Chronic ethanol increases lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced tumor necrosis factor alpha production.
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.
Shi
Liang
L
Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106-4906, USA.
Kishore
Raj
R
McMullen
Megan R
MR
Nagy
Laura E
LE
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
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