(Received for publication, May 15, 1995)
From the
Sphingosylphosphorylcholine (SPC) is a potent mitogen for Swiss
3T3 cells, but the signaling mechanisms involved are poorly
characterized. Here, we report that addition of SPC induces a rapid and
transient activation of p42 mitogen-activated protein kinase
(p42) in these cells. SPC-induced p42
activation peaked at 5 min and was undetectable after 30 min of
incubation with SPC. The effect of SPC on p42
activation
was comparable to that induced by bombesin and platelet-derived growth
factor. As SPC strongly induced phosphorylation of the major protein
kinase C (PKC) substrate 80K/MARCKS in either intact or permeabilized
cells, we examined whether PKC could be involved in SPC-induced
p42
activation. Here, we demonstrate that p42
activation by SPC was dependent on PKC activity as shown by
inhibition of PKC with the bisindolylmaleimide GF 109203X or
down-regulation of PKC by prolonged treatment of Swiss 3T3 cells with
phorbol esters. Activation of both PKC and p42
by SPC
was markedly inhibited by treatment with pertussis toxin, implicating a
G protein(s) of the G
/G
subfamily in the action
of SPC. SPC-induced rapid activation of a downstream target of
p42
, p90 ribosomal S6 kinase
(p90
), also required PKC and a pertussis
toxin-sensitive G protein. In addition, SPC-induced mitogenesis was
dependent on a G
protein in Swiss 3T3 cells. SPC also
induced p42
activation and DNA synthesis in secondary
cultures of mouse embryo fibroblasts through a pertussis
toxin-sensitive pathway. As G proteins link many cell surface receptors
to effector proteins, we hypothesize, therefore, that SPC could bind to
a receptor that mediates at least some of its biological effects in
Swiss 3T3 cells and mouse embryo fibroblasts.
Lipid-derived messengers are implicated in intercellular
communication and intracellular signaling, and the mechanisms involved
are attracting increasing interest(1) . Sphingolipids and their
breakdown products have emerged as active participants in the
regulation of cell growth, differentiation, transformation, and
cell-cell contact (reviewed in (2) and (3) ).
Lysosphingolipids such as sphingosylphosphorylcholine (SPC) ()are potential derivatives of sphingolipids. SPC has been
demonstrated in normal mouse cerebrum and human meningiomas (4) as well as in tissues of patients with Niemann Pick
disease, a lipid storage disorder(5) . Furthermore, SPC has
been identified as a growth-promoting factor for Swiss 3T3
cells(6, 7) , but the molecular basis of this
mitogenic effect is poorly understood.
The best characterized early
cellular response induced by SPC is the direct induction of
Ca mobilization from internal stores in a variety of
cell lines thereby acting as a potential second
messenger(7, 8, 9, 10) . Recent data
from our laboratory demonstrated that Ca
mobilization
in the absence of inositol phosphate production can stimulate DNA
synthesis in Swiss 3T3 cells only in synergistic combination with other
growth-promoting agents(11) . In contrast, SPC, like bombesin
and PDGF, can induce a mitogenic response in these cells in the absence
of any other growth factor ( (6) and (7) and this
paper). It is increasingly recognized that the initiation of DNA
synthesis is triggered by multiple signal transduction pathways that
act synergistically in mitogenic
stimulation(12, 13, 14) . We therefore
reasoned that the ability of SPC to act as a sole mitogen may be due to
the stimulation of other signaling pathways in addition to direct
Ca
mobilization.
Mitogen-activated protein kinases
(MAPKs), also known as extracellular signal-related kinases, are a
family of protein-serine/threonine kinases that play a role as
important intermediates in signal transduction pathways. MAPKs are
stimulated by a variety of polypeptide growth factors signaling via
tyrosine kinase receptors that activate
p21(15, 16) . The pathway
activated by these agents is a protein kinase cascade, which involves
c-Raf-1, MEK, p44
, and
p42
(15, 16) . Activation of seven
transmembrane receptors also leads to p44
and
p42
activation, but the mechanism(s) involved are less
clear, though both Raf-1 activation and PKC stimulation have been
implicated(17, 18, 19) . Activated MAPKs
directly phosphorylate and activate various enzymes, including the
90-kDa S6 protein kinase named p90
(20) .
Both, MAPK and p90
regulate gene expression by
transcription factor phosphorylation and have been strongly implicated
in the stimulation of cell
proliferation(15, 20, 21) .
Here, we
demonstrate that addition of SPC to quiescent cultures of Swiss 3T3
cells leads to rapid and transient activation of p42 and
p90
through a novel, pertussis toxin-sensitive
and PKC-dependent signal transduction pathway that can be distinguished
from that utilized by other single mitogens for these cells, such as
bombesin and PDGF.
Figure 1:
SPC stimulates DNA
synthesis in Swiss 3T3 cells. Confluent and quiescent Swiss 3T3 cells
in 35-mm dishes were stimulated with various concentrations of SPC.
[H]Thymidine incorporation was measured as
described under ``Experimental Procedures.'' Shown are the
mean values ± S.E. of four independent experiments. Inset, quiescent Swiss 3T3 cells were stimulated with 15
µM SPC, 10% fetal bovine serum (FBS), or 15
µM LPA. Detection of BrdUrd incorporated into cellular DNA
was performed as described under ``Experimental Procedures,''
and data are expressed as the percentage of BrdUrd-positive nuclei in
at least three independent microscopic fields per condition. Shown are
the mean values ± S.E. of three independent experiments. Where
no error bar is shown, it lies within the dimensions of the
symbol.
Figure 2:
SPC
induces MAPK activation. A, upper panel, confluent
and quiescent Swiss 3T3 cells were incubated with 10 µM SPC for various times and lysed in 2 SDS sample buffer;
the lysates were then subjected to SDS-PAGE followed by Western
blotting with anti-p42
antibody. Shown is a
representative experiment of three independent experiments. Lower
panel, in parallel experiments we performed immune complex assays
for p42
activation as described under
``Experimental Procedures.'' Cells were treated for various
times with 10 µM SPC. Results are expressed as cpm/1.5
10
cells, and the data shown are the means ±
S.E. of four independent experiments each performed in duplicate. B, confluent and quiescent Swiss 3T3 cells were treated for 5
min with various concentrations of SPC. Subsequently, p42
immune complex assays were performed. Results are expressed as
cpm/1.5
10
cells, and shown are the mean values
± S.E. of three independent experiments each performed in
duplicate. Where no error bar is shown, it lies within the dimensions
of the symbol. Inset, confluent and quiescent Swiss 3T3 cells
were treated for 5 min with 10 µM SPC (S), 20
ng/ml PDGF (P), or 10 nM bombesin (B) and
further treated as described above. Data are expressed as cpm
10
/1.5
10
cells, and shown are
the means of two independent experiments each performed in
duplicate.
The results obtained with the mobility shift assay
were substantiated by immune complex assays of p42 activity using a myelin basic protein peptide as a substrate. As
shown in Fig. 2A, lowerpanel, 10
µM SPC induced a marked stimulation of p42
activity, which peaked within 5 min of incubation. p42
activity returned toward basal levels after 30 min of incubation
with SPC and did not change afterward for up to 3 h of incubation. This
result was in good agreement with the data obtained in the mobility
shift assays. SPC induced MAPK activation in a concentration-dependent
manner. Half-maximum and maximum stimulation of p42
activity in immune complex assays were achieved at 6 and 10
µM SPC, respectively (Fig. 2B). The level
of p42
activity induced by 10 µM SPC was
comparable to that promoted by 20 ng/ml PDGF or 10 nM bombesin
in Swiss 3T3 cells (Fig. 2B, inset).
Next,
we examined the signaling pathways responsible for SPC-mediated
p42 activation in Swiss 3T3 cells.
Figure 3:
A, SPC-induced MAPK activation is
independent of [Ca]. Upperpanel, confluent, quiescent Swiss 3T3 cells were
incubated with 30 nM thapsigargin (TG) or 3 mM EGTA for 30 min as indicated (+) or received an equivalent
amount of solvent(-). The cells were stimulated with 10
µM SPC for 5 min and lysed; the lysates were then
subjected to SDS-PAGE followed by Western blotting with
anti-p42
antibody. Shown is a representative of three
independent experiments. Lower panel, immune complex assays
for p42
activation were performed in parallel using the
same conditions as described above. Results are expressed as cpm
10
/1.5
10
cells, and
shown are the mean values of two independent experiments each performed
in duplicate. B, SPC-induced MAPK activation is independent of
the integrity of the actin cytoskeleton. Upperpanel,
Swiss 3T3 cells were pretreated with 2 µM cytochalasin D (CytoD, +) or received an equivalent amount of
solvent(-). The cells were stimulated with 10 µM SPC
for 5 min and lysed; the lysates were then subjected to SDS-PAGE
followed by Western blotting with anti-p42
antibody. Lower panel, for immune complex assays for p42
activation, quiescent Swiss 3T3 cells were incubated with 1.2 or
2.4 µM cytochalasin D (CytoD) for 2 h.
Control cells received an equivalent amount of solvent(-). Cells
were subsequently stimulated with 10 µM SPC for 5 min and
further processed as described under ``Experimental
Procedures.'' Data are expressed as cpm
10
/1.5
10
cells, and shown are
the mean values of two independent experiments each performed in
duplicate.
Recently, it has
been reported that MAPK activation in response to integrin-mediated
cell adhesion is dependent on the integrity of the actin
cytoskeleton(32, 33) . This effect is probably
mediated by Grb2-SOS association to tyrosine-phosphorylated
p125(34) . As SPC induces p125
tyrosine phosphorylation as well as dramatic changes in the
organization of the actin cytoskeleton (35) , we examined a
possible link between these events and SPC-induced p42
activation. Treatment with cytochalasin D, at concentrations that
completely disrupted the actin cytoskeleton and blocked p125
tyrosine phosphorylation(35) , had no effect on
SPC-induced p42
activation as shown by mobility shift
assays and immune complex assays (Fig. 3B, upper and lowerpanels). Therefore, SPC stimulated
p42
activation by a pathway independent of either
Ca
fluxes or p125
tyrosine
phosphorylation, both of which have been implicated in the activation
of p74
(31, 34) .
Next, we examined
directly whether SPC could activate p74 in Swiss 3T3
cells. Confluent and quiescent Swiss 3T3 cells were treated with 10
µM SPC for various times and lysed. The lysates were
immunoprecipitated with a polyclonal anti-p74
antibody,
and the activity of this kinase in the immune complex was measured by a
sensitive assay based on the sequential activation of MEK and MAPK. SPC
induced a rapid and transient increase in p74
activity,
which was first detectable after 1 min and reached a maximum 2.3-fold
increase after 3 min of incubation (Fig. 4, inset).
Interestingly, the level of p74
activity measured in
SPC-treated cells reached only 15% of that induced by addition of 20
ng/ml PDGF (Fig. 4). PDGF at a low concentration of 0.5 ng/ml
induced a level of p74
activity comparable to that in
response to SPC. However, at this concentration, PDGF only weakly
induced MAPK activity (data not shown). As 10 µM SPC was
as potent as 20 ng/ml PDGF in stimulating p42
activity (Fig. 2B), it therefore seemed unlikely that
p74
was the major mediator of SPC-induced p42
activation in Swiss 3T3 cells.
Figure 4:
SPC induces p74 activation in Swiss 3T3 cells. Quiescent Swiss cells were
treated with 10 µM SPC for various times (inset)
or with 10 µM SPC or 20 ng/ml PDGF for 3 min and lysed;
p74
activity was then assayed as described
under ``Experimental Procedures.'' Data are expressed as
cpm/1.5
10
cells and are the mean ± S.E. of
at least three independent experiments each performed in duplicate.
Where no error bar is shown, it lies within the dimensions of the
symbol.
A rapid increase in the
phosphorylation of a M 80,000 acidic cellular
protein termed 80K/MARCKS has been shown to reflect the activation of
phorbol ester-sensitive PKC isoforms in intact Swiss 3T3
fibroblasts(39, 40, 41) . As shown in Fig. 5A, leftpanel, 10 µM SPC induced a rapid and sustained increase in the phosphorylation
of 80K/MARCKS in [
P]P
-labeled Swiss
3T3 cells, which was first detectable 1 min after addition of SPC and
peaked within 3 min of incubation. Pretreatment of the cells with the
bisindolylmaleimide GF 109203X, a selective inhibitor of the PKC
isoforms expressed in Swiss 3T3 cells(26, 42) ,
abolished PDB-induced 80K/MARCKS phosphorylation and markedly reduced
SPC-induced 80K/MARCKS phosphorylation (Fig. 5A, rightpanel), substantiating the role of PKC in the
action of SPC.
Figure 5:
A,
effect of SPC on 80K/MARCKS phosphorylation. Swiss 3T3 cells in 35-mm
dishes were incubated in phosphate-free DMEM with 50 µCi/ml
[P]P
. After 12 h of incubation at 37
°C, cells were treated with 10 µM SPC for various
times (leftpanel). In parallel experiments, cells
were pretreated with 3.5 µM GF 109203X (GF) for 1
h (+) or received an equivalent amount of solvent(-) and
were then challenged for 10 min with 200 nM PDB or 10
µM SPC, as indicated (rightpanel).
Cells were subsequently lysed, and lysates were immunoprecipitated with
anti-80K/MARCKS antibody and further analyzed by SDS-PAGE prior to
autoradiography. The results shown are representative of three
independent experiments. The position of 80K/MARCKS is indicated by an arrow. B, effect of GDP
S on the stimulation of
80K/MARCKS phosphorylation by SPC. Quiescent cells were incubated for 1
min in permeabilization medium containing 10 µCi/ml
[
-
P]ATP and 50 µM ATP,
without(-) or with 10 µM SPC or 6 nM bombesin (Bom), in the absence(-) or presence
(+) of 100 µM GDP
S. The result shown is
representative of four independent experiments. C, SPC
stimulates activation of 80K/MARCKS by a pertussis toxin-sensitive
pathway. Swiss 3T3 cells in 35-mm dishes were incubated in
phosphate-free DMEM with 50 µCi/ml
[
P]P
. After 12 h of incubation at 37
°C, cells were treated with 30 ng/ml pertussis-toxin (PTX)
for another 3 h (+). Control cells received an equivalent amount
of solvent(-). Subsequently, the cells were stimulated with 200
nM PDB, 10 µM SPC, or 10 nM bombesin (Bom) for 10 min and lysed. The lysates were
immunoprecipitated with anti-80K/MARCKS antibody and further analyzed
by SDS-PAGE prior to autoradiography. The results shown are
representative of three independent experiments. The position of
80K/MARCKS is indicated by an arrow.
To corroborate these results, we examined the effect
of SPC on 80K/MARCKS phosphorylation in digitonin permeabilized Swiss
3T3 cells. This procedure allows direct access to intracellular
compartments while leaving PKC-mediated phosphorylation of 80K/MARCKS
unaffected(43) . As shown in Fig. 5B, SPC at 10
µM induced a 3.5 ± 0.6 -fold stimulation (n = 4) of the phosphorylation of 80K/MARCKS in permeabilized
Swiss 3T3 cells. Interestingly, SPC-induced 80K/MARCKS phosphorylation
was prevented by GDPS, which inhibits G protein activity. This
result closely resembled the effect of GDP
S on 80K/MARCKS
phosphorylation in response to bombesin, which is shown for comparison (Fig. 5B). GDP
S inhibited 80K/MARCKS
phosphorylation by SPC in a concentration-dependent manner with a
maximum inhibition occurring at 100 µM GDP
S (data not
shown). Thus, the data suggest that a G protein is involved in the
signaling pathway leading to PKC activation in response to SPC.
To
characterize the nature of this G protein, quiescent Swiss 3T3 cells
were labeled with [P]P
and treated
with 30 ng/ml pertussis toxin for 3 h prior to stimulation with 10
µM SPC. Pertussis toxin ADP-ribosylates and thereby
inactivates G proteins of the G
/G
subfamily(44) . As shown in Fig. 5C,
SPC-induced 80K/MARCKS phosphorylation was markedly inhibited by 52
± 1.6% by pertussis-toxin (n = 4). In contrast,
PDB or bombesin-induced 80K/MARCKS phosphorylation was not affected by
pertussis toxin. These data demonstrate that a G protein of the
G
/G
subfamily mediates SPC-induced PKC
activation.
Figure 6:
SPC induces activation of MAPK by a
PKC-dependent pathway. A, upperpanel,
confluent and quiescent Swiss 3T3 cells were incubated with the
selective PKC inhibitor GF 109203X (GF) for 1 h (+), and
control cells received an equivalent amount of solvent (-). Cells
were subsequently stimulated with 200 nM PDB or 10 µM SPC for 5 min and lysed, and the lysates were subjected to
SDS-PAGE followed by Western blotting with anti-p42 antibody. Lowerpanel, in parallel experiments,
we performed immune complex assays for p42
activity as
described under ``Experimental Procedures.'' Cells were
treated with 3.5 µM GF 109203X (GF, +, hatchedbars) for 1 h or received an equivalent
amount of solvent (-, filledbars) and then
stimulated for 5 min with 200 nM PDB or 10 µM SPC
as indicated. Results are expressed as cpm/1.5
10
cells, and shown are the means ± S.E. of three independent
experiments each performed in duplicate. B, cells were treated
with 800 nM PDB for 48 h (+, hatchedbars) or received an equivalent amount of solvent
(-, filledbars) and then stimulated for 5 min
with 200 nM PDB or 10 µM SPC as indicated. Immune
complex assays for p42
activity were then performed as
described under ``Experimental Procedures.'' Results are
expressed as cpm/1.5
10
cells, and shown are the
mean values ± S.E. of three independent experiments each
performed in duplicate.
As shown above, SPC stimulated PKC activation via a
pertussis toxin-sensitive G protein, and, in turn, MAPK activation was
dependent on PKC. Therefore, we reasoned that SPC-induced p42 activation should also be dependent on G
. To examine
this prediction, quiescent Swiss 3T3 cells were incubated with various
concentrations of pertussis toxin for 3 h and then treated with 10
µM SPC for 5 min. As shown in Fig. 7, upperpanel, pertussis toxin prevented a subsequent activation
of p42
by SPC in a concentration-dependent manner.
Stimulation of p42
by SPC was attenuated at a
concentration of pertussis toxin as low as 0.3 ng/ml, and a maximum
inhibition of the MAPK mobility shift was achieved at 30 ng/ml.
Figure 7:
SPC induces activation of MAPK through a
pertussis toxin-sensitive pathway. Upperpanel,
confluent and quiescent Swiss 3T3 cells were incubated with various
concentrations of pertussis toxin (PTX) for 3 h or received an
equivalent amount of solvent (-). Cells were then stimulated with
10 µM SPC for 5 min and lysed, and the lysates were
subjected to SDS-PAGE followed by Western blotting with
anti-p42 antibody. Shown is a representative of four
independent experiments. Lower panel, in parallel experiments
immune complex assays for p42
activity were performed as
described under ``Experimental Procedures.'' Cells were
treated with 30 ng/ml pertussis toxin (PTX, +, hatchedbars) for 3 h or received an equivalent
amount of solvent (-, filledbars) and then
stimulated for 5 min with 10 µM SPC or 10 nM
bombesin (Bom) as indicated. Results are expressed as cpm/1.5
10
cells, and shown are the mean values ±
S.E. of three independent experiments each performed in
duplicate.
The
inhibitory effect of pertussis toxin was selective as shown by immune
complex assays of p42 activity. Pretreatment of the
cells for 3 h with 30 ng/ml pertussis toxin prior to stimulation did
not inhibit bombesin or PDB-stimulated p42
activity but
reduced SPC-induced p42
activity by 75% (Fig. 7, lowerpanel and data not shown). These results
indicate that SPC stimulates MAPK by a pertussis toxin-sensitive
pathway.
One of the major downstream targets of MAPK is
p90(20) . To examine whether SPC could also
stimulate p90
activity, lysates of quiescent Swiss 3T3
cells treated with 10 µM SPC for various times were
immunoprecipitated with a polyclonal anti-p90
antibody
and, the immunoprecipitates were further analyzed by an immune complex
kinase assay. As shown in Fig. 8A, SPC rapidly
stimulated p90
activity, reaching a maximum after 5 min
of incubation. Pretreatment of Swiss 3T3 cells with either GF 109203X
or pertussis toxin markedly reduced p90
activation in
response to 10 µM SPC (Fig. 8B).
Figure 8:
SPC stimulates p90 activity through a PKC-dependent and pertussis
toxin-sensitive pathway. A, quiescent Swiss 3T3 cells were
stimulated with 10 µM SPC for various times. Cells were
subsequently lysed and further processed as described under
``Experimental Procedures.'' Results are expressed as cpm
10
/1.5
10
cells. Shown
are the mean values ± S.E. of three independent experiments each
performed in duplicate. Where no error bar is shown, it lies within the
dimensions of the symbol. B, quiescent Swiss 3T3 cells were
incubated with 3.5 µM GF 109203X (GF, +) for
1 h or 30 ng/ml pertussis toxin (PTX, +) for 3 h, or they
received an equivalent amount of solvent(-). Subsequently, the
cells were stimulated with 10 µM SPC for 5 min, lysed, and
further processed as described under ``Experimental
Procedures.'' Results are expressed as cpm
10
/1.5
10
cells, and shown are
the mean values ± S.E. of three independent experiments each
performed in duplicate.
In
conclusion, SPC stimulates MAPK and p90 activation
predominantly by a signaling pathway involving a pertussis
toxin-sensitive G protein and PKC activation.
Figure 9:
Effect of SPC on DNA synthesis,
p42, and p74
activity in
secondary cultures of MEF. A, SPC stimulates DNA synthesis in
MEF. Confluent secondary cultures of MEF in 35-mm dishes were
stimulated with 10 µM of SPC in the presence (+) or
absence(-) of 30 ng/ml pertussis-toxin (PTX).
[
H]Thymidine incorporation was measured as
described under ``Experimental Procedures.'' Shown are the
mean values of two independent experiments each performed in duplicate. B, SPC induces p42
activation in MEF. Confluent
secondary cultures of MEF were incubated with 10 µM SPC or
20 ng/ml PDGF for 5 min, and immune complex assays for p42
activation were performed as described under ``Experimental
Procedures.'' Results are expressed as cpm
10
/1.5
10
cells, and shown are
the means of two independent experiments each performed in duplicates. C, SPC induces p74
activation in MEF.
Confluent secondary cultures of MEF were treated with 10 µM SPC or 20 ng/ml PDGF for 3 min and lysed; p74
activity was then assayed as described under
``Experimental Procedures.'' Data are expressed as cpm
10
/1.5
10
cells and are
the means of two independent experiments each performed in duplicates. D, SPC stimulates MAPK by a pathway dependent on PKC activity
and a pertussis toxin-sensitive G
protein. Confluent
secondary cultures of MEF were incubated with 3.5 µM GF
109203X (GF, +) for 1 h or 30 ng/ml pertussis toxin (PTX, +) for 3 h; or they received an equivalent amount
of solvent(-). Subsequently, the cells were stimulated with 10
µM SPC for 5 min, lysed, and further processed as
described under ``Experimental Procedures.'' Shown are the
mean values of two independent experiments each performed in
duplicates.
The results presented here demonstrate, for the first time,
that SPC induces a rapid and striking activation of p42,
p90
, and PKC. Our results reveal that SPC-induced
activation of the MAPK cascade has unique features in terms of its
kinetics and the signaling pathways that mediate this process in Swiss
3T3 cells as well as in secondary cultures of MEF.
The time course
of the p42 activation by extracellular stimuli has been
the subject of considerable attention. It has been proposed that agents
that can act as single mitogens induce persistent stimulation of the
MAPK cascade(45) . In contrast, our results show that SPC,
which induces DNA synthesis in the absence of any other exogenously
added growth factor as efficiently as bombesin and PDGF, stimulated a
transient rather than persistent activation of p42
.
Thus, the SPC results demonstrate that a persistent stimulation of the
MAPK cascade is not a necessary prerequisite for the induction of DNA
synthesis in Swiss 3T3 cells.
MAPK activation is known to be
triggered by a variety of signaling pathways(15) . Our studies
demonstrate that SPC-induced p42 activation is mediated
predominantly by phorbol ester-sensitive PKCs as judged by both
inhibition and down-regulation of these enzymes. None of the previous
reports on SPC signaling demonstrated stimulation of PKC. Indeed,
previous studies using concentrations of SPC 10 times higher than those
used in this paper suggested that SPC is an inhibitor of PKC
activity(46) . In the present study, we found that SPC markedly
and rapidly increases the phosphorylation of the major PKC substrate
80K/MARCKS either in intact or in permeabilized cells. Interestingly,
recent results demonstrated that this protein is in fact phosphorylated
by PKCs
,
, and
(39) . All of these PKC
isoforms are expressed in Swiss 3T3 cells, inhibited by GF 109203X, and
down-regulated by prolonged treatment with PDB in these
cells(26) . Thus, SPC stimulates transient p42
activation through a PKC-dependent signaling pathway.
In the
present study, we provide two independent lines of evidence that SPC
stimulation of PKC involves an intermediary G protein: a) the
stimulation of 80K/MARCKS in permeabilized cells was severely inhibited
by the G protein antagonist GDPS and b) the increase in 80K/MARCKS
phosphorylation in intact cells was inhibited by prior exposure to
pertussis toxin, which ADP-ribosylates and inactivates G
and G
. The concentration of pertussis toxin required
to block PKC activation has previously been demonstrated to
ADP-ribosylate a 40-kDa G
protein in intact Swiss 3T3
cells(47) . If PKC mediates activation of p42
in
response to SPC, treatment of cells with pertussis toxin should be
expected to prevent p42
activation. This prediction was
verified experimentally. We found that SPC-mediated activation of
p42
was profoundly inhibited by prior treatment of Swiss
3T3 cells with pertussis toxin.
A 90-kDa S6 protein kinase, named
p90, is directly regulated by MAPKs that phosphorylate
and partially reactivate dephosphorylated
p90
(20) . The results presented here demonstrate
that SPC induces a transient activation of p90
activity.
As expected from the results obtained with p42
, SPC
stimulation of p90
is mediated by PKC via a pertussis
toxin-sensitive signal transduction pathway.
As both p42 and p90
are localized in the cytoplasm and in the
nucleus and possess the potential for directly regulating gene
expression by transcription factor
phosphorylation(15, 20, 21) , these signaling
pathways are likely to contribute to SPC-induced mitogenesis in
fibroblasts. We demonstrate in this paper that SPC-induced mitogenesis
is also dependent on a G
protein in Swiss 3T3 cells. These
findings are not restricted to Swiss 3T3 cells. We verified that SPC
induced p42
activation and DNA synthesis in secondary
cultures of MEF through a largely pertussis toxin-sensitive pathway.
The signaling pathways leading to p42 activation in
response to SPC can be distinguished from those utilized by bombesin
and PDGF, which, like SPC, are potent mitogens for Swiss 3T3 cells.
Bombesin-induced p42
activation is predominantly
mediated by PKC (19) (
)but independent of a G
signaling pathway in Swiss 3T3 cells. PDGF causes accumulation of
p21
-GTP, which then initiates activation of a kinase
cascade comprising p74
, MEK, and MAPKs(15) . In
agreement with these reports, we found that PDGF induces a marked
increase in p74
and p42
activity in
Swiss 3T3 cells. In contrast, SPC is as potent as PDGF in stimulating
p42
activity but elicits only a small increase in
p74
activation. Furthermore, the stimulation of the
MAPK cascade by PDGF is entirely resistant to pertussis toxin treatment
(data not shown).
Stimulation of p42 activation by
SPC is also different from that induced in response to integrins. In
contrast to integrin signaling(32) , SPC-induced p42
activation is independent of the integrity of the actin
cytoskeleton. However, SPC-induced tyrosine phosphorylation of
p125
and changes in the organization of the actin
cytoskeleton were strongly inhibited by cytochalasin D(35) .
Furthermore, the signaling mechanism by which SPC induces MAPK
activation appears to differ from that utilized by other ligands that
act on G
-coupled receptors such as thrombin and LPA, which
stimulate MAPK activation independently of PKC (48, 49) .
It is known that SPC directly releases
Ca from inositol 1,4,5-trisphosphate-sensitive stores
in a variety of permeabilized cell preparations(8) .
Consequently, it has been proposed that SPC acts intracellularly,
perhaps as a direct mediator of the effects of sphingosine and
sphingosine-1-phosphate(9, 10) . In contrast, our
results demonstrate that SPC elicits PKC and p42
activation as well as p125
tyrosine phosphorylation (35) through Ca
-independent pathways in
intact Swiss 3T3 cells. The rapidity and diversity of the early
responses elicited by SPC are strongly reminiscent to those induced by
signaling molecules that act via G protein-coupled receptors. Our
results showing that treatment with pertussis toxin decreased SPC
stimulation of PKC, p42
, p90
, and DNA
synthesis in Swiss 3T3 cells implicate a heterotrimeric G protein(s) of
the G
/G
subfamily in the action of SPC. As G
proteins link many cell surface receptors to effector proteins, the
requirement of a functional G
has been regarded as
indicative of a receptor-mediated signal transduction pathway. We
hypothesize, therefore, that SPC binds to a receptor that mediates at
least some of its biological effects in Swiss 3T3 cells and MEF.
SPC has been demonstrated in tissues of patients with Niemann Pick disease, a lipid storage disorder caused by abnormal sphingomyelin metabolism(5) . Although initial reports of SPC synthesis in normal tissues (50, 51) have subsequently been challenged(52) , recent evidence obtained with NMR spectroscopy confirmed the existence of SPC in normal and tumor tissues(4) . Future experiments should elucidate whether SPC levels are regulated by signaling molecules and whether SPC can be secreted and act in an autocrine/paracrine manner. It is also conceivable that SPC could function as a surrogate agonist in intact cells, usurping a seven-transmembrane receptor intended for a different physiological ligand. In view of the potency of the mitogenic effects induced by SPC in serum-free medium and the striking early responses shown in the present and in the accompanying paper(35) , we conclude that SPC provides an attractive agonist to explore the molecular mechanisms underlying the regulation of cytoskeletal architecture and cell proliferation.