The human interleukin-6 (IL-6) promoter contains two regulatory elements, a kappa-B enhancer and a NFIL-6 (C/EBP-beta) binding site, which have been reported to be essential for inducibility of the IL-6 gene. We show that the kappa-B element alone is sufficient to confer inducibility on the IL-6 gene in cells treated with either IL-1-beta or TNF-alpha. Gel-retardation analysis of nuclear extracts from IL-1-beta or TNF-alpha-treated cells using specific antibodies has shown that at least rive retarded complexes bind to the IL-6-kappa-B element in addition to NF-kappa-B. Furthermore, apart from p50 (NF-kappa-B1) and p65 (RelA), no other members of the Rel family are present in these complexes. Comparative analysis with the kappa-B enhancer of the immunoglobulin kappa chain gene shows that three of these complexes bind specifically to the IL-6-kappa-B enhancer: a complex of p50/NFIL6, a p65 homodimer, and a non-Rel-related constitutive protein. Finally, transfection experiments, in which NF-kappa-B subunits, NFIL-6, and NFIL-6-beta (C/EBP-delta), were overexpressed in cells transfected with mutated IL-6 enhancer elements linked to a reporter gene show that interaction between members of the two families of factors is required for activation of the IL-6 gene in the absence of the NFIL-6 binding site. We conclude that the kappa-B enhancer of the IL-6 promoter is the IL-1-beta and TNF-alpha responsive element and that its activity is dependent on the direct interaction of NF-kappa-B with non-Rel transcription factors.
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