RESUMO
G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα(q)-coupled designer receptor (Rα(q)). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα(q) with clozapine-N-oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα(q) with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of an SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα(q)-coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα(q) stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα(q) stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα(q) stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα(q)-coupled receptor signaling.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Genes Precoces/genética , Sistema de Sinalização das MAP Quinases/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , Clozapina/análogos & derivados , Clozapina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Receptor Muscarínico M3/biossíntese , Elemento de Resposta Sérica , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Ativação Transcricional , Quinases raf/metabolismoRESUMO
It has been suggested that reduced glutamate receptor expression protects glioma cells from glutamate toxicity. GluR2 is the critical subunit of the GluR2 subtype of AMPA glutamate receptors as this subunit determines the Ca(2+) permeability of the receptor. The gene encoding the GluR2 subtype of AMPA receptors has been described as a target gene for the transcription repressor REST. However, we recently showed that the GluR2 gene is not regulated by REST in several neuronal and neuroendocrine cell lines, due to a repressive chromatin environment. Here, we show that the GluR2 gene has an open chromatin configuration in human glioma cells. Overexpression of REST reduced GluR2 mRNA levels while shRNA-mediated depletion of REST or expression of a REST mutant, that contained a transcriptional activation domain, enhanced GluR2 expression. Incubation with trichostatin A (TSA), a histone deacetylase inhibitor, induced acetylation of histone 4 of the GluR2 locus in glioma cells, leading to an upregulation of GluR2 expression. Together, these data suggest that REST is responsible for the reduced expression of GluR2 in glioma cells. The transcription factor Sp1 additionally binds under physiological conditions to the GluR2 gene in human glioma cells and expression of a dominant-negative mutant of Sp1 reduced expression of GluR2. Thus, the regulation via Sp1 represents a further control point for GluR2 expression in glioma cells. Together, we show that the GluR2 gene is embedded into an open chromatin configuration in glioma cells and expression of GluR2 is controlled by REST and Sp1.
Assuntos
Cromatina/metabolismo , Receptores de AMPA/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp1/metabolismo , Acetilação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Epigênese Genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Conformação Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de AMPA/genética , Proteínas Repressoras/genética , Fator de Transcrição Sp1/genética , Ativação Transcricional , Regulação para CimaRESUMO
Elevated extracellular Ca(2+) concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.
Assuntos
Cálcio/metabolismo , Receptores de Detecção de Cálcio/genética , Transdução de Sinais/fisiologia , Transcrição Gênica , Ativação Transcricional , Animais , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Receptores de Detecção de Cálcio/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
1321N1 astrocytoma cells are frequently used to analyze stimulus-induced intracellular signaling. These experiments require genetic manipulation of the cells and several chemical and physical methods have been employed in the past. Recently, microporation has been suggested as the best method to transfect 1321N1 astrocytoma cells. Here, we demonstrate that lentiviral gene transfer into 1321N1 cells is highly efficient, cheap and non-toxic. In addition, lentiviral gene transfer efficiently facilitates stable expression of small hairpin RNAs. Finally, lentiviral gene transfer can be used to implant promoter/luciferase reporter genes into the chromatin of the cells, allowing promoter studies using templates that are embedded into the nucleosomal structure of the chromatin.