Received for publication October 6, 2004.
Revised March 10, 2005.
Accepted for publication March 17, 2005.

Potential Role of CREB in Ethanol-induced NR2B Gene Transcription in Fetal Mouse Cortical Cells

Abstract

We have previously shown that chronic ethanol treatment causes an upregulation of NMDA receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have treated fetal cortical neurons chronically with ethanol and studied its effect on CREB and ERK levels by Western blot and ELISA. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Chronic ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, while it did increase ERK phosphorylation in medium containing serum, without affecting total ERK levels. CREB phosphorylation was increased by ethanol treatment in both media, irrespective of the presence of serum. Electrophoretic mobility shift assay, using a 25-bp long double-stranded DNA fragment containing the CRE-like sequence of the NR2B promoter as [³²P]-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from chronic ethanol-treated cells. A 467-bp long DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient co-transfection assay of mouse cortical neurons, and ethanol treatment increased this activity. Introducing site-directed mutation in the CRE sequence significantly reduced the reporter activity relative to the wild-type construct, and it also abolished the stimulatory effect by ethanol. Our results indicate that CREB is likely involved in mediating ethanol-induced upregulation of NR2B gene.

Key words: Ion channel regulation, CREB, Promoter analysis, Regulation of gene expression, Regulation - transcriptional, Alcohol, Excitotoxicity, neurodegeneration

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