NF-κB Subunit-Specific Regulation of the Interleukin-8 Promoter

Abstract

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 α TNF-α. Several cis elements, including a binding site for the inducible transcription factor NF-κB, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-κB 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 κB site of the IL-8 promoter and was inhibited by addition of purified IκBα 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 κB binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the κB element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the κB/Rel family examined, the IL-8 κB 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 κB-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-κB 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 κB/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a κB-regulated promoter in which the classical NF-κB complex is unable to activate transcription from the κB element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression.