The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization.
FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13-acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the IL-8 promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (c-Rel, p65, p50, and p49). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which NF-kappa B binding was also decreased by FK506, indicating that both IL-8 kappa B-like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization.
Okamoto
S
S
Department of Pharmacology, Kanazawa University, Japan.
Mukaida
N
N
Yasumoto
K
K
Rice
N
N
Ishikawa
Y
Y
Horiguchi
H
H
Murakami
S
S
Matsushima
K
K
D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D015458:Leukemia, T-Cell; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D017384:Sequence Deletion; D013489:Suppression, Genetic; D013601:T-Lymphocytes / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D016209:Interleukin-8; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D015759:Ionomycin; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016559:Tacrolimus