RESUMO
Ca(2+) and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca(2+) and cAMP signals, including some that are Ca(2+)-responsive, some that are cAMP-responsive and some that detect coincident Ca(2+) and cAMP signals. Because Ca(2+) and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca(2+) are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca(2+) buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP(3) levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP(3) metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca(2+) stores. Our data indicate that Ca(2+) release from IP(3)-sensitive pools is required for cAMP-induced transcription in hippocampal neurons.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Hipocampo/metabolismo , Inositol/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Transcrição Gênica , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Hipocampo/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Ratos , Ratos Wistar , Elemento de Resposta Sérica/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fatores de Regulação Miogênica/metabolismo , Neurônios/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Cálcio/metabolismo , Células Cultivadas , Ciclosporina/farmacologia , Inibidores Enzimáticos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Fatores de Transcrição MEF2 , Camundongos , Mutação/fisiologia , Fatores de Regulação Miogênica/genética , Fatores de Transcrição NFATC/metabolismo , Neurônios/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , Cloreto de Potássio/farmacologia , Regiões Promotoras Genéticas/genética , Ratos , Fatores de Tempo , Transfecção/métodosRESUMO
Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1, and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remains largely unclear. The recent discovery of EBV-encoded viral microRNA (miRNA) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNA. Using large-scale cloning analysis on EBV-positive NPC cells, two novel EBV miRNA, now named miR-BART21 and miR-BART22, were identified. These two EBV-encoded miRNA are abundantly expressed in most NPC samples. We found two nucleotide variations in the primary transcript of miR-BART22, which we experimentally confirmed to augment its biogenesis in vitro and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. More importantly, we determined that the EBV latent membrane protein 2A (LMP2A) is the putative target of miR-BART22. LMP2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells; down-modulation of LMP2A expression by miR-BART22 may permit escape of EBV-infected cells from host immune surveillance. Taken together, we demonstrated that two newly identified EBV-encoded miRNA are highly expressed in NPC. Specific sequence variations on the prevalent EBV strain in our locality might contribute to the higher miR-BART22 expression level in our NPC samples. Our findings emphasize the role of miR-BART22 in modulating LMP2A expression, which may facilitate NPC carcinogenesis by evading the host immune response.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , MicroRNAs/genética , Neoplasias Nasofaríngeas/virologia , Proteínas da Matriz Viral/biossíntese , Sequência de Bases , Northern Blotting , Western Blotting , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Evasão Tumoral , Proteínas da Matriz Viral/genéticaRESUMO
Deleted in liver cancer 2 (DLC2) is a candidate tumor suppressor frequently found to be deleted in hepatocellular carcinoma. In this study, we determined the subcellular localization of DLC2. Co-localization and biochemical fractionation studies revealed that DLC2 localized to mitochondria. In addition, the DLC2-containing cytoplasmic speckles were in proximity to lipid droplets. A DLC2 mutant containing the steroidogenic acute regulatory protein-related lipid transfer (START) domain only showed a localization pattern identical to that of DLC2. Taken together, we have provided the first evidence for mitochondrial localization of DLC2 through the START domain. These findings might have implications in liver physiology and carcinogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas Ativadoras de GTPase , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/genética , Mitocôndrias Hepáticas/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Hepatocellular carcinoma (HCC) is a major malignancy in many parts of the world, especially in Asia and Africa. Loss of heterozygosity (LOH) on the long arm of chromosome 13 has been reported in HCC. In search of tumor suppressor genes in this region, here we have identified DLC2 (for deleted in liver cancer 2) at 13q12.3 encoding a novel Rho family GTPase-activating protein (GAP). DLC2 mRNA is ubiquitously expressed in normal tissues but was significantly underexpressed in 18% (8/45) of human HCCs. DLC2 is homologous to DLC1, a previously identified tumor suppressor gene at 8p22-p21.3 frequently deleted in HCC. DLC2 encodes a novel protein with a RhoGAP domain, a SAM (sterile alpha motif) domain related to p73/p63, and a lipid-binding StAR-related lipid transfer (START) domain. Biochemical analysis indicates that DLC2 protein has GAP activity specific for small GTPases RhoA and Cdc42. Expression of the GAP domain of DLC2 sufficiently inhibits the Rho-mediated formation of actin stress fibers. Introduction of human DLC2 into mouse fibroblasts suppresses Ras signaling and Ras-induced cellular transformation in a GAP-dependent manner. Taken together, our findings suggest a role for DLC2 in growth suppression and hepatocarcinogenesis.