title: Characterization of IFN-gamma regulation of the complement factor B gene in macrophages.
abstract: Complement factor B (Bf) is involved in the activation of the alternative complement cascade. Bf is induced by IFN-gamma; however, the mechanisms of Bf gene regulation have not been well characterized in general, and not in macrophages specifically. Northern analysis reveals that IFN-gamma induces a dose- and time-dependent increase in Bf mRNA expression in primary macrophages and macrophage cell lines. MH-S cells transfected with reporter constructs containing truncated regions of the Bf promoter reveal that IFN-gamma responsiveness lies between -154 and -53 bp on the Bf promoter. This region of the Bf promoter contains both an IFN-gamma-activation site (GAS) and an interferon-stimulated response element (ISRE). Site-directed mutagenesis of the GAS binding site or the ISRE binding site in this region of the Bf promoter partially inhibits IFN-gamma responsiveness. Mutagenesis of both the GAS and ISRE cis elements totally abrogates IFN-gamma responsiveness of the Bf promoter. Electrophoretic mobility shift assays reveal nuclear binding complexes involving both Bf-GAS and Bf-ISRE oligonucleotide sequences upon IFN-gamma stimulation. In competition assays, both Bf-GAS and Bf-ISRE oligonucleotides, but not mutant Bf-GAS nor mutant Bf-ISRE oligonucleotides, compete for the DNA binding. Supershift analysis reveals that Stat1-GAS and IRF-1-ISRE nuclear binding complexes contribute to induction of Bf by IFN-gamma. Western analysis confirms an IFN-gamma-stimulated increase in tyrosine phosphorylation of Stat1. These findings suggest that both GAS and ISRE cis binding sites have an additive effect on IFN-gamma-stimulated Bf gene expression and that both are required for full expression of Bf by IFN-gamma. Stat1 and IRF-1 take part in IFN-gamma-stimulated Bf gene induction in macrophages through their respective cis binding elements.
authors.affiliation: Department of Medicine, Division of Critical Care Medicine and Immunology Research Group, University of Calgary, Calgary, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Huang
authors.forename: P M
authors.initials: PM
authors.lastname: Krein
authors.forename: B W
authors.initials: BW
authors.lastname: Winston
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D011415:Complement Factor B / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D020218:Response Elements; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D010750:Phosphoproteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D015534:Trans-Activators; D007371:Interferon-gamma; D011415:Complement Factor B