©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ceramide As a Modulator of Endocytosis (*)

Chii-Shiarng Chen (§) , Anne G. Rosenwald (¶) , Richard E. Pagano (**)

From the (1) Carnegie Institution of Washington, Baltimore, Maryland 21210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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),() 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.


EXPERIMENTAL PROCEDURES

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.

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-Cer; then the final BSA concentration was 50 µM) and the final ethanol concentration (from the preparation of CerBSA 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 lipidBSA complex (C-CerBSA 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%.

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-CerBSA 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).

To study the effect of C-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.

Cells were washed three times with phosphate-buffered saline (PBS) lacking divalent cations, scraped in PBS, and pelleted by centrifugation at 200 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).

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 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).


RESULTS

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.

We tested a number of other lipids for their effects on internalization of HRP (). All short-chain analogs of Cer tested, including C-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.

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.

Differential Effects of C-Cer on Fluid-phase versus Receptor-mediated Endocytosis

We also tested the effect of C-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).

C-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-Cer on the Distribution of HRP and LDL in Endosomal Fractions

To determine the effect of C-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-Cer

Upon incubation with C-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.




DISCUSSION

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 [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.

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-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.

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 (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.

Among the changes observed in cells in response to different agonists, including tumor necrosis factor 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.

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.

  
Table: Inhibition of fluid-phase endocytosis by C -Cer is dependent on cell density

Subconfluent (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

Subconfluent cultures of CHO-K1 cells were pretreated with 25 µM sphingolipidBSA (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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Mayo Clinic and Foundation, Guggenheim 6, Rochester, MN 55905.

Supported by Grant DRG-1067 from the Damon Runyon-Walter Winchell Cancer Research Fund. Current address: Laboratory of Biological Chemistry, DTP, DCT, NCI, National Institutes of Health, Bldg. 37, Room 5D-02, Bethesda, MD 20892.

**
Supported by Grant R37 GM22942 from the U.S. Public Health Service. To whom correspondence and reprint requests should be addressed. Current address: Mayo Clinic and Foundation, Guggenheim 6, Rochester, MN 55905. Tel.: 507-284-2301; Fax: 507-284-4521; E-mail: pagano.richard@mayo.edu.

The abbreviations used are: Cer, ceramide (N-acyl-D-erythro-sphingosine); C-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.

C.-S. Chen, A. G. Rosenwald, O. C. Martin, and R. E. Pagano, unpublished observations.

We observed that the HRP content in postnuclear supernatants prepared from cells which had been chased in the presence of up to 25 µM C-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.


ACKNOWLEDGEMENTS

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.


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