Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-alpha, IFN-gamma, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4 alpha (HNF-4 alpha) binding site that is evolutionarily conserved in several species suggesting that HNF-4 alpha has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4 alpha-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4 alpha are not known. We investigated the regulation of NHE3 gene expression by HNF-4 alpha in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4 alpha-null (Hnf4 alpha(Delta IEpC)) and control (Hnf4 alpha(fl/fl)) mice. HNF-4 alpha knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4 alpha directly interacts with the HNF-4 alpha motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4 alpha motif decreased, whereas ectopic overexpression of HNF-4 alpha increased, NHE3 promoter activity. Furthermore, loss of HNF-4 alpha in Hnf4 alpha(Delta IEpC) mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4 alpha in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4 alpha directly regulates NHE3 promoter activity and its basal expression in the intestine.