RESUMEN
The central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.
Asunto(s)
Acuaporina 1/genética , Acuaporina 5/genética , Condrocitos/metabolismo , Degeneración del Disco Intervertebral/genética , Núcleo Pulposo/metabolismo , Factores de Transcripción/genética , Adulto , Animales , Acuaporina 1/metabolismo , Acuaporina 5/metabolismo , Condrocitos/patología , Femenino , Regulación de la Expresión Génica , Humanos , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Núcleo Pulposo/patología , Concentración Osmolar , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: These studies investigated cytokine and chemokine receptor profiles in nucleus pulposus (NP) cells, and the effects of receptor stimulation on mRNA levels of extracellular matrix (ECM) components, degrading enzymes and cytokine and chemokine expression. METHOD: Immunohistochemistry (IHC) was performed to localise expression of CD4, CCR1, CXCR1 and CXCR2 in human NP tissue samples. Effects of cytokine and chemokine stimulation was performed to investigate effects related to ECM remodelling and modulation of cytokine and chemokine mRNA expression. RESULTS: IHC identified CD4, CCR1, CXCR1 and CXCR2 expression by NP cells. Differential expression profiles were observed for CD4 and CXCR2 in tissue samples from degenerate and infiltrated IVDs. In vitro stimulations of primary human NP cultures with IL-16, CCL2, CCL3, CCL7 or CXCL8 did not identify any modulatory effects on parameters associated with ECM remodelling or expression of other cytokines and chemokines. Conversely, IL-1 was seen to modulate ECM remodelling and expression of all other cytokines and chemokines investigated. CONCLUSION: This study demonstrates for the first time that NP cells express a number of cytokine and chemokine receptors and thus could respond in an autocrine or paracrine manner to cytokines and chemokines produced by NP cells, particularly during tissue degeneration. However, this study failed to demonstrate regulatory effects on ECM genes and degradative enzymes or other cytokines and chemokines for any target investigated, with the exception of IL-1. This suggests that IL-1 is a master regulator within the IVD and may exert regulatory potential over a plethora of other cytokines and chemokines.
Asunto(s)
Interleucina-1beta/inmunología , Degeneración del Disco Intervertebral/inmunología , Receptores de Citocinas/metabolismo , Adulto , Anciano , Células Cultivadas , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Matriz Extracelular/fisiología , Regulación de la Expresión Génica/inmunología , Humanos , Degeneración del Disco Intervertebral/patología , Vértebras Lumbares , Persona de Mediana Edad , Receptores de Quimiocina/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Interleukin-1ß (IL-1ß) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. METHODS: Effects of WIN-55 were studied on IL-1ß stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. RESULTS: Treatment with WIN-55 alone or in combination with IL-1ß, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1ß, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. CONCLUSION: Cannabinoid WIN-55 can reduce both basal and IL-1ß stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Benzoxazinas/farmacología , Condrocitos/efectos de los fármacos , Interleucina-1beta/antagonistas & inhibidores , Metaloproteinasas de la Matriz/biosíntesis , Morfolinas/farmacología , Naftalenos/farmacología , Inhibidores Tisulares de Metaloproteinasas/biosíntesis , Alginatos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Interleucina-1beta/farmacología , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/genética , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patología , Receptores de Cannabinoides/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/biosíntesisRESUMEN
ADAMs and ADAMTSs are multi-domain proteins characterised by the presence of both metalloproteinase and disintegrin-like domains. ADAM proteins are usually type 1 transmembrane proteins, and ADAMTSs are secreted from cells. The dysregulated expression of ADAMs and ADAMTSs has been reported in a wide range of human cancers, where, in many cases, they are implicated as positive regulators of cancer progression. Proteolytically active ADAMs act as ectodomain sheddases, which release extracellular regions of membrane-bound proteins (e.g., adhesion molecules, growth factors, cytokines, chemokines and receptors). Certain ADAMTSs break down extracellular matrix (ECM) proteoglycans (e.g., aggrecan, brevican and versican). Through these actions they are able to sculpt the tumour microenvironment and modulate key processes involved in cancer progression, including cell proliferation, migration and angiogenesis. Members of both groups of protein can also act to inhibit or slow cancer progression: ADAMs can interact with specific integrins to elicit inhibitory effects on cancer dissemination, and certain ADAMTSs possess antiangiogenic activity, which prevents an increase in tumour size. This review covers recent developments in the involvement of ADAM and ADAMTS proteins in human cancer.
Asunto(s)
Proteínas ADAM/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias/enzimología , Adhesión Celular/fisiología , Proliferación Celular , Progresión de la Enfermedad , Humanos , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/enzimologíaRESUMEN
The ECM (extracellular matrix) is a complex molecular framework that provides physical support to cells and tissues, while also providing signals for cell growth, migration, differentiation and survival. The ECM of the CNS (central nervous system) is unusual in that it is rich in CSPGs (chondroitin sulfate proteoglycans), hyaluronan and tenascins. The CSPGs are widely expressed throughout the developing and adult CNS and have a role in guiding or limiting neurite outgrowth and cell migration. Alterations in the synthesis or breakdown of the ECM may contribute to disease processes. Here, we examine changes in the brain-specific CSPGs, brevican and phosphacan, following transient middle cerebral artery occlusion, a model of stroke in the rat. We have investigated their expression at various time points as well as their spatial relationship with ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs 4). The co-localization of ADAMTS or its activity may indicate a functional role for this matrix-protease pair in degeneration/regeneration processes that occur in stroke.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Infarto de la Arteria Cerebral Media/metabolismo , Lectinas Tipo C/genética , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Fosfatasas/genética , Animales , Brevicano , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Modelos Animales de Enfermedad , Lectinas Tipo C/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Ratas , Proteínas Tirosina Fosfatasas Clase 5 Similares a ReceptoresRESUMEN
ADAM-17, a disintegrin and metalloproteinase, is the major proteinase responsible for the cleavage of membrane-bound tumour necrosis factor (TNF) as well as being an active sheddase of other cytokines, cytokine receptors, growth factors and adhesion molecules. TNF is a major proinflammatory cytokine that has been identified as having a pathogenic role in inflammatory diseases within the CNS including multiple sclerosis (MS). Here we report the cellular origin and distribution of ADAM-17 expression within clinically and neuropathologically confirmed MS and normal control white matter, assessed by immunohistochemistry, western blotting and PCR. ADAM-17 expression was associated with the blood vessel endothelium, activated macrophages/microglia and parenchymal astrocytes in MS white matter. Increased levels of ADAM-17 immunoreactivity were displayed in active lesions with evidence of recent myelin breakdown. Further studies into the functional role of ADAM-17 in the pathogenesis of MS and other inflammatory conditions are required.
Asunto(s)
Proteínas ADAM/metabolismo , Esclerosis Múltiple Crónica Progresiva/metabolismo , Esclerosis Múltiple Crónica Progresiva/patología , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Esclerosis Múltiple Recurrente-Remitente/patología , Proteínas ADAM/genética , Proteína ADAM17 , Anciano , Anciano de 80 o más Años , Astrocitos/patología , Células Endoteliales/patología , Femenino , Humanos , Inmunohistoquímica , Macrófagos/patología , Masculino , Microglía/patología , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Vaina de Mielina/inmunología , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) -1, -4 and -5 proteases have been identified in the CNS at the mRNA level. These glutamyl endopeptidases, inhibited by tissue inhibitor of metalloproteinases (TIMP)-3, are key enzymes in the degradation of the aggregating chondroitin sulphate proteoglycans (CSPGs), and may therefore play a role in CNS extracellular matrix (ECM) changes in multiple sclerosis (MS). We have investigated ADAMTS and TIMP-3 expression in normal and MS CNS white matter by real-time RT-PCR, western blotting and immunohistochemistry. We report for the first time the presence of ADAMTS-1, -4 and -5 in normal and MS white matter. Levels of ADAMTS-1 and -5 mRNA were decreased in MS compared to normal tissue, with no significant change in ADAMTS-4 mRNA levels. Protein levels of ADAMTS-4 were significantly higher in MS tissue compared to normal tissue. Immunohistochemical studies demonstrated that ADAMTS-4 was associated predominantly with astrocytes with increased expression within MS lesions. TIMP-3 mRNA was significantly decreased in MS compared to controls. These studies suggest a role for ADAMTS-4 in the pathogenesis of MS. Further studies on the activity of ADAMTS-4 will enable a better understanding of its role in the turnover of the ECM of white matter in MS.
Asunto(s)
Proteínas ADAM/genética , Esclerosis Múltiple Crónica Progresiva/metabolismo , Esclerosis Múltiple Crónica Progresiva/fisiopatología , Fibras Nerviosas Mielínicas/enzimología , Inhibidor Tisular de Metaloproteinasa-3/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS1 , Proteína ADAMTS4 , Proteína ADAMTS5 , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Encéfalo/enzimología , Encéfalo/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/patología , Fibras Nerviosas Mielínicas/patología , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Regulación hacia ArribaRESUMEN
ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzymes are a recently described group of metalloproteinases. The substrates degraded by ADAMTS-1, -4 and -5 suggest that they play a role in turnover of extracellular matrix in the central nervous system (CNS). ADAMTS-1 is also known to exhibit anti-angiogenic activity. Their main endogenous inhibitor is tissue inhibitor of metalloproteinases (TIMP)-3. The present study was designed to investigate ADAMTS-1, -4 and -5 and TIMP-3 expression after experimental cerebral ischaemia and to examine whether cytokines known to be up-regulated in stroke could alter their expression by astrocytes in vitro. Focal cerebral ischaemia was induced by transient middle cerebral artery occlusion in the rat using the filament method. Our results demonstrate a significant increase in expression of ADAMTS-1 and -4 in the occluded hemisphere but no significant change in TIMP-3. This was accompanied by an increase in mRNA levels for interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra) and tumour necrosis factor (TNF). ADAMTS-4 mRNA and protein were up-regulated by TNF in primary human astrocyte cultures. The increased ADAMTS-1 and -4 in experimental stroke, together with no change in TIMP-3, may promote ECM breakdown after stroke, enabling infiltration of inflammatory cells and contributing to brain injury. In vitro studies suggest that the in vivo modulation of ADAMTS-1 and -4 may be controlled in part by TNF.
Asunto(s)
Proteínas ADAM/metabolismo , Astrocitos/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Infarto de la Arteria Cerebral Media/fisiopatología , Procolágeno N-Endopeptidasa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas ADAM/genética , Proteína ADAMTS1 , Proteína ADAMTS4 , Animales , Astrocitos/metabolismo , Western Blotting/métodos , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica/métodos , Masculino , Procolágeno N-Endopeptidasa/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is an animal model of inflammatory demyelination, a pathological event common to multiple sclerosis (MS). During CNS inflammation there are alterations in the extracellular matrix (ECM). A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS)-1, -4 and -5 are proteases present in the CNS, which are able to cleave the aggregating chondroitin sulphate proteoglycans, aggrecan, phosphacan, neurocan and brevican. It is therefore important to investigate changes in their expression in different stages of EAE induction. We have investigated expression of ADAMTS-1, -4, -5 and tissue inhibitor of metalloproteinase (TIMP)-3, by real-time RT-PCR. We have also examined protein expression of ADAMTS-1, -4 and -5 by western blotting and immunocytochemistry in spinal cord from animals at different stages of disease progression. Our study demonstrated a decrease in ADAMTS-4 mRNA and protein expression. TIMP-3 was decreased at the mRNA level although protein levels were increased in diseased animals compared to controls. Our study identifies changes in ADAMTS expression during the course of CNS inflammation which may contribute to ECM degradation and disease progression.
Asunto(s)
Proteínas ADAM/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Médula Espinal/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteínas ADAM/análisis , Proteínas ADAM/genética , Enfermedad Aguda , Animales , Inmunohistoquímica , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/química , Inhibidor Tisular de Metaloproteinasa-3/análisis , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
ADAM 17, also known as TACE, is an important sheddase for a number of proteins, including tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha), L-selectin, p75, and p55 TNF receptors, and interleukin-1 receptor II (IL-1RII). The presence of ADAM 17 mRNA in adult mouse and rat CNS was recently reported (Karkkainen et al. Mol Cell Neurosci 15:547-560, 2000). However, the cellular origin of ADAM 17 remains unknown. In this study, we have used an anti-ADAM 17 antibody in an immunohistochemical study of its distribution in human adult CNS tissue. Cells with astrocytic and endothelial morphology were ADAM 17-positive. This finding was further confirmed using double immunofluorescence with antibodies against GFAP and von Willebrand factor, which label astrocytes and endothelial cells, respectively. This study demonstrates that ADAM 17 is expressed by astrocytes and endothelial cells in normal brain tissue and may have a role in normal brain function.
Asunto(s)
Astrocitos/enzimología , Encéfalo/enzimología , Circulación Cerebrovascular , Endotelio Vascular/enzimología , Metaloendopeptidasas/metabolismo , Proteínas ADAM , Proteína ADAM17 , Anciano , Anciano de 80 o más Años , Encéfalo/citología , Endotelio Vascular/citología , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Distribución TisularRESUMEN
The effect of mannosamine, an inhibitor of glycosylphosphatidylinositol (GPI) anchor formation, on chondrocyte-mediated cartilage proteoglycan breakdown was investigated using cartilage explant cultures. Mannosamine inhibited interleukin 1alpha-, tumour necrosis factor alpha- and retinoic acid-stimulated proteoglycan release from bovine nasal and articular cartilage, and retinoic acid-stimulated proteoglycan release from human cartilage. Its effects on two GPI-anchored proteins [the urokinase receptor, which binds urokinase-type plasminogen activator (uPA) to cell surfaces, and alkaline phosphatase] were also studied using bovine chondrocytes. Enzyme histochemistry and zymography demonstrated cell-associated uPA-like serine proteinase activity and PA activity respectively which was not reduced by treatment of chondrocytes with mannosamine at concentrations effective at inhibiting cartilage proteoglycan breakdown. Similarly, the activity of cell-associated alkaline phosphatase was not reduced, except at mannosamine concentrations much higher than those used to inhibit proteoglycan breakdown. These results demonstrate that inhibition of proteoglycan breakdown by mannosamine is too potent to be explained by an effect on GPI-anchor formation.
Asunto(s)
Cartílago/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Hexosaminas/metabolismo , Proteoglicanos/metabolismo , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Cartílago/efectos de los fármacos , Bovinos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Técnicas de Cultivo , Femenino , Hexosaminas/farmacología , Humanos , Interleucina-1/farmacología , Ácido Láctico/metabolismo , Tabique Nasal/efectos de los fármacos , Tabique Nasal/metabolismo , Proteoglicanos/efectos de los fármacos , Activador de Tejido Plasminógeno/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The presence of metalloproteinase activity in endometrial flushings obtained from premenopausal women, during the proliferative and secretory phases of the menstrual cycle, control post-menopausal women and women with post-menopausal bleeding (PMB) with or without adenocarcinoma was analysed by zymography. In addition, quantitative measurements of matrix metalloproteinase 2 (MMP-2), MMP-3, MMP-9 and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the flushings were obtained by ELISA. The zymography results showed eight bands of activity, with molecular weights ranging from 51 to 208 kDa in the flushings from pre-menopausal women and post-menopausal women, particularly those with adenocarcinoma. Both zymography and ELISA showed that MMP-2 and MMP-9 were the major metalloproteinases found in the flushings and only low concentrations of MMP-3 were found. Concentrations of MMP-2 in pre-menopausal women were higher in flushings obtained during the secretory phase of the menstrual cycle than those obtained in the proliferative phase (P < 0.05), suggesting that it may play a role in embryo implantation. Concentrations of MMP-2 (P < 0.001), MMP-9 (P < 0.05) and TIMP-1 (P < 0.001) in the flushings from post-menopausal control women were lower than those from pre-menopausal women. Concentrations of MMP-2 (P < 0.05) and TIMP-1 (P < 0.05) were higher in flushings from women with PMB without carcinoma compared with post-menopausal controls and concentrations of MMP-9 (P < 0.01) and TIMP-1 (P < 0.05) in flushings from women with adenocarcinoma were higher than in post-menopausal controls. Among subjects with PMB, concentrations of MMP-9 in women with adenocarcinoma were higher than those without carcinoma (P < 0.05). Our results show that concentrations of MMP-2, MMP-9 and TIMP-1, but not MMP-3, are associated with endometrial activity and, therefore, may have a role in the breakdown of endometrial tissue. In addition, the increased concentrations of MMP-9 in flushings of women with adenocarcinoma indicate that this particular proteinase is associated with the presence of endometrial neoplastic cells.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias Endometriales/enzimología , Endometrio/enzimología , Menopausia/metabolismo , Metaloendopeptidasas/análisis , Inhibidor Tisular de Metaloproteinasa-1/análisis , Adulto , Anciano , Anciano de 80 o más Años , Colagenasas/análisis , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Fase Folicular/metabolismo , Gelatinasas/análisis , Humanos , Fase Luteínica/metabolismo , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 3 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz , Persona de Mediana Edad , Estadísticas no ParamétricasRESUMEN
Suramin is an anti-neoplastic drug. Its actions include the inhibition of binding of urokinase-type plasminogen activator (uPA) to its receptor, an event which may prevent cartilage breakdown. The aim of this work was to determine the effect of suramin on cartilage resorption. Cartilage expiants, stimulated with interleukin-1alpha, tumour necrosis factor-alpha or retinoic acid were incubated with suramin. Release of incorporated (35)S-sulphate from pre-labelled expiants was used as a measure of proteoglycan breakdown and toluidine blue staining was used to visualise proteoglycan loss.Suramin inhibited the resorption of cytokine and retinoic acid-stimulated bovine nasal cartilage at concentrations between 100-1000 microM. These findings were confirmed by histochemistry. Though reversibility studies indicated that suramin toxicity could not be excluded above 100 muM, retention of suramin in the expiants may have contributed to this. There was no significant effect on lactate production up to 500 muM. The observed inhibition of cartilage resorption may reflect actions of suramin on the PA/plasmin system or on cytokine action.
RESUMEN
The role played by serine proteinases with trypsin-like specificity in chondrocyte-mediated cartilage proteoglycan breakdown was investigated by use of a selective proteinase inactivator, 7-amino-4-chloro-3-(-3-isothiureidopropoxy)isocoumarin, in explant culture systems. This compound was a rapid inactivator of urokinase-type plasminogen activator. It potently inhibited interleukin 1- and tumor necrosis factor-stimulated proteoglycan release from both nasal and articular cartilage. Its less potent inhibition of basal and retinoic acid-stimulated release appeared to be due to cytotoxic effects. The functional half-life of the inactivator in culture medium was 95 min, and its concentration in cartilage was 2.5-fold higher than in the surrounding medium. Following spontaneous hydrolysis the breakdown products of the inactivator were unable to inhibit proteoglycan release. Trypsin-like activity was demonstrated by enzyme histochemistry to be chondrocyte-associated and inhibited by the serine proteinase inactivator. Cell-associated and secreted plasminogen activator activity was detected by zymography. These results suggest the involvement of a serine proteinase(s) with trypsin-like specificity, possibly urokinase-type plasminogen activator, in chondrocyte-mediated cartilage proteoglycan breakdown occurring as a result of stimulation with proinflammatory cytokines. Basal proteoglycan breakdown may occur via a different pathway. Our findings point to a pathological role for serine proteinase(s) in the development of cartilage diseases such as arthritis, possibly in a cascade which results in the activation of the enzyme(s) directly responsible for proteoglycan breakdown. It remains to be shown whether the target serine proteinase is urokinase-type plasminogen activator.
Asunto(s)
Cartílago/efectos de los fármacos , Cartílago/metabolismo , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Proteoglicanos/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Animales , Bovinos , Cumarinas/farmacología , Medios de Cultivo , Técnicas de Cultivo , Citocinas/farmacología , Humanos , Mediadores de Inflamación/farmacología , Isocumarinas , Cinética , Serina Endopeptidasas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
Transforming growth factor beta (TGF beta) has previously been shown to have actions on chondrocytes and cartilage both in vitro and in vivo which suggest a role in connective tissue repair. In particular, some of its actions have been shown to be antagonistic to those of interleukin 1 (IL-1). In this study, the effects of TGF beta on prostaglandin E (PGE) production and caseinase activity, in the presence and absence of IL-1, in human articular chondrocytes were investigated. TGF beta 1 and TGF beta 2 were shown to modulate IL-1 beta-stimulated PGE production and caseinase activity. Both TGF beta 1 and beta 2 inhibited IL-1 beta-stimulated PGE production in the absence of serum and augmented it in the presence of serum. TGF beta 1 and TGF beta 2 inhibited IL-1-stimulated caseinase activity with and without serum. In general, the TGF beta s had little or no effect on basal PGE or caseinase levels. TGF beta s may be important modulators of chondrocyte metabolism, their effects on PGE production may depend on cytokine interactions; furthermore, their effects on caseinase activity may help prevent cartilage breakdown.
Asunto(s)
Cartílago Articular/metabolismo , Interleucina-1/farmacología , Metaloendopeptidasas/metabolismo , Prostaglandinas E/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Anciano , Anciano de 80 o más Años , Cartílago Articular/citología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Interacciones Farmacológicas , Matriz Extracelular/metabolismo , Humanos , Persona de Mediana Edad , RadioinmunoensayoRESUMEN
Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Colágeno/metabolismo , Proteínas de la Matriz Extracelular , Interleucina-1/farmacología , Proteoglicanos/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Agrecanos , Células Cultivadas , Humanos , Inmunohistoquímica , Lectinas Tipo C , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismoRESUMEN
This is a study of the regulation of human articular chondrocyte proliferation by transforming growth factor beta (TGF beta) and interleukin-1 beta (IL-1 beta) in vitro. Human articular chondrocytes were cultured at different cell densities on plastic and on a collagen substratum, in the presence and absence of serum. The effects of TGF beta and IL-1 beta on proliferation of chondrocytes, as determined by [3H]thymidine incorporation, under these conditions of culture were examined. TGF beta was found to have both stimulatory and inhibitory effects on chondrocytes in vitro. Interactions between TGF beta and growth factors present in serum influence the modulation of chondrocyte proliferation by TGF beta. IL-1 beta caused a significant reduction of the TGF beta-stimulated increase in chondrocyte proliferation. The complex inter-relationships between TGF beta and IL-1 beta on chondrocytes have implications for cartilage repair.
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Cartílago Articular/efectos de los fármacos , Interleucina-1/farmacología , Timidina/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Cartílago Articular/metabolismo , División Celular , Células Cultivadas , ADN/análisis , HumanosRESUMEN
OBJECTIVE: To investigate the pattern of cytokine gene expression in human articular chondrocytes in culture in response to interleukin-1 beta (IL-1 beta). The effect of serum and variations in culture conditions was also studied. METHODS: Messenger RNA was extracted from cells, reverse-transcribed to complementary DNA, and amplified by the polymerase chain reaction (PCR), using specific oligonucleotide primers. The PCR products were validated by restriction analysis with specific enzymes and by Southern blot analysis. RESULTS: In cultured articular chondrocytes, IL-1 beta, IL-1 alpha, granulocyte colony-stimulating factor (CSF), and granulocyte-macrophage CSF cytokine genes were expressed only after induction by IL-1 beta. However, IL-6, IL-8, and macrophage CSF genes were expressed constitutively. The expression of IL-1 beta was dose and time dependent. CONCLUSION: Using PCR, it was possible to demonstrate gene expression for several cytokines in human articular chondrocytes in culture. It was evident that some cytokine genes were expressed constitutively and some were inducible by IL-1 beta.
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Cartílago Articular/citología , Citocinas/genética , Interleucina-1/farmacología , Secuencia de Bases , Southern Blotting , Células Cultivadas , Electroforesis en Gel de Agar , Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , ARN/genéticaRESUMEN
Cathepsin B and L activity was studied histochemically in arthritic rat ankle joints using specific synthetic substrates in a post coupling method on unfixed and undecalcified cryostat sections of rat ankle joints. Activity was strongly increased in chondrocytes and cells of the inflamed synovium with the development of arthritis induced by the synthetic adjuvant CP20961. Activity reached a maximum 20 days after induction of arthritis and decreased as the rats entered natural remission. Cathepsin B and L were at their highest level when macrophages were present in the joint space, as shown by using monoclonal antibody markers for rat macrophages (ED1 and ED2) in a biotin-avidin immunoperoxidase assay. This suggests that the macrophage infiltrate may have stimulated proteinase production in chondrocytes through cytokine release. The profile of appearance of cysteine proteinases suggests their involvement in the breakdown of cartilage and bone in the arthritic joint.
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Artritis Experimental/enzimología , Cartílago Articular/enzimología , Catepsina B/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Endopeptidasas , Membrana Sinovial/enzimología , Adyuvantes Inmunológicos , Animales , Articulación del Tobillo , Catepsina L , Diaminas/toxicidad , Modelos Animales de Enfermedad , Macrófagos , Ratas , Ratas Endogámicas Lew , Linfocitos TRESUMEN
Transforming growth beta (TGF-beta) has been proposed to have a role in bone remodeling by affecting the differentiation and activity of osteoblasts and osteoclasts and by inhibiting the production of proteinases, such as plasminogen activators (PAs). Studies on PAs have largely been based on data from nonhuman and fetal cell lines, however. The purpose of this study was to investigate the effect of TGF-beta on the PA activity of normal human osteoblast-like cells and to compare this with its action on the human osteosarcoma cell line MG-63. The action of interleukin-1 beta (IL-1 beta) was also assessed because it has been shown to increase PA activity in other connective tissue cell types. Normal osteoblast-like cells had low to undetectable basal urokinase (uPA) and tissue plasminogen activator (tPA) activity, which was significantly stimulated by TGF-beta 1. This action was shown to be dependent on transcription and new protein synthesis. TGF-beta 2 had a similar action. IL-1 beta did not stimulate PA activity. In contrast, the MG-63 cell line had high basal tPA and uPA activities. TGF-beta 1 decreased basal PA activity, the effect being most marked for uPA activity. IL-1 beta stimulated uPA and tPA activity. TGF-beta 1 inhibited IL-1 beta-stimulated uPA activity, but the effect on tPA was more variable. This study has shown that TGF-beta has opposite effects on the PA activity of the two osteoblast-like cell types studied. Care must therefore be used before extrapolating data from one cell type to another.(ABSTRACT TRUNCATED AT 250 WORDS)