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 transcript ion of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-tr eated murine macrophages. We performed functional studies on the 5' fl anking region of the iNOS gene linked to a CAT reporter gene to identi fy the cis-acting element(s) responsible for the activation of iNOS tr anscription by picolinic acid. Transient transfection assays showed th at the full-length iNOS promoter in the murine macrophage cell line AN A-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 t o confer inducibility by picolinic acid but not by IFN-gamma or LPS. E lectrophoretic mobility shift assays confirmed that picolinic acid alo ne induced a specific binding activity to the iNOS-HRE. Furthermore, w e found that the INOS-HRE activity was inducible by hypoxia and that h ypoxia in combination with IFN-gamma activated the iNOS promoter in tr ansient transfection assays and induced iNOS transcription and mRNA ex pression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provi de the first evidence that iNOS is a hypoxia-inducible gene.