In vitro and in vivo characterization of the minimal promoter region of  the human thiamin transporter <i>SLC19A2</i>

Reidling, Jack C.; Said, Hamid M.

doi:10.1152/ajpcell.00076.2003

Cited by 41 publications

(72 citation statements)

References 21 publications

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“…These findings, however, do not eliminate the possibility that other cisand trans-acting elements may also contribute to minimal promoter activity. Previously, we (27) have shown that the minimal promoter region of the other thiamin transporter SLC19A2 contains several cis-regulatory elements important for promoter activity, including SP1. In this regard, it is interesting to note that SP1 is involved in the regulation of basal promoter activity of both thiamin transporters, and thus this NF appears be important for regulation of both thiamin transporter genes.…”

Section: Discussionmentioning

confidence: 99%

“…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). In addition, we have identified the minimal region required for basal activity of the SLC19A2 promoter and identified a role of cis-regulatory elements for gut-enriched Krupple-like factor, nuclear factor-1 (NF1), and stimulating protein-1 (SP1) in the regulation of the promoter activity and confirmed the promoter activity in vivo using transgenic mice.…”

Section: Slc19a3; Thiamin Transporter; Transcriptional Regulationmentioning

confidence: 99%

“…Routine biochemicals and cell culture reagents were all of molecular biology quality and were purchased from Fisher Scientific (Tustin, CA) and Sigma (St. Louis, MO). SP1-containing plasmid was kindly provided by Dr. Robert Tjian (University of California, Berkeley, CA), the pPacSP3 construct was kindly provided by Dr. Guntram Suske (Philipps-Universität, Marburg, Germany), and pPacSP1 and pPac0 were made in our laboratory (27).…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

“…The 2,016-bp cloned 5Ј-regulatory region of the SLC19A3 gene fused to the firefly luciferase reporter gene was used to generate transgenic founders utilizing the expertise of the transgenic mouse facility of our university as described by us recently (27). Pups were genotyped by performing a PCR with specific primers for the human SLC19A3 promoter (forward 5Ј-CGGGGTACCGTGCAGAGTGATTATAAC-3Ј) and luciferase gene (reverse 5Ј-ATGCGAGAATCTCACGCAG-3Ј) that would yield a 592-bp product.…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

“…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).…”

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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: Discussionmentioning

confidence: 99%

Section: Slc19a3; Thiamin Transporter; Transcriptional Regulationmentioning

confidence: 99%

Section: Chemicals and Reagentsmentioning

confidence: 99%

Section: Chemicals and Reagentsmentioning

confidence: 99%

mentioning

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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Gastrointestinal Handling of Water‐Soluble Vitamins

Said

Nexø

2018

Comprehensive Physiology

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Developmental maturation of intestinal and renal thiamin uptake: Studies in wild‐type and transgenic mice carrying human THTR‐1 and 2 promoters

Reidling¹,

Nabokina²,

Balamurugan³

et al. 2005

Journal Cellular Physiology

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Thiamin (B1) is an essential micronutrient for normal growth and development. Mammals obtain thiamin through intestinal absorption, while in the kidney thiamin is reabsorbed to prevent its loss in the urine, both processes are specialized, carrier-mediated and involve thiamin transporters-1 and 2 (THTR-1 and THTR-2, respectively; products of the SLC19A2 and SLC19A3 genes). Although thiamin appears to play an important role in neonatal growth, little is currently known about the possible regulation of intestinal and renal thiamin uptake during developmental maturation. We addressed these issues by examining intestinal and renal thiamin uptake and expression of THTR-1 and THTR-2 during early stages of life. We utilized wild-type mice (mice express orthologues of both thiamin transporters) and transgenic mice expressing human SLC19A2 or SLC19A3 promoter-reporter transgenes as a model system and examined carrier-mediated thiamin uptake, mTHTR-1 and 2 protein and mRNA levels and luciferase activity in suckling (13 days), weanling (25-27 days), and adult (60-65 days) mice. Carrier-mediated thiamin uptake by jejunal and renal brush border membrane vesicles (BBMV) both decreased with maturation (suckling>weanling>adult) and were associated with a reduction in mTHTR-1 and mTHTR-2 protein, mRNA levels, and the activity of human SLC19A2 and SLC19A3 promoter-reporter constructs in the intestines and kidneys of transgenic mice. These results are the first to demonstrate that intestinal and renal thiamin uptake are developmentally regulated during early stages of life, mediated through mTHTR-1 and mTHTR-2, and suggest the possible involvement of transcriptional regulatory mechanism(s) in this regulation.

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In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2

Cited by 41 publications

References 21 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

Gastrointestinal Handling of Water‐Soluble Vitamins

Developmental maturation of intestinal and renal thiamin uptake: Studies in wild‐type and transgenic mice carrying human THTR‐1 and 2 promoters

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