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

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5' regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity. promoter analysis in vitro and in vivo; thiamin transport; transgenic mice     

Common human 5' dopamine transporter (SLC6A3) haplotypes yield varying expression levels in vivo

1. Individuals display significant differences in their levels of expression of the dopamine transporter (DAT; SLC6A3). These differences in DAT are strong candidates to contribute to individual differences in motor, mnemonic and reward functions. To identify "cis"-acting genetic mechanisms for these individual differences, we have sought variants in 5' aspects of the human DAT gene and identified the haplotypes that these variants define. 2. We report (i) significant relationships between 5' DAT haplotypes and human individual differences in ventral striatal DAT expression assessed in vivo using [(11)C] cocaine PET and (ii) apparent confirmation of these results in studies of DAT expression in postmortem striatum using [(3)H] carboxyflurotropane binding. 3. These observations support the idea that cis-acting variation in 5' aspects of the human DAT/SLC6A3 locus contributes to individual differences in levels of DAT expression in vivo. 5' DAT variation is thus a good candidate to contribute to individual differences in a number of human phenotypes.     

SLC19A3 encodes a second thiamine transporter ThTr2.

Recently, a new family of facilitative carriers has been cloned consisting of the reduced folate (SLC19A1) and the thiamine (SLC19A2) transporters. Despite a high level of sequence identity and similarity there is essentially no functional overlap between these carriers. The former transports folates and the latter thiamine. In this paper we describe the function of SLC19A3, another member of this transporter family most recently cloned, after transient transfection of the cDNA into HeLa cells. Uptake of [3H]thiamine, but not of methotrexate nor folic acid, was enhanced in SLC19A3 transfectants relative to vector control. Similarly, in the transfectants thiamine transport increased with an increase in pH with peak activity at pH approximately 7.5. While [3H]thiamine uptake was markedly inhibited by nonlabeled thiamine it was not inhibited by several organic cations in 100-fold excess. Hence this carrier has a high degree of specificity for vitamin B1. The data indicate that SLC19A3 has the characteristics of SLC19A2 (ThTr1) and represents a second thiamine transporter (ThTr2) in this family of facilitative carriers.     

Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis

SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant (P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.     

Characterization of the organic cation transporter SLC22A16: a doxorubicin importer

Specific efflux transporters, such as P-glycoprotein, have been shown to confer drug resistance by decreasing the intracellular accumulation of anticancer drugs. Understanding influx transporters, as well as efflux transporters, is essential to overcome this resistance. We report the expression profile and pharmacological characterization of an organic cation transporter, SLC22A16. The results of our experiments indicate that SLC22A16 is a mediator of doxorubicin uptake in cancer cells. Quantitative real-time RT-PCR analyses show that SLC22A16 is expressed in primary samples taken from patients with acute leukemia. Xenopus oocytes injected with SLC22A16 cRNA import doxorubicin, a widely used anticancer drug for hematological malignancies, in a saturable and dose-dependent manner. The apparent Km value for doxorubicin import was 5.2+/-0.4 microM. In cytotoxic assays, stable transfectants of leukemic Jurkat cells overexpressing SLC22A16 cells became significantly more sensitive to doxorubicin (2 microM) treatment. Characterization of SLC22A16 will help in designing novel therapies targeting hematological malignancies.     

pH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent K_m of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.     

Stimulation of the amino acid transporter SLC6A19 by JAK2

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation ^V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na⁺ coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, ^V617FJAK2 or inactive ^K882EJAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I_le), which was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual decline of I_le. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I_le following inhibition of carrier insertion by brefeldin A (5 μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.     

Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5)

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na⁺-glutamine_ex/glutamine_in transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K⁺ gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na⁺. Internal Na⁺ exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.     

Characterization of a new degradable polymer scaffold for regeneration of the dermis: In vitro and in vivo human studies

Full thickness skin wounds in humans heal with scars, but without regeneration of the dermis. A degradable poly(urethane urea) scaffold (PUUR), Artelon® is already used to reinforce soft tissues in orthopaedics, and for treatment of osteoarthritis of the hand, wrist and foot. In this paper we have done in vitro experiments followed by in vivo studies to find out whether the PUUR is biocompatible and usable as a template for dermal regeneration. Human dermal fibroblasts were cultured on discs of PUUR, with different macrostructures (fibrous and porous). They adhered to and migrated into the scaffolds, and produced collagen. The porous scaffold was judged more suitable for clinical applications and 4 mm Ø, 2 mm-thick discs of porous scaffold (12% w/w or 9% w/w polymer solution) were inserted intradermally in four healthy human volunteers. The implants were well tolerated and increasing ingrowth of fibroblasts was seen over time in all subjects. The fibroblasts stained immunohistochemically for procollagen and von Willebrand factor, indicating neocollagenesis and angiogenesis within the scaffolds. The PUUR scaffold may be a suitable material to use as a template for dermal regeneration.Key words: dermal regeneration, tissue engineering, polymer scaffold, wound healing, in vitro, in vivo, guided tissue regeneration, human, burns     

Characterization of in vitro models of SLC30A10 deficiency

BioMetals - Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux...     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na⁺-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na⁺-coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na(+)-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na(+) -coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.