RESUMEN
BACKGROUND: Osteosarcoma (OS) is the most frequent primary malignant bone tumour in children and adolescents with a high propensity for lung metastasis. Chemokines and chemokine receptors have been described to have an important role in many malignancies including OS. The aim of this study was to investigate the expression of CXCR7 receptor in OS tissues and its role in the progression of the disease in the lungs. METHODS: Immunohistochemistry was used to study CXCR7 expression in primary tumours and metastatic tissues from patients with OS. Its contribution to tumour expansion in the lungs has been also assessed using animal models and synthetic-specific CXCR7 ligands. RESULTS: CXCR7 was expressed on human primary bone tumours and on lung metastases. Its expression was predominantly located on tumour-associated blood vessels. Mice challenged with OS cells and systematically treated with synthetic CXCR7 ligands presented a significant reduction of lung nodules compared with untreated mice. CONCLUSION: This study shows that CXCR7 has a critical role in OS progression in the lungs, where are expressed CXCR7 ligands, especially CXCL12. Moreover, we highlight that synthetic CXCR7 ligands could represent a powerful therapeutic tool to impede lung OS progression.
Asunto(s)
Neoplasias Óseas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Osteosarcoma/metabolismo , Osteosarcoma/secundario , Receptores CXCR/biosíntesis , Animales , Neoplasias Óseas/patología , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Osteosarcoma/genética , Osteosarcoma/patología , Receptores CXCR/genéticaRESUMEN
BACKGROUND: Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Chemokine-receptor pairs have a critical role in determining the metastatic progression of tumours. Our hypothesis was that disruption of CXCR7/CXCR7 ligands axis could lead to a decrease in CRC metastases. METHODS: Primary tumours and metastatic tissues from patients with CRC were tested for the expression of CXCR7 and its ligands. Relevance of CXCR7/CXCR7 ligands for CRC metastasis was then investigated in mice using small pharmacological CXCR7 antagonists and CRC cell lines of human and murine origins, which - injected into mice - enable the development of lung and liver metastases. RESULTS: Following injection of CRC cells, mice treated daily with CXCR7 antagonists exhibited a significant reduction in lung metastases. However, CXCR7 antagonists failed to reduce the extent of liver metastasis. Moreover, there were subtle differences in the expression of CXCR7 and its ligands between lung and liver metastases. CONCLUSION: Our study suggests that the activation of CXCR7 on tumour blood vessels by its ligands may facilitate the progression of CRC within lung but not within liver. Moreover, we provide evidence that targeting the CXCR7 axis may be beneficial to limit metastasis from colon cancer within the lungs.
Asunto(s)
Carcinoma/metabolismo , Carcinoma/secundario , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Receptores CXCR/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-8/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.
Asunto(s)
Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Receptores CXCR3/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular , Neoplasias del Colon/tratamiento farmacológico , Humanos , Ligandos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Trasplante de Neoplasias , Especificidad de Órganos , Receptores CXCR3/metabolismo , Tasa de SupervivenciaRESUMEN
Pentoxifylline (PTX) has been shown to protect the liver against normothermic ischemia-reperfusion (I-R) injury. The aims of this study were to investigate the action of PTX on tumor necrosis factor alpha (TNFalpha) gene transcription following normothermic liver I-R as well as to evaluate the resulting effects on liver function and survival. A segmental normothermic liver ischemia was induced for 90 minutes. Rats were divided into three groups: group 1, control, Ringer lactate administration; group 2, PTX treatment; group 3, sham-operated control rats. PTX (50 mg/kg) was injected intravenously 30 minutes before induction of ischemia and 30 minutes before reperfusion. The nonischemic liver lobes were resected at the end of ischemia. Survival rates were compared and serum activities of TNFalpha, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured. Liver histology was assessed 6 hours after reperfusion. Liver TNFalpha mRNA was assessed by polymerase chain reaction amplification at different times after reperfusion. PTX treatment significantly decreased serum activities of TNFalpha and inhibited liver expression of TNFalpha mRNA. The extent of liver necrosis and serum levels of liver enzymes were significantly decreased by PTX treatment, resulting in a significant increase in 7-day survival compared with nontreated control rats. In conclusion, PTX inhibits liver TNFalpha gene transcription, decreases serum TNFalpha levels, and reduces liver injury following normothermic I-R.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Circulación Hepática , Pentoxifilina/farmacología , Daño por Reperfusión/prevención & control , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética , Animales , Circulación Hepática/efectos de los fármacos , Masculino , Modelos Animales , Ratas , Ratas Long-Evans , Vasodilatadores/farmacologíaRESUMEN
The lung is one target organ to which solid tumors frequently metastasize. Given the systemic adverse effects of currently available treatments, developing effective strategies of drug/gene delivery directly to the lungs is therefore needed. Aerosol delivery is a non-invasive gene transfer approach to target the airways. Here, we sought to evaluate the potential to deliver a fractalkine (FKN)-encoding plasmid formulated with the tetrafunctional amphiphilic block copolymer 704 through aerosolization in two models of pulmonary metastases. FKN is a chemokine recently described as a good candidate to stimulate a strong antitumor immune response in various forms of cancers. Here, we have assessed the effect of single and repeated aerosolizations of FKN-encoding plasmid formulated with 704 on the development of experimental lung metastases of mouse colon carcinoma and osteosarcoma. For this purpose, we have designed FKN-Fc sequences encoding an optimized version of the chemokine. Repeated intratracheal administrations of 704/FKN-Fc markedly inhibited growth of experimental lung metastases of CT-26 and K7M2 cells. Our results showed that tetrafunctional amphiphilic block copolymer 704 is a highly efficient synthetic vector for mediating local and safe gene transfer into the lung. In addition, FKN-Fc gene therapy of pulmonary nodules may provide a promising immunotherapeutic approach.
Asunto(s)
Quimiocina CX3CL1/genética , Terapia Genética/métodos , Fragmentos de Inmunoglobulinas/genética , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Administración por Inhalación , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Técnicas de Transferencia de Gen , Humanos , Inmunoterapia , Neoplasias Pulmonares/genética , Ratones , Nanosferas , Metástasis de la Neoplasia , Plásmidos/administración & dosificación , Plásmidos/genética , TransfecciónRESUMEN
BACKGROUND AND AIMS: Fractalkine, a chemokine that presents as both a secreted and a membrane-anchored form, has been described as having tumour-suppressive activities in standard subcutaneous models. Here, we investigate the antitumour effect of fractalkine, in its three molecular forms, in two orthotopic models of metastatic colon cancer (liver and lung) and in the standard subcutaneous model. METHODS: We have developed models of skin tumours, liver and pulmonary metastasis and compared the extent of tumour development between C26 colon cancer cells expressing either the native, the soluble, the membrane-bound fractalkine or none. RESULTS: The native fractalkine exhibits the strongest antitumour effect, reducing the tumour size by 93% in the skin and by 99% in the orthotopic models (p<0.0001). Its overall effect results from a critical balance between the activity of the secreted and the membrane-bound forms, balance that is itself dependent on the target tissue. In the skin, both molecular variants reduce tumour development by 66% (p<0.01). In contrast, the liver and lung metastases are only significantly reduced by the soluble form (by 96%, p<0.002) whereas the membrane-bound variant exerts a barely significant effect in the liver (p = 0.049) and promotes tumour growth in the lungs. Moreover, we show a significant difference in the contribution of the infiltrating leukocytes to the tumour-suppressive activity of fractalkine between the standard and the orthotopic models. CONCLUSIONS: Fractalkine expression by C26 tumour cells drastically reduces their metastatic potential in the two physiological target organs. Both molecular forms contribute to its antitumour potential but exhibit differential effects on tumour development depending on the target tissue.
Asunto(s)
Quimiocinas CX3C/fisiología , Neoplasias del Colon/metabolismo , Inmunoterapia/métodos , Proteínas de la Membrana/fisiología , Animales , Quimiocina CX3CL1 , Quimiocinas CX3C/genética , Quimiocinas CX3C/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Femenino , Terapia Genética/métodos , Vectores Genéticos , Inmunofenotipificación , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/prevención & control , Neoplasias Cutáneas/secundario , TransfecciónRESUMEN
Oxyuranus scutellatus toxin 1 (OS1) and toxin 2 (OS2) are two monochain phospholipases A2 isolated from the venom of Taipan. Their iodinated derivatives have been used to characterize phospholipase A2 receptors on rabbit skeletal muscle cells in culture. Both ligands recognize one family of binding sites on myotube membranes with a Bmax of 1.9 to 2.2 pmol/mg of protein and dissociation constant values of 7.4 pM for 125I-OS2 and 38 pM for 125I-OS1. Other snake venom phospholipases A2 are able to inhibit 125I-OS2 binding to the muscle receptor. Competition experiments with these unlabeled phospholipases A2 define a pharmacological profile of the muscle receptor very different from the previously described pharmacological profile of the neuronal phospholipase A2 receptors. The number of 125I-OS2 receptors in skeletal muscle cells increases during in vitro cell maturation but there is no clear relation between the increase of Bmax and the fusion of myoblasts into myotubes. The phospholipase A2 binding protein from myotubes has been identified both by cross-linking experiments and by purification studies. It is composed of only one subunit of Mr 180,000.
Asunto(s)
Músculos/análisis , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Receptores de Superficie Celular/análisis , Marcadores de Afinidad , Animales , Unión Competitiva , Diferenciación Celular , Membrana Celular/análisis , Células Cultivadas , Ácidos Cólicos , Cromatografía , Reactivos de Enlaces Cruzados , Venenos Elapídicos/análisis , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Desarrollo de Músculos , Fosfolipasas A2 , Conejos , Receptores de Superficie Celular/metabolismoRESUMEN
Two phagocytes-mediated activities of the sea bass Dicentrarchus labrax were examined after exposure to sublethal concentrations of copper and cadmium: (a) phagocytosis (measured by phagocytotic index), and (b) the production of reactive oxygen intermediates (luminol-dependent chemiluminescence) in response to bacteria Aeromonas salmonicida. In vivo exposure for 48 h to each metal separately by intraperitoneal injection did not affect the quantity of phagocytes of pronephros and their viability but inhibited, in dose-dependent manner, phagocytosis and chemiluminescence of these cells. The half-inhibition value was 250 micrograms kg-1 for copper and 1 mg kg-1 for cadmium. In vitro exposure to copper for 30 min had the same immunomodulatory effect on macrophage chemiluminescence as that observed in vivo, whereas treatment with cadmium under the same conditions had a dose-dependent effect opposite to that observed in vivo. Assessment of these two macrophage-mediated functions could therefore be used as early bioindicators of the marine pollution.
Asunto(s)
Lubina/inmunología , Cadmio/toxicidad , Cobre/toxicidad , Animales , Cadmio/administración & dosificación , Recuento de Células/efectos de los fármacos , Células Cultivadas , Cobre/administración & dosificación , Inyecciones Intraperitoneales , Riñón/citología , Riñón/efectos de los fármacos , Mediciones Luminiscentes , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacosRESUMEN
Endothelin, one of the most potent vasoconstrictor known, has been suggested to act as an endogenous agonist of L-type Ca2+ channels. In this paper we show that endothelin stimulates the metabolism of inositol phosphates and induces the mobilization of intracellular Ca2+ stores. The transient activation of Ca2+-sensitive K+ channel provokes an hyperpolarization of the membrane. It is followed by a sustained depolarization which is due to the opening of a non-specific cation channel which is permeable to Ca2+ and Mg2+. The depolarization then activates L-type Ca2+ channels. This mechanism of action explains why part of the endothelin-induced vasocontriction is eliminated by L-type Ca2+ channel blockers.
Asunto(s)
Canales de Calcio/fisiología , Péptidos/farmacología , Vasoconstricción/efectos de los fármacos , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Potenciales de Acción/efectos de los fármacos , Angiotensina II/farmacología , Animales , Aorta Torácica/fisiología , Calcio/farmacología , Bloqueadores de los Canales de Calcio , Canales de Calcio/efectos de los fármacos , Membrana Celular/fisiología , Electrofisiología , Endotelinas , Endotelio Vascular , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/metabolismo , Isradipino , Oxadiazoles/metabolismo , Oxadiazoles/farmacología , Fosfatidilinositoles/metabolismo , Ratas , Ratas Endogámicas , Vasopresinas/farmacologíaRESUMEN
We have reported recently that colchicine and other microtubule-disrupting agents stimulated interleukin-1 (IL-1) alpha and beta synthesis in human monocytes. In this study we found that unexpectedly colchicine failed to stimulate the expression of two other potent immune mediators, namely tumor necrosis factor-alpha and interleukin-6. Remarkably, taxol which induces stable microtubule bundles, antagonized the colchicine but not the LPS-induced IL-1 synthesis. These results suggested that the colchicine-mediated IL-1 induction was generated by microtubule disassembly. We next demonstrated that microtubule disruption triggered an elevation of intracellular levels of cAMP and a subsequent stimulation of protein kinase A. The use of different protein kinase inhibitors supported a role of the PKA, but not the PKC, in the colchicine-induced IL-1 production. Furthermore, elevation of intracellular cAMP levels by 8-bromo-cAMP, forskolin, or cholera toxin potentiated the effect of suboptimal concentration of colchicine on IL-1 synthesis. However, these agents alone were unable to induce IL-1 synthesis. Therefore, our data indicate that the cAMP/protein kinase A-signaling pathway is necessary but not sufficient to generate IL-1 synthesis by microtubule disruption. Thus, microtubule-disrupting drugs appear as useful tools to further characterize the molecular events which regulate IL-1 production in human monocytes.
Asunto(s)
Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Microtúbulos/fisiología , Monocitos/metabolismo , Proteínas Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Adenilil Ciclasas/metabolismo , Células Cultivadas , Colchicina/farmacología , AMP Cíclico/biosíntesis , AMP Cíclico/metabolismo , Activación Enzimática , Humanos , Indometacina/farmacología , Microtúbulos/efectos de los fármacos , Monocitos/ultraestructura , Paclitaxel/farmacologíaRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) is a major chemoattractant for monocytes and T lymphocytes. The MonoMac6 cell line was used to examine MCP-1 receptor-mediated signal transduction events in relation to MCP-1-mediated monocytic transendothelial migration. MCP-1 stimulates, with distinct time courses, extracellular signal-related kinases (ERK1 and ERK2) and stress-activated protein kinases (SAPK1/JNK1 and SAPK2/p38). SAPK1/JNK1 activation was blocked by piceatannol, indicating that it is regulated by Syk kinase, whereas SAPK2/p38 activation was inhibited by PP2, revealing an upstream regulation by Src-like kinases. In contrast, ERK activation was insensitive to PP2 and piceatannol. Pertussis toxin, a blocker of Go/Gi proteins, abrogated MCP-1-induced ERK activation, but was without any effect on SAPK1/JNK1 and SAPK2/p38 activation. These results underscore the major implication of Go/Gi proteins and nonreceptor tyrosine kinases in the early MCP-1 signaling. Furthermore, MCP-1-mediated chemotaxis and transendothelial migration were significantly diminished by a high concentration of SB202190, a broad SAPK inhibitor, or by SB203580, a specific inhibitor of SAPK2/p38, and abolished by pertussis toxin treatment. Altogether, these data suggest that coordinated action of distinct signal pathways is required to produce a full response to MCP-1 in terms of monocytic locomotion.
Asunto(s)
Quimiocina CCL2/farmacología , Monocitos/citología , Transducción de Señal/efectos de los fármacos , Línea Celular , Quimiotaxis/efectos de los fármacos , Toxina del Cólera/farmacología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Fibronectinas/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Proteína Quinasa 8 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Toxina del Pertussis , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Cordón Umbilical/citología , Factores de Virulencia de Bordetella/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos , Familia-src Quinasas/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
The vasoconstricting action of endothelin-1 (ET-1) is partly mediated by voltage-dependent L-type Ca2+ channels. Activation of the Ca2+ channels is indirect. ET-1 action involves (i) the hydrolysis of phosphatidylinositol and the release of Ca2+ from internal stores and (ii) the opening of a nonselective cation channel in the plasma membrane. The resulting depolarization triggers the activity of L-type Ca2+ channels.
Asunto(s)
Aorta/citología , Péptidos/farmacología , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Electrofisiología , Endotelinas , RatasRESUMEN
Organ injury after ischemia and reperfusion (I/R) remains one of the most important limiting factors in liver surgery and transplantation. Oxygen-free radical (OFR) generation is considered a major cause of this damage. JNK1/SAPK1, a member of MAPK family, regulates cell adaptation to stressful conditions. The aim of this study was to determine if hypoxia-reoxygenation (H/R) can activate JNK1/SAPK1 and if OFR are involved in this activation. Primary cultured rat hepatocytes isolated from other liver cells and blood flow were submitted to warm and cold H/R phases mimicking surgical and transplant conditions. JNK1/SAPK1 was activated by both warm and cold H/R. Deferoxamine (1 mM), di-phenyleneiodonium (50 microM) and N-acetylcysteine (10 mM) significantly inhibited this kinase activation.
Asunto(s)
Hipoxia de la Célula/fisiología , Hepatocitos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/fisiología , Animales , Células Cultivadas , Activación Enzimática , Hepatocitos/citología , Hepatocitos/enzimología , Masculino , Proteína Quinasa 8 Activada por Mitógenos , Ratas , Ratas Wistar , TemperaturaRESUMEN
Fractalkine displays features that distinguishes it from the other chemokines. In particular, besides its chemoattractant action it promotes, under physiologic flow, the rapid capture and the firm adhesion of a subset of leukocytes or intervenes in the neuron/microglia interaction. This study verified that indeed the human monocytic MonoMac6 cell line adheres to fibronectin-coated filters in response to soluble fractalkine (s-FKN). s-FKN stimulates, with distinct time courses, extracellular signal-related kinases (ERK1 and ERK2) and stress-activated protein kinases (SAPK1/JNK1 and SAPK2/p38). Both p60 Src and p72 Syk were activated under s-FKN stimulation with a rapid kinetic profile compatible with a downstream regulation on the mitogen-activated protein kinase (MAPK) congeners. The use of specific tyrosine kinase inhibitors revealed that the ERK pathway is strictly controlled by Syk, whereas c-Src up-regulated the downstream SAPK2/p38. In contrast, the SAPK1/JNK1 pathway was not regulated by any of these nonreceptor tyrosine kinases. The s-FKN-mediated increased adherence of MonoMac6 cells was partially inhibited by SB202190, a broad SAPKs inhibitor, PD98059, an MEK inhibitor, LY294002, a phosphatidyl inositol 3-kinase inhibitor, and a pertussis toxin-sensitive G protein. These data highlight that the integration of a complex array of signal transduction pathways is necessary to complete the full s-FNK-dependent adherence of human monocytic cells to fibronectin. (Blood. 2001;97:2031-2037)
Asunto(s)
Quimiocinas CX3C , Quimiocinas CXC/fisiología , Proteínas de la Membrana/fisiología , Monocitos/efectos de los fármacos , Receptores de Citocinas/fisiología , Receptores del VIH/fisiología , Transducción de Señal/fisiología , Receptor 1 de Quimiocinas CX3C , Adhesión Celular/efectos de los fármacos , Quimiocina CX3CL1 , Toxina del Cólera/farmacología , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/fisiología , Fibronectinas/metabolismo , Flavonoides/farmacología , Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/fisiología , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Monocitos/citología , Morfolinas/farmacología , Toxina del Pertussis , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Piridinas/farmacología , Receptores de Citocinas/efectos de los fármacos , Receptores del VIH/efectos de los fármacos , Quinasa Syk , Factores de Virulencia de Bordetella/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Prolonged liver ischemia followed by reperfusion (I/R) causes functional and structural damage to liver cells, resulting in necrosis and apoptosis. c-jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK(1)/SAPK(1)) is activated during I/R and participates in the onset of the apoptosis program. Excessive blood loss during surgery can hinder postoperative recovery. Intermittent portal triad clamping (PTC) is better tolerated than prolonged continuous ischemia. This study was designed to demonstrate that intermittent ischemia could improve postischemic survival rates by a decrease of JNK(1)/SAPK(1) and caspase 3 activation, which were involved in the apoptosis process. Rats were subjected to intermittent 1-hour ischemia (15-minute ischemia/5-minute reperfusion, 4 times), followed by 220-minute reperfusion, or to continuous ischemia (1 hour), followed by 240-minute reperfusion. Mortality rates were assessed on day 7. Serum aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase levels (LDH) were measured 6 hours after ischemia. This study was completed in primary cultured isolated rat hepatocytes, subjected to the same continuous or intermittent hypoxic conditions. The activation status of JNK(1)/SAPK(1) was evaluated by immunoprecipitation or Western blotting experiments. Apoptosis was assessed by measuring caspase activation and by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) reaction. Eighty percent of the intermittent-ischemia group was alive 7 days after surgery and serum enzyme levels were significantly decreased. Intermittent hypoxia or ischemia did not lead to JNK(1)/SAPK(1) activation, but at least 3 hypoxia-reoxygenation (H/R) sets were necessary to inhibit kinase activation. Consequently, caspase 3 activation and apoptosis were dramatically reduced. Intermittent ischemia is a powerful, protective way to reduce I/R damage of the liver, by reduction of JNK(1)/SAPK(1) activation associated with a down-regulation of caspase 3 activity, which leads to inhibition of hepatocyte apoptosis.
Asunto(s)
Hepatocitos/fisiología , Hipoxia/enzimología , Precondicionamiento Isquémico , Circulación Hepática , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oxígeno/metabolismo , Animales , Apoptosis , Caspasa 3 , Caspasas/metabolismo , Activación Enzimática , Enzimas/sangre , Masculino , Proteína Quinasa 8 Activada por Mitógenos , Ratas , Ratas Wistar , Análisis de SupervivenciaRESUMEN
A microtubule reorganization is often observed during cellular contacts that are associated to IL-1 production. Here, we show that in HL60 cells, vincristine, a microtubule-disrupting agent that induces a strong production of IL-1, triggers the activation of both extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK-1). While ERK activation is rapid and transient, peaking at 10 min, the JNK1 activation is delayed and more sustained reaching a maximum at 2 h. ERK activation was blocked by CP 118556, indicating it is regulated by a Src-like kinase, while JNK1 was inhibited by piceatannol, revealing an upstream regulation by Syk. Each kind of the nonreceptor tyrosine kinase blockers efficiently inhibits the vincristine-induced IL-1 production and diminishes the level of IL-1 transcripts, indicating that the ERK and JNK pathways act coordinately to elicit the transcription of the IL-1 gene. Furthermore, we found that pertussis toxin, a blocker of Go/Gi proteins, abrogated the vincristine-induced activation of both Src and Syk. Our data support a model where the status of microtubule polymerization influences the activity of Go or Gi proteins that control, in turn, two independent Src/ERK and Syk/JNK1 cascades that are both necessary to sustain IL-1 synthesis.
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Precursores Enzimáticos/fisiología , Interleucina-1/biosíntesis , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/inmunología , Familia-src Quinasas/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Precursores Enzimáticos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HL-60 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Microtúbulos/efectos de los fármacos , Microtúbulos/enzimología , Microtúbulos/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Toxina del Pertussis , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Quinasa Syk , Factores de Tiempo , Vincristina/antagonistas & inhibidores , Vincristina/toxicidad , Factores de Virulencia de Bordetella/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Low stringency screening of a Jurkat cDNA library with a rat brain K+ channel (RCK1) probe has resulted in the isolation of HLK3, a voltage-gated K+ channel. In Xenopus oocytes, the HLK3 clone directs the expression of a rapidly activating transient outward K+ current similar to the type n K+ current recorded in Jurkat T cells. The HLK3 gene is located on the short arm of human chromosome 1 (p13.3). Polymerase chain reaction was used to clone HIsK from Jurkat cDNA. The HIsK clone shares the same sequence with a previously described genomic clone (Murai, T., Kazikuka, A., Takumi, T., Ohkubo, H., and Nakanishi, S. (1989) Biochem. Biophys. Res. Commun. 161, 176-181). In Xenopus oocytes, it encodes a slowly activating, noninactivating K+ channel which cannot be recorded in Jurkat cells by conventional patch-clamp techniques. Transcripts of both clones are present at a similar level before and after activation of purified human T lymphocytes and Jurkat cells, reflecting a constitutive expression of K+ channel messages. This finding is in good agreement with the electrophysiological results for type n K+ current density on the same cells. HLK3 current is very sensitive to the scorpion toxin charybdotoxin (IC50 = 0.8 nM). HIsK current is totally insensitive to this toxin but is blocked by the antiarrhythmic clofilium (IC50 = 80 microM). While charybdotoxin has no effect on interleukin 2 mRNA induction, clofilium potently inhibits interleukin 2 mRNA expression upon mitogen-induced T cell activation. It is concluded that the HLK3 channel is not an important component of the T cell mitogenic response. Other targets for K+ channel blockers, such as the HIsK protein, could be involved in the activation process.
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Canales de Potasio/genética , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , División Celular/efectos de los fármacos , Caribdotoxina , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Clonación Molecular , ADN/genética , Expresión Génica , Humanos , Interleucina-2/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Canales de Potasio/efectos de los fármacos , Biosíntesis de Proteínas , Compuestos de Amonio Cuaternario/farmacología , ARN Mensajero/genética , Venenos de Escorpión/farmacología , Alineación de Secuencia , Linfocitos T/citología , Transcripción Genética , XenopusRESUMEN
Human monocyte-derived macrophages possess a NADPH oxidase that catalyzes superoxide formation upon phagocytosis. Extracellular ATP per se does not activate NADPH oxidase but potentiates superoxide generation triggered by opsonized zymosan. UTP can substitute for ATP with the same efficiency, suggesting that ATP mediates its effects specifically through P2U receptors. Extracellular UTP stimulates a rapid increase in cytoplasmic Ca2+ concentration in monocytic cells, which results from a release of intracellular Ca2+ stores. Moreover, UTP-induced calcium increase is sufficient to activate a charybdotoxin-sensitive Ca2+-dependent outward K+ channel (K(Ca)). The activity of this channel develops between 0.1 and 1.0 microM free cytoplasmic Ca2+ concentration; it is half-blocked by 10 nM charybdotoxin but insensitive to iberiotoxin. Under asymmetrical K+ conditions, this K(Ca) channel does not depend on membrane potential and is characterized by a linear single-current voltage relationship in the voltage range of -100 to +50 mV, giving a unitary conductance of 10 pico-Siemens. Interestingly, ATP/UTP-induced oxygen radicals release was inhibited by charybdotoxin in the same range of concentration as the UTP-induced K(Ca) channel. Furthermore, we show that ATP or UTP fail to enhance oxygen radicals production before K(Ca) channel is expressed (3 days). The electrogenic nature of the NADPH oxidase, i.e., its level of activation, being dependent on the plasmic membrane potential, might provide the causal link between the reactive oxygen intermediates generation and the opening of the K(Ca) channel.
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Adenosina Trifosfato/fisiología , Calcio/fisiología , Caribdotoxina/farmacología , Macrófagos/metabolismo , Canales de Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Uridina Trifosfato/fisiología , Antioxidantes/farmacología , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Espacio Extracelular/fisiología , Humanos , Macrófagos/fisiología , Monocitos/metabolismo , Monocitos/fisiología , Proteínas Opsoninas , Bloqueadores de los Canales de Potasio , Canales de Potasio/biosíntesis , Zimosan/metabolismo , Zimosan/farmacologíaRESUMEN
We have demonstrated previously that microtubule depolymerization by colchicine in human monocytes induces selective production of interleukin-1 (IL-1) (Manié, S., Schmid-Alliana, A., Kubar, J., Ferrua, B., and Rossi, B. (1993) J. Biol. Chem. 268, 13675-13681). Here, we provide evidence that disruption of the microtubule structure rapidly triggers extracellular signal-regulated kinase (ERK) activation, whereas it was without effect on SAPK2 activity, which is commonly acknowledged to control pro-inflammatory cytokine production. This process involves the activation of the entire cascade including Ras, Raf-1, MEK1/2, ERK1, and ERK2. Activation of ERKs is followed by their nuclear translocation. Although other SAPK congeners might be activated upon microtubule depolymerization, the activation of ERK1 and ERK2 is mandatory for IL-1 production as shown by the blocking effect of PD 98059, a specific MEK1/2 inhibitor. Additionally, we provide evidence that microtubule disruption also induces the activation of c-Src and Hck activities. The importance of Src kinases in the mediation of the colchicine effect is underscored by the fact that CP 118556, a specific inhibitor of Src-like kinase, abrogates both the colchicine-induced ERK activation and IL-1 production. This is the first evidence that ERK activation is an absolute prerequisite for induction of this cytokine. Altogether, our data lend support to a model where the status of microtubule integrity controls the level of Src activities that subsequently activate the ERK kinase cascade, thus leading to IL-1 production.