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J Biol Chem, Vol. 274, Issue 30, 21121-21127, July 23, 1999
,From the Glycobiology Program, Center for Cancer and Transplantation Biology, Children's Research Institute, Washington, D.C. 20010 and the Departments of Pediatrics and Biochemistry/Molecular Biology, The George Washington University School of Medicine, Washington, D. C. 20052 and the § Lipid Cell Biology Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Ceramide is a newly discovered second messenger
that has been shown to cause cell growth arrest and apoptosis. Here, we
present evidence that exogenously added C6-ceramide
induces enlargement of late endosomes and lysosomes. 10 µM C6-ceramide caused the formation of
numerous vesicles of varying sizes (2-10 µm) in fibroblasts (3T3-L1
and 3T3-F442A), without toxic effects. Vesicle formation induced by
C6-ceramide was time- and dose-dependent,
rapid, and reversible. Numerous small vesicles appeared within 8 h
of treatment with 10 µM C6-ceramide. They
enlarged with time, with large vesicles found in the perinuclear region
and small ones observed at the cell periphery. Within 24 h of
treatment, ~30% of the cells exhibited these vesicles. Removal of
ceramide from the culture medium caused disappearance of the vesicles,
which reappeared upon readdition of ceramide. Confocal
immunofluorescence microscopic analysis using an
anti-lysosome-associated membrane protein antibody identified the
enlarged vesicles as late endosomes/lysosomes. The fluorescent C6-NBD-ceramide, a vital stain for the Golgi apparatus,
did not stain these vesicles. The effect on vesicle formation was
influenced by ceramide structure;
D-erythro-C6-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as
sphingosine, sphingosine 1-phosphate,
N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C2-ceramide
GM3, (GM3,
N-acetylneuraminosyl- Sphingolipids and their metabolites, a new class of intracellular
second messengers, have been implicated in a spectrum of biological
processes, including cell growth, differentiation, and apoptosis (1).
It has been proposed that ceramide, generated by the hydrolysis of
sphingomyelin in the cell membrane through the sphingomyelin pathway
(2), mediates apoptosis through the SAPK cascade in response to tumor
necrosis factor- In addition to these well documented biological functions, ceramide may
also regulate protein secretion (6) and endocytosis (7). Vesicular
transport is essential for the biogenesis and maintenance of cellular
organelles and the transport of proteins and lipids. Using
C6-NBD-ceramide1
and 14C-labeled C6-ceramide as probes,
exogenous C6-ceramide has been shown to accumulate in the
Golgi apparatus (8-10), where it is converted to sphingomyelin and
glycosphingolipids (9, 11, 12). Incubation of vesicular stomatitis
virus-infected Chinese hamster ovary cells with C6-ceramide
decreased the rate of viral glycoprotein transport through the Golgi
apparatus and reduced the number of infectious virions released from
cells in a concentration-dependent manner (6). Short
incubation of Chinese hamster ovary cells with C6-ceramide
(C6-ceramide-bovine serum albumin complexes) at 4 °C
followed by horseradish peroxidase at 37 °C inhibited the uptake of
horseradish peroxidase by these cells and slowed horseradish peroxidase
and LDL transport from endosomes to lysosomes (7). These studies point
to a possible effect of ceramide on the process of vesicular transport.
Here, we present evidence that C6-ceramide induces
endocytic vesicle formation, causing enlarged late endosomes and
lysosomes in two mouse embryonic fibroblast cell lines, 3T3-L1 and
3T3-F442A.
Preparation of Ceramide and Other Sphingolipid Stock
Solutions--
N-Hexanoyl-D-erythro-sphingosine
(C6-ceramide),
N-acetyl-D-erythro-sphingosine
(C2-ceramide),
N-octanoyl-D-erythro-sphingosine (C8-ceramide), D-erythro-sphingosine,
N-hexanoyl-sphingosylphosphorylcholine (C6-Cer sphingomyelin), and N-acetylpsychosine
(C2-Cer cerebroside) were purchased from Matreya, Inc.
(Pleasant Gap, PA) and Molecular Probes, Inc. (Eugene, OR).
D-Erythro-sphingosine 1-phosphate was purchased from
Calbiochem (La Jolla, CA). C2-Cer GM3 was
synthesized and provided by Drs. Akira Hasegawa and Makoto Kiso of Gifu
University (Gifu, Japan). [Hexanoyl
1-14C]N-hexanoyl-D-erythro-sphingosine
and [octanoyl
1-14C]N-octanoyl-D-erythro-sphingosine
were purchased from American Radiolabeled Chemicals, Inc. (St. Louis,
MO). Ceramide and other sphingolipids were prepared in ethanol (except
for D-erythro-sphingosine 1-phosphate, which was dissolved
in methanol) at 4 mM and stored at Cell Culture--
The cell lines used in this study were
purchased from American Type Culture Collection (Manassas, VA). The
mouse embryonic fibroblast cell line 3T3-L1 was cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) with 10% fetal
bovine serum (Hyclone, Logan, UT); 3T3-F442A cells were cultured in
Iscove's modified Dulbecco's medium (Life Technologies, Inc.) with
10% fetal bovine serum. These fibroblasts were adherent to the plastic and grown as monolayers. For cell passage, cells were seeded at 2.4-4.0 × 103 cells/cm2 in culture
flasks, and the culture medium was changed every 3 days. Cell viability
was assessed by trypan blue dye exclusion.
Cell Proliferation Assays--
The effect of ceramide on
proliferation of 3T3-L1 cells was assessed by direct cell counting and
by [3H]thymidine uptake. To determine cell numbers,
3T3-L1 cells were seeded at 105 cells/25-cm2
flask (~4 × 103 cells/cm2) and cultured
for 24 h. The culture medium was then replaced with 7 ml of fresh
medium containing either C6-ceramide (10-20 µM) or ethanol. The cells were counted at 24-h intervals.
For the [3H]thymidine incorporation assay, 3T3-L1 cells
were plated at 103 cells/well in 96-well cell culture
clusters (area, 0.32 cm2; Costar). One day after plating,
the culture medium was replaced with 100 µl of fresh medium/well
containing either C6-ceramide (10-20 µM) or
ethanol. At 24-h intervals, triplicate cultures were pulsed for 3 h with 0.5 µCi of [3H]thymidine per well. The cells
were harvested, and cellular [3H]thymidine incorporation
was determined.
Induction of Vesicle Formation by
C6-ceramide--
3T3-L1 cells were seeded at
105 cells/25-cm2 flask or 2.2 × 10
5 cells/culture dish (100 × 20 mm; growth area, 55 cm2) in complete culture medium, and were exposed to either
C6-ceramide or other sphingolipids at final concentrations
of 5-20 µM on the following day. Cells cultured in
medium containing ethanol were used as controls. The cell number was
~2 × 105 cells/25-cm2 flask and
~4 × 105 cells/culture dish when
C6-ceramide or other sphingolipids were added to the
culture medium. The culture volume was 7 ml/25-cm2 flask
and 10 ml/culture dish (100 × 20 mm). To evaluate the possible effect of cell density on vesicle formation, cells were also examined at higher densities (0.5-1.5 × 106
cells/25-cm2 flask). Vesicle formation (the number of cells
bearing vesicles and the number of vesicles appearing in each cell) was
assessed at different time points, and the cells were photographed
under a phase-contrast microscope.
Measurement of Ceramide Uptake by 3T3-L1 Cells--
3T3-L1 cells
were plated in 96-well cell culture clusters (area, 0.32 cm2; Costar) at three densities: 1 × 103,
5 × 103, and 1 × 104 cells/well.
After an 18-h incubation, in which the cell number doubled, the culture
medium was replaced with 100 µl of fresh medium containing 10 µM 14C-labeled C6-ceramide
(N-[hexanoyl
1-14C]hexanoyl-D-erythro-sphingosine; specific
activity, 50 mCi/mmol) or 10 µM 14C-labeled
C8-ceramide (N-[octanoyl
1-14C]octanoyl-D-erythro-sphingosine; specific
activity, 53 mCi/mmol) (American Radiolabeled Chemicals Inc., St.
Louis, MO) per well. At different time points, cells were harvested,
and the uptake of 14C-labeled ceramide was determined.
Confocal Immunofluorescence Microscopy--
3T3-L1 cells were
seeded in two-well tissue culture slide chambers (2 × 104 cells/well) (Lab-Tek, Nunc, Inc., Naperville, IL),
which contained 2 ml of culture medium. The cells were cultured for
24 h in normal culture medium and then exposed to
C6-ceramide for 24 h. The cells were fixed in 3%
(w/v) paraformaldehyde in phosphate buffered saline for 30 min and then
processed as described previously (13). After the cells were
permeabilized by saponin (1 mg/ml), lysosomes were stained with the
anti-lysosome-associated membrane protein (LAMP) antibody, ABL-93 (14)
at 4 °C overnight, followed by rhodamine labeled goat anti-rat IgG
(Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at
room temperature. Cellular neutral lipids were detected by staining
with 0.01% Nile Red (Molecular Probes) in phosphate-buffered saline
for 10 min. In selected experiments, cells were pretreated with 10 µM C6-ceramide to induce the appearance of
vesicles and were then incubated at 37 °C for 15 min with 20 µM C6-NBD-ceramide (Molecular Probes). After
extensive washing with phosphate-buffered saline, cells were either
cooled to 4 °C and fixed or further incubated at 37 °C for 2 h and then fixed, to trace the intracellular localization of
C6-NBD-ceramide. All cells were mounted in buffered
glycerol containing para-phenylenediamine (15) to prevent
fading and analyzed with an LSM-410 laser scanning confocal imaging
system equipped with an Omnichrome krypton-argon laser (Carl Zeiss,
Thornwood, NY).
Effect of C6-ceramide on Proliferation of 3T3-L1
Cells--
Because ceramide has been shown to cause cell growth arrest
and apoptosis (1), we first examined the effect of
C6-ceramide on 3T3-L1 cell proliferation. The doubling time
for 3T3-L1 cells was determined to be ~16 h. As shown in Fig.
1A, C6-ceramide
had a dose-dependent inhibitory effect on cell growth.
Treatment of the cells with 10 µM C6-ceramide
had only a minor effect on cell growth, whereas 15 µM
C6-ceramide caused modest inhibition of growth of 3T3-L1
cells, without reducing cell viability, as assessed by trypan blue dye
exclusion. At 20 µM, cell growth was totally blocked, but
nearly all the adherent cells were viable. In parallel with this
inhibition of cell proliferation, the rate of DNA synthesis (measured
as [3H]thymidine uptake) of cells exposed to
C6-ceramide was inhibited (Fig. 1B). Finally,
when cells were treated with higher concentrations of
C6-ceramide (>20 µM) for prolonged periods
of time, there was detectable cell death, consistent with previous
reports (16).
Induction of Enlarged Vesicles by
C6-ceramide--
C6-ceramide has been shown to
regulate protein secretion and endocytosis under certain conditions (6,
7). To investigate the possible role of ceramide in vesicular
trafficking, we studied the effect of C6-ceramide on
vesicle formation in the mouse embryonic fibroblast cell line 3T3-L1.
When these cells were treated with 10 µM
C6-ceramide for 24 h, numerous vesicles of varying
sizes (2-10 µm) were visualized by phase-contrast microscopy (Fig.
2). Large vesicles were found in the
perinuclear region, whereas small ones were present at the cell
periphery (Fig. 2, B and C). The effect of
ceramide was dose-dependent. Within 24 h, 30% of the cells treated with 10 µM C6-ceramide bore
vesicles (Fig. 2D). This increased to 67 and 74% when the
cells were treated with 15 and 20 µM
C6-ceramide, respectively.
The effect of ceramide was also time-dependent. Sequential
observations revealed that initial vesicle formation was quite rapid,
with numerous small vesicles starting to appear within 8 h
following the treatment of the cells with 10 µM
C6-ceramide. These vesicles gradually became larger.
Vesicle formation reached its maximal level on day 2. The vesicles then
began disappearing, and after 4 days, most of the vesicles had
disappeared unless fresh ceramide was added to the culture medium.
When ceramide was removed from the culture medium and the cells were
cultured in fresh medium without ceramide, nearly all of the vesicles
disappeared within 24 h (Fig. 3).
Re-exposure of the cells to ceramide caused the vesicles to reappear
within 24 h, indicating that these cells can be repeatedly induced
to form enlarged vesicles and that this formation of enlarged vesicles
caused by C6-ceramide is reversible. We confirmed the above
effects of C6-ceramide on vesicle formation in another
mouse fibroblast cell line, 3T3-F442A. When treated with 10-20
µM C6-ceramide, these cells also demonstrated
enlarged vesicles, similar to those of the 3T3-L1 cells (not shown),
suggesting that exogenous C6-ceramide may induce such
vesicle formation in mouse fibroblasts in general.
Influence of Cell Density on Ceramide-induced Vesicle
Formation--
To evaluate the possible influence of cell density on
vesicle formation, we examined the effect of C6-ceramide on
vesicle formation over a range of cell densities (0.2-1.5 × 106 cells/25-cm2 flask). The vesicles were
prominent over the density range of 2-4 × 105
cells/25 cm2 when exogenous C6-ceramide was
added to the culture medium (Fig. 2). The vesicle formation induced by
C6-ceramide also occurred at higher densities. For example,
when 3T3-L1 cells were treated for 24 h with 15 µM
C6-ceramide at the density of 0.55-1.5 × 106 cells/25-cm2 flask (the assay starting and
ending cell densities, respectively), 67% of the cells bore vesicles
(Fig. 4B). Further studies
revealed that confluent and nearly confluent cells were less sensitive to the effect of C6-ceramide than were the more rapidly
proliferating cell populations, i.e. those at lower
densities (20-70% of confluence).
To determine whether this influence of cell density on vesicle
formation might reflect the relative availability of
C6-ceramide to the cells, we measured uptake of
radiolabeled C6-ceramide at three different cell densities:
2 × 103, 1 × 104, and 2 × 104 cells/well in 96-well cell culture clusters (area, 0.32 cm2). The uptake of C6-ceramide by 3T3-L1 cells
decreased with increasing cell density (Fig.
5A). At the density of 2 × 103 cells/well, 3T3-L1 cells incubated with 10 µM 14C-labeled C6-ceramide (1 nmol/well) underwent prominent vesicle formation and had a maximal
uptake of 7.5-9.8 pmol/103 cells. This uptake is in the
same range as the previously reported uptake of ceramide by other cell
types (17, 18). At the higher density of 2 × 104
cells/well, uptake of the 14C-labeled
C6-ceramide was lower (3.0-3.9 pmol/103 cells;
Fig. 5A), suggesting that the reduced ceramide effect at
high cell density is at least partially due to decreased uptake of
C6-ceramide molecules by the cells.
Characterization of C6-ceramide-induced Vesicles by
Confocal Immunofluorescence Microscopic Analysis--
We characterized
the vesicles induced by C6-ceramide using confocal
microscopic analysis. The mouse embryonic fibroblast cell line 3T3-L1
is one of the well established models that undergoes differentiation
into mature lipid-laden adipocytes (19). Under a phase contrast
microscope, the vesicles induced by C6-ceramide appeared
much like the fat droplets known to exist in differentiated 3T3-L1
cells. Thus, 3T3-L1 cells treated with C6-ceramide were first stained with Nile Red with the expectation that the vesicles would be identical to fat droplets. Surprisingly, however, although both control and ceramide-treated cells contained neutral lipids (Fig.
6, B and D), the
vesicles themselves were conspicuously devoid of neutral lipids.
Additionally, the distribution pattern of neutral lipids stained by
Nile Red (Fig. 6D) differed from the pattern of vesicles
visualized by phase contrast microscopy (Fig. 6C). All of
the vesicles were Nile Red negative, indicating that the vesicles
induced by C6-ceramide were not lipid droplets. Likewise, cell staining by periodic acid-Schiff reagent revealed the
vesicles to be unstained (not shown). Thus, these vesicles were
determined to be neither neutral lipid-containing nor glycol-containing cellular components.
We then considered the possibility that the vesicles were endocytic
vesicles. Early endosomes exist in the periphery of the cell and are
the first structures to receive endocytosed materials. The residence
time as early endosomes is on the order of minutes (20). The
internalized materials that do not recycle from early endosomes to the
cell plasma membrane are subsequently transported to late endosomes.
Late endosomes are formed by the fusion of early endosomes containing
materials coming in for digestion and the vesicles containing lysosomal
hydrolases coming out from the Golgi apparatus. After acquiring
lysosomal membrane proteins, such as LAMPs, late endosomes gradually
mature into lysosomes (20). Taking into account this knowledge, we used
the antibody against LAMP, ABL-93, to test whether the vesicles induced
by C6-ceramide were lysosomes. In fact, in 3T3-L1 cells
treated with C6-ceramide, the antibody specifically
stained the membrane of the vesicles. As indicated by the
arrows (Fig. 6F), the lysosomal membrane staining
appears as ring-shaped structures, which resulted from
immunolocalization of LAMP present in the lysosomal membrane and
colocalized with the lucent vesicles seen by phase microscopy (Fig.
6E). The control cells had small, scattered bodies, which is
the characteristic staining pattern of normal lysosomes (not shown).
Although specific, it should be noted that this LAMP staining cannot
distinguish lysosomes from late endosomes, because there is no clear
distinction between the two compartments, as discussed above. Thus,
some of these vesicles identified by LAMP staining may be late
endosomes. In any case, these studies clearly identify the
ceramide-induced vesicles as endocytic vesicles.
To determine whether the enlarged lysosomes/late endosomes were caused
by storage of C6-ceramide, we used
C6-NBD-ceramide as a fluorescent probe to trace the
localization of C6-ceramide (Fig.
7), because both
C6-NBD-ceramide and 14C-labeled
C6-ceramide have been shown to equilibrate into and accumulate in the Golgi apparatus (8-10). Following the appearance of
vesicles induced by 10 µM C6-ceramide, 3T3-L1
cells were either incubated for 15 min at 37 °C with 20 µM C6-NBD-ceramide or first incubated for 15 min at 37 °C with 20 µM C6-NBD-ceramide,
washed, and incubated at 37 °C for 2 h. After the 15-min
incubation, cells showed a diffuse pattern of
C6-NBD-ceramide staining (Fig. 7B) throughout
the cytoplasm, whereas after the additional 2-h incubation C6-NBD-ceramide fluorescence was concentrated in the
perinuclear region, the Golgi apparatus (Fig. 7D). These
results are consistent with previous studies showing that
C6-NBD-ceramide is a vital stain for the Golgi apparatus
(8). In both cases, the vesicles induced by C6-ceramide
were not stained by the fluorescent C6-NBD-ceramide.
Accompanying the appearance of the large vesicles in the cells treated
with ceramide was a profound morphological change. The cell size
doubled, and sometimes tripled (Fig. 2, B and C). It has been known that in most plant and fungal cells, each cell contains one or several, large, fluid-filled vacuoles, or versatile lysosomes. These vacuoles typically occupy more than 30%, and sometimes as much as 90%, of the cell volume (21). In animal cells,
vacuoles are related to lysosomes, which contain a variety of
hydrolytic enzymes with diverse functions. Thus, the increase in cell
size and volume in 3T3-L1 cells treated with ceramide possibly resulted
from the accumulation of enlarged lysosomes. Nevertheless, the
relationship between the present findings and the growth inhibitory
effect of C6-ceramide remains unclear.
Influence of Ceramide Structure on Induction of Vesicles--
To
further delineate the ceramide effect, we determined the influence of
the molecular structure of ceramide on induction of enlarged
lysosomes/late endosomes in 3T3-L1 cells. In these experiments, cells
were seeded at a density of 105 cells/25-cm2
flask in complete culture medium and cultured overnight. The cell
number was ~ 2 × 105 cells when ceramide
analogues were added to the culture medium at a range of ceramide
concentrations (10-20 µM). Vesicle formation was
assessed after exposure of the cells to ceramide for 24-48 h. These
studies revealed the stereospecificity of ceramide effect. Among the
stereoisomers of C6-ceramide,
D-erythro-C6-ceramide was the most active
isomer, whereas L-threo-C6-ceramide was
inactive. L-Erythro-C6-ceramide and
D-threo-C6-ceramide had a minor effect on
vesicle formation. Thus, the natural form of ceramide
(D-erythro-) appears to be the most active one. When
ceramide analogues with different fatty acyl chain length were compared
for their effect on vesicle formation,
D-erythro-C6-ceramide was the most active molecule among ceramide analogues tested,
D-erythro-C8-ceramide was less effective,
and D-erythro-C2-ceramide was not active
at all under these experimental conditions. For example, 3T3-L1 cells treated with 10 or 15 µM
D-erythro-C8-ceramide for up to 48 h did
not have any enlarged vesicle formation, and only <10% of the cells
bore vesicles when treated with 20 µM
D-erythro-C8-ceramide for 24-48 h. In
contrast, 30% and of the cells bore large vesicles when treated with
10 µM D-erythro-C6-ceramide for
24 h (Fig. 2D), and 67% of the cells treated with 15 µM for 24 h bore vesicles (Fig. 4B).
To determine whether the difference in effect of ceramide analogues on
vesicle formation was related to possible differences in their uptake,
we compared the uptake of 10 µM 14C-labeled
D-erythro-C6-ceramide and
D-erythro-C8-ceramide by 3T3-L1 cells. The
uptake was equal (Fig. 5). For example, at the density of 2 × 103 cells/well, both 14C-labeled
D-erythro-C6-ceramide and
14C-labeled D-erythro-C8-ceramide
were rapidly incorporated into the cells, reaching maximal levels of
9.8 and 9.0 pmol/103 cells, respectively. These studies
exclude reduced cellular uptake of C8-ceramide as the cause
of its lack of effect on vesicle formation.
Examination of Ceramide Metabolites on Induction of
Vesicles--
Ceramide has a central role in sphingolipid metabolism
and is actively metabolized in most cells. For example, in HL-60 cells, 3H-labeled C2-ceramide was rapidly incorporated
into the cells, and a substantial amount of
[3H]C2-ceramide remained intact even after
prolonged incubation at 37 °C. Only a small amount of
[3H]C2-ceramide was converted to
sphingomyelin but not to sphingosine (22). Similar results were
obtained using C6-ceramide in MOLT-4 cells (23). In B16
melanoma cells, C2- and C6-Cer were rapidly glycosylated and converted to short chain ceramide-containing cerebrosides and GM3, in addition to short chain
ceramide-containing sphingomyelin (12). Because these and other studies
have shown that ceramide can be metabolized to a number of molecules,
which have also been shown to have potential roles in cell function, we
investigated whether the effect of ceramide on vesicle formation could
be attributed to one of its metabolites. Under the conditions at which
C6-ceramide induced enlarged vesicles (treatment of 3T3-L1 cells for 1 or 2 days at 5-20 µM), the ceramide
metabolites sphingosine, sphingosine 1-phosphate, C6-Cer
sphingomyelin, C2-Cer cerebroside, and C2-Cer
GM3 were all inactive in causing vesicle formation (Fig. 4,
C-F). Thus, these ceramide metabolites, when added
exogenously, do not have the same effect as ceramide.
It has been shown that the small GTPases of the Rab family are actively
involved in vesicular docking and membrane fusion at various steps of
biosynthetic and endocytic transport (24-26). GTPases of the Rho
family regulate the actin cytoskeleton (27). A recent study suggests
that pinocytosis and membrane ruffling are regulated by distinct Ras
signal transduction pathways: activation of Rab5 via Ras stimulates
endosome fusion and pinocytosis, whereas activation of Rac via Ras
stimulates actin polymerization and membrane ruffling (28).
Because ceramide activates several protein kinases (3, 29), one
possibility is that exogenous C6-ceramide causes numerous
enlarged late endosomes/lysosomes by an effect of ceramide on the small
GTPases. For example, ceramide could activate one of the small GTPases,
such as a Rab protein, and thereby enhance or accelerate the vesicular
traffic to lysosomes. In turn, this could cause the appearance and
accumulation of enlarged late endosomes/lysosomes. Alternately,
ceramide could cause their persistence and enlargement by inhibiting
lysosome catabolism. Clearly, the mechanism of ceramide action will be
important to elucidate. Finally, the present study suggests that
C6-ceramide may be a useful probe for studying endocytic
vesicle transport and membrane fusion along the endocytic pathway in fibroblasts.
(2,3)-galactosyl-
(1,4)-glucosylceramide), were inactive in causing vesicle formation when added
exogenously. Together, these studies demonstrate that exogenous
C6-ceramide induces endocytic vesicle formation and causes
enlarged late endosomes and lysosomes in mouse fibroblasts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, Fas ligand, and radiation (3, 4). On the other
hand, ceramide catabolites, such as sphingosine 1-phosphate, stimulate
cell growth and suppress ceramide-mediated cell death (5).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
20 °C. In each
experiment, ceramide and sphingolipids were added directly into the
medium by diluting the stock solutions. The final concentration of
ethanol in the culture medium was 
0.2% (v/v).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effect of C6-ceramide on
proliferation of 3T3-L1 cells. 3T3-L1 cells were seeded at
105 cells/25-cm2 flask (A) or
103 cells/well in 96-well plates (B) and
cultured for 24 h in Dulbecco's modified Eagle's medium with
10% fetal bovine serum. The culture medium was then replaced with
fresh medium containing 10 µM ( 
), 15 µM
(
), or 20 µM (
) C6-ceramide or
ethanol (
). The cell number (A) and
[3H]thymidine uptake (B) were determined at
24-h intervals (see under "Materials and Methods"). The
[3H]thymidine incorporation was assayed as dpm per
103 cells seeded. Each value is the mean (±S.D.) of
triplicate cultures.
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Fig. 2.
Induction of vesicle formation in 3T3-L1
cells by C6-ceramide. 3T3-L1 cells were seeded at
105 cells/25-cm2 flask or at ~2 × 105 cells/culture dish (100 × 20 mm) and cultured for
24 h in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum. The medium was then replaced with fresh medium containing
either 0.1% ethanol (control, A) or 10 µM
C6-ceramide (B-D), and the cells were cultured
for 24 h (B and D) or 48 h
(A and C). The cell number was ~2 × 105 cells/25-cm2 flask and ~4 × 105 cells/culture dish when C6-cermide was
added to the culture medium. Magnification was × 400 for
A-C and × 200 for D.
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Fig. 3.
Reversibility of
C6-ceramide-induced vesicle formation. 3T3-L1
cells and 3T3-F442A cells were cultured as described in Fig. 2. After
the cells were exposed to 20 µM C6-ceramide
for 24 h and the vesicles appeared, the cells were washed and
cultured in fresh medium without ceramide for 24 h. Under this
condition, all of the vesicles originally induced by ceramide, as shown
in Fig. 2D, disappeared. A and B,
3T3-L1 cells; C and D, 3T3-F442A cells;
A and C, cells without ceramide treatment;
B and D, cells were first exposed to ceramide for
24 h, washed, and cultured in fresh medium for 24 h without
ceramide. All of the vesicles induced by C6-ceramide
disappeared in B and D. Original magnification, × 400.
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Fig. 4.
Effect of short chain ceramide
metabolites on vesicle formation in 3T3-L1 cells. 3T3-L1 cells
(2 × 105 cells/25-cm2 flask) were exposed
to 0.1% ethanol (control, A), 10 µM
sphingosine (C), 20 µM C6-Cer
sphingomyelin (D), 20 µM C2-Cer
cerebroside (E), or 20 µM C2-Cer
GM3 (F) for 48 h (except for E,
in which cells were cultured for 24 h). In B, 3T3-L1
cells were treated for 24 h with 15 µM
C6-ceramide at the density of 0.55-1.5 × 106 cells/25-cm2 flask (the assay starting and
ending density, respectively); 67% of the cells bore vesicles.
Magnification was × 200.
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Fig. 5.
Ceramide uptake by 3T3-L1 cells. The
uptake of C6-ceramide and C8-ceramide by 3T3-L1
cells was measured at three different cell densities: 2 × 103 ( ), 1 × 104 (), and 2 × 104 () cells/well in 96-well cell culture clusters
(area, 0.32 cm2). Each well contained 100 µl of culture
medium with either 10 µM 14C-labeled
C6-ceramide (A) or 10 µM
14C-labeled C8-ceramide (B). At
different time points, cells were harvested, and uptake of
14C-labeled ceramide was determined. The data are expressed
as pmol/103 cells. Each value is the mean (±S.D.) of
triplicate measurements.
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Fig. 6.
Confocal immunofluorescence microscopic
analysis of C6-ceramide-induced vesicles. 3T3-L1 cells
were seeded in two-well tissue culture slide chambers (2 × 104 cells/well) and were exposed to either 0.1% ethanol
(control, A and B) or 20 µM
C6-ceramide (C-F) for 24 h. The cells were
stained with Nile Red for neutral lipids (A-D) or with
ABL-93 anti-LAMP antibody for lysosomes (E and F)
and were examined in parallel by phase-contrast microscopy (left
panels) and confocal immunofluorescence microscopy (right
panels). Numerous neutral lipid droplets were detected, by Nile
Red staining, throughout the cytoplasm in both control (B)
and ceramide-treated cells (D). However, the phase lucent
vesicles in C6-ceramide treated cells (C) were
not stained by Nile Red (D), showing that vesicles induced
by C6-ceramide were not composed of neutral lipids. The
vesicles induced by C6-ceramide (E) were
positively stained with the anti-LAMP antibody (F). Note
that the ring-shaped structures (F) (indicated by
arrows) resulted from immunolocalization of LAMP present in
the lysosomal membrane and colocalized with the lucent vesicles seen by
phase microscopy (E).
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Fig. 7.
Distribution of
C6-NBD-ceramide in C6-ceramide-preteated 3T3-L1
cells. Following the appearance of vesicles induced by 10 µM C6-ceramide, 3T3-L1 cells were incubated
for 15 min at 37 °C with 20 µM
C6-NBD-ceramide and examined (A and
B) or washed and incubated for an additional 2 h at
37 °C before examination (C and D). The cells
were analyzed in parallel by Nomarski differential interference
contrast microscopy (left panels) and confocal
immunofluorescence microscopy (right panels). After the
15-min incubation, cells showed a diffuse pattern of
C6-NBD-ceramide staining, seen as green
throughout the cytoplasm (B), whereas after the additional
2-h incubation, C6-NBD-ceramide fluorescence was
concentrated in the region near the nucleus, the Golgi apparatus
(D). Note that the vesicles induced by
C6-ceramide appeared as black circles in
fluorescence micrographs (B and D). In both
cases, it is clear that the vesicles induced by C6-ceramide
were not stained by the fluorescent C6-NBD-ceramide.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Thomas August for the anti-LAMP antibody; we also thank Nancy K. Dwyer and Jessica Manela for assistance in this study.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health, NCI Grant CA61010 and by the Discovery Fund from the Children's Research Institute.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.
Recipient of an Avery Scholar Research Award from the Children's
Research Institute. To whom correspondence should be addressed: Center
for Cancer and Transplantation Biology, Children's Research Institute,
111 Michigan Ave. NW, Washington, D. C. 20010-2970. Tel.:
202-884-3898; Fax: 202-884-3929; E-mail: RLi@cnmc.org.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: C6-NBD-ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-D-erythro-sphingosine; C2-ceramide, N-acetyl-D-erythro-sphingosine; Cer, ceramide; C2-Cer cerebroside, N-acetylpsychosine; C6-ceramide, N-hexanoyl-D-erythro-sphingosine; C6-Cer sphingomyelin, N-hexanoyl-sphingosylphosphorylcholine; C8-ceramide, N-octanoyl-D-erythro-sphingosine; LAMP, lysosome-associated membrane protein.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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