Characterization of IFN-δ 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-γ; however, the mechanisms of Bf gene regulation have not been well characterized in general, and not in macrophages specifically. Northern analysis reveals that IFN-γ 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-γ responsiveness lies between -154 and -53 bp on the Bf promoter. This region of the Bf promoter contains both an IFN-γ-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-γ responsiveness. Mutagenesis of both the GAS and ISRE cis elements totally abrogates IFN-γ responsiveness of the Bf promoter. Electrophoretic mobility shift assays reveal nuclear binding complexes involving both Bf-GAS and Bf-ISRE oligonucleotide sequences upon IFN-γ 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-γ. Western analysis confirms an IFN-γ-stimulated increase in tyrosine phosphorylation of Stat1. These findings suggest that both GAS and ISRE cis binding sites have an additive effect on IFN-γ-stimulated Bf gene expression and that both are required for full expression of Bf by IFN-γ. Stat1 and IRF-1 take part in IFN-γ-stimulated Bf gene induction in macrophages through their respective cis binding elements.