Suppression of CYP2C11 Gene Transcription by Interleukin-1 Mediated by NF-κB Binding at the Transcription Start Site

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-κB-responsive element, nκB-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-1β, formed a protein complex with an oligonucleotide probe containing the nκB-RE1, and that this complex contained predominantly the p50 subunit of NF-κB. Binding of NF-κB to the nκB-RE1 probe was of lower affinity than to a probe containing the prototypic NF-κB enhancer of the immunoglobulin κ chain gene. Mutations in the 5′-end of the nκB-RE1, and to a lesser extent the 3′-end, reduced the affinity of NF-κB for this element. Introduction of the 5′-mutation into nκB-RE1 abolished the response of the −200-CYP2C11-chloramphenicol acetyltransferase reporter construct to interleukin-1 or lipopolysaccharide. We conclude that nκB-RE1 is a functional negative regulatory element that participates in the inflammatory suppression of CYP2C11.