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.
Kubo
M
M
Department of Inflammation Biology and Immunology, Syntex Discovery Research, Palo Alto, CA 94304.
Kincaid
R L
RL
Webb
D R
DR
Ransom
J T
JT
D000818:Animals; D001483:Base Sequence; D019703:Calcineurin; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D015847:Interleukin-4 / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D015847:Interleukin-4; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium