title: Stat proteins play a role in tumor necrosis factor alpha gene expression.
abstract: Trauma produces dysfunction in immunity, which appears to be partially related to alterations in the cytokine response. Signal transducer and activator of transcription proteins (STATs) mediate activation of several cytokine genes. However, the effect of STAT proteins on tumor necrosis factor-alpha (TNFalpha) activation is not fully defined. We identified binding sites for STAT 3 and STAT 5/6 within the promoter region of TNFalpha and hypothesize that alterations in these sites would affect TNFalpha expression. The TNFalpha promoter was inserted into the luciferase reporter vector, and binding sites for STAT 3, STAT 5/6, and activator protein-1 (AP-1) were mutated using site-directed mutagenesis. Murine macrophages were transfected with the resultant plasmids, then incubated with and without lipopolysaccharide (LPS) or IFNalpha. Gene expression was measured by dual luciferase assay. Mutation of the STAT 3 binding site was associated with decreased LPS-inducible activity. Mutation of the AP-1 and STAT 5/6 consensus binding sites alone had no effect on TNFalpha expression. However, combined mutation of both STAT 5/6 and AP-1 was associated with increased LPS-inducible activity. Mutations of the STAT binding sites in the promoter region of TNFalpha affect TNFalpha gene expression. These results suggest a regulatory role for STATs in TNF gene transcription.
authors.affiliation: Department of Surgery, The University of Texas Medical Branch, Galveston 77555-1173, USA.
authors.forename: V L
authors.initials: VL
authors.lastname: Chappell
authors.forename: L X
authors.initials: LX
authors.lastname: Le
authors.forename: L
authors.initials: L
authors.lastname: LaGrone
authors.forename: W J
authors.initials: WJ
authors.lastname: Mileski
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins; D009154:Mutation; D011401:Promoter Regions, Genetic; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D008894:Milk Proteins; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D050800:STAT6 Transcription Factor; C494087:Stat3 protein, mouse; C494100:Stat6 protein, mouse; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma