title: Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1.
abstract: BACKGROUND
  High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel disease (IBD). Na(+) absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-α (TNF-α) on the expression and activity of NHE2, a Na(+) /H(+) exchanger (NHE) that is involved in transepithelial Na(+) absorption in intestinal epithelial cells.
  
  
  METHODS
  NHE2 regulation was examined in TNF-α-treated C2BBe1 cells by reverse-transcription polymerase chain reaction (RT-PCR), reporter gene assays, and Western blot analysis. NHE isoform activities were measured as ethyl-isopropyl-amiloride- and HOE694-sensitive (22) Na-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies.
  
  
  RESULTS
  TNF-α treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA, and protein levels; and inhibited both NHE2 and NHE3 mediated (22) Na-uptake. 5'-deletion analysis of the NHE2 promoter-reporter constructs identified basepair -621 to -471 as the TNF-α-responsive region (TNF-RE). TNF-α activated NF-κB subunits, p50 and p65, and their DNA-binding to a putative NF-κB motif within TNF-RE. Mutations in the NF-κB motif abolished NF-κB-DNA interactions and abrogated TNF-α-induced repression. Ectopic overexpression of NF-κB resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-κB blocked the inhibitory effect of TNF-α.
  
  
  CONCLUSIONS
  The human NHE2 isoform is a direct target of transcription factor NF-κB. TNF-α-mediated activation of NF-κB decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF-κB in the modulation of Na(+) absorption during intestinal inflammatory conditions such as IBD where a high level of TNF-α is detected.
authors: Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, USA.
authors: Md Ruhul
authors: MR
authors: Amin
authors: Temitope
authors: T
authors: Orenuga
authors: Sangeeta
authors: S
authors: Tyagi
authors: Pradeep K
authors: PK
authors: Dudeja
authors: Krishnamurthy
authors: K
authors: Ramaswamy
authors: Jaleh
authors: J
authors: Malakooti
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D008156:Luciferases / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D016328:NF-kappa B; D012333:RNA, Messenger; D012097:Repressor Proteins; C081517:SLC9A2 protein, human; D017923:Sodium-Hydrogen Exchangers; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases