Transcription factors of the NFAT family are thought to play a major role in regulating the expression of cytokine genes and other inducible genes during the immune response. The role of NFAT1 was investigated by targeted disruption of the NFAT1 gene. Unexpectedly, cells from NFAT1 -/- mice showed increased primary responses to Leishmania major and mounted increased secondary responses to ovalbumin in vitro. In an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased in NFAT1 -/- mice. These results suggest that NFAT1 exerts a negative regulatory influence on the immune response.

The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.The nuclear factor of activated T cells (NF-AT), a multisubunit protein, is thought to regulate transcriptional induction of the interleukin 2 (IL-2) gene and other cytokine genes in antigenstimulated T cells (1-3). An NF-AT binding activity is detected in cytosolic extracts of resting T cells and in nuclear extracts of cells that have been stimulated with T-cell-receptor ligands or with ionomycin (4-7). The immunosuppressive drugs cyclosporin A (CsA) and FK506 block the appearance of this DNA-binding activity in nuclear extracts (8, 9) and inhibit the transcription of several cytokine genes in activated T cells (10-16). The identification of the protein phosphatase calcineurin as the immediate target of the immunosuppressive drugs (17-21) led to the proposal that calcineurin is central in the signal transduction pathway leading to transcription of the IL-2 gene and other cytokine genes in T cells and that CsA and FK506 exert a major portion of their immunosuppressive effect by preventing the nuclear translocation of a cytosolic subunit of NF-AT (3,(22)(23)(24) (8,(29)(30)(31)(32)(33)(34).A protein (NFAT1/NFATp) that meets the defining criteria of the preexisting cyclosporin-sensitive subunit of NF-AT has been purified from cytosolic extracts of a murine T-cell clone (35), and cDNAs encoding three protein isoforms related by alternative splicing have been isolated (36). Additional family members (NFATc, NFATx/NFAT4, NFAT3) encoded by separate genes have been identified (37)(38)(39). The presence of the mRNAs for NFAT1, NFATc, and NFATx/NFAT4 in T cells or thymus (36)(37)(38)(39) indicates that these proteins could be involved in controlling the expression of the IL-2 gene and other cytokine genes in T cells. NFAT1 is expressed in certain cells of the immune system in addition to T cells (40) and could contribute to cytokine gene expression in these other cell types.Here we have used specific antisera against NFAT1 to examine the early steps in its activation, which include changes in its phosphorylation state, its subcellular localization, and its DNA-binding activity. MATERIALS AND METHODSCells. Splenic and peritoneal cells were collected from CB6F1 mice. T-cell blasts (>98% CD3+) were obtained as described (41). Purified splenic B cells (>96% B220+) were obtained by a...

The insulinotropic hormone glucagon-like peptide-1 (7-36)-amide (GLP-1) has potent effects on glucose-dependent insulin secretion, insulin gene expression, and pancreatic islet cell formation and is presently in clinical trials as a therapy for type 2 diabetes mellitus. We report on the effects of GLP-1 and two of its long-acting analogs, exendin-4 and exendin-4 WOT, on neuronal proliferation and differentiation, and on the metabolism of two neuronal proteins in the rat pheochromocytoma (PC12) cell line, which has been shown to express the GLP-1 receptor. We observed that GLP-1 and exendin-4 induced neurite outgrowth in a manner similar to nerve growth factor (NGF), which was reversed by coincubation with the selective GLP-1 receptor antagonist exendin (9-39). Furthermore, exendin-4 could promote NGF-initiated differentiation and may rescue degenerating cells after NGF-mediated withdrawal. These effects were induced in the absence of cellular dysfunction and toxicity as quantitatively measured by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays, respectively. Our findings suggest that such peptides may be used in reversing or halting the neurodegenerative process observed in neurodegenerative diseases, such as the peripheral neuropathy associated with type 2 diabetes mellitus and Alzheimer's and Parkinson's diseases. Due to its novel twin action, GLP-1 and exendin-4 have therapeutic potential for the treatment of diabetic peripheral neuropathy and these central nervous system disorders.

NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.