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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA011953-04
Application #
6629623
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Guo, Qingbin
Project Start
2000-03-01
Project End
2006-02-28
Budget Start
2003-03-01
Budget End
2006-02-28
Support Year
4
Fiscal Year
2003
Total Cost
$185,000
Indirect Cost
Name
Texas Tech University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Sanchez, Alma C; Davis, Randall L; Syapin, Peter J (2007) The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells. Brain Res 1179:16-27
Davis, Randall L; Sanchez, Alma C; Lindley, Daniel J et al. (2005) Effects of mechanistically distinct NF-kappaB inhibitors on glial inducible nitric-oxide synthase expression. Nitric Oxide 12:200-9
Davis, Randall L; Syapin, Peter J (2004) Ethanol increases nuclear factor-kappa B activity in human astroglial cells. Neurosci Lett 371:128-32
Davis, Randall L; Syapin, Peter J (2004) Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis. Neurosci Lett 362:220-5
Davis, Randall L; Syapin, Peter J (2004) Acute ethanol exposure modulates expression of inducible nitric-oxide synthase in human astroglia: evidence for a transcriptional mechanism. Alcohol 32:195-202
Sanchez, Alma C; Davis, Randall L; Syapin, Peter J (2003) Identification of cis-regulatory regions necessary for robust Nos2 promoter activity in glial cells: indirect role for NF-kappaB. J Neurochem 86:1379-90
Davis, Randall L; Dertien, Janet; Syapin, Peter J (2002) Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells. Alcohol Clin Exp Res 26:1404-11
Syapin, P J; Militante, J D; Garrett, D K et al. (2001) Cytokine-induced iNOS expression in C6 glial cells: transcriptional inhibition by ethanol. J Pharmacol Exp Ther 298:744-52