NF-kappa B subunit-specific regulation of the interleukin-8 promoter.
Interleukin-8 (IL-8), a chemotactic cytokine for T lymphocytes and neutrophils, is induced in several cell types by a variety of stimuli including the inflammatory cytokines IL-1 and tumor necrosis factor alpha TNF-alpha. Several cis elements, including a binding site for the inducible transcription factor NF-kappa B, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-kappa B subunits to bind to, and activate transcription from, the IL-8 promoter. A nuclear complex was induced in phorbol myristate acetate-treated Jurkat T cells which bound specifically to the kappa B site of the IL-8 promoter and was inhibited by addition of purified I kappa B alpha to the reaction mixture. Only antibody to RelA (p65), but not to NFKB1 (p50), NFKB2 (p50B), c-Rel, or RelB was able to abolish binding, suggesting that RelA is a major component in these kappa B binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the kappa B element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the kappa B/Rel family examined, the IL-8 kappa B site bound only to RelA and to c-Rel and NFKB2 homodimers, but not to NFKB1 homodimers or heterodimers of NFKB1-RelA. Transient transfection analysis demonstrated a kappa B-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-kappa B subunits indicated that RelA and c-Rel, but neither NFKB1 nor heterodimeric NFKB1-RelA, was able to activate transcription from the IL-8 promoter. Furthermore, cotransfection of NFKB1 and RelA, although able to support activation from the human immunodeficiency virus type 1 long terminal repeat, failed to activate expression from the IL-8 promoter. Antisense oligonucleotides to RelA, but not NFKB1, inhibited phorbol myristate acetate-induced IL-8 production in Jurkat T lymphocytes. These data demonstrate the differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a kappa B-regulated promoter in which the classical NF-kappa B complex is unable to activate transcription from the kappa B element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression.
Kunsch
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Human Genome Sciences, Rockville, Maryland 20850.
Rosen
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CA
D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D016325:HIV Long Terminal Repeat; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051996:Transcription Factor RelA; D015533:Transcriptional Activation
D016209:Interleukin-8; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D051996:Transcription Factor RelA; D004247:DNA; D013755:Tetradecanoylphorbol Acetate