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 initiation site is 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 reduced 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 carrying multiple copies of each element, we then found that the NF-AT construct was most effective in responding to PMA activation and to cAMP inhibition. Electrophoretic mobility shift assay revealed that, after exposure of cells to Bt2cAMP, NF-AT binding complex changed in amount or in mobility as a function of time. Furthermore, overexpression of the cytoplasmic component of NF-AT abrogated the inhibitory action of cAMP. These results indicate that the NF-AT site is a target of the inhibitory action of cAMP. In addition, binding of the NF-kappa B (p50/p65) heterodimer to the NF-kappa B site was inhibited by cAMP. Taken together, our data show that cAMP in EL-4 cells inhibits mouse IL-2 gene transcription through cis regulatory elements that include the NF-AT site as well as the NF-kappa B site.
Tsuruta
L
L
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Lee
H J
HJ
Masuda
E S
ES
Koyano-Nakagawa
N
N
Arai
N
N
Arai
K
K
Yokota
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection / Q000235:genetics
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D000242:Cyclic AMP