title: Chronic ethanol increases lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced tumor necrosis factor alpha production.
abstract: Increased production of tumor necrosis factor alpha (TNFalpha) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor alpha (TNFalpha) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFalpha promoter activity contribute to increased TNFalpha expression. Binding of Egr-1 to the TNFalpha promoter was increased by 2.5-fold after ethanol exposure, whereas NFkappaB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFkappaB site to LPS-stimulated TNFalpha promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFalpha mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulated Egr-1 promoter-driven CAT expression and transcription of Egr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFalpha. Increased Egr-1 promoter activity and TNFalpha mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFalpha production after chronic ethanol exposure.
authors.affiliation: Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106-4906, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.forename: Raj
authors.initials: R
authors.lastname: Kishore
authors.forename: Megan R
authors.initials: MR
authors.lastname: McMullen
authors.forename: Laura E
authors.initials: LE
authors.lastname: Nagy
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D024202:Electrophoretic Mobility Shift Assay; D000431:Ethanol / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D017874:Immediate-Early Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D015342:DNA Probes; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D008070:Lipopolysaccharides; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol