Transcriptional Regulation of SLC26A3 (DRA) by CDX2

Abstract

BackgroundSLC26A3 (Down Regulated in Adenoma, DRA) plays a key role in mammalian intestinal NaCl absorption via mediating apical membrane Cl−/HCO3− exchange. DRA function and expression is significantly decreased in diarrheal diseases associated with infections and inflammatory bowel diseases (IBD). Since, DRA is now being touted as an important therapeutic target for infectious and IBD associated diarrhea, it is critical to understand the detailed mechanisms of regulation of DRA expression at the molecular and cellular level in health and disease. Earlier studies showed that, the intestinal epithelial caudal‐related homeobox transcription factor 2 (CDX2) is an essential regulator of intestinal epithelial homeostasis and is also involved in differentiation. Interestingly, DRA expression is higher in differentiated cells and in silico analysis of DRA promoter showed 2 consensus sites for CDX2 binding.AimCurrent studies were, therefore, undertaken to test the hypothesis that CDX2 regulates DRA transcription.MethodsCdx2Fl/Fl;Villin‐CreERT2 mice, [where tamoxifen (TAM) deletes Cdx2 in intestine], were used for these studies and Caco2 cells were used as an in vitro model to study the overexpression and silencing effects of CDX2 by transfecting the cDNA and siRNA, respectively. mRNA and protein levels were assessed by real time RT‐PCR and immunoblotting/immuno‐staining, respectively. DRA promoter activity was determined by luciferase assay. Direct binding of CDX2 to DRA promoter region was assessed by chromatin immuno‐precipitation (CHIP) and electrophoretic mobility shift assay (EMSA).ResultsCDX2− /− mice showed marked reduction in DRA mRNA in ileum (Arbitrary units: WT=2.73 ± 1.04, KO=0.029 ± 0.02 (P<0.05), proximal colon (PC) WT=1.87 ± 0.53, KO=0.51 ± 0.08; P <0.05) and distal colon (DC) (WT=1.75 ± 0.46, KO=0.02 ±0.02, P<0.05) as well as in protein levels as compared to wild type mice. Similarly, silencing of CDX2 in Caco2 cells resulted in a marked (~ 50%) decrease in DRA mRNA and protein levels, whereas, ectopic over‐expression of CDX2 significantly up‐regulated DRA expression. CDX2 overexpression also stimulated DRA promoter activity by ~10‐fold, indicating its role in transcriptional regulation. We next utilized EMSA and ChIP assays to demonstrate direct binding of CDX2 to one putative cis elements identified in the 645 to 663 (+) region of the DRA promoter.ConclusionOur studies, for the first time, demonstrate a direct transcriptional regulation of DRA by CDX2. Given that CDX2 has been shown to be downregulated in UC (ulcerative colitis) and CD (crohn's disease) patients, CDX2 may at least partly be responsible for the observed decrease in DRA expression in IBD.Support or Funding InformationSupported by NIDDK/Department of Veteran Affairs