Inhibition of NF-κB binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes

Pranoti Mandrekar, Gary Bellerose, G. Szabó

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Background: Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-κB in monocytes in the presence of ongoing IκBα degradation, suggesting regulation of NF-κB activation downstream of IκBα degradation. DNA binding of NF-κB 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 NFκB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 μM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IκBα 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, IκBα. 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-κB activation.

Original languageEnglish
Pages (from-to)1872-1879
Number of pages8
JournalAlcoholism: Clinical and Experimental Research
Volume26
Issue number12
Publication statusPublished - Dec 1 2002

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Glucocorticoid Receptors
Cytoplasmic and Nuclear Receptors
Monocytes
Alcohols
Response Elements
Glucocorticoids
Chemical activation
Lipopolysaccharides
Ligands
Transcriptional Activation
Degradation
Electrophoretic Mobility Shift Assay

Keywords

  • Dexamethasone
  • Ethanol
  • IκB
  • Inflammation
  • Macrophage
  • TNFα

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Toxicology

Cite this

Inhibition of NF-κB binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes. / Mandrekar, Pranoti; Bellerose, Gary; Szabó, G.

In: Alcoholism: Clinical and Experimental Research, Vol. 26, No. 12, 01.12.2002, p. 1872-1879.

Research output: Contribution to journalArticle

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abstract = "Background: Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-κB in monocytes in the presence of ongoing IκBα degradation, suggesting regulation of NF-κB activation downstream of IκBα degradation. DNA binding of NF-κB 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 NFκB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 μM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IκBα 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, IκBα. 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-κB activation.",
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N2 - Background: Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-κB in monocytes in the presence of ongoing IκBα degradation, suggesting regulation of NF-κB activation downstream of IκBα degradation. DNA binding of NF-κB 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 NFκB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 μM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IκBα 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, IκBα. 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-κB activation.

AB - Background: Acute alcohol treatment blocks inflammatory cytokines via inhibition of NF-κB in monocytes in the presence of ongoing IκBα degradation, suggesting regulation of NF-κB activation downstream of IκBα degradation. DNA binding of NF-κB 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 NFκB activation was studied in the same extracts by gel shift analysis (EMSA). Cells were stimulated with 1 μM of Dex to be used as positive control for GR activation. GR/GRE binding was also determined in nuclear extracts by EMSA. IκBα 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, IκBα. 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-κB activation.

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