The κB Enhancer of the Human Interleukin-6 Promoter Is Necessary and Sufficient to Confer an IL-1β and TNF-α Response in Transfected Human Cell Lines: Requirement for Members of the C/EBP Family for Activity

Abstract

The human interleukin-6 (IL-6) promoter contains two regulatory elements, a κB enhancer and a NFIL-6 (C/EBPβ) binding site, which have been reported to be essential for inducibility of the IL-6 gene. We show that the κB element alone is sufficient to confer inducibility on the IL-6 gene in cells treated with either IL-1β or TNF-α. Gel-retardation analysis of nuclear extracts from IL-1β or TNF-α-treated cells using specific antibodies has shown that at least five retarded complexes bind to the IL-6κB element in addition to NF-κB. Furthermore, apart from p50 (NF-κB1) and p65 (RelA), no other members of the Rei family are present in these complexes. Comparative analysis with the κB enhancer of the immunoglobulin κ chain gene shows that three of these complexes bind specifically to the IL-6κB enhancer: a complex of p50/NFIL6, a p65 homodimer, and a non-Rel-related constitutive protein. Finally, transfection experiments, in which NF-κB subunits, NFIL-6, and NFIL-6β (C/EBPδ), 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 κB enhancer of the IL-6 promoter is the IL-1β and TNF-α responsive element and that its activity is dependent on the direct interaction of NF-κB with non-Rel transcription factors.