Induction of Ref-1 ensures AP-1 activation in intracellular oxidative environment of IL-2-stimulated BA/F3 beta cells

Our previous study of interleukin-2 (IL-2) signaling found that redox facto r-1 (Ref-l) mRNA was upregulated by IL-2. in this study,we further studied the function of Ref-l in the potential redox regulation of IL-2 signaling i n BA/F3 beta cells. Western blot analysis confirmed that IL-2 stimulation i ncreases Ref-l protein. Flow cytometric assay by using 2',7'-dichlorofluore scin diacetate indicated that IL-2 stimulation results in an oxidative shif t of intracellular environment. However, IL-2-induced activator protein-1 ( AP-1) is oxidation-sensitive. Gel shift assays of nuclear extracts immunode pleted of Ref-l protein demonstrated that IL-2-induced AP-1 DNA binding is dependent on the presence of Ref-l. This was further confirmed by the resto ration of AP-1 DNA binding upon the re-addition of immunoprecipitated Ref-l . Additionally, reporter gene assays showed that AP-1 transcriptional activ ity was enhanced by the overexpression of Ref-l and attenuated by the intro duction of antisense Ref-l. These results suggest that the induction of Ref -l ensures AP-1 activation in the intracellular oxidative environment of IL -2-stimulated BA/F3 beta cells, (C) 2000 Academic Press.