Abstract
Proliferating human medullary thymocytes can exhibit characteristic T helper cell type 1 cytokine responses exemplified by the immediate early expression of interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and lymphotoxin-beta. Here we report that cAMP-mediated attenuation of the transcription of T helper-1-specific cytokine genes in human medullary thymocytes correlates with the induction of the cAMP-mediated transcriptional repressor ICER (inducible cAMP early repressor). We show that ICER binds specifically to several NFAT/AP-1 (nuclear factor of activated T cells/activating protein-1) composite DNA sites essential for the activation of the interleukin (IL)-2 promoter as well as to a homologous DNA motif present in the proximal segment of the interferon-gamma promoter. In the presence of the minimal NFAT DNA-binding domain, which is sufficient for both DNA binding and AP-1 complex formation, ICER and NFAT form NFAT/ICER ternary complexes on several NFAT/AP-1 DNA composite sites previously identified as essential for the expression of the immunoregulatory cytokines such as IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. In extracts prepared from human medullary thymocytes treated with forskolin and ionomycin, these composite sites bind endogenously expressed ICER either singly or in complexes. Moreover, in Jurkat cells, ectopically expressed ICER represses transcription from NFAT-mediated, phorbol ester/ionophore-activated IL-2, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha promoters. We present evidence that ICER interactions with NFAT/AP-1 composite DNA sites correlate with its ability to repress transcription. These findings provide further insight into the mechanisms involved in cAMP-mediated transcriptional attenuation of cytokine expression.
Highlights
It is well established that cAMP signaling is inhibitory to T cell proliferation and effector functions
We propose that at least part of this transcriptional attenuation is based on cAMP-mediated expression of the transcriptional repressor ICER and a subsequent blockade of NFAT/AP-1 composite DNA sites essential for T helper-1 cytokine expression
Cyclic AMP-mediated Attenuation of T Helper-1 Cytokine Transcription in Human Medullary Thymocytes Correlates with cAMP-mediated Induction of the Transcriptional Repressor ICER—To test the proposed mechanism involved, we first sought evidence for the presence of ICER protein in human medullary thymocytes after forskolin treatment to explore whether ICER may interact with important NFAT/AP-1 enhancer motifs of the IL-2 and IFN␥ promoters
Summary
It is well established that cAMP signaling is inhibitory to T cell proliferation and effector functions. Earlier reported studies of fibroblasts showed that elevated levels of intracellular cAMP inhibit upstream signal transduction pathways involved in cell growth and differentiation (4, 5). Lular cAMP inhibit extracellular signal-regulated kinases 1 and 2 and c-Jun NH2-terminal kinases involved in the signal transduction of mitogen-activated protein kinase pathways, T cell extracellular signal-regulated kinases 1 and 2 are insensitive to elevated levels of intracellular cAMP (6). The cAMP-mediated inhibition of c-Jun NH2-terminal kinase in T cells shows delayed kinetics, an observation that correlates with the induction of the cAMP-inducible early repressor ICER1 (7). The notion that a newly synthesized transcriptional repressor rather than inhibition of upstream signal transduction pathways could be involved in the cAMP-mediated transcriptional attenuation of T helper-1 cytokine expression was further strengthened by the reported alleviation of cAMP-mediated inhibition of IL-2 expression in the presence of inhibitors of both RNA and protein synthesis (6). The highest affinity of ICER binding was found on a CD28-responsive element (CD28RE; Ϫ160 NFAT/AP-1 composite site) and Ϫ90 site, which is the motif in the IL-2 promoter that has striking sequence homology with the conserved proximal region (base pairs Ϫ73 to Ϫ48) of both the human and mouse promoters of the IFN␥ genes (21, 22)
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