HOW DOES ALCHOHOL SUPPRESS GLIAL NOS-2 GENE EXPRESSION

遗传学对酒精滥用风险因素和行为易感性及其他酒精效应的影响已得到充分证实。相反的情况在多大程度上是正确的，即风险因素和酒精影响是由酒精引起的基因调节干扰决定的，目前尚不清楚，但有必要了解。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。急性和慢性暴露于乙醇的大鼠神经胶质细胞的Nos2基因产物，一氧化氮合酶-2（iNOS），一个重要的诱导蛋白在NS的表达减少。iNOS蛋白和mRNA水平均被乙醇降低，Nos2启动子激活的荧光素酶基因报告活性也是如此。因此，乙醇通过降低Nos2基因的转录激活来抑制神经胶质中iNOS的表达。因此，乙醇通过降低Nos2基因的转录激活来抑制神经胶质中iNOS的表达。这被认为对CNS有不利影响。有趣的是，在戒断过程中抑制的逆转是持久的，表明可能的长期后果。 具体的目标将调查乙醇抑制Nos2基因转录的机制和测试的假设，即乙醇抑制Nos2基因的启动子区域通过特定的顺式作用元件的Nos2基因的表达。目的1建立稳定表达修饰的大鼠Nos 2启动子荧光素酶基因报告载体的细胞系，以描述乙醇介导的转录抑制的启动子区域。目的2将表征乙醇对相关反式作用因子如NF-κ B的影响。目的二利用体内DNA足迹法验证乙醇对活细胞中相关转录因子Nos2启动子结合活性的影响。将检查暴露于急性和慢性乙醇的细胞因子刺激的C6神经胶质细胞，以及经历乙醇戒断的慢性暴露细胞。标准的分子生物学技术将被用来构建修改后的大鼠Nos2启动子报告基因载体，并研究转录因子的变化，结合到顺式作用区域，介导的乙醇抑制。DNA足迹法将使用高灵敏度的连接介导的PCR方法。这些结果将对理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影响的基础。

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.