RESUMEN
The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.
Asunto(s)
Adenocarcinoma/patología , Adenoma/patología , Anticarcinógenos/farmacología , Colon/citología , Neoplasias del Colon/patología , Células Epiteliales/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Adenocarcinoma/enzimología , Adenoma/enzimología , Butiratos/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Movimiento Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Colon/enzimología , Neoplasias del Colon/enzimología , Regulación hacia Abajo , Inducción Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Flavonoides/farmacología , Humanos , Lisofosfolípidos/farmacología , Malus/química , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fenoles/farmacología , Extractos Vegetales/farmacología , Polifenoles , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/farmacología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Té/química , Transcripción Genética/efectos de los fármacos , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Rapamycin has been shown to reduce neointimal thickening in the setting of balloon angioplasty and chronic graft vessel disease. This study was designed to test the effect of oral rapamycin on atherosclerotic plaque progression and the possible mechanism involved. Apolipoprotein E (apoE) knockout mice were fed either a diet supplemented with cholesterol or with cholesterol and rapamycin. At 4 and 8 weeks, quantitative analyses of plaque area and macrophage numbers were determined. Plasma cholesterol, triglyceride, and whole-blood rapamycin levels were measured. Rapamycin could be detected in the blood of mice (117+/-7 pg/mL). In mice fed with rapamycin, atherosclerotic lesions covered 22% of the aortic arch as compared with 41% in cholesterol-fed mice. The macrophage count was significantly lower in the rapamycin-fed mice as compared with cholesterol-fed mice. Rapamycin, in a dose-dependent manner, inhibited monocyte chemotaxis elicited by stromal cell-derived factor-1. Lesions in the cholesterol-fed mice had complex atherosclerotic plaque with acellular core, cholesterol clefts, and an abundant collection of monocytes/macrophages. Lesions in the rapamycin-fed mice were mainly composed of monocytes/macrophages. Oral rapamycin is effective in slowing the progression of atherosclerosis. Along with its multitude actions, attenuation of monocyte chemotaxis may be one more way by which rapamycin attenuates plaque progression.
Asunto(s)
Apolipoproteínas E/genética , Arteriosclerosis/prevención & control , Inmunosupresores/farmacología , Sirolimus/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/metabolismo , Arteriosclerosis/sangre , Arteriosclerosis/patología , Peso Corporal/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Suplementos Dietéticos , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Inmunosupresores/administración & dosificación , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/fisiología , Sirolimus/administración & dosificación , Triglicéridos/sangreRESUMEN
G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Bioensayo , Neoplasias Óseas , Brefeldino A/farmacología , Células COS , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Osteosarcoma , Receptores CXCR4/agonistas , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine whose receptor, CXCR4, is distributed in specific brain areas including hypothalamus. SDF-1alpha has recently been found to play important roles in neurons, although direct modulation of voltage-gated ionic channels has never been shown. In order to clarify this issue, we performed patch-clamp experiments in fetal mouse hypothalamic neurons in culture. SDF-1alpha (10 nm) decreased the peak and rising slope of the action potentials and spike discharge frequency in 22% of hypothalamic neurons tested. This effect was blocked by the CXCR4 antagonist AMD 3100 (1 microm) but not by the metabotropic glutamate receptor antagonist MCPG (500 microm), indicating a direct action of SDF-1alpha on its cognate receptor. This effect involved a depression of both inward and outward voltage-dependent currents of the action potential. We confirmed these effects in the human neuroblastoma cell line SH-SY5Y, which endogenously expresses CXCR4. Voltage-clamp experiments revealed that SDF-1alpha induced a 20% decrease in the peak of the tetrodotoxin-sensitive sodium current and tetraethylammonium-sensitive delayed rectifier potassium current, respectively. Both effects were concentration dependent, and blocked by AMD 3100 (200 nm). This dual effect was reduced or blocked by 0.4 mm GTPgammaS G-protein pre-activation or by pre-treatment with the G-protein inhibitor pertussis toxin (200 ng/mL), suggesting that it is mediated via activation of a G(i/o) protein. This study extends the functions of SDF-1alpha to a direct modulation of voltage-dependent membrane currents of neuronal cells.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Quimiocinas CXC/farmacología , Glicina/análogos & derivados , Neuronas/efectos de los fármacos , Porinas/metabolismo , Animales , Bencilaminas , Cloruro de Cadmio/farmacología , Células Cultivadas , Quimiocina CXCL12 , Ciclamas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Glicina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Compuestos Heterocíclicos/farmacología , Humanos , Hipotálamo/citología , Inmunohistoquímica/métodos , Ratones , Neuroblastoma , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Porinas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , ARN Mensajero/biosíntesis , Receptores CXCR4/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Bloqueadores de los Canales de Sodio/farmacología , Tetraetilamonio/farmacología , Tetrodotoxina/farmacología , Canales Aniónicos Dependientes del VoltajeRESUMEN
The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19-32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Comunicación Autocrina/inmunología , Calreticulina/fisiología , Movimiento Celular/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Trombospondina 1/fisiología , Adyuvantes Inmunológicos/metabolismo , Secuencia de Aminoácidos , Antígenos CD/fisiología , Sitios de Unión/inmunología , Antígeno CD47 , Calreticulina/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Inhibición de Migración Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Colágeno Tipo I , Heparina/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Transporte de Proteínas/inmunología , Linfocitos T/metabolismo , Trombospondina 1/biosíntesis , Trombospondina 1/metabolismo , Zinc/fisiologíaRESUMEN
OBJECTIVE: To investigate the anti-HIV effects of ampelopsin and its interaction with HIV-1 coreceptor CXCR4. METHODS: Through anti-virus experiments in vitro, the inhibitory effect of ampelopsin on HIV-1 infection was verified. Chemotaxis assay was performed to show the ability to induce PBMCs migration by ampelopsin, RANTES and SDF-1alpha. Fluorescence labelling monoclonal antibody was utilized to observe the interaction of ampelopsin and CXCR4. Mice immunosuppressant model was also established to detail the role ampelopsin played in regulating cellular immunological functions. RESULTS: Ampelopsin could protect sensitive cells against HIV-1 infection and dramatically reduce HIV-1 antigen P24 expression. HIV-1SF33 attaching to MT-4 cells was interfered by ampelopsin, and the EC50 was 0.175 mg/mL for cellular protection and 0.024 mg/mL for P24 inhibition. At co-cultivating phase, EC50 was 0.229 mg/mL and 0.197 mg/mL respectively. Furthermore, the EC50 was 0.179 mg/mL and 0.348 mg/mL in acute infection. Human PBMCs migration was induced after being challenged with ampelopsin or chemokines, and synergistic action was observed during co-treatment. Ampelopsin alone resulted in maximal chemotaxis at 1 mg/mL. HIV-1 co-receptor CXCR4 on the surface of PBMCs was decreased by internalization, which indicated the effect of ampelopsin on CXCR4. About 70% CXCR4 was reduced by ampelopsin at 1 mg/mL. Ampelopsin also augmented cellular immunological functions in immunosuppressive mice. CONCLUSION: Ampelopsin displays a strong inhibitive role during HIV-1 absorption, incubation and acute infection. These results are coincident with its immune enhancement.
Asunto(s)
Fármacos Anti-VIH/farmacología , Flavonoides/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Leucocitos Mononucleares/efectos de los fármacos , Receptores CXCR4/efectos de los fármacos , Ampelopsis/química , Animales , Línea Celular , Quimiocina CCL5/farmacología , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Quimiotaxis de Leucocito , Regulación hacia Abajo , Medicamentos Herbarios Chinos , Flavonoides/economía , Flavonoides/aislamiento & purificación , VIH-1/metabolismo , Humanos , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Raíces de Plantas/química , Receptores CXCR4/antagonistas & inhibidores , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines. METHODS: We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis. RESULTS: One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment. CONCLUSIONS: Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology.
Asunto(s)
Quimiocinas CXC/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Western Blotting , Señalización del Calcio/efectos de los fármacos , Quimiocina CXCL10 , Quimiocina CXCL12 , Quimiotaxis/efectos de los fármacos , ADN Complementario/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores CXCR3 , Receptores CXCR4/efectos de los fármacos , Receptores de Quimiocina/efectos de los fármacos , Receptores de Quimiocina/genética , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnica de Sustracción , Linfocitos T/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
The effect of (-)-epigallocatechin 3-gallate (EGCG), a major polyphenol of green tea, on neutrophil migration has been studied using multiwell-type Boyden chambers in vitro and a fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA)-induced rat allergic inflammation model in vivo. EGCG inhibited rat neutrophil chemotaxis toward cytokine-induced neutrophil chemoattractant-1 (CINC-1) in a concentration-dependent manner. In addition, CINC-1-induced neutrophil chemotaxis was suppressed by the pretreatment of rat neutrophils with EGCG at the concentration over 15 microg/mL. EGCG caused concentration-dependent suppression of the transient increase in CINC-1-induced intracellular free calcium level in both rat neutrophils and rat CXC chemokine receptor 2 (CXCR2)-transfected HEK 293 cells. EGCG inhibited CINC-1 production by IL-1beta-stimulated rat fibroblasts (NRK-49F cells) and lipopolysaccharide-stimulated rat macrophages at the concentration over 50 microg/mL, a comparatively high concentration. Oral administration of EGCG (1.0 mg or 1.5 mg/rat) at 1 h before the challenge with FITC-OVA suppressed neutrophil infiltration into the air pouch (inflammatory site) in the air-pouch type FITC-OVA-induced allergic inflammation in rats. Chemokine levels in the pouch fluids, however, were not influenced by EGCG administration. The results suggest that EGCG suppressed neutrophil infiltration by a direct action on neutrophils, but not by indirect actions, including the suppression of chemokine production at the inflammatory site.
Asunto(s)
Camellia sinensis/química , Catequina/análogos & derivados , Catequina/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Animales , Quimiocina CXCL1 , Quimiocinas/biosíntesis , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/farmacología , Inflamación/inmunología , Inflamación/patología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Ratas , Ratas WistarRESUMEN
During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Comunicación Celular/inmunología , Matriz Extracelular/enzimología , Matriz Extracelular/inmunología , Glucuronidasa/fisiología , Proteínas Recombinantes/farmacología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiología , Adyuvantes Inmunológicos/metabolismo , Adhesión Celular/inmunología , Comunicación Celular/fisiología , Línea Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Quimiotaxis de Leucocito/inmunología , Colágeno Tipo IV/fisiología , Matriz Extracelular/fisiología , Fibronectinas/metabolismo , Fibronectinas/fisiología , Quinasa 2 de Adhesión Focal , Glucuronidasa/metabolismo , Humanos , Ácido Hialurónico/fisiología , Interfase/inmunología , Rodamiento de Leucocito/inmunología , Activación de Linfocitos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/inmunología , Especificidad por Sustrato/inmunología , Subgrupos de Linfocitos T/enzimología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
OBJECTIVE: Stromal-derived factor-1 (SDF-1/CXCL12) is chemotactic for lympho/hematopoietic stem cells. We have previously shown that increasing peripheral blood (PB) levels of SDF-1 with adenovectors expressing human SDF-1 complementary DNA (ad-SDF-1) leads to hematopoietic stem cell mobilization as well as migration of megakaryocytes and thrombocytosis in mice. Herein, we studied the in vivo effects of ad-SDF-1 and of an analogue peptide of SDF-1 (CTCE-0214) on human hematopoiesis in a xenotransplant model. MATERIALS AND METHODS: Sublethally irradiated (300 cGY) NOD/SCID mice transplanted with human cord blood mononuclear cells (CB MNC) were injected with ad-SDF-1 (10(9) plaque forming units, i.v., x 1) or CTCE-0214 (10 mg/kg/dose, i.v. q 24 hours x 7). Effects on megakaryocytopoiesis (CD41+ cells and platelets) as well as stem cell mobilization were monitored. RESULTS: CB MNC in NOD/SCID mice are able to differentiate into CD41+ cells and platelets, peaking at week 9 at a mean of 3.7 x 10(3)/microL. i.v. injection of ad-SDF-1 increased human CD41+ cells by day 4 in PB and was followed by an increase in human platelet production by day 5, with return to baseline by day 30. Human colony-forming cells (CFC) were mobilized from bone marrow to spleen (by day 6-13) and to PB (by day 13). Human CD34+ and CD33+ cells were mobilized by this treatment as well. A novel SDF-1 peptide agonist (CTCE-0214) also mobilized human CFC and enhanced human thrombopoiesis. CONCLUSION: SDF-1 and its analogue may be of clinical value in stimulating platelet recovery after chemo/radiation treatment as well as in stem cell mobilization.
Asunto(s)
Plaquetas/citología , Quimiocinas CXC/farmacología , Hematopoyesis/efectos de los fármacos , Movilización de Célula Madre Hematopoyética/métodos , Megacariocitos/efectos de los fármacos , Adenoviridae/genética , Animales , Diferenciación Celular/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/agonistas , Quimiocinas CXC/sangre , Quimiocinas CXC/genética , Evaluación Preclínica de Medicamentos , Sangre Fetal/citología , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Quimera por Radiación , Proteínas Recombinantes de Fusión/fisiología , Trombopoyetina/genética , Trombopoyetina/farmacología , Transducción Genética , Trasplante HeterólogoRESUMEN
Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.
Asunto(s)
Sistema Nervioso Central/metabolismo , Quimiocinas CXC/metabolismo , Ácido Glutámico/metabolismo , Receptores AMPA/metabolismo , Receptores de Interleucina-8B/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/inmunología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/inmunología , Células Cultivadas , Sistema Nervioso Central/inmunología , Corteza Cerebelosa/efectos de los fármacos , Corteza Cerebelosa/inmunología , Corteza Cerebelosa/metabolismo , Quimiocinas CXC/inmunología , Quimiocinas CXC/farmacología , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Ácido Glutámico/farmacología , Humanos , Canales Iónicos/genética , Canales Iónicos/inmunología , Neuronas/efectos de los fármacos , Neuronas/inmunología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores AMPA/genética , Receptores AMPA/inmunología , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/inmunología , Sinapsis/inmunologíaRESUMEN
CXC chemokine receptor 4 (CXCR4), the high-affinity receptor for stroma-derived factor 1alpha (SDF-1alpha), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum-free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte colony-stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF-1alpha, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL-3 and erythropoietin, SDF-1alpha induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF-1alpha significantly reduced the number of plurifocal erythroid colonies (erythroid blast-forming units; BFU-E), whereas it did not affect granulocyte-macrophage colony-forming units (CFU-GM). We also demonstrated that the inhibitory effect of SDF-1alpha on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF-1alpha plays a role as a negative regulator of erythropoiesis.
Asunto(s)
Antígenos CD34 , Quimiocinas CXC/farmacología , Eritropoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores CXCR4/metabolismo , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12 , Depresión Química , Proteína Ligando Fas , Sangre Fetal/citología , Glicoforinas/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Factor de Células Madre/farmacologíaRESUMEN
BACKGROUND: The chemokine receptor CXCR4 (a receptor for the Cys-X-Cys class of chemokines) is a CD4-associated coreceptor for T-cell-tropic strains of human immunodeficiency virus 1 (HIV-1) and represents a target for antiviral therapy. Infection by T-tropic HIV-1 can be blocked by stromal-cell-derived factor-1 (SDF-1), the natural ligand of CXCR4. The broad variety of cells expressing CXCR4 and the perturbations observed in mice deficient for SDF-1 suggest that antiviral compounds antagonizing the signalling activity of CXCR4 might have severe side effects in vivo. Compounds that interfere selectively with HIV entry and not with SDF-1 signalling would therefore be useful. RESULTS: A series of peptides, each of 13 residues, spanning the whole SDF-1alpha sequence were tested for their ability to block HIV-1 infection. The antiviral and signalling properties of SDF-1 were retained by a peptide corresponding to its amino terminus. Removal of the first two residues resulted in an antiviral antagonist of the SDF-1-CXCR4 signalling pathway. We prepared 234 single-substitution analogues and identified one antiviral analogue that had drastically reduced agonistic or antagonistic properties. The antiviral peptides competed with the monoclonal antibody 12G5 for CXCR4 binding. Their antiviral activity seems to be due to receptor occupancy rather than induction of receptor endocytosis. CONCLUSIONS: The amino terminus of the SDF-1 chemokine is sufficient for signal transduction via CXCR4 and for inhibition of HIV-1 entry, but these activities could be dissociated in a peptide analogue. This peptide represents a lead molecule for the design of low molecular weight antiviral drugs.