant collagen was impaired. We conclude that type I collagen cleavage is required for efficient healing of myocardial infarcts and is critical for both dynamic positioning of collagen-producing cells and hierarchical assembly of collagen fibrils. This seemingly paradoxical requirement for collagen cleavage in fibrotic repair should be considered when designing potential strategies to inhibit matrix degradation in cardiac disease.
Assuntos
Colágeno Tipo I/metabolismo , Colagenases/fisiologia , Fibroblastos/enzimologia , Infarto do Miocárdio/enzimologia , Cicatrização/fisiologia , Animais , Movimento Celular , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Colagenases/genética , Constrição , Vasos Coronários , Fibroblastos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genéticaRESUMO
Thrombin activates immunocompetent microglia and increases release of inflammatory cytokines under intracerebral hemorrhage (ICH) insults. Also, thrombin injection into the striatum evokes acute necrosis and delayed apoptosis of neurons. A nucleoprotein high-mobility group box 1 (HMGB1) that is released from necrotic cells has been suggested to behave like a cytokine and cause over-facilitation of immune functions. Here we examined the effect of glycyrrhizin, known as an inhibitor of HMGB1, on thrombin-induced injury in rat cortico-striatal slice cultures and in vivo rat ICH model. In slice cultures, thrombin-induced a drastic increase in propidium iodide fluorescence indicating necrotic cell death in the cortical region, and robust shrinkage of the striatal tissue. Glycyrrhizin (10-100 µM) attenuated thrombin-induced cortical injury in a concentration-dependent manner. The protective effect of glycyrrhizin was not mediated by glucocorticoid receptors or modulation of nitric oxide production, but was reversed by exogenous HMGB1 application. The injury induced by a high concentration of HMGB1 was suppressed by glycyrrhizin. In vivo, unilateral injection of type IV collagenase into rat striatum induced ICH associated with brain edema formation, contralateral paralysis and neuron death. Once daily intraperitoneal administration of glycyrrhizin attenuated ICH-induced edema in both the cortex and the basal ganglia, and improved behavioral performance of rats in forelimb placing. Moreover, glycyrrhizin partially but significantly ameliorated ICH-induced neuron loss inside hematoma. These findings suggest that an HMGB1 inhibitor glycyrrhizin is a potential candidate for a remedy for ICH.
Assuntos
Anti-Inflamatórios/farmacologia , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/patologia , Ácido Glicirrízico/farmacologia , Proteína HMGB1/antagonistas & inibidores , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Edema Encefálico/induzido quimicamente , Edema Encefálico/tratamento farmacológico , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Bovinos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Hemorragia Cerebral/induzido quimicamente , Colagenases/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Avaliação Pré-Clínica de Medicamentos , Ácido Glicirrízico/metabolismo , Ácido Glicirrízico/uso terapêutico , Proteína HMGB1/fisiologia , Hemostáticos/farmacologia , Masculino , Terapia de Alvo Molecular , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Trombina/farmacologia , Técnicas de Cultura de TecidosRESUMO
Osteoarthritic cartilage destruction is caused by an imbalance between anabolic and catabolic factors. Here, we show that hypoxia-inducible factor-2alpha (HIF-2alpha, encoded by EPAS1) is a catabolic transcription factor in the osteoarthritic process. HIF-2alpha directly induces the expression in chondrocytes of genes encoding catabolic factors, including matrix metalloproteinases (MMP1, MMP3, MMP9, MMP12 and MMP13), aggrecanase-1 (ADAMTS4), nitric oxide synthase-2 (NOS2) and prostaglandin-endoperoxide synthase-2 (PTGS2). HIF-2alpha expression was markedly increased in human and mouse osteoarthritic cartilage, and its ectopic expression triggered articular cartilage destruction in mice and rabbits. Moreover, mice transgenic for Epas1 only in chondrocytes showed spontaneous cartilage destruction, whereas heterozygous genetic deletion of Epas1 in mice suppressed cartilage destruction caused by destabilization of the medial meniscus (DMM) or collagenase injection, with concomitant modulation of catabolic factors. Our results collectively demonstrate that HIF-2alpha causes cartilage destruction by regulating crucial catabolic genes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Cartilagem/metabolismo , Osteoartrite/metabolismo , Fatores de Transcrição/fisiologia , Animais , Cartilagem/fisiopatologia , Condrócitos/metabolismo , Condrócitos/fisiologia , Colagenases/metabolismo , Colagenases/fisiologia , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Genes/genética , Genes/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Osteoartrite/fisiopatologia , CoelhosRESUMO
There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed 'quickly', while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue.
Assuntos
Colágeno/fisiologia , Colagenases/fisiologia , Animais , Fenômenos Biomecânicos , Bovinos , Colágeno/química , Colágeno/ultraestrutura , Técnicas In Vitro , Microscopia de Interferência , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Complexos Multiproteicos/ultraestrutura , Estresse MecânicoRESUMO
Matrix metalloproteinases (MMPs), a family of proteinases including collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs, affect the breakdown and turnover of extracellular matrix (ECM). Moreover, they are major physiologic determinants of ECM degradation and turnover in the glomerulus. Renal hypertrophy and abnormal ECM deposition are hallmarks of diabetic nephropathy (DN), suggesting that altered MMP expression or activation contributes to renal injury in DN. Herein, we review and summarize recent information supporting a role for MMPs in the pathogenesis of DN. Specifically, studies describing dysregulated activity of MMPs and/or their tissue inhibitors in various experimental models of diabetes, including animal models of type 1 or type 2 diabetes, clinical investigations of human type 1 or type 2 diabetes, and kidney cell culture studies are reviewed.
Assuntos
Nefropatias Diabéticas/etiologia , Metaloproteinases da Matriz/fisiologia , Animais , Colagenases/fisiologia , Modelos Animais de Doenças , Gelatinases/fisiologia , Humanos , Metaloproteinase 3 da Matriz/fisiologia , Metaloproteinase 7 da Matriz/fisiologia , Metaloproteinases da Matriz Associadas à Membrana/fisiologia , Inibidores Teciduais de Metaloproteinases/fisiologiaRESUMO
The objective of this study was to test the hypothesis that enzymatic degradation by collagenase significantly reduces dynamic moduli and increases compressive strains of bovine articular cartilage under physiological compressive stress levels and loading frequencies. Twenty-seven distal femoral cartilage plugs (3 mm diameter) were loaded in a custom apparatus under load control, with a load up to 40 N and loading frequencies of 0.1, 1, 10, and 40 Hz, before and after incubation in physiological buffered saline containing various concentrations of collagenase (0, 2, and 10 U/mL). Collagenase digestion reduced the equilibrium Young's modulus by 49% with 2 U/mL and 61% with 10 U/mL, while the decrease in dynamic modulus at 40 Hz was in the range of 13-20% with 2 U/mL and 24-33% with 10 U/mL, relative to respective controls. The amplitudes of dynamic compressive strains increased from 22 +/- 6% to 26 +/- 8% at 0.1 Hz and 9.6 +/- 3.3% to 13.5 +/- 3.2% at 40 Hz, with 10 U/mL collagenase. This experimental study serves to confirm that collagen contributes significantly to the dynamic compressive properties of cartilage, by demonstrating that collagenase digestion impairs these properties, under stress amplitudes and frequencies which are representative of physiological loading conditions.
Assuntos
Cartilagem Articular/fisiologia , Colágeno/fisiologia , Colagenases/fisiologia , Força Compressiva/fisiologia , Glicosaminoglicanos/fisiologia , Modelos Biológicos , Suporte de Carga/fisiologia , Animais , Bovinos , Simulação por Computador , Elasticidade , Técnicas In Vitro , Estresse MecânicoRESUMO
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.
Assuntos
Colágeno/metabolismo , Colagenases/fisiologia , Endocitose , Fibroblastos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Células Cultivadas , Metaloproteinase 14 da Matriz/fisiologia , Camundongos , Conformação ProteicaRESUMO
Although dexamethasone (Dex) substantially enhances the osteoblastic phenotype in osteogenic cells, including human periodontal ligament (PDL) cells, the basis for this response remains poorly understood. Since the accretion of a collagenous matrix is important for an osteoblastic response and dexamethasone is known to decrease collagenase expression, we examined whether osteoblastic differentiation mediated by Dex is linked to a decrease in collagenase expression in PDL cells. Early passage human PDL cells were exposed to Dex, or ascorbic acid (AA) or beta-glycerophosphate (betaGP) alone, or in various combinations in serum-free media for 3 or 5 days. Cells exposed to Dex alone or any combinations of treatments that included Dex demonstrated increased core binding factor alpha 1 (Cbfa1), alkaline phosphatase (AP), osteonectin (ON), osteopontin (OP), bone sialoprotein (BSP) and collagen I (alpha1) expression when compared to control cells or those exposed to AA or betaGP. The induction of these osteoblastic markers was accompanied by a decrease in collagenase-1 expression. Collagenase activity showed a statistically significant strong negative relationship to Cbfa1 (Pearson's r=-0.97), AP (r=-0.87), OP (r=-0.95) and BSP (r=-0.82) in 5-day cultures, and moderately strong relationship to ON (r=-0.74) from 3 days culture. Dex also produced a dose-dependent increase in AP that was paralleled by a decrease in collagenase activity (r=-0.98). Addition of collagenase inhibitors increased AP expression while concomitantly suppressing collagenase activity. Conversely, addition of exogenous collagenase decreased the AP phenotype of the cells, which was more marked in the absence then in the presence of Dex. The findings indicate that Dex enhances specific markers of osteoblastic differentiation in PDL cells by decreasing collagenase expression, and suggest that endogenous collagenase may regulate osteoblastic differentiation of these cells.
Assuntos
Colagenases/fisiologia , Dexametasona/farmacologia , Inibidores de Metaloproteinases de Matriz , Osteoblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Fosfatase Alcalina/análise , Fosfatase Alcalina/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular , Colágeno Tipo I/análise , Colágeno Tipo I/metabolismo , Colagenases/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Expressão Gênica , Humanos , Sialoproteína de Ligação à Integrina , Metaloproteinase 1 da Matriz/genética , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteonectina/análise , Osteonectina/metabolismo , Osteopontina/análise , Osteopontina/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Sialoglicoproteínas/análise , Sialoglicoproteínas/metabolismoRESUMO
Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.
Assuntos
Microfibrilas/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Colagenases/fisiologia , Elasticidade , Membranas Extraembrionárias/metabolismo , Membranas Extraembrionárias/ultraestrutura , Fibrilina-1 , Fibrilinas , Guanidina/farmacologia , Humanos , Hidrólise , Microfibrilas/química , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/ultraestrutura , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Estrutura Terciária de ProteínaRESUMO
We present a model to describe the biased diffusion of a collagenase along collagen fibrils. Based on the structures of collagen fibril and collagenase, the interaction is described by a one-dimensional potential that is symmetric in the region of no cleavage and asymmetric in the cleavage region. We show that the mean velocity of the unidirectional diffusion of the collagenase depends on the three parameters: the asymmetric ratio of the local potential in the cleavage region, the chemical reaction rate of proteolysis and the jumping rate of collagenase between two neighboring tracks. We calculate the correlation function and the mean transport velocity for both wild-type and mutant collagenases along collagen fibrils, the results of which are consistent with the previous experiments.
Assuntos
Colágeno/metabolismo , Colagenases/fisiologia , Simulação por Computador , Matriz Extracelular/metabolismo , Modelos Químicos , Animais , DifusãoRESUMO
We have previously shown that stimulation of extracellular signal-regulated protein kinase (ERK) by bradykinin (BK) in murine inner medullary collecting duct (mIMCD)-3 cells is mediated by epidermal growth factor receptor (EGFR) transactivation. The mechanism of EGFR transactivation seemed to be novel, because it does not require phospholipase C, Ca(2+), calmodulin, protein kinase C, G alpha(i) subunits, or EGFR-B(2) receptor heterodimerization. In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in B(2) receptor-induced EGFR transactivation using their broad-spectrum inhibitors batimastat and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (Galardin) (GM-6001). Selective inhibitors for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) blocked BK-induced EGFR phosphorylation and ERK activation, whereas inhibitors for MMP-1, -2, -3, -7, or -9 were without effect. Transfection of mIMCD-3 cells with MMP-8 small interfering RNA (siRNA) resulted in approximately 50% decrease of BK-induced ERK activation. A neutralizing antibody against MMP-13 as well as transfection with MMP-13 siRNA produced a similar effect. Inhibition of both collagenases resulted in approximately 65% decrease of BK-induced ERK activation, supporting roles for both enzymes. Stimulation of mIMCD-3 cells with 10 nM BK increased the activity of collagenases in concentrated culture media within 10 min. Moreover, recombinant MMP-13 and MMP-8, when applied to mIMCD-3 cells for 10 min without BK, stimulated tyrosine phosphorylation of EGFR and caused approximately 250% increase over basal ERK phosphorylation comparable with BK-induced ERK activation. Collagenases-induced ERK activation was inhibited by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478) and thus dependent on EGFR tyrosine kinase activity. This study demonstrates a novel role for collagenase-2 and -3 in signaling of the G(q)-coupled BK B(2) receptor in mIMCD-3 cells.
Assuntos
Colagenases/fisiologia , Receptores ErbB/metabolismo , Túbulos Renais Coletores/metabolismo , Metaloproteinase 8 da Matriz/fisiologia , Receptor B2 da Bradicinina/fisiologia , Ativação Transcricional , Animais , Bradicinina/farmacologia , Cálcio/metabolismo , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Túbulos Renais Coletores/citologia , Metaloproteinase 13 da Matriz , Camundongos , Fosforilação , RNA Interferente Pequeno/farmacologia , Fator de Crescimento Transformador alfa/fisiologiaRESUMO
OBJECTIVES: Retinoic acid (RetA) and oncostatin M (OSM) have both been shown to mediate potent effects with respect to extracellular matrix integrity. This study assesses the effects of a RetA + OSM combination on cartilage catabolism. METHODS: Animal and human cartilage samples were used to assess the ability of RetA + OSM to promote the release of collagen and proteoglycan fragments, which was determined by measuring glycosaminoglycan and hydroxyproline, respectively. Total collagenolytic and tissue inhibitor of metalloproteinases (TIMP) inhibitory activities were determined by bioassay, whilst gene expression of matrix metalloproteinases (MMPs) and TIMP-1 were determined by northern blotting. Immunohistochemistry was used to assess the presence of MMP-1 and -13 in resorbing cartilage explants. RESULTS: Both agents alone induced proteoglycan release from bovine cartilage, whilst RetA-induced collagen release was variable. Reproducible and synergistic collagenolysis was observed with RetA + OSM, which appeared to be due to MMP-13. Similar collagen release was observed from porcine cartilage. Conversely, no collagen release was seen with human articular cartilage. In primary human chondrocytes, RetA + OSM failed to induce MMP-1 or -13 but caused a significant increase in TIMP-1 expression. CONCLUSIONS: These novel observations show that the combination of RetA + OSM has profound effects on cartilage matrix turnover, but these effects are species-specific. A better understanding of the mechanism by which this combination differentially regulates MMP and TIMP expression in human chondrocytes could provide valuable insight into new therapeutic strategies aimed at the prevention of cartilage destruction.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Colagenases/fisiologia , Citocinas/farmacologia , Tretinoína/farmacologia , Animais , Cartilagem Articular/enzimologia , Cartilagem Articular/metabolismo , Bovinos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Condrócitos/metabolismo , Colágeno/metabolismo , Colagenases/metabolismo , Sinergismo Farmacológico , Humanos , Mediadores da Inflamação/farmacologia , Metaloproteinase 13 da Matriz , Oncostatina M , Proteoglicanas/metabolismo , Proteínas Recombinantes/farmacologia , Especificidade da Espécie , Suínos , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
CD166 is an Ig superfamily molecule that binds homotypically to itself and heterotypically to CD6. Interactions between CD6 and CD166 are important during immune development and in alloreactivity. CD166 is expressed at increased levels in selected cancers and in rheumatoid arthritis synovium. Knowledge that CD166 was expressed in normal human salivary epithelium led to these studies of CD166 and CD6 in diseased mouse salivary glands, that resemble pathology seen in the human disease, Sjögren's syndrome. We showed that in mouse salivary epithelium CD166 was expressed but that expression of CD166 did not necessarily predict its function. Recombinant soluble CD6-Ig bound to CD6 ligands (CD6L) on transformed and freshly isolated salivary epithelial cells. Cross-blocking studies showed that binding of CD6-Ig to salivary epithelium was in part dependent on CD166, but that CD6-Ig binding may also involve additional CD6L. Binding of CD6-Ig was sensitive to trypsin digestion but resistant to digestion by collagenase and sialidase. Anti-CD166 ab precipitated CD166 from salivary epithelium pre- and post-treatment with the pro-inflammatory cytokine IFN-gamma. In contrast CD6-Ig only precipitated CD166 from IFN-gamma treated cells. More extensive colocalization between CD166 and the actin cytoskeleton was observed in sialoadenitis epithelium compared to control. We conclude that during sialoadenitis, CD166 undergoes a gain of function, resulting in closer association with the actin cytoskeleton and increased capacity to bind CD6. We suggest that altered CD166 function may contribute to the pro-inflammatory milieu during sialoadenitis seen in Sjögren's syndrome.