The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1.
The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.
Banerji
S S
SS
Department of Molecular Immunology, Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Parsons
J N
JN
Tocci
M J
MJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007376:Interleukin-2 / Q000235:genetics*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects; D012190:Retroviridae / Q000187:drug effects / Q000235:genetics; D016385:TATA Box / Q000187:drug effects; D016559:Tacrolimus; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D003208:Concanavalin A; D015759:Ionomycin; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate; D016559:Tacrolimus