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.
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ABSTRACT |
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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Cells and Cell CultureNormal 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 SLsNatural 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 StainingCells 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 (ex = 360 nm;
em = 460 ± 50 nm).
Analysis of Cholesterol, Cholesterol Esters, and SLsMonolayer cultures were grown in 60-mm dishes (7080% 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-1Cells were grown to 6070% 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 DextranTo 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 LBPATo 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 MethodsFor 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 (4050% 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 6070% 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).
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RESULTS |
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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; 515 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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Accumulation of Endogenous GlcCer Induces Cholesterol Uptake in Normal HSFsTo 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 CellsTo 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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LDLR Expression but Not LDL Internalization Is Increased in
SL-treated CellsWe 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 2050%
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 25-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 3080%
increase in LDLR expression was also seen when longer incubations
(e.g. 2448 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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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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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 CellsWe 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 1.52-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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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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Intracellular Localization of Exogenously Supplied GM1 and Accumulated CholesterolThe 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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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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DISCUSSION |
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Internalization of Exogenous SLs and Accumulation in EndosomesWhen 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 CholesterolIncubation 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 SLsSL 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 2.54.5-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 SequestrationHow 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.
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FOOTNOTES |
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Present address: Luther College, 700 College Dr., Decorah, IA 52101.
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-(-D-galactopyranosyl)-(1
4)-S-(
-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.
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REFERENCES |
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