RESUMEN
In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.
Asunto(s)
Pulpa Dental/citología , Dentinogénesis/genética , Glicoproteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Compuestos de Aluminio , Animales , Compuestos de Calcio , Resinas Compuestas , Recubrimiento de la Pulpa Dental/métodos , Combinación de Medicamentos , Glicoproteínas/deficiencia , Técnicas para Inmunoenzimas , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Diente Molar/cirugía , Óxidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Silicatos , Microtomografía por Rayos XRESUMEN
Extracellular matrix metalloproteinase inducer (EMMPRIN), known for its ability to induce matrix metalloproteinase (MMP) expression, was proposed to play a role in the adverse cardiac extracellular matrix remodeling. After observing an age-associated increase in cardiac EMMPRIN expression in both mice and rats, the role and mechanism of action of EMMPRIN was investigated in the myocardial age-associated changes using 3, 12 and 24 month old EMMPRIN knock-out (KO) vs. wild-type (WT) mice, by cardiac echocardiography, Western blots, immunohistochemistry, ELISA and histology. Adilated cardiomyopathy characterized by a decreased ejection fraction and an enlargement of left ventricular chamber (LV) associated with LV hypertrophy, occurred in KO mice as soon as 12 month old. The increase in interstitial collagen deposition during aging in WT mice could not be detected in KO mice. This may be related to the reduced activation (48% reduction; P < 0.05) and signaling (smad2/3 nuclear translocation) of TGF-ß in the 12 month old KO mice which paralleled with a greater reduction in the TGF-ß known activating enzymes such as MT1-MMP and MMP-1 (33% and 37% reduction respectively, between 3 and 12 month old in KO mice; P < 0.05) as well as uPA. These findings demonstrate that EMMPRIN gene silencing is associated with an aberrant extracellular matrix remodeling, characterized by the absence of a detected age-associated fibrosis and consequently to dilated cardiopathy, indicating that a fine regulation of EMMPRIN is essential for the coordinated ECM remodeling during aging.
Asunto(s)
Envejecimiento/fisiología , Basigina/metabolismo , Matriz Extracelular/metabolismo , Remodelación Ventricular/fisiología , Animales , Basigina/genética , Colágeno/metabolismo , Femenino , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Ratas , Ratas WistarRESUMEN
The histological classification of testicular germ cell tumours (TGCTs) to seminoma or non-seminomatous germ cell tumours is at present the main criterion for the clinical outcome and selection of the treatment strategy. In view of the need to identify novel prognostic biomarkers for TGCTs, we investigated the expression of the matrix metalloproteinases MMP-2 and MMP-9 in testicular tumour tissues and cell lines of both seminoma and non-seminoma origin. Immunohistochemistry and zymography analysis of tumoural tissues showed significantly higher levels of MMP-2 and MMP-9 compared with normal testis with the active forms detected only in the tumour tissues. Three cell lines representative of the different tumour types, JKT-1 seminoma, NCCIT teratocarcinoma and NTERA2/D1 embryonal carcinoma were also evaluated for their expression of these MMPs using qPCR and zymography and for their invasive properties. The more invasive non-seminomatous teratocarcinoma and embryonal cells expressed considerably more MMP-2 and MMP-9 compared with seminoma cells exhibiting lower invasiveness. Furthermore, an inverse relation was observed between invasiveness and the expression of endogenous inhibitors TIMP-1 and TIMP-2. The MMP inhibitor Marimastat inhibited invasion in all cell lines, the highest inhibition was observed in the more invasive NTERA2/D1 and NCCIT cells, which presented the highest ratio of MMP-2 and MMP-9 vs. TIMP-1 and TIMP-2. These results highlight the importance of MMP-2 and MMP-9 in the invasiveness of testicular tumours and suggest that their levels, vs. those of TIMP-1 and TIMP-2, may represent potential biomarkers for testicular malignancy.
Asunto(s)
Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Invasividad Neoplásica , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias Testiculares/patología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Inmunohistoquímica , Masculino , Neoplasias de Células Germinales y Embrionarias/enzimología , Neoplasias de Células Germinales y Embrionarias/metabolismo , Reacción en Cadena de la Polimerasa , Neoplasias Testiculares/enzimología , Neoplasias Testiculares/metabolismoRESUMEN
The activation of epidermal growth factor receptor (EGFR) by its ligands constitutes an important step in the metastatic process but the clinical response to its inhibition in breast cancer patients has so far been very low. In this work, we investigated the role of the EGFR ligand amphiregulin (AR) in modulating EGFR activation. For this, transformed epithelial mammary tumor cells NS2T2A1 were used in which AR or EGFR expression was down-regulated by antisense cDNA technique. This down-regulation was associated with a significant inhibition of matrix metalloproteinase-9 production as well as cell proliferation, but this inhibition was only minimally reversed by exogenously added AR or EGF. EGFR protein levels were not affected but EGFR-tyrosine phosphorylation in response to EGF was markedly reduced. Thus, the inhibition of AR expression, which impairs EGFR response to its exogenously available ligands, may represent an alternative anti-EGFR therapeutic strategy in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores ErbB/metabolismo , Glicoproteínas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Anfirregulina , Elementos sin Sentido (Genética) , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Familia de Proteínas EGF , Factor de Crecimiento Epidérmico/farmacología , Femenino , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Metaloproteinasa 9 de la Matriz/biosíntesis , Inhibidores de la Metaloproteinasa de la Matriz , Fosforilación , Tirosina/metabolismoRESUMEN
Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.
Asunto(s)
Adhesivos/metabolismo , Dentina/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Resinas Sintéticas/metabolismo , Adolescente , Niño , Dentina/química , Dentina/ultraestructura , Proteínas de la Matriz Extracelular/genética , Humanos , Sialoproteína de Unión a Integrina , Metaloproteinasa 3 de la Matriz/análisis , Metaloproteinasa 3 de la Matriz/genética , Peso Molecular , Osteopontina/análisis , Osteopontina/química , Osteopontina/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo , Factores de TiempoAsunto(s)
Síndrome de Behçet/diagnóstico , Trombomodulina/sangre , Biomarcadores/sangre , Femenino , Humanos , MasculinoRESUMEN
The objective of this review is to summarize our understanding of the role of host matrix metalloproteinases (MMPs) in the caries process and to discuss new therapeutic avenues. MMPs hydrolyze components of the extracellular matrix and play a central role in many biological and pathological processes. MMPs have been suggested to play an important role in the destruction of dentin organic matrix following demineralization by bacterial acids and, therefore, in the control or progression of carious decay. Host-derived MMPs can originate both from saliva and from dentin. They may be activated by an acidic pH brought about by lactate release from cariogenic bacteria. Once activated, they are able to digest demineralized dentin matrix after pH neutralization by salivary buffers. Furthermore, the degradation of SIBLINGs (Small Integrin-binding Ligand N-linked Glycoproteins) by the caries process may potentially enhance the release of MMPs and their activation. This review also explores the different available MMP inhibitors, natural or synthetic, and suggests that MMP inhibition by several inhibitors, particularly by natural substances, could provide a potential therapeutic pathway to limit caries progression in dentin.
Asunto(s)
Caries Dental/enzimología , Metaloproteinasas de la Matriz/fisiología , Dentina/enzimología , Progresión de la Enfermedad , Matriz Extracelular/enzimología , Glicoproteínas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Inhibidores de Proteasas/uso terapéutico , Saliva/enzimología , Inhibidores Tisulares de Metaloproteinasas/uso terapéutico , Desmineralización Dental/enzimologíaRESUMEN
Metalloproteinases (MMPs) are essential regulators during various phases of the angiogenic process. These include the degradation of the basement membrane and the extracellular matrix, the mobilisation and activation of growth factors and the production of fragments with pro- or anti-angiogenic activity. In addition to their role in migration and invasion, MMPs can influence endothelial cell proliferation and survival by modifying the balance between angiogenic and anti-angiogenic molecules.
Asunto(s)
Metaloendopeptidasas/metabolismo , Neovascularización Fisiológica , Animales , Membrana Basal , División Celular , Supervivencia Celular , Endotelio Vascular/citología , Matriz Extracelular , HumanosRESUMEN
The dentino-enamel junction is not an simple inert interface between two mineralized structures. A less simplistic view suggests that the dentino-enamel junctional complex should also include the inner aprismatic enamel and the mantle dentin. At early stages of enamel formation, fibroblast growth factor (FGF)-2 is stored in and released from the inner aprismatic enamel, possibly under the control of matrix metalloproteinase (MMP)-3. The concentration peak for MMP-2 and -9 observed in the mantle dentin coincided with a very low labeling for TIMP-1 and -2, favoring the cross-talk between mineralizing epithelial and connective structures, and as a consequence the translocation of enamel proteins toward odontoblasts and pulp cells, and vice versa, the translocation of dentin proteins toward secretory ameloblasts and cells of the enamel organ. Finally, in X-linked hypophosphatemic rickets, large interglobular spaces in the circumpulpal dentin were the major defect induced by the gene alteration, whereas the mantle dentin was constantly unaffected. Altogether, these data plead for the recognition of the dentino-enamel junctional complex as a specific entity bearing its own biological characteristics.
Asunto(s)
Esmalte Dental/embriología , Dentina/embriología , Dentina/metabolismo , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/fisiología , Dentina/crecimiento & desarrollo , Embrión de Mamíferos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
Apigenin is a widely distributed plant flavonoid and was proposed as an antitumor agent. In this study, we investigated the apigenin effects on the protease-mediated invasiveness in an estrogen-insensitive breast tumor cell line MDA-MB231. The results show that apigenin at 22.8-45.5 microM (2.5-10 micrograms/ml) strongly inhibited, in a dose-dependent manner, tumor cell invasion through Matrigel, cell migration, and cell proliferation. We show that apigenin treatment from 22.8 microM (2.5 micrograms/ml) led to a partial decrease in urokinase-plasminogen activator expression and to a total inhibition of phorbol 12-myristate 13-acetate-induced matrix metalloproteinase-9 secretion. We also demonstrate in the apigenin-treated cells a defective adhesion to Matrigel and a G2-M cell cycle arrest. Taken together, our results demonstrate that apigenin is a pleiotropic effector affecting protease-dependent invasiveness and associated processes and proliferation of tumor cells.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Ciclo Celular/efectos de los fármacos , Endopeptidasas/biosíntesis , Flavonoides/farmacología , Apigenina , Neoplasias de la Mama/patología , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Femenino , Citometría de Flujo , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Invasividad Neoplásica , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
Immunohistochemical studies using a polyclonal antibody, raised against the recombinant form of dentin matrix protein 1 (DMP1), show that DMP1 was detected mainly in odontoblasts in cultured mouse embryonic tooth germs. However, in restricted areas, DMP1 staining was also observed in secretory ameloblasts, in the stratum intermedium and stellate reticulum, but only when the odontoblasts located in front of them were unstained. When the embryonic tooth germs were cultured in the presence of inositol hexasulfate, a casein kinase I and II inhibitor, staining of odontoblasts was weak or nil, whereas, in contrast, ameloblasts and enamel organ were strongly immunolabelled, suggesting an enhanced translocation of DMP1 after secretion to the secretory ameloblasts and/or stratum intermedium and stellate reticulum. Moreover, DMP1--was shown to be a good substrate for gelatinase A (MMP-2), but not to gelatinase B (MMP- 9). We hypothesized that DMP1--or the sub-fractions cleaved by the MMP--could behave as diffusible signaling molecule (s) rather than as a true dentin extracellular matrix component.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inositol/análogos & derivados , Inositol/farmacología , Isoenzimas/antagonistas & inhibidores , Fosfoproteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas , Germen Dentario/efectos de los fármacos , Ameloblastos/efectos de los fármacos , Ameloblastos/ultraestructura , Animales , Especificidad de Anticuerpos , Caseína Quinasas , Colorantes , Electroforesis en Gel de Poliacrilamida , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/ultraestructura , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/farmacología , Metaloproteinasa 9 de la Matriz/farmacología , Ratones , Odontoblastos/efectos de los fármacos , Odontoblastos/ultraestructura , Técnicas de Cultivo de Órganos , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Germen Dentario/ultraestructuraRESUMEN
TIMP-2 is a natural matrix metalloproteinase (MMP) inhibitor that prevents the degradation of extracellular matrix proteins. It abolishes the hydrolytic activity of all activated members of the metalloproteinase family and in particular that of MT1-MMP, MMP-2, and MMP-9, which are selective for type IV collagenolysis. Since MMPs have been implicated in both cancer progression and angiogenesis, we generated a recombinant adenovirus to deliver human TIMP-2 (AdTIMP-2) and evaluated its anticancer efficiency in three murine models. Our results demonstrated that overexpression in vitro of TIMP-2 inhibited the invasion of both tumor and endothelial cells without affecting cell proliferation. Its in vivo efficiency has been evaluated in murine lung cancer LLC, and colon cancer C51 in syngeneic mice as well as in human breast cancer MDA-MB231 in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three types significantly reduced tumor growth rates by 60--80% and tumor-associated angiogenesis index by 25--75%. Lung metastasis of LLC tumor was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.
Asunto(s)
Adenoviridae/genética , Terapia Genética/métodos , Inhibidor Tisular de Metaloproteinasa-2/genética , Animales , Apoptosis , Western Blotting , Colágeno/metabolismo , Medios de Cultivo Condicionados/farmacología , ADN Complementario/metabolismo , Combinación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Laminina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Metástasis de la Neoplasia , Neovascularización Patológica , Proteoglicanos/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
We examined the spatial distribution of MMP-2 on the surface of human endothelial cells using immunofluorescence and confocal microscopy. Staining endothelial cells with MMP-2-specific antibodies revealed a punctate labeling at the basolateral side of the cell periphery, which colocalized with patches of caveolin-1, a major constituent of the caveolae. This colocalization was confirmed by immunogold electron microscopy. MT1-MMP, TIMP-2, and the alphavbeta3 integrin exhibited a similar pattern of staining, with pericellular patches that colocalized with either MMP-2 or caveolin-1. The presence of MT1-MMP and TIMP-2 in caveolae patches could be seen only after treatment with concanavalin A, which induced MMP-2 activation but had no noticeable effect on the pattern or intensity of MMP-2 immunostaining. In contrast, MMP-9 and TIMP-1 staining showed a pattern completely different from that of MMP-2 and TIMP-2, with positive spots uniformly distributed throughout the cell body. Our data show that MMP-2, its activator the MT1-MMP, and its proposed receptor, the alphavbeta3 integrin, are all targeted to the same membrane microdomains on the endothelial cell, thereby restricting matrix proteolysis to a limited microenvironment at the cell surface.
Asunto(s)
Caveolas/química , Caveolinas/análisis , Endotelio Vascular/química , Metaloproteinasa 2 de la Matriz/análisis , Adulto , Caveolina 1 , Células Cultivadas , Endotelio Vascular/citología , Humanos , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/análisis , Receptores de Vitronectina/análisisRESUMEN
Thrombomodulin (TM) is an endothelial cell surface proteoglycan with anticoagulant functions, also implicated in cell proliferation, cell-cell adhesion and differentiation. In this study we determined circulating plasma TM (pTM) levels in human foetuses at different stages of pregnancy, at birth and in childhood. TM levels increased with gestational age, the median level reaching a peak of approximately 165 ng/ml between the 23rd and 26th week, thereafter decreasing gradually, reaching a value of 108 ng/ml at birth. pTM continues to decrease progressively during childhood, reaching in the 5-15 years group a median of 56 ng/ml which approaches the adult value. The pTM peak was statistically significant and represents a specific foetal phenomenon as it was independent of the corresponding maternal values. As a whole, the pTM pattern during foetal maturation appears totally different from that of protein C, prothrombin and other coagulation activators and inhibitors and thus, TM may play in the foetus another role in addition to its well-known anticoagulant function.
Asunto(s)
Coagulación Sanguínea , Feto/metabolismo , Trombomodulina/sangre , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Femenino , Humanos , Recién Nacido , EmbarazoRESUMEN
Angiogenesis and the formation of new blood vessels requires coordinated regulation of matrix proteolysis and endothelial cell migration. Cellular proteolytic capacity is the balance between secreted matrix metalloproteinases (MMP) and their inhibitors (TIMPs). We have examined the regulation of the gelatinase/TIMP balance by transforming growth factor-beta1 (TGF-beta1) and phorbol myristate acetate (PMA) in bovine endothelial cells. The low constitutive expression of gelatinase A/MMP-2 was upregulated by TGF-beta1 in a dose-dependent manner. Gelatinase B/MMP-9 was only detected upon treatment with either PMA or TGF-beta1. However, addition of both factors together revealed a striking synergistic effect causing upregulation of MMP-9 and downregulation of TIMPs, thereby increasing the net MMP-9/TIMP balance and the gelatinolytic capacity. These effects were observed at both the protein and mRNA levels. We demonstrate that changes in different members of the Jun oncogene family with distinct transactivation properties may account for this synergistic effect. We investigated the contribution of these changes in gelatinolytic balance to endothelial cell migration and invasion. The endothelial cells showed increased cell motility in response to PMA, but the addition of TGF-beta1 had an inhibitory effect. Hence, regulation of the MMP-9/TIMP balance failed to correlate with the migratory or invasive capacity. These results question a direct role for MMP-9 in endothelial cell motility and suggest that gelatinases may contribute in alternative ways to the angiogenic process.
Asunto(s)
Endotelio Vascular/fisiología , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Neovascularización Patológica , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Bovinos , Movimiento Celular/fisiología , Endotelio Vascular/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Degradation of extracellular matrix takes place in areas of cell-matrix contacts and is partly carried out by the action of matrix metalloproteinases (MMP). MMP-2 is a member of the MMP family that has been associated with breast-cancer metastasis. In the present study, we investigated the association of MMP-2 to the surface of breast-cancer cells and revealed an MMP-2-binding site that is expressed on sparsely plated cells and which is progressively lost as the cells approach confluence. Gelatin zymography, immunostaining and flow cytometry of MDA-MB-231 cells from sparse cultures demonstrated binding both of latent and of activated exogenous MMP-2, while little or no binding of MMP-2 was observed in confluent culture. Analysis of the expression of MTI-MMP, TIMP-2 and alpha(v) integrin, 3 proteins shown to play a role in cell-surface association of MMP-2, revealed enhanced levels of these proteins in confluent MDA-MB-231 cells. Thus, the reduced MMP-2 binding to confluent cells is not related to a deficiency in these MMP-2-binding proteins. Taken together, these studies suggest that MMP-2 binding to the surface of breast-cancer cells is regulated by cell-cell interactions and that tumor cells invading from the main tumor mass can up-regulate their MMP-2-binding capacity to acquire greater invasive capacity.
Asunto(s)
Neoplasias de la Mama/enzimología , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Antígenos CD/análisis , Neoplasias de la Mama/patología , Recuento de Células , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Integrina alfaV , Metaloproteinasa 2 de la Matriz , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Células Tumorales CultivadasRESUMEN
The interaction between tumor cells and platelets facilitates the formation of metastasis in a way depending on the platelet aggregating ability of the tumor cell, but the mechanism remains to be elucidated. We have shown, by zymography and Western blot, that platelets greatly increased the secretion to the culture medium of MMP-9 by human mammary tumor cells MDA-MB231. This increase, which was dependent on protein synthesis, was caused by the platelet aggregates interacting with the tumor cells and not by the soluble factors released during platelet activation. Platelet subcellular fractionation allowed the localization of the inducing factor to the membrane fraction of the platelet granules, thus requiring platelet aggregation in order to become accessible on the platelet surface.
Asunto(s)
Plaquetas/fisiología , Colagenasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Neoplasias de la Mama , Fraccionamiento Celular , Gránulos Citoplasmáticos/fisiología , Femenino , Humanos , Membranas Intracelulares/fisiología , Metaloproteinasa 9 de la Matriz , Activación Plaquetaria , Trombina/farmacología , Células Tumorales CultivadasRESUMEN
Progelatinase A is a matrix metalloproteinase involved in the turnover of extracellular matrix (ECM). We report that the ECM produced by bovine corneal endothelial (BCE) cells contains progelatinase A free of tissue inhibitor of metalloproteinase (TIMP2). The matrix-bound progelatinase A can be activated by APMA to generate a 62 kDa and a 45 kDa species with enzymatic activity and is inhibited by TIMP2. The bound progelatinase can be released after treatment of the ECM with gelatinase B. These studies suggest that the ECM can function as a reservoir for progelatinase A which may be readily available for cells in processes such as metastasis, angiogenesis, inflammation and wound healing.
Asunto(s)
Endotelio Corneal/enzimología , Precursores Enzimáticos/metabolismo , Matriz Extracelular/enzimología , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Animales , Bovinos , Células Cultivadas , Colagenasas , Endotelio Corneal/citología , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/análisis , Precursores Enzimáticos/antagonistas & inhibidores , Gelatinasas/análisis , Gelatinasas/antagonistas & inhibidores , Metaloproteinasa 9 de la Matriz , Metaloendopeptidasas/análisis , Metaloendopeptidasas/antagonistas & inhibidores , Acetato Fenilmercúrico/análogos & derivados , Acetato Fenilmercúrico/farmacología , Proteínas/análisis , Proteínas/farmacología , Inhibidor Tisular de Metaloproteinasa-2RESUMEN
Interaction of tumor cells with platelets facilitates metastasis of tumor cells. It has been proposed that platelets protect tumor cells against the host's immune defense and enhance tumor-cell extravasation. In the present work we show that platelets increase the invasiveness of 3 mammalian cell lines (MCF-7, ZR-51 and MDA-MB231) through extracellular matrix, and propose this as an additional mechanism by which platelets facilitate metastasis. Since gelatinase and urokinase have both been implicated in degradation of the extracellular matrix and cell migration, and therefore in tumor invasion, we have also analyzed whether the interaction of platelets with tumor cells can modify the secretion of these proteases by tumor cells. MDA-MB231, which was the most invasive cell line among the 3 tested and was the most potent in inducing platelet aggregation, secreted the highest level of urokinase and was the only one in which gelatinase was detected. While platelets had no significant effect on the urokinase activity expressed by these cells, they induced in MDA-MB231 an important increase in the secretion of gelatinase, which can be reproduced by both platelet membrane and platelet releasate of activated platelets. This increase in gelatinase could be responsible, at least in part, for the increased invasiveness of these cells, since added TIMP-1 significantly reduced the number of cells which traversed matrigel.
Asunto(s)
Plaquetas/fisiología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Gelatinasas/metabolismo , Activadores Plasminogénicos/metabolismo , Humanos , Técnicas In Vitro , Invasividad Neoplásica , Activación Plaquetaria , Agregación Plaquetaria , Células Tumorales CultivadasRESUMEN
When porcine endothelial cells in culture are incubated in the presence of human platelets, a 90kDa neutral proteinase activity is generated on casein gel (PECAP-Platelet Endothelial Cell Activated Protease). This activity was undetected when platelet extract or serum free EC conditioned medium were analysed under similar conditions. The optimum pH, isoelectric point, molecular weight and inhibitory profile of this activity were similar to Glu-plasmin. However, the low plasminogen content (less than 50ng/ml) in the conditioned medium of endothelial cells incubated with platelet could not contribute alone to this activity and the presence of a plasmin potentiating factor was suggested. This factor was separated from plasminogen by lysine-Sepharose chromatography.