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
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
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DOI：
10.1124/mol.104.007872
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发表时间：
2005-06-01
影响因子：
3.6
通讯作者：
Ticku, MK
中科院分区：
文献类型：
--
作者：
Rani, CSS;Qiang, M;Ticku, MK
我们以前已经证明，长期的乙醇处理导致培养的胎鼠皮质神经元N-甲基-D-天冬氨酸(NMDA)受体2B亚单位(NR2B)数量和功能的上调。为了研究这种NR2B基因转录所涉及的细胞内信号通路，我们用乙醇长期处理胚胎皮质神经元，并通过Western印迹和酶联免疫吸附试验研究其对cAMP反应元件结合蛋白(CREB)和细胞外信号调节蛋白(ERK)水平的影响。我们发现，在乙醇处理5天的细胞中，磷酸化的CREB显著增加，而总CREB蛋白没有变化。长期乙醇处理不增加无血清培养上清液中总ERK和磷酸化ERK的水平，但增加含血清培养上清液中ERK的磷酸化水平，但不影响总ERK水平。无论血清存在与否，乙醇处理均可增加CREB的磷酸化。以含有NR2B启动子的环状AMP反应元件(Cre)样序列的25个碱基对(BP)的双链DNA片段为P-32标记探针的凝胶迁移率改变分析表明，从长期乙醇处理的细胞中分离的核蛋白与Cre的特异性结合增加。将含有Cre序列的NR2B启动子的467bpDNA片段克隆到荧光素酶载体中，在瞬时共转染小鼠皮质神经元的实验中显示出高报告活性，乙醇处理提高了这一活性。在Cre序列中引入定点突变显著降低了报告基因相对于野生型载体的活性，并取消了乙醇的刺激作用。我们的结果表明，CREB可能参与了乙醇诱导的NR2B基因的上调。
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 P-32-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.