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-alpha (TNF-alpha) 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-alpha-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-amilorideand HOE694-sensitive Na-22-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies. Results: TNF-alpha treatment of C2BBe1 cells led to repression of NHE2 promoter activity, mRNA, and protein levels; and inhibited both NHE2 and NHE3 mediated 22Na-uptake. 5`-deletion analysis of the NHE2 promoter-reporter constructs identified basepair -621 to -471 as the TNF-alpha-responsive region (TNF-RE). TNF-alpha activated NF-kappa B subunits, p50 and p65, and their DNA-binding to a putative NF-kappa B motif within TNF-RE. Mutations in the NF-kappa B motif abolished NF-kappa B-DNA interactions and abrogated TNF-alpha-induced repression. Ectopic overexpression of NF-kappa B resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-kappa B blocked the inhibitory effect of TNF-alpha. Conclusions: The human NHE2 isoform is a direct target of transcription factor NF-kappa B. TNF-alpha-mediated activation of NF-kappa B decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF-kappa B in the modulation of Na+ absorption during intestinal inflammatory conditions such as IBD where a high level of TNF-alpha is detected.