Bacterial lipopolysaccharide inhibits colonic carrier-mediated uptake of thiamin pyrophosphate: roles for TLR4 receptor and NF-κB/P38/JNK signaling pathway.

This study investigated the effect of the bacterial endotoxin lipopolysaccharide (LPS) on colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1 that is generated by gut microbiota. We used three complementary models in our study: in vitro (human-derived colonic epithelial NCM460), ex vivo (human differentiated colonoid monolayers), and in vivo (mouse colonic tissue). The results showed that exposure of NCM460 cells to LPS leads to a significant inhibition of carrier-mediated TPP uptake as well as in decreased expression of the colonic TPP transporter (cTPPT) protein, mRNA, and heterologous nuclear RNA (hnRNA) compared with untreated controls. Similarly, exposure of human differentiated colonoid monolayers and mice to LPS caused significant inhibition in colonic carrier-mediated TPP uptake and in cTPPT protein, mRNA, and hnRNA expression. The effect of LPS on colonic TPP uptake and cTTPT expression was also found to be associated with a significant reduction in activity of the SLC44A4 promoter as well as in decreased expression of the nuclear factor Elf-3 (E74-like ETS transcription factor 3), which is needed for promoter activity. Finally, we found that knocking down the Toll-like receptor 4 (TLR4) and blocking the nuclear factor kappa B (NF-κB), JNK, and p38 signaling pathways with the use of pharmacological inhibitors lead to significant abrogation in the degree of LPS-mediated inhibition in TPP uptake and cTPPT expression. These results demonstrated that exposure of colonic epithelia to LPS inhibits colonic TPP uptake via transcriptional mechanism(s) and that the effect is mediated via TLR4 receptor and NF-κB/p38/JNK signaling pathways.NEW & NOTEWORTHY This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1. Three complementary models were used: in vitro (human NCM460 cells), ex vivo (human colonoids), and in vivo (mice). The results showed LPS to significantly suppress TPP uptake and the expression of its transporter, and that these effects are mediated via the membrane TLR4 receptor, and involve the NF-κB/p38/JNK signaling pathways.

Effect of chronic alcohol exposure on gut vitamin B7 uptake: involvement of epigenetic mechanisms and effect of alcohol metabolites.

Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.

Thiamine mimetics sulbutiamine and benfotiamine as a nutraceutical approach to anticancer therapy.

Malignant cells frequently demonstrate an oncogenic-driven reliance on glycolytic metabolism to support their highly proliferative nature. Overexpression of pyruvate dehydrogenase kinase (PDK) may promote this unique metabolic signature of tumor cells by inhibiting mitochondrial function. PDKs function to phosphorylate and inhibit pyruvate dehydrogenase (PDH) activity. Silencing of PDK expression has previously been shown to restore mitochondrial function and reduce tumor cell proliferation. High dose Vitamin B1, or thiamine, possesses antitumor properties related to its capacity to reduce PDH phosphorylation and promote its enzymatic activity, presumably through PDK inhibition. Though a promising nutraceutical approach for cancer therapy, thiamine's low bioavailability may limit clinical effectiveness. Here, we have demonstrated exploiting the commercially available lipophilic thiamine analogs sulbutiamine and benfotiamine increases thiamine's anti-cancer effect in vitro. Determined by crystal violet proliferation assays, both sulbutiamine and benfotiamine reduced thiamine's millimolar IC50 value to micromolar equivalents. HPLC analysis revealed that sulbutiamine and benfotiamine significantly increased intracellular thiamine and TPP concentrations in vitro, corresponding with reduced levels of PDH phosphorylation. Through an ex vitro kinase screen, thiamine's activated cofactor form thiamine pyrophosphate (TPP) was found to inhibit the function of multiple PDK isoforms. Attempts to maximize intracellular TPP by exploiting thiamine homeostasis gene expression resulted in enhanced apoptosis in tumor cells. Based on our in vitro evaluations, we conclude that TPP serves as the active species mediating thiamine's inhibitory effect on tumor cell proliferation. Pharmacologic administration of benfotiamine, but not sulbutiamine, reduced tumor growth in a subcutaneous xenograft mouse model. It remains unclear if benfotiamine's effects in vivo are associated with PDK inhibition or through an alternative mechanism of action. Future work will aim to define the action of lipophilic thiamine mimetics in vivo in order to translate their clinical usefulness as anticancer strategies.

Biotin Supplementation Ameliorates Murine Colitis by Preventing NF-κB Activation.

BACKGROUND & AIMS: Biotin is a water-soluble vitamin that is indispensable for human health. Biotin deficiency can cause failure-to-thrive, immunodeficiency, alopecia, dermatitis, and conjunctivitis. We previously reported that biotin deficiency also can lead to severe colitis in mice, which is completely reversed with supplementation. Our aim in this study was to determine if high-dose biotin supplementation can provide a therapeutic benefit in a preclinical model for inflammatory bowel disease (IBD) and to identify the molecular mechanism by which this occurs. METHODS: Mice were challenged with dextran sodium sulfate to induce colitis and were treated with 1 mmol/L biotin to induce or maintain remission. Clinical response was monitored by the Disease Activity Index and fecal calprotectin levels. The colon tissue was investigated for histology, length, as well as expression of inflammatory cytokines (interleukin 6, tumor necrosis factor-α, interleukin 1ß), intestinal permeability, tight junctions (zonula occludens-1 and claudin-2), and the transcription factor nuclear factor-κB (NF-κB). RESULTS: Biotin therapy led to delayed onset and severity of colitis as well as accelerated healing. There was improvement in the Disease Activity Index, fecal calprotectin levels, colon length, and histology. In addition, biotin-treated mice had reduced expression of inflammatory cytokines, reduced intestinal permeability, and reduced activation of NF-κB. CONCLUSIONS: Oral supplementation with biotin provides benefit for maintenance and induction of remission in the dextran sodium sulfate preclinical model for IBD. Biotin does this by reducing the activation of NF-κB, which prevents the production of inflammatory cytokines and helps maintain the integrity of the intestinal barrier. Clinically, the NF-κB pathway is important in the development of IBD and this finding suggests that biotin may have therapeutic potential for patients with IBD.

Functional analysis of the third identified SLC25A19 mutation causative for the thiamine metabolism dysfunction syndrome 4.

Thiamine metabolism dysfunction syndrome-4 (THMD4) includes episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy. Until now only two mutations (G125S and S194P) have been reported in the SLC25A19 gene as causative for this disease and a third mutation (G177A) as related to the Amish lethal microcephaly. In this work, we describe the clinical and molecular features of a patient carrying a novel mutation (c.576G>C; Q192H) on SLC25A19 gene. Functional studies on this mutation were performed explaining the pathogenetic role of c.576G>C in affecting the translational efficiency and/or stability of hMTPPT protein instead of the mRNA expression. These findings support the pathogenetic role of Q192H (c.576G>C) mutation on SLC25A19 gene. Moreover, despite in other patients the thiamine supplementation leaded to a substantial improvement of peripheral neuropathy, our patient did not show a clinical improvement.

Mutations in SLC5A6 associated with brain, immune, bone, and intestinal dysfunction in a young child.

The human sodium-dependent multivitamin transporter (hSMVT) is a product of the SLC5A6 gene and mediates biotin, pantothenic acid, and lipoate uptake in a variety of cellular systems. We report here the identification of mutations R94X, a premature termination, and R123L, a dysfunctional amino acid change, both in exon 3 of the SLC5A6 gene in a child using whole genome-scanning. At 15 months of age, the child showed failure to thrive, microcephaly and brain changes on MRI, cerebral palsy and developmental delay, variable immunodeficiency, and severe gastro-esophageal reflux requiring a gastrostomy tube/fundoplication, osteoporosis, and pathologic bone fractures. After identification of the SLC5A6 mutations, he responded clinically to supplemental administration of excess biotin, pantothenic acid, and lipoate with improvement in clinical findings. Functionality of the two mutants was examined by 3H-biotin uptake assay following expression of the mutants in human-derived intestinal HuTu-80 and brain U87 cells. The results showed severe impairment in biotin uptake in cells expressing the mutants compared to those expressing wild-type hSMVT. Live cell confocal imaging of cells expressing the mutants showed the R94X mutant to be poorly tolerated and localized in the cytoplasm, while the R123L mutant was predominantly retained in the endoplasmic reticulum. This is the first reporting of mutations in the SLC5A6 gene in man, and suggests that this gene is important for brain development and a wide variety of clinical functions.

Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption.

Riboflavin (RF) is indispensable for normal cell metabolism, proliferation, and growth. The RFVT-3 protein (product of the Slc52a3 gene) is expressed in the gut with the expression being restricted to the apical membrane domain of the polarized intestinal epithelial cells. The relative contribution of RFVT-3 to total carrier-mediated RF uptake in the native intestine, however, is not clear. We addressed this issue in the current investigation using a conditional (intestinal-specific) RFVT-3 knockout (cKO) mouse model developed by the Cre/Lox approach. All RFVT-3 cKO mice were found to be RF deficient and showed a significant growth and development retardation; also, nearly two-thirds of them died prematurely between the age of 6 and 12 wk. In vivo (intestinal and colonic loops) and in vitro (native isolated intestinal epithelial cells) uptake studies showed a severe inhibition in carrier-mediated RF uptake in the cKO mice compared with control littermates. We also observed a significant increase in the level of expression of oxidative stress-responsive genes in the intestine of the cKO mice compared with control littermates. Supplementation of the RFVT-3 cKO mice with pharmacological doses of RF led to a complete correction of the growth retardation and to normalization in the level of expression of the oxidative stress-responsive genes in the gut. These results show, for the first time, that the RFVT-3 system is the main transporter involved in carrier-mediated RF uptake in the native mouse small and large intestine, and that its dysfunction impairs normal RF body homeostasis.

Molecular Mechanisms Mediating the Adaptive Regulation of Intestinal Riboflavin Uptake Process.

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.

Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter.

Colonic microbiota synthesize a considerable amount of thiamine in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes. Nothing, however, is known about the molecular identity of this system. Here we report on the molecular identification of the colonic TPP uptake system as the product of the SLC44A4 gene. We cloned the cDNA of SLC44A4 from human colonic epithelial NCM460 cells, which, upon expression in ARPE19 cells, led to a significant (p < 0.01, >5-fold) induction in [(3)H]TPP uptake. Uptake by the induced system was also found to be temperature- and energy-dependent; Na(+)-independent, slightly higher at acidic buffer pH, and highly sensitive to protonophores; saturable as a function of TPP concentration, with an apparent Km of 0.17 ± 0.064 µM; and highly specific for TPP and not affected by free thiamine, thiamine monophosphate, or choline. Expression of the human TPP transporter was found to be high in the colon and negligible in the small intestine. A cell surface biotinylation assay and live cell confocal imaging studies showed the human TPP transporter protein to be expressed at the apical membrane domain of polarized epithelia. These results show, for the first time, the molecular identification and characterization of a specific and high-affinity TPP uptake system in human colonocytes. The findings further support the hypothesis that the microbiota-generated TPP is absorbable and could contribute toward host thiamine homeostasis, especially toward cellular nutrition of colonocytes.

Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: a role for NF-Y in regulating basal transcription.

Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5'-regulatory region of the SLC25A19 gene (1,080 bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5'- and 3'-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between -131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.

Identification and functional characterization of the Caenorhabditis elegans riboflavin transporters rft-1 and rft-2.

Two potential orthologs of the human riboflavin transporter 3 (hRFVT3) were identified in the C. elegans genome, Y47D7A.16 and Y47D7A.14, which share 33.7 and 30.5% identity, respectively, with hRFVT3. The genes are tandemly arranged, and we assign them the names rft-1 (for Y47D7A.16) and rft-2 (for Y47D7A.14). Functional characterization of the coding sequences in a heterologous expression system demonstrated that both were specific riboflavin transporters, although the rft-1 encoded protein had greater transport activity. A more detailed examination of rft-1 showed its transport of riboflavin to have an acidic pH dependence, saturability (apparent Km = 1.4 ± 0.5 µM), inhibition by riboflavin analogues, and Na(+) independence. The expression of rft-1 mRNA was relatively higher in young larvae than in adults, and mRNA expression dropped in response to RF supplementation. Knocking down the two transporters individually via RNA interference resulted in a severe loss of fertility that was compounded in a double knockdown. Transcriptional fusions constructed with two fluorophores (rft-1::GFP, and rft-2::mCherry) indicated that rft-1 is expressed in the intestine and a small subset of neuronal support cells along the entire length of the animal. Expression of rft-2 is localized mainly to the intestine and pharynx. We also observed a drop in the expression of the two reporters in animals that were maintained in high riboflavin levels. These results report for the first time the identification of two riboflavin transporters in C. elegans and demonstrate their expression and importance to metabolic function in worms. Absence of transporter function renders worms sterile, making them useful in understanding human disease associated with mutations in hRFVT3.

Effect of folate oversupplementation on folate uptake by human intestinal and renal epithelial cells.

BACKGROUND: Folic acid [corrected] plays an essential role in cellular metabolism. Its deficiency can lead to neural tube defects. However, optimization of body folate homeostasis can reduce the incidence of neural tube defects and may decrease the risk of Alzheimer and cardiovascular diseases and cancer. Hence, food fortification and intake of supplemental folate are widespread. OBJECTIVE: We examined the effects of long-term folate oversupplementation on the physiologic markers of intestinal and renal folate uptake processes. DESIGN: Human-derived intestinal Caco-2 and renal HK-2 epithelial cells were maintained (5 generations) in a growth medium oversupplemented (100 micromol folic acid/L) or maintained under sufficient conditions (0.25 and 9 micromol folic acid/L). RESULTS: Carrier-mediated uptake of (3)H-folic acid (2 micromol/L) at buffer pH 5.5 (but not buffer pH 7.4) by Caco-2 and HK-2 cells maintained under the folate-oversupplemented condition was significantly (P<0.01) and specifically lower than in cells maintained under the folate-sufficient condition. This reduction in folic acid uptake was associated with a significant decrease in the protein and mRNA levels of the human reduced-folate carrier (hRFC) and a decrease in the activity of the hRFC promoter. It was also associated with a decrease in mRNA levels of the proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) and folate receptor (FR). CONCLUSIONS: Long-term oversupplementation with folate leads to a specific and significant down-regulation in intestinal and renal folate uptake, which is associated with a decrease in message levels of hRFC, PCFT/HCP1, and FR. This regulation appears to be mediated via a transcriptional mechanism, at least for the hRFC system.

Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin is not a substrate for hTHTR2.

The water-soluble micronutrient thiamine is required for normal tissue growth and development in humans. Thiamine is accumulated into cells through the activity of two cell surface thiamine transporters (hTHTR1 and hTHTR2), which are differentially targeted in polarized tissues. Mutational dysfunction of hTHTR1 is associated with the clinical condition of thiamine-responsive megaloblastic anemia: the symptoms of which are alleviated by thiamine supplementation. Recently, two hTHTR2 mutants (G23V, T422A) have been discovered in clinical kindreds manifesting biotin-responsive basal ganglia disease (BBGD): the symptoms of which are alleviated by biotin administration. Why then does mutation of a specific thiamine transporter isoform precipitate a disorder correctable by exogenous biotin? To investigate the suggestion that hTHTR2 can physiologically function as a biotin transporter, we examined 1) the cell biological basis of hTHTR2 dysfunction associated with the G23V and T422A mutations and 2) the substrate specificity of hTHTR2 and these clinically relevant mutants. We show that the G23V and T422A mutants both abrogate thiamine transport activity rather than targeting of hTHTR2 to the cell surface. Furthermore, biotin accumulation was not detectable in cells overexpressing either the full length hTHTR2 or the clinically relevant hTHTR2 mutants, yet was demonstrable in the same assay using cells overexpressing the human sodium-dependent multivitamin transporter, a known biotin transporter. These results cast doubt on the most parsimonious explanation for the BBGD phenotype, namely that hTHTR2 is a physiological biotin transporter.

Targeting and trafficking of the human thiamine transporter-2 in epithelial cells.

Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.

Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells.

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.

Expression and promoter analysis of SLC19A2 in the human intestine.

The molecular mechanism and regulation of the intestinal uptake process of dietary thiamine is not well understood. Previous studies have established the involvement of a carrier-mediated system for thiamine uptake in the human intestine. Recently a human thiamine transporter, SLC19A2, was cloned from a number of human tissues. Little, however, is known about expression of the SLC19A2 message along the native human gastrointestinal tract, and no analysis of its promoter in intestinal tissue is available. Therefore, the current studies were aimed at investigating the expression of SLC19A2 in the human gastrointestinal tract and at analyzing the promoter of this potential intestinal thiamine transporter. First we cloned SLC19A2 cDNA from a human intestinal cell line (Caco-2) by reverse transcriptase-polymerase chain reaction, then used this cDNA as a probe in Northern blot analysis. SLC19A2 message was found to be expressed in all gastrointestinal tissues in the following order: liver>stomach>duodenum>jejunum>colon>cecum>rectum>ileum. SLC19A2 was also expressed at the protein level in Caco-2 cells and in native human small intestine by Western blot analysis. We also cloned the 5'-regulatory region of the SLC19A2 gene and confirmed activity of its promoter following transfection into intestinal epithelial Caco-2 cells. Furthermore, we identified the minimal promoter region required for basal activity of SLC19A2 in these cells which was found to be mainly encoded in a sequence between -356 and -36, and included multiple cis-regulatory elements. Transcription initiation sites of the SLC19A2 gene in intestinal epithelial Caco-2 cells were also identified by 5'-rapid amplification of cDNA ends. These results demonstrate that SLC19A2 is expressed in various regions of the human gastrointestinal tract. In addition, the results provide the first characterization of the SLC19A2 promoter. These findings raise the possibility that SLC19A2 may play a role in the normal intestinal thiamine absorption process.