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

Abstract

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 ...

Hypoxia induced upregulation and function of the thiamine transporter, SLC19A3 in a breast cancer cell line

“…Recently, it has been shown that basal expression of SLC19A3 is under the transcriptional control of SP1 and SP3. 55 Evidence suggests that hypoxia can increase SP1 expression, increase SP1 nuclear localization, and decrease expression of the dual transcriptional activator/repressor, SP3. [56][57][58] Thus, it is feasible that HIF-1α may enhance the action of SP1/ SP3 to increase transcription of SLC19A3.…”

Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations

“…Mouse-derived pancreatic beta-TC-6 insulinoma cells (passages [10][11][12][13][14][15][16][17][18][19][20][21][22] were obtained from American Type Tissue Collection (ATCC; Rockville, MD) and maintained in DMEM growth medium in the presence of 15 nM thiamin. Uptake was examined (unless otherwise stated) at 37°C in Krebs-Ringer buffer (in mM: 133 NaCl, 4.93 KCl, 1.23 MgSO 4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES; pH 8.0) by a rapid filtration technique (14), as described by us previously (10,30).…”

“…Pancreas was minced and digested with HEPES (25 mM) containing collagenase V (1 mg/ml) and DNase I (0.1 mg/ml) for 40 min at 37°C. The digested tissue was filtered through a nylon mesh with a pore size of 600 m, and islet cells were purified on a discontinuous Ficoll gradient in the order of 25,23,20, and 11% in HBSS. The gradient was centrifuged for 15 min at 2,500 RPM and 4°C, and islets were taken from the interface of 20 and 11% Ficoll gradient.…”

“…The SLC19A2 (Ϫ2250 to Ϫ36) and SLC19A3 (Ϫ1957 to ϩ59) promoter-luciferase full-length reporter constructs used in this study were generated and described by us previously (20,26,28). Pancreatic beta-TC-6 cells were cotransfected in 12-well plates at ϳ50 -80% confluency with 2 g of each promoter construct and 100 ng of the Renilla transfection control plasmid Renilla luciferase-thymidine kinase (pRL-TK) (Promega, Madison, WI).…”

Changes in Thiamin Contents in Porcine Muscles and Liver during Growth

“…8) Some molecular species of the thiamin transporter have been discovered and their biochemical properties investigated. [9][10][11] It has been also clarified that the lack of a thiamin transport system induced a thiamin uptake deficiency, 8) therefore, a carrier-assisted thiamin transport system is required for maintaining the thiamin contents in animal organs. In addition, the expression of thiamin transporting protein mRNA is different among animal species and organs.…”