title: Potential role of cAMP response element-binding protein in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene transcription in fetal mouse cortical cells.
abstract: 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 (32)P-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.
authors.affiliation: Department of Pharmacology, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900, USA. kadapakkam@uthscsa.edu
authors.forename: C S Sheela
authors.initials: CS
authors.lastname: Rani
authors.forename: Mei
authors.initials: M
authors.lastname: Qiang
authors.forename: Maharaj K
authors.initials: MK
authors.lastname: Ticku
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D005333:Fetus; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000187:drug effects / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology
chemical_list: C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D000431:Ethanol