(Received for publication, June 1, 1995; and in revised form, August 25, 1995)
From the
C- and C
-ceramides (N-acetylsphingosine and N-hexanoylsphingosine,
respectively) abolished the stimulation of DNA synthesis by sphingosine
1-phosphate in rat fibroblasts. This inhibition by ceramide was
partially prevented by insulin. C
-ceramide did not alter
the stimulation of DNA synthesis by insulin and decreased the
sphingosine-induced stimulation by only 16%. The ceramides did not
significantly modify the actions of sphingosine or sphingosine
1-phosphate in decreasing cAMP concentrations. C
- and
C
-ceramides blocked the activation of phospholipase D by
sphingosine 1-phosphate, and this inhibition was not affected by
insulin. Okadaic acid decreased the activation of phospholipase D by
sphingosine 1-phosphate and did not reverse the inhibitory effect of
C
-ceramide on this activation. Therefore, this effect of
C
-ceramide is unlikely to involve the stimulation of
phosphoprotein phosphatase activity. Sphingosine did not activate
phospholipase D activity significantly after 10 min.
C
-ceramide stimulated the conversion of exogenous
[
H]sphingosine 1-phosphate to sphingosine and
ceramide in fibroblasts. Ceramides can inhibit some effects of
sphingosine 1-phosphate by stimulating its degradation via a
phosphohydrolase that also hydrolyzes phosphatidate. Furthermore,
C
- and C
-ceramides stimulated ceramide
production from endogenous lipids, and this could propagate the
intracellular signal. This work demonstrates that controlling the
production of ceramide versus sphingosine and sphingosine
1-phosphate after sphingomyelinase activation could have profound
effects on signal transduction.
The activation of the SM ()cycle plays an important
role in signal transduction(1, 2, 3) .
Stimulation of cells with 1,25-dihydroxyvitamin
D
(4) , TNF-
, or
-interferon(1, 2, 3, 5) activates a neutral sphingomyelinase that hydrolyzes
membrane SM to generate intracellular ceramides. The latter event is
linked to the induction of cell differentiation and the inhibition of
cell growth in HL-60 human leukemia
cells(1, 2, 3, 4, 5) .
Other stimulators of SM hydrolysis in different cell types include
interleukin-1(1, 2, 3, 6, 7) ,
dexamethasone(8) , and complement proteins(9) . The
other product of SM hydrolysis, phosphorylcholine, was thought not to
play a role in signal transduction(10) . However, Lacal et
al. (11) showed that phosphorylcholine is essential for late
events in the induction of DNA synthesis by platelet-derived growth
factor. The stimulation of a neutral sphingomyelinase by TNF-
may
involve phospholipase A
and the release of arachidonic
acid(12) . TNF-
also stimulates an acidic sphingomyelinase
through the production of 1,2-diacylglycerol via stimulation of a
phosphatidylcholine-specific phospholipase C.
There is abundant
evidence that ceramides are second messengers for cell regulation. The
generation of intracellular ceramides or the addition of cell-permeable
ceramides leads to the rapid induction of a nuclear transcription
factor system, NF-B(13, 14) , although a recent
report presented evidence that dissociates NF-
B activation from an
increase in cell ceramide concentrations(15) . Incubation of
cells with exogenous bacterial sphingomyelinase to generate
intracellular ceramides (a) inhibits the phorbol ester-induced
differentiation in HL-60 cells(16) , (b) inhibits
hydroxymethylglutaryl-CoA reductase(17, 18) , (c) increases the binding, internalization, and degradation of
low density lipoproteins and the synthesis of cholesterol esters in
cultured human fibroblasts(19) , and (d) stimulates PA
and lyso-PA metabolism in rat fibroblasts(20) . Exogenous
cell-permeable ceramides mimic the action of 1,25-dihydroxyvitamin
D
, TNF-
, and
-interferon(4) . In
addition, cell-permeable ceramides exhibit specific antiproliferative
effects in HL-60 cells(2) , Madin-Darby canine kidney
cells(21) , and rat fibroblasts stimulated with PA or
lyso-PA(20) . C
-ceramide can also induce
internucleosomal DNA fragmentation, which leads to programmed cell
death or apoptosis(22) , and this process can be initiated by
TNF-
(23) . Ceramide levels are increased significantly in
T lymphocytes that are infected with the human immunodeficiency virus
and that undergo apoptosis(24) . Ceramides also play a role in
inflammatory processes, and they modulate the secretion of
prostaglandin E
in response to interleukin-1 (7) .
Furthermore, ceramides stimulate the secretion of interleukin-2 in
lymphocytes(6) .
Several intracellular targets for ceramide
action have been described. C-ceramide decreases the mRNA
for c-myc, an important step in cell
differentiation(5) . C
-ceramide also stimulates a
specific serine/threonine protein phosphatase in mammalian cell
extracts (25) and in Saccharomyces
cerevisiae(26) . Ceramides stimulate specific kinases (2) and the phosphorylation of the epidermal growth factor
receptor in A431 human epidermoid carcinoma cells (27) and act
as competitive inhibitors of diacylglycerol kinase in HL-60
cells(28) . Sphingomyelinase and cell-permeable ceramides
activate mitogen-activated protein kinase in HL-60 cells(29) ,
and this effect has also been demonstrated for TNF-
in human
fibroblasts(30) . We have also demonstrated that cell-permeable
ceramides inhibit the activation of PLD by several
agonists(20) . Further details of the SM cycle and the actions
of ceramides have been described by Hannun (1) .
The role of
sphingolipids in regulating cell functions has been described by
Merrill (31, 32) and Merrill and Jones(33) .
Sphingosine inhibits protein kinase C (34) and
Mg-dependent and -independent phosphatidate
phosphohydrolase
activities(35, 36, 37, 38, 39) .
Sphingosine also activates PLD in NG108-15, rat pituitary, and NIH-3T3
cells(40) , and it stimulates an 80-kDa diacylglycerol
kinase(41) . These combined actions increase the accumulation
of PA relative to diacylglycerol (42) , which could also
decrease protein kinase C activation. Sphingosine also stimulates cell
proliferation in fibroblasts (43) and induces intracellular
Ca
mobilization(44, 45) . Some of
these effects of sphingosine have been attributed to the formation of
SPP(46) . SPP stimulates PLD activity in Swiss 3T3
fibroblasts(46) . It is a mitogenic agent (47) and may
be a second messenger that mediates the proliferative effects of
platelet-derived growth factor and fetal calf serum(48) .
However, conversion of sphingosine to SPP is not necessarily essential
for the stimulation of mitogenesis. Treatment of fibroblasts with
fumonisin B
, an inhibitor of sphingosine (sphinganine) N-acyltransferase(49) , enhanced
[
H]thymidine incorporation into DNA, but did not
cause any accumulation of SPP, suggesting that sphingoid bases per
se can also stimulate DNA synthesis(50) .
Sphingosine-induced Ca
mobilization may also be
independent of conversion to SPP(45) .
The agonist-stimulated breakdown of SM can potentially generate ceramides, sphingosine, and SPP. Therefore, we studied the interactions of these compounds on signal transduction and cell activation in cultured fibroblasts to define more precisely the effects and mechanisms of action of these putative second messengers. Cell-permeable ceramides increase the production of ceramide from endogenous lipids and stimulate the conversion of SPP to sphingosine and ceramide. This work emphasizes that regulating the balance in the production of ceramide versus sphingosine and SPP after sphingomyelinase activation could have profound effects on signal transduction and cell division.
[H]SPP was prepared by
deacylation of [
H]ceramide 1-phosphate
essentially according to Desai et al.(46) .
[
H]Ceramide 1-phosphate was synthesized from N-octanoyl[
H]sphingosine essentially as
described previously (59) by using Escherichia coli diacylglycerol kinase. [
H]Ceramide
1-phosphate was treated with 6 M HCl/1-butanol (1:1, v/v) for
60 min at 100 °C to produce [
H]SPP. This
compound was purified by thin-layer chromatography (47) using
1-butanol/acetic acid/water (3:1:1, v/v/v). The product also
cochromatographed with nonradioactive SPP in the solvent systems
described above.
For the analysis of the sphingolipid metabolites of
SPP, we developed a thin-layer system that separated SPP, sphingosine,
SM, ceramide, and ceramide 1-phosphate efficiently in one dimension.
After lipids were extracted and dried down, they were spotted on
glass-backed Silica Gel 60 plates, which were developed sequentially
with three different solvent systems. Solvent A consisted of
chloroform/methanol/NH0H (65:35:7.5, v/v/v). In this
system, the solvent was run to 11.5 cm from the origin, which was set
up at 1.5 cm from the bottom of the plate. Solvent B consisted of
chloroform/methanol/acetic acid (9:1:1, v/v/v), and the chromatograph
was developed to the top of the plate. Solvent C consisted of
1-butanol/acetic acid/water (3:1:1, v/v/v), and the solvent mixture was
run to 14 cm from the origin. In all cases, plates were allowed to dry
before they were developed with the next solvent mixture. The position
of non-phosphate-containing sphingolipids was identified by staining
with 0.2% 2`,7`-dichlorofluorescein in 95% ethanol and viewing the
lipid spots under light at 366 nm. Phospholipids were identified by
spraying the plates with molybdenum blue reagent(58) . In all
cases, authentic standards were used to establish R
values. Under the conditions used, the final R
values for the sphingolipids were 0.15 for SM, 0.33 for SPP, 0.50
for ceramide 1-phosphate, 0.61 for sphingosine, and 0.83 for ceramide.
Radioactive sphingolipids were detected with a Bioscan System 200
Imaging Scanner, and they were quantitated by liquid scintillation
counting after scraping from the plate.
Figure 1:
Effect of ceramide concentration on the
stimulation of DNA synthesis by sphingosine 1-phosphate. Rat1
fibroblasts were incubated for 22 h in serum-free DMEM in the absence
() or presence (
) of 2 µM SPP and the
indicated concentrations of C
-ceramide.
[
H]Thymidine was present during the last 6 h of
incubation. Basal incorporation of [
H]thymidine
corresponded to 23,102 ± 2,616 dpm/dish in 20 independent
determinations. Results are expressed relative to the control value
with no additions. These are given as the means ± S.E. (where
large enough to be shown) of three independent experiments performed in
duplicate. Similar results were obtained in an additional experiment in
which C
-ceramide was substituted with
C
-ceramide.
Figure 2:
Effect of C-ceramide on the
synthesis of DNA induced by increasing concentrations of sphingosine
1-phosphate. Rat1 fibroblasts were incubated for 22 h in serum-free
DMEM with increasing concentrations of SPP in the absence (
and
) or presence (
and
) of 10 µM C
-ceramide and in the absence (
and
) or
presence (
and
) of 1 µg/ml insulin.
[
H]Thymidine was present during the last 6 h of
incubation. Results are expressed relative to the control value and are
given as the means ± S.E. (where large enough to be shown) of
three to five independent experiments performed in
duplicate.
Figure 3:
Effect of C-ceramide on the
synthesis of DNA induced by sphingosine and high concentrations of
sphingosine 1-phosphate. Rat1 fibroblasts were incubated for 22 h in
serum-free DMEM with increasing concentrations of sphingosine (
and
) or SPP (
and
) in the absence (
and
or presence (
and
) of 10 µM
C
-ceramide. [
H]Thymidine was present
during the last 6 h of incubation. Results are expressed relative to
the control value and are given as the means ± S.E. of three to
five independent experiments performed in duplicate for the studies
with sphingosine and as the means ± range of two independent
experiments performed in duplicate for the studies with SPP. Error
bars are given where large enough to be
shown.
Sphingosine also stimulates the
incorporation of [H]thymidine into
DNA(20, 43) , and this effect has been suggested to be
mediated by its conversion to SPP in Swiss 3T3
fibroblasts(44) . However, in rat fibroblasts, sphingosine is
more mitogenic than SPP, and 20 µM sphingosine caused a
4-fold stimulation of DNA synthesis. Concentrations greater than 25
µM sphingosine were toxic to the fibroblasts. Furthermore,
C
-ceramide, which blocks the effect of SPP completely ( Fig. 1and Fig. 2), had only a marginal effect on the
inhibition of DNA synthesis induced by sphingosine (Fig. 3).
Sphingosine and SPP appear to stimulate DNA synthesis by different
mechanisms, and therefore, they might be expected to increase this
process in an additive manner. However, the combination of an optimum
SPP concentration of 2 µM with 20 µM
sphingosine stimulated DNA synthesis
11% less than did sphingosine
alone. This effect is compatible with the results of Spiegel et
al.(44) . It is difficult to rationalize these results
except to postulate that the addition of extra SPP to cells already
exposed to 20 µM sphingosine causes cell damage, as does
extra sphingosine.
Figure 4:
Effect of C-ceramide on the
stimulation of PLD by sphingosine 1-phosphate. In A, Rat2
fibroblasts were labeled with [
H]myristate for 2
h and then incubated with 0.5% albumin in the absence (
) or
presence (
) of 50 µM C
-ceramide for an
additional 2 h. The fibroblasts were then treated for 10 min with
increasing concentrations of SPP, as indicated, in the presence of 200
mM ethanol. PLD activities were determined through the
formation of [
H]phosphatidylethanol. The results
were calculated as a percentage of radioactivity in phosphatidylcholine (20) and then expressed as the -fold stimulation relative to
the incubations that contained neither ceramide nor SPP. In B,
incubations contained 1 µM SPP, and the concentration of
C
-ceramide was varied as shown. Values are the means
± range of two independent experiments performed in duplicate,
except for the 50 µM C
-ceramide point, which
is the mean ± S.E. of five independent experiments performed in
duplicate.
During the course of these experiments
on PLD, we detected that the addition of cell-permeable ceramides to
the cells that were labeled with [H]myristate
increased the production of [
H]ceramide. This
increase was
2.5- and 8.7-fold for C
- and
C
-ceramides, respectively, and the presence of SPP had no
significant effect on these values (Table 2). Furthermore, we
established that the presence of ethanol in the incubations did not
significantly modify the production of
[
H]ceramides. Ceramide-induced production of
endogenous ceramides was also observed when the cells were prelabeled
with [
H]palmitate rather than
[
H]myristate (data not shown).
Some of the
effects of ceramides on cell signaling are caused by the stimulation of
phosphoprotein phosphatase activity, and these effects can be blocked
by okadaic acid(25, 26) . Okadaic acid (0.25
µM) increased (p < 0.025) the relative PLD
activity by 1.8-fold (Fig. 5A). However, there was no
significant increase when C-ceramide was also present.
Okadaic acid at 0.5 and 1.0 µM strongly inhibited the
activation of PLD by SPP. This effect was also seen to a smaller extent
when C
-ceramide was present with SPP (Fig. 5A). As already shown in Table 2, the
presence of C
-ceramides in the incubations stimulated the
production of endogenous [
H]ceramide (Fig. 5B). Neither okadaic acid nor SPP significantly
changed the relative concentration of [
H]ceramide
either in the presence or absence of C
-ceramide. We
concluded that the inhibition by C
-ceramide of the
stimulation of PLD activity by SPP does not appear to involve the
stimulation of a phosphoprotein phosphatase since this effect is not
reversed by okadaic acid.
Figure 5:
Effect of okadaic acid on the stimulation
of PLD by sphingosine 1-phosphate and the production of endogenous
ceramides by C-ceramide. Rat2 fibroblasts were labeled with
[
H]myristate for 2 h and then incubated for 2 h
in the absence (
and
) or presence (
and
) of
50 µM C
-ceramide. The media were changed, and
the cells were then incubated for 10 min in the presence of ethanol
with no added lipids (
) or with 50 µM
C
-ceramide (
), 1 µM SPP (
), or 50
µM C
-ceramide plus 0.5 µM SPP
(
) with the concentrations of okadaic acid indicated. A,
relative PLD activity; B, relative formation of
[
H]ceramide. Results are the means ± S.E.
of three independent experiments. Similar effects were observed at 15
µM C
-ceramide in the absence of albumin (data
not shown).
Figure 6:
Effect of C-ceramide on the
metabolism of [
H]sphingosine 1-phosphate by rat
fibroblasts. Rat2 fibroblasts were preincubated for 2 h with 0.5%
albumin in the absence (
) or presence (
) of 50 µM C
-ceramide; 2 µM [
H]sphingosine 1-phosphate (3.9 Ci/mol) was
then added, and the incubation was continued for the times indicated.
Sphingolipids were analyzed as described under ``Experimental
Procedures''. A-E show the radioactivity in SPP,
sphingosine (Sph), ceramide (Cer), SM, and total
sphingolipids in the fibroblasts, respectively. Results are from a
representative experiment and were confirmed in a second independent
experiment.
The
mechanism of the ceramide-induced increase in SPP degradation was
studied further in cell-free preparations. Sonicates of the Rat2
fibroblasts exhibited an SPP-dependent formation of P that
was optimum at 0.4-0.5 mM SPP (Fig. 7A).
This activity was inhibited by
90% with 5-10 mM NaF (Fig. 7B). The reaction was not dependent upon the
presence of Mg
or Ca
, and
preincubation with N-ethylmaleimide did not alter the activity
significantly. The phosphohydrolase is membrane-associated since it was
recovered in the particulate fraction of cell sonicates after
ultracentrifugation (data not shown). These characteristics are similar
to those of plasma membrane phosphatidate
phosphohydrolase(35, 42) . Phosphatidate
phosphohydrolase activity in fibroblasts is increased by cell-permeable
ceramides or by treatment with sphingomyelinase(20) . The
presence of a protein in rat fibroblasts similar to rat liver
phosphatidate phosphohydrolase was confirmed by Western blot analysis (Fig. 8) using affinity-purified antibody generated against the
homogeneous liver phosphatidate phosphohydrolase(61) . The
predominant immunoreactive protein in both rat fibroblasts and liver
homogenates has an apparent molecular mass of 51-53 kDa. If the
same enzyme were hydrolyzing both PA and SPP in Rat2 fibroblasts, then
SPP should inhibit the activity of phosphatidate phosphohydrolase. This
prediction was confirmed since increasing concentrations of SPP
decreased PA hydrolysis by >80% (Fig. 8). The
ceramide-induced hydrolysis of SPP (Fig. 6) is therefore
explained by the stimulation of a phosphatidate phosphohydrolase (20) that also degrades SPP.
Figure 7:
Effects of sphingosine 1-phosphate
concentrations and F on sphingosine 1-phosphate
phosphohydrolase activity. The activity was measured by quantitating
the formation of P
by membranes prepared from rat
fibroblasts as described under ``Experimental Procedures.'' A, SPP dose-dependent effect on P
production; B, effect of increasing concentrations of F
on phosphohydrolase activity. Results are from single experiments
that were each confirmed in an independent
experiment.
Figure 8:
Inhibition of a phosphatidate
phosphohydrolase activity in rat fibroblasts by sphingosine
1-phosphate. A, inhibition by SPP of the conversion of
[H]PA to [
H]diacylglycerol
by sonicates of Rat2 fibroblasts. Results are from a single experiment
that was reproduced in two other independent experiments. B,
Western blot analysis of Rat2 fibroblasts and rat liver homogenate.
Samples containing 100 µg of protein from sonicated fibroblasts (lane 1) or liver homogenate (lane 2) were probed
with anti-phosphatidate phosphohydrolase antibody and detected using
ECL. The migration of molecular mass markers (in kilodaltons) is
indicated to the right of B.
We recently demonstrated that cell-permeable ceramides
(C- and C
-ceramides) specifically inhibit the
mitogenic effects of PA, lyso-PA, and ceramide 1-phosphate in rat
fibroblasts(20, 66) . We have now shown a
ceramide-dependent inhibition of the stimulation of DNA synthesis by
SPP ( Fig. 1and Fig. 2). Maximum effects on DNA synthesis
were obtained at 2 µM SPP, which was
25 times less
than for PA or lyso-PA (Fig. 2)(20) . Higher
concentrations (>5 µM) of SPP failed to stimulate
[
H]thymidine incorporation into DNA or were
inhibitory (Fig. 3). Microscopic examination of cells incubated
with 10 and 20 µM SPP did not reveal any obvious toxic
effects. These high concentrations of SPP could generate sufficient
quantities of ceramides to block the stimulation of DNA synthesis by
SPP (Fig. 6). Moreover, C
-ceramide enhanced the
inhibition of DNA synthesis at 10 µM SPP (Fig. 3),
and this could have resulted from a more rapid conversion of SPP to
ceramide (Fig. 6) together with ceramide production from
endogenous lipids (Table 2). Cell-permeable ceramides do not
inhibit DNA synthesis induced by insulin alone(20) , and
insulin also reverses the inhibitory effect of C
-ceramide
on the SPP-induced synthesis of DNA.
C-ceramide was much
less effective in inhibiting DNA synthesis induced by sphingosine than
that by SPP even when compared at equivalent stimulations of
2-fold ( Fig. 1and Fig. 2versus 3).
Although some of the mitogenic effects of sphingosine may involve its
conversion to SPP(44) , our results indicate that sphingosine per se may also affect DNA synthesis by a different mechanism
than that of SPP. Furthermore, although both sphingosine and SPP
increase cellular Ca
concentrations(45, 46, 67) , the effect
of sphingosine depends on phosphoinositide breakdown, whereas that of
SPP does not(45) . Ca
ions have also been
involved in the induction of early events of mitogenesis(44) .
However, cell-permeable ceramides have no effect on either
phosphoinositide turnover or intracellular Ca
concentration (45) , suggesting that the inhibition of
sphingosine- or SPP-induced DNA synthesis by ceramides is independent
of Ca
mobilization. Our results also imply that the
ceramide inhibition of DNA synthesis is independent of effects on
adenylate cyclase.
Stimulation of fibroblasts with as little as 10
nM SPP causes a significant increase in PLD activity (Fig. 4). It seemed possible that cell-permeable ceramides might
inhibit PLD by stimulating the activity of a phosphoprotein
phosphatase(25, 26) . However, okadaic acid alone
stimulated PLD activity slightly and did not reverse the effects of
C-ceramide in inhibiting PLD activation by SPP. Therefore,
the effect of C
-ceramide in this case cannot be explained
by stimulation of an okadaic acid-sensitive phosphoprotein phosphatase.
Furthermore, the strong inhibition by okadaic acid of the activation of
PLD by SPP indicates that this process may be dependent upon the
stimulation of a phosphatase activity. The need for a relatively high
concentration of okadaic acid (0.5-1.0 µM) indicates
that phosphatase 1 as well as phosphatase 2A may be involved.
By contrast to the effects of SPP, sphingosine did not activate PLD significantly after 10 min. However, treating the fibroblasts for 2 h with 20 µM sphingosine increased PLD activity by 85 ± 28% in three independent experiments. This longer time course for PLD activation by sphingosine was also reported by Lavie and Liscovitch (40) and could involve the formation of SPP(44, 46) .
Although externally added PA, lyso-PA(20, 62) , or SPP is a potent mitogen for fibroblasts, the activation of PLD (and presumably the generation of PA at the inner surface of the plasma membrane) is not sufficient in itself to stimulate DNA synthesis(20) . For example, endothelin is also a potent stimulator of PA generation through PLD, but it is a poor mitogen(68) . Therefore, an inhibition of PLD activation by ceramides may not in itself be sufficient to account for their antimitogenic effects.
Exogenous SPP is rapidly dephosphorylated and
metabolized by rat (Fig. 6) and human (65) fibroblasts.
We have determined that the dephosphorylation of SPP is catalyzed by a
F-sensitive, membrane-associated phosphohydrolase
that does not require bivalent cations and is not inhibited by N-ethylmaleimide. These characteristics are similar to those
of the plasma membrane phosphatidate phosphohydrolase (35) . We
have also shown that exogenous PA is rapidly dephosphorylated by intact
rat fibroblasts and that pretreatment of the cells with short-chain
ceramides enhances the dephosphorylation of both PA (20) and
SPP (Fig. 6). Furthermore, SPP inhibits
[
H]PA hydrolysis in homogenates of Rat2
fibroblasts (Fig. 8A). Experiments with phosphatidate
phosphohydrolase purified from rat liver demonstrate that this
inhibition is competitive and that purified phosphatidate
phosphohydrolase hydrolyzes SPP. (
)Affinity-purified
antibody generated against purified rat liver phosphatidate
phosphohydrolase (61) immunoprecipitates phosphatidate
phosphohydrolase activity from homogenates of rat fibroblasts (data not
shown) and identifies only one immunoreactive 51-53-kDa protein
on Western blot analysis of cell lysates (Fig. 8B).
Collectively, these observations establish that a common enzyme
catalyzes the hydrolysis of SPP and PA. Consequently, we can explain
the effects of cell-permeable ceramides in stimulating the hydrolysis
of exogenous PA and SPP by activating a common phosphohydrolase that
mitigates the mitogenic activity of these bioactive phospholipids.
Furthermore, cell-permeable ceramides inhibit the PA-induced (20) and SPP-induced (Fig. 4) production of PA by PLD.
C-ceramide not only destroys the SPP signal, but also
potentiates an antagonistic one by increasing ceramide production from
SPP (Fig. 6) and endogenous sphingolipids ( Fig. 5and Table 2). The latter observation is compatible with previous work
with A431 human epidermoid carcinoma cells(27) , and this
effect could represent a major route of transmission for the
intracellular signals from exogenous cell-permeable ceramides. Neither
the basal nor the stimulated production of endogenous ceramides was
affected significantly by SPP or okadaic acid.
Insulin could be an
important factor in controlling ceramide action since it can attenuate
the inhibition of the SPP-induced DNA synthesis by ceramides (Fig. 2). However, insulin did not have any significant effect
on the inhibition of the SPP-induced PLD activation by ceramide (data
not shown). The stimulation of DNA synthesis is regulated by several
independent mechanisms, and not all of these are controlled by
ceramides. For example, the incorporation of thymidine into DNA that
was induced by insulin (Fig. 2), platelet-derived growth factor,
or epidermal growth factor (data not shown) was not inhibited by
ceramide. Furthermore, the sphingosine-induced synthesis of DNA is
decreased by only 16% by C
-ceramide (Fig. 3).
In conclusion, the effects of ceramides in decreasing PLD activity
were not blocked by okadaic acid, and the latter inhibited the
activation of PLD by SPP. The antimitogenic effects of cell-permeable
ceramides were not caused by reversing the SPP-induced decrease in
cAMP. Insulin could play an important role in ceramide action since it
counteracts the inhibition of the SPP-induced DNA synthesis by
ceramides. SPP and sphingosine may function through different
mechanisms to stimulate DNA synthesis since C-ceramide
inhibits the stimulation of DNA synthesis by SPP completely, but has
little effect on DNA synthesis induced by sphingosine. Ceramides
stimulate the degradation of SPP to sphingosine and ceramide and
stimulate the production of ceramides from endogenous lipids. The
increased degradation of SPP can now be attributed to the stimulation
of a phosphohydrolase that also degrades PA. Our results emphasize that
the balance in the production of ceramide versus sphingosine
and SPP after sphingomyelinase activation could determine the patterns
of cell signaling that control the extent of DNA synthesis, cell
division, and cell death.