title: TNF-alpha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells.
abstract: OBJECTIVE
  NOD2, a member of the NOD1/Apaf-1 family, was recently identified as the first susceptibility gene for Crohn's disease. The aim of this report was to describe the regulation and functional significance of NOD2 expression in intestinal epithelial cells.
  
  
  METHODS
  Expression of NOD2 messenger RNA was determined by reverse-transcription polymerase chain reaction (RT-PCR); NOD2 protein was detected by Western blot. Promoter activity was assessed by reporter gene assays and DNA-binding of NF-kappaB by electrophoretic mobility shift assays. IL-8 production was investigated by RT-PCR and enzyme-linked immunosorbent assay.
  
  
  RESULTS
  TNF-alpha induced an up-regulation of NOD2 in epithelial cell lines (HT-29, SW620, SW948, HeLa S3) and in primary colonic epithelial cells. A synergism was seen by cotreatment with IFN-gamma. Two NF-kappaB binding sites were identified in the promoter. Deletion of either site or overexpression of dominant negative IkappaBalpha led to reduced levels of TNF-alpha/IFN-gamma-stimulated reporter gene activity. The identified kappaB3 site was bound by NF-kappaB as determined by gelshift assays. Elevated amounts of NOD2 protein were also found in colonic epithelial cells from patients with IBD. LPS induced high levels of IL-8 production in SW620 cells overexpressing NOD2.
  
  
  CONCLUSIONS
  TNF-alpha(/IFN-gamma) treatment up-regulates the expression of the NOD2 gene in intestinal epithelial cells and subsequently increases their LPS susceptibility. Together with the mutation-derived truncation and functional change of the NOD2 protein, this could be part of the complex pathophysiology of barrier disruption as it is observed in inflammatory bowel diseases.
authors.affiliation: 1st Department of Medicine, Christian-Albrechts-University Kiel, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstiel
authors.forename: Massimo
authors.initials: M
authors.lastname: Fantini
authors.forename: Karen
authors.initials: K
authors.lastname: Bräutigam
authors.forename: Tanja
authors.initials: T
authors.lastname: Kühbacher
authors.forename: Georg H
authors.initials: GH
authors.lastname: Waetzig
authors.forename: Dirk
authors.initials: D
authors.lastname: Seegert
authors.forename: Stefan
authors.initials: S
authors.lastname: Schreiber
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000235:genetics*; D004357:Drug Synergism; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D016209:Interleukin-8 / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D002352:Carrier Proteins; D016209:Interleukin-8; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma