RT Journal Article
SR Electronic
T1 Potential Role of cAMP Response Element-Binding Protein in Ethanol-Induced <em>N</em>-Methyl-<span class="sc">d</span>-aspartate Receptor 2B Subunit Gene Transcription in Fetal Mouse Cortical Cells
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 2126
OP 2136
DO 10.1124/mol.104.007872
VO 67
IS 6
A1 C. S. Sheela Rani
A1 Mei Qiang
A1 Maharaj K. Ticku
YR 2005
UL http://molpharm.aspetjournals.org/content/67/6/2126.abstract
AB We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-d-aspartate (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 subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Long-term ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, whereas 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-base pair (bp) double-stranded DNA fragment containing the cyclic AMP response element (CRE)-like sequence of the NR2B promoter as 32P-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from cells undergoing long-term ethanol treatment. A 467-bp DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient cotransfection 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 probably involved in mediating ethanol-induced up-regulation of NR2B gene.