A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter

Giovanni Melillo, Tiziana Musso, Antonio Sica, Lynn S. Taylor, George W. Cox, Luigi Varesio

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Abstract

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-γ-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-γ 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-γ 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 to combination with IFN-γ activated the iNOS promoter in transient transfection assays and induced iNOS transcription an 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.

Original languageEnglish
Pages (from-to)1683-1693
Number of pages11
JournalJournal of Experimental Medicine
Volume182
Issue number6
DOIs
Publication statusPublished - Dec 1 1995

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Nitric Oxide Synthase Type II
Genes
Hypoxia
Macrophages
Reporter Genes
Transfection
Sequence Deletion
5' Flanking Region
Electrophoretic Mobility Shift Assay
Sequence Homology
Genetic Promoter Regions
Tryptophan
picolinic acid

ASJC Scopus subject areas

  • Immunology

Cite this

A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. / Melillo, Giovanni; Musso, Tiziana; Sica, Antonio; Taylor, Lynn S.; Cox, George W.; Varesio, Luigi.

In: Journal of Experimental Medicine, Vol. 182, No. 6, 01.12.1995, p. 1683-1693.

Research output: Contribution to journalArticle

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