Role of AP-1 in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene up-regulation in mouse cortical neurons.
Activator protein 1 (AP-1) has been reported to regulate the gene expression in a wide variety of cellular processes in response to stimuli. In this study, we investigated the DNA-protein binding activities and promoter activity in the N-methyl-D-aspartate R2B (NR2B) gene AP-1 site in normal and ethanol-treated cultured neurons. The identity of the AP-1 site as the functional binding factor is suggested by the specific binding of nuclear extract derived from cultured cortical neurons to the labeled probes and the specific antibody-induced supershift. Mutations in the core sequence resulted in a significantly reduced promoter activity and the ability to compete for the binding. Moreover, treatment of the cultured neuron with 75 mm ethanol for 5 days caused a significant increase in the AP-1 binding activity and promoter activity. The AP-1 DNA-binding complex in control and ethanol-treated nuclear extract was composed of c-Fos, FosB, c-Jun, JunD, and phosphorylated CREB (p-CREB). Western blot analysis showed that p-CREB and FosB significantly increased, whereas c-Jun decreased. The DNA affinity precipitation assay indicated that FosB, p-CREB, and c-Jun increased in the AP-1 complex following ethanol treatment. These results suggest that AP-1 is an active regulator of the NR2B transcription and ethanol-induced changes may result at multiple levels in the regulation including AP-1 proteins expression, CREB phosphorylation and perhaps reorganization of dimmers.
Qiang
Mei
M
Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA. qiang@uthscsa.edu
Ticku
Maharaj K
MK
D024506:5' Flanking Region / Q000502:physiology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000166:cytology*; D047369:Chromatin Immunoprecipitation / Q000379:methods; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004622:Embryo, Mammalian; D000431:Ethanol / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016296:Mutagenesis / Q000502:physiology; D009474:Neurons / Q000187:drug effects*; D016760:Proto-Oncogene Proteins c-fos / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000276:immunology; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000187:drug effects*
D000906:Antibodies; D002492:Central Nervous System Depressants; D017362:Cyclic AMP Response Element-Binding Protein; C121001:NR2B NMDA receptor; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016194:Receptors, N-Methyl-D-Aspartate; D018808:Transcription Factor AP-1; D000431:Ethanol; D004247:DNA; D008156:Luciferases