Inhibition of NF-kappa B binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes.
BACKGROUND
Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-kappaB in monocytes in the presence of ongoing IkappaBalpha degradation, suggesting regulation of NF-kappaB activation downstream of IkappaBalpha degradation. DNA binding of NF-kappaB has been suggested to be regulated by other nuclear regulatory factors, including the glucocorticoid receptor (GR). Here, we show for the first time that acute alcohol (25 mM) exposure modulates GR activation in monocytes.


METHODS
Human peripheral blood monocytes were treated with lipopolysaccharide (LPS) in the presence or absence of alcohol (25 mM) for 1 hour. Nuclear GR levels were estimated by Western blotting and NFkappaB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 microM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IkappaBalpha mRNA known to be induced by GR/GRE activation was studied in total RNA extracts by the SuperArray method (SuperArray Inc., Bethesda, MD).


RESULTS
LPS is a potent inducer of GR nuclear translocation and GR binding to the glucocorticoid response element (GRE). Acute alcohol treatment both induced (p < 0.05) and augmented (p < 0.05) LPS-stimulated GR nuclear levels. However, alcohol inhibits LPS-induced (nonligand bound) GR/GRE binding activity in monocytes. This inhibition of GR transactivation by alcohol was further confirmed by decreased expression (40%) of a target gene, IkappaBalpha. Thus, alcohol treatment increases nonligand-bound nuclear GR, but inhibits its transactivation function. Ligand-induced GR/GRE binding was decreased in alcohol-treated monocytes. Inhibition of ligand-induced GR/GRE binding by alcohol exposure is likely due to cytoplasmic retention of the GR.


CONCLUSIONS
Our results show that acute alcohol exposure inhibits GR in monocytes by differently affecting ligand- and nonligand-induced GR nuclear translocation. These data also suggest that acute alcohol regulates GR activation in monocytes concomitant to inhibition of NF-kappaB activation.
Mandrekar
Pranoti
P
Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School, Worcester 01605, USA. pranoti.mandrekar@umassmed.edu
Bellerose
Gary
G
Szabo
Gyongyi
G
D000293:Adolescent; D000328:Adult; D000431:Ethanol / Q000008:administration & dosage*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D018709:Statistics, Nonparametric; D014178:Translocation, Genetic / Q000187:drug effects / Q000502:physiology
D016328:NF-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D011965:Receptors, Glucocorticoid; D000431:Ethanol