Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-kappa B binding at the transcription start site

Inflammatory cytokines cause the down-regulation of multiple cytochrome P45 0 mRNAs, but the transcriptional mechanisms involved are not known. We inve stigated the role of a putative negative NF-kappa B-responsive element, n k appa B-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 extrac ts from livers of rats treated with bacterial lipopolysaccharide, or from h epatocytes treated with interleukin-1 beta, formed a protein complex with a n oligonucleotide probe containing the n kappa B-RE1, and that this complex contained predominantly the p50 subunit of NF-kappa B. Binding of NF-kappa B to the n kappa B-RE1 probe was of lower affinity than to a probe contain ing the prototypic NF-kappa B enhancer of the immunoglobulin kappa chain ge ne. Mutations in the 5'-end of the n kappa B-RE1, and to a lesser extent th e 3'-end, reduced the affinity of NF-kappa B for this element. Introduction of the 5'-mutation into n kappa B-RE1 abolished the response of the -200-C YP2C11-chloramphenicol acetyltransferase reporter construct to interleukin- 1 or lipopolysaccharide. We conclude that n kappa B-RE1 is a functional neg ative regulatory element that participates in the inflammatory suppression of CYP2C11. (C) 2000 Academic Press.