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.