title: Functional analysis of interleukin 6 response elements (IL-6REs) on the human gamma-fibrinogen promoter: binding of hepatic Stat3 correlates negatively with transactivation potential of type II IL-6REs.
abstract: Several families of transcription factors play important roles in modulating liver-specific gene expression during an acute phase response (APR). Stat3/APR factor is the main transactivator of gene expression by the interleukin (IL)-6 family of cytokines signaling through gp130. During an APR, fibrinogen (FBG) genes are coordinately up-regulated by IL-6 and glucocorticoids. Except for rat gammaFBG, attempts to demonstrate direct binding of IL-6-activated Stat3 to FBG CTGGGAA promoter elements have not been successful. Herein we show the presence of three functional type II IL-6 response elements (IL-6REs) on the human gammaFBG promoter and that the magnitude of Stat3 binding to these elements correlates negatively with their functional activity in reporter gene assays. Stat3-specific binding to gammaFBG IL-6REs was confirmed by cross-competition with alpha2-macroglobulin IL-6RE and specific interactions with anti-Stat3 in electrophoretic mobility shift assays. All type II IL-6REs contributed to full promoter activity; however, transactivation from Site II at -306 to -301 was strongest. In contrast to a previous report, IL-6 failed to induce activation of serum amyloid A-activating factor-1/c-Myc-associated zinc finger protein (SAF-1/MAZ), and mutation of the SAF-1RE had little effect on IL-6 induction of gammaFBG promoter activity. In the absence of a functional glucocorticoid receptor response element, dexamethasone potentiated IL-6-induced gammaFBG promoter activity 2-fold, requiring promoter-proximal Site I and Site II; the promoter-distal Site III had no effect on dexamethasone potentiation of IL-6-induced promoter activity. Notably the propensity for Stat3 binding to human gammaFBG IL-6REs was low compared with Stat3 binding to the alpha2-macroglobulin IL-6RE. Together these data suggest that Stat3 transactivation via IL-6REs on FBG promoters likely involves participation of additional transcription factors and/or coactivators to achieve optimal coordinated up-regulation during an APR.
authors.affiliation: Department of Medicine, University of Rochester School of Medicine and Dentistry Rochester, New York 14642, USA.
authors.forename: Hai Ou
authors.initials: HO
authors.lastname: Duan
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Simpson-Haidaris
mesh_terms: D000210:Acute-Phase Reaction; D015153:Blotting, Western; D002130:Calcium Phosphates / Q000378:metabolism; D002455:Cell Division; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000235:genetics*; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050796:STAT3 Transcription Factor; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
chemical_list: D002130:Calcium Phosphates; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D015850:Interleukin-6; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D003907:Dexamethasone; D005340:Fibrinogen; D004247:DNA; C020243:calcium phosphate; D015500:Chloramphenicol O-Acetyltransferase; C485829:calcium phosphate, dibasic, anhydrous