Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

Ting-Yu Chin and Sheau-Huei Chueh

Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In cultured porcine aortic smooth muscle cells, sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced a rapid dose-dependent increase in the cytosolic Ca2+ concentration ([Ca2+]i) and also stimulated inositol 1,4,5-trisphosphate (IP3) generation. Pretreatment of cells with pertussis toxin blocked the SPC-induced IP3 generation and [Ca2+]i increase but had no effect on the action of ATP or BK. In addition, SPC stimulated the mitogen-activated protein kinase (MAPK) and increased DNA synthesis, whereas neither ATP nor BK produced such effects. Both the SPC-induced MAPK activation and DNA synthesis were pertussis toxin sensitive. SPC-induced MAPK activation was blocked by treatment of cells with the phospholipase C inhibitor, U-73122, or the intracellular Ca2+-ATPase inhibitor, thapsigargin, but not by removal of extracellular Ca2+. Lysophosphatidic acid induced cellular responses similar to SPC in a pertussis toxin-sensitive manner in terms of [Ca2+]i increase, IP3 generation, MAPK activation, and DNA synthesis. Platelet-derived growth factor (PDGF) also induced a [Ca2+]i increase, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in [Ca2+]i. SPC-induced MAPK activation was inhibited by pretreatment of cells with staurosporine, W-7, or calmidazolium. Our results suggest that, in porcine aortic smooth muscle cells, MAPK is not activated by the increase in [Ca2+]i unless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role of Ca2+ in pertussis toxin-sensitive G protein-mediated MAPK activation.

cytosolic calcium concentration; lysophosphatidic acid; ATP; bradykinin; platelet-derived growth factor; porcine aortic smooth muscle cells

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACTIVATION OF mitogen-activated protein kinase (MAPK) leads to the phosphorylation of various proteins that regulate gene transcription factor phosphorylation and has been strongly implicated in the stimulation of cell proliferation (2, 28, 35). MAPK is activated by a variety of extracellular stimuli, including peptide growth-promoting factors acting on tyrosine kinase receptors and heterotrimeric G protein-coupled receptor agonists. Although the activation of the MAPK pathway by receptors with tyrosine kinase activity is well defined, the signaling mechanism leading to MAPK activation via G protein-coupled receptors is less clear. Several G protein-coupled receptors, including lysophosphatidic acid (LPA) (15, 16), M1 and M2 muscarinic ACh (5, 33), ANG II (10), alpha 2A-adrenergic (13), D2 dopamine (11), and bradykinin (BK) (9) receptors, have been shown to mediate mitogenic signals. The mitogenic signaling mechanism used by a given G protein-coupled receptor agonist to stimulate MAPK depends on the class of G protein and second messenger in a given cell type. For example, in COS-7 cells transfected with alpha 1B-adrenergic or M1 muscarinic receptors, protein kinase C plays an essential role in the mitogenic signaling pathway following activation of pertussis toxin-insensitive, Gq-coupled receptors (5, 11, 13). After stimulation of pertussis toxin-sensitive, Gi-coupled receptors, Gbeta gamma is responsible for the activation of MAPK, using p21ras and p74raf as downstream mediators, the effect being independent of protein kinase C (4, 5, 11, 13-16).

Recently, sphingosylphosphorylcholine (SPC) has been shown to act as an extracellular signal. For example, in common with peptide growth factors, SPC rapidly activates MAPK and stimulates DNA synthesis in Swiss 3T3 fibroblasts (7, 8, 29). Thus exogenous SPC acts as a mitogen. In addition to its mitogenic role, SPC stimulates arachidonic acid release in Swiss 3T3 fibroblasts (7) and induces the production of superoxide anion and inositol 1,4,5-trisphosphate (IP3); the latter compound subsequently causes an increase in the cytosolic Ca2+ concentration ([Ca2+]i) (26, 31). Although the gene product of edg-1 has been identified as a receptor for sphingosine 1-phosphate (S1P; Ref. 21), no receptor for SPC has yet been cloned. It is presumed that SPC interacts with a seven-transmembrane-spanning domain, G protein-coupled receptor, since all SPC-stimulated signaling events are inhibited by pertussis toxin treatment, demonstrating the involvement of a G protein that leads to stimulation of phospholipases A2 and C (26, 29, 31).

In this study, we examined the SPC signal transduction mechanism induced in both Ca2+ signaling and mitogenic signaling in quiescent porcine aortic smooth muscle cells. These cells are mitogenically highly responsive not only to platelet-derived growth factor (PDGF) but also to SPC and LPA. Furthermore, they express functional purinoceptors and BK receptors, stimulation of which results in marked phospholipid breakdown but fails to stimulate mitogenesis. We further demonstrate that the MAPK activation induced by SPC, but not by PDGF, is dependent on the [Ca2+]i increase, although PDGF also causes an increase in [Ca2+]i. Using pertussis toxin, we were able to dissociate the action of SPC and LPA from those of ATP, BK, and PDGF in terms of MAPK stimulation. It appears that SPC-induced MAPK activation in porcine aortic smooth muscle cells requires a functional phospholipase C cascade.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. DMEM, fetal bovine serum, and trypsin-EDTA were purchased from Life Technologies (Grand Island, NY). Fura 2-AM was obtained from Molecular Probes (Eugene, OR). ATP, BK, PDGF, digitonin, EGTA, SPC, leupeptin, aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, FITC-conjugated monoclonal anti-alpha -smooth muscle actin antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG were purchased from Sigma (St. Louis, MO). Rabbit polyclonal anti-MAPK antiserum [extracellular-regulated protein kinase 2(C-14)-R], recognizing p42MAPK, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-active phosphospecific MAPK IgG was provided by Promega (Madison, WI). Pertussis toxin, cholera toxin, thapsigargin, phorbol 12-myristate 13-acetate (PMA), staurosporine, W-7, calmidazolium, PD-098059, suramin, and U-73122 were provided by Research Biochemicals International (Natick, MA). [3H]thymidine (25 Ci/mmol), the D-myo-[3H]IP3 assay system, the p42/p44 MAPK enzyme assay system, horseradish peroxidase-linked donkey anti-rabbit IgG, and enhanced chemiluminescence Western blotting analysis system were obtained from Amersham (Buckinghamshire, UK). All other chemicals were analytical grade and obtained from Merck (Darmstadt, Germany).

Culture of smooth muscle cells. Porcine aortic smooth muscle cells were prepared using the explant method of Ross (27), as previously described (30). Explants of porcine aorta obtained from a local slaughterhouse were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 37°C in an atmosphere of 95% air and 5% CO2. Over the next 10 days, smooth muscle cells slowly migrated onto the dish surface. After they reached confluency, cells were subcultured by incubation with Ca2+- and Mg2+-free 0.05% trypsin-EDTA solution and maintained for five passages in culture. The cells were identified as smooth muscle cells by their typical "hill-and-valley" growth pattern and by staining with an anti-alpha -smooth muscle actin antibody (Sigma) (data not shown). For [Ca2+]i measurement experiments, cells were plated on glass coverslips 48 h before use. Quiescent cells were obtained by culturing cells in 0.1% fetal bovine serum medium for 24 h. In some experiments, cells were preincubated with pertussis toxin (50 ng/ml) or cholera toxin (1 µg/ml) for 18 h.

[Ca2+]i measurement. [Ca2+]i was measured using the fluorescent indicator, fura 2, as previously described (12). After two washes with a buffer containing (in mM) 150 NaCl, 5 KCl, 5 glucose, 1 MgCl2, 2.2 CaCl2, and 10 HEPES, pH 7.4 (loading buffer), cells were incubated with 5 µM fura 2-AM, in the same buffer, at 37°C for 20 min. Then, the fura 2 was removed by washing and the cells were incubated at 37°C for 10 min to convert the fura 2-ester to the free acid form under the action of nonselective esterase. The coverslip was then mounted in a modified Cunningham chamber (6) attached to the stage of a Nikon Diaphot inverted microscope, equipped with a Nikon ×40 fluor objective. The fluorescence of the fura 2-loaded cells was monitored using a dual-excitation spectrofluorometer with a photomultiplier-based detection system (Spex Industries, Edison, NJ) coupled to the microscope through a fiber-optic cable. Single cells were excited alternately with 340- and 380-nm light, and the emitted fluorescent light was collected by the objective through a 510-nm long-wave pass filter. Agonists were introduced by adding the solution to one side of the Cunningham chamber and draining it through to the other side with filter paper. Only one cell per coverslip, selected by a pinhole diaphragm placed in the image plane in front of the photomultiplier, was used for experiments. Instrument operation, data acquisition, and analysis were performed using DM3000 software (Spex Industries). The fluorescence ratio obtained at 340 and 380 nm (F340/F380) was used as an index of [Ca2+]i. Some experiments were performed in a nominally Ca2+-free condition, with Ca2+ being omitted from the loading buffer during [Ca2+]i measurement after fura 2 loading in the presence of Ca2+. All experiments were performed using at least 29 cells. The results of one representative experiment are illustrated graphically, and the mean ± SD values for the ratio increase, calculated for n cells of different batches, are given in the text.

Measurement of IP3 generation. Briefly, after near-confluency was reached in six-well plates, cells were pretreated with either the phospholipase C inhibitor, U-73122 (10 µM), for 5 min or the LPA receptor inhibitor, suramin (100 µM), for 15 min. Cells were then washed and incubated with the indicated agonists at 37°C for 15 s in a volume of 1 ml of loading buffer. The amount of IP3 produced in each well of cells was determined using the D-myo-[3H]IP3 assay system (Amersham), according to the manufacturer's instructions. Some experiments were performed using pertussis toxin-treated cells. All experiments were performed at least five times in triplicate, using different batches of cells; the data presented are means ± SD for n independent experiments. Statistical differences between means were assessed using Student's t-test.

Measurement of DNA synthesis. After 70% confluency was reached in six-well plates, quiescent cells were incubated with buffer, SPC, LPA, PDGF, or serum, either with or without PD-098059 (30 µM), for 20 h in serum-free medium. During the last 5 h of incubation, 1 µCi/ml [3H]thymidine was added to the medium and [3H]thymidine incorporation into DNA was quantified by measuring TCA-insoluble radioactivity as previously described (19). Some experiments were performed using pertussis toxin-pretreated cells. The values shown are means ± SD of n independent experiments. Statistical differences between means were assessed using Student's t-test.

Immunoblotting. After stimulation with the indicated agonists in 1 ml of loading buffer in six-well plates at 37°C for 5 min, quiescent cells were rapidly detached from each well and centrifuged. The cell pellets were suspended in 200 µl of ice-cold lysis buffer containing 125 mM sucrose, 10 mM HEPES, 1 mM EGTA, 20 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM dithiothreitol, and 200 µM phenylmethylsulfonyl fluoride, pH 7.0. After homogenization by mechanical shearing of the cells by 20 passages through a Hamilton syringe, the homogenates were centrifuged at 25,000 g for 30 min. Aliquots of supernatant (10 µl) were mixed with an equal volume of twofold-concentrated SDS-PAGE buffer and were boiled for 8 min at 95°C. After electrophoresis in duplicate, the proteins were then electrophoretically transferred to nitrocellulose paper. One blot was immunoblotted using a rabbit polyclonal anti-MAPK antiserum that detects p42MAPK. The immunoreactive proteins were visualized by the alkaline phosphatase enzyme reaction. The other blot was detected using a rabbit anti-active phosphospecific MAPK IgG. Bands corresponding to MAPK were visualized with enzyme-linked chemiluminescence. All experiments were repeated at least four times with similar results.

Assay of MAPK. Quiescent cells in six-well plates were stimulated by the indicated agonists at 37°C for 5 min in 1 ml of loading buffer. After they were washed, cells were rapidly detached, centrifuged, and suspended in 200 µl of ice-cold homogenization buffer containing 10 mM Tris, 50 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin, pH 7.4. After homogenization by mechanical shearing of the cells by 20 passages through a Hamilton syringe, the material was centrifuged at 25,000 g for 30 min and the supernatant was assayed for MAPK activity. MAPK activity in aliquots of cell lysate (15 µl containing 10-20 µg of protein) was quantified using an assay kit (Amersham) that measures the incorporation of [gamma -32P]ATP into a specific synthetic peptide MAPK substrate, with MAPK activity being expressed as picomoles per minute per milligram protein. All experiments were performed at least five times, and similar results were obtained. The data shown are means ± SD of n independent experiments. Statistical differences between mean values were assessed using Student's t-test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

As shown in Fig. 1A, [Ca2+]i, expressed as F340/F380, increased from the basal level of 1.31 ± 0.02 (n = 144) to a peak level of 2.01 ± 0.12, 2.16 ± 0.09, 2.31 ± 0.15, and 1.94 ± 0.1 (n = 36) in response to the addition of 10 µM SPC (trace a), 100 µM ATP (trace b), 10 µM BK (trace c), or 100 µM LPA (trace d), respectively. After the peak level was reached, [Ca2+]i declined to the basal level within 200 s in BK-, SPC-, and LPA-stimulated cells, whereas it remained at a sustained level of 1.76 ± 0.04 (n = 36) in ATP-stimulated cells. These increases were reduced by about 17, 34, 12, and 13%, respectively, when nominally Ca2+-free extracellular buffer was used during [Ca2+]i measurements (traces e-h). The concentration dependence of the [Ca2+]i changes seen on addition of SPC, ATP, BK, and LPA is shown in Fig. 1B. Removal of extracellular Ca2+ did not affect the EC50 for SPC, with respective values in the presence or absence of Ca2+ of 1 µM and 2 µM (Fig. 1Ba). The same was true for ATP, BK, and LPA for which the EC50 values were ~3 µM, 27 nM, and 10 µM, respectively (Fig. 1B, b-d).


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Fig. 1.   Effect of extracellular Ca2+ on cytosolic Ca2+ concentration ([Ca2+]i) increases induced by sphingosylphosphorylcholine (SPC), ATP, bradykinin (BK), or lysophosphatidic acid (LPA). A: SPC (10 µM), ATP (100 µM), BK (10 µM), or LPA (100 µM) was added as indicated (arrowheads) to fura 2-loaded porcine aortic smooth muscle cells bathing in normal loading buffer (traces a-d) or nominally Ca2+-free loading buffer (traces e-h). B: [Ca2+]i increases induced by various concentrations of SPC, ATP, BK, or LPA in the presence (open circle ) or absence (bullet ) of extracellular Ca2+. F340/F380, 340 nm-to-380 nm fluorescence ratio. Data are means ± SD of 36 cells for each concentration of agonist.

Our results indicate that the [Ca2+]i increase in response to stimulation by SPC, BK, or LPA was predominantly attributable to intracellular Ca2+ release. The results shown in Fig. 2A provide further support for this concept; after depletion of the intracellular Ca2+ pools by treatment with thapsigargin, subsequent exposure to SPC, BK, or LPA failed to generate any further [Ca2+]i increase, even in the presence of extracellular Ca2+ (traces a, c, and d). However, ATP could still induce a significant [Ca2+]i increase after the same treatment, although the amplitude was reduced by ~69%. PDGF, an MAPK-activating agent, also induced a [Ca2+]i increase that was insensitive to extracellular Ca2+, the increased [Ca2+]i being 0.81 ± 0.02 and 0.76 ± 0.03 (n = 29) in the presence and absence of extracellular Ca2+, respectively (Fig. 2B).


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Fig. 2.   Effect of thapsigargin (TG) or extracellular Ca2+ on the agonist-induced [Ca2+]i increase in aortic smooth muscle cells. A: [Ca2+]i change in response to 10 µM SPC (trace a), 100 µM ATP (trace b), 10 µM BK (trace c), or 100 µM LPA (trace d), 5 min after cells were treated with 1 µM TG. B: [Ca2+]i change in response to 10 ng/ml platelet-derived growth factor (PDGF) in the presence (trace a) or absence (trace b) of extracellular Ca2+ was measured. Experiments were repeated at least 6 times (at least 29 cells in total for each agonist), and similar results were obtained.

To determine whether exogenously added SPC exerts its effect through a membrane receptor, we next examined the toxin susceptibility of the effects of SPC, ATP, BK, and LPA. As shown in Fig. 3A, pretreatment of cells with pertussis toxin (50 ng/ml) for 18 h markedly inhibited the SPC- and LPA-induced [Ca2+]i rise (traces a and f and traces d and i, respectively) but only slightly inhibited the ATP- or BK-induced [Ca2+]i increase (traces b and g and traces c and h, respectively). Furthermore, PDGF also produced a pertussis toxin-resistant increase in [Ca2+]i (traces e and j). The inhibitory effect was dependent on the concentration of pertussis toxin because, as the concentration of toxin increased, the SPC- or LPA-induced increase in [Ca2+]i decreased. In contrast, the ATP-, BK-, or PDGF-induced [Ca2+]i increases were essentially resistant to the action of pertussis toxin. Maximal inhibition of the SPC-induced [Ca2+]i increase (~87%) was seen at a toxin concentration of 50 ng/ml (Fig. 3Ba), whereas a concentration of 50 ng/ml only resulted in a 13% inhibition of the ATP-induced [Ca2+]i increase (Fig. 3Bb).


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Fig. 3.   Effect of pertussis toxin (PTX) on SPC-, ATP-, BK-, LPA-, and PDGF-induced [Ca2+]i increases in porcine aortic smooth muscle cells. A: [Ca2+]i increase induced by SPC (10 µM), ATP (100 µM), BK (10 µM), LPA (100 µM), and PDGF (10 ng/ml) in control cells (traces a-e) or cells pretreated with 50 ng/ml pertussis toxin at 37°C for 18 h (traces f-j). B: effect of different concentrations of pertussis toxin. Experiments were performed as in A. Results are expressed as percentage of control values obtained using cells without pertussis toxin pretreatment. Data are means ± SD of 26-32 cells for each concentration of pertussis toxin.

A similar toxin pretreatment effect was seen when IP3 generation was measured. Stimulation of cells with SPC, ATP, BK, or LPA caused an increase in IP3 concentration from the basal level of 2.6 ± 0.2 pmol/mg (n = 6) to 12.5 ± 0.5, 20.2 ± 0.7, 18.0 ± 0.2, and 8.9 ± 0.2 pmol/mg (n = 6), respectively (Fig. 4A). When cells were pretreated with pertussis toxin, the basal and ATP- or BK-induced IP3 levels were not significantly different from those seen in control cells, whereas SPC- or LPA-induced IP3 generation dropped to 4.8 ± 0.2 (n = 6) and 4.6 ± 1.1 pmol/mg (n = 6), a decrease of about 92 and 85% (P < 0.001 and P < 0.001), respectively (Fig. 4B). Our data indicate that, in porcine aortic smooth muscle cells, SPC or LPA induces a [Ca2+]i increase via activation of phospholipase C, which is coupled to a pertussis toxin-sensitive G protein, whereas the phospholipases C activated by purinoceptor and BK receptor agonists are coupled to a pertussis toxin-insensitive G protein. Indeed, none of these four agonists could induce a significant increase of IP3 generation following inhibition of phospholipase C by U-73122 (P < 0.001 for all cases) (Fig. 4C). As shown in Fig. 4D, in addition to inhibiting the action of LPA on IP3 generation, suramin, an LPA receptor antagonist (25), also inhibited the action of SPC (P < 0.001), whereas it had no effect on the actions of ATP and BK.


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Fig. 4.   Effect of pertussis toxin, U-73122, or suramin on SPC-, ATP-, BK-, and LPA-induced inositol 1,4,5-trisphosphate (IP3) generation in porcine aortic smooth muscle cells. Control cells (A) or cells pretreated with 50 ng/ml pertussis toxin (at 37°C for 18 h; B), 10 µM U-73122 (at 37°C for 5 min; C) or 100 µM suramin (at 37°C for 15 min; D) were used for IP3 measurement. Cells were stimulated with buffer (control), 10 µM SPC, 100 µM ATP, 10 µM BK, or 100 µM LPA at 37°C for 15 s. IP3 was then extracted and determined using a radioreceptor assay. Data are means ± SD of 6 independent experiments. * Significant decrease in IP3 generation following pertussis toxin (P < 0.001), U-73122 (P < 0.001), or suramin (P < 0.001) pretreatment.

In addition to causing an increase in [Ca2+]i, SPC and LPA acted as mitogens and enhanced DNA synthesis (Fig. 5). As shown in Fig. 5A, on addition of SPC or PDGF, DNA synthesis remained at the basal level for the first 10 h and then gradually increased. In contrast, the mitogenic effect of serum was seen immediately, with a 268% increase in the first 10 h, followed by a continuous increase up to 20 h. Over 20 h, DNA synthesis increased 283, 219, 460, or 866% after stimulation with SPC, LPA, PDGF, or serum, respectively (Fig. 5Ba). The SPC- or LPA-induced cell proliferation was mediated via receptor activation, since their effects were markedly inhibited (about 80 and 90%; P < 0.001 and P < 0.001, respectively) after pretreatment of cells with pertussis toxin (Fig. 5Bb). The mitogenic effect of serum was also sensitive to pertussis toxin, whereas that of PDGF was rather resistant (decrease of about 66 and 12%, respectively; P < 0.001 for serum values). Although ATP, BK, SPC, and LPA all produced a similar increase in [Ca2+]i, neither ATP nor BK was as effective as SPC or LPA in stimulating DNA synthesis (data not shown). It seems that the mitogenic effect of SPC, LPA, PDGF, and serum occurs via the activation of MAPK, since the enhanced DNA synthesis was diminished after inhibition of MAPK kinase activation by PD-098059 (P < 0.001 for all cases) (Fig. 5Bc).


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Fig. 5.   Effect of pertussis toxin or PD-098059 on SPC-, LPA-, PDGF-, and serum-induced [3H]thymidine incorporation in porcine aortic smooth muscle cells. A: [3H]thymidine incorporation into DNA induced by buffer (bullet ), 10 µM SPC (), 10 ng/ml PDGF (black-down-triangle ), or 10% serum (down-triangle). [3H]thymidine was added to the medium during last 5 h of incubation. Results are means ± SD of 5 independent experiments. B: experiments were performed as in A with [3H]thymidine incorporation into DNA being measured 20 h after stimulation either with (c) or without (a) 30 µM PD-098059. Experiments were also performed using pertussis toxin-pretreated cells (b). Data are means ± SD of 6 independent experiments. * Significant decrease in [3H]thymidine incorporation following pertussis toxin pretreatment (P < 0.001) or PD-098059 treatment (P < 0.001).

The results described above show that, in porcine aortic smooth muscle cells, SPC and LPA are able to increase [Ca2+]i and stimulate DNA synthesis, both events being sensitive to pertussis toxin. In contrast, although ATP and BK both induced a [Ca2+]i increase, their effects were pertussis toxin resistant, and neither ligand was able to induce DNA synthesis. In addition, the mitogenic effect of PDGF was rather insensitive to pertussis toxin. We then studied the effect of mitogens on the activity of MAPK. In a first approach, immunoblot analysis was used to measure agonist-induced MAPK activation. As shown in Fig. 6A, stimulation of cells with SPC, PDGF, and serum induced a significant increase in phosphorylated MAPK (p42MAPK/p44MAPK), whereas no difference was seen in the total amount of MAPK in quiescent and mitogen-stimulated cells as determined using anti-active (pTEpY) MAPK antibody and anti-p42MAPK antibody, respectively. Using a synthetic peptide, which is more specific than myelin basic protein as a substrate for MAPK, we further measured the phosphorylation ability of MAPK. As shown in Fig. 6B, the activity of MAPK increased following the stimulation by SPC, PDGF, and serum; it reached the peak at 3 min in SPC-stimulated cells, whereas, at 5 min, it still continuously increased in PDGF- or serum-stimulated cells.


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Fig. 6.   Agonist-induced phosphorylation of mitogen-activated protein kinase (MAPK). Cells were stimulated with 10% serum (down-triangle), 10 ng/ml PDGF (black-down-triangle ), 10 µM SPC (), or buffer (bullet ) at 37°C for 5 min (A) or indicated time (B). Immunoblotting was performed using anti-active MAPK (phosphorylated MAPK) or anti-p42MAPK antibody (A). MAPK activity was determined using a synthetic substrate (B). Data represent means ± SD of 4 experiments from different batches of cells.

The basal MAPK activity of 55 ± 6 pmol · min-1 · mg-1 (n = 6) was only marginally increased by ATP or BK but showed a significant increase of 602, 498, and 343% 5 min after stimulation by serum, PDGF or SPC, respectively (Fig. 7A). In the absence of extracellular Ca2+, SPC-stimulated MAPK activity was unaffected (Fig. 7B), whereas depletion of intracellular Ca2+ pools using thapsigargin, either in the presence (Fig. 7C) or absence (Fig. 7D) of extracellular Ca2+, caused a marked inhibition (P < 0.001) to basal levels. In contrast, the serum- or PDGF-induced activation of MAPK was independent of either depletion of intracellular Ca2+ pool or removal of extracellular Ca2+ (Fig. 7, A-D). The very slight activation of MAPK seen with ATP or BK was unaffected by [Ca2+]i. Furthermore, the SPC-induced MAPK activation was completely abolished after preexposure of cells to pertussis toxin, whereas the same treatment only partially inhibited the action of serum or PDGF (Fig. 7E). As seen for stimulation of DNA synthesis (Fig. 5), the serum effect was more sensitive than that of PDGF to pertussis toxin (reduction of 56 and 13%, respectively). These results show an identical susceptibility of the serum, PDGF, or SPC effects to pertussis toxin pretreatment in terms of both MAPK activation and DNA synthesis, suggesting that stimulation of DNA synthesis and MAPK activation occurs via the same pathway.


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Fig. 7.   MAPK activity in aortic smooth muscle cells. MAPK activity was measured in control cells (A and B) or cells pretreated with 1 µM TG for 5 min (C and D) or 50 ng/ml pertussis toxin for 18 h (E). Quiescent cells were stimulated with buffer (control), 10% serum, 10 ng/ml PDGF, 10 µM SPC, 100 µM ATP, or 10 µM BK at 37°C for 5 min. In some experiments, extracellular Ca2+ was removed (B and D). Cell lysates were then prepared, and their MAPK activity was measured. Data are means ± SD of 6 independent experiments. * Significant decrease in MAPK activity following TG (P < 0.001) or pertussis toxin (P < 0.001) pretreatment.

As described above, SPC-induced MAPK activation is dependent on a [Ca2+]i increase resulting from intracellular Ca2+ release. Consistent with this, inhibition of phospholipase C by U-73122 to prevent generation of IP3 (Fig. 4) abolished the SPC-induced activation of MAPK (Fig. 8). We further demonstrated that Ca2+-dependent protein phosphorylation, i.e., activation of protein kinase C or Ca2+/calmodulin-dependent protein kinase II, was involved in the process of SPC-induced MAPK activation. As shown in Fig. 8A, activation of protein kinase C by PMA caused significant MAPK activation, whereas the protein kinase inhibitor, staurosporine, and the calmodulin inhibitors, W-7 and calmidazolium, had no effect on the basal MAPK activity. PMA also had an additive stimulatory effect on the MAPK stimulatory activity of SPC (P < 0.001), whereas staurosporine, W-7, and calmidazolium totally blocked the SPC-induced MAPK activation (Fig. 8B). The effect of PMA on MAPK activation either with or without SPC was inhibited by staurosporine by ~91 and 78%, respectively, further indicating a role for protein phosphorylation (Fig. 8).


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Fig. 8.   Effect of phosphorylation on SPC-induced MAPK activation. MAPK activity was measured in control cells or cells pretreated with 100 nM staurosporine, 10 µM W-7, or 10 µM calmidazolium for 1 h; then cells were stimulated with buffer (A) or 10 µM SPC (B) at 37°C for 5 min. In some experiments, 100 nM phorbol 12-myristate 13-acetate (PMA) or 10 µM U-73122 was added at same time as the buffer or SPC. MAPK activity induced by LPA (100 µM) in control cells was also measured (A). Data are means ± SD of 6 independent experiments. * Significant reduction (P < 0.001) in SPC-induced MAPK activity.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we have examined the effect of SPC on the mediation of mitogenic signals in porcine aortic smooth muscle cells. In these cells, SPC not only caused an increase in [Ca2+]i and IP3 generation but also activated MAPK and DNA synthesis, all four events being pertussis toxin sensitive. Thus the ability of SPC to stimulate MAPK activation is clearly mediated by a pertussis toxin-sensitive G protein-coupled pathway. It is believed that, following stimulation of heterotrimeric G protein-coupled receptors, Gbeta gamma is responsible for the Gi-coupled receptor-mediated MAPK activation, whereas the Gq-coupled pathway uses phospholipase C-dependent protein kinase C activation (13, 18). The inhibition of SPC-mediated MAPK activation by thapsigargin or a phospholipase C inhibitor (Figs. 7 and 8) suggests that SPC-induced, G protein-coupled MAPK activation requires an increase in [Ca2+]i.

In vascular smooth muscle cells, it has recently been reported that blockade of the expression of protein kinase C-zeta inhibits ANG II-induced MAPK activation, whereas it has no effect on the action of PDGF, suggesting the difference in signal transduction by ANG II and PDGF (23). In the current study, although PDGF does cause an increase in [Ca2+]i, its effect on MAPK activation is pertussis toxin resistant and insensitive to changes in [Ca2+]i. Our results also suggest that SPC and PDGF stimulate MAPK activation via distinct signaling pathways.

In response to stimulation of either the N-methyl-D-aspartate (NMDA) or alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, Ca2+ influx is the predominant pathway involved in the [Ca2+]i increase. In rat hippocampal neurons, it has previously been shown that activation of the NMDA glutamate receptor causes MAPK activation, which requires Ca2+ influx from the extracellular medium (1). Similarly, in rat cortical neurons, AMPA-mediated MAPK activation is dependent on extracellular Ca2+ (34). Our current results are consistent with these previous reports in terms of Ca2+ dependence of MAPK activation.

We have compared the action of SPC with that of ATP or BK, which activate their own pertussis toxin-insensitive G protein-coupled receptors to trigger phosphoinositide hydrolysis and [Ca2+]i increase but, unlike SPC, do not activate MAPK and are not mitogenic by themselves. Thus [Ca2+]i increase is required but not sufficient for the activation of MAPK in porcine aortic smooth muscle cells. It has been previously shown that ANG II-induced MAPK activation requires an unimpaired phospholipase C and full intracellular Ca2+ pools in vascular smooth muscle cells. However, in these cells, the Ca2+ ionophore, A-23187, also causes MAPK activation (10). One possible mechanism for Ca2+-mediated MAPK activation is shown by the recent finding that, in PC-12 cells, Pyk2, a member of the Fak family of tyrosine kinase, transduces the Ca2+ signal from a Gi- or Gq-coupled receptor resulting in the formation of the Src-Grb2-Sos complex that activates the MAPK signaling pathway (9, 22).

SPC (lysosphingomyelin) is structurally similar to detergents, and it is possible that the observed Ca2+ effects might be due to a general increase in cell permeability. However, the results of the pertussis toxin sensitivity experiments, the inhibition by thapsigargin and U-73122, and the insensitivity to extracellular levels of Ca2+ preclude this possibility. Furthermore, the concentration of SPC used in the present study (10 µM) is much lower than that (300 µM) required to increase the general permeability of the plasma membrane in NG108-15 cells (17). In the present study, removal of extracellular Ca2+ had little effect on the SPC-induced [Ca2+]i increase and MAPK activation (Figs. 1 and 7), suggesting that the SPC-induced Ca2+ signaling mechanism is mainly due to IP3-dependent Ca2+ release. Our results further indicate that the SPC-induced MAPK stimulation depends on intracellular Ca2+ release. Recently, a cDNA clone (1869 nt), encoding a SPC-gated Ca2+-permeable channel in the endoplasmic reticulum, has been identified and named SCaMPER (sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) (24). The possibility that SPC may enter cells and interact with SCaMPER to increase Ca2+ can be excluded by the fact that inhibition of phospholipase C by U-73122 blocks the SPC-induced MAPK activation, showing that SPC-induced MAPK activation in porcine aortic smooth muscle cells requires functional phospholipase C cascade. This is inconsistent with previous reports, in which it has been shown that the activation of MAPK and the phosphoinositide hydrolysis caused by the expression of Gbeta gamma in COS-7 cells are two independent signaling pathways (13) and that, in Rat-1 fibroblasts, LPA-induced phospholipase C and MAPK activation represent parallel, rather than sequential, events (15).

Recently, the gene product of edg-1 has been identified as a receptor for S1P (21), whereas that of edg-2/vzg-1/rec1.3 has been identified as a receptor for LPA (14). These two genes display 37% homology and are members of a common orphan receptor subfamily. It has been shown that S1P shares a common receptor with LPA, stimulation of which results in an increase in [Ca2+]i in platelets (32) and activates a muscarinic K+ current in atrial myocytes (3). In the current study, LPA induces similar cellular responses to SPC in a pertussis toxin-sensitive manner in terms of [Ca2+]i increase, IP3 generation, MAPK activation, and DNA synthesis. Thus, in porcine aortic smooth muscle cells, either SPC or LPA acts on a single receptor or they activate their own pertussis toxin-sensitive G protein-coupled receptors, both of which activate a common signaling pathway.

In conclusion, the results presented in this study provide evidence in support of a role for Ca2+ in mediating MAPK activation induced by a pertussis toxin-sensitive, heterotrimeric G protein-coupled receptor agonist. Thus the components of the phospholipase C cascade interact with those of the MAPK signaling pathway. In contrast, PDGF-induced MAPK activation is independent of [Ca2+]i changes. Our data further suggest that SPC and PDGF stimulate MAPK activation via distinct mechanisms.

    ACKNOWLEDGEMENTS

We thank Dr. Thomas Barkas for helpful discussion.

    FOOTNOTES

This work was supported by grants from the National Science Council (NSC86-2314-B016-015), Academia Sinica (IBMS-CRC86-S01), and National Defense Medical Center (DOD-88-10), Taiwan, Republic of China.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address reprint requests to S.-H. Chueh.

Received 8 January 1998; accepted in final form 4 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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