The serotonin (5-HT) transporter (SERT) facilitates clearance of extracellular 5-HT by its uptake and internalization. Decreased expression of SERT and consequent high 5-HT levels have been implicated in various diarrheal disorders. Thus, appropriate regulation of SERT is critical for maintenance of 5-HT homeostasis in health and disease. Previous studies demonstrated that SERT is regulated via posttranslational and transcriptional mechanisms. However, the role of epigenetic mechanisms in SERT regulation is not known. Current studies investigated the effects of histone deacetylase (HDAC) inhibition on SERT expression and delineated the mechanisms. Treatment of Caco-2 cells with the pan-HDAC inhibitors butyrate (5 mM) and trichostatin (TSA, 1 μM) decreased SERT mRNA and protein levels. Butyrate- or TSA-induced decrease in SERT was associated with decreased activity of human SERT (hSERT) promoter 1 (upstream of exon 1a), but not hSERT promoter 2 (upstream of exon 2). Butyrate + TSA did not show an additive effect on SERT expression, indicating that mechanisms involving histone hyperacetylation may be involved. Chromatin immunoprecipitation assays demonstrated enrichment of the hSERT promoter 1 (flanking nt -250/+2) with tetra-acetylated histone H3 or H4, which was increased (~3-fold) by butyrate. Interestingly, specific inhibition of HDAC2 (but not HDAC1) utilizing small interfering RNA decreased SERT mRNA and protein levels. The decrease in SERT expression by HDAC inhibition was recapitulated in an in vivo model. SERT mRNA levels were decreased in the ileum and colon of mice fed pectin (increased availability of butyrate) compared with controls fed a fiber-free diet (~50-60%). Our results identify a novel role of HDAC2 as a regulator of SERT gene expression in intestinal epithelial cells.
[downregulated in adenoma (DRA)] is a Cl Ϫ /HCO 3 Ϫ exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3=-untranslated region (3=-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3=-UTR (pmirGLO-3=-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3=-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3=-UTR of DRA abrogated the effect of miR-494 on 3=-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.
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