SYNERGISTIC TRANSCRIPTIONAL ACTIVATION OF THE IL-8 GENE BY NF-KAPPA-BP65 (RELA) AND NF-IL-6

Transcriptional activation of the IL-8 gene by several inflammatory me diators, including the cytokines IL-1 and TNF-alpha, is mediated throu gh sequences located between nucleotide -94 and -71 of the IL-8 promot er. Because adjacent binding sites for the inducible transcription fac tors NF-kappa B and NF-IL-6 are located within this region, we examine d the functional interaction of these two transcription factor familie s in IL-8 gene regulation. Maximal transcriptional activation by PMA i n Jurkat T lymphocytes was shown to require intact binding sites for b oth NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis in dicates 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 additi on, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds speci fically to the NF-kappa B site. When incubated together, RelA and NF-I L-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 a nd RelA. Transient cotransfection analyses indicate that the cooperati ve association of NF-IL-6 and RelA with the IL-8 promoter results in s ynergistic transcriptional activation. Mutational analyses of RelA dem onstrate that the C-terminal transactivation domain and the DNA bindin g domain are required for synergistic activation with NF-IL-6. In addi tion, 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 fam ily can functionally cooperate in transcriptional activation of the IL -8 gene and suggest a common mechanism for inducible regulation of cyt okine gene expression.