IL-4-induced expression of the IL-1 receptor antagonist gene is mediated by STAT6

IL-4 alone or in cooperation with LPS can induce the expression of the gene encoding the secreted-type IL-1 receptor antagonist (sIL-1ra) in mononuclear phagocytes. To determine the nuclear signaling mechanisms involved in this response, the region flanking the transcription start site of the human sIL-1ra gene was placed upstream of the luciferase reporter gene, and the function of specific sequence elements was analyzed following transient transfection in the macrophage-like cell line RAW264.7. A region located between -250 and -200 bases relative to the transcription start site was necessary for response to IL-4 alone and for cooperation between IL-4 and LPS. This 50-bp region contains two inverted repeat elements that represent potential binding sites for members of the signal transducer and activator of transcription (STAT) gene family (STAT-binding elements (SBEs)). Site-directed mutagenesis of the distal SBE abolished IL-4 responsiveness, and multiple copies of this motif were able to confer IL-4 sensitivity to luciferase expression in the context of a heterologous (herpes virus thymidine kinase) promoter. Mutation of the proximal SBE in the intact IL-1ra promoter had little or no effect on response to IL-4, and this sequence motif was inactive when examined alone. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the distal SBE identified a single binding activity that was detected in nuclei within 15 min of IL-4 treatment and that was recognized by Ab to STAT6. These results indicate that IL-4-induced STAT6 is required for IL-4-induced transcriptional activation of the sIL-1ra gene.