©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of a Pertussis Toxin-sensitive G Protein in the Mitogenic Signaling Pathways of Sphingosine 1-Phosphate (*)

Kimberly A. Goodemote (§) , Mark E. Mattie , Alvin Berger (¶) , Sarah Spiegel (**)

From the (1) Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 Gor 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.


INTRODUCTION

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),() 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 Cafrom 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.

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 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.


EXPERIMENTAL PROCEDURES

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 Cameasurements, 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 10cells/cmin 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 CaConcentration

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 NaHCO, 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 10cpm; 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.


RESULTS

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/Gphase (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 Gprotein.


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 ().

As previously reported, addition of SPP to Fura-2 loaded Swiss 3T3 cells caused an increase in intracellular free Ca(Zhang et al., 1991; Mattie et al., 1994). Preincubation of cells with PT inhibited the early increase in intracellular free Cainduced 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 Pfor 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).




DISCUSSION

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) (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).

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 Gprotein may be involved. However, the inhibition of adenylate cyclase may not be associated with Gregulation 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 Gresults 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.

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, GTPS stimulated, whereas GTPS inhibited sphingosine-induced inositol phosphates accumulation in permeabilized cells (Chao et al., 1994).

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 InsPlevels in Swiss 3T3 fibroblasts, complete inhibition of inositol phosphate formation by TPA did not inhibit SPP-mediated calcium responses, indicating that formation of InsPis 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 InsPantagonist, inhibited calcium release induced by exogenous InsPbut 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).

Of the SPP signaling pathways examined, only accumulation of PA was insensitive to PT, indicating that accumulation of PA may be dissociated from G-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.

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 Gproteins 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 Gproteins (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

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 [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.



FOOTNOTES

*
This work was supported in part by Research Grant 1RO1 GM43880 from the National Institutes of Health and Grant 3018M from The Council for Tobacco Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported by the Georgetown Summer Research Fellowship.

Supported by a postdoctoral fellowship from the National Cancer Institute (F32CA09249).

**
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Georgetown University Medical Center, 357 Basic Science Building, 3900 Reservoir Road NW, Washington, D.C. 20007. Tel.: 202-687-1432; Fax: 202-687-02600

The abbreviations used are: SPP, sphingosine 1-phosphate; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; InsP, inositol (1,4,5)-trisphosphate; PA, phosphatidic acid; PT, pertussis toxin; TPA, 12- O-tetradecanoylphorbol 13-acetate; GTPS, guanosine 5`- O-(3-thiotriphosphate); GTPS, guanosine 5`- O-(2-thiodiphosphate); [Ca], intracellular Ca; AP-1, activator protein-1.


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