Transcriptional inhibition of the interleukin-8 gene by interferon is mediated by the NF-kappa B site.
The cytokine interleukin-8 (IL-8) is an important mediator of neutrophil, lymphocyte, and basophil chemotaxis and activation. Earlier we demonstrated that beta interferon (IFN-beta) can inhibit tumor necrosis factor (TNF)-induced IL-8 gene expression at the transcriptional level, apparently by a novel mechanism. To define the cis-acting elements and trans-acting factors involved in this inhibition, DNA constructs containing portions of the 5'-flanking region of the IL-8 gene were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into human diploid FS-4 fibroblasts. The region spanning positions -98 to +44 was sufficient to confer both inducibility by TNF and inhibition by simultaneous treatment with IFN-beta. Inhibition of TNF- or IL-1-induced CAT activity by IFN-beta or IFN-alpha was also observed when a DNA fragment containing only the NF-IL-6 and NF-kappa B sites (positions -94 to -70) was placed upstream of the homologous or a heterologous minimal promoter. A construct containing three copies of the NF-kappa B element in front of the CAT gene also was inducible by TNF, and this stimulatory effect too was inhibited by IFN-beta, indicating that the NF-kappa B element is sufficient to confer inhibition by IFN-beta. This inhibitory effect was specific for the NF-kappa B site of the IL-8 gene since it was less marked with constructs containing three copies of the NF-kappa B site from the HLA-B7 gene. Gel shift assays with a probe containing the NF-kappa B and NF-IL-6 binding sites of the IL-8 gene (positions -101 to -63) showed that IFN-beta treatment did not block the activation of NF-kappa B proteins or their ability to bind to the NF-kappa B site. However, nuclear extracts from cells treated with TNF in the presence of IFN-beta gave rise to an additional band that appears to contain protein components from the NF-kappa B and NF-IL-6 families. NF-kappa B site-mediated suppression of IL-8 gene expression by IFN-beta represents a hitherto unknown mechanism and target of IFN action.
Department of Microbiology, New York University Medical Center, New York 10016.
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Oliveira
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Mukaida
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Matsushima
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Vilcek
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology; D016899:Interferon-beta / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D007375:Interleukin-1; D016209:Interleukin-8; D046911:Macromolecular Substances; D016328:NF-kappa B; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016899:Interferon-beta; D007371:Interferon-gamma