From the
The effects of ceramide analogs on the uptake of markers for
fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low
density lipoprotein, LDL) endocytosis were studied in Chinese hamster
fibroblasts.
N-Hexanoyl-D-erythro-sphingosine
(C
In the last 10 years, a number of sphingolipid metabolites have
been shown to act as second messengers. Sphingosine was among the first
of these, when it was demonstrated that protein kinase C can be
inhibited by this lipid (reviewed in Hannun and Bell(1989, 1993),
Kolesnick(1991), and Merrill et al.(1993)). Ceramide
(Cer),
Cells were
treated with short-chain Cer analogs as complexes with defatted BSA as
described (Pagano and Martin, 1988; Rosenwald and Pagano, 1993a). The
BSA concentration was constant at 25 µM (except when cells
were treated with 50 µM C
Quantification of LDL
internalization was determined as described by Goldstein et
al.(1983). Briefly, cells were cultured in medium containing 5%
LPDS rather than fetal bovine serum for 24-48 h prior to the
experiment. Cells were then washed once with HMEM/BSA, incubated with
HMEM/BSA for 10 min at 4 °C, followed by 0-50 µM
C
To study the effect of
C
Cells
were washed three times with phosphate-buffered saline (PBS) lacking
divalent cations, scraped in PBS, and pelleted by centrifugation at 200
Endocytic vesicles thus prepared were analyzed on Percoll
gradients prepared in the following manner: 6.62 ml of 90% Percoll was
mixed with 25.68 ml of 0.25 M sucrose containing 1 mM
EDTA (pH 7.0) and 2.7 ml of postnuclear supernatant (17% final Percoll
concentration). This mixture was layered onto a 4-ml cushion of 2.5
M sucrose with 10 mM EDTA (pH 6.8). The samples were
centrifuged using a Beckman vTi50 rotor for 1 h at 25,000
We tested a number of other lipids for their
effects on internalization of HRP (). All short-chain
analogs of Cer tested, including C
Two
other agents which alter intracellular concentrations of sphingolipids
were also tested for their effects on HRP internalization
(). When cells were pretreated with sphingomyelinase to
hydrolyze cell surface sphingomyelin to Cer (Spence, 1993), HRP
internalization was inhibited. With increasing amounts of
sphingomyelinase, progressively more inhibition was seen. Cells were
also pretreated with the glycosphingolipid synthesis inhibitor, PDMP
(Radin et al., 1993). PDMP at these concentrations inhibits
both glucosylceramide synthase (Radin et al., 1993) and
sphingomyelin synthase (Rosenwald et al., 1992) and increases
the intracellular Cer concentration (Betts et al., 1994). When
cells were treated with increasing amounts of PDMP, HRP internalization
was increasingly affected. These two results suggest that increases in
endogenous long-chain Cer, as well as exogenous short-chain Cer, may
inhibit internalization of HRP.
C
The pathways by which various molecules are internalized by
cells have been studied extensively in the last several years (for a
recent review, see Trowbridge et al.(1993)). The mechanisms
regulating internalization of molecules from the extracellular space to
the cell interior, however, are less well understood. In this paper, we
demonstrate that one means of regulation may be through alterations of
the intracellular concentrations of the lipid second messenger, Cer.
Short-chain analogs of Cer decreased both fluid-phase endocytosis
and receptor-mediated endocytosis, as measured by internalization of
HRP and LDL, respectively. These effects were not due to decreased cell
viability since incorporation of [
Previous results have demonstrated that short-chain analogs of Cer
are taken up effectively by cells (Lipsky and Pagano, 1983, 1985;
Rosenwald and Pagano, 1993b) and metabolized, chiefly to the
corresponding analogs of sphingomyelin and glucosylceramide (for a
recent review, see Rosenwald and Pagano (1993b)). At present we cannot
rule out the possibility that the effects documented here are caused by
a metabolite of Cer. However, it appears that breakdown of Cer to
sphingosine, another lipid second messenger, is not responsible for the
effects seen on endocytosis, although another Cer metabolite may be the
primary agent responsible for the effects seen. Future experiments will
address this question.
Several lines of evidence suggest that
C
It has been proposed that a ``sphingomyelin
cycle'' exists in cells to transmit extracellular signals (Hannun,
1994; Hannun and Bell, 1993; Hannun et al., 1993; Kolesnick,
1992; Kolesnick and Golde, 1994; Mathias and Kolesnick, 1993). In this
model, when extracellular signals, such as tumor necrosis factor
Among the changes
observed in cells in response to different agonists, including tumor
necrosis factor
In summary, we have shown that
endocytosis is affected by Cer. The results from the present study and
our previous work (Rosenwald and Pagano, 1993a) suggest that membrane
traffic is responsive to intracellular concentrations of Cer in at
least three different pathways: constitutive secretion, fluid-phase
endocytosis, and receptor-mediated endocytosis. Alterations in the
intracellular Cer concentration may therefore serve as a general
modulator of membrane traffic events.
Subconfluent
(
Subconfluent cultures of CHO-K1 cells were
pretreated with 25 µM sphingolipid
We thank Dr. Robert Murphy (Carnegie-Mellon
University, Pittsburgh, PA) for useful suggestions about the separation
of endosomes and lysosomes on Percoll gradients and members of the
Pagano laboratory for their helpful comments on this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Cer) decreased the uptake of HRP in a dose-dependent
manner. Internalization was inhibited >40% with 25 µM
C
-Cer, relative to controls, and was apparent within 5 min.
Internalization of HRP was also inhibited by other Cer analogs and by
treatment with agents that raise levels of endogenous Cer
(sphingomyelinase or the glycosphingolipid synthesis inhibitor,
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)), but not by
N-hexanoyl-D-erythro-sphinganine
(C
-dihydro-Cer) or sphingosine. Internal-ization of LDL was
also inhibited by C
-Cer in a concentration-dependent
manner, but was less pronounced than the effect on HRP internalization
(10% versus 40% inhibition with 25 µM
C
-Cer), suggesting that ceramide might affect fluid-phase
and receptor-mediated endocytosis to different extents.
C
-Cer also slowed HRP and LDL transport from endosomes to
lysosomes as studied by analysis of endocytic vesicles on Percoll
density gradients and induced a redistribution of endocytic organelles
as determined by fluorescence microscopy of intact cells using
appropriate markers. This resulted in decreased degradation of
I-labeled LDL in the presence of C
-Cer. These
results suggest that endogenous ceramide may modulate endocytosis.
(
)
the precursor for all cellular
sphingolipids, has also been shown to act as a second messenger in a
number of pathways, including those induced by tumor necrosis factor
, vitamin D
, interleukin 1
, and interleukin 4
(reviewed in Hannun(1994), Hannun et al.(1993), Kolesnick
(1992), and Mathias and Kolesnick(1993)). Studies from this laboratory
have suggested that membrane traffic may also be affected by
alterations in the intracellular levels of Cer. Previously, we
demonstrated that short chain analogs of Cer inhibit the movement of
vesicular stomatitis virus glycoprotein through the medial and
trans compartments of the Golgi apparatus and its subsequent
transport to the cell surface. In addition, the number of viral
particles released from cells treated with Cer analogs is markedly
reduced compared to untreated cells (Rosenwald and Pagano, 1993a). In
the current work, we explored the possibility that Cer also modulates
endocytosis. Short-chain analogs of Cer inhibited endocytosis in a
concentration-dependent manner. Both fluid-phase and receptor-mediated
endocytosis were decreased by treatment with Cer, although to different
extents. These findings, in conjunction with our earlier work, suggest
that Cer may be a general modulator of membrane traffic.
Materials
D-erythro-Sphingosine,
N-hexanoyl-D-erythro-sphingosine-(C-Cer),
N-acetyl-D-erythro-sphingosine
(C
-Cer),
N-hexanoyl-D-erythro-sphinganine
(C
-dihydro-Cer), and
DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
(PDMP) were from Matreya, Inc. (Pleasant Gap, PA). Human low density
lipoprotein (LDL; 1.019-1.063 g/ml), human lipoprotein-deficient
serum (LPDS; lipoproteins of density < 1.21 g/ml have been removed),
and
I-labeled human LDL (
0.2-0.5
µCi/µg) were from Biotechnology Research Institute (Rockville,
MD).
N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine
(C
-NBD-Cer), 3,3`-dioctadecylinodocarbocyanine low density
lipoprotein (DiI-LDL), and fluorescein (FITC)-dextran (10 kDa) were
from Molecular Probes. Percoll was from Pharmacia (Uppsala, Sweden).
Triton X-100 (10% solution) was from Pierce. Horseradish peroxidase
(HRP type II), sphingomyelinase from Staphylococcus aureus,
bovine serum albumin (BSA), and o-dianisidine were from Sigma.
Cells and Cell Culture
Chinese hamster ovary cells
(CHO-K1; American Type Culture Collection; CCL-61) were grown and
maintained as described (Rosenwald and Pagano, 1993a). Depletion of
cellular cholesterol was performed as described except the medium
contained 5% lipoprotein-deficient serum (LPDS) rather than 10% (Martin
et al., 1993). Since subconfluent cells were found to be more
sensitive to the effects of C-Cer (see ), cells
were plated 3 days prior to use at
4-6
10
cells/35-mm culture dish, unless otherwise noted, and were
70-80% confluent at the time of the experiments.
-Cer; then the final
BSA concentration was 50 µM) and the final ethanol
concentration (from the preparation of Cer
BSA complexes) was
0.25%. Cells maintained normal morphology, and the cellular protein
content of dishes was unaffected by treatment with C
-Cer
under all experimental conditions.
Endocytosis Assays
The internalization of HRP was
quantified using the method of Marsh et al.(1987). Briefly,
the monolayers were washed once with cold HEPES-buffered Eagle's
minimal essential medium without indicator, containing 1.3 mM
CaCl, 0.8 mM MgSO
, and 2% bovine serum
albumin (BSA) (HMEM/BSA). Subsequently, the cells were incubated for 10
min at 4 °C in HMEM/BSA, then treated with a lipid
BSA complex
(C
-Cer
BSA unless otherwise noted) for 5 min at 4
°C. Concentrated HRP in HMEM/BSA was added to 1 mg/ml (final
concentration), and the cells were then incubated at either 4 °C or
37 °C for 5-120 min. Subsequently, the HRP-containing medium
was removed and the monolayers were washed once with cold HMEM/BSA.
Cells were then scraped, transferred to BSA-coated glass tubes, washed
once with HMEM/BSA, and then washed three times with HMEM (without
BSA). The cell pellet was lysed with 0.5 ml of lysis buffer (10
mM Tris-HCl, pH 7.4, 0.05% Triton X-100), then homogenized and
sonicated for 10 s. HRP content and protein content of the homogenate
were determined according to the methods of Marsh et al.(1987)
and Lowry et al.(1951), respectively. Nonspecific
cell-associated HRP (amount of uptake at 4 °C) was subtracted from
each point and was less than 12%.
-Cer
BSA for 5 min.
I-LDL (10
µg/ml; specific activity
580 cpm/ng protein) in the presence
or absence of 500 µg/ml unlabeled LDL was then added to the cells,
and incubations were continued for up to 30 min at 37 °C. Cells
were then chilled to 4 °C, scraped, and washed as described for HRP
internalization, and the cell-associated radioactivity was determined
in a
counter. Background values (obtained in the presence of
excess unlabeled LDL) were less than 12% of experimental values
(obtained in the absence of excess LDL).
-Cer on LDL degradation, cells were pulse-labeled with 10
µg/ml
I-LDL for 10 min at 37 °C, then cooled to 4
°C, washed, and chased in HMEM containing 25 µM
defatted BSA for 0-160 min at 37 °C. C
-Cer (25
µM) was added either at the beginning of the chase or
after 15 min of chase. The cells were then chilled, scraped, and
pelleted by centrifugation. The supernatant fractions were then
precipitated with trichloroacetic acid, and the radioactivity in the
acid-soluble fraction was determined as described (Goldstein and Brown,
1974).
Isolation and Characterization of Endosome
Fractions
For preparation of HRP- or
I-LDL-containing endosomes, subconfluent cell monolayers
were prepared by plating 1.5
10
cells in 150-mm
culture dishes 3 days prior to the experiment and refed with media
containing 5% LPDS for the last 24 h. Cells were washed once with cold
HMEM/BSA, treated with either 3-4 mg of HRP/ml or 10 µg/ml
I-LDL in HMEM, then incubated for 10 min at 37 °C
(pulse conditions). The cells were then chilled and washed with
HMEM/BSA. Cells were then warmed for 0 or 20 min at 37 °C in the
presence of 25 µM defatted BSA in HMEM (for LDL
experiments this chase medium also contained an excess of unlabeled LDL
(500 µg/ml)). To study the effect of Cer on the distribution of
these markers, C
-Cer (0-25 µM in HMEM
containing 25 µM defatted BSA) was added to the chase
medium. The cells were then chilled and washed three times with
HMEM/BSA. All subsequent steps were performed at 4 °C.
g for 5 min. The cells were washed once with PBS, then
with 0.25 M sucrose containing 1 mM EDTA (pH 7.0).
The cells were resuspended, homogenized using a ball-bearing
homogenizer (12 passes using a ball-bearing with a clearance of 0.00045
inch) (Balch and Rothman, 1985), and centrifuged for 10 min at 950
g to remove nuclei. Vesicle latency in the postnuclear
supernatant fraction was
72% as determined by measurements of HRP
activity in the absence and presence of detergent (Storrie and Madden,
1990).
g. Thirty-six 0.9-ml fractions were collected from the top
using a Buchler Auto Densi-Flow II C fraction collector. Density across
the gradient was determined by refractive index of a parallel tube.
-Galactosidase activity was used as a lysosomal marker (Storrie
and Madden, 1990). HRP activity (Steinman et al., 1974) and
I radioactivity were determined for each fraction.
Fluorescence Microscopy
Fluorescence microscopy
was performed with a model IM-35 inverted microscope equipped with
epifluorescence optics (Carl Zeiss, Inc., Thornwood, NY). For
microscopy experiments, cells were grown on glass coverslips as
described (Pagano et al., 1989). Cells were labeled with
DiI-LDL and viewed as described (Barak and Webb, 1981). Internalization
of fluoresceinated-dextran (FITC-dextran) was performed as described
for sulforhodamine-dextran (Koval and Pagano, 1989).
Effects of Short-chain Ceramide Analogs on Horseradish
Peroxidase Internalization
C-Cer inhibited
internalization of the fluid-phase marker, HRP, in a dose-dependent
manner (Fig. 1A); at 25 µM
C
-Cer, HRP internalization was inhibited by more than 40%
relative to controls. The inhibition of HRP internalization by 25
µM C
-Cer was apparent within 5 min
(Fig. 1B). Control experiments demonstrated that
C
-Cer had no direct effect on the enzymatic activity of HRP
(data not shown). Generally, cells were treated with C
-Cer
for 5 min at 4 °C prior to warming at 37 °C in the presence of
HRP to permit internalization. However, control experiments
demonstrated that addition of C
-Cer for 0-10 min at 4
°C prior to warming with HRP gave similar results (data not shown).
Therefore, the effect of C
-Cer on internalization of HRP
was immediate and without a lag.
Figure 1:
Effect of
C-Cer on the uptake of HRP by CHO cells. A, cells
were pretreated with 0-50 µM C
-Cer for 5
min at 4 °C, and then HRP (1 mg/ml final concentration) was added
and the cells were further incubated for 30 min at 37 °C. The cells
were then chilled, scraped, and washed, and cellular protein and HRP
content was determined (see ``Experimental Procedures'').
Uptake data were corrected for background, normalized to cellular
protein, and expressed as percent of control values determined in the
absence of C
-Cer. Each data point represents the mean
± S.D. of three measurements. B, cells treated with
either 0 or 25 µM C
-Cer and incubated with HRP
as above, but for 5-120 min at 37 °C. Data points are the
averages of duplicate determinations.
Inhibition of fluid-phase
endocytosis by C-Cer was found to be dependent upon cell
density (). Confluent cells (
100% coverage) were
insensitive to the effects of C
-Cer, compared to
subconfluent cells (70-80% coverage); internalization of HRP by
confluent cultures was unaffected by up to 25 µM
C
-Cer, while HRP internalization by subconfluent cultures
was inhibited by more than 40% ( Fig. 1and ).
Moreover, in the absence of C
-Cer, confluent cells
internalized less HRP than subconfluent cells. This suggests that
confluent cells were not only less active in terms of membrane traffic
from the cell surface, but also less sensitive to the effects of
short-chain Cer analogs. Subsequent experiments were performed on
subconfluent cells.
-Cer,
C
-NBD-Cer, and C
-Cer, inhibited uptake by
40-60% at 25 µM. These analogs were also found to
inhibit secretion (Rosenwald and Pagano, 1993a). Sphingosine (up to 10
µM) did not inhibit internalization of HRP (data not
shown) and had no effect on secretion (Rosenwald and Pagano, 1993a).
The dihydro analog of C
-Cer was not an effective inhibitor
of HRP internalization at 25 µM. In contrast, short-chain
analogs of dihydro-Cer were found to inhibit secretion,
(
)
suggesting that the two processes, endocytosis and
secretion, may be regulated by partially distinct mechanisms.
Differential Effects of C
We also
tested the effect of C-Cer on
Fluid-phase versus Receptor-mediated Endocytosis
-Cer on the internalization of LDL, a
molecule which enters cells by receptor-mediated endocytosis. Cells
were grown under cholesterol depletion conditions for 48 h (with LPDS,
rather than fetal bovine serum) to up-regulate the levels of surface
LDL receptors. As a control, the internalization of HRP by cells in
lipid-replete versus lipid-depleted conditions was compared.
The amount of HRP taken up in LPDS-treated cells was approximately 70%
of the amount taken up by fetal bovine serum-grown cells. However, the
magnitude of the C
-Cer effect on HRP internalization
relative to control values (i.e. in the absence of
C
-Cer) was similar in LPDS- and fetal bovine serum-treated
cells (data not shown).
-Cer inhibited internalization
of LDL in a dose-dependent manner (Fig. 2A).
Interestingly, the magnitude of the effect on receptor-mediated
endocytosis was much less pronounced than that seen on fluid-phase
endocytosis. For example, at 10 µM C
-Cer, HRP
internalization was
71%, while LDL internalization was
98% of
the control values obtained during a 30-min incubation at 37 °C
(Fig. 2A). This differential effect could also be seen
at the earliest time measured (5 min of warming;
Fig. 2B). These results suggest that the two modes of
endocytosis were differentially affected by C
-Cer. In
control experiments, we found that preincubation of cells for 0, 5, or
10 min at 4 °C with C
-Cer prior to warming to 37 °C
(in the presence of C
-Cer) was without effect, again
suggesting that the effect of C
-Cer was immediate (data not
shown).
Figure 2:
Comparison of the effects of
C-Cer on uptake of LDL versus HRP. Subconfluent
monolayer cultures were grown in medium containing 5% LPDS for 48 h
prior to use. A, cells were incubated with 0 (control) to 50
µM C
-Cer for 5 min at 4 °C.
I-LDL (10 µg/ml; with or without 500 µg/ml
unlabeled LDL) or HRP (1 mg/ml) was added and the cells were then
incubated for 30 min at 37 °C, and the amount of cell-associated
I-LDL or HRP was determined (see ``Experimental
Procedures''). Appropriate background values were subtracted for
each data point (for HRP, incubation at 4 °C; for
I-LDL, incubation in the presence of excess cold LDL at
37 °C). Each value represents the mean ± S.D. of four
determinations. B, cells were incubated with 0 (control) or 25
µM C
-Cer for 5 min at 4 °C. HRP (solid
bars) or
I-LDL (stippled bars) was then
added, and incubations were carried out for 5, 10, or 15 min at 37
°C. Values represent means of duplicate
measurements.
Effects of C
To determine the effect
of C-Cer on the Distribution of
HRP and LDL in Endosomal Fractions
-Cer on transit of material within the endosomal
pathway, we followed the distribution of HRP and LDL in endocytic
vesicles in the presence or absence of C
-Cer by analysis of
endosomal fractions on Percoll density gradients. First, a pulse-chase
experiment was performed in the absence of C
-Cer to
document the movement of HRP and LDL from endosomes to lysosomes
(Fig. 3A). After a 10-min pulse (no chase), most of the
HRP resided in fractions 6-8 which were relatively light in
density (1.044-1.053 g/ml) and are referred to as early endosomes
(Wilson et al., 1993). After a 20-min chase, some of the HRP
was observed to move into fractions 32-34 of higher density
(1.08-1.16 g/ml) which colocalized with the lysosomal marker,
-galactosidase (Storrie and Madden, 1990). It should be noted that
the total amount of HRP activity on the gradient decreased with
increasing chase times (Fig. 3A), presumably as a result
of HRP recycling back to the cell surface and release into the
extracellular medium. Chase of material from early endosomes to
lysosomes was also observed when cells were pulsed with
I-LDL for 10 min, then chased in the presence of excess
unlabeled cold LDL for 20 min (Fig. 3A). The effect of
C
-Cer on the distribution of HRP activity was analyzed by
incubating cells in the presence of up to 25 µM
C
-Cer during a 20-min chase following the initial 10-min
pulse with HRP (Fig. 3B). In the presence of increasing
amounts of C
-Cer, progressively less HRP activity was found
in the lysosomes and relatively more activity was found in the early
endosomes.
(
)
Consistent with the results shown in
Fig. 2B, the effect of C
-Cer on the
transport of HRP from early endosomes to lysosomes was more pronounced
than that seen for LDL (Fig. 3C). These results suggest
that C
-Cer slowed transport of HRP and LDL from early
endosomes to lysosomes.
Figure 3:
Effect of C-Cer on the
movement of HRP and LDL from endosomes to lysosomes as determined by
Percoll density gradient fractionation. Cells (1.5
10
/150-mm dish) were plated 3 days prior to the experiment
and labeled with HRP or
I-LDL as described below.
Postnuclear supernatants were then prepared and fractionated on 17%
Percoll gradients (see ``Experimental Procedures'').
Fractions 6-8 correspond to early endosomes and fractions
32-34 to lysosomes (see text). A, cells were
pulse-labeled for 10 min with either HRP (
,
) or
I-LDL (
,
) and then chased for 0 min
(open symbols) or 20 min (closed symbols) in the
absence of C
-Cer. B, cells were pulse-labeled with
HRP for 10 min and chased in the presence of 0-25 µM
C
-Cer for 20 min (
, early endosomes;
,
lysosomes). C, cells were pulse-labeled for 10 min with HRP or
I-LDL and chased for 20 min with or without 25
µM C
-Cer. The amount of HRP or
I-LDL found in early endosomes and lysosomes was then
determined and expressed as a percent of the control values found for
each fraction in the absence of
C
-Cer.
Since C-Cer slowed transport of
I-LDL to the lysosomes, we next examined the effect of
C
-Cer on LDL degradation. We first determined by
pulse-chase analysis that the t for both the disappearance of
LDL from the cells (as measured by cell-associated radioactivity) and
the appearance of LDL degradation products (as measured by release of
trichloroacetic acid-soluble radioactivity in the media) was 15 min
(data not shown). When we compared the addition of 25 µM
C
-Cer at 0 min of chase to the addition of
C
-Cer at 15 min of chase (in both cases after a 10-min
pulse with
I-LDL), we found that the Cer effect was
apparent only when Cer was added at the beginning of the chase, when
the LDL was in transit to the lysosomes (Fig. 4). Similar results
at 0 min of chase were achieved with as little as 10 µM
C
-Cer, while no effect was seen at 15 min of chase even
when 50 µM C
-Cer was used (data not shown).
Finally, we note that we were unable to detect any change in the pH of
the lysosomes in the presence of C
-Cer using two
independent methods (Gluck et al., 1982; Ohkuma and Poole,
1978) (data not shown).
Figure 4:
Effect of C-Cer on
I-LDL degradation. Cell monolayers were incubated with
media containing 5% LPDS for 24 h, then pulse-labeled with
I-LDL (10 µg/ml;
580 cpm/ng protein) for 10 min
at 37 °C in the presence of HMEM containing 25 µM
defatted BSA. Cells were then cooled to 4 °C, washed with HMEM
containing 25 µM defatted BSA, and then warmed to 37
°C for 0-160 min. In some samples, 25 µM
C
-Cer was added either at the beginning of the chase or
after 15 min of chase. Degradation of
I-LDL was
determined as described under ``Experimental Procedures.''
, control, no additions;
, C
-Cer addition at
zero time of chase; and
, C
-Cer addition after 15
min of chase.
Redistribution of Endocytic Organelles in the Presence of
C
Upon incubation with
C-Cer
-Cer, changes in the intracellular distribution of
endosomes and lysosomes were observed. Cells were pulse-labeled for 24
h with FITC-dextran, then chased for 1.5 h to label lysosomal
compartments (Koval and Pagano, 1989). After treatment with or without
C
-Cer for 5 min at 4 °C, DiI-LDL (2 µg/ml final
concentration) was added for 10 min at 37 °C, and the cells were
washed and chased for either 20 min or 80 min prior to fluorescence
microscopy ( Fig. 5and Fig. 6). After 20 min of chase in
the absence of C
-Cer (Fig. 5, A and
B), the lysosomes stained by FITC-dextran were observed in the
periphery of the cells (Fig. 5B), while DiI-LDL-labeled
endosomes were seen throughout the cell (Fig. 5A).
Little staining of the cell surface was seen, demonstrating the
effectiveness of the chase. In contrast, in cells treated with 25
µM C
-Cer, both endosomes and lysosomes were
found at the cell periphery (Fig. 5, C and D).
Similar observations were made with chase times as short as 10 min
(data not shown), suggesting that Cer exerted its effects on
trafficking rapidly. After 80 min of chase (Fig. 6) in the
absence of C
-Cer treatment (Fig. 6, A and
B), extensive colocalization of FITC-dextran and DiI-LDL was
observed, but, in the presence of 25 µM C
-Cer,
DiI-LDL-containing endosomes were observed at the cell periphery
(Fig. 6C), while FITC-dextran-containing lysosomes were
seen in the perinuclear region of the cell (Fig. 6D) and
little colocalization was seen. Thus, treatment of cells with
C
-Cer altered the distribution of lysosomes and endosomes
in both the short term and the long term and prevented delivery of
endosomal contents to the lysosomes.
Figure 5:
Redistribution of DiI-LDL-labeled
endosomes during a short-term chase in the presence of
C-Cer. Subconfluent cells were cultured in medium
containing 5% LPDS for 24 h. The cells were then refed with 5% LPDS
medium containing 0.5 mg/ml FITC-dextran and incubated for an
additional 24 h. The FITC-dextran was chased into lysosomes by washing
the cells with HMEM and incubating them for 90 min at 37 °C in
culture medium. The cells were then treated without (A and
B) or with (C and D) 25 µM
C
-Cer for 5 min at 4 °C. DiI-LDL (2 µg/ml) was then
added for 10 min at 37 °C. The cells were then washed and chased
for 20 min at 37 °C. The doubly labeled cells were then
photographed using rhodamine and fluorescein filter combinations to
view the distribution of DiI-LDL (A and C) and
FITC-dextran (B and D) fluorescence in the same
cells. Bar represents 10 µm.
Figure 6:
Redistribution of DiI-LDL-labeled
endosomes during a long-term chase in the presence of
C-Cer. Cells were treated exactly as in Fig. 5, except that
the DiI-LDL was chased for 80 (rather than 20) min at 37 °C. Cells
were treated with 0 (A and B) or 25 µM
(C and D) C
-Cer; DiI-LDL (A and
C) and FITC-dextran (B and D) fluorescence
is shown for the same cells. Note the peripheral distribution and
partial colocalization of DiI-LDL (A) and FITC-dextran
(B) fluorescence in the absence of C
-Cer treatment
(arrowheads), while, in the presence of C
-Cer, the
labeled lysosomes (D) were found in the perinuclear region
(arrows), while endosomes containing DiI-LDL were at the cell
periphery (C). Bar represents 10
µm.
S]methionine
into cellular protein and the ability of CHO-K1 cells to exclude trypan
blue were unaffected by the concentrations of short-chain Cer analogs
used (Rosenwald and Pagano, 1993a). Interestingly, fluid-phase
endocytosis was much more sensitive to the concentration of short-chain
Cer than was receptor-mediated endocytosis, suggesting differential
regulation of these two processes in vivo ( Fig. 2and
Fig. 3
). The effects on fluid-phase endocytosis were seen only
with the Cer analogs and not the dihydro-Cer analog or sphingosine
(), suggesting that the effects were specific. We also
found that incubation of cells with exogenous sphingomyelinase or with
the glucosphingolipid synthesis inhibitor, PDMP, two treatments which
increase the intracellular concentration of endogenous Cer, also
inhibited fluid-phase endocytosis (). These results
support the observations seen with exogenous short-chain Cer analogs
and suggest that the processes of endocytosis may be regulated by
alterations in the endogenous, long-chain Cer concentration.
-Cer affects the transport of LDL to the
degradative compartment, rather than the degradation process itself:
(i) C
-Cer inhibited vesicular traffic of HRP and LDL from
the endosomes to lysosomes which was studied by cell fractionation
using Percoll density gradients (Fig. 3); (ii) C
-Cer
slowed LDL degradation when it was added at the beginning of
the chase when LDL was in transit to the lysosomes, but had no effect
when it was added later (Fig. 4); (iii) C
-Cer
inhibited the delivery of DiI-LDL from endosomes to FITC-dextran
containing lysosomes as seen by fluorescence microscopy of living cells
( Fig. 5and Fig. 6); and (iv) C
-Cer had no
effect on lysosomal pH which could directly affect lysosomal
degradation.
(Dressler et al., 1992; Kim et al., 1991; Mathias
et al., 1991),
-interferon (Kim et al., 1991),
interleukin-1
(Mathias et al., 1993), and others,
interact with their receptors, a sphingomyelinase is activated which
results in increases in the intracellular Cer concentration. Many, if
not all, of the effects of these extracellular signals can be
duplicated by treatment of cells with either exogenous sphingomyelinase
or exogenous short-chain analogs of Cer, such as those used in this
study (Hannun, 1994). Changes in the internal concentration of Cer,
whether from exogenous or endogenous sources, can activate a
proline-directed, membrane-associated kinase (Mathias et al.,
1991; Joseph et al., 1993) and a cytosolic ceramide-activated
protein phosphatase (Dobrowsky and Hannun, 1992). This phosphatase,
likely phosphatase 2A, is inhibitable by okadaic acid (Dobrowsky and
Hannun, 1992). Interestingly, it has been observed that upon treatment
of cells with okadaic acid, pinocytosis increases (Kreienbuhl et
al., 1992) and staurosporine, a potent kinase inhibitor, inhibits
receptor-mediated endocytosis of asialoglycoprotein receptor ligands
(Fallon and Danaher, 1992). Thus, downstream targets of these
ceramide-mediated phosphorylation/dephosphorylation reactions may be
responsible for regulation of endocytosis.
and
-interferon, are alterations in the
cytoskeletal architecture (Bar-Sagi and Feramisco, 1986; West et
al., 1989). An example of this is the ``ruffling''
phenomenon observed in phagocytic cells. In this study, we observed
changes in the positioning of both endosomes and lysosomes within cells
in response to Cer. Since these organelles are tethered to microtubules
(Matteoni and Kreis, 1987; Hamm-Alvarez et al., 1993),
alterations observed in membrane traffic and organelle position in
cells may reflect changes in microtubule arrays within cells upon
changes in the intracellular Cer concentration. Alternatively, changes
in the function of microtubule motors may occur in response to rises in
Cer. Alterations in vesicle movements have been observed in the
presence of okadaic acid (Hamm-Alvarez et al., 1993),
suggesting that the ceramide-activated protein phosphatase may be
involved in regulation at this step.
Table:
Inhibition of fluid-phase endocytosis by
C -Cer is dependent on cell density
70-80% confluency) or confluent (
100% confluency)
cultures of CHO-K1 cells were seeded 3 days prior to the experiment,
and the effects of 0, 10, or 25 µM C
-Cer on
internalization of HRP following a 30-min incubation at 37 °C was
then determined (see ``Experimental Procedures'').
Table:
Effect of various agents and treatments on
fluid-phase endocytosis
BSA (prepared as a
1:1 molar complex as described for C
-Cer
BSA) for 5
min at 4 °C, and HRP (1 mg/ml, final concentration) was added. The
cells were then warmed for 30 min at 37 °C, and chilled, and HRP
uptake was determined (see ``Experimental Procedures''). To
study the effect of sphingomyelinase on internalization of HRP, cells
were pretreated with S. aureus sphingomyelinase
(0.013-0.1 unit in 25 µM defatted BSA) for 20 min at
37 °C, washed, and incubated with HRP as above. To study the effect
of PDMP on the internalization of HRP, cells were incubated with
10-40 µM PDMP in the presence of 25 µM BSA for 20 min at 37 °C prior to the addition of HRP. Data are
presented as percent of control in the absence of any addition or
treatment, but in the presence of 25 µM defatted BSA.
-Cer,
N-acetyl-D-erythro-sphingosine;
C
-Cer,
N-hexanoyl-D-erythro-sphingosine;
C
-NBD-Cer,
N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine;
CHO-K1, Chinese hamster ovary cells; DiI,
3,3`-dioctadecylinodocarbocyanine; BSA, bovine serum albumin;
FITC-dextran, fluorescein isothiocyanate-conjugated dextran; HMEM, 10
mM HEPES-buffered Eagle's minimum essential medium, pH
7.4, without indicator and with 1.3 mM CaCl
and
0.8 mM MgSO
; HMEM/BSA, HMEM containing 2% BSA;
HRP, horseradish peroxidase; LDL, low density lipoprotein; LPDS,
lipoprotein-deficient serum; PDMP,
1-phenyl-2-decanoylamino-3-morpholino-1-propanol.
-Cer was the same as that prepared from cells chased in
the absence of C
-Cer (data not shown), suggesting that
C
-Cer had little effect on recycling of HRP back to the
cell surface.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.