FC-EPSILON-RI-MEDIATED INDUCTION OF NUCLEAR FACTOR OF ACTIVATED T-CELLS

Nuclear factor of activated T-cells (NFAT) is a transcriptional activa tor that binds to the interleukin-2 promoter and is believed to be res ponsible for T-cell-specific interleukin-2 gene expression. Here we de monstrate using electrophoretic mobility shift assays that nuclear NFA T can be induced in the rat basophilic leukemia (RBL 2H3) mast cell li ne and rat bone marrow-derived mast cells upon cross-linkage of the hi gh affinity receptor (Fc epsilon RI) for immunoglobulin E (IgE). Recep tor-dependent activation of NFAT was mimicked by the combination of th e protein kinase C activator phorbol myristate acetate and the calcium ionophore ionomycin. The induced binding activity was specific for th e NFAT recognition motif because competition with nonradioactive NFAT oligonucleotide abolished the DNA binding activity, whereas nonradioac tive oligonucleotides recognized by the transcription factors NF kappa B, glucocorticoid receptors, and TFIID did not, An oligonucleotide re presenting the AP-1 recognition sequence also blocked the NFAT DNA bin ding activity, as did a combination of anti-Fos and anti-Jun antibodie s. Using electrophoretic mobility shift assays, AP-1-binding proteins were found to be induced in RBL-2H3 cells under the same conditions as was the NFAT binding activity. Together these data suggest that the N FAT complex in mast cells contains Fos and dun proteins as does NFAT i n T-cells, The appearance of nuclear NFAT binding activity was depende nt in part upon calcium mobilization, as buffering the antigen-induced calcium rise with intracellular BAPTA strongly inhibited NFAT activat ion. Prevention of calcium influx with external EGTA also inhibited NF AT activation, indicating that release of calcium from internal stores was insufficient for sustained activation of mast cell NFAT. Cyclospo rin A, a potent inhibitor of the calmodulin-dependent phosphatase calc ineurin, blocked the induction of NFAT-DNA binding activity, implicati ng calcineurin as a key signaling enzyme in this pathway, These result s suggest that NFAT is present in the mast cell line RBL-2H3 and in pr imary bone marrow-derived mast cells, is similar in subunit compositio n to the T-cell NFAT, and may play a role in calcium-dependent signal transduction in mast cells.