Tumor necrosis factor-α represses the expression of NHE2 through NF-κB activation in intestinal epithelial cell model, C2BBe1
✍ Scribed by Md. Ruhul Amin; Temitope Orenuga; Sangeeta Tyagi; Pradeep K. Dudeja; Krishnamurthy Ramaswamy; Jaleh Malakooti
- Publisher
- John Wiley and Sons
- Year
- 2011
- Tongue
- English
- Weight
- 974 KB
- Volume
- 17
- Category
- Article
- ISSN
- 1078-0998
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✦ Synopsis
Background: High levels of proinflammatory cytokines are linked to pathogenesis of diarrhea in inflammatory bowel disease (IBD). Na þ absorption is compromised in IBD. The studies were designed to determine the effect of tumor necrosis factor-a (TNFa) on the expression and activity of NHE2, a Na þ /H þ exchanger (NHE) that is involved in transepithelial Na þ absorption in intestinal epithelial cells.
Methods: NHE2 regulation was examined in TNF-a-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 22 Na-uptake. In vitro and in vivo protein-DNA interactions were assessed by gel mobility shift assays and chromatin immunoprecipitation studies.
Results: TNF-a 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 0 -deletion analysis of the NHE2 promoter-reporter constructs identified basepair À621 to À471 as the TNF-a-responsive region (TNF-RE). TNF-a activated NF-jB subunits, p50 and p65, and their DNA-binding to a putative NF-jB motif within TNF-RE. Mutations in the NF-jB motif abolished NF-jB-DNA interactions and abrogated TNFa-induced repression. Ectopic overexpression of NF-jB resulted in repression of NHE2 expression. Two functionally distinct inhibitors of NF-jB blocked the inhibitory effect of TNF-a.
Conclusions:
The human NHE2 isoform is a direct target of transcription factor NF-jB. TNF-a-mediated activation of NF-jB decreases the expression and activity of NHE2 in the intestinal epithelial cell line, C2BBe1. These findings implicate NF-jB in the modulation of Na þ absorption during intestinal inflammatory conditions such as IBD where a high level of TNF-a is detected.