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1.
J Neurochem ; 141(1): 100-110, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28144998

RESUMEN

Microglia are widely accepted as surveillants in the central nervous system that are continually searching the local environment for signs of injury. Following an inflammatory situation, microglia alter their morphology, extend ramified processes, and undergo cell body hypertrophy. Extracellular nucleotides are recognized as a danger signal by microglia. ADP acting on P2Y12 receptors induce process extension of microglia thereby attracting microglia to the site of adenosine tri-phosphate/ADP leaking or release. However, the question whether ADP/P2Y12 receptor signaling directly stimulates the production or release of inducible factors such as cytokines remains unclear. In this study, we found that CC chemokine ligand 3 (CCL3) is induced by ADP-treated primary microglia. Pharmacological characterization using pertussis toxin, a P2Y12 receptor inhibitor, and a calcium chelator revealed that CCL3 induction was caused by P2Y12 receptor-mediated intracellular calcium elevation. Next, nuclear factor of activated T-cell dephosphorylation and nuclear translocalization were observed. Calcineurin, an inhibitor for nuclear factor of activated T cell, suppressed CCL3 induction. These data indicate that microglial P2Y12 receptors are utilized to trigger an acute inflammatory response in microglia via rapid CCL3 induction after ADP stimulation.


Asunto(s)
Quimiocina CCL3/biosíntesis , Microglía/metabolismo , Factores de Transcripción NFATC/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Transducción de Señal/fisiología , Adenosina Difosfato/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Quimiocina CCL3/genética , Relación Dosis-Respuesta a Droga , Expresión Génica , Microglía/efectos de los fármacos , Factores de Transcripción NFATC/agonistas , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos
2.
Methods ; 112: 75-83, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-27327144

RESUMEN

MHC-multimers are reagents used for the detection and enumeration of antigen-specific T cells (ASTs). These reagents exploit the mechanism by which T cell receptors (TCR) on cytotoxic CD8 T cells recognize specific antigens in the context of a major histocompatibility complex (MHC) molecule during antigen presentation. MHC-multimers are fluorescently-labeled dextran polymers that carry MHC Class I molecules and peptide sequences that can be modified to represent specific cognate sequences of the antigen of interest with dextramers having a 10-fold multiplicity of the MHC/peptide combination within a single multimer. Since the binding of antigen-specific dextramers mimics antigen presentation to the TCR, the present study sought to determine whether this TCR engagement on the AST was sufficient to elicit a functional T cell response. The effect of binding of CMV specific dextramers on the activation of the NFAT signal transduction cascade was assessed in peripheral blood from bone marrow transplant recipients previously determined to be positive for CMV-ASTs (CASTs). NFAT activation was quantified by measuring nuclear translocation of NFAT1 in CD8+ CASTs and CD8+ non-CASTs by imaging flow cytometry. Our results demonstrate that an increase in the nuclear localization of NFAT1 was detectable in the CASTs following the CMV-dextramer binding and could be observed as early as 10min post-exposure. The NFAT1 activation correlated with a downstream functional response in the form of interferon gamma production. Sample preparation, temperature, and duration of dextramer exposure were important parameters affecting the dextramer-induced NFAT activation with 2h exposure in whole blood at room temperature being the optimal of the conditions tested. Intra- and inter-individual heterogeneity was observed with regards to the NFAT activation in the CASTs. Importantly, no effect of the dextramers was observed in the CD8+ non-CASTs, and therefore dextramer negative cell populations. Exposure to PMA/ionomycin following dextramer exposure resulted in a homogeneous NFAT activation in both the dextramer-positive but NFAT1 nonresponsive CAST and non-CAST cells. Thus, the data demonstrate that binding of antigen-specific dextramers to ASTs specifically results in activation of NFAT, that the NFAT activation correlates with a downstream functional response and that the response can be heterogeneous. This functional parameter may provide insight to the issue whether enumeration alone of ASTs is a sufficient parameter to assess an individual's immune status against a specific antigen.


Asunto(s)
Citometría de Flujo/métodos , Citometría de Imagen/métodos , Complejo Mayor de Histocompatibilidad , Factores de Transcripción NFATC/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Presentación de Antígeno , Citomegalovirus/inmunología , Citometría de Flujo/instrumentación , Colorantes Fluorescentes/química , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Citometría de Imagen/instrumentación , Interferón gamma/farmacología , Ionomicina/farmacología , Leucemia/inmunología , Leucemia/patología , Leucemia/terapia , Activación de Linfocitos/efectos de los fármacos , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/genética , Ficoeritrina/química , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T/genética , Coloración y Etiquetado/métodos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/patología , Acetato de Tetradecanoilforbol/farmacología , Receptores de Trasplantes
3.
J Biol Chem ; 291(7): 3385-94, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26710850

RESUMEN

The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and ß-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.


Asunto(s)
Proteínas Angiogénicas/agonistas , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Transducción de Señal , Regulación Alostérica , Sustitución de Aminoácidos , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Arrestinas/química , Arrestinas/genética , Arrestinas/metabolismo , Secuencia Conservada , Genes Reporteros , Células HEK293 , Humanos , Ligandos , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador alfa/química , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Ubiquitinación , beta-Arrestinas
4.
J Biol Chem ; 291(3): 1148-61, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26472929

RESUMEN

Bone remodeling is controlled by dual actions of osteoclasts (OCs) and osteoblasts (OBs). The calcium-sensitive nuclear factor of activated T cells (NFAT) c1 transcription factor, as an OC signature gene, regulates differentiation of OCs downstream of bone morphogenetic protein-2 (BMP-2)-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1, we analyzed bones from OB-specific BMP-2 knock-out mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OBs. We showed that BMP-2, via the receptor-specific Smad pathway, regulates expression of NFATc1 in OBs. Phosphatidylinositol 3-kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under the basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by the calcium-dependent serine/threonine phosphatase calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. 1,2Bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester, an inhibitor of intracellular calcium abundance, blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity, resulting in NFATc1 nuclear translocation. Cyclosporin A, which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription, confirming its autoregulation. Together, these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk among Smad, phosphatidylinositol 3-kinase/Akt, and Ca(2+) signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.


Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción NFATC/agonistas , Osteoblastos/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Proteína Morfogenética Ósea 2/genética , Calcineurina/química , Calcineurina/metabolismo , Quelantes del Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad5/agonistas , Proteína Smad5/genética , Proteína Smad5/metabolismo
5.
PLoS One ; 8(11): e80113, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24244623

RESUMEN

Nerve growth factor is a neurotrophin that promotes cell growth, differentiation, survival and death through two different receptors: TrkA(NTR) and p75(NTR). Nerve growth factor serum concentrations increase during many inflammatory and autoimmune diseases, glomerulonephritis, chronic kidney disease, end-stage renal disease and, particularly, in renal transplant. Considering that nerve growth factor exerts beneficial effects in the treatment of major central and peripheral neurodegenerative diseases, skin and corneal ulcers, we asked whether nerve growth factor could also exert a role in Cyclosporine A-induced graft nephrotoxicity. Our hypothesis was raised from basic evidence indicating that Cyclosporine A-inhibition of calcineurin-NFAT pathway increases nerve growth factor expression levels. Therefore, we investigated the involvement of nerve growth factor and its receptors in the damage exerted by Cyclosporine A in tubular renal cells, HK-2. Our results showed that in HK-2 cells combined treatment with Cyclosporine A + nerve growth factor induced a significant reduction in cell vitality concomitant with a down-regulation of Cyclin D1 and up-regulation of p21 levels respect to cells treated with Cyclosporine A alone. Moreover functional experiments showed that the co-treatment significantly up-regulated human p21promoter activity by involvement of the Sp1 transcription factor, whose nuclear content was negatively regulated by activated NFATc1. In addition we observed that the combined exposure to Cyclosporine A + nerve growth factor promoted an up-regulation of p75 (NTR) and its target genes, p53 and BAD leading to the activation of intrinsic apoptosis. Finally, the chemical inhibition of p75(NTR) down-regulated the intrinsic apoptotic signal. We describe two new mechanisms by which nerve growth factor promotes growth arrest and apoptosis in tubular renal cells exposed to Cyclosporine A.


Asunto(s)
Apoptosis/efectos de los fármacos , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Sinergismo Farmacológico , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Receptores de Factor de Crecimiento Nervioso/agonistas , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Proteína p53 Supresora de Tumor/agonistas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína Letal Asociada a bcl/agonistas , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo
6.
J Pharmacol Exp Ther ; 346(1): 54-66, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23639801

RESUMEN

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²âº release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.


Asunto(s)
Plaquetas/metabolismo , Señalización del Calcio , Endotelio Vascular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Compuestos de Azabiciclo/farmacología , Benzoatos/farmacología , Plaquetas/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Antagonistas de Receptores de Cannabinoides/farmacología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Expresión Génica , Células HEK293 , Humanos , Ligandos , Lisofosfolípidos/antagonistas & inhibidores , Lisofosfolípidos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Piperazinas/antagonistas & inhibidores , Piperazinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/ética , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/metabolismo , Sulfonas/antagonistas & inhibidores , Sulfonas/farmacología , Cicatrización de Heridas/efectos de los fármacos
7.
J Immunol ; 190(5): 2345-53, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23365084

RESUMEN

NFAT transcription factors control the proliferation and survival of peripheral lymphocytes. We have reported previously that the short isoform NFATc1/αA whose generation is induced by immune receptor stimulation supports the proliferation and inhibits the activation-induced cell death of peripheral T and B cells. We will show in this study that in novel bacterial artificial chromosome transgenic mice that express EGFP under the control of entire Nfatc1 locus the Nfatc1/Egfp transgene is expressed as early as in double-negative thymocytes and in nonstimulated peripheral T and B cells. Upon immune receptor stimulation, Nfatc1/Egfp expression is elevated in B, Th1, and Th2 cells, but only weakly in T regulatory, Th9, and Th17 cells in vitro whose generation is affected by TGFß. In naive lymphocytes, persistent immune receptor signals led to a 3-5 increase in NFATc1/αA RNA levels during primary and secondary stimulation, but a much stronger induction was observed at the protein level. Whereas anti-CD3(+)CD28 stimulation of primary T cells induces both NFATc1/αA and their proliferation and survival, anti-IgM stimulation of B cells induces NFATc1/αA and proliferation, but activation-induced cell death after 3-d incubation in vitro. The anti-IgM-mediated activation-induced cell death induction of B cells in vitro is suppressed by anti-CD40-, LPS-, and CpG-mediated signals. In addition to inducing NF-κB factors, together with anti-IgM, these signals also support the generation of NFATc1/αA. According to these data and the architecture of its promoter region, the Nfatc1 gene resembles a primary response gene whose induction is affected at the posttranscriptional level.


Asunto(s)
Linfocitos B/efectos de los fármacos , Factores de Transcripción NFATC/genética , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacos , Animales , Anticuerpos/farmacología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Cromosomas Artificiales Bacterianos/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Genes Reporteros , Proteínas Fluorescentes Verdes , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/inmunología , Factores de Transcripción NFATC/agonistas , Factores de Transcripción NFATC/inmunología , Regiones Promotoras Genéticas , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología
8.
Biochem Biophys Res Commun ; 416(3-4): 318-24, 2011 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-22108054

RESUMEN

Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, ß-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.


Asunto(s)
Artocarpus/química , Mastocitos/inmunología , Lectinas de Plantas/inmunología , Receptores de IgE/inmunología , Animales , Línea Celular , Expresión Génica , Inmunoglobulina E/inmunología , Mastocitos/efectos de los fármacos , FN-kappa B/agonistas , Factores de Transcripción NFATC/agonistas , Fosforilación , Lectinas de Plantas/metabolismo , Lectinas de Plantas/farmacología , Ratas , Tirosina/metabolismo
9.
Cell Mol Life Sci ; 65(17): 2752-62, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18668201

RESUMEN

Nuclear factor of activated T cells 3 (NFAT3) activities have been implicated in many biological processes, such as breast cancer, cardiac hypertrophy, learning and memory, and adipocyte differentiation. However, how protein factors regulate NFAT3 transcriptional activity is poorly understood. Here, we report that regardless of estrogen, overexpression of estrogen receptor alpha and beta (ERalpha and ERbeta) suppresses NFAT3 transcriptional activity, whereas knockdown of endogenous ERalpha and ERbeta enhances the activity. Estrogen further enhances ER inhibition of NFAT3-dependent transcription. ERalpha and ERbeta interact with NFAT3 independently of the NFAT agonists phorbol myristate acetate (PMA) and ionomycin, and ERalpha is recruited to an NFAT3 target gene promoter. Phosphorylation of ERalpha at different sites differentially affects ERalpha modulation of NFAT3 transcriptional activity. These results suggest that ER may play a critical role in regulation of NFAT3 transcriptional activity.


Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Regulación de la Expresión Génica/genética , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Transcripción Genética/genética , Línea Celular , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Humanos , Factores de Transcripción NFATC/agonistas , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , Transcripción Genética/efectos de los fármacos
10.
Mol Cancer Res ; 4(11): 811-20, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17114339

RESUMEN

Down syndrome candidate region 1 (DSCR1) is one of more than 50 genes located in a region of chromosome 21 that has been implicated in Down syndrome. DSCR1 can be expressed as four isoforms, one of which, isoform 4 (DSCR1-4), has recently been found to be strongly induced by vascular endothelial growth factor A (VEGF-A(165)) and to provide a negative feedback loop that inhibits VEGF-A(165)-induced endothelial cell proliferation in vitro and angiogenesis in vivo. We report here that another DSCR1 isoform, DSCR1-1L, is also up-regulated by VEGF-A(165) in cultured endothelial cells and is strongly expressed in several types of pathologic angiogenesis in vivo. In contrast to DSCR1-4, the overexpression of DSCR1-1L induced the proliferation and activation of the transcription factor NFAT in cultured endothelial cells and promoted angiogenesis in Matrigel assays in vivo, even in the absence of VEGF-A. Similarly, small interfering RNAs specific for DSCR1-1L and DSCR1-4 had opposing inhibitory and stimulatory effects, respectively, on these same functions. DSCR1-4 is thought to inhibit angiogenesis by inactivating calcineurin, thereby preventing activation and nuclear translocation of NFAT, a key transcription factor. In contrast, DSCR1-1L, regulated by a different promoter than DSCR1-4, activates NFAT and its proangiogenic activity is inhibited by cyclosporin, an inhibitor of calcineurin. In sum, DSCR1-1L, unlike DSCR1-4, potently activates angiogenesis and could be an attractive target for antiangiogenesis therapy.


Asunto(s)
Calcineurina/metabolismo , Proteínas Musculares/fisiología , Factores de Transcripción NFATC/agonistas , Neovascularización Patológica/genética , Transporte Activo de Núcleo Celular , Animales , Inhibidores de la Calcineurina , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Proteínas de Unión al ADN , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Laminina/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Chaperonas Moleculares , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Fragmentos de Péptidos/farmacología , Proteínas Gestacionales/antagonistas & inhibidores , Proteínas Gestacionales/genética , Proteínas Gestacionales/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteínas/fisiología , Proteoglicanos/metabolismo , Interferencia de ARN , ARN Largo no Codificante , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transfección , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/farmacología
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