Inflammatory cytokines cause the down-regulation of multiple cytochrome P450 mRNAs, but the transcriptional mechanisms involved are not known. We investigated the role of a putative negative NF-kappaB-responsive element, nkappaB-RE1, in the down-regulation of the CYP2C11 gene in rat hepatocytes. This sequence spans the transcription start site of CYP2C11, from positions -2 to +8. Electrophoretic mobility shift assays showed that nuclear extracts from livers of rats treated with bacterial lipopolysaccharide, or from hepatocytes treated with interleukin-1beta, formed a protein complex with an oligonucleotide probe containing the nkappaB-RE1, and that this complex contained predominantly the p50 subunit of NF-kappaB. Binding of NF-kappaB to the nkappaB-RE1 probe was of lower affinity than to a probe containing the prototypic NF-kappaB enhancer of the immunoglobulin kappa chain gene. Mutations in the 5'-end of the nkappaB-RE1, and to a lesser extent the 3'-end, reduced the affinity of NF-kappaB for this element. Introduction of the 5'-mutation into nkappaB-RE1 abolished the response of the -200-CYP2C11-chloramphenicol acetyltransferase reporter construct to interleukin-1 or lipopolysaccharide. We conclude that nkappaB-RE1 is a functional negative regulatory element that participates in the inflammatory suppression of CYP2C11.
Copyright 2000 Academic Press.