Interaction of interferon regulatory factor-1 and nuclear factor kappa B during activation of inducible nitric oxide synthase transcription

We investigated the molecular mechanism for the synergistic induction of in ducible nitric oxide synthase transcription by TNF-alpha and IFN-gamma. Sin ce TNF-alpha and IFN-gamma stimulate cells in part by activating NF-kappa B and IRF-1, we hypothesized that these two transcription factors interact w ith each other. IRF-1 and NF-kappa B co-localize in the nucleus of stimulat ed macrophages. Co-immunoprecipitation experiments show that LRF-1 and NF-k appa B interact in stimulated but not resting cells. Super-shift experiment s show that LRF-1 and NF-kappa B interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical i nteraction between IRF-1 and NF-kappa B proteins in vivo. We next suggested that this interaction between IRF-1 and NF-kappa B bends the DNA of the iN OS promoter region. Using a cyclization assay, we demonstrate that nuclear extracts from stimulated cells accelerate the rate of conversion of a linea r to circular DNA, compared to extracts from resting cells. However, stimul ated nuclear extracts cannot affect the rate of cyclization of a promoter w ith a mutant IRE or kappa B site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-kappa B cannot induce cyclization. We conclude tha t IRF-1 and NF-kappa B interact in vivo, and that this interaction physical ly bends the indicible nitric oxide synthase promoter DNA. This interaction may explain the mechanism by which IFN-gamma synergistically augments indu cible nitric oxide synthase transcription. (C) 1999 Academic Press.