Our previous study of interleukin-2 (IL-2) signaling found that redox factor-1 (Ref-1) mRNA was upregulated by IL-2. In this study, we further studied the function of Ref-1 in the potential redox regulation of IL-2 signaling in BA/F3beta cells. Western blot analysis confirmed that IL-2 stimulation increases Ref-1 protein. Flow cytometric assay by using 2',7'-dichlorofluorescin diacetate indicated that IL-2 stimulation results in an oxidative shift of intracellular environment. However, IL-2-induced activator protein-1 (AP-1) is oxidation-sensitive. Gel shift assays of nuclear extracts immunodepleted of Ref-1 protein demonstrated that IL-2-induced AP-1 DNA binding is dependent on the presence of Ref-1. This was further confirmed by the restoration of AP-1 DNA binding upon the re-addition of immunoprecipitated Ref-1. Additionally, reporter gene assays showed that AP-1 transcriptional activity was enhanced by the overexpression of Ref-1 and attenuated by the introduction of antisense Ref-1. These results suggest that the induction of Ref-1 ensures AP-1 activation in the intracellular oxidative environment of IL-2-stimulated BA/F3beta cells.
Copyright 2000 Academic Press.