Up-regulation of Neutral Glycosphingolipid Synthesis upon Long Term Inhibition of Ceramide Synthesis by Fumonisin B1*

Irit Meivar-Levy and Anthony H. FutermanDagger

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

    ABSTRACT
Top
Abstract
Introduction
References

In a previous study we observed that long term (5 days) incubation with fumonisin B1 (FB1), an inhibitor of acylation of sphingoid long chain bases to (dihydro)ceramide, resulted in morphological and biochemical changes in 3T3 fibroblasts (Meivar-Levy, I., Sabanay, H., Bershadsky, A. D., and Futerman, A. H. (1997) J. Biol. Chem. 272, 1558-1564). Among these were changes in the profile of synthesis of sphingolipids (SLs) and glycosphingolipids (GSLs). Whereas [3H]globotriaosylceramide ([3H]Gb3) comprised 1.9% of the total [3H]SLs and [3H]GSLs synthesized in control cells, it comprised 16.5% in FB1-treated cells. We now demonstrate by in vitro analysis that inhibition of ceramide synthesis by FB1 for 5 days results in up-regulation of the activities of three enzymes in the pathway of Gb3 synthesis, namely glucosylceramide, lactosylceramide, and Gb3 synthases; up-regulation is due to an increase in Vmax, with no change in Km values toward lipid substrates. Moreover, molecular analysis (reverse transcriptase-polymerase chain reaction) of glucosylceramide synthase indicated that this enzyme is up-regulated at the transcriptional level. No changes in either the Vmax or Km values of sphingomyelin or of GM3 synthase were detected after FB1 treatment. Analysis of SL and GSL synthesis in cultured cells using [4,5-3H]sphinganine as a metabolic precursor demonstrated that at low substrate concentrations, Gb3 synthesis is favored over GM3 synthesis and glucosylceramide synthesis is favored over sphingomyelin synthesis, whereas the opposite is true at high substrate concentrations. These data demonstrate that GSL synthesis and in particular Gb3 synthesis are tightly regulated in fibroblasts, presumably so as to maintain constant levels of Gb3 on the cell surface.

    INTRODUCTION
Top
Abstract
Introduction
References

Sphingolipids (SLs)1 and glycosphingolipids (GSLs) are ubiquitous and essential components of eukaryotic cell membranes (1). Significant variation exists in the types and levels of both acidic and neutral GSLs between different cells, and although most attention has been paid to the sialic-acidic containing GSLs, the gangliosides (2), neutral GSLs are also found at relatively high levels in a number of tissues (1, 3). Among these is globotriaosylceramide (Galalpha 1-4Galbeta 1-4Glcbeta 1-ceramide; Gb3). Gb3 is expressed in many types of human blood cells, including erythrocytes, lymphocytes, and platelets, and its expression is elevated in some lymphomas (4, 5). Sequential changes in the expression of Gb3 and of other GSLs occurs during B cell differentiation due to sequential activation of the corresponding glycosyltransferases (6). No single function has been ascribed to Gb3, although it has been implicated as a differentiation antigen for B lymphocytes, as the Pk blood group antigen (7), and as a marker for apoptosis of germinal center B-cells (8). In addition, although not a physiological function, Gb3 acts as the cell surface receptor for Shiga toxin (9-11).

In a recent study, we demonstrated that upon inhibition of SL and GSL synthesis by fumonisin B1 (FB1), an inhibitor of (dihydro)ceramide synthesis (12), levels of Gb3 synthesis were not reduced (13). Upon incubation of 3T3 fibroblasts for 5 days with 20 µM FB1, the incorporation of [4,5-3H]sphinganine (a precursor of the synthesis of all SLs and GSLs (14)) into [3H]SLs was reduced by ~70% (13). However, whereas [3H]sphingomyelin (SM) and total [3H]GSL synthesis decreased by 75 and 72%, respectively, [3H]Gb3 synthesis unexpectedly increased after FB1 treatment by ~2.3-fold. This resulted in different profiles of [3H]SL synthesis, with [3H]Gb3 comprising 1.9% of the total [3H]SLs synthesized in control cells but 16.5% in FB1-treated cells (13).

In the current study, we examine the molecular mechanisms underlying the differences in profiles of SL and GSL synthesis after long term inhibition of ceramide synthesis by FB1. By in vitro analyses, we demonstrate that three glycosyltransferases in the metabolic pathway leading to Gb3 synthesis are up-regulated. In addition, in vivo analysis demonstrates that differences in Km values also result in a preference for Gb3 synthesis when ceramide is synthesized at low levels. That fibroblasts specifically maintain cellular Gb3 levels, in preference over other GSLs and SLs, adds weight to the idea that Gb3 plays one or more essential, although as yet unidentified, physiological functions in fibroblasts and presumably in other cells.

    EXPERIMENTAL PROCEDURES

Materials

FB1 was from the Division of Food Science and Technology (CSIR, Pretoria, South Africa). D-Sphinganine and lactosylceramide (LacCer) were from Matreya (Pleasant Gap, PA). n-Hexanoic acid [1-14C]n-hydroxysuccinimide ester (55 mCi/mmol) was from American Radioactive Chemicals (St. Louis, MO). CMP[14C]sialic acid (294 mCi/mmol) and UDP[U-14C]Gal (261 mCi/mmol) were from Amersham International plc (Amersham, UK). TitanTM one tube RT-PCR system was from Boehringer Mannheim. Silica Gel 60 plates were from Merck. Ultima Gold was from Packard (Meriden, CT). Other chemicals were from Sigma, and solvents (analytical grade) were from Bio-Lab Laboratories Ltd. (Jerusalem, Israel).

Cell Culture

NIH 3T3 fibroblasts were cultured in Dulbecco's modified medium containing 10% calf serum and maintained in a water-saturated atmosphere of 5% CO2. Cells were dissociated with trypsin/EDTA and plated in either 100-mm culture dishes for in vitro experiments or 60-mm culture dishes for in vivo experiments, both at densities of ~5 × 104 cells/ml of medium. Medium was not changed before harvesting.

In Vitro Analysis of SL Synthesis

Preparation and Analysis of Lipids-- N-(1-[14C]Hexanoyl)-D-erythro-sphingosine ([14C]hexanoyl Cer) and [14C]hexanoyl GlcCer were synthesized by N-acylation of sphingosine and glucosylsphingosine, respectively, using the N-hydroxysuccinimide ester of 1-[14C]hexanoic acid (15). [14C]Hexanoyl-lipids were prepared as a complex with defatted bovine serum albumin (molar ratio, 1:1) (16).

GlcCer Synthase (UDP-Glucose:N-Acylsphingosine beta 1-1-Glucosyltransferase)-- Cells were washed three times with phosphate-buffered saline, harvested using a rubber policeman, and homogenized in a hand-held Potter-Elvehjem homogenizer in 1 ml of 25 mM KCl and 50 mM Tris (pH 7.4) (TK buffer). Homogenates were used fresh. GlcCer synthesis was assayed exactly as described (17). The standard reaction mixture contained 50 µg of protein (determined according to Ref. 18), 5 mM UDP-Glc, 10 µM [14C]hexanoyl Cer, 10 mM MnCl2, in total volume of 500 µl of TK buffer. The reaction was terminated after 30 min at 37 °C by addition of 1.5 ml chloroform/methanol (1:2 v/v). Lipids were extracted (19) and separated on TLC using chloroform/methanol/water (65:25:4 v/v/v) as developing solvent. TLC plates were exposed to a 14C-sensitive imaging plate, lipids were recovered from the plates by scraping, and radioactivity was determined by liquid scintillation counting.

SM Synthase (N-Acylsphingosine:Phosphatidylcholine Phosphocholinetransferase)-- SM synthase (20) was assayed as described for GlcCer synthase, except that 100 µg of protein was used for a 2-h incubation and no UDP-Glc or MnCl2 were added to the reaction mixture.

LacCer Synthase (UDP-Galactose:Glucosylceramide beta 1-4-Galactosytransferase)-- Cells were homogenized in 1 ml of 250 mM sucrose/10 mM Tris (pH 7.4). The assay was based on a published procedure (21) with some modifications. Briefly, 200 µg of a cell homogenate was incubated with 10 µM [14C]hexanoyl GlcCer, 10 mM MgCl2, 50 mM NaCl, and 5 mM UDP-Gal in total volume of 500 µl of 250 mM sucrose/10 mM Tris (pH 7.4) for 2 h at 37 °C. [14C]Hexanoyl lipids were extracted and analyzed as described above.

Gb3 Synthase (UDP-Gal:Lactosylceramide alpha 1-4-Galactosyltransferase)-- Cells were harvested using a rubber policeman and pelleted by centrifugation (10,000 × gav, 30 min, 4 °C). The cell pellet was homogenized in 500 µl of 50 mM sodium cacodylate buffer (pH 5.9). Gb3 synthase activity was assayed as described (22) with some modifications. The reaction mixture contained 500 µg of cell homogenate, 5-50 µg of LacCer, 0.3% Triton X-100, 10 mM MnCl2, 1.4 µM UDP[14C]Gal (0.375 µCi), 50 mM sodium cacodylate buffer (pH 5.9) in a total volume of 1 ml. Incubations were performed for 4 h at 37 °C with vigorous shaking, and reactions were terminated by addition of 3 ml chloroform/methanol (2:1 v/v) and extracted (22). Lipids were separated by TLC using chloroform/methanol/CaCl2 (60:35:8 v/v/v) as developing solvent. TLC plates were exposed to a 14C-sensitive imaging plate, lipids were recovered from the plates by scraping, and radioactivity was determined by liquid scintillation counting.

GM3 Synthase (CMP-Sialic Acid:Lactosylceramide alpha 2-3-Sialytransferase)-- A cell pellet, prepared as described for assay of Gb3 synthase, was homogenized in 500 µl of 250 mM sodium cacodylate buffer (pH 6.15). Ganglioside GM3 synthase (6)2 was assayed using 500 µg of a cell homogenate, 5-50 µg of LacCer, 0.2% Triton CF-54, 10 mM MnCl2, 0.85 µM CMP[14C]sialic acid (0.25 µCi) (diluted 1:5 in CMP-sialic acid), 250 mM sodium cacodylate buffer (pH 6.15), in total volume of 1 ml. Incubations were performed for 4 h at 37 °C with vigorous shaking, and reactions terminated by addition of 3 ml chloroform/methanol (2:1 v/v). Lipids were extracted and analyzed as described above for Gb3 synthase.

In Vivo Analysis of SL Metabolism

[4,5-3H]Sphinganine was synthesized by reduction of D-erythro-sphingosine with NaB[3]H4 (10 Ci/mmol) (14, 23, 24). After 24 h of incubation with [4,5-3H]sphinganine, cells were washed with phosphate-buffered saline, removed by scraping with a rubber policeman, and centrifuged (15,000 × gav, 30 min, 4 °C). Protein was determined (18), and [3H]SLs/[3H]GSLs were extracted and analyzed exactly as described (13). Upon metabolism of [4,5-3H]dihydroceramide to [3H]ceramide, we assume that 50% of the 3H radioactivity is lost due to dehydrogenation of the 4,5-double bond; this was taken into account when quantifying [3H]GSL synthesis as described previously (14).

RT-PCR Analysis of GlcCer Synthase

Partial GlcCer synthase cDNA (~400 base pairs) based on the sequence of rat GlcCer synthase (28) was obtained from NIH 3T3 fibroblasts by RT-PCR using the following GlcCer synthase primers: 5'-TTGTTCGGCTTCGTGCTCTT-3' (forward primer) and 5'-GACTCGTATTCCGCTATCAC-3' (reverse primer). Total RNA was isolated by the TRI-Reagent protocol (Molecular Research Center Inc., Cincinnati, OH). RT-PCR products were resolved on 1.5% agarose-gels.

    RESULTS

Five major SLs and GSLs are synthesized by 3T3 fibroblasts, namely SM, GlcCer, LacCer, Gb3, and GM3 (13). Using [4,5-3H]sphinganine as a precursor of SL and GSL synthesis, we previously observed that residual levels of synthesis of each lipid differed after 5 days incubation with FB1 (20 µM) (13), and unexpectedly, Gb3 synthesis was not inhibited to any extent. No differences in residual levels of SL or GSL synthesis were observed after incubation with FB1 for short times (i.e. 1 h).3 We have now systematically determined the activity of the five enzymes responsible for the synthesis of each lipid by in vitro analyses and analyzed whether differences in Km values between the different enzymes might account for the change in the profile of SL and GSL synthesis.

In Vitro Analysis of SL Synthesis-- Cultured fibroblasts were incubated with FB1 (20 µM) for various times, removed from the culture dishes, and homogenized, and SM and GlcCer synthases were assayed in vitro using a short acyl chain radioactive analogue of ceramide, [14C]hexanoyl Cer (15, 17, 20). After 3 h of incubation of fibroblasts with FB1, there was no difference in the activity of GlcCer synthase compared with control cells, but after 1 day, a 30% increase was detected (Fig. 1). As time of incubation with FB1 increased, the activity of GlcCer synthase increased to a maximum of ~3-fold higher in cells incubated with FB1 for 5 days compared with untreated cells (Fig. 1). Addition of FB1 had no effect on GlcCer synthase activity or on the activity of any of the other enzymes assayed below, when added directly to the reaction mixture. The activity of SM synthase was much lower than GlcCer synthase in vitro, and no change in the level of SM synthase was detected even after 5 days of incubation with FB1 (Fig. 1).


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Fig. 1.   In vitro analysis of SM and GlcCer synthases. A, GlcCer synthase was assayed in vitro using 50 µg of protein from homogenates obtained from control (black-square) or FB1-treated fibroblasts (). SM synthase was assayed in cells treated with (open circle ) or without (bullet ) FB1 using 100 µg of protein. Each point is the mean ± S.E. (n = 6); the error bar is smaller than the size of the symbol in cases were no error bars can be seen. B, TLC plate showing a typical experiment analyzing GlcCer and SM synthases. The two left-hand lanes show the activity of GlcCer synthase, and the two right-hand lanes show SM synthase, both assayed after 5 days of incubation with FB1. Con, control; FB1, FB1-treated.

To determine whether the increase in activity of GlcCer synthase was due to changes in the Vmax or in the Km of the reaction, assays were performed using conditions in which the initial reaction rate (Vo) was linear with respect to time and to protein concentration and was not limited by substrate availability. After 5 days incubation with FB1, the Vmax of GlcCer synthase was 333 pmol/min/mg of protein, compared with 167 pmol/min/mg of protein in control cells (Fig. 2), but the Km values with respect to [14C]hexanoyl Cer were unchanged (6.3 µM in FB1-treated cells and 6.5 µM in control cells (Fig. 2)). No change was detected in either the Vmax or Km value of SM synthase after 5 days of incubation with FB1 (not shown).


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Fig. 2.   Kinetic analysis of GlcCer synthase activity. GlcCer synthase was assayed in homogenates (50 µg of protein) from control (black-square) or FB1-treated () fibroblasts. A, Michaelis-Menten analysis of enzyme activity. B, double reciprocal plot. Each point is the mean ± S.E. of 2-5 duplicate analyses of GlcCer synthase activity.

Because the cDNA encoding GlcCer synthase has recently been isolated (29), we analyzed the molecular mechanisms by which GlcCer synthase activity was regulated. RT-PCR analysis revealed that GlcCer synthase mRNA expression was increased in cells incubated with FB1 for 3 (not shown) or 5 days (Fig. 3) but not for 3 h (not shown). This demonstrates that GlcCer synthase is up-regulated at the transcriptional level upon long term incubation with FB1.


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Fig. 3.   RT-PCR analysis of GlcCer synthase. RNA was extracted from fibroblasts after 5 days of incubation with FB1. RT-PCR products were resolved on 1.5% agarose gels using the indicated amounts of total RNA. Oligonucleotide markers (400-700 base pairs) are shown in the left-hand lane. Note that RT-PCR analysis was repeated three times, and in every case, more RNA expression was observed in FB1-treated than in control cells, although the extent of the increase differed between individual experiments.

We next examined the activity of LacCer synthase using [14C]hexanoyl GlcCer as substrate. The Vmax of LacCer synthase also increased upon incubation with FB1 for 5 days, from 143 fmol/min/mg of protein in control cells to 250 fmol/min/mg of protein in FB1-treated cells (Fig. 4). The Km values with respect to [14C]hexanoyl GlcCer were unchanged (1.25 µM in FB1-treated cells and 1.57 µM in control cells (Fig. 4)). Similarly, the activity of Gb3 synthase was 2.7-fold higher in FB1-treated versus control cells (Fig. 5),4 but in contrast, there was no increase in the Vmax of GM3 synthase after FB1 treatment for 5 days (Fig. 6), although a small but statistically insignificant reduction in the activity of GM3 synthase was observed after FB1 treatment.


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Fig. 4.   Kinetic analysis of LacCer synthase activity. A, Michaelis-Menten analysis of LacCer synthase in homogenates (200 µg of protein) from control (black-square) or FB1-treated () fibroblasts. B, double reciprocal plot. Each point is the mean ± S.E. of 2-4 duplicate analyses of LacCer synthase activity.


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Fig. 5.   Analysis of Gb3 synthase activity. Gb3 synthase was assayed in homogenates (500 µg of protein) from control (Con) or FB1-treated fibroblasts. Data are the means ± S.E. (n = 6). The inset is part of a TLC plate showing [14C]Gb3 synthesis in control and FB1-treated cells.


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Fig. 6.   Kinetic analysis of GM3 synthase activity. GM3 synthase was assayed in homogenates (500 µg of protein) from control (black-square) or FB1-treated cells (). Data are the means ± S.E. (n = 4). The inset is part of a TLC plate showing [14C]GM3 synthesis in control and FB1-treated cells.

Incubation with FB1 causes depletion of ceramide from the synthetic pathway but also accumulation of sphinganine (12). Incubation of cultured cells directly with sphinganine (10 µM added each day for 5 days) had no effect on enzyme activity measured in vitro (GlcCer, SM, and LacCer synthases), and co-incubation of FB1 together with sphinganine (10 µM added each day for 5 days) did not change the extent of enzyme up-regulation induced by FB1 (not shown). These results suggest that the effects observed are due to inhibition of ceramide synthesis rather than accumulation of sphinganine.

In Vivo Analysis of [3H]GSL and [3H]SM Synthesis-- The data presented above demonstrate that long term inhibition (5 days) of ceramide synthesis results in up-regulation of the activity of three glycosyltransferases that use ceramide or downstream metabolites of ceramide as substrate, GlcCer, LacCer, and Gb3 synthases, but does not affect SM or GM3 synthases. We next examined whether flux to different branches of the SL and GSL synthesis pathway (see Fig. 8) is correlated with substrate levels by directly incubating cultured cells with varying amounts of [4,5-3H]sphinganine, a metabolic precursor of the five lipids analyzed above.

Upon incubation with 30 nM [4,5-3H]sphinganine, [3H]Gb3 comprised 1.9% of the total [3H]SLs and [3H]GSLs synthesized (see also Ref. 13), but at 0.6 nM [4,5-3H]sphinganine, [3H]Gb3 comprised 9.0% (Table I). Because [3H]GM3 comprised ~25% of total [3H]SLs and [3H]GSLs synthesized at both high and low [4,5-3H]sphinganine concentrations (Table I), the ratio of [3H]Gb3 to [3H]GM3 synthesis increased ~4-fold as [4,5-3H]sphinganine decreased (Fig. 7A). In addition, whereas [3H]SM comprised 59% of the total [3H]SLs synthesized at 30 nM [4,5-3H]sphinganine, it comprised only 43% at 0.6 nM [4,5-3H]sphinganine (Table I), and as a result the ratio of [3H]GSLs to [3H]SM synthesis increased by ~2-fold as [4,5-3H]sphinganine decreased (Fig. 7B). These results demonstrate that the amount of substrate shunted to one or the other branch of the synthesis pathway varies depending on substrate concentration, with a preference for Gb3 versus GM3 synthesis at low substrate levels.

                              
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Table I
The relationship between of [4,5-3H]sphinganine concentration and [3H]SL/[3H]GSL synthesis
3T3 fibroblasts were incubated with varying concentrations of [4,5-3H]sphinganine for 24 h (13) prior to extraction and analysis of [3H]SLs and [3H]GSLs. Data are shown as a percentage of total [3H]SL and [3H]GSL synthesis, and the numbers in parentheses show the absolute amounts of [3H]SL and [3H]GSLs synthesized in fmol/µg of protein (means ± S.E. from three independent, duplicate experiments).


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Fig. 7.   Synthesis of [3H]GSLs and [3H]SM in cultured fibroblasts. Ratios of synthesis of each lipid or lipid class are calculated from the total amount of synthesis as given in Table I (fmol/µg protein) and expressed as the ratios of [3H]Gb3 synthesis versus [3H]GM3 synthesis (A) or [3H]GSL synthesis (the sum of synthesis of each individual GSL) versus [3H]SM synthesis (B).

Analysis of GSL Turnover-- To determine whether GSL turnover is also affected upon long term incubation with FB1, we analyzed acid glucosylceramidase activity in an in vitro assay using [14C]hexanoyl GlcCer (30) as substrate. No significant change in activity was observed for up to 9 days of incubation with FB1 (e.g. glucosylceramidase activity in control cells after 4 days of incubation was 516 ± 1 pmol/min/mg of protein and in FB1-treated cells was 529 ± 45 pmol/min/mg of protein, and after 7 days it was 618 ± 139 and 635 ± 90 in control and FB1-treated cells, respectively).

    DISCUSSION

The data presented above demonstrate that although only a minor GSL compared with GM3, fibroblasts strive to maintain constant levels of Gb3. When levels of metabolic precursors are low, Gb3 levels are preserved by two mechanisms (Fig. 8), namely (i) up-regulation of the activity of the three glycosyltransferases in the pathway of Gb3 synthesis and (ii) shunting LacCer to Gb3 synthesis rather than to GM3 synthesis. Together, this results in maintenance of Gb3 levels even when levels of metabolic precursors are low. In addition, GSL synthesis is preferred to SM synthesis when ceramide synthesis is low (Fig. 8) (see also Ref. 31). A similar dual mechanism of regulation has been suggested for GM2 and GD2 synthases in human cancer cell lines (32), where expression of GD2 synthase is regulated by levels of its immediate precursor, GD3. Regulation of glycolipid expression by substrate availability has also been observed during development and oncogenesis (33, 34).


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Fig. 8.   Proposed regulation of GSL and SM synthesis in 3T3 fibroblasts. When ceramide is synthesized at low levels, GlcCer synthesis (bold line) is favored over SM synthesis (dashed line), and Gb3 synthesis (bold line) is favored over GM3 synthesis (dashed line). Long term inhibition of ceramide synthesis by incubation with FB1 for >3 days results in up-regulation of the three glycosyltransferases in the pathway of Gb3 synthesis (shown in bold italics) but no increase in the activity of either GM3 or SM synthases. PC, phosphatidylcholine.

Molecular analysis demonstrated that GlcCer synthase is up-regulated at the transcriptional level resulting in increased production of the glycosyltransferase rather than by post-translational modification of existing enzyme. This is supported by the observation that relatively long times of incubation with FB1 (>1 day) were required to detect changes in Vmax values. Similarly, GlcCer synthase is induced during keratinocyte differentiation (35) due to transcriptional up-regulation (36). Interestingly, upon long term incubation with FB1, the activity of three glycosyltransferases (GlcCer, LacCer, and Gb3 synthases) is increased, presumably by coordinate regulation at the transcriptional level. At this time, it is not possible to determine whether Gb3 synthase is also regulated at the transcriptional level because this enzyme has not been cloned, although a partial purification of enzyme activity was reported from rat liver (37).

We do not know whether up-regulation of the glycosyltransferase activities is a result of inhibition of ceramide synthesis per se or of reduction of GM3 and Gb3 levels. It is well documented that cells respond in various ways to changes in ceramide levels, but most cellular responses are due to increased production of ceramide at the cell surface due to degradation of SM in ceramide-mediated signaling pathways (38) rather than ceramide depletion in the endoplasmic reticulum where it is synthesized (23, 39).5 Alternatively, fibroblasts may respond to changes in levels of the lipid products (GM3 and Gb3). Depletion of GM3 in fibroblasts results in changes in the actin cytoskeleton and in a block in cell proliferation and DNA synthesis (13), and one of these effects could be an initial signaling event in glycosyltransferase up-regulation. Although we cannot distinguish between biochemical effects due to depletion of GM3 compared with Gb3, it is nevertheless apparent that fibroblasts ascribe particular importance to maintaining Gb3 levels.

That fibroblasts go to such length to maintain cellular Gb3 levels implies that Gb3 plays a key role in fibroblast function. Gb3 is expressed in a restricted set of hematopoietic cells and is considered both a differentiation antigen (40) and more recently as a regulator of apoptosis during differentiation of the hematopoietic system (41). No single function has been ascribed to Gb3 in fibroblasts. We assume that the function of Gb3 is related to its localization at the cell surface and are testing the possibility that Gb3 is found in GSL-enriched domains on the cell surface, as may be the case for both GM3 (42) and GM1 (43). Because a number of transducer molecules also appear to be found in these GSL-enriched domains, such as c-Src, Ras, Rho, and focal adhesion kinase, the presence of Gb3 in these domains might suggest a regulatory role in signaling events related to cytoskeletal organization; indeed, we have recently shown that GM3 mediates events associated with assembly of the actin cytoskeleton (13). This possible role has not been tested for Gb3, but if Gb3 is also enriched in these domains, it will be of importance to determine the mode of interaction of these two structurally similar GSLs with both signaling molecules and with the underlying cytoskeleton.

    ACKNOWLEDGEMENTS

We thank Alexander D. Bershadsky (Weizmann Institute of Science) for many helpful discussions and Zehava Levy from the laboratory of Dr. Mike Fainzilber for help with the RT-PCR analysis.

    FOOTNOTES

* This work was supported by the Minerva Foundation (Munich, Germany).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 972-8-9342704; Fax: 972-8-9344112; E-mail: bmfuter{at}weizmann.weizmann.ac.il.

    ABBREVIATIONS

The abbreviations used are: SL, sphingolipid; FB1, fumonisin B1; Gb3, globotriaosylceramide; GlcCer, glucosylceramide; GSL, glycosphingolipid; LacCer, lactosylceramide; SM, sphingomyelin; RT-PCR, reverse transcriptase-polymerase chain reaction.

2 Gangliosides are named according to Svennerholm (44).

3 I. Meivar-Levy and A. H. Futerman, unpublished observations.

4 Assays for Gb3 synthesis were not performed at saturating levels of UDP[14C]Gal because the low specific activity of this enzyme precluded detection of activity when UDP[14C]Gal was diluted with UDP-Gal.

5 Generation of ceramide at the cell surface after treatment with an endoglycoceramidase results in a transient increase in GlcCer synthase activity in B16 melanoma cells and in 3T3 fibroblasts, but this appears to be due to post-translational modification of enzyme activity (45).

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Introduction
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