We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-alpha and IFN-gamma. Since TNF-alpha and IFN-gamma stimulate cells in part by activating NF-kappaB and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-kappaB co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that IRF-1 and NF-kappaB interact in stimulated but not resting cells. Super-shift experiments show that IRF-1 and NF-kappaB interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-kappaB proteins in vivo. We next suggested that this interaction between IRF-1 and NF-kappaB bends the DNA of the iNOS promoter region. Using a cyclization assay, we demonstrate that nuclear extracts from stimulated cells accelerate the rate of conversion of a linear to circular DNA, compared to extracts from resting cells. However, stimulated nuclear extracts cannot affect the rate of cyclization of a promoter with a mutant IRE or kappaB site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-kappaB cannot induce cyclization. We conclude that IRF-1 and NF-kappaB interact in vivo, and that this interaction physically bends the indicible nitric oxide synthase promoter DNA. This interaction may explain the mechanism by which IFN-gamma synergistically augments inducible nitric oxide synthase transcription.
Copyright 1999 Academic Press.