The Ca2+ Calmodulin-Activated, Phosphoprotein Phosphatase Calcineurin Is Sufficient For Positive Transcriptional Regulation Of The Mouse Il-4 Gene

We have studied the TCR mediated signal transduction pathways involved in transcriptional regulation of the mouse IL-4 gene. The sequences extending from base pair -766 to + 63 of the IL-4 gene were inserted upstream of a luciferase indicator gene. Transcriptional activity was observed when the construct, [pIL-4(- 766)], was transfected into either the IL-4 producing cell line, EL-4, or the IL-4 non-producing T cell hybridoma, 68-41, but not in the L929 fibroblast cell line. By analysis of deletion mutants of pIL-4(- 766), we identified a transcriptional regulatory element that is tightly associated with a signal coming from the TCR and which controls inducible activation of the IL-4 promoter. By analysis of deletion mutants of pIL-4(- 766), this latter element was found between base pairs - 147 to - 17. Electrophoretic mobility shift assays indicated that expression of a nuclear binding protein with binding sites between base pairs - 84 and - 55 could be induced. By competition and mutation analysis, the binding motif of this protein was determined to be AAAATTTTCC. Stimulation with ionomycin alone was sufficient to induce activity in pIL-4(- 766). Cyclosporin A inhibited both the IL-4 promoter activity and activation of the inducible nuclear protein. Transient over-expression of a constitutively active form of the Ca2+/Calmodulin-regulated protein phosphatase, calcineurin was sufficient to cause activation of pIL-4(- 766) without any additional stimulus. These results indicate that the signaling requirements for activation of upstream positive regulatory elements of the IL-4 gene are distinct from those of the IL-2 gene. Ca2+ Mobilization is sufficient to activate the IL-4 promoter, whereas IL-2 gene transcription requires both Ca2+ mobilization and protein kinase C activation.