CYCLIC-AMP INHIBITS EXPRESSION OF THE IL-2 GENE THROUGH THE NUCLEAR FACTOR OF ACTIVATED T-CELLS (NF-AT) SITE, AND TRANSFECTION OF NF-AT CDNAS ABROGATES THE SENSITIVITY OF EL-4 CELLS TO CYCLIC-AMP

cAMP inhibits PMA-induced IL-2 production at the transcriptional level in EL-4, a mouse lymphoma line. The region of the mouse IL-2 promoter covering positions from -321 to +46 relative to the transcription ini tiation site ip required for activation by PMA and inhibition by cAMP. This region contains the nuclear factor of activated T cells (NF-AT), nuclear factor-kappa B (NF-kappa B), AP-1, and Oct binding sites, and the role of each element in responding to PMA and/or cAMP signals was characterized. The IL-2 promoter carrying mutations in each element r educed response to PMA while it retained sensitivity to cAMP, thereby suggesting that multiple elements contribute to positive and negative responses to PMA and cAMP, respectively. Using reporter plasmid carryi ng multiple copies of each element, we then found that the NF-AT const ruct was most effective in responding to PMA activation and to cAMP in hibition. Electrophoretic mobility shift assay revealed that, after ex posure of cells to Bt(2)cAMP, NF-AT binding complex changed in amount or in mobility as a function of time. Furthermore, overexpression of t he cytoplasmic component of NF-AT abrogated the inhibitory action of c AMP. These results indicate that the NF-AT site is a target of the inh ibitory action of cAMP. In addition, binding of the NF-kappa B (p50/p6 5) heterodimer to the NF-kappa B site was inhibited by cAMP. Taken tog ether, our data show that cAMP in EL-4 cells inhibits mouse IL-2 gene transcription through cis regulatory elements that include the NF-AT s ite as well as the NF-kappa B site.