Pharmacology and Neuroscience
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 CNS. 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. This is proposed to have detrimental effects in the CNS. Interestingly, reversal of the suppression during withdrawal is protracted, indicating 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-k B. Aim 3 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.