HOW DOES ALCHOHOL SUPPRESS GLIAL NOS-2 GENE EXPRESSION

遗传学对酒精滥用风险因素以及对行为和其他酒精影响的易感性的影响已经得到了充分的证实。相反，风险因素和酒精效应是由酒精诱导的基因调控紊乱决定的，这在多大程度上是正确的，这一点不太清楚，但理解起来是必要的。确定酒精对中枢神经系统基因调控的影响将有助于确定酒精滥用和酒精中毒的分子机制。急性和慢性乙醇暴露可降低NOS2基因产物一氧化氮合酶-2(INOS)的表达，iNOS是NS中一种重要的诱导蛋白。乙醇使iNOS蛋白和mRNA水平降低，NOS2启动子激活的荧光素酶基因报告活性也降低。因此，乙醇通过降低NOS2基因的转录活性来抑制神经胶质细胞中iNOS的表达。因此，乙醇通过降低NOS2基因的转录活性来抑制神经胶质细胞中iNOS的表达。这被认为对中枢神经系统有不利影响。有趣的是，撤军期间的镇压逆转是旷日持久的，表明可能产生长期后果。这些特定的目的将研究乙醇抑制NOS2基因转录的机制，并验证乙醇通过NOS2基因启动子区域的特定顺式作用元件抑制NOS2表达的假说。目的1建立稳定表达大鼠NOS2启动子荧光素酶基因报告载体的细胞系，以确定乙醇介导转录抑制的启动子区域。目的2研究乙醇对核因子-kappaB等相关反式作用因子的影响。目的2将利用体内DNA足迹来验证乙醇改变活细胞中相关转录因子的NOS2启动子结合活性。细胞因子刺激的C6神经胶质细胞暴露在急性和慢性乙醇中，以及慢性暴露的细胞经历乙醇戒断。标准的分子生物学技术将被用来构建修饰的大鼠NOS2启动子-报告基因载体，并研究与介导乙醇抑制的顺式作用区域结合的转录因子的变化。DNA足迹将使用高灵敏的连接介导的聚合酶链式反应方法。这些结果将对了解酒精对中枢神经系统基因表达的影响具有广泛的意义，这被认为是酒精慢性行为和病理生理效应的基础。

The influence of genetics on alcohol abuse risk factors and susceptibility to behavioral and other alcohol effects is well established. The degree to which the converse is true, that risk factors and alcohol effects are determined by alcohol-induced disturbances in gene regulation, is less clear, but necessary to understand. Determining alcohol effects on CNS gene regulation will help identify molecular mechanisms at play in alcohol abuse and alcoholism. Acute and chronic exposure of rat glial cells to ethanol reduces expression of the Nos2 gene product, nitric oxide synthase-2 (iNOS), an important inducible protein in the NS. Both iNOS protein and mRNA levels are reduced by ethanol, as is Nos2 promoter- activated luciferase gene reporter activity. Thus, ethanol inhibits iNOS expression in glia by reducing transcriptional activation of the Nos2 gene. Thus, ethanol inhibits iNOS expression in glia by reducing transcriptional activation of the Nos2 gene. This is proposed to have detrimental effects in the CNS. Interestingly, reversal of the suppression during withdrawal is protracted, indicated possible long-term consequences. The specific aims will investigate the mechanisms underlying ethanol inhibition of Nos2 gene transcription and test the hypothesis that ethanol suppresses Nos2 expression through specific cis-acting elements in the promoter region of the Nos2 gene. Aim 1 will develop cell lines stably expressing modified rat Nos2 promoter luciferase gene reporter vectors to delineate the promoter region where transcriptional inhibition by ethanol is mediated. Aim 2 will characterize the ethanol effect on relevant trans-acting factors such as NF-kappaB. Aim 2 will use in vivo DNA footprinting to verify that ethanol alters Nos2 promoter binding activity of relevant transcription factors in living cells. Cytokine-stimulated C6 glial cells exposed to acute and chronic ethanol, and chronically exposed cells undergoing ethanol withdrawal will be examined. Standard molecular biology techniques will be used to construct the modified rat Nos2 promoter-reporter gene vectors and to study changes in transcription factors that bind to the cis-acting region that mediates the ethanol suppression. DNA footprinting will use the highly sensitive ligation mediated PCR method. The results will have broad implications for understanding ethanol effects on CNS gene expression, which is proposed to underlie chronic behavioral and pathophysiologic effects of alcohol.