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Epigenetic Modulation of Intestinal NHE3 Expression by DNA Methylation

NHE3 plays an important role in intestinal Na+ absorption and its down‐regulation has been implicated in inflammatory diarrhea. Recent evidence also indicates a novel role of epigenetic mechanisms, such as DNA methylation in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. To address this, methyl‐CpG binding domain‐based capture assay was performed in Caco2 cells treated with the pro‐inflammatory cytokines. IL6 (100 ng/ml) or IL1β (50 ng/ml) for 48h increased DNA methylation at the NHE3 gene promoter, indicating that DNA methylation may contribute to the decrease in NHE3 expression during intestinal inflammation. In parallel, in vitro methylation of NHE3 promoter (p‐1507/+131) cloned into a CpG free lucia vector decreased the promoter activity. Conversely, DNA methyltransferase (DNMT) inhibitor, 5‐azacytidine (10 μM, 24h) caused a significant decrease in DNA methylation of NHE3 gene. Also, 5‐aza increased NHE3 expression in Caco2 & duodenal HUTU‐80 cells by ~2‐3 fold (p<0.05). Treatment of mice with 5‐aza for 3 wks resulted in an increase in NHE3 expression in the ileum and colon (~2 fold, p<0.05). Further, the role of the demethylase GADD45β was examined. siRNA knockdown of GADD45β in Caco2 cells decreased NHE3 mRNA expression by ~50% (p<0.05). Immunostaining showed a decrease in NHE3 expression in the ileum & colon of GADD45β ‐/‐ mice. These findings suggest that changes in DNA methylation may be involved in the inhibition of NHE3 gene expression in intestinal inflammation contributing to the pathophysiology of IBD‐associated diarrhea. (Supported by VA/NIH)