RESUMEN
Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.
Asunto(s)
Carcinoma Hepatocelular/genética , Transición Epitelial-Mesenquimal/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Neoplasias Hepáticas/genética , MicroARNs/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Carcinoma Hepatocelular/patología , Embrión de Pollo , Progresión de la Enfermedad , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Oncogenes/genética , Oncogenes/fisiología , Células Tumorales Cultivadas , Regulación hacia Arriba/genéticaRESUMEN
Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation of pro-apoptotic genes through the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and antiapoptotic proteins through distinct phosphorylation events. This review analyses our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis.
Asunto(s)
Apoptosis , MAP Quinasa Quinasa 4/metabolismo , Transducción de Señal , Apoptosis/genética , Núcleo Celular/enzimología , Humanos , Mitocondrias/enzimología , FosforilaciónAsunto(s)
Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismoRESUMEN
G proteins provide signal-coupling mechanisms to heptahelical cell surface receptors and are critically involved in the regulation of different mitogen-activated protein kinase (MAPK) networks. The four classes of G proteins, defined by the G(s), G(i), G(q) and G(12) families, regulate ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by different mechanisms. The alpha- as well as betagamma-subunits are involved in the regulation of these MAPK modules in a context-specific manner. While the alpha- and betagamma-subunits primarily regulate the MAPK pathways via their respective effector-mediated signaling pathways, recent studies have unraveled several novel signaling intermediates including receptor tyrosine kinases and small GTPases through which these G-protein subunits positively as well as negatively regulate specific MAPK modules. Multiple mechanisms together with specific scaffold proteins that can link G-protein-coupled receptors or G proteins to distinct MAPK modules contribute to the context-specific and spatio-temporal regulation of mitogen-activated protein signaling networks by G proteins.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiologíaRESUMEN
Mitogen-activated protein kinases (MAPKs) regulate critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Recent studies have shown that a novel class of scaffold proteins mediates the structural and functional organization of the three-tier MAPK module. By linking the MAP3K, MAP2K and MAPK into a multienzyme complex, these MAPK-specific scaffold proteins provide an insulated physical conduit through which signals from the respective MAPK can be transmitted to the appropriate spatiotemporal cellular loci. Scaffold proteins play a determinant role in modulating the signaling strength of their cognate MAPK module by regulating the signal amplitude and duration. The scaffold proteins themselves are finely regulated resulting in dynamic intra- and inter-molecular interactions that can modulate the signaling outputs of MAPK modules. This review focuses on defining the diverse mechanisms by which these scaffold proteins interact with their respective MAPK modules and the role of such interactions in the spatiotemporal organization as well as context-specific signaling of the different MAPK modules.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Humanos , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismoRESUMEN
BACKGROUND: Galphaq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. OBJECTIVES: We studied Galphaq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. METHODS AND RESULTS: PMA-treated HEL cells showed enhanced Galphaq expression. Reporter (luciferase) gene studies on 5' upstream construct (up to -116 bp from ATG) revealed a negative regulatory site at -238/-202 and two positive sites at -203/-138 and -1116/-731. The positive regulatory region -203/-138 contained overlapping Sp1/AP-2/EGR-1 consensus sites. Gel shift studies on Galphaq oligonucleotides 1 (-203/-175) and 2 (-174/-152) using HEL cell extracts demonstrated protein binding that was due to early growth response factor EGR-1 at two sites. Mutations in either EGR-1 site markedly decreased the gene activity, indicating functional relevance. Mutation of consensus E-Box motif (-185/-180) had no effect. Reduction in the expression of endogenous EGR-1 with antisense oligonucleotide to EGR-1 inhibited PMA-induced Galphaq transcription. Correspondingly, Egr-1 deficient mouse platelets also showed approximately 50% reduction in the Galphaq expression relative to wild-type platelets. CONCLUSIONS: These studies suggest that Galphaq gene is regulated during PMA-induced megakaryocytic differentiation by EGR-1, an early growth response transcription factor that regulates a wide array of genes and plays a major role in diverse activities, including cell proliferation, differentiation and apoptosis, and in vascular response to injury and atherosclerosis.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Megacariocitos/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Plaquetas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Ensayo de Cambio de Movilidad Electroforética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Eliminación de Gen , Genes Reporteros , Humanos , Luciferasas , Sustancias Luminiscentes , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
G-proteins play an important role in platelet signal transduction and regulate responses upon activation of G-protein coupled receptors (GPCR). We have previously reported a patient with impaired platelet responses associated with deficiency in platelet Galphaq. To understand the molecular basis for this defect, the cDNA sequence encoding Galphaq (1080 bp) was obtained by reverse-transcription and polymerase chain reaction of platelet RNA; the cDNA sequence showed no mutations in the patient. Platelet Galphaq mRNA levels were decreased by >50% compared to normal subjects; platelet Galphai2 mRNA levels were normal. Neutrophil calcium mobilization and elastase secretion, upon activation with several agonists, and neutrophil Galphaq mRNA and protein levels were normal. These studies demonstrate that the patient has a defect in Galphaq gene expression in platelets but not neutrophils, possibly due to defects in transcriptional regulation or mRNA stability, and suggest a hematopoietic-lineage specific defect.