Sphingolipid Storage Induces Accumulation of Intracellular Cholesterol by Stimulating SREBP-1 Cleavage*

Vishwajeet Puri, John R. Jefferson {ddagger}, Raman Deep Singh, Christine L. Wheatley, David L. Marks and Richard E. Pagano §

From the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905

Received for publication, January 10, 2003 , and in revised form, March 20, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We showed previously that the intracellular transport of sphingolipids (SLs) is altered in SL storage disease fibroblasts, due in part to the secondary accumulation of free cholesterol. In the present study we examined the mechanism of cholesterol elevation in normal human skin fibroblasts induced by treatment with SLs. When cells were incubated with various natural SLs for 44 h, cholesterol levels increased 25–35%, and cholesterol esterification was reduced. Catabolism of the exogenous SLs was not required for elevation of cholesterol because (i) a non-hydrolyzable and a degradable SL analog elevated cellular cholesterol to similar extents, and (ii) incubation of cells with various SL catabolites, including ceramide, had no effect on cholesterol levels. Elevated cholesterol was derived primarily from low density lipoproteins (LDL) and resulted from up-regulation of LDL receptors induced by cleavage of the sterol regulatory element-binding protein-1. Upon SL treatment, cholesterol accumulated with exogenous SLs in late endosomes and lysosomes. These results suggest a model in which excess SLs present in endocytic compartments serve as a "molecular trap" for cholesterol, leading to a reduction in cholesterol at the endoplasmic reticulum, induction of sterol regulatory element-binding protein-1 cleavage, and up-regulation of LDL receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sphingolipid storage diseases (SLSDs)1 are metabolic disorders that generally result from a defective lysosomal hydrolase or activator protein leading to accumulation of endogenous lipids in the lysosomes in many different cell types (1). We showed previously that a fluorescent glycosphingolipid (GSL) analog (BODIPY-lactosylceramide; LacCer) is internalized from the plasma membrane (PM) and transported to the Golgi complex in normal human skin fibroblasts (HSFs) but is mistargeted to endosomes and lysosomes in cells from multiple SLSDs, suggesting a common mechanism of cellular dysfunction in these biochemically distinct diseases (2). In a subsequent study we showed that these SLSD cells accumulate unesterified cholesterol in the late endosomes and lysosomes (3), and this cholesterol plays an important role in the altered sorting and targeting of the GSL analog in SLSD fibroblasts (3, 4, 5, 6). Similarly, cholesterol has been reported to accumulate secondary to sphingomyelin storage in sphingomyelinase-deficient (Niemann-Pick A disease) human and mouse cells (7, 8). The mechanism by which intracellular cholesterol is elevated and/or redistributes in SLSDs with primary defects in SL catabolism is unknown.

In the current study, we show that incubation of normal HSFs with exogenous, naturally occurring SLs resulted in a dramatic increase and redistribution in cellular cholesterol reminiscent of that seen in SLSD cells. These observations led us to study further the relationship between SL accumulation and cellular cholesterol. We provide evidence that accumulation of SLs in endocytic vesicles serves as a molecular trap for cholesterol, binding and immobilizing the sterol as it circulates through the cell. Importantly, although the cell has high levels of free cholesterol that can be readily detected by lipid analysis, the protein machinery that regulates cholesterol homeostasis behaves as if cellular cholesterol levels are low. This leads to an induction of sterol regulatory element-binding protein (SREBP-1) cleavage, transcriptional activation of the LDL receptor (LDLR), and a reduction in cholesterol esterification.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Lipids were obtained from Matreya (State College, PA). Filipin was purchased from Polysciences Inc. (Warrington, Pennsylvania). DiI-LDL was from Intracel (Frederick, MD). Lyso-Lac-S-Cer was synthesized by the Organic Synthesis Core of the Mayo Clinic as described (9). Lyso-LacCer and Lyso-Lac-S-Cer were N-acylated with BODIPY fatty acid as described (10), and the corresponding products, BODIPY-LacCer and BODIPY-Lac-S-Cer, were purified by TLC and/or high pressure liquid chromatography. Monoclonal antibodies against human EEA1 were from Transduction Laboratories. A monoclonal antibody against lysobisphosphatidic acid (LBPA) was a kind gift from Dr. Toshihide Kobayashi (RIKEN Frontier Research System, Saitama, Japan). Unless otherwise indicated, all other reagents were from Sigma.

Cells and Cell Culture—Normal and SLSD skin fibroblasts were obtained from the Coriell Institute (Camden, NJ) or Human Genetic Mutant Cell repository and cultured in Eagle's MEM (EMEM) plus 10% FBS as described (2, 3, 11).

Incubation of Cells with Exogenous SLs—Natural SLs were dissolved in 2:1 ethanol/Me2SO to make a 5 mM stock. An aliquot of this stock solution was added to the culture medium (EMEM plus 10% FBS) to make a final concentration of 40 µM SL. The cells were incubated in this medium for various times at 37 °C in a CO2 incubator. Control cultures were incubated with the same amount of ethanol/Me2SO but containing no SL. Cell viability for each treatment was >90%, as assessed by trypan blue staining.

Filipin Staining—Cells were fixed in 3% formaldehyde in PBS for 30 min followed by incubation in PBS containing 125 µg/ml filipin for 30 min at room temperature (12). Samples were then observed by fluorescence microscopy ({lambda}ex = 360 nm; {lambda}em = 460 ± 50 nm).

Analysis of Cholesterol, Cholesterol Esters, and SLs—Monolayer cultures were grown in 60-mm dishes (70–80% confluency) and scraped in 1 ml of PBS. 100 µl of this suspension was used for protein determination (Bradford protein assay), and the remaining 900 µl was used for quantitation of cholesterol, cholesterol esters, or SLs. Cholesterol and cholesterol esters were extracted as described (13), and the lower organic phase containing these lipids was collected and dried under N2. The extracted lipids were then separated by TLC using CHCl3/C2H5OC2H5/CH3COOH, 65:15:1 (v/v/v) as the developing solvent for cholesterol, or heptane/isopropyl ether/acetic acid, 60:40:4 (v/v/v) for cholesterol and cholesterol esters. TLC plates were dried and then stained overnight with iodine. Cholesterol and cholesterol esters were quantified by densitometry and comparison to cholesterol and cholesterol linoleate standards run on the same TLC plate.

For SL extraction and analysis (14, 15), 900 µl of cell suspension (see above) was mixed with 10 µl of pyridine, followed by the addition of 4.5 ml of CH3OH and 4.5 ml of CHCl3, and stirred overnight at 40 °C. The sample was then centrifuged for 10 min at 500 x g to remove insoluble cellular debris. The supernatant was collected, dried, resuspended in 900 µl of CHCl3/CH3OH (1:1), and saponified by addition of 100 µlof0.5 M KOH (stirred for 2 h at 40 °C). After neutralizing with 10 µl of 5 M acetic acid, the sample was dried, resuspended in synthetic upper phase (CH3OH/H2O/CHCl3, 94:96:6 (v/v/v)), and desalted by passage through a Waters Sep-Pak C18 column (Milford, MA). SLs were separated by TLC along with appropriate standards using CHCl3/CH3OH/15 mM CaCl2 (65:35:8; v/v/v) as the developing solvent. Primulin was used as a detection reagent (16), and lipids were quantified by scanning densitometry.

Western Blotting for LDLR or SREBP-1—Cells were grown to 60–70% confluency and treated with lipids for the indicated times. Two hours before completion of the experiment 25 µg/ml N-acetyl-leucyl-leucyl-norleucinal was added to inhibit proteolysis of SREBP. Cells were scraped in Western lysis buffer (10 mM Tris-Cl, 100 mM NaCl, and 1% SDS, pH 7.6) containing protease inhibitors (CompleteTM, Roche Molecular Biochemicals), lysed, and homogenized by passing 10 times through a 22-gauge needle. Aliquots of each sample (40 µg of protein) were run on 7 (for LDLR) or 10% SDS-PAGE gels (for SREBP-1) (17) and transferred to polyvinylidene difluoride membranes in transfer buffer with 20% methanol. Blots were probed with monoclonal LDLR antibody (Oncogene Research Products, San Diego) or monoclonal SREBP-1 antibody (Pharmingen) followed by peroxidase-labeled anti-mouse secondary antibody (Amersham Biosciences) and detected using Renaissance chemiluminescence reagent plus (PerkinElmer Life Sciences).

Colocalization of Exogenous GM1 with Cholesterol and Fluorescent Dextran—To study the distribution of exogenously supplied GM1 relative to cellular cholesterol, HSFs were incubated with 40 µM bovine GM1 in culture medium for 44 h at 37 °C as above. The cells were then fixed with 3% formaldehyde in PBS for 30 min, permeabilized with 0.05% Triton X-100 in PBS (2 min), and then incubated in PBS containing 125 mg/ml filipin and 7.5 mg/ml AlexaFluor 488 CtxB (Molecular Probes, Eugene, OR) for 30 min (all incubations were at room temperature). The samples were then washed, mounted, and observed under the fluorescence microscope.

To study the localization of GM1, HSFs were first incubated with 40 µM GM1 for 20 h at 37 °C as above. 2 mg/ml Cascade blue dextran (10 kDa, lysine fixable; Molecular Probes, Eugene, OR) was then added to the GM1-containing medium, and the samples were further incubated for 22 h at 37 °C. The samples were then washed with culture medium containing GM1 (but not dextran) and chased for 2 h at 37 °C to label the lysosomes with dextran (18). (All 37 °C incubations were in a CO2 incubator.) The cells were then fixed, permeabilized, and stained with AlexaFluor 488 CtxB as above, except that filipin was not present. The samples were then observed under the fluorescence microscope, and separate images were acquired for CtxB and dextran. Overlap of CtxB with the dextran-positive lysosomes was calculated as described (2, 3, 19).

Filter sets and exposure times for these double label experiments were selected such that there was no crossover between AlexaFluor 488 CtxB and filipin or blue dextran fluorescence.

Colocalization of Cholesterol with EEA1 and LBPA—To study the localization of cholesterol in SL-treated cells, HSFs were incubated with 40 µM bovine globoside in culture medium for 44 h at 37 °C. Cells were then stained with filipin as described above, followed by immunofluorescence using antibodies against EEA1 (labels early endosomes) or LBPA (labels late endosomes) (20).

Other Methods—For inhibition of glucocerebrosidase, cells were treated with Conduritol B-epoxide (CBE) (21). Briefly, an aliquot of a 50 mM stock of CBE was added to cells (~40–50% confluent) to give a final concentration of 50 µM, and the cultures were incubated for 24 h at 37 °C in CO2 incubator prior to the experiment.

For studies with fluorescent LDL, cells were grown to 60–70% confluency, incubated with 0.5 µg/ml DiI-LDL (Intracel, Frederick, MD) for 30 min at 4 °C in 10 mM Hepes buffered MEM, pH 7.4 (HMEM). to label the plasma membrane, and washed. The samples were then incubated for 5 min at 37 °C in HMEM to internalize the DiI-LDL, acid-stripped to remove any DiI-LDL remaining on the cell surface (22), and observed under the fluorescence microscope. Fluorescence microscopy and image analysis were performed as described previously (4, 19).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of Exogenous SLs on Cholesterol in Normal HSFs— Cells were incubated with 40 µM bovine LacCer or SM for 44 h (see "Experimental Procedures"), and the intracellular distribution of cholesterol was examined by filipin staining. Increased filipin fluorescence was observed following incubation with either lipid, relative to untreated control cells (Fig. 1a). In addition, the pattern of filipin staining was remarkably similar to that seen in various SLSD cell types (3). Similar results to those shown in Fig. 1a were also obtained when normal HSFs were incubated with C6-SM, a short chain non-fluorescent SM analog, or natural globoside, or natural glucosylceramide (GlcCer) (data not shown). In addition to filipin staining, we also studied the intracellular distribution of NPC1, a protein that contains a sterol sensing domain and normally resides in Rab7 and Rab9 containing late endosomes but which redistributes to lysosomes upon accumulation of cholesterol (12, 24). Following incubation of normal HSFs with natural LacCer or SM, we observed a more intense staining pattern of NPC1 by immunofluorescence, as reported previously (3) for SLSD cell types (data not shown). We also found that when normal HSFs were incubated with exogenous SLs as above and then pulse-labeled with fluorescent LacCer, the LacCer analog was targeted to punctate endosomal structures, rather than to the Golgi apparatus as seen in untreated control cells (data not shown). Finally, we quantified the increase in cellular cholesterol by lipid analysis when HSFs were incubated with natural LacCer or SM for various times (Fig. 1b). After 44 h, the increase in cholesterol was nearly the same using either SL; however, at shorter times, cholesterol was seen to increase more rapidly using LacCer than SM. Incubation of HSFs with natural LacCer or SM also resulted in an increase in other classes of SLs (Table I).



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FIG. 1.
Exogenous SLs elevate cholesterol levels in normal HSFs. HSFs were incubated for various times in culture medium containing 10% FBS and 40 µM bovine SM or LacCer. a, increase in intracellular cholesterol detected by filipin staining. Following a 44-h incubation with the indicated lipid, cells were washed, fixed, stained with filipin, and observed under the fluorescence microscope. Bar, 10 µm. b, increase in cellular cholesterol determined by lipid analysis. Cells were incubated for the indicated times with LacCer or SM and washed, and the amount of cholesterol was quantified by lipid extraction and analysis.

 

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TABLE I
Modulation of cellular SLs and cholesterol in normal HSFs

Samples were untreated (control) or incubated for 44 h with 40 µM bovine LacCer or SM for 44 h (see "Experimental Procedures"), or for 24 h with 50 µM of the glucocerebrosidase inhibitor, CBE. Cellular lipids were extracted, and the SL and cholesterol content were quantified (see "Experimental Procedures"). Results are mean ± S.D. from at least three independent experiments.

 

The increase in cellular cholesterol upon treatment with exogenous SLs could potentially be due to the formation of SL metabolites. In order to test this, cells were incubated with various concentrations of ceramide (Cer; 5–15 µM), lyso-LacCer (50 µM), gluco-psycosine (50 µM), or psycosine (50 µM) for 44 h. These treatments had no effect on cellular cholesterol levels as determined by filipin staining or lipid extraction and analysis (data not shown).

Exogenous SLs Increase LDL Cholesterol Uptake in HSFs— The cholesterol increase in SL-treated cells could be due to an increase in the uptake of LDL cholesterol and/or to an increase in cholesterol synthesis. To test the former possibility, HSFs were incubated with exogenous SM or LacCer for 44 h in a medium containing 5% lipoprotein-deficient serum (LPDS). Filipin staining of HSFs incubated with SM in the presence of LPDS was virtually identical to that in untreated control cultures grown in 10% FBS indicating no change in cholesterol distribution, whereas incubation with SM in FBS-containing medium showed enhanced staining indicative of cholesterol accumulation (Fig. 2a). It should be noted that the increase in cell-associated SM during a 44-h incubation in the presence of LPDS (~60% increase) was similar to that seen when incubations were carried out in FBS-containing medium (~77% increase). Similar results were obtained using bovine LacCer in LPDS medium instead of SM (data not shown). These data show that in cells treated with exogenous SLs, most of the cholesterol that accumulated was derived from lipoproteins present in the FBS-containing culture medium.



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FIG. 2.
Elevation of intracellular cholesterol by SLs requires serum lipoproteins. a, HSFs were incubated with 40 µM bovine SM in medium containing 10% FBS or LPDS for 44 h. Samples were then washed, fixed, and stained for cholesterol using filipin. b, HSFs were incubated with 50 µM CBE for 24 h to induce endogenous GlcCer accumulation (see Table I) and then stained with filipin as in a. Note the absence of cholesterol accumulation in both a and b when incubations were carried out in LPDS. Bars, 10 µm.

 

Accumulation of Endogenous GlcCer Induces Cholesterol Uptake in Normal HSFs—To determine whether accumulation of endogenous SLs in normal HSFs also results in an increase in cellular cholesterol, cells were treated with 50 µM CBE, an inhibitor of lysosomal glucocerebrosidase (25), for 24 h. This resulted in approximately a 2-fold increase in endogenous GlcCer levels (Table I), a dramatic increase in filipin staining (Fig. 2b), and a 27% increase in unesterified cholesterol determined by lipid extraction and analysis (Table I). On the other hand, when the cells were treated with CBE in medium containing 5% LPDS, there was no change in cellular cholesterol relative to untreated control samples (Fig. 2b), although CBE treatment in the presence of LPDS still induced approximately a 2-fold increase in GlcCer (data not shown).

Protein Synthesis Is Required for Cholesterol Uptake in SL-treated Cells—To determine whether protein synthesis was required for the elevation in cellular cholesterol induced by exogenous SLs, HSFs were incubated with 40 µM SM for 44 h in the presence or absence of 17.5 µM cycloheximide, and subsequently fixed and stained using filipin as in Fig. 1. No increase in filipin staining was observed in the presence of cycloheximide, whereas in the absence of this drug there was a dramatic increase in staining (Fig. 3a) and approximately a 25% increase in total cellular cholesterol as determined by lipid extraction and analysis. Similar results to those shown in Fig. 3a were obtained using bovine LacCer in place of SM (data not shown). These results demonstrate that protein synthesis is required for the increase in cellular cholesterol that occurs during incubation of HSFs with exogenous SLs.



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FIG. 3.
Elevation of intracellular cholesterol by exogenous SLs requires protein synthesis and is due to an increase in LDL receptors. a, HSFs were incubated with 40 µM bovine SM for 40 h in the presence or absence of 17.5 µM cycloheximide (CHX) in medium containing 10% FBS. Samples were then fixed and stained with filipin as in Fig. 1. Note the absence of cholesterol accumulation in cells treated with SM and CHX. b, HSFs were incubated with 40 µM bovine SM, LacCer, or GM1 in 10% FBS for 24 h. The samples were then washed and incubated with 0.5 µg/ml DiI-LDL for 30 min at 10 °C in HMEM to label LDLR at the PM. Identically treated specimens were further incubated for 5 min at 37 °C in HMEM to stimulate endocytosis, chilled, and acid-stripped to remove any DiI-LDL remaining at the cell surface. Samples were then observed under the fluorescence microscope, and the amount of cell-associated fluorescence was quantified by image analysis. Each value is the mean ± S.D. of at least 10 cells in three independent experiments.

 

LDLR Expression but Not LDL Internalization Is Increased in SL-treated Cells—We used fluorescent LDL (DiI-LDL) to study the cell surface expression and internalization of the LDLR in HSFs under various conditions. When HSFs were incubated with DiI-LDL at low temperature to bind LDLR at the PM, we observed a 20–50% increase in cells treated with SLs for 24 h relative to untreated cells (Fig. 3b). We also quantified the amount of DiI-LDL internalization during 5 min of endocytosis and found an increase of ~50% in SL-treated cells relative to control HSFs (Fig. 3b). This increase in LDL uptake in SL-treated cells was most likely due to the increased number of LDLR at the PM because there was little or no difference in the uptake data (relative to controls) after normalization for the number of receptors at the PM (data not shown). To further investigate the increase in LDLR induced by SL treatment, we also studied LDLR expression by Western blot analysis. We found a 2–5-fold increase in LDLR expression in cells treated with SLs for 5 h relative to control cells depending on the SL used (Fig. 4). This variation in receptor expression may reflect differences in the elevation of cell-associated SLs which varied from a 1.6 to a 2.1-fold increase in the treated cells, depending on the SL type (data not shown). A 30–80% increase in LDLR expression was also seen when longer incubations (e.g. 24–48 h) with the exogenous SLs were used (data not shown). CBE treatment (50 µM; 24 h) also resulted in an increase (~2-fold) of LDLR expression (data not shown). These results suggested that SL accumulation induced the up-regulation of LDLR in normal HSFs, resulting in an increased uptake of LDL and high levels of intracellular cholesterol.



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FIG. 4.
Expression of LDLR in SL-treated cells. Normal HSFs were incubated with the indicated natural SL (40 µM) for 5 h in culture medium containing 10% FBS. Cells were then washed and lysed, and equal amounts of protein were loaded on gels for determination of LDLR expression by Western blotting and densitometry.

 

SL Treatment Results in Sterol Regulatory Element-mediated Gene Transcription of LDLRTo investigate the mechanism of up-regulation of the LDLR by exogenous SLs, we next studied SREBP-1 expression in normal, cholesterol-depleted, and SL-treated cells. As shown in Fig. 5a, SREBP was cleaved to the mature 68-kDa fragment (mSREBP) when HSFs were incubated for 5 h with exogenous LacCer, globoside, GM1, or SM, similar to that seen when cells were incubated in LPDS medium, whereas little SREBP-1 cleavage was seen in control samples that were not incubated with exogenous SLs. Similar results were also obtained using HSFs following a 24-h treatment with 50 µM CBE to increase endogenous GlcCer (data not shown). These results show that SLs could potentially play an important role in regulating the transcriptional processing of SREBP-1. We also attempted to study the cleavage of SREBP-2; however, using commercial antibodies, we were unable to consistently detect this isoform in our samples.



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FIG. 5.
Exogenous SLs induce SREBP-1 cleavage in normal HSFs. Cells, grown in 10% FBS, were untreated (Control) or incubated for 5 h with 40 µM of the indicated natural SL (a), or LacCer versus Lac-S-Cer (non-degradable analog) in 10% FBS (b). Samples were then washed and lysed, and equal amounts of protein were loaded on gels to detect SREBP-1 cleavage by Western blotting. Each of the SLs induced cleavage of the SREBP precursor (P) into the mature 68-kDa fragment (M, mSREBP), similar to that seen when HSFs were grown in LPDS (see a) to deplete cellular cholesterol.

 

Table I shows that Cer levels were elevated in SL-treated HSFs. To test the possibility that Cer might activate SREBP-1 cleavage, HSFs were incubated with bovine Cer, C2-Cer, C8-Cer, or sphingomyelinase under various conditions; however, no activation of SREBP was found (data not shown). To test further the possibility that degradation of the exogenous SLs was required for induction of SREBP-1 cleavage, we used a non-degradable analog of LacCer, C8-Lac-S-Cer (9, 15). In preliminary studies we found that incubation of HSFs with C8-Lac-S-Cer elevated cellular cholesterol to a similar extent as that seen using bovine LacCer (data not shown). As shown in Fig. 5b, both Lac-S-Cer and bovine LacCer resulted in cleavage of SREBP-1 to produce the transcriptionally active 68-kDa fragment, demonstrating that the intact SL can modulate the transcriptional regulation of LDLR. Interestingly, although no degradation of Lac-S-Cer could be detected, cellular Cer was elevated ~1.8-fold in this experiment (data not shown).

SRE-mediated Gene Transcription and High Levels of LDLR in SLSD Cells—We showed previously (3) that SLSD fibroblasts have elevated levels of cellular cholesterol. Because these cell types also accumulate endogenous SLs (e.g. see Table II), we examined SREBP-1 activation and LDLR expression to determine whether they were similar to normal HSFs incubated with exogenous SLs. As shown in Fig. 6, there is an enhanced cleavage of SREBP-1 and approximately a 1.5–2-fold increase in the levels of LDLR in Niemann Pick type A, Niemann Pick type C, and GM1 gangliosidosis cell types relative to normal HSFs grown in the presence of 10% fetal calf serum. Other SLSD cell types have not yet been studied.


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TABLE II
SL composition in NP-C, NP-A, and GM1 gangliosidosis cell types

Cells were grown in 60-mm dishes ({approx}70% confluency) in complete medium containing 10% FBS. SLs were extracted and quantified as described under "Experimental Procedures." Results are mean ± S.D. from at least three independent experiments. ND, not determined. Control values for normal HSFs are given in Table I.

 


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FIG. 6.
Activation of SREBP cleavage and expression levels of LDLR in SLSD cells. Normal HSFs (Control) or fibroblasts from patients with Niemann Pick type C (NPC), Niemann Pick type A (NPA), or GM1 gangliosidosis (GM1) fibroblasts were grown in 10% FBS, washed, and lysed, and equal amounts of protein were loaded on gels for determination of SREBP cleavage (as in Fig. 5) (a) or LDLR expression levels (b) (as in Fig. 4).

 

Decreased Cholesterol Esterification in SL-treated Cells— The activation of SREBP-1 in SL-treated cells suggests that even though total cholesterol levels are high, cholesterol is unable to reach the ER pool that regulates SREBP-1 transport to the Golgi apparatus for cleavage. To test further the concept that cholesterol transport to the ER is inhibited in SL-treated cells, we quantified total cholesterol ester and free cholesterol levels in SL-treated cells. In cells incubated with 40 µM bovine globoside in media with 10% FBS for 20 h, there was >60% decrease in total cholesterol esters compared with untreated controls, with a concomitant increase in free cholesterol (Table III). As expected, in cells incubated with 5% LPDS for 20 h, cholesterol ester levels were significantly reduced for both control and globoside-treated samples. Similar results to those in Table III were obtained when cells were incubated with bovine SM or GM1 in media with 10% FBS for 20 or 44 h (data not shown).


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TABLE III
Modulation of cellular cholesterol and cholesterol ester content in normal HSFs following incubation with an exogenous SL

Cells growth in 60-mm culture dishes were incubated without (control) or with 40 µM bovine globoside for either 20 h in EMEM 10% FBS or for 5 h in EMEM, 10 % FBS, followed by 20 h in McCoy's medium, 5% LPDS. Cellular lipids were then extracted, and the cholesterol and cholesterol ester content were quantified (see "Experimental Procedures"). Results are mean ± S.D. from at least three independent experiments.

 

Intracellular Localization of Exogenously Supplied GM1 and Accumulated Cholesterol—The data presented above show that incubation of HSFs with exogenous SLs causes a reduction in cholesterol delivery to the ER and an accumulation of cellular free cholesterol. Thus, we next attempted to determine the intracellular location where cholesterol is "trapped" in SL-treated cells. First, we incubated HSFs with 40 µM bovine GM1 for 44 h and then studied its intracellular distribution with respect to cellular cholesterol. In this experiment, fluorescent CtxB was used to examine the distribution of the GM1, and filipin staining was used to assess the distribution of cholesterol (see "Experimental Procedures"). As shown in Fig. 7a (upper panels), the distribution of CtxB and filipin were virtually identical, demonstrating that the exogenously supplied GM1 and cellular cholesterol were present in the same intracellular compartments. In control experiments in which cells were not incubated with GM1, little CtxB staining could be detected, and filipin staining was very low relative to HSFs incubated with GM1 (Fig. 7a, lower panels).



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FIG. 7.
Distribution of exogenously supplied GM1 with respect to cholesterol and a lysosomal marker. HSFs were incubated with 40 µM bovine GM1 for 44 h in culture medium containing 10% FBS. a, distribution of GM1 and cholesterol, as determined by fluorescent CtxB (green) and filipin staining (red) (see "Experimental Procedures"). In control experiments in which cells were not incubated with GM1, little CtxB staining could be detected, and filipin staining was very low relative to HSFs incubated with GM1. b, distribution of GM1 with respect to a lysosomal marker. HSFs were coincubated with GM1 (as in a) and fluorescent dextran. The distribution of GM1 was examined by CtxB staining and compared with that of the fluorescent dextran (see "Experimental Procedures"). Quantitative analysis indicated that about 25% of the GM1 was present in the dextran-labeled lysosomes. Bars, 10 µm.

 

In a related experiment, we also attempted to identify the intracellular compartment that contained the exogenous GM1. HSFs were incubated with fluorescent dextran under conditions which result in labeling of the lysosomes (18). The distribution of GM1 was then examined by CtxB staining and compared with that of the fluorescent dextran (Fig. 7b). By using image analysis to quantify the overlap of the two markers (19), we determined that ~25% of the GM1 was in dextran-positive lysosomes.

Finally, we studied the localization of intracellular cholesterol in HSFs incubated with 40 µM bovine globoside as above. As shown in Fig. 8, only ~5% of cholesterol-laden endocytic vesicles colocalized with the early endosome marker EEA1, whereas there was more than a 70% colocalization with LBPA, a marker for late endosomes (20). These studies suggest that in SL-treated cells most of the intracellular cholesterol resides in the late endosomes/lysosomes along with the accumulated exogenous lipid.



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FIG. 8.
Localization of intracellular cholesterol in HSFs following incubation with exogenous globoside. a, cells were incubated with 40 µM bovine globoside for 44 h in culture medium containing 10% FBS. Samples were then washed, and the intracellular distribution of cholesterol was determined by filipin staining (red pseudocolor). The distribution of cholesterol with respect to markers for early (EEA1) and late (LBPA) endosomes was determined in the same cells by immunofluorescence (green). Note the extensive overlap indicated by the yellow puncta, seen for filipin and LBPA (panel II), but not for filipin and EEA1 (panel I). b, quantitative analysis of the overlap of cholesterol-laden endocytic vesicles with EEA1 or LBPA.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study we show that incubation of exogenous, natural SLs with normal HSFs resulted in a significant increase in cellular cholesterol. This cholesterol accumulation was similar to that seen in Niemann Pick type C and various other SLSD cell types (Table II) (3, 26), leading us to further study the relationship between SL accumulation and cellular cholesterol. The data from our current study suggest a multistep process linking SL accumulation and cholesterol homeostasis. The evidence for a number of these steps is discussed below.

Internalization of Exogenous SLs and Accumulation in Endosomes—When exogenous natural SLs are added to culture medium, the lipids are taken up by an endocytic process; however, the process of lipid internalization is not well characterized. The various SLs used in the present study have different water solubilities, depending on the nature of the polar head group or fatty acid chain length. The more water-soluble lipids can readily integrate into the PM bilayer and are internalized with other endogenous lipid constituents present in the PM. In contrast, less soluble SLs may form lipid vesicles or aggregates as they are dispersed in culture medium; and these dispersions, rather than lipid monomers, would then be internalized by the cells. The mechanism by which lipids are internalized from the PM may vary in these two cases. Previous work from our laboratory (4, 22) has shown that GSL analogs, following their integration into the PM, are internalized from the cell surface by a clathrin-independent mechanism involving caveolae. The mechanism(s) by which lipid vesicles or aggregates are internalized is not known.

Another point of interest with respect to the current study concerns the endocytic compartment(s) in which the exogenous SLs accumulate. In the case of GM1, whose distribution can be readily examined using labeled CtxB, we found that there was almost complete colocalization between GM1 and intracellular cholesterol (Fig. 7a). However, only ~25% of the lipid accumulated in dextran-stained lysosomes (Fig. 7b). Finally, we found that in globoside-treated cells, intracellular cholesterol was highly colocalized with the late endosome marker LBPA (Fig. 8). These studies show that a significant fraction of both the internalized exogenous SL and cholesterol accumulates in late endosomes and lysosomes.

SL Accumulation Leads to Elevated Cholesterol—Incubation of various naturally occurring SLs with normal HSFs over a period of several days resulted in elevated cellular cholesterol (Fig. 1 and Table I). As shown in Table I, there was an increase in the cell-associated exogenous lipid and cholesterol, relative to untreated control cells, as well as an increase in Cer. To rule out the possibility that a degradation product of the exogenous lipids, rather than the lipid itself, was responsible for the increase in cholesterol, we added various SL breakdown products (psychosine, glucopsychosine, sphingosine, Cer, and lyso-LacCer) to HSFs, but no effect on cellular cholesterol levels was seen. Furthermore, addition of the non-degradable LacCer analog, Lac-S-Cer (9, 15), also increased cellular cholesterol. There was also an elevation of cellular cholesterol when endogenous glucosylceramide was increased by addition of the glucosylceramidase inhibitor CBE (Table I). Together these results suggest that an excess of SLs, whether derived from exogenous or endogenous sources, induces an increase in cellular cholesterol. At present it is not known whether there is a direct correlation between the amount of SL accumulation and the amount of cholesterol increase (refer to Table I). Similarly, the time course for the increase in cholesterol is different for different exogenous lipids as illustrated by the data in Fig. 1b. This could reflect differences in (a) the solubility of the exogenous lipids, (b) lipid uptake mechanisms, (c) SL degradation rates, or (d) a combination of these factors. Finally, our data suggest that the exogenously supplied SLs and cholesterol accumulate in the same endocytic compartments as demonstrated when labeled CtxB and filipin were used to probe the distribution of GM1 and cholesterol in GM1-treated cells (Fig. 7a). The extensive colocalization of these probes in punctate endocytic structures suggests that there may be a direct physical interaction of the accumulated SLs with cellular cholesterol.

Source of Elevated Cholesterol Induced by SLs—SL loading increased the binding and internalization of fluorescent LDL (Fig. 3b), and no increase in intracellular cholesterol was seen when SL loading was performed in medium containing LPDS in place of FBS (Fig. 2a). These results suggest that the majority of the increase in intracellular cholesterol in response to SL loading comes from an increase in the uptake of LDL rather than from de novo synthesis. This conclusion is consistent with our findings that SL loading induced SREBP-1 cleavage and caused a 2.5–4.5-fold increase in LDLR protein expression depending on the exogenous SL (Fig. 4). Thus, elevation in cellular SLs modulated the transcriptional regulation of the LDLR. SREBP-1 cleavage was induced by all the exogenous SLs tested, as well as by elevation of endogenous glucosylceramide (by CBE treatment). We recognize that SREBP-1 cleavage should also lead to a stimulation of do novo cholesterol synthesis (27), although we have not tested this directly. However, because cholesterol did not accumulate in SL-treated cells cultured in the absence of LDL, de novo synthesis of cholesterol appears to be insufficient to cause a significant elevation of cholesterol under our experimental conditions. In addition, we note that SREBP cleavage and LDLR expression were increased in three different SLSD cell types, in agreement with the concept that SL accumulation (from uptake of exogenous SLs or from inhibition of SL degradation) induces an elevation in cholesterol through the LDL pathway. Thus, our studies suggest that SREBP-1 cleavage contributes to elevated cholesterol in SL-treated normal fibroblasts and in SLSD cells.

Cholesterol Mobilization and Sequestration—How is the accumulation of SLs in endosomal compartments linked to an increase in cholesterol through the LDLR pathway? At first glance this seems paradoxical because cells incubated with SLs have high levels of cholesterol and elevated cholesterol should inhibit LDLR expression (27). We suggest that the accumulation of SLs in endosomal structures functions as a "molecular trap" for cholesterol, binding and immobilizing cholesterol as it circulates (28) through the cell. Indeed, such a mechanism has been proposed to account for defective cholesterol trafficking in acid sphingomyelinase-deficient macrophages (8). Molecular trapping of cholesterol would lead to an accumulation of cholesterol in endosomal compartments (e.g. as in Fig. 7a) and a reduction in the level of free cholesterol in the ER. Our studies showing that incubation of fibroblasts with SLs leads to a reduction in cholesterol esterification (Table III) support the idea that cholesterol transport to the ER is inhibited in these cells. Reduction in free cholesterol in the ER thus would induce SREBP-1 translocation to the Golgi apparatus where proteolysis occurs, releasing the transcriptionally active mature 68-kDa fragment (mSREBP-1) that subsequently activates transcription of the genes encoding the LDLR and 3-hydroxy-3-methylglutaryl-CoA reductase (27, 29, 30, 31). The converse situation, in which SM depletion by sphingomyelinase treatment leads to increased cholesterol translocation to the ER and a subsequent suppression of SREBP cleavage, has also been reported (32). Our model is in agreement with many observations in the literature showing interactions of SLs with cholesterol in various model and natural membrane systems (33, 34, 35, 36, 37). The "strength" of the interaction between cholesterol and different SLs could influence the reduction in free cholesterol in the cell and thus explain the variations seen in cellular cholesterol levels (Table I) and LDLR expression (Fig. 3b) using different SLs.

Finally we note that incubation of exogenous SLs with HSFs resulted in a significant elevation of Cer (Table I). Several studies (17, 23, 38) have demonstrated that Cer, derived from cleavage of SM, may either increase or decrease SREBP cleavage. However, in our studies Cer played no obvious role in mediating SREBP activation because SREBP-1 was cleaved similarly in cells treated with either the non-hydrolyzable Lac-S-Cer analog (which cannot be degraded to Cer) or with natural LacCer. In addition, treatment of cells with various SL metabolites, including Cer, did not result in cholesterol accumulation. Furthermore, incubation of HSFs with sphingomyelinase to generate natural Cer at the PM or incubation with natural or cell-permeable Cer analogs did not induce SREBP-1 cleavage or up-regulate the LDLR, consistent with a previous report (32). In the future, it will be of interest to examine the source of the increase in cellular Cer in response to SL loading and the consequences of this elevated Cer.


    FOOTNOTES
 
* This work was supported by United States Public Health Service Grant GM60934 (to R. E. P.), a grant from the Ara Parseghian Medical Research Foundation (to R. E. P. and D. M.), and an American Heart Association fellowship (to V. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Luther College, 700 College Dr., Decorah, IA 52101. Back

§ To whom correspondence should be addressed: Mayo Clinic and Foundation, Stabile 8, 200 First St., S.W., Rochester, MN 55905-0001. Tel.: 507-284-8754; Fax: 507-266-4413; E-mail: Pagano.richard{at}mayo.edu.

1 The abbreviations used are: SLSD, sphingolipid storage disease; BODIPY, boron dipyrromethene difluoride; CBE, Conduritol B-epoxide; Cer, ceramide; CtxB, cholera toxin, B subunit; DiI-LDL, DiI-labeled LDL; ER, endoplasmic reticulum; GlcCer, glucosylceramide; GSL, glycosphingolipid; HSFs, human skin fibroblasts; LacCer, lactosylceramide; Lac-S-Cer, O-({beta}-D-galactopyranosyl)-(1 -> 4)-S-({beta}-D-glucopyranosyl)-(1 -> 1)-(2R,3R,4E)-2-octanamido-3-hydroxy-4-octadecene-1-thiol; LDL, low density lipoprotein; LDLR, LDL receptor; LPDS, lipoprotein-deficient serum; LBPA, lysobisphosphatidic acid; PM, plasma membrane; SL, sphingolipid; SM, sphingomyelin; SREBP, sterol regulatory element-binding protein; EMEM, Eagle's minimal essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs, B., Kinzler, K. W., and Vogelstein, B. (eds) (2001) The Metabolic & Molecular Bases of Inherited Disease, Vol. III, 8th Ed., pp. 3371-3894, McGraw-Hill, New York
  2. Chen, C. S., Patterson, M. C., Wheatley, C. L., O'Brien, J. F., and Pagano, R. E. (1999) Lancet 354, 901-905[CrossRef][Medline] [Order article via Infotrieve]
  3. Puri, V., Watanabe, R., Dominguez, M., Sun, X., Wheatley, C. L., Marks, D. L., and Pagano, R. E. (1999) Nat. Cell Biol. 1, 386-388[CrossRef][Medline] [Order article via Infotrieve]
  4. Puri, V., Watanabe, R., Singh, R. D., Dominguez, M., Brown, J. C., Wheatley, C. L., Marks, D. L., and Pagano, R. E. (2001) J. Cell Biol. 154, 535-547[Abstract/Free Full Text]
  5. Marks, D. L., and Pagano, R. E. (2002) Trends Cell Biol. 12, 605-613[CrossRef][Medline] [Order article via Infotrieve]
  6. Pagano, R. E., Puri, V., Dominguez, M., and Marks, D. L. (2000) Traffic 1, 807-815[CrossRef][Medline] [Order article via Infotrieve]
  7. Schuchman, E. H., and Desnick, R. J. (2001) in The Metabolic & Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs, B., Kinzler, K. W., and Vogelstein, B., eds) Vol. III, 8th Ed., pp. 3589-3610, McGraw-Hill Inc., New York
  8. Leventhal, A. R., Chen, W., Tall, A. R., and Tabas, I. (2001) J. Biol. Chem. 276, 44976-44983[Abstract/Free Full Text]
  9. Albrecht, B., Putz, U., and Schwarzmann, G. (1995) Carbohydr. Res. 276, 289-308[CrossRef][Medline] [Order article via Infotrieve]
  10. Schwarzmann, G., and Sandhoff, K. (1987) Methods Enzymol. 138, 319-341[Medline] [Order article via Infotrieve]
  11. Martin, O. C., and Pagano, R. E. (1994) J. Cell Biol. 125, 769-781[Abstract]
  12. Neufeld, E. B., Wastney, M., Patel, S., Suresh, S., Cooney, A. M., Dwyer, N. K., Roff, C. F., Ohno, K., Morris, J. A., Carstea, E. D., Incardona, J. P., Strauss, J. F., III, Vanier, M. T., Patterson, M. C., Brady, R. O., Pentchev, P. G., and Blanchette-Mackie E. J. (1999) J. Biol. Chem. 274, 9627-9635[Abstract/Free Full Text]
  13. Martin, O. C., Comly, M. E., Blanchette-Mackie, E. J., Pentchev, P. G., and Pagano, R. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2661-2665[Abstract/Free Full Text]
  14. van Echten, G., Iber, H., Stotz, H., Takatsuki, A., and Sandhoff, K. (1990) Eur. J. Cell Biol. 51, 135-139[Medline] [Order article via Infotrieve]
  15. Schwarzmann, G., Hofmann, P., Pütz, U., and Albrecht, B. (1995) J. Biol. Chem. 270, 21271-21276[Abstract/Free Full Text]
  16. Skipski, V. P. (1975) Methods Enzymol. 35, 396-425[Medline] [Order article via Infotrieve]
  17. Worgall, T. S., Johnson, R. A., Seo, T., Gierens, H., and Deckelbaum, R J. (2002) J. Biol. Chem. 277, 3878-3885[Abstract/Free Full Text]
  18. Koval, M., and Pagano, R. E. (1990) J. Cell Biol. 111, 429-442[Abstract]
  19. Pagano, R. E., Watanabe, R., Wheatley, C., and Dominguez, M. (2000) Methods Enzymol. 312, 523-534[Medline] [Order article via Infotrieve]
  20. Kobayashi, T., Beuchat, M.-H., Lindsay, M., Frias, S., Palmiter, R. D., Sakuraba, H., Parton, R. G., and Gruenberg, J. (1999) Nat. Cell Biol. 1, 113-118[CrossRef][Medline] [Order article via Infotrieve]
  21. Sillence, D. J., Puri, V., Marks, D. L., Butters, T. D., Dwek, R. K., Pagano, R. E., and Platt, F. M. (2002) J. Lipid Res. 43, 1837-1845[Abstract/Free Full Text]
  22. Sharma, D. K., Choudhury, A., Singh, R. D., Wheatley, C. L., Marks, D. L., and Pagano, R. E. (2003) J. Biol. Chem. 278, 7564-7572[Abstract/Free Full Text]
  23. Lawler, J. F., Yin, M., Diehl, A. M., Roberts, E., and Chatterjee, S. (1998) J. Biol. Chem. 273, 5053-5059[Abstract/Free Full Text]
  24. Patel, S. C., Suresh, S., Kumar, U., Hu, C. Y., Cooney, A., Blanchette-Mackie, E. J., Neufeld, E. B., Patel, R. C., Brady, R. O., Patel, Y. C., Pentchev, P. G., and Ong, W.-Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1657-1662[Abstract/Free Full Text]
  25. Radin, N. S., and Vunnan, R. R. (1981) Methods Enzymol. 72, 673-684[Medline] [Order article via Infotrieve]
  26. Lange, Y., Ye, J., Rigney, M., and Steck, T. (2000) J. Biol. Chem. 275, 17468-17475[Abstract/Free Full Text]
  27. Brown, M. S., and Goldstein, J. L. (1997) Cell 89, 331-340[Medline] [Order article via Infotrieve]
  28. Fielding, C. J., and Fielding, P. E. (1997) J. Lipid Res. 38, 1503-1521[Abstract]
  29. DeBose-Boyd, R. A., Brown, M. S., Li, W. P., Nohturfft, A., Goldstein, J. L., and Espenshade, P. J. (1999) Cell 99, 703-712[Medline] [Order article via Infotrieve]
  30. Wang, X., Sato, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994) Cell 77, 53-62[Medline] [Order article via Infotrieve]
  31. Yokoyama, C., Wang, X., Briggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein, J. L., and Brown, M. S. (1993) Cell 75, 187-197[Medline] [Order article via Infotrieve]
  32. Scheek, S., Brown, M. S., and Goldstein, J. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11179-11183[Abstract/Free Full Text]
  33. Bittman, R., Kasireddy, C. R., Mattjus, P., and Slotte, J. P. (1994) Biochemistry 33, 11776-11781[Medline] [Order article via Infotrieve]
  34. Chatterjee, S. (1993) J. Biol. Chem. 268, 3401-3406[Abstract/Free Full Text]
  35. Slotte, J. P., and Bierman, E. L. (1988) Biochem. J. 250, 653-658[Medline] [Order article via Infotrieve]
  36. Thomas, P. D., and Poznansky, M. J. (1988) Biochem. J. 251, 55-61[Medline] [Order article via Infotrieve]
  37. Ridgway, N. D. (2000) Biochim. Biophys. Acta 1484, 129-141[Medline] [Order article via Infotrieve]
  38. Diomede, L., Albani, D., Bianchi, M., and Salmona, M. (2001) Eur. Cytokine Netw. 12, 625-630[Medline] [Order article via Infotrieve]