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Cytokines and bacterial lipopolysaccharides (LPSs) stimulate nitric oxide production in macrophages by inducing transcription of the gene coding for the inducible isoform of nitric oxide synthase (iNOS). We have cloned the mouse iNOS gene promoter and analysed its structural features and its response to interferon-gamma (IFN-gamma) and Escherichia coli LPS in RAW 264.7 mouse macrophage-like cells. Transcription of a recombinant reporter gene including the promoter and 4 kb of its 5'-flanking DNA, linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, is stimulated by IFN-gamma and, more efficiently, by LPS upon transient transfection in RAW 264.7 cells. Two upstream DNA regions are required for maximal promoter activation of LPS: the first maps between positions -1541 and -775 and the other between -420 and -47, with respect to the major transcriptional start site of the iNOS gene. The upstream-most region also mediates promoter trans-activation by IFN-gamma. As reported earlier for transcription of the endogenous iNOS gene, combined stimulation of RAW 264.7 cells with IFN-gamma and LPS results in lower activation of the transfected promoter, when compared with LPS alone. NG-Monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase activity, enhances iNOS gene mRNA induction and promoter activation by IFN-gamma and LPS, indicating that nitric oxide can influence negatively the reponsiveness of this gene to inducers. These results suggest the possibility of a negative regulatory feedback exerted by iNOS on the transcriptional activation of its own gene.

biochemical journal

Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-<i>γ</i>, bacterial lipopolysaccharide and <i>N</i>G-monomethyl-<scp>l</scp>-arginine

Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-γ, bacterial lipopolysaccharide and NG-monomethyl-l-arginine