Signal requirements for interleukin 4 promoter activation in human T cells

We have studied the signal requirements for human IL-4 promoter activation in Jurkat T cells by the use of DNA transfection assays with vectors carrying the IL-4 promoter linked to a reporter gene. Stimulation with calcium (Ca2+) ionophores (ionomycin), but not with phorbol esters (phorbol myristate acetate, PMA) or cyclic AMP elevating agents (prostaglandin E2, PGE2), induced the transcriptional activity of the IL-4 promoter by approximately 3-fold. Costimulation with ionomycin and PGE2 resulted in the same level of IL-4 promoter activity as the stimulation with ionomycin alone. In contrast, costimulation with ionomycin and PMA decreased the activity of the IL-4 promoter by approximately 40% compared to stimulation with ionomycin alone. Induction of Il-4 promoter by ionomycin was partially inhibited (approximately 50% inhibition) in the presence of as high as 2 microgram/ml cyclosporin A (CsA), an inhibitor of the Ca+/calmodulin-dependent phosphatase calcineurin. Under the same conditions, only 0.1 microgram/ml of CsA inhibited by >95% the transactivation of the IL-2 promoter in response to ionomycin and PMA. Transfection of a deletion mutant of the calcineurin catalytic subunit (delta CaM-AI) known to have Ca2+-independent, constitutive phosphatase activity increased IL-4 promoter activity by approximately 14-fold. Stimulation with ionomycin of cells transfected with low doses of delta CaM-AI, further induced IL-4 promoter activity by approximately 2-fold. These results identify the Ca2+-signaling system as a key component of the signal transduction pathway leading to IL-4 promoter activation in Jurkat T cells and suggest a major role of calcineurin in its regulation.