(Received for publication, June 3, 1996, and in revised form, September 30, 1996)
From the Department of Membrane Research and Biophysics and
Department of Molecular Cell Biology, Weizmann Institute
of Science, Rehovot, Israel 76100
Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3, the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was ~45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB1 blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.
Sphingolipids (SLs)1 are almost ubiquitous components of eukaryotic cell membranes where they play a variety of roles (1-3). An important approach to defining the precise roles of SLs is to inhibit their synthesis, by either genetic approaches, such as the production of mutants defective in SL synthesis (4), or by specific chemical inhibitors (5, 6). Unfortunately, little information is available using the first approach due to the lack of success in purifying the enzymes of SL synthesis, and the small number of mutants that have been obtained. However, specific inhibitors of SL synthesis have become available recently (5), including the mycotoxin, fumonisin B1 (FB1) (7). FB1 inhibits acylation of the sphingoid long chain bases sphinganine (dihydrosphingosine) and sphingosine to dihydroceramide and ceramide, respectively.
A number of studies have examined the effects of FB1 on the growth of cultured cells in attempts to determine the cellular basis for the diseases associated with FB1 (reviewed in Merrill et al. (8)). It has been shown that FB1 stimulates DNA synthesis in confluent cultures of fibroblasts (9), but inhibits the proliferation of renal epithelial cells (10) and the growth of Saccharomyces cerevisiae (11). In cultured hippocampal neurons, FB1 disrupts axonal growth by affecting the formation or stabilization of axonal branches (12, 13) and disrupts dendrite growth in cerebellar Purkinje neurons (14). Together, these studies suggest that SL synthesis is required for cell growth and morphogenesis.
To further study the roles of SLs in cell morphogenesis, we have now analyzed the effects of FB1 on Swiss 3T3 fibroblasts cultured at subconfluent densities. We previously demonstrated that the delivery of new membrane to the leading edge of these cells is required for pseudopodial activity and for directional migration (15), and we now show that long term incubation with FB1 causes profound changes in a number of morphological processes associated with the actin cytoskeleton. Remarkably, all the effects of FB1 on cell morphology could be reversed by addition of low concentrations of ganglioside GM3. These results suggest that SLs may be involved in the assembly of both new membrane and of the underlying cytoskeleton.
FB1 was from the Division of Food Science and Technology, CSIR, Pretoria, South Africa, or from Sigma. Ganglioside GM3, ganglioside GM1, short acyl chain analogs of ceramide (N-hexanoyl-D-erythro-sphingosine, N-hexanoyl-L-threo-sphingosine, and N-hexanoyl-D-erythro-dihydrosphingosine), D-sphinganine, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) were from Matreya (Pleasant Gap, PA). 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin, mitomycin C, 4,6-diamidino-2-phenylindole (DAPI), staurosporine, and D-erythro-sphingosine were from Sigma. Silica Gel 60 plates were from Merck. Lissamine-rhodamine-conjugated goat anti-mouse and rhodamine-conjugated goat anti-rabbit antibodies were from Jackson ImmunoResearch Laboratories Inc. [6-3H]Thymidine was from Amersham International plc, Amersham, UK. Other chemicals were from Sigma, and solvents (analytical grade) were from Bio-Lab Laboratories Ltd., Jerusalem, Israel.
Cell CultureSwiss mouse 3T3 cells 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 60-mm culture dishes for biochemical experiments, or on glass coverslips for morphological analysis, both at densities of ~2 × 104 cells/ml of medium.
Drugs and LipidsFB1 was added to cells from a 1 mM stock solution in 20 mM HEPES, pH 7.4; in most experiments FB1 was added to cultures immediately after plating. Mitomycin C was prepared as a 1 mg/ml stock solution in 50% ethanol. Staurosporine was prepared as a 20 µM stock solution in Me2SO. H-7 was prepared as a 30 mM stock solution in H2O. Gangliosides GM3 and GM1 were prepared as 0.5 mM stock solutions in methanol, dried under a stream of N2, dissolved in medium, sonicated, and added to cultures to give final concentrations of 25 or 100 nM.
SL Metabolism[4,5-3H]Sphinganine was synthesized by reduction of D-erythro-sphingosine with NaB[3]H4 (10 Ci/mmol) (16-18) and used to analyze [3H]SL synthesis (16). Briefly, [4,5-3H]sphinganine (5 × 106 cpm) was added to the culture medium of 3T3 cells. After 24 h, 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 (19), and [3H]SLs were extracted and analyzed exactly as described previously (16), except that CHCl3/CH3OH/H2O (65:25:4, v/v/v) was used as the developing solvent for TLC. [3H]GM3 and [3H]GM1 were synthesized by tritium-labeling of the sphingoid long chain bases using NaB[3]H4 (18, 20).
Immunofluorescence and Fluorescence MicroscopyMicrotubule
distribution was examined using a monoclonal anti--tubulin antibody
(clone DM1A, Sigma), intermediate filaments were
examined using a polyclonal anti-vimentin antibody (provided by Dr.
Benny Geiger, Department of Molecular Cell Biology, Weizmann Institute
of Science), and actin distribution was examined using TRITC-conjugated
phalloidin. Cells were observed using Plan Apochromat 40×/1.3 n.a.,
63×/1.4 n.a., and 100×/1.3 n.a. oil objectives of a Zeiss Axiovert 35 microscope with an appropriate filter for rhodamine fluorescence. Cells
labeled with DAPI were examined using a Plan Neofluar 20 × 0.5 n.a.
objective of a Zeiss Axiophot microscope with a filter for DAPI
fluorescence.
Projected cell area (i.e. the area occupied by the cell on the substrate) was determined after labeling cells with TRITC-conjugated phalloidin, and capturing images of labeled cells via an Applitec MSV-700L CCD camera to a Macintosh 840AV computer and using NIH imaging software. Dispersion and elongation indices of cell outlines were calculated according to Dunn and Brown (15, 21). Cell outlines were identified and analyzed using software provided by Dr. Z. Kam, Department of Molecular Cell Biology, Weizmann Institute of Science.
Scanning Electron MicroscopyFibroblasts, grown on 13-mm glass coverslips, were fixed in glutaraldehyde (2%, w/v) and paraformaldehyde (3%, w/v), followed by postfixation in 1% (w/v) osmium tetroxide in 0.15 M Na-cacodylate, pH 7.4 (22). Cells were rinsed in distilled water, treated with 1% tannic acid, rinsed again, treated with 2% (w/v) uranyl acetate in distilled water for 30 min, and then rinsed again. Specimens were dehydrated in a graded series of acetone, and critical point drying was performed using a Pelco critical point dryer. Specimens were coated with gold using a S150 Sputter Coater (Edwards) and examined using a Jeol JSM 6400 scanning electron microscope.
Thymidine IncorporationCells were plated in 24 multiwell dishes at a density of 5 × 103 cells/well, and incubated for 2 h with 0.5 µCi of [6-3H]thymidine (50 Ci/mmol). Cells were washed in phosphate-buffered saline, incubated with trichloracetic acid (5%, 4 °C, 20 min), washed with ethanol, and dissolved in 0.1 M NaOH. Radioactivity was determined by liquid scintillation counting in a Packard Tri-Carb 1500 scintillation counter using Ultima gold scintillation fluid (Packard):water (8:1, v/v).
To
determine the extent of inhibition of SL synthesis by FB1
in Swiss 3T3 fibroblasts, various concentrations of FB1
were added to the medium at the time of plating. A
dose-dependent inhibition of [3H]SL synthesis
was observed (Fig. 1A), with significant
inhibition obtained using 20 µM FB1. Using
this concentration, [3H]SL synthesis was inhibited to a
similar extent when analyzed on each of the first 5-6 days in culture,
and even short incubations with FB1 (i.e. 1 h) resulted in similar levels of inhibition. When expressed as a
function of the amount of protein per culture dish on day 5, the
incorporation of [4,5-3H]sphinganine into total
[3H]SLs was inhibited by >70% at 20 µM
FB1 and above (Fig. 1B). The extent of
inhibition appeared less when expressed as incorporation of
[4,5-3H]sphinganine per µg protein (compare Fig. 1,
A and B), since FB1-treated cells did
not proliferate (see below), and the amount of protein per dish was
much lower in FB1-treated cells, particularly at high
concentrations of FB1 (20 or 50 µM).
Analysis of the [3H]SL profile demonstrated that levels of individual [3H]SLs remaining after FB1-treatment varied (Fig. 1C). Whereas [3H]glucosylceramide ([3H]GlcCer), [3H]sphingomyelin ([3H]SM), and [3H]GM3 were reduced by 73-78% (see also Fig. 1A), [3H]lactosylceramide ([3H]LacCer) was reduced by only 37%, and [3H]ceramide trihexoside ([3H]Gb3) increased by 2.3-fold (Fig. 1C). This resulted in different profiles of [3H]SLs in control compared to FB1-treated cells. [3H]Gb3 comprised 1.9% of the total [3H]SLs in control cells, but 16.5% in FB1-treated cells. [3H]GlcCer comprised 8.7 and 7.7%, [3H]LacCer 4.5 and 8.9%, [3H]SM 58.7 and 48.1%, and [3H]GM3 comprised 26.2% in control cells versus 18.8% in FB1-treated cells.
Cell Spreading Is Reduced by FB1 TreatmentInhibition of SL synthesis by FB1 (20 µM) for 5 days caused profound changes in cell
morphology. Whereas control cells displayed typical fibroblast
morphology, with a leading edge and a trailing edge (Fig.
2A), FB1-treated cells were less
well spread on the substrate and displayed reduced pseudopodial
activity (Fig. 2B). After incubation with FB1
for 5 days, the projected cell area was reduced by ~45% (Table
I), and there was a significant decrease in the
morphometric index (21) of dispersion (dispersion index of control
cells was 0.91 ± 0.09, and of FB1-treated cells was 0.55 ± 0.09, n = 40), but not of elongation
(elongation index of control cells was 1.23 ± 0.1 and of
FB1-treated cells was 1.10 ± 0.08, n = 40). Elongation is a measure of the extent to which shape must be
compressed along its longitudinal axis in order to minimize its
difference from a circle, while dispersion is invariant to stretching,
compressing, or shearing the shape in any direction; both of these
indices are equal to zero for a circle. Elongation is considered as a
measure of cell bipolarity, whereas dispersion is a measure of
multipolarity (21). Thus, the formation of protrusions is reduced in
FB1-treated cells, while cells remain elongated to a
similar extent to control cells.
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The effects of FB1 on morphology were completely reversed 24 h after its removal from the medium, or by addition of exogenous ganglioside GM3 to the medium for 24 h, even in the presence of FB1 (Table I); GM3 itself had no effect on projected cell area. Neither of the bioactive intermediates, sphinganine or sphingosine, which both accumulate upon long term treatment with FB1 (8, 23), had any effect on morphology even for up to 5 days incubation, and even after multiple additions (Table I). Short acyl chain analogues of ceramide, whose level is depleted upon FB1 treatment (8), and a short acyl chain analogue of dihydroceramide were also unable to reverse the effects of FB1 (Table I).
Since in fibroblasts, short acyl chain analogues of ceramide are metabolized mainly to GlcCer and SM (not shown; see also Meivar-Levy et al. (24)), and since sphinganine, sphingosine, and ceramides do not significantly affect cell morphology, these data together demonstrate that the ability of GM3 to restore cell morphology is due to depletion of an essential higher order glycosphingolipid, probably GM3, and not due to accumulation or depletion of bioactive intermediates. This is supported by observations that exogenously added GM3 was metabolized to only a limited extent by fibroblasts during a 24-h incubation (see also Chigorno et al. (25)). Upon incubation of control and FB1-treated cells with 100 pmol [3H]GM3 on day 4, 68-72% of the [3H] radioactivity remained in [3H]GM3 on day 5, with most of the remainder in [3H]GlcCer (5-6%), [3H]SM (5%),2 [3H]LacCer (6%), and [3H]Gb3 (3-4%). A related ganglioside, GM1, was able to partially restore the effects of FB1 (Table I). After incubation of cells with [3H]GM1, ~10% was degraded to [3H]GM3; the small amount of GM3 formed from GM1 may be responsible for the partial ability of GM1 (Table I) to restore the effects of FB1 on cell morphology.
FB1-Treatment Affects the Organization of the Actin CytoskeletonThe effects of FB1 were further analyzed
by examination of the distribution of cytoskeletal elements.
FB1 had no apparent effect on the radial distribution of
microtubules or of intermediate filaments (not shown), but changes in
the actin cytoskeleton were observed, with far fewer actin-rich
"stress fibers," and loss of actin-rich lamellipodia at the leading
edge (compare Fig. 3, A with C and
E).
In addition to its roles in organization of the leading edge in
interphase cells, the actin cytoskeleton also plays an important role
in cell division, particularly in the formation of the contractile ring
during cytokinesis. Incubation with FB1 interfered with
cytokinesis as demonstrated by the appearance of binuclear cells (Fig.
3, B, D, and F). The percent of
binuclear cells was 3-4% in control cells, but 11-12% in
FB1-treated cells (Fig. 4). Addition of
GM3 on day 4 in culture partially reversed the effects of
FB1 on cytokinesis (Fig. 4), but neither sphingosine or
sphinganine had any effect (not shown).
FB1 Interferes with Protrusional Activity and Microvilli Formation
A number of protein kinase inhibitors, such
as staurosporine and H-7, induce changes in fibroblast morphology (26,
27). Upon incubation with H-7, fibroblasts acquire numerous long, thin processes (Fig. 5A) that result from the
inability of cells to retract their trailing edge (26); at the leading
edge, lamellipodial activity is unaffected and may even be enhanced
(Fig. 5A). Pretreatment with FB1 (20 µM) for 5 days significantly decreased the number of long
processes formed upon H-7 or staurosporine-treatment (Fig. 5), and
GM3 almost completely restored the ability of staurosporine to induce long processes (Fig. 5).
Fibroblasts were also incubated with mitomycin C, a drug that inhibits DNA replication and arrests cells in the S phase of the cell cycle. Since other biosynthetic processes are not affected, including synthesis of new membrane and new cytoskeletal components, cells continue to grow and acquire much larger sizes than untreated cells. The projected area of cells treated with 0.1 µg/ml mitomycin C for 5 days was 4.3-fold greater than that of untreated cells (Table II), and mitomycin C-treated cells displayed a large number of small microvilli at the cell surface that were generally located near the center of the cell (Fig. 6A). The projected area of FB1-treated cells was only 2.4-fold greater after incubation with mitomycin C (Table II), and FB1-treated cells displayed far fewer microvilli (Fig. 6B). The addition of GM3 on day 4 to cells that had been treated with both mitomycin C and FB1 resulted in a significant increase in projected cell area (Table II), demonstrating that GM3 is able to partially restore the inhibitory effects of FB1 on cell spreading after mitomycin C treatment.
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Cell Proliferation and DNA Synthesis Is Blocked by FB1
Little cell proliferation was observed for up to
10 days in the presence of 20 µM FB1,
although cells remained viable throughout this period. The number of
cells in untreated cultures increased by ~23-fold during the first 7 days in culture, but only increased by 2.6-fold in
FB1-treated cultures (Fig. 7A).
The block in cell proliferation was reversible, since cell number
increased after removal of FB1 on day 4, and attained
values similar to untreated cells (Fig. 7A). The block of
cell proliferation could not be explained solely by inhibition of
cytokinesis, since only 11-12% of the cells were binuclear after
FB1-treatment (see Fig. 4). Indeed, analysis of
[3H]thymidine incorporation demonstrated that DNA
synthesis was also significantly inhibited by FB1-treatment
(Fig. 7B). The effects of FB1 on DNA synthesis
(Fig. 7B) could be reversed by removing FB1 from
the medium, but in contrast to its effects on cell morphology, addition
of GM3 had no effect on either cell proliferation (not shown) or on [3H]thymidine incorporation (Fig.
7B). Addition of either sphingosine or sphinganine directly
to the culture medium had no effect on cell proliferation, either in
the absence or presence of FB1 (not shown).
Inhibition of ceramide synthesis by FB1 causes a number of responses in various cells (8). We now demonstrate that the reduction in complex SL synthesis that occurs upon incubation of Swiss 3T3 cells with FB1 results in major changes in the actin cytoskeleton and in processes related to, or dependent on, the actin cytoskeleton and that these effects can be reversed by addition of ganglioside GM3.
Characterization of the Biochemical Effects of FB1 on Swiss 3T3 FibroblastsOf all the inhibitors tested (5), FB1 has proved particularly useful in manipulating levels of SL synthesis (7, 8, 28). As a consequence of FB1 treatment, sphingosine and sphinganine levels are elevated, and ceramide is depleted (7). All three of these molecules can themselves disrupt cell morphology and proliferation (10, 29), and it is therefore essential when using FB1 to distinguish between effects caused by changes in levels of these bioactive intermediates or effects caused by depletion of complex sphingolipids (8). The inability of sphingoid long chain bases and of ceramide, and the ability of GM3 to reverse the effects of FB1 even in the presence of FB1, strongly suggests that the effects we observed in sparse cultures of 3T3 cells are due to depletion of complex SLs, as appears to be the case for changes in cell morphology and growth upon FB1 treatment of cultured hippocampal neurons (12, 13). The major SLs in 3T3 cells are GM3 and SM, and both of these lipids are depleted to a similar extent after incubation with FB1. Surprisingly, levels of the neutral glycosphingolipid, Gb3, are elevated after FB1 treatment. In cultured cerebellar neurons (28), SM synthesis was more sensitive to FB1 than glycosphingolipid synthesis. Since the activities of SM synthase and of glycosyltransferases were not directly affected by FB1, it was suggested that different enzymes in the metabolic pathway are sensitive to a different extent to reduction in the levels of their respective substrates (28). Our data suggest that Gb3 synthase has a relatively high Km value, which renders it relatively insensitive to changes in levels of its substrate, lactosylceramide.
The Ability of GM3 to Reverse the Effects of FB1Incubation with FB1 affects a number of cellular processes, including cell spreading, microvilli formation, cytokinesis, formation of long processes, and disruption of DNA synthesis and cell proliferation. GM3 restores the disruptive effects of FB1 on cell morphology, but not on proliferation. Four pieces of evidence suggest that GM3 itself is responsible for the restoration of cell morphology. (i) GM3 is synthesized at far higher levels than either lactosylceramide or Gb3, rendering it more suitable to mediate interactions between the plasma membrane and the actin cytoskeleton (see below). Moreover, Gb3 synthesis is not inhibited by FB1 treatment. It should however be noted that no role has yet been ascribed to Gb3, and the differences in the sensitivity of Gb3 and GM3 synthesis to FB1-treatment may suggest that levels of Gb3 must be maintained at a constant level in the cell for it to perform whatever function it may play. (ii) GM3 is only degraded to a limited extent by fibroblasts (see also Chigorno et al. (25)). (iii) The lack of effects of short acyl chain analogues of ceramide (which are metabolized mainly to GlcCer and SM, but not to lactosylceramide, GM3 and Gb3), suggest that GlcCer cannot be responsible for the restoration of cell morphology. (iv) Neither SM or ceramide can be responsible for restoration since incubation with PDMP (which inhibits the synthesis of GlcCer and of higher order glycosphingolipids, but enhances SM synthesis (30) and elevates ceramide levels), results in a similar decrease in projected cell area to that observed with FB1, and addition of exogenous GM3 together with PDMP restores normal cell morphology.3
Although exogenous GM3 could restore all of the disruptive effects of FB1 on cell morphology, including cytokinesis, it was completely inactive in reversing the inhibition of cell proliferation and DNA synthesis. Other studies have also demonstrated inhibition of cell proliferation by FB1 (10, 11, 31). Cell growth is also arrested in rabbit skin fibroblasts (32) and in Swiss 3T3 cells (33) by PDMP, with growth arrested at the G1/S and G2/M transitions in 3T3 cells (33). In contrast, FB1 stimulates thymidine incorporation in 3T3 cells (9); however, the conditions used in this study (9) were completely different from those used in the current study, inasmuch as we examined cell proliferation and DNA synthesis during the logarithmic phase of growth, whereas Schroeder et al. (9) examined stimulation of DNA synthesis in quiescent cultures. Although we cannot draw any definitive conclusions about the mechanism(s) by which FB1 inhibits cell proliferation in sparse cultures of 3T3 cells, the inability of GM3 to restore proliferation demonstrates that GM3 is not the limiting factor in the regulation of cell proliferation. Elucidation of the possible roles of bioactive sphingolipid intermediates in regulating proliferation (2) requires further study.
The Relationship between GM3 and the Actin CytoskeletonThe most interesting conclusion from the current study is that GM3 synthesis is required for a variety of processes that depend on the assembly of new membrane and of the underlying actin cytoskeleton. These processes are exemplified by the reduction in the number of microvilli in mitomycin C-treated cells (Fig. 6). Microvilli are surface extensions containing an actin core, and are found on the surface of many cells, particularly on cells that require a large surface area to function. Normal fibroblasts have few microvilli, but after mitomycin C-treatment, their levels are greatly increased, presumably since synthesis and assembly of actin and new membrane continues, even though cell division is arrested. The fact that FB1-treated cells have far fewer microvilli indicates that an intimate relationship exists between GM3 and the actin cytoskeleton. Other examples of this relationship are illustrated by the reduction in projected cell area (Table I), pseudopodial activity (Fig. 2), the formation of long processes (Fig. 5), and the inhibition of cytokinesis (Figs. 3 and 4).
How might ganglioside GM3 be related to the actin cytoskeleton? Since gangliosides are highly enriched in the plasma membrane (34), the effects described above are presumably due to alteration in a plasma membrane function. It is not known whether the effects depend on alterations in a physical property of the membrane, or alternatively on the assembly of new membrane, but it should be noted that all of the processes described above require membrane synthesis and assembly. For instance, protrusional activity at the leading edge can be blocked by treatments that inhibit the synthesis or delivery of new membrane (15, 35). During the assembly of new membrane, actin-binding proteins such as ezrin, radixin, and moesin (whose recruitment to the plasma membrane is required for microvilli formation) (36, 37) should be recruited to the membrane, and it may be that GM3 is involved in their recruitment. Consistent with GM3 playing a role in the recruitment or activity of membrane proteins that direct actin assembly are studies showing that GM3 is functionally associated with integrins (38). These and similar studies (39) focused mainly on the involvement of GM3 in integrin-mediated cell adhesion. However, the data reported in the current study suggest that GM3 plays a wider role since it appears necessary for mediating events associated with assembly of the actin cytoskeleton and of new membrane.