Threshold levels of individual NFAT factors appear to be critical for apoptosis induction in effector T cells. In these cells, the short isoform A of NFATc1 is induced to high levels due to the autoregulation of the NFATc1 promoter P1 by NFATs. P1 is located within a CpG island in front of exon 1, represents a DNase I hypersensitive chromatin site, and harbors several sites for binding of inducible transcription factors, including a tandemly arranged NFAT site. A second promoter, P2, before exon 2, is not controlled by NFATs and directs synthesis of the longer NFATc1/B+C isoforms. Contrary to other NFATs, NFATc1/A is unable to promote apoptosis, suggesting that NFATc1/A enhances effector functions without promoting apoptosis of effector T cells.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL‐2) induction, while induction of the genes coding for the lymphokines IL‐4 and IL‐5 is enhanced. We show that the differential effect of PKA activity on induction of the IL‐2 and IL‐4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL‐2 promoter. A kappa B site is missing in the IL‐4 promoter. Mutations preventing factor binding to the IL‐2 kappa B site result in a loss of PKA‐mediated suppression of IL‐2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL‐2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA‐stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF‐kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c‐Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B‐alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA‐mediated suppression of NF‐kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.

We show here that the immediate upstream region (from position -12 to -270) of the murine interleukin 4 (Il-4) gene harbors a strong cell-type specific transcriptional enhancer. In T lymphoma cells, the activity of the Il-4 promoter/enhancer is stimulated by phorbol esters, Ca++ ionophores and agonists of protein kinase A and inhibited by low doses of the immunosuppressant cyclosporin A. The Il-4 promoter/enhancer is transcriptionally inactive in B lymphoma cells and HeLa cells. DNase I footprint protection experiments revealed six sites of the Il-4 promoter/enhancer to be bound by nuclear proteins from lymphoid and myeloid cells. Among them are four purine boxes which have been described to be important sequence motifs of the Il-2 promoter. They contain the motif GGAAA and are recognized by the inducible and cyclosporin A-sensitive transcription factor NFAT-1. Three of the Il-4 NFAT-1 sites are closely linked to weak binding sites of Octamer factors. Several purine boxes and an AT-rich protein-binding site of the Il-4 promoter are also recognized by the high mobility group protein HMG I(Y). Whereas the binding of NFAT-1 and Octamer factors enhance the activity of the Il-4 promoter, the binding of HMG I(Y) suppresses its activity and, therefore, appears to be involved in the suppression of Il-4 transcription in resting T lymphocytes.

The transcription factor NF-ATc is synthesized in three prominent isoforms. These differ in the length of their C terminal peptides and mode of synthesis. Due to a switch from the use of a 3' polyA site to a more proximal polyA site, NF-ATc expression switches from the synthesis of the two longer isoforms in naive T cells to that of short isoform A in T effector cells. The relative low binding affinity of cleavage stimulation factor CstF-64 to the proximal polyA site seems to contribute to its neglect in naive T cells. These alternative polyadenylation events ensure the rapid accumulation of high concentrations of NF-ATc necessary to exceed critical threshold levels of NF-ATc for gene induction in effector T cells.

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