Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression

Am J Physiol Gastrointest Liver Physiol. 2018 Mar 1;314(3):G309-G318. doi: 10.1152/ajpgi.00293.2017. Epub 2017 Nov 22.

Abstract

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 μM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

Keywords: 5-azacytidine; SLC9A3; deoxyribonucleic acid demethylation; deoxyribonucleic acid methyltransferase; growth arrest and deoxyribonucleic acid damage-inducible 45b.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antigens, Differentiation / genetics
  • Antigens, Differentiation / metabolism
  • Azacitidine / pharmacology
  • Caco-2 Cells
  • Colon / drug effects
  • Colon / metabolism*
  • DNA Methylation* / drug effects
  • DNA Modification Methylases / antagonists & inhibitors
  • DNA Modification Methylases / metabolism
  • Enzyme Inhibitors / pharmacology
  • Epigenesis, Genetic* / drug effects
  • Gene Expression Regulation
  • Humans
  • Ileum / drug effects
  • Ileum / metabolism*
  • Male
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Promoter Regions, Genetic
  • RNA Interference
  • Sodium-Hydrogen Exchanger 3 / genetics*
  • Sodium-Hydrogen Exchanger 3 / metabolism

Substances

  • Antigens, Differentiation
  • Enzyme Inhibitors
  • GADD45B protein, human
  • Gadd45b protein, mouse
  • SLC9A3 protein, human
  • Sodium-Hydrogen Exchanger 3
  • DNA Modification Methylases
  • Azacitidine