Identification and Characterization of the Human and Mouse SLC19A3 Gene: A Novel Member of the Reduced Folate Family of Micronutrient Transporter Genes

Eudy, James D.; Spiegelstein, Ofer; Barber, Robert C.; Wlodarczyk, Bogdan J.; Talbot, Jeffrey T.; Finnell, Richard H.

doi:10.1006/mgme.2000.3112

Cited by 119 publications

(108 citation statements)

References 25 publications

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“…Interestingly, the highest level of the SLC19A3 promoter-luciferase transgene expression was found in mouse brain. This observation is in agreement with the previously reported high expression of mouse Slc19a3 in the brain (9). Expression of the human SLC19A3 promoter-luciferase transgene in vivo was also found to be higher in mouse ileum compared with jejunum.…”

Section: Discussionsupporting

confidence: 92%

“…More recent investigations in our laboratory (24,25) have shown that the process of thiamin uptake in the intestine is regulated during ontogeny and in thiamin deficiency and that this regulation involves transcriptional regulatory mechanisms. With this knowledge in mind and because expression of these two hTHTRs is tissue/ cell specific (9,26,27), understanding the basal and regulated transcriptional activity of the involved genes is obviously important. To achieve this aim, we have recently cloned the 5Ј-regulatory region of the human SLC19A2 gene and confirmed its promoter activity first in vitro and then in vivo using transgenic mice (26,27).…”

Section: Slc19a3; Thiamin Transporter; Transcriptional Regulationmentioning

confidence: 99%

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Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

Nabokina¹,

Said²

2004

American Journal of Physiology-Gastrointestinal and Liver Physi

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Nabokina, Svetlana M., and Hamid M. Said. Characterization of the 5Ј-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies. Am J Physiol Gastrointest Liver Physiol 287: G822-G829, 2004. First published June 24, 2003 10.1152/ ajpgi.00234.2004.-Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5Ј-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5Ј-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between Ϫ77 and ϩ59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cisregulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position Ϫ48/Ϫ45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene. SLC19A3; thiamin transporter; transcriptional regulationTHIAMIN, A WATER-SOLUBLE vitamin, plays an essential role in normal cellular functions via its involvement in key metabolic reactions (2). Thiamin deficiency leads to a variety of clinical abnormalities including neurological and cardiovascular disorders (2, 33, 34, 36), whereas optimization of its level appears to have the potential for preventing diabetic retinopathy and blocking tissue damage caused by hyperglycemia of diabetes (13). Thiamin deficiency and suboptimal levels represent significant nutritional problems (18) and occur in a large percentage of alcoholic (17,36,37) and diabetic patients (32) and in patients with celiac and renal diseases (20,23,35). Thus studies that lead to an improvement in our understanding of the mechanisms involved in the maintenance of normal thiamin body homeostasis are of significance.Because it cannot be synthesized in the body, thiamin must be obtained from exogenous sources by absorption in the intestine, and thus the gut plays a critical role in maintaining normal thiamin body ...

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Section: Discussionsupporting

confidence: 92%

Section: Slc19a3; Thiamin Transporter; Transcriptional Regulationmentioning

confidence: 99%

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

Nabokina¹,

Said²

2004

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…10). These results indicate that fedratinib directly affects the uptake of thiamine via hTHTR2, which is expressed at the intestinal epithelium and other tissues including the brain (Eudy et al, 2000). Although both hTHTR1 and hTHTR2 are expressed on the apical side of Caco-2 cells (Said et al, 2004), hTHTR2 appears to play a more important role in thiamine uptake than hTHTR1 based on the report that the intestinal thiamine uptake was significantly reduced in the THTR2-deficient mice but was preserved in the THTR1-deficient mice (Reidling et al, 2010).…”

Section: Discussionmentioning

confidence: 83%

“…Two human thiamine transporters (hTHTR1 and hTHTR2) have been demonstrated to actively transport thiamine across the cell membrane and are widely expressed in various tissues including the intestine, liver, brain, and kidney (Dutta et al, 1999;Eudy et al, 2000;Said et al, 2004;Bukhari et al, 2011;Larkin et al, 2012). Based on the structural similarity between thiamine and a substructure of fedratinib, it was hypothesized that fedratinib interferes with the oral absorption of thiamine via inhibition of thiamine transport.…”

Section: Introductionmentioning

confidence: 99%

The Janus Kinase 2 Inhibitor Fedratinib Inhibits Thiamine Uptake: A Putative Mechanism for the Onset of Wernicke’s Encephalopathy

Zhang

Diamond

et al. 2014

Drug Metab Dispos

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The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

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“…Overall these results suggest the possible conservation of targeting mechanisms between these closely related cousins within the major facilitator superfamily of transporters (35). It remains to be seen whether this homology extends to hTHTR2, the protein product of the recently characterized SLC19A3 gene (36,37), which encodes a second human thiamine transporter with even greater amino acid residue identity to hTHTR1 (ϳ48% (36, 37)). …”

mentioning

confidence: 78%

Cell Biology of the Human Thiamine Transporter-1 (hTHTR1)

Subramanian¹,

Marchant²,

Parker³

et al. 2003

Journal of Biological Chemistry

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The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH 2 -terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.Thiamine (vitamin B1) is a water-soluble micronutrient that is essential for many cellular functions relating to growth and development. For example, thiamine pyrophosphate (the coenzyme form) is important for normal carbohydrate metabolism and energy production (1, 2). Thiamine is not synthesized by humans and other mammals and is obtained from dietary sources via absorption into intestinal epithelia. Deficiencies in cellular thiamine accumulation lead to cardiovascular and neurological disorders and are associated with the inherited condition of thiamine-responsive megaloblastic anemia (TRMA) 1 (1, 3-7). Recent molecular analyses have identified the protein product of the SLC19A2 gene in humans (chromosome 1q23.3) as the site of mutations that link with TRMA inheritance (2, 7-10). SLC19A2 encodes a saturable, high affinity human thiamine transporter known as hTHTR1 (2, 7-10), which is expressed in many tissues, including duodenal epithelium (11). The functional properties of hTHTR1 mimic the biochemical characteristics of thiamine uptake in intestinal preparations (reviewed in Refs. 12 and 13). The full-length hTHTR1 protein encodes a 497-amino acid polypeptide with 12 predicted transmembrane-spanning segments and cytoplasmic NH 2 -and COOH-terminal regions (2, 8). Several clinically identified mutations have been characterized in hTHTR1 (2, 7-10, 14), including ten prematurely truncated mutants, resulting from either point mutation, deletion, or insertion of nucleotides (reviewed in Refs. 7 and 15...

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Identification and Characterization of the Human and Mouse SLC19A3 Gene: A Novel Member of the Reduced Folate Family of Micronutrient Transporter Genes

Cited by 119 publications

References 25 publications

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

The Janus Kinase 2 Inhibitor Fedratinib Inhibits Thiamine Uptake: A Putative Mechanism for the Onset of Wernicke’s Encephalopathy

Cell Biology of the Human Thiamine Transporter-1 (hTHTR1)

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