ABSTRACT
Organic anion-transporting polypeptide 1A2 (OATP1A2) (gene symbol, SLCO1A2) mediates cellular uptake of a wide range of endogenous substrates, as well as drugs and xenobiotics. OATP1A2 is expressed in several tissues, including apical membranes of small intestinal epithelial cells. Given its role in intestinal drug absorption, a detailed analysis of the mechanisms that regulate SLCO1A2 gene expression is potentially of great pharmacological relevance. We show here that treatment of human intestine-derived Caco-2 cells with vitamin D(3) markedly increased endogenous OATP1A2 mRNA and protein levels. Suppression of endogenous vitamin D receptor (VDR) expression with siRNAs significantly reduced this induction. Two alternative promoter regions exist in genomic databases for the SLCO1A2 gene. One putative VDR response element (VDRE) that was predicted to interact efficiently with VDR-retinoid X receptor α (RXRα) was identified in silico within SLCO1A2 promoter variant 1. This VDRE served as a strong binding site for the recombinant VDR-RXRα heterodimers in vitro and was potently activated by VDR in the presence of vitamin D(3) in heterologous promoter assays. In reporter assays using native promoter constructs, SLCO1A2 promoter variant 1 was strongly induced by VDR, and site-directed mutagenesis of a single VDRE within this region abolished this activation. Native VDR-RXRα also interacted with this element both in vitro and in living cells. We showed that expression of the SLCO1A2 gene is induced by vitamin D(3) at the transcriptional level through the VDR. Our results suggest that pharmacological administration of vitamin D(3) may allow modulation of intestinal absorption of OATP1A2 transport substrates.
Subject(s)
Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Binding Sites/genetics , Caco-2 Cells , Cells, Cultured , Cholecalciferol/metabolism , Gene Expression/genetics , Humans , Intestinal Absorption/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutagenesis, Site-Directed/methods , Promoter Regions, Genetic , Protein Transport/genetics , RNA, Messenger/genetics , Retinoid X Receptors/genetics , Retinoid X Receptors/metabolism , Transcriptional Activation , Vitamin D Response ElementABSTRACT
The apical sodium-dependent bile acid transporter (ASBT) is expressed abundantly in the ileum and mediates bile acid absorption across the apical membranes. Caudal-type homeobox proteins CDX1 and CDX2 are transcription factors that regulate genes involved in intestinal epithelial differentiation and proliferation. Aberrant expression of both ASBT and CDXs in Barrett's esophagus (BE) prompted us to study, whether the expression of the ASBT gene is regulated by CDXs. Short interfering RNA-mediated knockdown of CDXs resulted in reduced ASBT mRNA expression in intestinal cells. CDXs strongly induced the activity of the ASBT promoter in reporter assays in esophageal and intestinal cells. Nine CDX binding sites were predicted in silico within the ASBT promoter, and binding of CDXs to six of them was verified in vitro and within living cells by electrophoretic mobility shift assays and chromatin immunoprecipitation assays, respectively. RNAs were extracted from esophageal biopsies from 20 BE patients and analyzed by real-time PCR. Correlation with ASBT expression was found for CDX1, CDX2, and HNF-1α in BE biopsies. In conclusion, the human ASBT promoter is activated transcriptionally by CDX1 and CDX2. Our finding provides a possible explanation for the reported observation that ASBT is aberrantly expressed in esophageal metaplasia that also expresses CDX transcription factors.
