From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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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.
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 (Gal 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.
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
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
Gb3 Synthase (UDP-Gal:Lactosylceramide
GM3 Synthase (CMP-Sialic Acid:Lactosylceramide
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.
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).
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).
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.
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.
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.
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).
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).
INTRODUCTION
Top
Abstract
Introduction
References
1-4Gal
1-4Glc
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).
EXPERIMENTAL PROCEDURES
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.
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.
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.
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.
RESULTS
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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 ( ) or FB1-treated fibroblasts (
). SM synthase
was assayed in cells treated with (
) or without (
)
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.
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Fig. 2.
Kinetic analysis of GlcCer synthase
activity. GlcCer synthase was assayed in homogenates (50 µg of
protein) from control ( ) 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.
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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.
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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 ( ) 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 ( ) 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.
The relationship between of [4,5-3H]sphinganine concentration
and [3H]SL/[3H]GSL synthesis
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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).
DISCUSSION
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed. Tel.: 972-8-9342704;
Fax: 972-8-9344112; E-mail: bmfuter{at}weizmann.weizmann.ac.il.
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ABBREVIATIONS |
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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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REFERENCES |
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