Role of AP-1 in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene up-regulation in mouse cortical neurons

J Neurochem. 2005 Dec;95(5):1332-41. doi: 10.1111/j.1471-4159.2005.03464.x.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • 5' Flanking Region / physiology
  • Animals
  • Antibodies / pharmacology
  • Blotting, Western / methods
  • Central Nervous System Depressants / pharmacology*
  • Cerebral Cortex / cytology*
  • Chromatin Immunoprecipitation / methods
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • DNA / metabolism
  • Electrophoretic Mobility Shift Assay / methods
  • Embryo, Mammalian
  • Ethanol / pharmacology*
  • Gene Expression / drug effects*
  • Luciferases / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mutagenesis / physiology
  • Neurons / drug effects*
  • Proto-Oncogene Proteins c-fos / immunology
  • Proto-Oncogene Proteins c-jun / immunology
  • Receptors, N-Methyl-D-Aspartate / genetics
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism
  • Transcription Factor AP-1 / genetics
  • Transcription Factor AP-1 / metabolism*
  • Transcriptional Activation / physiology
  • Transfection / methods
  • Up-Regulation / drug effects*


  • Antibodies
  • Central Nervous System Depressants
  • Cyclic AMP Response Element-Binding Protein
  • NR2B NMDA receptor
  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • Receptors, N-Methyl-D-Aspartate
  • Transcription Factor AP-1
  • Ethanol
  • DNA
  • Luciferases