Synergistic transcriptional activation of the IL-8 gene by NF-kappa B p65 (RelA) and NF-IL-6.
Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Because adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region, we examined the functional interaction of these two transcription factor families in IL-8 gene regulation. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression.
Kunsch
C
C
Department of Molecular Biology, Human Genome Sciences, Rockville, MD 20850.
Lang
R K
RK
Rosen
C A
CA
Shannon
M F
MF
D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000008:administration & dosage* / Q000378:metabolism / Q000494:pharmacology; D004357:Drug Synergism; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000008:administration & dosage*; D009687:Nuclear Proteins / Q000008:administration & dosage* / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins