Interaction of Interferon Regulatory Factor-1 and Nuclear Factor κB during activation of inducible nitric oxide synthase transcription

Abstract

We investigated the molecular mechanism for the synergistic induction of inducible nitric oxide synthase transcription by TNF-α and IFN-γ. Since TNF-α and IFN-γ stimulate cells in part by activating NF-κB and IRF-1, we hypothesized that these two transcription factors interact with each other. IRF-1 and NF-κB co-localize in the nucleus of stimulated macrophages. Co-immunoprecipitation experiments show that IRF-1 and NF-κB interact in stimulated but not resting cells. Super-shift experiments show that IRF-1 and NF-κB interact while binding to their respective DNA binding sites. These results demonstrate the existence of a physical interaction between IRF-1 and NF-κB proteins in vivo. We next suggested that this interaction between IRF-1 and NF-κB 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 κB site. Furthermore, stimulated nuclear extracts depleted of IRF-1 and NF-κB cannot induce cyclization. We conclude that IRF-1 and NF-κB 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-γ synergistically augments inducible nitric oxide synthase transcription.