(Received for publication, October 31, 1996, and in revised form, May 13, 1997)
From the The folate receptor, like many
glycosylphosphatidylinositol-anchored proteins, is found
associated with membrane domains that are insoluble in Triton X-100 at
low temperature and that are enriched in cholesterol and sphingolipids.
Depletion of cellular cholesterol has been shown to inhibit vitamin
uptake by this receptor (Chang, W.-J., Rothberg, K. G., Kamen, B. A., and Anderson, R. G. W. (1993) J. Cell Biol.
118, 63-69), suggesting that these domains regulate this
process. In this study, the importance of sphingolipids for folate
receptor function was investigated in Caco-2 cells using fumonisin
B1, a mycotoxin that inhibits the biosynthesis of these
lipids. The folate receptor-mediated transport of
5-methyltetrahydrofolate was almost completely blocked in cells in
which sphingolipids had been reduced by ~40%. This inhibition was
dependent on the concentration and duration of the treatment with the
mycotoxin and was mediated by the sphingolipid decrease. Neither
receptor-mediated nor facilitative transport was inhibited by fumonisin
B1 treatment, indicating that the effect of sphingolipid depletion was specific for folate receptor-mediated vitamin uptake. A
concurrent loss in the total amount of folate binding capacity in the
cells was seen as sphingolipids were depleted, suggesting a causal
relationship between folate receptor number and vitamin uptake. These
findings suggest that dietary exposure to fumonisin B1
could adversely affect folate uptake and potentially compromise cellular processes dependent on this vitamin. Furthermore, because folate deficiency causes neural tube defects, some birth defects unexplained by other known risk factors may be caused by exposure to
fumonisin B1.
The folate vitamins play an essential role as cofactors in many
biochemical reactions involving one-carbon metabolism. These include
the biosynthesis of purines and thymidine, the regeneration of
methionine from homocysteine, and histidine metabolism. Cellular processes dependent upon folate can be compromised if dietary levels of
this vitamin are insufficient or if its transport into cells is
affected. Two different systems are used for folate uptake into cells.
The first uses a high capacity, low affinity transmembrane transporter
known as the reduced folate carrier. The second involves a
glycosylphosphatidylinositol
(GPI)1-anchored protein referred to as the
folate receptor (1, 2). This high affinity receptor is responsible for
the transport of folate into cells of the placenta, kidney, breast, and
other tissues with elevated requirements for this vitamin.
The mechanism by which the GPI-anchored folate receptor transports
vitamin into the cytosol has received considerable attention in recent
years. The immunochemical localization of several GPI-anchored proteins, including the folate receptor, to uncoated membrane invaginations called caveolae (3) led to the suggestion that the uptake
of folate is mediated by these structures by a process termed
potocytosis (4). In this and other studies, caveolae were equated with
membrane domains that could be isolated based on their insolubility in
Triton X-100 at 4 °C (5, 6) and that are enriched in cholesterol and
sphingolipids (7). More recent evidence has suggested that the Triton
X-100-insoluble domains may include caveolae, but are primarily other
membrane regions in which the GPI-anchored proteins (including the
folate receptor) reside (8). Characterization of the protein components of caveolae isolated using new, detergent-free purification schemes has
supported the conclusion that GPI-anchored proteins are not enriched in
these structures (9, 10). Collectively, this evidence suggests that the
folate receptor is not in caveolae, and therefore, potocytosis may not
be the mechanism by which vitamin transport occurs.
Recent evidence suggests that uptake mediated by the folate receptor
involves endocytosis (11, 12). However, the association of the folate
receptor with Triton X-100-insoluble domains does appear to be
important to its function. Depletion of cellular cholesterol through
inhibition of its biosynthesis inhibited receptor-mediated folate
uptake (13). Interpreting these results in the context of potocytosis,
Rothberg et al. (14) suggested that this occurred because
the clustering of the folate receptor in caveolae was disrupted. In
terms of the effect on the endocytosis of the folate receptor,
cholesterol depletion has been found to accelerate the rate at which
this protein was recycled to the cell
surface.2 How this results in an inhibition
of folate uptake is unclear.
The importance of sphingolipids, the other lipids enriched in Triton
X-100-insoluble domains, for folate receptor function has not yet been
investigated, although several studies have probed the importance of
these lipids for other GPI-anchored proteins. Inhibition of
sphingolipid biosynthesis influenced both the localization of
GPI-anchored proteins to these Triton X-100-insoluble domains (15) and
the transport of newly synthesized GPI-anchored proteins to the Golgi
in yeast (16) and to the appropriate membrane surface in
polarized epithelial cells (17). Therefore, the localization, transport, and targeting of these lipids and GPI-anchored proteins appear to be linked (18).
In this study, the effects of changes in the cellular levels of
sphingolipids on folate receptor-mediated vitamin uptake were investigated in Caco-2 cells. Originally isolated from a human colon
adenocarcinoma, Caco-2 cells were chosen for this study because their
folate receptor (2) and the association of GPI-anchored proteins with
Triton X-100-insoluble domains in these cells (19) have been
characterized. Cellular sphingolipids were depleted using fumonisin
B1. A mycotoxin produced by the fungus Fusarium moniliforme (20), fumonisin B1 blocks sphingolipid
biosynthesis by inhibiting the reaction catalyzed by sphingosine
N-acyltransferase (ceramide synthase) (21). The results
presented here indicate that sphingolipids play an important role in
folate receptor function and that fumonisin B1 could
influence cellular folate status through its effects on these membrane
lipids.
Fetal bovine serum was purchased from Atlanta
Biologicals, Inc. RPMI 1640 medium and folate-free RPMI 1640 medium
were from Life Technologies, Inc. [3,5,7,9-3H]Folic acid
(25-30 Ci/mmol, 99% pure) was obtained from American Radiolabeled
Chemicals. 5-[3 Caco-2 cells were purchased from the American
Type Culture Collection and were routinely grown in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and
100 µg/ml streptomycin. Cells were plated at 2.5 × 105 cells/25-cm flask or at 8 × 105
cells/125-mm flask for quantitation of folate binding and uptake or for
analysis of lipids, respectively. After 2 days of growth, the medium
was changed to folate-free RPMI 1640 medium and 10% fetal bovine serum
such that the cells were maintained under these conditions for a total
of 5 days. Treatments were done for the indicated times during this
5-day period such that they ended at the time of analysis.
Lovastatin-treated cells were grown in folate-free RPMI 1640 medium
supplemented with lipoprotein-depleted fetal bovine serum prepared as
described by Goldstein et al. (22). In all cases, the cells
were confluent by the end of the experiment, but continued to divide at
nearly the same rate as subconfluent cells. Neither the growth rate (as
measured by cell counting and [3H]thymidine uptake)
nor the viability of the cells (as assessed by trypan blue
exclusion) was affected by the treatments (data not shown).
Triton
X-100-insoluble domains were isolated using the two-step sucrose
gradient method described by Arreaza et al. (23). Cells were
washed twice with phosphate-buffered saline (8 g/liter NaCl, 0.2 g/liter KCl, 1.15 g/liter Na2HPO4, and 0.2 g/liter KH2PO4) and lysed by the addition of
cold TNE buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA) containing 1% Triton
X-100 to the dish. After 20 min of incubation on ice, the cells were
scraped from the dish with a rubber policeman and homogenized. The
lysate was then adjusted to 40% sucrose with TNE buffer containing
80% sucrose, placed in an ultracentrifuge tube, and overlaid with 5.5 ml of TNE buffer containing 38% sucrose followed by 2 ml of TNE buffer
containing 5% sucrose. The samples were ultracentrifuged at
120,000 × g at 37 °C for 15-20 h. The Triton
X-100-insoluble membranes visible at the interface between the 5 and
38% sucrose layers were harvested with a syringe, diluted ~5-fold
with TNE buffer, and pelleted by ultracentrifugation (1 h at
120,000 × g at 4 °C).
The
internalization of the reduced folate derivative into the cytosol was
quantitated as described by Smart et al. (24). Briefly,
cells were incubated with 5 nM
5-[3H]methyltetrahydrofolate (0.5 µCi) in folate-free
medium for various amounts of time at 37 °C. The medium was then
removed, and the cells were washed four times with cold
phosphate-buffered saline before the addition of 1.5 ml of lysis buffer
(10 mM Tris-HCl (pH 8.0), 20 µg/ml leupeptin, 20 µg/ml
aprotinin, and 1 µM 5-methyltetrahydrofolate) to each
flask. The cells were lysed by placing the flasks at Internalization of
125I-labeled diferric transferrin was measured on cells
plated in 6-well dishes as described (26) with the following minor
modifications. Cells were incubated with medium A (RPMI 1640 medium
containing 0.2% bovine serum albumin) for 15 min at 37 °C to
deplete endogenous transferrin. The cells were then washed once with
this medium and incubated in medium A containing 3 µg/ml
125I-transferrin at 37 °C in 5% CO2 for 2, 4, 6, or 8 min. At the end of the incubation, the cells were placed on
ice; the 125I-transferrin-containing medium was removed;
and prechilled 0.2 N acetic acid in 0.2 M NaCl
was added to each well. After 2 min on ice, this solution was removed,
and the cells were washed three times with 150 mM NaCl, 20 mM Hepes (pH 7.4), 1 mM CaCl2, 5 mM KCl, and 1 mM MgCl2. The cells
were then solubilized with 0.1 N NaOH in phosphate-buffered
saline, and an aliquot was counted in a The binding of
folic acid in solubilized cells and Triton X-100-insoluble fractions
was quantitated as described by Antony et al. (27). For the
total folate binding capacity, cells were solubilized 25 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% octyl glucoside. The Triton X-100-insoluble pellet obtained as
described above was solubilized in this octyl glucoside-containing buffer for quantitation of folate receptors in these domains. The
different samples were incubated with 5 nM
[3H]folate (0.5 µCi) for 20 min at 37 °C to allow
ligand binding to the receptor (total volume of 1 ml/tube). The samples
were then cooled on ice for 5 min, after which 40 mg of dextran-coated charcoal was added to each tube to absorb unbound radiolabel. After
mixing and the addition of 1 ml of 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% octyl
glucoside, the samples were incubated on ice for 10 min, followed by
centrifugation for 30 min at 30,000 × g at 4 °C.
Aliquots of the supernatant were then counted by scintillation
counting. Nonspecific binding was determined in each experiment by
measuring binding in the presence of 2.5 µM
[3H]folate (500-fold excess).
Lipids were extracted and purified from
either whole cells or Triton X-100-insoluble domains using the method
of Ariga et al. (28). Cells (2.5 × 108) or
the appropriate fraction (isolated from 2.5 × 108
cells) was sequentially extracted with chloroform/methanol (2:1, v/v),
chloroform/methanol (1:1, v/v), and chloroform/methanol/water (30:60:8,
v/v). The extracts were then pooled and applied to a DEAE-cellulose
column. The neutral lipids were eluted in chloroform/methanol/water (30:60:8, v/v) and passed through a second DEAE-cellulose column to
remove contaminants. The acidic lipids were eluted with chloroform, methanol, and 0.8 M sodium acetate (30:60:8, v/v). The
neutral lipids were further fractionated on a silica column from which fatty acids and cholesterol were eluted with chloroform and neutral glycosphingolipids and phospholipids were eluted with
chloroform/methanol (80:20, v/v). Glycolipids and gangliosides were
purified of any contaminating lipids by base hydrolysis followed by
re-chromatography on DEAE-cellulose.
The lipids were identified and quantitated as described by Brown and
Rose (7). Identifications were made by comigration on high performance
TLC plates with standards; stability (sphingolipids) or lability
(glycerolipids) in base; and reactivity with molybdate reagent
(phospholipids), orcinol (glycolipids), or resorcinol (gangliosides).
Phospholipids, cholesterol, sulfatides, and cerebrosides were
visualized by charring with cupric acetate and quantitated by
densitometric scanning and comparison to standards. Gangliosides were
detected using resorcinol and quantitated by measuring sialic acid.
Neutral glycolipids were visualized with orcinol and quantitated by
densitometric scanning and comparison to standards. The thin layer
chromatography systems used to resolve the various lipids were as
follows: chloroform/methanol/ammonium hydroxide (60:35:8, v/v) for
phospholipids; chloroform/methanol/acetic acid/formic acid/water
(42:18:7.2:2.4:1.2, v/v) for neutral glycolipids; and chloroform,
methanol, and 0.25% KCl (50:45:10, v/v) for gangliosides.
Cells were treated with either fumonisin
B1 (20 µg/ml (27.7 µM) for 2 days) or
lovastatin (25 µM for 3 days) to decrease the cellular
sphingolipids or cholesterol, respectively. The specificity and
effectiveness of these treatments were assessed by quantitating the
major lipids both from whole Caco-2 cells and from the Triton X-100-insoluble domains. These results are shown in Table
I. Consistent with the previous results of Brown and
Rose (7), the Triton X-100-insoluble domains were found to be enriched
in sphingolipids and cholesterol, containing ~93% of the former and 80% of the latter. Fumonisin B1 treatment significantly
reduced the levels of all the measured sphingolipids in both whole
cells and the Triton X-100-insoluble domains. Lovastatin treatment
specifically decreased cholesterol among the total lipids, but was
found to affect the levels of several other lipids (both glycerolipids and sphingolipids) in the Triton X-100-insoluble domains. Overall, both
treatments resulted in an ~20% decrease in the
cholesterol/sphingolipid content of the Triton X-100-insoluble
domains.
Table I.
Lipid composition of control and fumonisin B1- and
lovastatin-treated cells
Department of Radiation Oncology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
,5
,7,9-3H]Methyltetrahydrofolate (30 Ci/mmol, 97.8% pure) was purchased from Moravek Biochemicals, Inc.
125I-Labeled diferric transferrin was from NEN Life Science
Products. Lovastatin was a generous gift from Merck. The high
performance Silica Gel 60 TLC plates were obtained from Whatman.
DEAE-cellulose, charcoal, all lipid standards, and other chemicals were
from Sigma. The reagents for the bicinchoninic acid protein assay were
purchased from Pierce.
80 °C for 15 min and thawed on ice. The cells were then collected and centrifuged
for 20 min at 100,00 × g in an Optima TL
ultracentrifuge to separate the membrane (pellet) and cytosolic
(supernatant) fractions. The radioactivity in each fraction was
quantitated by scintillation counting. Nonspecific uptake was measured
using 2.5 µM 5-[3H]methyltetrahydrofolate
and subtracted from the total radioactivity to give the specific
uptake. The results were normalized to protein determined using the
bicinchoninic acid assay of Smith et al. (25).
-counter to quantitate the
internalized transferrin. Surface transferrin was determined by
incubating cells with prechilled medium A containing 3 µg/ml
125I-transferrin on ice for 30 min, followed by four washes
with 150 mM NaCl, 20 mM Hepes (pH 7.4), 1 mM CaCl2, 5 mM KCl, and 1 mM MgCl2.
Fumonisin B1-induced Depletion of Cellular
Sphingolipids
Lipid
Whole cells
Triton
X-100-insoluble fraction
Control
Fumonisin
B1
Lovastatin
Control
Fumonisin
B1
Lovastatin
nmol/108
cells
nmol/108 cells
Cholesterol
1727 ± 88
1829
± 84 (0)
1365 ± 60 (21)
1388 ± 112
1536
± 86 (0)
921 ± 76 (34)
TGa
475
± 41
388 ± 15 (18)
508 ± 16 (0)
240
± 29
243 ± 23 (0)
204 ± 56 (15)
FA
1119
± 101
1136 ± 23 (0)
1022 ± 172 (9)
806
± 89
857 ± 163 (0)
777 ± 143 (4)
PC
2762
± 142
3129 ± 258 (0)
2537 ± 110 (8)
1234
± 129
1242 ± 213 (0)
1015 ± 86 (18)
PE
1814
± 50
1943 ± 49 (0)
1777 ± 26 (2)
884
± 95
1058 ± 97 (0)
664 ± 31 (25)
PI
896
± 53
934 ± 89 (0)
945 ± 25 (0)
234
± 50
243 ± 99 (0)
176 ± 24 (25)
PS
898
± 59
850 ± 82 (5)
1043 ± 25 (0)
266
± 27
212 ± 36 (20)
280 ± 42 (0)
CL
429
± 25
424 ± 45 (1)
477 ± 25 (0)
40 ± 13
40
± 18 (0)
34 ± 5 (15)
SM
1230 ± 38
809
± 30 (34)
1167 ± 26 (5)
1210 ± 55
762
± 18 (37)
1081 ± 80 (11)
Ceramides
271
± 19
117 ± 17 (57)
254 ± 37 (6)
239
± 26
110 ± 28 (54)
208 ± 35 (13)
Sulfatides
226 ± 28
91 ± 16 (60)
284
± 31 (0)
172 ± 19
66 ± 9 (62)
199 ± 27 (0)
Glc-Cer
100 ± 10
36 ± 10 (64)
93
± 8 (7)
87 ± 5
46 ± 23 (47)
74 ± 5 (15)
Gal-Cer
81 ± 8
40 ± 13 (51)
84
± 11 (0)
71 ± 9
42 ± 14 (41)
61 ± 12 (14)
Lac-Cer
157 ± 25
119 ± 31 (24)
144
± 22 (8)
147 ± 28
111 ± 36 (24)
119 ± 31 (19)
Gangliosides
45 ± 4
23 ± 7 (49)
52
± 3 (0)
37 ± 4
21 ± 4 (43)
40 ± 8 (0)
a
TG, triglyceride; FA, fatty acid; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PI,
phosphatidylinositol; PS, phosphatidylserine; CL, cardiolipin; SM,
sphingomyelin; Cer, ceramide.
The consequences of these changes in membrane lipid
composition on folate receptor function were evaluated by measuring the rate of uptake of 5-methyltetrahydrofolate by fumonisin B1-
and lovastatin-treated cells. As shown in Fig. 1, the
rate of uptake of the vitamin was roughly linear in untreated Caco-2
cells grown in folate-free medium with either normal (Fig. 1,
A and B, closed circles) or
lipoprotein-depleted (Fig. 1B, triangles) serum.
Uptake was inhibited by ~90% in the fumonisin B1-treated
cells (Fig. 1A). A similar level of inhibition, which has
been reported previously by others (13), was observed in the
lovastatin-treated cells (Fig. 1B). Uptake of the vitamin
was unaffected in cells treated with fumonisin B1 for only
1 h, indicating that the inhibition was not mediated by the
mycotoxin alone. Treatment with either various concentrations of
fumonisin B1 for 2 days (Fig. 2A)
or 20 µg/ml for various amounts of time (Fig. 2B)
demonstrated that the inhibition of 5-methyltetrahydrofolate uptake was
both concentration- and time-dependent. The sphingolipid
levels of these cells also decreased in a concentration- and
time-dependent manner (data not shown), establishing that
the inhibition of 5-methyltetrahydrofolate uptake in the fumonisin
B1-treated cells was mediated by the changes in the
sphingolipid composition.
Coupled with the previous reports (13, 14) of the effects of lowering
cellular cholesterol on folate transport, the finding that depletion of
cellular sphingolipids by fumonisin B1 inhibited this
process suggests that the cholesterol/sphingolipid-enriched domains are
involved in this effect. To determine if fumonisin B1
specifically inhibits processes dependent on these domains, the effect
of this mycotoxin on other types of uptake systems was assessed.
Facilitative transport was measured by quantitating 2-deoxyglucose
uptake. Treatment with fumonisin B1 (20 µg/ml for 2 days)
had no effect on the rate of uptake of this glucose analog by Caco-2
cells (data not shown). Receptor-mediated endocytosis was assessed by
measuring transferrin uptake. Both the rates of internalization (Fig.
3) and externalization (data not shown) of
125I-transferrin were found to be very similar in control
and fumonisin B1-treated cells. The only difference found
was in the amount of surface transferrin binding, which was 2.7 times
more in the sphingolipid-depleted groups. While the reason for this
difference is unclear, it is responsible for the line representing the
fumonisin B1-treated rate of uptake being offset from that
of the control cells in Fig. 3. Therefore, transport processes not
thought to involve sphingolipid-enriched domains (receptor-mediated
endocytosis via clathrin-coated pits and facilitative transport,
respectively) were not compromised by changes in the cellular
sphingolipid levels.
Lipid Depletion-induced Changes in Cellular Folic Acid Binding Capacity
Previous studies of the effect of depletion of cellular
cholesterol on folate uptake suggested that this process was
compromised because the clustering of the folate receptor in
cholesterol/sphingolipid-rich domains in the plasma membrane was
disrupted (13, 14). To determine if the decrease in cellular
sphingolipids caused by fumonisin B1 affected the folate
receptor in a similar manner, the amount of this protein localized in
the Triton X-100-insoluble domains was determined. This was
accomplished by quantitating the high affinity binding of folic acid in
either solubilized whole Caco-2 cells or Triton X-100-insoluble
domains. Because folic acid is essentially bound irreversibly by the
folate receptor (Kd = 0.4 nM (29)), the
amount of this ligand bound is an approximate measure of the amount of
this protein in the cell or fraction. As shown in Fig.
4, ~80% of the folate binding was localized to the
Triton X-100-insoluble domains in untreated cells. Surprisingly, the
total amount of folate receptor in the cell, but not its localization,
was affected in both the fumonisin B1- and
lovastatin-treated cells. While ~50% of the total folate receptor was lost with either sphingolipid or cholesterol depletion, ~80% of the remaining folate binding was found in the Triton
X-100-insoluble domain. As with the inhibition of
5-methyltetrahydrofolate uptake, the decrease in total folate binding
was dependent on both the duration of the treatment (Fig.
5A) and the concentration of fumonisin B1 used (Fig. 5B). While these results indicate
that the decrease in folate binding is mediated by the changes in lipid
content in the cell, the possibility that fumonisin
B1 directly affected binding of the vitamin was further
ruled out by the finding that the mycotoxin was unable to alter this
parameter when added directly to the binding assay. Therefore, changes
in the cholesterol or sphingolipid levels appear to decrease the total
amount of folate receptor in the cells, but do not change its
enrichment in Triton X-100-insoluble domains.
Relationship between Folate Receptor Function and Number
These results indicate that both the fumonisin
B1 and lovastatin treatments induce a loss or
down-regulation of the folate receptor. The relationship between the
change in folate receptor number and the inhibition of
5-methyltetrahydrofolate uptake was investigated by comparing these two
parameters (Fig. 6). A linear relationship
(r = 0.967) between the amount of folate receptor in
the cell and the ability to transport 5-methyltetrahydrofolate into the
cytoplasm was found. Therefore, it seems more likely that the
inhibition of vitamin uptake is caused by the loss of the folate
receptor than by a change in the membrane localization of the
protein.
Treatment of Caco-2 cells with fumonisin B1 resulted in almost complete inhibition of uptake of 5-methyltetrahydrofolate by the folate receptor. Consistent with it being mediated by the depletion of cellular sphingolipids, this inhibition was dependent on the concentration of mycotoxin used and the duration of the treatment. Fumonisin B1 did not perturb either 2-deoxyglucose or transferrin uptake, indicating that the effect of sphingolipid depletion was specific for folate receptor-mediated transport. The inhibition caused by lowering the cellular sphingolipid levels was very similar to that previously observed when cellular cholesterol was depleted by inhibition of hydroxymethylglutaryl-CoA reductase (13). Therefore, it seems likely that decreases in the cellular levels of these two lipids perturb folate receptor function by the same mechanism and involve the Triton X-100-insoluble domains.
Quantitation of the amount of folic acid binding activity in the Triton X-100-insoluble domains in fumonisin B1- and lovastatin-treated cells revealed that the lipid depletion did not cause redistribution of the folate receptor. Approximately 80% of the cellular folic acid binding capacity was localized to these cholesterol/sphingolipid-enriched domains in control and lipid-depleted cells. While localization was unaffected, the total amount of folic acid binding capacity was decreased by ~50% in fumonisin B1- and lovastatin-treated cells. That folate receptor function and number decrease in parallel as sphingolipids are depleted from Caco-2 cells suggests that there is a causal relationship between these two parameters. Growth of cells in medium containing low concentrations of folate, which presumably increases the need to take up this vitamin, has been found to induce folate receptor function (29). Therefore, inhibition of folate uptake, or compromised folate receptor function, could lead to a drop in the amount of this protein in the cell. Alternatively, a decrease in the level of folate receptor in the cell could result in inhibition of vitamin uptake. The data presented here do not address the question of whether the decrease in folate receptor number caused inhibition of vitamin uptake or vice versa. Both parameters appear to decrease with roughly similar rates (Figs. 2B and 5A). Curiously, conditions that resulted in nearly complete inhibition of 5-methyltetrahydrofolate uptake (20 µg/ml fumonisin B1 for 2 or 3 days) led to the loss of only half of the folate receptors in the cell. If vitamin uptake is compromised because of the decrease in the number of receptors, then why did the remaining folate receptors not support an intermediate level of folate transport into the cytosol? Perhaps the remaining folate receptors are not functional, or the altered lipid composition has affected some other critical component of the folate uptake pathway (e.g. folate polyglutamation).
This study clearly demonstrates the importance of sphingolipids for folate receptor-mediated vitamin uptake. However, the mechanism by which this process is affected is not clear. The finding that depletion of cellular cholesterol also inhibits folate receptor function (13) suggests that this effect is mediated by changes in the membrane domains enriched in cholesterol and sphingolipids to which the GPI-anchored folate receptor has been localized. Alternatively, folate receptor number and function could be altered in response to changes in the level of one or more intermediates in the synthesis of these lipids that have a signaling role in the cell. Recent evidence has implicated several sphingolipids, including sphingosine, sphingosine 1-phosphate, ceramide, and sphingomyelin, in signal transduction (reviewed in Refs. 30-33). Fumonisin B1 inhibition of sphingolipid biosynthesis should result in elevation of cellular long-chain bases and decreased levels of ceramide (34). In preliminary experiments, supplementation of fumonisin B1-treated cells (20 µg/ml for 2 days) with 1 µM C6-ceramide for 1 additional day (in the presence of fumonisin B1) has been found to reverse the effects on the folate receptor.3 Because long-chain bases should still be elevated, this result suggests that increases in the levels of sphinganine and/or sphingosine are not responsible for the effect on the folate receptor. However, whether it is the loss of mature sphingolipids or ceramide, which has been suggested to play a role in the regulation of endocytosis (35), that mediates the effects of fumonisin B1 cannot be determined from these experiments. Unraveling which of these is the critical factor will provide important information regarding the regulation of vitamin uptake mediated by the GPI-anchored folate receptor and will be addressed in future studies.
While fumonisin B1 has proven to be a useful reagent for probing the role of sphingolipids in various cellular processes (34), the fact that it is a naturally occurring compound raises the possibility that these same events could be adversely affected by dietary exposure to this mycotoxin. F. moniliforme, the fungus that produces fumonisin B1, is a common contaminant of corn. Exposure to fumonisin B1 causes a variety of animal diseases and has been linked to an increased incidence of esophageal cancer in humans in areas of southern Africa and China (36-39). Investigation into the consequences of fetal exposure to this mycotoxin using either mice or hamsters has shown that it causes developmental toxicity (40, 41). The mouse fetuses that survived to birth had gross skeletal and visceral abnormalities (42). Inhibition of folate uptake through fumonisin B1-induced depletion of sphingolipids could lead to an intracellular deficiency in this vitamin. Since folate deficiency during the first trimester of pregnancy is associated with an increased risk of neural tube defects in the developing fetus (43-45), it is possible that some instances of high rates of occurrence of these birth defects unexplained by known causes might be linked to dietary exposure to fumonisin B1. For instance, high rates of neural tube defects have been observed in Cameron County, TX from 1990 to 1991 (46, 47) and in Harris County, TX from 1989 to 1991 (48, 49). The prevalence of these birth defects was high among Hispanics (48), for whom corn and corn products are expected to represent a sizable portion of their diet. The occurrence of a high number of clusters of the fatal equine disease caused by fumonisin B1 in Texas in 1989 established that the corn crop was contaminated with this mycotoxin during this period. Coupled with the present finding that fumonisin B1-induced depletion of cellular sphingolipids blocked folate uptake, this evidence suggests that there should be further investigation into the possibility that this mycotoxin may contribute to some birth defects not accounted for by other known risk factors.
We thank Dr. Alfred Merrill for the gift of fumonisin B1 and for helping to bring this problem to our attention and Drs. David Lambeth and Satyajit Mayor for helpful discussions regarding this research.