Pharmacogenomic analysis of mechanisms mediating ethanol regulation of dopamine β-hydroxylase
Pharmacogenomic analysis of mechanisms mediating ethanol regulation of dopamine β-hydroxylase
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DOI：
10.1074/jbc.m305040200
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发表时间：
2003-10-03
影响因子：
4.8
通讯作者：
Miles, MF
中科院分区：
文献类型：
--
作者：
Hassan, S;Duong, B;Miles, MF
我们之前表明乙醇调节人神经母细胞瘤细胞中的多巴胺β-羟化酶（DBH）mRNA和蛋白质水平（Thibault, C.、Lai, C.、Wilke, N.、Duong, B.、Olive, M. F.、Rahman, S.、Dong, H.、Hodge, C. W.、Lockhart, D. J.和Miles, M. F. (2000) Mol. Pharmacol. 58、1593-1600）。 DBH 催化去甲肾上腺素合成，一些研究表明去甲肾上腺素在乙醇介导的行为中发挥作用。在这里，我们对 SH-SY5Y 细胞中 DBH 表达的乙醇调节机制进行了详细分析。瞬时转染分析表明，乙醇 (25-200 mM) 会导致 DBH 基因转录出现浓度和时间依赖性增加。逐步删除在 DBH 基因启动子的 -262 至 -142 bp 区域中鉴定出乙醇响应序列。该区域的 cAMP 响应元件 (CRE) 序列的诱变消除了乙醇响应性，同时保持了对佛波酯的响应性。显性失活 CRE 结合蛋白的共表达大大降低了 DBH 的乙醇诱导。蛋白激酶 A、酪蛋白激酶 II 和 MAPK 抑制剂可降低乙醇诱导的 DBH 启动子活性。使用微阵列进行的药物基因组学研究表明，蛋白激酶 A、MEK 和酪蛋白激酶 II 抑制剂可阻断 DBH 和大部分乙醇反应基因的诱导。这些基因具有不同的功能分组，包括 MAPK 和磷脂酰肌醇信号级联的多个成员。实时 PCR 分析验证了选定的微阵列结果。综上所述，这些结果表明乙醇对 DBH 的调节需要功能性 CRE 及其结合蛋白，并且可能需要多个激酶途径的相互作用。这种机制还可能介导神经细胞中复杂基因子集的乙醇反应性。这些研究可能对乙醇的行为反应或乙醇相关神经疾病的机制产生影响。
We previously showed that ethanol regulates dopamine beta-hydroxylase (DBH) mRNA and protein levels in human neuroblastoma cells (Thibault, C., Lai, C., Wilke, N., Duong, B., Olive, M. F., Rahman, S., Dong, H., Hodge, C. W., Lockhart, D. J., and Miles, M. F. (2000) Mol. Pharmacol. 58, 1593-1600). DBH catalyzes norepinephrine synthesis, and several studies have suggested a role for norepinephrine in ethanol-mediated behaviors. Here, we performed a detailed analysis of mechanism(s) underlying ethanol regulation of DBH expression in SH-SY5Y cells. Transient transfection analysis showed that ethanol (25-200 mM) caused concentration- and time-dependent increases in DBH gene transcription. Progressive deletions identified ethanol-responsive sequences in the -262 to -142 bp region of the DBH gene promoter. Mutagenesis of cAMP-response element (CRE) sequences in this region abolished ethanol responsiveness while maintaining responsiveness to phorbol esters. Co-expression of dominant-negative CRE-binding protein greatly reduced ethanol induction of DBH. Inhibitors of protein kinase A, casein kinase II, and MAPK reduced ethanol induction of DBH promoter activity. Pharmacogenomic studies with microarrays showed that protein kinase A, MEK, and casein kinase II inhibitors blocked induction of DBH and a large subset of ethanol-responsive genes. These genes had diverse functional groupings, including multiple members of the MAPK and phosphatidylinositol signaling cascades. Real-time PCR analysis validated select microarray results. Taken together, these results suggest that ethanol regulation of DBH requires a functional CRE and its binding protein and may require interaction of multiple kinase pathways. This mechanism may also mediate ethanol responsiveness of a complex subset of genes in neural cells. These studies may have implications for behavioral responses to ethanol or mechanisms underlying ethanol-related neurological disease.