Signal transduction of T cell activation and regulation of IL-3 and GM-CSF gene expression

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The CLE2/GC-box (at positions -95 - -73, which is homologous to the NF-kB binding site) and CLE0 (at positions -54 - -40) of the mouse GM-CSF promoter are essential for transcriptional activation in response to phorbol 12-myristate 13 acetate (PMA) and calcium ionophore (A23187). GM-kB defines a binding site for a protein induced by PMA which is related to NF-kB.CLE0 binding proteins, which are responsive to PMA/calcium signal and also sensitive to cyclosporin A, is composed of AP1 and NFAT, a T-cell-specific trnascription factor. CLE0-like elements are found in the promoters of IL-3, IL-4, IL-5, as well as in the IL-2 and GM-CSF promoters. NFAT or related factors may account for the coordinate induction of these cytokine genes during T-cell activation.The CT/GC-rich sequence (at positions -76 to -57) of the human IL-3 gene is one of the upstream elements that mediate the response to HTLV-I Tax. We isolated cDNAs encoding DB1, a novel zinc-finger DNA binding protein, as well as previou … More sly described EGR1 and EGR2, all of which bind to this CT/GC-rich sequence. DB1 has six Cys_2/His_2-type zinc finger motifs and a glutamine-stretch which is often found in other transcription factors. Electrophoretic mobility shift assay (EMSA), using specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T cell activation-dependent manner. Forced expression of EGR1, EDR2, or DB1 proteins in Jurkat cells increased the transcription activity of the IL-3 promoter in the presence of upstream elements and T cell activation signals. These results suggest that these zinc finger proteins support the constitutive and inducible promoter activity of the IL-3 gene through the CT/GC-rich sequence.Mouse thymoma cell line EL-4 produces IL-5 as well as IL-2, IL-3, IL-4, IL-10 and GM-CSF in response to PMA.Although the production of IL-5 by PMA alone is very weak, co-stimulation with PMA and cAMP significantly increased the level of the IL-5 mRNA and the protein. Transient transfection assay employing EL-4 cells revealed that the IL-5 promoter was synergistically activated by PMA and cAMP.Co-transfection of the catalytic subunit of protein kinase A (PKA) mimicked cAMP response. These results indicated that IL-5 production upon T cell activation in vivo is mediated by a signal(s) that modulate cAMP-PKA pathway. Activation of the IL-5 promoter in response to cAMP and PMA was dependent on two distinct regions located at nucleotide positions -970 to -930 (region I) and -141 to -80 (region II). In contrast, cAMP almost completely inhibited the PMA-dependent activation of endogenous IL-2 and GM-CSF genes as well as that of the transfected IL-2 promoter. These results indicate that the IL-5 gene is positively regulated by cAMP in a manner opposite to IL-2 and GM-CSF genes in EL-4 cells. Less