Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Although the reduced folate carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share ~40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ~50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia.
SLC19A transporters; thiamine-responsive megaloblastic anemia; thiamine pyrophosphate; thiamine homeostasis; vitamin B1 uptake
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INTRODUCTION |
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THIAMINE (VITAMIN B1), in its coenzyme form thiamine pyrophosphate, plays a critical role in oxidative phosphorylation and in the pentose phosphate pathway. Dietary thiamine deficiency causes beriberi and Wernicke encephalopathy; the latter is often encountered in chronic alcoholism (17). Mutations in the thiamine transporter gene (THTR-1, SCL19A2) are the cause of an autosomal recessive disorder, thiamine-responsive megaloblastic anemia (TRMA), associated with diabetes mellitus and sensoneural deafness (5, 9, 14, 19, 25, 30). This transporter is highly expressed in skeletal muscle and to a lesser extent in heart and placenta. Expression is very low in small intestine and kidney despite higher thiamine uptake in these tissues than muscle (27), suggesting that an alternative transport route(s) for thiamine is present in these organs. Recently, a gene (SLC19A3) homologous to SLC19A2 was identified (7), and its function was established as a second thiamine transporter, THTR-2 (23).
Thiamine is present in human tissues and fluids mainly in three forms: thiamine, thiamine monophosphate (TMP), and thiamine pyrophosphate (TPP). Thiamine is converted to TPP by thiamine pyrophosphokinase, and TPP is hydrolyzed by thiamine pyrophosphatase to TMP, which is further hydrolyzed to thiamine by thiamine monophosphatase (26). No specific metabolic role is known for TMP. Whereas TPP exists intracellularly exclusively, thiamine and TMP are present both intracellularly and extracellularly. In plasma, TMP is present at levels ~80% that of thiamine, whereas TMP concentrations exceed that of thiamine by 65% in cerebrospinal fluid (34). Blood thiamine level is normal in patients with TRMA, suggesting that the majority of intestinal thiamine transport is not dependent on THTR-1 (28).
The reduced folate carrier RFC1 is a major transporter for physiological folate in plasma, 5-methyltetrahydrofolate, as well as in antifolates (22). Inactivation of RFC1 in mice results in early embryonic lethality, but in animals brought to term by administration to dams of high levels of folic acid, death occurs rapidly due to failure of hematopoietic tissues (38). RFC1, THTR-1, and THTR-2 are a family of carriers within the major facilitator superfamily (29). RFC1 shares protein sequence identity of ~40% with both THTR-1 and THTR-2 but is functionally distinct from the thiamine transporters. RFC1 does not transport thiamine, and THTR-1 and THTR-2 do not transport folates (6, 37). However, RFC1 transports TPP, the major thiamine intracellular metabolite, and recent studies from this laboratory (37) demonstrated that the level of RFC1 expressed in murine leukemia cells is inversely related to the extent of net TPP accumulation. Hence, low RFC1 expression augments TPP accumulation in these cells, whereas high expression suppresses it.
Because RFC1 is an anion exchanger and has been shown to transport TPP, the current studies were designed to assess whether TMP is also a substrate for RFC1 in murine leukemia cells. The data indicate that RFC1 mediates transport of TMP, which is then rapidly hydrolyzed to thiamine and subsequently phosphorylated to TPP in these cells. These results provide an additional facet of the complex role that RFC1 may play in thiamine homeostasis.
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MATERIALS AND METHODS |
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Chemicals. [3',5',7-3H]methotrexate (MTX) (5.7 Ci/mmol) and [3H]thiamine hydrochloride (20 Ci/mmol) were obtained from Amersham (Arlington Heights, IL), and [3H]TMP (generally labeled, 2.1 Ci/mmol) was custom-made by Moravek Biochemicals (Brea, CA). Unlabeled MTX was provided by Lederle (Carolina, Puerto Rico), and thiamine, TMP, and TPP were purchased from Sigma. Tritiated MTX was purified by high-performance liquid chromatography (HPLC) before use (10). Tritiated thiamine and TMP were used immediately after purity was confirmed by HPLC (37).
Cells lines and culture conditions. The MTXrA line was selected from murine leukemia L1210 cells with a loss of RFC1 function due to a point mutation in the carrier (2, 31). R16 cells have high-level expression of RFC1 obtained by transfection of murine RFC1 cDNA into MTXrA (2, 39). Cells were grown in RPMI 1640 medium containing 5% bovine calf serum (HyClone), 2 mM glutamine, 20 µM 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO2. R16 cells were grown with G418 at a concentration of 750 µg/ml to ensure stable, high-level expression of RFC1.
Transport studies. Cells were harvested, washed twice with HEPES-buffered saline (HBS; 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose, pH 7.4), and resuspended in HBS to 2.0 × 107 cells/ml. Cell suspensions were incubated at 37°C for 25 min, after which uptake was initiated by the addition of [3H]thiamine, [3H]TMP, or [3H]MTX. Samples were taken at the indicated times. Uptake was terminated by injection of 1 ml of the cell suspension into 10 ml of ice-cold HBS. Cells were collected by centrifugation, washed twice with ice-cold HBS, dried, and digested with 1 N NaOH in an 8-ml vial. After the liquid scintillation fluor was added, radioactivity was assessed in a liquid scintillation spectrometer. For influx experiments, the tritiated compound with or without unlabeled chemicals was added to the cell suspension to initiate uptake. Intracellular tritium was expressed in units of nanomoles per gram dry weight of cells. One milligram of dry cells corresponds to 6 × 106 L1210 cells.
Determination of rates of TMP hydrolysis. [3H]TMP was exposed to 250 µl of cell suspension or to the supernatant obtained by centrifugation of the cell suspension. Either the concentration of [3H]TMP, the density of the cell suspension, or the interval of incubation was varied. TMP hydrolysis was stopped by the addition of 15 µl of 100% trichloroacetic acid. After a brief centrifugation, the supernatant was extracted three times with water-saturated ethyl ether. Unlabeled thiamine, TMP, and TPP, each at a final concentration of 1 mM, were added to the mixture, and 20 µl were loaded on a 20 × 20-cm thin-layer chromatography plate with a 250-µm layer of silica gel (Whatman). The plate was developed by a solvent mixture of diethanolamine-methanol-formic acid-67 mM dibasic sodium phosphate (1:15:1.5:5) for 2.5 h (20). Under these conditions, TPP (Rf = 0.26), TMP (Rf = 0.37), and thiamine (Rf = 0.51) were well separated. The TMP and thiamine spots were traced under ultraviolet light, removed from plates, and added to scintillation vials. The silica gel was incubated in NaOH (2 N, 0.5 ml) for 15 min at room temperature, and tritium was measured on a liquid scintillation spectrometer after introduction of the fluor. When TMP hydrolysis was complete, radioactivity recovered as thiamine was equivalent to radioactivity recovered from TMP in the absence of TMP hydrolysis, indicating a 100% conversion of TMP to thiamine.
Measurement of intracellular thiamine metabolites. R16 cells were exposed to 1 µM [3H]TMP for 1 min after a 25-min incubation in HBS. Cells were washed twice with ice-cold HBS. Half of the cell pellet was processed for dry weight and total cell tritium, as described in Transport studies. The other half was resuspended in 250 µl of HBS, and 15 µl of 100% trichloroacetic acid were added. The digest was processed for thin-layer chromatography as described above to assess levels of thiamine, TMP, and TPP. Cell levels were expressed in units of picomoles per gram dry weight, according to the percentages of total tritium recovered.
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RESULTS |
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Influx of TMP in murine leukemia cells.
Initial uptake of TMP was assessed in three murine leukemia cell lines
with different levels of RFC1 expression: 1) L1210 wild-type cells; 2)
MTXrA, a variant of L1210 cells that has lost RFC1 activity
due to a point mutation (A130P) in the third transmembrane domain
(2); and 3) R16 cells derived by transfection of murine
RFC1 into MTXrA cells with carrier expression and activity
nine times greater than that of L1210 cells (39). As
illustrated in Fig. 1, influx of TMP in
MTXrA cells was ~20% that of L1210 cells, whereas TMP
influx in R16 was approximately fivefold higher than that of L1210
cells. Thus influx of TMP correlated with the level of RFC1 expression
in these cell lines. The high ordinate intercept in R16 cells could be
attributed in part to a rapid deviation from initial rates as simulated
by the interrupted line.
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Inhibitory effect of MTX and thiamine on [3H]TMP
influx.
TMP influx in L1210, MTXrA, and R16 cells was assessed in
the presence of 100 µM MTX, an inhibitor of RFC1 but not the thiamine transporter. At this MTX concentration, RFC1-mediated TMP influx (1 µM) should be inhibited >90% in L1210 cells, based on an MTX influx
Km for RFC1 of ~5.8 µM (39) and
a TMP Ki of 25 µM (see Affinity of RFC1
for TMP). TMP influx in these cell lines was also
determined in the presence of 10 µM thiamine, an inhibitor of THTR-1
but not RFC1. At this thiamine concentration (1 µM), 90% of
[3H]thiamine influx activity should be blocked based on
the thiamine influx Km of 0.96 ± 0.17 µM
that we determined in L1210 cells. As shown in Fig.
2, 100 µM MTX abolished ~90% of TMP
influx in R16 cells over a 25-s interval of uptake, whereas 10 µM
thiamine had no inhibitory effect at all, indicating that virtually all TMP influx in R16 cells was mediated exclusively by RFC1 under these
conditions. Because RFC1 is highly expressed in this cell line, the
component of TMP influx mediated by this process is maximized. MTX also
inhibited >50% of TMP influx in L1210 cells with lesser RFC1
expression but had no effect at all on TMP influx in MTXrA
cells, which lack functional RFC1. In the presence of 10 µM thiamine,
however, TMP influx in L1210 and MTXrA cells was reduced by
20 and 37%, respectively. As indicated in Chemical instability
of TMP in vitro, the emergence of a thiamine-inhibitable TMP influx component in these cells, with lower or absent RFC1 expression, was subsequently found to be due to the rapid hydrolysis of
[3H]TMP to [3H]thiamine.
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Comparison of TMP and thiamine influx in L1210 cells at
physiological substrate concentrations.
Because TMP and thiamine are present in plasma and cerebrospinal fluid
at a concentration range of 3-50 nM (34), the
relative contribution of TMP and thiamine delivery to cells was
assessed by measuring influx of these two substrates at 50 nM over
40 s. TMP influx was ~70% that of thiamine influx (Table
1). More than half of TMP influx was
blocked by 100 µM MTX, and half of the remaining activity was further
inhibited by 10 µM thiamine. The latter is attributed to inhibition
of influx of thiamine derived from the hydrolysis of TMP (see
Chemical instability of TMP in vitro). Thiamine
influx at 50 nM was decreased by ~75% in the presence of unlabeled
thiamine. Thus, in L1210 cells, influx of TMP via RFC1 was
approximately half the carrier-mediated influx of thiamine at a
physiological substrate concentration.
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Chemical instability of TMP in vitro.
Chemical stability of TMP was assessed under the same conditions as in
the influx experiments. Cells in HBS at a density of 2 × 107 cells/ml were incubated in the transport buffer for 25 min, after which [3H]TMP was added and the identity of
extracellular radiolabel was assessed by thin-layer chromatography. As
indicated in Fig. 3A, 80% of
[3H]TMP was hydrolyzed to thiamine by 1 min of
incubation. By 3 min, no TMP could be detected. When supernatant was
separated from the suspension after 25 min of incubation, after which
[3H]TMP was added, hydrolysis of TMP was also detected
but at a reduced rate. There was no detectable degradation of TMP in
HBS alone over a 5-min incubation. These findings suggest that TMP hydrolysis is mediated by phosphatases associated with the L1210 cell
membrane and secreted into the suspension buffer.
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Affinity of RFC1 for TMP.
In measuring the affinity of RFC1 for TMP, R16 cells were used to
maximize the component of influx attributable to TMP uptake via RFC1
because this carrier is highly expressed in this cell line. Also, MTX
influx can be measured over a very short interval, minimizing
hydrolysis of TMP. As shown in Fig. 4,
[3H]MTX influx was decreased by 35, 49, and 59% in the
presence of 15, 30, and 45 µM TMP, respectively. Assuming that TMP
inhibits MTX influx competitively as described for TPP
(37), the influx Ki of TMP for RFC1
was calculated to be 25.9 ± 0.9 µM based on an MTX influx
Km of 5.8 µM in R16 cells (39).
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Formation of [3H]TMP metabolites in R16 cells.
Most of the thiamine transported into L1210 cells is converted to TPP,
the active cofactor for several enzymes (37). As indicated
in Fig. 5, about half of TMP transported
into R16 cells was converted to thiamine within 1 min; 15% was
identified as TPP and 35% remained as TMP. MTX, at a concentration of
100 µM, suppressed intracellular levels of thiamine, TMP, and TPP;
the greatest suppression was observed for thiamine. In contrast, 10 µM thiamine had no effect on total cell tritium, with only negligible effects on cell levels of tritiated TMP or TPP. These observations indicate that [3H]thiamine appearing in cells exposed to
[3H]TMP was due to TMP hydrolysis to thiamine within
cells and not to influx of thiamine from the extracellular compartment.
These data also demonstrate that transport of TMP via RFC1 results in the delivery of a precursor of the active thiamine cofactor TPP into
cells.
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DISCUSSION |
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Recent studies from this laboratory demonstrated that TPP is a good substrate for RFC1 and that its transport into cells correlates with the level of RFC1 expression (37). Because TPP is present exclusively in the intracellular compartment where it is formed by thiamine pyrophosphokinase, RFC1 action with respect to this substrate is asymmetrical. This asymmetry results in the unidirectional export of TPP, depression of the intracellular TPP level, and the hydrolysis of TPP to thiamine in the extracellular space. On the basis of this consideration, cells that express high levels of RFC1 would be at a disadvantage under conditions of thiamine deficiency.
We now demonstrate that RFC1 also transports TMP, a normal
constituent of plasma and cerebrospinal fluid, present at
concentrations nearly equivalent to thiamine. Once transported into
cells, TMP is converted to thiamine by thiamine monophosphatase,
followed by phosphorylation of thiamine to TPP by thiamine
pyrophosphokinase (Fig. 6). Hence, the
level of RFC1 expression will determine the rate of delivery of this
thiamine precursor into cells. Under conditions of thiamine deficiency,
when blood levels of thiamine and TMP are low, this added transport via
RFC1 might not be important. However, when the thiamine carrier THTR-1
is not functional as occurs in TRMA (33), RFC1 could be an
important alternative route for thiamine uptake into cells, along with
other potential transport pathways for thiamine such as THTR-2
(23) (Fig. 6). Indeed, the pattern of RFC1 tissue
expression may be one of the determinants of the selective organ
and metabolic defects associated with TRMA-macrocytic anemia,
sensoneural deafness, and diabetes mellitus. It is interesting in this
regard that dietary thiamine deficiency leads to cardiac (beriberi) and
brain (Wernicke's syndrome) abnormalities, whereas TRMA leads to
functional defects in only a limited number of other tissues.
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The high level of expression of RFC1 in the apical brush border of the choroid plexus suggests that this may be an important route of TMP transport, because TMP is present at higher levels than is thiamine in cerebrospinal fluid (35). High expression of RFC1 in the brush border of small intestinal cells could be a route for absorption of TMP and TPP present in the gut (35). However, because of the high phosphatase level in intestinal brush border, these phosphorylated compounds are probably hydrolyzed to thiamine before they are absorbed. Our results suggest that at least part of TMP transport across rat everted jejunal sacs reported previously could be mediated by RFC1 (11). However, RFC1 expressed in intestine has a low pH optimum (4, 18, 24), and the extent to which TMP could be transported by this mechanism under these conditions remains unclear. On the other hand, the transport activity of THTR-1 and THTR-2 is optimal at high pH (6), so that the extent to which thiamine is transported via this carrier is also uncertain at the pH present in the microenvironment of intestinal villi (21).
Unlike THTR-1, RFC1 is an anion exchanger. Folates are bivalent anions with negative charges at the two carboxyl groups of the glutamate moiety. RFC1 is inhibited by a variety of inorganic and organic anions, including the organic phosphates such as the adenine nucleotides (12, 16, 36). It is the asymmetrical distribution of organic phosphates across cell membranes, with their high intracellular electrochemical potential, that is thought to be the energy source for the uphill transport of folates into many mammalian cells (12, 16, 36). Because TPP and TMP have comparable affinities for RFC1 but differ in total charge, the data suggest that the thiamine moiety may play an important role in binding to the carrier. On the other hand, thiamine itself is not transported by RFC1 at all, likely due to thiamine's positive charge, which is reversed by phosphorylation. Further evidence for the role of this moiety in binding to RFC1 comes from the observation that other organic anions, such as ADP and AMP, that share the same charge have a much lower affinity for RFC1 (15). It remains to be determined whether there are critical charged residues in RFC1 vs. THTR-1 and THTR-2 that determine the charge specificity of these carriers for folates and thiamine as has been demonstrated for the rat organic anion transporter rOAT3 (8).
These studies demonstrate the high degree of chemical instability of TMP in cell suspensions. Hence, it is unclear why this substance is present at levels comparable to thiamine in plasma. It is possible that TMP is bound to circulating proteins that protect it from degradation and/or that this component is restored by the slow but constant exit of TMP and/or TPP from cells with subsequent hydrolysis by thiamine pyrophosphatase (Fig. 6). These findings indicate that previous suggestions that thiamine and TMP share the same transport route, based on TMP competition with radiolabeled thiamine for transport, are erroneous (1, 3, 13, 32). It is highly likely that apparent inhibitory effects are due to rapid hydrolysis of TMP to thiamine that, in turn, inhibits radiotracer thiamine uptake into cells. It is clear that any studies focused on the transport of TMP or TPP must be designed to minimize, and correct for, the rapid degradation of these compounds to thiamine.
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ACKNOWLEDGEMENTS |
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This work was supported by National Cancer Institute Grant CA-82621.
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FOOTNOTES |
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Address for reprint requests and other correspondence: I. D. Goldman, Albert Einstein College of Medicine Cancer Research Center, Chanin 2, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: igoldman{at}aecom.yu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 13, 2002;10.1152/ajpcell.00547.2001
Received 15 November 2001; accepted in final form 8 February 2002.
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