Identification of a salicylate-responsive region in the human IL-4 promoter.
(A) Jurkat cells transiently transfected with the indicated human IL-4 promoter deletional constructs were treated with ASA (■, 10−3 M), SA (▨, 10−3 M), or FBP (▪, 10−5 M) before 20-hour stimulation with 0.5 μg/mL A23187. The mean ± SEM percentage of control A23187-induced CAT expression (broken horizontal line) in 3 independent experiments is shown. The asterisk indicates P < .05 (Wilcoxon test) relative to DMSO-treated controls. (B) Schematic representation of the human IL-4 promoter and the sequence required for its inhibition by salicylates. Open and solid boxes indicate the relative positions of positive and negative regulatory elements, respectively, that have been identified. ISRE indicates IFN stimulation-response element; MARE, c-Maf response element; NRE, negative regulatory element; and OAP, octamer-associated protein. Schematically shown below are 3 oligonucleotides (IL-4.155, IL-4.135, and IL-4.115), spanning putative promoter elements in the salicylate-targeted region, that were used as probes in EMSAs. TRE indicates tetradecanoylphorbol acetate–response element. (C) Jurkat cells were treated with ASA (10−3 M; lanes 4 and 8) or DMSO carrier (lanes 2, 3, 6, and 7) before stimulation with A23187 (0.5 μg/mL) for 2 hours. Nuclear extracts were incubated with labeled IL-4.155 (data not shown), IL-4.135, or IL-4.115 oligonucleotides. The arrow at left indicates an A23187-induced IL-4.135–binding complex whose formation was markedly reduced in cells treated with ASA (lanes 3 and 4).
