Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-kappaB 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-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.
Iber
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Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Chen
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Cheng
P Y
PY
Morgan
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ET
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D016384:Consensus Sequence / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects*; D007375:Interleukin-1 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D020218:Response Elements / Q000235:genetics; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics
D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase