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INTRODUCTION |
Glycosphingolipids
(GSLs)1 and sphingomyelin
(SM) are characteristic components of vertebrate plasma membranes and
have the same hydrophobic residue, ceramide, which consists of a
sphingosine and a fatty acid. GSLs have been defined as tumor antigens,
receptors for microbes and their toxins, and possible modulators of
cell proliferation, differentiation, and cell-cell interactions (1-3). Recently, ceramide has emerged as a novel second messenger for intracellular signaling pathways responding to various cytokines and
stress (4). Several lines of evidence indicate that a signaling ceramide is produced from SM by the action of endogenous neutral (5)
and acid sphingomyelinase (SMase; EC3.1.4.12) (6), or by de
novo synthesis (7). However, little is known about the mechanism
of regulation of intracellular level of ceramide, which should be
strictly regulated within cells.
Endoglycoceramidase (EC3.2.1.123) is a GSL-specific enzyme from
Rhodococcus sp. that hydrolyzes the glycosidic linkage of ceramide and sugar chains of various GSLs (8). The cell surface GSLs of
various erythrocytes (9) and cultured mammalian cells (10) were
hydrolyzed by the purified rhodococcal endoglycoceramidase (11) with
the assistance of its protein activator (12). We found that treatment
of B16 melanoma cells with a microbial endoglycoceramidase activated
GSL synthesis via transient up-regulation of UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1, glucosylceramide synthase; EC2.4.1.80) (13). As a result, cell surface
NeuAc
2,3Gal
1,4Glc
1,1ceramide (GM3), the end product of GSL
synthesis in B16 cells, was restored quickly when the enzyme was
removed from the culture medium (13).
In this study, we examined the effects of bacterial SMase on the
metabolism of GSLs and SM using B16 cells and their GSL-deficient mutant counterpart GM95 cells, which lack GlcT-1 (14). Although GSLs
were quickly restored after endoglycoceramidase treatment, restoration
of SM was not observed after treatment with bacterial SMase in B16
melanoma cells. Interestingly, ceramides generated from not only GSLs
but also SM by the microbial enzymes were primarily glucosylated by
GlcT-1, metabolized to GM3, and then transported to the plasma
membrane. Ceramide was accumulated during SMase treatment in GM95 or
B16 cells in the presence of
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (15) or N-butyldeoxynojirimycin (16), potent
inhibitors of GlcT-1. These results suggest a biological role of GlcT-1
in the regulation of intracellular ceramide content.
Because excess generation of ceramide is toxic to cells, GlcT-1 seems
to function for expulsion of ceramide from the cell. This regulation,
regarded as a putative detoxification mechanism, may function as a
defense against an unexpected increase of ceramide, which could be
caused by various forms of stress, e.g. infections with
pathogenic microorganisms that produce SMase or endoglycoceramidase. This paper also indicates the biological role of ceramide as a modulator of the overall synthesis of GSLs by regulating GlcT-1 at both
the transcriptional and post-translational levels.
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EXPERIMENTAL PROCEDURES |
Materials--
Monoclonal antibody M2590 and fluorescein
isothiocyanate-conjugated goat anti-mouse IgM were purchased from Cosmo
Bio Co., SMases from Staphylococcus aureus and
Bacillus cereus were obtained from Sigma and Funakoshi Co.,
respectively. D-Threo-PDMP and
N-hexanoylsphingosine (C6-ceramide) were purchased from
Matreya, and thin layer chromatography (TLC) plates (Silica Gel 60)
were from Merck. 14C-Labeled Gal, serine, choline, and
[32P]ATP were obtained from DuPont NEN, and
[3H]thymidine, [14C]palmitic acid, and
[14C]UDP-glucose were from American Radiolabeled
Chemicals. sn-1,2-Diacylglycerol kinase was kindly provided
by Dr. T. Okazaki (Kyoto University, Kyoto, Japan). All other chemicals
were of the highest grade available.
Cell Culture and SMase Treatment--
All experiments using B16
melanoma and the GlcT-1-deficient mutant GM95 cells (14) were conducted
at 37 °C in minimum essential medium (MEM) supplemented with 5%
fetal bovine serum (FBS) in a humidified 95% air, 5% CO2
incubator. SMase from S. aureus was used for all experiments
except that shown in Fig. 2, in which the enzyme from B. cereus was used.
Metabolic Labeling--
Cells were incubated in 200 µl of MEM
supplemented with 5% FBS containing 1 µCi of [14C]Gal,
[14C]serine, or [14C]choline for the times indicated.
Extraction and Determination of Radiolabeled
Sphingolipids--
Cells (1 × 105) were harvested by
centrifugation (2000 rpm for 5 min) and washed twice with
phosphate-buffered saline (PBS). Sphingolipids were extracted with 750 µl of i-propanol/hexane/water (55:35:10, v/v) in a
sonicator for 20 min. After centrifugation at 13,000 rpm for 5 min, the
supernatants obtained were dried under N2 gas, dissolved in
25 µl of chloroform/methanol (2:1, v/v), and applied to TLC plates,
which were developed with chloroform/methanol/0.02% CaCl2
(5:4:1, v/v) for GSLs and chloroform/methanol/acetic
acid/H2O (50:30:8:5, v/v) for SM. Radiolabeled
sphingolipids separated on TLC plates were analyzed and quantified
using a BAS1000 imaging analyzer (Fuji Film). Identification of
C6-ceramide GSLs was done by the method described by Komori and Ito
(17).
Two-dimensional TLC--
The radiolabeled total lipid extracts
from cells were spotted onto TLC plates (10 × 10 cm). The first
chromatographic run was performed with chloroform/methanol/formic
acid/H2O (65:25:8.9:1.1, v/v). The second run was performed
with chloroform/methanol/5 N NH4OH (50:40:10,
v/v) perpendicular to the original direction. Then the third run was
performed with diethylether in the opposite direction to the second run
to separate ceramides from other neutral lipids. Spots on the TLC
plates were identified using authentic standards (18).
Determination of SM Content--
Total lipids, extracted from
5-7 × 106 cells with a mixture of
i-propanol/hexane/water (55:35:10, v/v), were applied to TLC plates, which were then developed with chloroform/methanol/acetic acid/H2O (50:30:8:5, v/v). SM was visualized with Coomasie
Brilliant Blue (19) and quantified using a Shimadzu CS-9300
chromatoscanner with reflection mode set at 580 nm.
Determination of Ceramide Content--
Ceramide was extracted
from cells (2 × 106) with 3 ml of chloroform/methanol
(1:2, v/v), and 0.8 ml of water was added and mixed well. The organic
and aqueous phases were subsequently separated by addition of 1 ml of
chloroform and 1 ml of water followed by vigorous shaking and
centrifuged at 3000 rpm. The organic phase was carefully removed and
transferred to a new tube, and the samples were dried under
N2 gas. The amount of ceramide was measured using sn-1,2-diacylglycerol kinase as described (20). Ceramide
1-phosphate and phosphatidic acid were separated by TLC using
chloroform/acetone/methanol/acetic acid/H2O (10:4:3:2:1,
v/v) as a solvent system.
Cell Surface GM3 Staining with Monoclonal Antibody
M2590--
B16 cells (1 × 106) were incubated on ice
for 30 min with 100 µl of monoclonal antibody M2590 specific to GM3
(NeuAc) (21). After washing twice with PBS, B16 cells were treated with
100 µl of secondary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse IgM) on ice for 30 min and then analyzed by flow cytometry (FACScan, Becton Dickinson).
Measurement of DNA Synthesis--
Cells (2 × 104) were treated with 20 milliunits of SMase in 200 µl
of MEM supplemented with 5% FBS in 96-well plates at 37 °C for
14 h. After incubation, 10 µl of PBS containing 0.1 µCi of
[3H]thymidine was added to the medium. After incubation
at 37 °C for 4 h, cells were collected using a Combi 11025 cell
harvester (Skatron), and the incorporation of
[3H]thymidine into DNA was quantified by liquid
scintillation counting.
Assay of GlcT-1--
GlcT-1 activity was determined according to
the method of Basu et al. (22) with slight modifications. To
prepare cell lysates, cells were washed with 1 ml of PBS and suspended
in 50 µl of 10 mM Tris-HCl, pH 7.5, containing 2 mM KCl and 5 mM MgCl2. Standard incubation mixture (50 µl) contained 0.5% Triton X-100, 500 µM [14C]UDP-glucose (0.02 µCi/reaction),
0.3 mM ceramide (type III), and cell lysate (500 µg as
protein) in 20 mM Tris-HCl, pH 7.5. After incubation at
32 °C for 2 h, 100 µl of chloroform/methanol (2:1, v/v) was
added to terminate the reaction, and the lower layer was applied to TLC
plates, which were then developed with chloroform/methanol/12
mM MgCl2 (65:25:4, v/v). The
[14C]glucosylceramide produced was determined with an
imaging analyzer (BAS1000, Fuji Film).
Assay of SM Synthase--
Fifty µg of
C67-nitrobenz-2-oxa-1,3-diozole-ceramide was mixed with 500 µg of
phosphatidylcholine and 10 µg of sulfatide in 1 ml of distilled water
to form liposomes. To prepare cell lysates, cells (5 × 105) were washed with 1 ml of PBS and were suspended in 50 µl of 10 mM Tris-HCl, pH 7.5. The incubation mixture
contained 10 µl of liposomes, 500 µM CDP-choline, 1 mM EDTA, and cell lysate (50 µg as protein) in 50 µl of
16 mM Tris-HCl, pH 7.5. After incubation at 32 °C for 15 min, 100 µl of chloroform/methanol (2:1, v/v) was added to terminate
the reaction, and the lower layer was applied to TLC plates, which were
then developed with chloroform/methanol/12 mM
MgCl2 (65:25:4, v/v). The NBD-SM produced was determined
with a Shimadzu CS-9300 chromatoscanner (excitation, 475 nm; emission, 525 nm). The activity measured by this method appeared to show the
total activity of phosphatidylcholine-specific phospholipase C and
CDP-choline:ceramide cholinephosphotransferase.
Protein Determination--
Protein contents of cell extracts
were determined by the BCA method (Pierce) with bovine serum albumin as
the standard.
Isolation of Total RNA and Northern Blotting Analysis--
Total
RNA was isolated from cells with an RNeasy mini kit (Qiagen). Aliquots
of 30 µg of total RNA were subjected to electrophoresis on 1%
agarose gels containing 18% formaldehyde, and transblotted onto nylon
membranes (Hybond-N, Amersham Pharmacia Biotech). The 1.2-kb
BamHI-XhoI cDNA fragment of the mouse GlcT-1 gene
was labeled with [
-32P]dCTP (6000 Ci/mmol, Amersham
Pharmacia Biotech) by the Multiprime DNA labeling system (Amersham
Pharmacia Biotech) and used as a probe. Hybridization was carried out
at 42 °C for 24 h in 5 × SSPE (750 mM NaCl,
43.3 mM NaH2PO4, 6.25 mM EDTA) containing 50% formamide, 4% SDS, 5 × Denhardt's solution, salmon sperm DNA (100 µg/ml), and
32P-labeled probe (23). After hybridization, the membrane
was washed continuously with 2 × SSPE containing 0.5% SDS,
1 × SSPE containing 0.5% SDS, and 0.1 × SSPE containing
0.5% SDS at 50 °C for 40 min at each step. The membranes were also
hybridized by the method described above but with a
-actin cDNA
probe for normalization of mRNA levels. mRNA levels were
quantified using an imaging analyzer (BAS1000, Fuji Film).
Determination of Fluorescent C6-NBD
Sphingolipids--
NBD-labeled C6-ceramide was added to cells (1 × 106) and incubated at 37 °C in a CO2
incubator for the times indicated. Cells were harvested by
centrifugation (2000 rpm for 5 min) and washed twice with PBS.
Sphingolipids were extracted with 750 µl of
i-propanol/hexane/water (55:35:10, v/v) in a sonicator for
20 min. After centrifugation at 13,000 rpm for 5 min, the supernatants
obtained were dried under N2 gas, dissolved in 25 µl of
chloroform/methanol (2:1, v/v), and applied to TLC plates, which were
then developed with chloroform/methanol/12 mM
MgCl2 (65:25:4, v/v). The NBD-sphingolipids produced were
determined with a Shimadzu CS-9300 chromatoscanner (excitation, 475 nm;
emission, 525 nm).
 |
RESULTS |
Effects of SMase Treatment on Sphingolipid Metabolism in B16
Cells--
We previously reported that endoglycoceramidase treatment
of B16 melanoma cells stimulated GSL synthesis (13). Because ceramide is the common lipid backbone of GSLs and SM, we examined the metabolism of SM-derived ceramide using bacterial SMase instead of
endoglycoceramidase. The ceramide portions of SM and GSLs of B16 cells
were metabolically labeled with [14C]serine, washed with
fresh MEM, and then treated with SMase from S. aureus.
Two-dimensional TLC revealed that SMase treatment markedly reduced the
content of [14C]SM to 49% of that in controls, whereas
that of [14C]GM3 simultaneously increased to 197% of the
control level (Fig. 1A). To
avoid the influence of de novo synthesis of ceramide, the
same experiment was conducted but in the presence of Fumonisin B1, an
inhibitor for acyl-CoA:sphinganine N-acyltransferase (24). Even in the presence of Fumonisin B1, the increase in GM3 concomitant with the decrease in SM was observed after SMase treatment (Fig. 1A), indicating that the increase in GM3 level was not
attributable to an increase in de novo synthesis of
ceramide. During SMase treatment, [14C]Gal uptake into
ceramide monohexoside (CMH) and GM3 was increased in B16 cells (Fig.
1B). However, the level of sphingosine was not changed after
SMase treatment (data not shown). These results strongly suggest that
increased GM3 was metabolized from SM-derived ceramide by direct
glycosylation but not from sphingoid base salvaged from hydrolysis of
ceramide. The increase of GM3 was also confirmed by flow cytometry
using a GM3-specific monoclonal antibody, M2590, indicating that the
increased GM3 was actually transported to the cell surface (Fig.
1C).

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Fig. 1.
Effects of SMase treatment on sphingolipid
metabolism in B16 cells. A, two-dimensional TLC of
14C-labeled sphingolipids of B16 cells. B16 cells (1 × 105) were incubated in MEM supplemented with 5% FBS
containing 1 µCi of [14C]serine for 24 h, washed
with fresh MEM, and recultured in MEM containing 20 milliunits of SMase
for 18 h in the presence or absence of Fumonisin B1 (100 µM). Total 14C-labeled lipids were extracted,
separated by TLC, and analyzed with an imaging analyzer as described
under "Experimental Procedures." A, results in the
absence of Fumonisin B1. B, de novo synthesis of
GSLs in B16 cells during SMase treatment. B16 cells (1 × 105) were metabolically labeled with 1 µCi of
[14C]Gal in the presence or absence of 20 milliunits of
SMase in 200 µl of MEM supplemented with 5% FBS at 37 °C for
18 h. 14C-labeled GSLs were extracted, separated by
TLC, and analyzed with an imaging analyzer. Values are the means ± S.D. for triplicate determinations. PSL, photo-stimulated
luminescence/mm2. C, cytofluorometric analysis
of cell surface GM3. B16 cells (1 × 106) were
incubated with 100 milliunits of SMase in 1 ml of MEM supplemented with
5% FBS at 37 °C for 18 h. Cells with or without
(Control) SMase treatment were incubated with M2590
monoclonal antibody followed by a second incubation with fluorescein
isothiocyanate-conjugated goat anti-mouse IgM and analyzed by flow
cytometry as described under "Experimental Procedures."
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An increase in GSL synthesis during SMase treatment was also observed
in not only B16 cells but also other cell lines, as shown by the
synthesis of CMH in HL60 human myelocytic leukemia cells, which
increased by not less than 2-fold during SMase treatment.
Effects of SMase on GlcT-1 and SM Synthase Activities, GSL
Synthesis, and GlcT-1 mRNA--
The GlcT-1 activity and de
novo synthesis of GSLs in B16 cells were examined with or without
SMase treatment. Interestingly, GlcT-1 activity in the cell lysate
increased by ~2-fold by the enzyme treatment, whereas SM synthase
activity was not affected (Fig.
2A). It was confirmed that
GM95 cells completely lacked GlcT-1 activity (data not shown). The
activation of GlcT-1 seems to occur post-translationally, because even
in the presence of cycloheximide an increase in GSL synthesis was
observed after SMase treatment (Fig. 2B). On the other hand,
Northern blotting analysis indicated that SMase treatment slightly
activated transcription of the GlcT-1 gene (Fig. 2C).
Treatment of B16 cells with C6-ceramide also increased GlcT-1 mRNA
expression in a concentration-dependent manner (Fig.
2D). These results indicate that up-regulation of GSL
synthesis by SMase treatment occurs at the level of both transcription and post-translation of the GlcT-1 gene, and also that ceramide could
modulate the overall synthesis of GSLs via regulation of GlcT-1.

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Fig. 2.
Effects of SMase on GlcT-1 and SM synthase
activities, GSL synthesis, and GlcT-1 mRNA expression.
A, GlcT-1 and SM synthase activities. B16 cells (6 × 106) were treated with 500 milliunits of
Bacillus SMase in 5 ml of MEM supplemented with 5% FBS.
After incubation at 37 °C for 18 h, cells were washed with PBS,
and the activities of GlcT-1 (500 µg as protein) and SM synthase (50 µg as protein) in cell lysates were determined by the method
described under "Experimental Procedures." B, GSL
synthesis after SMase treatment in the presence or absence of
cycloheximide. After treatment with 20 milliunits of
Bacillus SMase for 3 h, B16 cells were washed with
fresh MEM and metabolically labeled with 1 µCi of
[14C]Gal for 3 h in the same medium without SMase.
GSLs were extracted and analyzed by the method described in the legend
to Fig. 1B. PSL, photo-stimulated
luminescence/mm2. C and D, Northern
blotting analysis of GlcT-1 mRNA. B16 cells (6 × 106) were treated with 500 milliunits of
Bacillus SMase for 3 h (C) or C6-ceramide at
the indicated concentration for 12 h (D) in 5 ml of MEM
supplemented with 5% FBS. Total RNA was isolated from cells using an
RNeasy mini kit (Qiagen) and analyzed by the method described under
"Experimental Procedures." *, p < 0.05 by
t test versus control values (n = 3). In D, values are the means for duplicate determinations.
Cont, control.
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SM Restoration--
The content of GM3 in B16 cells was reduced by
endoglycoceramidase treatment but recovered quickly after the enzyme
was removed from the culture medium (13). However, no restoration in SM content was observed after removal of SMase when B16 cells were prelabeled with [14C]choline (Fig.
3). GM95 cells also showed no rapid
restoration of SM (Fig. 3). It should be noted that this restoration
was estimated by a base exchange reaction between ceramide and
phosphatidylcholine, which is considered to be the major pathway of SM
synthesis (25). However, a portion of SM (<25%) appeared to recover
in B16 cells after removal of the enzyme and reculture for 9 h
when the SM mass was estimated by Coomasie Brilliant Blue staining.
This increase in SM seemed to be derived from de novo
synthesis of ceramide and not from the recycling of ceramide.

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Fig. 3.
SM content after SMase treatment. B16
and GM95 cells (1 × 105) were metabolically labeled
with 1 µCi of [14C]choline for 24 h, washed, and
then treated with 20 milliunits of SMase in 200 µl of MEM containing
5% FBS at 37 °C for 3 h. Cells were washed twice with fresh
MEM and recultured in the same medium without the enzyme for 6 or
9 h. The total lipids were extracted, separated by TLC, and
determined with an imaging analyzer as described under "Experimental
Procedures." Values are the means ± S.D. for triplicate
determinations.
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Accumulation of Ceramide after SMase Treatment--
The ceramide
content of B16 cells, which was measured by
sn-1,2-diacylglycerol kinase assay, increased by ~2-fold
after SMase treatment for 3 h but gradually decreased after
removal of the enzyme and returned to the basal level after 6-9 h. In
contrast, in GlcT-1-deficient mutant GM95 cells the accumulation of
ceramide by SMase treatment was much higher than in the parental cells and was maintained after the enzyme was removed from the culture medium
(Fig. 4A). Interestingly,
SMase treatment of B16 cells in the presence of PDMP, a potent
inhibitor of GlcT-1, markedly increased the accumulation of ceramide
(Fig. 4B). Ceramide generation in B16 cells by bacterial
SMase was also enhanced 2.6 times by addition of
N-butyldeoxynojirimycin at 200 µM, which is an
another inhibitor of GlcT-1 (16). These results suggest that GlcT-1 functions to remove the excess ceramide generated by bacterial SMase.

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Fig. 4.
Effects of SMase treatment on ceramide
content. A, ceramide contents in B16 and GM95 cells after
SMase treatment. Cells (6 × 106) were treated with
500 milliunits of SMase in 5 ml of MEM supplemented with 5% FBS at
37 °C for 3 h, washed twice with fresh MEM, and recultured in
the same medium without the enzyme for 6 or 9 h. Ceramide content
was determined by the method described under "Experimental
Procedures." Values are the means ± S.D. for triplicate
determinations. B, ceramide content in B16 cells after SMase
treatment with or without PDMP. B16 cells (6 × 106)
were pretreated at 37 °C for 1 h with 40 µM PDMP
in 5 ml of MEM supplemented with 5% FBS, and the medium was changed to
the same medium containing 500 milliunits of SMase and followed by
reculture for 3 h. Ceramide content was determined by the method
described under "Experimental Procedures." Values are the
means ± S.D. for triplicate determinations.
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Effects of Ceramide on DNA Synthesis--
Although ceramide
functions as a novel class of second messenger (4), the excess
generation of ceramide must be harmful to the cell. As shown in Fig.
5A, DNA synthesis of GM95
cells measured by the incorporation of [3H]thymidine was
significantly inhibited by bacterial SMase treatment, whereas this
enzyme treatment seemed to have no effect on that of B16 cells (Fig.
5A). However, in the presence of PDMP, SMase treatment
appeared to be harmful for B16 cells (Fig. 5A). The degree
of inhibition of DNA synthesis was consistent with that of accumulation
of ceramide (Fig. 4). Hidari et al. (26) reported that the
treatment of GM95 cells with the bacterial SMase disrupted cell-substratum adhesion, and the cells were peeled off from dishes. However, in this experiment a much lower concentration of SMase was
used to avoid disrupting the adhesion. The effects of the short chain
ceramide, C6-ceramide, on DNA synthesis of B16 and GM95 cells were also
examined. Incubation with 50 µM C6-ceramide for 3 h
strongly suppressed DNA synthesis in GM95 cells but not in B16 cells
(Fig. 5B). This result is consistent with that using SMase
(Fig. 5A).

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Fig. 5.
Effects of ceramide on DNA synthesis.
A, effects of SMase treatment on DNA synthesis. Cells
(2 × 104) were pretreated with 20 milliunits of SMase
in 200 µl of MEM supplemented with 5% FBS. After incubation at
37 °C for 14 h, 10 µl of PBS containing 0.1 µCi of
[3H]thymidine was added to the cultures and incubated at
37 °C for 4 h. Cells were harvested, and incorporation of
[3H]thymidine into DNA was quantified by liquid
scintillation counting. Values are the means ± S.D. for
triplicate determinations. B, effects of C6-ceramide on DNA
synthesis. Cells (2 × 104) were incubated at 37 °C
for 3 h in MEM supplemented with 5% FBS containing 50 µM C6-ceramide and 0.1 µCi of
[3H]thymidine. The incorporation of
[3H]thymidine into DNA was measured by the method
described above. Values are the means ± S.D. for triplicate
determinations. DPM, disintegrations per minute.
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Conversion of Short Chain Ceramide to GSLs and SM--
When
C6-ceramide was added to B16 cell cultures in the presence of
[14C]Gal, C6-CMH and C6-GM3 were generated, both of which
were also released into the medium (Fig.
6A). However, glycosylation of the short chain ceramide was not found in cultures of GM95 cells (Fig.
6A). To examine the ratio for conversion of ceramide to GSLs
and SM, NBD-C6-ceramide was added to cultures of B16 and GM95 cells.
The NBD-C6-ceramide was exhausted in B16 cells much more rapidly than
in GM95 cells (Fig. 6B). In B16 cells, NBD-C6-ceramide was
converted to NBD-C6-CMH and NBD-C6-SM at the same rate by 15 min, but
thereafter the generation of NBD-C6-CMH was much faster than that of
NBD-C6-SM. In GM95 cells, NBD-C6-ceramide was converted to NBD-C6-SM,
but not to NBD-C6-GSLs, and the conversion reached a plateau by 15 min
when 70% of NBD-C6-ceramide still remained in the cells. These results
indicated that the exclusion of ceramide in B16 cells was mainly
achieved by glycosylation. The preference for glycosylation of ceramide
seems to be restricted to ceramide generated from GSLs (13) and SM on
the plasma membrane, because the de novo synthesis of GM3
and SM in B16 cells was almost 1:1 when [14C]serine was
used as a precursor and chased for 30 min and also 18 h.

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Fig. 6.
Conversion of short chain ceramide to
sphingolipids. A, generation of C6-GM3 in B16 cells. B16 or
GM95 cells (2 × 105) were incubated at 37 °C for
3 h in 200 µl of MEM supplemented with 5% FBS containing 1 µCi of [14C]Gal and 50 µM C6-ceramide.
The total lipids were extracted separately from cells and medium and
analyzed by TLC as described under "Experimental Procedures."
Lane 1, control without short chain ceramide (cells);
lane 2, 50 µM C6-ceramide (cells); lane
3, control without short chain ceramide (culture supernatant);
lane 4, 50 µM C6-ceramide (culture
supernatant). B, time course for conversion of
NBD-C6-ceramide to NBD-sphingolipids. B16 cells were treated with 5 nmol of C6-NBD-ceramide, followed by incubation at 37 °C for the
indicated times. Total lipids were extracted from cells and analyzed by
TLC. 100% represents the total fluorescence of sphingolipids
(NBD-ceramide (Cer) + NBD-CMH + NBD-SM). Details are
described under "Experimental Procedures." Values are the means for
duplicate determinations.
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In conclusion, this study has clearly demonstrated the biological role
of GlcT-1 in regulation of the intracellular ceramide content;
i.e. genetic and pharmacological blockade of GlcT-1 results in accumulation of ceramide and inhibition of DNA synthesis after SMase
treatment in B16 melanoma cells.
 |
DISCUSSION |
Recently, Zhang et al. (27) reported the expression of
a recombinant B. cereus SMase in Molt-4 leukemia cells.
After the transfection of the gene and stable expression of the SMase,
intracellular ceramide content increased, resulting in induction of
apoptosis. However, exogenously added B. cereus SMase,
despite causing a greater elevation of ceramide level, did not induce
apoptosis in Molt-4 cells (27). This result suggested the existence of two distinct SM pools, one of which is accessible by endogenous SMase
and the other by exogenous SMase. The former seems to be responsible
for transduction of the apoptotic signal, and the turnovers of these
two pools might be somewhat different. On the other hand, some reports
indicated that exogenous bacterial SMase had biological effects on
cells. For example, the Streptomyces SMase enhanced the
action of subthreshold vitamin D3 in inducing HL60 cell differentiation
(28), and the enzyme from S. aureus induced apoptosis of
U937 cells (29). In the present study, treatment of B16 cells with
bacterial SMase inhibited DNA synthesis under conditions of genetic or
pharmacological blockade of GlcT-1. These results suggest that the
localization and topology of SM and their susceptibility to SMase
differ according to cell type.
Luberto and Hannun (30) reported that treatment of human lung
fibroblast WI38 cells with bacterial SMase resulted in a decrease in SM
level and concomitant generation of ceramide. This ceramide level
decreased very slowly in the cells, but there was little restoration of
SM content. In contrast, SV40-transformed cells, in which the activity
of SM synthase (phosphatidylcholine-specific phospholipase C) was found
to be 3-fold higher than that in parental cells, cleared ceramide much
more rapidly and regenerated SM. The authors argued the potential
significance of SM synthase for regulation of intracellular ceramide
levels in the fibroblasts. We showed, on the other hand, that in B16
melanoma cells the ceramides generated from SM as well as GSLs (13) by
microbial enzymes were primarily glucosylated by GlcT-1 and metabolized
to GM3. This discrepancy may be attributable to the balance between
GlcT-1 and SM synthase activities in cells, which is genetically
defined depending on the origin of cells or their phenotype and might be affected by other environmental factors.
The present study revealed that ceramide generated on the outer leaflet
of the plasma membrane by bacterial SMase was directly, but not via the
sphingoid-base salvage pathway (31), metabolized to GSLs in B16
melanoma cells. Because the catalytic domain of GlcT-1 is located on
the cytosolic side of the Golgi membrane (32), the generated ceramide
must be translocated to the outer leaflet of the Golgi membrane before
it becomes accessible to the enzyme. Although the transportation of
ceramide to the Golgi membrane remains unclear, our findings suggest
that the transport of ceramide in protein-directed (33) and
vesicle-independent manners (34) is significant.
Many pathogenic and opportunistic microbes produce SMases, some of
which have been identified as hemolysins and cytotoxins (35). These
observations indicate that cell surface SM of vertebrates might be
exposed to the action of microbial SMase, which may result in the
elevation of the intracellular ceramide level. Because the excess
generation of ceramide must be toxic for the cell, the exclusion of
ceramide from the cell by glycosylation can be regarded as a mechanism
of defense against infection by SMase-producing pathogens. It is
interesting to note that many opportunistic pathogens can also produce
endoglycoceramidase extracellularly (13).
Lavie et al. (36, 37) reported that multidrug-resistant
human breast cancer cells exhibited marked accumulation of
glucosylceramide compared with the parental cells. The reverse
multidrug resistance drug tamoxifen was found to inhibit GlcT-1,
resulting in a decrease in the level of glucosylceramide and an
increase in that of ceramide. This drug as well as
1-phenyl2-palmitoyoamino-3-morpholino-1-propanol, an inhibitor of
GlcT-1, sensitized the multidrug-resistant cells to some anticancer
drugs. These results suggested that GlcT-1 is involved in regulation of
ceramide levels, which may affect the sensitivity of cancer cells to
anticancer drugs.
We conclude that GlcT-1, distributed ubiquitously in vertebrate cells,
functions to regulate the level of intracellular ceramide by
glycosylation of the ceramide when it is present in excess.