Mechanisms of transcriptional modulation of the human anion exchanger SLC26A3 gene expression by IFN-γ

Abstract

Two members of the SLC26 gene family, SLC26A3 or DRA (downregulated in adenoma) and SLC26A6 (putative anion transporter 1, PAT1), are known to play a major role in apical Cl(-)/OH(-) (HCO(3)(-)) exchange process in the human intestine. We have previously shown the inhibitory effects of IFN-gamma (30 ng/ml, 24 h) on both SLC26A3 and A6 expression and promoter activity. We also demonstrated that the effects of IFN-gamma on SLC26A6 gene expression were mediated via IRF-1 transcription factor. However, the molecular mechanisms underlying the transcriptional modulation of SLC26A3 gene expression by IFN-gamma in the intestine are not known. The present studies were, therefore, designed to elucidate the signaling mechanisms and transcription factor(s) involved in mediating the inhibitory effects of IFN-gamma on DRA promoter (p--1183/+114) activity. Deletion analysis indicated that the IFN-gamma response element is located within the -1183 to -790 region, and sequence analysis of this region revealed the presence of potential gamma-activated site (GAS), a binding site (-933/-925 bp) for signal transducer and activator of transcription factor 1 (STAT1). Mutations in the potential GAS element abrogated the inhibitory effects of IFN-gamma. These studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3 gene expression by IFN-gamma in the human intestine.

Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells

“…Previous studies from our laboratory have shown the transcriptional regulation of DRA gene (2,14,39,41,43). In the present study our data show for the first time a novel regulatory pathway of down regulation of DRA by miR-494 via mechanisms involving translational repression through its interactions with the DRA mRNA 3=-UTR.…”

Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice

“…For flux experiments, Caco-2 cells grown in modified Eagle's medium (40) were plated on 12-well Transwell collagen-coated inserts (43) and used at day 21 postplating. Cells were treated from the apical side with L. acidophilus, L. rhamnosus, or L. casei CS diluted in a ratio of 1:10 or 1:50 in serum-free cell culture medium for 24 h. In separate sets of experiments, cells were pretreated with the specific PI3K inhibitor LY-294002 (50 M) or ERK1/2 MAPK inhibitor PD-98059 (30 M) for 1 h and then coincubated with L. acidophilus or L. rhamnosus CS diluted in a ratio of 1:10 in serum-free cell culture medium for another 24 h.…”

“…Caco-2 cells were transfected with MDR1 promoter fragment cloned upstream of the luciferase reporter gene in pGL2-basic and ␤-galactosidase expression vectors by electroporation using the Amaxa Nucleofactor System, as described previously (43). MDR1 promoter activity was expressed in terms of relative luciferase activity normalized to ␤-galactosidase activity.…”

Transcriptional modulation of SLC26A3 (DRA) by sphingosine-1-phosphate

“…Caco-2 cells were transiently transfected with full-length DRA promoter and different deletion constructs cloned upstream of the luciferase reporter gene by electroporation utilizing an Amaxa Nucleofector system as previously described by us (36). p-Cytomegalovirus (CMV)-␤, ␤-galactosidase mammalian expression vector (BD Biosciences, Clontech, Palo Alto, CA), was also transfected that served as an internal control for transfection efficiency.…”

“…The double-stranded oligonucleotide was end labeled with digoxigenin-11, and binding reactions were performed as previously described (14,36); 6% nondenaturing gels were used to separate the DNA-protein complexes by electrophoresis. Electrotransferred complexes on the membrane were detected by use of antidigoxigenin antibody.…”