Both basal and enhancer κB elements are required for full induction of the mouse inducible nitric oxide synthase gene

The transcriptional regulatory region of the mouse inducible nitric oxide synthase (iNOS) gene has two KB elements, one enhancer-linked (KBII) and the other (KBI) proximal to its core promoter. Mutation of κBII substantially reduced the extent to which the iNOS promoter could be induced by LPS and interfered with augmented responsiveness of the promoter to LPS+IFN-γ. Mutation of KBI had a quantitatively less dramatic negative effect on LPS responsiveness and this construct still showed augmented responsiveness to LPS+IFN-γ. When both KB elements were mutated, inducibility by LPS and, in particular, by LPS+IFN-γ was paradoxically restored, compared with the mutated KBII alone, suggesting cooperative interactions among the transcription factors that trans-activate the iNOS gene. In vivo footprint analysis showed that both KB elements were bound by protein complexes when macrophages were stimulated with LPS ± IFN-γ. Furthermore, KBI was bound even in untreated cells, suggesting that KB binding proteins might also have a negative influence on expression of the gene. Both KBI and KBII were bound by NF-KB/Rel proteins found in nuclear extracts prepared from macrophages treated with LPS ± IFN-γ, although the specificity of binding to each element was different. Our results show that, while NF-KB/Rel proteins are required for maximal expression of the iNOS gene, alone they are not alone sufficient. Furthermore, the results reported here show that the augmentative effect of IFN-γ on the LPS-induced expression of the iNOS gene is not mediated through increased activation of NF-KB/Rel.