Interaction of interferon regulatory factor-1 and nuclear factor kappaB during activation of inducible nitric oxide synthase transcription.
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.
Saura
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Division of Cardiology, Department of Medicine, School of Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD, 21205, USA.
Zaragoza
C
C
Bao
C
C
McMillan
A
A
Lowenstein
C J
CJ
D000818:Animals; D001665:Binding Sites; D002460:Cell Line / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D016328:NF-kappa B; D010750:Phosphoproteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse