title: Interferon-gamma augments CARD4/NOD1 gene and protein expression through interferon regulatory factor-1 in intestinal epithelial cells.
abstract: Although intestinal epithelial cells appear to be functionally hyporesponsive to normal intestinal flora, human intestinal epithelial cells can respond to enteroinvasive bacteria and induce an inflammatory response. This initial inflammatory response leads to the recruitment of polymorphonuclear leukocytes to the affected site in vitro and in vivo. CARD4/NOD1 is a potential cytosolic receptor for peptidoglycan in mammalian cells that resembles pathogen-resistant proteins of plants. In this context, CARD4/NOD1 is a candidate for a recognition protein of intracellular bacteria or peptidoglycan in intestinal epithelial cells. In this study, we demonstrate that CARD4/NOD1 is constitutively expressed in intestinal epithelial cell lines and isolated primary intestinal epithelial cells. Interferon-gamma (IFN gamma), which is a potent pro-inflammatory cytokine in intestinal mucosal inflammation, activates CARD4/NOD1 mRNA transcription in a time- and dose-dependent manner and results in augmentation of CARD4/NOD1 protein in SW480 cells. Promoter analysis of CARD4/NOD1 indicates that interferon regulatory factor-1 (IRF-1) binding motif (-791 to -782) is essential for the effect of IFN gamma. Nuclear extracts from SW480 cells treated with IFN gamma show specific binding of oligonucleotides corresponding to this IRF-1-binding motif, which was supershifted by anti-IRF-1 antibody in electrophoretic mobility shift assay. Overexpression of IRF-1 protein activates the CARD4/NOD1 promoter but not the deletion mutant of the IRF-1-binding site in a co-transfection assay of IRF-1 expression plasmid with CARD4/NOD1 promoter. These studies suggest that the Th1 cytokine, IFN gamma, activates CARD4/NOD1 transcription and regulate innate immune mechanisms in the condition of intestinal mucosal inflammation.
authors.affiliation: Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Tadakazu
authors.initials: T
authors.lastname: Hisamatsu
authors.forename: Manabu
authors.initials: M
authors.lastname: Suzuki
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Podolsky
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D001419:Bacteria / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology*; D003106:Colon / Q000473:pathology; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009092:Mucous Membrane / Q000378:metabolism; D053474:Nod1 Signaling Adaptor Protein; D010457:Peptidoglycan / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C119067:NOD1 protein, human; D053474:Nod1 Signaling Adaptor Protein; D010457:Peptidoglycan; D010750:Phosphoproteins; D012333:RNA, Messenger; D007371:Interferon-gamma; D008156:Luciferases