A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.
Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid- but not LPS-induced CAT activity by more than 70%. Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-gamma or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRE. Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-gamma activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004357:Drug Synergism; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D000860:Hypoxia / Q000235:genetics*; D007371:Interferon-gamma / Q000008:administration & dosage*; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics*; D010848:Picolinic Acids / Q000008:administration & dosage*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D010848:Picolinic Acids; D012333:RNA, Messenger; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; C030614:picolinic acid