Tumor necrosis factor‐α represses the expression of NHE2 through NF‐κB activation in intestinal epithelial cell model, C2BBe1

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. (Inflamm Bowel Dis 2011;)