From the
Sphingosine 1-phosphate, a sphingolipid metabolite, was
previously reported to increase DNA synthesis in quiescent Swiss 3T3
fibroblasts and to induce transient increases in intracellular free
calcium (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G.,
and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). In
the present study, pretreatment of Swiss 3T3 fibroblasts with pertussis
toxin reduced sphingosine 1-phosphate-induced DNA synthesis.
Sphingosine 1-phosphate decreased cellular cAMP levels and also caused
a drastic decrease in isoproterenol- and forskolin-stimulated cAMP
accumulation. Pertussis toxin treatment prevented the inhibitory effect
of sphingosine 1-phosphate on cAMP accumulation, suggesting that a
pertussis toxin-sensitive G
Recent evidence suggests that sphingolipid metabolites may
function as a new class of intracellular second messengers involved in
cell growth regulation and signal transduction (Hannun and Bell, 1989;
Merrill, 1991; Spiegel et al., 1993; Kolesnick and Golde,
1994). Previously, we reported that sphingosine 1-phosphate
(SPP),
Pertussis toxin (PT)-sensitive guanylate nucleotide-binding proteins
(G proteins) have an important role in the control of cell growth
(Hildebrandt et al., 1986; Letterio et al., 1986;
Chambard et al., 1987; Van Corven et al., 1989;
Pouyssegur and Seuwen, 1992). Many groups have shown that PT, which
catalyzes the ADP-ribosylation of the
As previously reported, addition of SPP
to Fura-2 loaded Swiss 3T3 cells caused an increase in intracellular
free Ca
Previous studies suggested that the mitogenic effects of
sphingosine were mediated, at least in part, via its conversion to SPP
(Zhang et al., 1991; Olivera and Spiegel, 1993). SPP is a more
potent mitogen than sphingosine itself, consistent with the fact that
only a small fraction of exogenous sphingosine is converted to SPP
intracellularly (Olivera et al., 1994). Furthermore,
inhibition of the phosphorylation of sphingosine with an inhibitor of
sphingosine kinase, DL- threo-dihydrosphingosine, not
only inhibited DNA synthesis induced by sphingosine (Olivera and
Spiegel, 1993), it also eliminated its ability to stimulate DNA binding
activity of AP-1 (Su et al., 1994). For these reasons, we have
focused our attention on SPP, rather than sphingosine.
We have
examined the involvement of PT-sensitive GTP-binding proteins in the
signal transduction pathways underlying the mitogenic actions of SPP.
DNA synthesis and cellular proliferation stimulated by SPP were reduced
by PT. PT also induced ADP-ribosylation of a 41-kDa membrane protein
(Spiegel, 1989), suggesting involvement of a GTP-binding protein that
is a substrate for PT in mitogenesis induced by SPP. Sphingolipid
metabolites may act through a subclass of G proteins to induce several
second messenger cascades. These include inhibition of cAMP
accumulation; activation of phospholipase C leading to increases in
inositol
(1, 4, 5) -trisphosphate levels
(InsP
Similar to the effect
of sphingosine (Zhang et al., 1990b), SPP drastically
decreased cellular cAMP levels which was evident after a brief
treatment. Adrenaline-, propranolol-, and forskolin-stimulated
increases in cAMP levels were also lowered in S49 lymphoma cells by
sphingosine treatment (Johnson and Clark, 1990), which suggests that
sphingoid bases could either inhibit adenylate cyclase or activate
phosphodiesterase, independently of receptor function. In Swiss 3T3
fibroblasts, activation of phosphodiesterase appears an unlikely
mechanism since SPP inhibited cAMP accumulation in the presence of
phosphodiesterase inhibitors. PT prevented the inhibitory effect of SPP
on cAMP accumulation, suggesting that a PT-sensitive G
Sphingolipid metabolites enhance
phosphatidylinositol turnover by stimulating phospholipase C activity,
and the activation of this process is modulated by a G protein that is
a substrate for PT. Sphingosine stimulated polyphosphoinositides
hydrolysis in Swiss 3T3 fibroblasts (Zhang et al., 1990b), in
primary cultured astrocytes (Ritchie et al., 1992), and in
primary human skin fibroblasts (Chao et al., 1994). We
observed that mitogenic concentrations of SPP also stimulated
production of inositol phosphates in Swiss 3T3 cells (Mattie et
al., 1994), which was attenuated by PT. In agreement, it was
previously shown that treatment of human foreskin fibroblasts with PT
partially inhibited sphingosine-mediated inositol phosphates
accumulation (Chao et al., 1994). Furthermore, GTP
Independent of phosphoinositol turnover, SPP was found to
stimulate a more rapid release of intracellular calcium than
sphingosine in skin fibroblasts (Chao et al., 1994). Recently,
we found that mobilization of calcium by SPP also proceeded by a
previously undescribed mechanism, independent of calcium influx and
inositol lipid hydrolysis (Mattie et al., 1994). Although SPP
increased InsP
Of the SPP
signaling pathways examined, only accumulation of PA was insensitive to
PT, indicating that accumulation of PA may be dissociated from
G
Does
endogenously formed SPP activate certain G protein effector systems at
the inner leaflet of the plasma membrane? It is likely that SPP taken
up by cells exerts its actions intracellularly since SPP is formed
intracellularly in response to sphingosine, platelet-derived growth
factor, and serum (Olivera and Spiegel, 1993). The response to these
mitogens was decreased when administered with the sphingosine kinase
inhibitor, DL- threo-dihydrosphingosine, which
decreases synthesis of SPP (Olivera and Spiegel, 1993). Moreover, the
amount of SPP formed intracellularly in response to these mitogens was
nearly the same as that taken up by the cells after treatment with
mitogenic concentrations of SPP (Zhang et al., 1991; Olivera
and Spiegel, 1993). However, the possibility that exogenous SPP has
additional effects cannot be excluded. For example, SPP may perturb the
lipid bilayer in such a manner that the G proteins are selectively
activated in a receptor-independent fashion. Alternatively, SPP could
bind to and directly activate a specific cell-surface receptor that is
coupled to G proteins similar to the recently described
lysophosphatidic acid receptor (Van Corven et al., 1993).
What role do G
Confluent and
quiescent cultures of Swiss 3T3 cells were incubated at 37 °C in
DMEM/Waymouth (1:1) supplemented with BSA (20 µg/ml), transferrin
(5 µg/ml), and insulin (4 µg/ml), in presence or absence of
pertussis toxin (20 ng/ml). After 2 h, cells were exposed to the
indicated mitogens and [
[
or G
-like protein
may be involved in sphingosine 1-phosphate-mediated inhibition of cAMP
accumulation. Mitogenic concentrations of sphingosine 1-phosphate
stimulated production of inositol phosphates which was inhibited by
pertussis toxin, while the response to bradykinin was not affected.
Furthermore, calcium release induced by sphingosine 1-phosphate, but
not by bradykinin, was also attenuated by pertussis toxin treatment.
However, sphingosine 1-phosphate-induced phosphatidic acid accumulation
was unaffected by pertussis toxin. The increase in specific DNA binding
activity of activator protein-1, which was induced by treatment of
quiescent Swiss 3T3 fibroblasts with sphingosine 1-phosphate, was also
inhibited by pertussis toxin. These results suggest that some of the
sphingosine 1-phosphate-induced signaling pathways are mediated by G
proteins that are substrates for pertussis toxin.
(
)
induces cell proliferation of Swiss 3T3
fibroblasts via a protein kinase-C-independent pathway (Zhang et
al., 1991). SPP triggers intracellular signaling mechanisms which
includes calcium mobilization from internal sources (Ghosh et
al., 1990; Zhang et al., 1991) and activation of
phospholipase D (Zhang et al., 1990b; Desai et al.,
1992), both of which may be important events in the control of cellular
proliferation. SPP has appropriate properties that make it suitable to
function as an intracellular second messenger: it elicits diverse
cellular responses (Zhang et al., 1991; Desai et al.,
1992; Sadahira et al., 1992); it is rapidly produced from
sphingosine by a specific kinase and degraded by a specific lyase
(Stoffel et al., 1973; Van Veldhoven et al., 1991;
Buehrer and Bell, 1992); its levels can be transiently increased by
specific growth factors (Olivera and Spiegel, 1993); it releases
Ca
from internal sources in an inositol
trisphosphate-independent manner (Ghosh et al., 1994; Mattie
et al., 1994); and finally, it elevates phosphatidic acid
levels (Desai et al., 1992) and activates DNA binding activity
of AP-1 (Su et al., 1994) which may link sphingolipid
signaling pathways to cellular ras-mediated signaling pathways.
subunit of a structurally
similar subset of GTP-binding proteins (reviewed in Neer and Clapham
(1988)), inhibited DNA synthesis in cultured fibroblasts induced by
fetal calf serum (Hildebrandt et al., 1986) and several growth
promoting agents, including bombesin (Letterio et al., 1986),
thrombin (Chambard et al., 1987), lysophosphatidic acid (Van
Corven et al., 1989), and the B subunit of cholera toxin
(Spiegel, 1989). Microinjection of anti-
antibodies
into Balb/c 3T3 fibroblasts decreased thrombin-induced DNA synthesis
(Lamorte et al., 1993). Furthermore, expression of
in Balb/c fibroblasts decreased proliferation and
also led to agonist-specific changes in growth regulation (Cui et
al., 1991). In addition, in rat 1a fibroblasts, expression of the
constitutively activated mutant
subunit decreased
doubling time, diminished the requirement for serum, and induced
transformation (Gupta et al., 1992). In agreement, expression
of GTPase-deficient forms of
and
,
but not
, induced a loss of contact inhibition and
anchorage dependence and decreased doubling time of NIH 3T3 fibroblasts
(Hermouet et al., 1993). In this study, the possibility that a
pertussis toxin-sensitive GTP-binding protein plays a role in signaling
pathways and cellular proliferation induced by SPP was examined.
Materials
PT was from List Biological Labs
(Campbell, CA). [ methyl-H]Thymidine (55
Ci/mmol), myo-[2-
H]inositol (15
Ci/mmol), [ adenylate-
P]NAD, and
[
-
P]ATP were from Amersham. Insulin and
transferrin were from Collaborative Research (Lexington, MA).
Bradykinin, and Streptomyces chromofuscus phospholipase D
(type VI, 3000 units/mg) were from Sigma. Fura-2/acetoxymethyl ester
(fura-2/AM) was from Molecular Probes Inc. (Eugene, OR). SPP was
prepared by enzymatic digestion of sphingosylphosphorylcholine with
phospholipase D (Zhang et al., 1991).
Cell Culture
Swiss 3T3 cells from American Type
Culture Collection (CCL 92) were cultured as described previously
(Spiegel, 1989). For measurement of DNA synthesis and phosphoinositide
breakdown, cells were seeded and grown on multicluster plastic tissue
culture dishes (24 16-mm wells, Costar, Cambridge, MA). For
Ca
measurements, cells were seeded and grown on glass
coverslips contained in 6-well cluster tissue culture dishes. Cells
were subcultured at a density of 1.5
10
cells/cm
in DMEM supplemented with 2 mM glutamine and 10% calf serum, refed with the same medium after 2
days and used 5 days later when cells were confluent and quiescent
(Spiegel and Panagiotopoulos, 1988).
Assay of DNA Synthesis
DNA synthesis was measured
by [H]thymidine incorporation (Spiegel and
Panagiotopoulos, 1988).
Flow Cytometric Analysis
Analysis of cell cycle
distribution was performed by propidium iodide staining of cellular DNA
using an FACStarflow cytofluorometer (Becton Dickinson,
San Jose, CA).
Assay of Cyclic AMP
Cells were incubated for 20
min at 37 °C in DMEM containing the phosphodiesterase inhibitors
3-isobutyl-1-methylxanthine (0.5 mM) and Ro20-1724 (0.2
mM), with and without 10 µM isoproterenol, 10
µM SPP or vehicle. Medium was then aspirated and cAMP
extracted from cells with 1 ml of 0.1 M HCl containing 0.1
mM CaCland measured by radioimmunoassay (Zhang
et al., 1990b).
Measurement of Phosphoinositide
Breakdown
Confluent cultures of Swiss 3T3 fibroblasts were
prelabeled for the final 3 days of culture with
myo-[2-H]inositol (10 µCi/ml) in
6-well clusters. Cells were then rinsed twice with 10 mM LiCl
in 20 mM HEPES-buffered DMEM supplemented with 30 µg/ml
BSA, incubated for 5 min at 37 °C, and then treated with the
mitogenic agents. To increase sensitivity of the assays, measurements
were performed in the presence of 10 mM LiCl, an inhibitor of
inositol 1-phosphate and inositol bisphosphate phosphatases (Berridge,
1993). At the end of the stimulation period, medium was removed and
reactions terminated by addition of chloroform, methanol, 4 N HCl (100:200:2, v/v). After extraction, inositol phosphates in the
aqueous phase were isolated on Dowex AG 1X8 ion-exchange columns
(Mattie et al., 1994).
Measurement of Cytoplasmic Free Ca
Cells were grown on glass coverslips and
loaded with the fluorescent calcium-sensitive dye, fura-2/AM (5
µM) for 45 min at 37 °C in DMEM supplemented with 60
µg/ml BSA. Subsequently, cells were washed with Locke's
buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCOConcentration
, 1.2 mM MgCl
, 2.3 mM CaCl
, 5.6 mM glucose, 30 µg/ml BSA, 5
mM HEPES, pH 7.4) and mounted in a 35-mm holder maintained at
37 °C. Changes in fura-2 fluorescence were monitored in single
cells by dual excitation imaging using an Attofluor Digital
Fluorescence Microscopy System (Atto Instruments Inc., Rockville, MD).
[Ca
]
was determined
from the ratio of fura-2 fluorescence emission after excitation at
wavelengths of 334 and 380 nm (Mattie et al., 1994).
Phosphatidic Acid Determination
Confluent and
quiescent cultures of 3T3 cells were washed with DMEM and incubated in
this medium containing P
(100 µCi/ml) for
24 h. Cells were then treated with SPP or vehicle alone, after which
the medium was rapidly removed, and lipids extracted (Zhang et
al., 1990b). Phosphatidic acid was analyzed by thin-layer
chromatography (TLC) using the organic phase of the mixture of
isooctane:ethyl acetate:acetic acid:water (50:110:20:100) (Zhang et
al., 1990b). In this system, phosphatidic acid
( R
= 0.1) was separated from other
phospholipids ( R
= 0). Lipid
standards were visualized with molybdenum blue. Phospholipids were
located by autoradiography and radioactivity quantified by liquid
scintillation counting of corresponding silica gel areas.
Electrophoretic Mobility Shift Assays
(EMSA)
Nuclear extracts were prepared from quiescent Swiss 3T3
fibroblasts (Dignam et al., 1983; Su et al., 1994).
DNA-protein binding reactions for EMSA were performed in 25 µl of
buffer containing 10 mM HEPES, pH 7.9, 50 mM KCl, 0.1
mM EDTA, 0.25 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, 10% glycerol, 1 µl (1 µg)
of poly(dI-dC), and 5-10 µl (5 µg) of nuclear extracts
with and without oligonucleotide competitors. Following a 15-min
preincubation at 25 °C, 0.3 ng of double-stranded, spin-column
purified, P-end labeled AP-1 consensus oligonucleotide
probe (1
10
cpm; Stratagene, La Jolla, CA) was
added for an additional 15 min at 25 °C. 3 µl of indicator dye
was then added and the DNA-protein complexes resolved by
electrophoresis on a 1.5-mm thick, 6% polyacrylamide gel
(acrylamide:bisacrylamide, 60:1) containing 0.5
TBE (1
TBE: 89 mM Tris borate, pH 8.0, 2 mM EDTA), at 300 V
for 30 min and then 150 V for 30 min at room temperature with tap water
cooling. Gels were dried and visualized by autoradiography. Assays in
the presence of oligonucleotide competitors were performed in the same
fashion.
Pertussis Toxin Inhibits SPP-induced DNA
Synthesis
We previously showed that SPP stimulates mitogenesis
in Swiss 3T3 fibroblasts (Zhang et al., 1991; Mattie et
al., 1994). In agreement, FACS analysis of the cell cycle
progression revealed that in quiescent Swiss 3T3 fibroblasts, most of
the cells were in G/G
phase (85%), and only a
small fraction was in S phase (3.5%). SPP treatment increased the
number of cells in S phase 2-fold (Fig. 1). To investigate the
possibility that G proteins may be involved in the proliferative
response induced by SPP, quiescent Swiss 3T3 fibroblasts were treated
with PT prior to addition of SPP. PT pretreatment inhibited the
SPP-induced mitogenic response (). In agreement with
previous studies (Hildebrandt et al., 1986; Letterio et
al., 1986; Chambard et al., 1987; Spiegel, 1989; Van
Corven et al., 1989), PT also inhibited DNA synthesis induced
by FBS, while it had no effect on DNA synthesis induced by TPA.
SPP-induced DNA synthesis was inhibited by PT in a dose-dependent
manner. Substantial inhibition was observed when cells were exposed to
0.1 ng/ml PT and maximal effects observed at 10 ng/ml. Significant
stimulation of DNA synthesis was still evident at the highest
concentration of PT tested (100 ng/ml). Even in the presence of
insulin, which greatly potentiated the mitogenic response to SPP, the
inhibitory effects of PT were essentially the same (data not shown).
Figure 1:
Flow cytometric analysis of cell cycle
distribution in Swiss 3T3 fibroblasts stimulated with SPP. Confluent
and quiescent cultures of Swiss 3T3 cells were washed with DMEM and
incubated at 37 °C in DMEM supplemented with BSA (20 µg/ml) and
transferrin (5 µg/ml), in the presence ( hatched bars) or
absence ( open bars) of 10 µM SPP. After 24 h,
cells were analyzed by FACS as described under ``Experimental
Procedures.'' Results of the cell cycle study (mean ± S.D.;
n = 3) are expressed as percent of cells in the
indicated cell cycle stages in each treatment group. Asterisk indicates a significant difference from control by two-tail,
unpaired t test, p <
0.01.
SPP Inhibits cAMP Accumulation in a Pertussis
Toxin-dependent Manner
In many cell types, Gproteins that are substrates for PT are coupled to adenylate
cyclase (Ui, 1986). Previously, it was shown that incubation of Swiss
3T3 fibroblasts with sphingosine decreased cellular cAMP levels (Zhang
et al., 1990b). As the mitogenic effect of sphingosine is
mediated mainly via conversion to SPP, we examined effects of SPP on
levels of cAMP in Swiss 3T3 cells. The phosphodiesterase inhibitors
isobutylmethylxanthine and Ro20-1724 were included to insure that
changes in cAMP levels were not mediated by effects on
phosphodiesterase activity. As illustrated in Fig. 2 A,
SPP decreased cellular cAMP levels within 1 min. Similar to the effects
of sphingosine, SPP also significantly decreased
isoproterenol-stimulated cAMP accumulation (Fig. 2 B).
SPP also inhibited forskolin-stimulated cAMP accumulation, although
with less efficacy. The extent of SPP-induced decrease in cAMP was
diminished by prior exposure of cells to PT (Fig. 2 C).
In this experiment, incubation medium was supplemented with
isoproterenol to allow accurate estimation of cAMP suppression in
intact cells. PT prevented the inhibitory effect of SPP on cAMP
accumulation stimulated by isoproterenol. SPP-mediated inhibition of
cAMP accumulation in Swiss 3T3 cells may therefore involve a
PT-sensitive G
protein.
Figure 2:
Effects
of SPP and pertussis toxin on cAMP accumulation. Confluent and
quiescent cultures of Swiss 3T3 cells were exposed to 5 µM SPP () or vehicle alone (
) in the presence of 0.5
mM 3-isobutyl-1-methylxanthine and 0.2 mM Ro20-1724 for the indicated times and levels of cAMP were
then measured. In panel B, cells were incubated for 15 min
with vehicle alone, SPP (5 µM), isoproterenol
( ISO, 10 µM), forskolin ( FOR, 10
µM), and combinations in the absence ( open bars)
or presence of 0.5 mM 3-isobutyl-1-methylxanthine and 0.2
mM Ro20-1724 ( hatched bars) and cAMP
accumulation was measured. In panel C, cells were pretreated
for 12 h in the presence ( hatched bars) or absence ( open
bars) of 20 ng/ml PT. Cells were then washed and stimulated with
vehicle alone, SPP (5 µM), or cholera toxin (1 µg/ml),
and cAMP responses were measured after a 15-min incubation in the
presence of 3-isobutyl-1-methylxanthine, Ro20-1724, and
isoproterenol (10 µM).
Pertussis Toxin Attenuates SPP-mediated Phospholipase C
Activity and Cytosolic Free Ca
Previous
studies have shown that SPP increases inositol phosphate levels
probably by activating phospholipase C (Mattie et al., 1994).
In agreement, addition of SPP to quiescent Swiss 3T3 fibroblasts
increased levels of inositol phosphates (). Pretreatment
with PT alone had no effect on basal inositol phosphate levels of
G-arrested Swiss 3T3 fibroblasts, but attenuated
SPP-induced inositol phosphate accumulation. In contrast, PT had no
appreciable effect on bradykinin-induced formation of inositol
phosphates ().
(Zhang et al., 1991; Mattie et
al., 1994). Preincubation of cells with PT inhibited the early
increase in intracellular free Ca
induced by SPP by
80% without any effect on bradykinin-induced calcium release
(), indicating absence of nonspecific effects on cellular
calcium homeostatic mechanisms.
Pertussis Toxin Does Not Effect SPP-stimulated
Phosphatidic Acid Accumulation
We previously demonstrated that
the mitogenic effects of SPP are accompanied by an increase in levels
of phosphatidic acid (PA), mainly through activation of phospholipase D
(Desai et al., 1992). A mitogenic concentration of SPP caused
a 2-fold increase in [P]phosphatidic acid levels
in Swiss 3T3 cells prelabeled to isotopic equilibrium with
P
(Fig. 3). However, unlike its effects
on the other signaling pathways examined, PT did not induce major
changes in the level of phosphatidic acid induced by SPP
(Fig. 3).
Figure 3:
Effects of pertussis toxin on SPP-induced
phosphatidic acid accumulation. Confluent and quiescent cultures of
Swiss 3T3 cells were prelabeled with P
for 24
h with ( hatched bars) or without ( open bars) 20 ng/ml
PT for the last 12 h of incubation. Cells were then stimulated with
vehicle, 10 µM SPP, or 10% FBS for 1 h. Lipids were
separated by TLC and [
P]phosphatidic acid was
analyzed. Inset, autoradiogram from a representative
experiment demonstrating the separation of phosphatidic acid ( upper
band) from other phospholipids ( lower band); and that PT
pretreatment had no effect on SPP-stimulated phosphatidic acid
accumulation.
Pertussis Toxin Attenuates SPP-induced DNA Binding
Activity of AP-1
Recently, we found that SPP stimulated AP-1 DNA
binding activity as demonstrated by appearance of a distinct and
specific complex in EMSA (Su et al., 1994). In agreement,
treatment of Swiss 3T3 fibroblasts with SPP for 3 h resulted in
increased AP-1 binding activity (Fig. 4). Binding was specific as
incubation with a 10-fold molar excess of cold AP-1 probe decreased
binding (Fig. 4). PT not only inhibited DNA synthesis induced by
SPP by >60% (), it also significantly decreased the
SPP-induced stimulation of DNA binding activity of AP-1 (Fig. 4).
In sharp contrast, PT had no effect on DNA binding activity of AP-1
induced by 100 nM TPA treatment for 3 h (data not shown).
Figure 4:
Effects of pertussis toxin on SPP-induced
DNA binding activity of AP-1. Swiss 3T3 fibroblasts were pretreated in
the presence ( lanes 3 and 5) or absence ( lanes 2,
4, and 6) of 20 ng/ml pertussis toxin for 12 h. Cells
were then washed and stimulated for 3 h with vehicle alone ( lanes 2 and 3) or 10 µM SPP ( lanes
4-6). Nuclear extracts were then prepared and DNA binding
activity of AP-1 analyzed by EMSA. Upper and lower bands are the AP-1 protein complex and free AP-1 probe, respectively.
Lane 1 contains only P-labeled AP-1
oligonucleotide in the absence of nuclear extract. Unlabeled AP-1 DNA
was used as a competitor at 10-fold molar excess with an SPP-treated
sample ( lane 6).
) (Mattie et al., 1994); release of calcium
from InsP
-sensitive and -insensitive intracellular pools
(Zhang et al., 1991; Chao et al., 1994; Ghosh et
al., 1994; Mattie et al., 1994); and activation of
phospholipase D leading to formation of phosphatidic acid (Zhang et
al., 1990a; Desai et al., 1992).
protein may be involved. However, the inhibition of adenylate
cyclase may not be associated with G
regulation of
mitogenesis. In Swiss 3T3 cells, cAMP has been shown unequivocally to
act as a positive effector of proliferation (Rozengurt, 1986); thus, it
is unlikely that sphingosine- or SPP-induced decreases in cAMP play a
significant role in the induction of proliferation. Furthermore, in Rat
1a cells overexpression of G
results in
transformation without a corresponding inhibition of cAMP accumulation
(Johnson et al., 1994). However, in several other cell types
including Rat-1 and human foreskin fibroblasts, cAMP is most likely a
negative effector of mitogenesis, and therefore it seems reasonable to
assume that reduction of cAMP may have growth promoting effects in
these cells (Gomez et al., 1994). The relevance of changes in
cAMP levels to mitogenic effects of sphingosine or SPP in these cell
types is presently unclear.
S
stimulated, whereas GTP
S inhibited sphingosine-induced inositol
phosphates accumulation in permeabilized cells (Chao et al.,
1994).
levels in Swiss 3T3 fibroblasts, complete
inhibition of inositol phosphate formation by TPA did not inhibit
SPP-mediated calcium responses, indicating that formation of InsP
is not required for release of calcium by SPP (Mattie et
al., 1994). Moreover, in permeabilized cells (Mattie et
al., 1994) as well as in endoplasmic reticulum from smooth muscle
cells (Ghosh et al., 1994), heparin, an InsP
antagonist, inhibited calcium release induced by exogenous
InsP
but did not affect calcium release induced by SPP.
Recently, PA was found to mobilize calcium from internal sources in
Jurkat T cells through a mechanism independent of phosphoinositide
turnover or calcium influx (Breittmayer et al., 1991). Thus,
it is possible that PA production by sphingolipid metabolites may be
involved in this uncharacterized pathway by which SPP can release
calcium from internal sources. This is unlikely, however, since PT
markedly inhibited calcium mobilization induced by SPP without
affecting its ability to stimulate PA formation. The lack of
involvement of PA in SPP-induced mobilization of calcium is also
supported by the fact that sphingosine-induced increases in PA levels
were not stereospecific since four stereoisomers of sphingosine were
equally effective in increasing PA. However, only the
D-erythro stereoisomers released calcium from internal sources
and were mitogenic (Olivera et al., 1994).
-regulated mitogenesis. Hence, our results suggest that
intracellular PA formation may not be sufficient to mediate
sphingolipid metabolite-induced cell proliferation in Swiss 3T3
fibroblasts. However, since there was still significant stimulation of
DNA synthesis induced by SPP after pretreatment with maximally
effective concentrations of PT, PA could still be contributing to the G
protein-independent mechanism for cellular proliferation.
proteins play in controlling mitogenesis?
Recently, it was found that a common feature of G
-coupled
stimulation of DNA synthesis is the activation of mitogen-activated
protein kinases. The mitogen-activated protein kinase cascade results
in the activation of several important transcription factors, including
AP-1 (Karin and Smeal, 1992). PT inhibited the ability of SPP to
stimulate DNA binding activity of AP-1. In hamster fibroblasts, PT,
which is known to inhibit thrombin mitogenicity, efficiently inhibited
activation of mitogen-activated protein kinase independent of its
effect on G
-mediated sustained inhibition of adenylate
cyclase (Kahan et al., 1992). In agreement, PT-sensitive
activation of p21 by thrombin and lysophosphatidic acid in Rat-1 and
hamster lung fibroblasts was not attributable to known PT-sensitive G
proteins pathways, including stimulation of phospholipases, inhibition
of adenylate cyclase, or modulation of ion channels (Van Corven et
al., 1993). Instead, pharmacological evidence suggested that an
intermediary protein tyrosine kinase may be involved in p21 activation
(Van Corven et al., 1993). This interesting result defines a
novel signaling pathway involved in the action of certain G
proteins (Van Corven et al., 1993). It is also possible
that the G
-linked effector is not a tyrosine kinase and
that regulation of tyrosine kinase activity is indirect and involves an
intermediate second messenger.
Table: Effects of pertussis toxin treatment on DNA
synthesis induced by sphingosine 1-phosphate
H]thymidine incorporation
measured. Values are means ± S.D. of triplicate determinations
from a representative experiment of seven experiments performed.
Mitogen concentrations: SPP, 10 µM; FBS, 10% (v/v); and
TPA, 100 nM.
Table: Effect of pertussis
toxin pretreatment on inositol phosphate formation and calcium
responses induced by sphingosine 1-phosphate and bradykinin
H]Inositol-labeled Swiss 3T3 cells were
washed and incubated in DMEM supplemented with 30 µg/ml BSA with
either vehicle (control), pertussis toxin (20 ng/ml), or heat
inactivated toxin for 12 h at 37 °C. Cells were washed and
incubated with 20 mM HEPES-buffered DMEM containing 10 mM LiCl and 30 µg/ml BSA for 5 min. Cells were then treated with
SPP (5 µM), bradykinin (1 µM), or BSA
(Vehicle) for an additional 5 min and levels of
[
H]inositol phosphates measured. Data represent
means ± S.D. of ([
H]inositol
phosphates/total [
H]inositol lipids)
100.
For [Ca
]
measurements,
Swiss 3T3 fibroblasts were pretreated with either vehicle, pertussis
toxin (20 ng/ml), or heat inactivated toxin for 12 h. Levels of
[Ca
]
were measured in
fura-2-loaded Swiss 3T3 fibroblasts incubated in Locke's buffer
and stimulated with 5 µM SPP, 1 µM BK, or 10
µM ionomycin. Ionomycin was added immediately following
wash and addition of 2.5 mM EGTA. Values are means of peak
changes in [Ca
]
. Basal
calcium levels, determined 10 s prior to the addition of agonist, were
120 ± 60 nM.
, inositol (1,4,5)-trisphosphate;
PA, phosphatidic acid; PT, pertussis toxin; TPA,
12- O-tetradecanoylphorbol 13-acetate; GTP
S, guanosine
5`- O-(3-thiotriphosphate); GTP
S, guanosine
5`- O-(2-thiodiphosphate); [Ca
],
intracellular Ca
; AP-1, activator protein-1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.