Molecular Mechanisms Mediating the Adaptive Regulation of Intestinal Riboflavin Uptake Process

Abstract

The intestinal absorption process of vitamin B2 (riboflavin, RF) is carrier-mediated, and all three known human RF transporters, i.e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. We have previously shown that the intestinal RF uptake process is adaptively regulated by substrate level, but little is known about the molecular mechanism(s) involved. Using human intestinal epithelial NCM460 cells maintained under RF deficient and over-supplemented (OS) conditions, we now show that the induction in RF uptake in RF deficiency is associated with an increase in expression of the hRFVT-2 & -3 (but not hRFVT-1) at the protein and mRNA levels. Focusing on hRFVT-3, the predominant transporter in the intestine, we also observed an increase in the level of expression of its hnRNA and activity of its promoter in the RF deficiency state. An increase in the level of expression of the nuclear factor Sp1 (which is important for activity of the SLC52A3 promoter) was observed in RF deficiency, while mutating the Sp1/GC site in the SLC52A3 promoter drastically decreased the level of induction in SLC52A3 promoter activity in RF deficiency. We also observed specific epigenetic changes in the SLC52A3 promoter in RF deficiency. Finally, an increase in hRFVT-3 protein expression at the cell surface was observed in RF deficiency. Results of these investigations show, for the first time, that transcriptional and post-transcriptional mechanisms are involved in the adaptive regulation of intestinal RF uptake by the prevailing substrate level.

Sodium Butyrate Enhances Intestinal Riboflavin Uptake via Induction of Expression of Riboflavin Transporter-3 (RFVT3)

“…Since NaB is known to exert epigenetics effects (via acting as histone deacetylase inhibitor and its suppression of histone de-acetylation lead to accumulation of multi-acetylated forms of histone, which in turn alters the compactness of chromatin therefore affecting DNA folding and gene expression [49, 50]), we examined possible involvement of epigenetic mechanisms (e.g., histone modifications) in the effect of NaB on expression of the RFVT3. We also examined possible involvement of DNA methylation (specifically trimethylation of histone 3 lysine 27; H3K27me3) since this histone modification also influence gene expression [40, 51, 52]. For this, we performed a ChIP-qPCR analysis and focused on the SLC52A3 promoter region (−199 to +8) since it is essential for driving basal activity of the promoter and is involved the regulation of the RF uptake process in intestinal epithelial cells under other conditions [40, 53].…”

“…We also examined possible involvement of DNA methylation (specifically trimethylation of histone 3 lysine 27; H3K27me3) since this histone modification also influence gene expression [40, 51, 52]. For this, we performed a ChIP-qPCR analysis and focused on the SLC52A3 promoter region (−199 to +8) since it is essential for driving basal activity of the promoter and is involved the regulation of the RF uptake process in intestinal epithelial cells under other conditions [40, 53]. The results showed that treating Caco-2 cells with NaB (1 mM; 24 hrs) to be associated with a significant (P < 0.01) increase in the histone acetylation marker (H3Ac) at SLC52A3 promoter region compared to untreated control cells (Fig.…”

“…Briefly, Caco-2 cells were incubated in KR buffer (133 mM NaCl, 4.93 mM KCl, 1.23 mM MgSO4, 0.85 mM CaCl 2 , 5 mM glucose, 5 mM glutamine, 10 mM HEPES, and 10 mM MES, pH 7.4) at 37°C in the presence of 3 H-RF (25 nM). After incubation, cells were subjected for radioactivity measurement in a liquid scintillation counter (Beckman Coulter) as described before [20, 40]. …”

“…To determine the RFVT3 mRNA and hnRNA expression levels, total RNA from Caco-2 cells and mouse colonoids treated with NaB and untreated controls was extracted as described [26, 40]. Total RNA (2 μg) was reverse transcribed to cDNA using i-Script reverse transcriptase (RT) kit (Bio-Rad, CA).…”

“…NaB-treated and untreated Caco-2 cells were subjected for ChIP analysis as described before [40, 47]. The cross-linked chromatin was incubated overnight with 1–2 μg of the specific antibody [H3, H3Ac, H3K27me3 and IgG (Millipore, Billerica, MA)] and the immunoprecipitated samples were subjected to DNA purification and analyzed by qPCR using SLC52A3 promoter specific primers (forward 5ꞌGGGTTCGCTCAGTGAAGGTA-3ꞌ and reverse 5ꞌ-AGCAGGAGTGTGTCCGTTGTG-3ꞌ) to amplify the region −199 to +8 relative to transcriptional start site (relative to TSS as +1) using the PCR conditions as described before [40].…”