Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China
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ABSTRACT |
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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
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INTRODUCTION |
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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), 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
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, G
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.
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MATERIALS AND METHODS |
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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--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--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
[-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.
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RESULTS |
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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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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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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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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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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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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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The basal MAPK activity of 55 ± 6 pmol · min1 · 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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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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DISCUSSION |
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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, G 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- 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 -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 G 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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Thomas Barkas for helpful discussion.
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FOOTNOTES |
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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.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bading, H.,
and
M. E. Greenberg.
Stimulation of protein tyrosine phosphorylation by NMDA receptor activation.
Science
253:
912-914,
1991[Medline].
2.
Bokemeyer, D.,
A. Sorokin,
and
M. J. Dunn.
Multiple intracellular MAP kinase signaling cascades.
Kidney Int.
49:
1187-1198,
1996[Medline].
3.
Bunemann, M.,
B. Brandts,
D. Meyer zu Heringdorf,
D. J. van Koppen,
K. H. Jakobs,
and
L. Pott.
Activation of muscarinic K+ current in guinea-pig atrial myocytes by sphingosine-1-phosphate.
J. Physiol. (Lond.)
489:
701-707,
1995[Abstract].
4.
Cook, S. J.,
B. Rubinfeld,
I. Albert,
and
F. McCormick.
RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts.
EMBO J.
12:
3475-3485,
1993[Abstract].
5.
Crespo, P.,
N. Xu,
W. F. Simonds,
and
J. S. Gutkind.
Ras-dependent activation of MAP kinase pathway mediated by G-protein subunits.
Nature
369:
418-420,
1994[Medline].
6.
Cunningham, A. J.,
and
A. Szenberg.
Further improvements in the plaque technique for detecting single antibody-forming cells.
Immunology
14:
599-601,
1968[Medline].
7.
Desai, N. N.,
R. O. Carlson,
M. E. Mattie,
A. Olivera,
N. E. Buckley,
T. Seki,
G. Brooker,
and
S. Spiegel.
Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts.
J. Cell Biol.
121:
1385-1395,
1993[Abstract].
8.
Desai, N. N.,
and
S. Spiegel.
Sphingosylphosphorylcholine is a remarkably potent mitogen for a variety of cell lines.
Biochem. Biophys. Res. Commun.
181:
361-366,
1991[Medline].
9.
Dikic, I.,
G. Tokiwa,
S. Lev,
S. A. Courtneidge,
and
J. Schlessinger.
A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.
Nature
383:
547-550,
1996[Medline].
10.
Eguchi, S.,
T. Matsumoto,
E. D. Motley,
H. Utsunomiya,
and
T. Inagami.
Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells.
J. Biol. Chem.
271:
14169-14175,
1996
11.
Faure, M.,
T. A. Voyno-Yasenetskaya,
and
H. R. Bourne.
cAMP and subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells.
J. Biol. Chem.
269:
7851-7854,
1994
12.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
13.
Hawes, B. E.,
T. van Biesen,
W. J. Koch,
L. M. Luttrell,
and
R. J. Lefkowitz.
Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation.
J. Biol. Chem.
270:
17148-17153,
1995
14.
Hecht, J. H.,
J. A. Weiner,
S. R. Post,
and
J. Chun.
Vzg-1 encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex.
J. Cell Biol.
135:
1071-1083,
1996[Abstract].
15.
Hordijk, P. L.,
I. Verlaan,
E. J. van Corven,
and
W. H. Moolenaar.
Protein tyrosine phosphorylation induced by lysophosphatidic acid in rat-1 fibroblasts.
J. Biol. Chem.
269:
645-651,
1994
16.
Howe, L. R.,
and
C. J. Marshall.
Lysophosphatidic acid stimulates mitogen-activated protein kinase activation via a G-protein-coupled pathway requiring p21ras and p74raf-1.
J. Biol. Chem.
268:
20717-20720,
1993
17.
Huang, W. C.,
and
S. H. Chueh.
Calcium mobilization from the intracellular mitochondrial and nonmitochondrial stores of the rat cerebellum.
Brain Res.
718:
151-158,
1996[Medline].
18.
Inglese, J.,
W. J. Koch,
K. Touhara,
and
R. J. Lefkowitz.
G interactions with PH domains and Ras-MAPK signaling pathways.
Trends Biochem. Sci.
20:
151-156,
1995[Medline].
19.
Jacobs, L. S.,
and
M. Kester.
Sphingolipids as mediators of effects of platelet-derived growth factor in vascular smooth muscle cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C740-C747,
1993
20.
Koch, W. J.,
B. E. Hawes,
L. F. Allen,
and
R. J. Lefkowitz.
Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G activation of p21ras.
Proc. Natl. Acad. Sci. USA
91:
12706-12710,
1994
21.
Lee, M.-J.,
J. R. Van Brocklyn,
S. Thangada,
C. H. Liu,
A. R. Hand,
R. Menzeleev,
S. Spiegel,
and
T. Hla.
Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.
Science
279:
1552-1555,
1998
22.
Lev, S.,
H. Moreno,
R. Martinez,
P. Canoll,
E. Peles,
J. M. Musacchio,
G. D. Plowman,
B. Rudy,
and
J. Schlessinger.
Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions.
Nature
376:
737-745,
1995[Medline].
23.
Liao, D.-F.,
B. Monia,
N. Dean,
and
B. C. Berk.
Protein kinase C- mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells.
J. Biol. Chem.
272:
6146-6150,
1997
24.
Mao, C.,
S. H. Kim,
J. S. Almenoff,
X. L. Rudner,
D. M. Kearney,
and
L. A. Kindman.
Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum.
Proc. Natl. Acad. Sci. USA
93:
1993-1996,
1996
25.
Moolenaar, W. H.
Lysophosphatidic acid, a multifunctional phospholipid messenger.
J. Biol. Chem.
270:
12949-12952,
1995
26.
Okajima, F.,
and
Y. Kondo.
Pertussis toxin inhibits phospholipase C activation and Ca2+ mobilization by sphingosylphosphorylcholine and galactosylsphingosine in HL60 leukemia cells.
J. Biol. Chem.
270:
26332-26340,
1995
27.
Ross, R.
The smooth muscle cells. II. Growth of smooth muscle in culture and formation of elastic fibers.
J. Cell Biol.
50:
172-186,
1971
28.
Seger, R.,
and
E. G. Krebs.
The MAPK signaling cascade.
FASEB J.
9:
726-735,
1995
29.
Seufferlein, T.,
and
E. Rozengurt.
Sphingosylphosphorylcholine activation of mitogen-activated protein kinase in Swiss 3T3 cells requires protein kinase C and pertussis toxin-sensitive G protein.
J. Biol. Chem.
270:
24334-24342,
1995
30.
Song, S. L.,
and
S. H. Chueh.
Antagonistic effect of Na+ and Mg2+ on P2z purinoceptor-associated pores in dibutyryl cyclic AMP-differentiated NG108-15 cells.
J. Neurochem.
67:
1694-1701,
1996[Medline].
31.
Van Koppen, C. J.,
D. Meyer zu Heringdorf,
C. Zhang,
K. T. Laser,
and
K. H. Jakobs.
A distinct Gi protein-coupled receptor for sphingosylphosphorylcholine in human leukemia HL-60 cells and human neutrophils.
Mol. Pharmacol.
19:
956-961,
1996.
32.
Yatomi, Y.,
S. Yamamura,
F. Ruan,
and
Y. Igarashi.
Sphingosine 1-phosphate induces platelet activation through an extracellular action and shares a platelet surface receptor with lysophosphatidic acid.
J. Biol. Chem.
272:
5291-5297,
1997
33.
Wan, Y.,
T. Kurosaki,
and
X. Y. Huang.
Tyrosine kinases in activation of the MAP kinase cascade by G-protein-coupled receptors.
Nature
380:
541-544,
1996[Medline].
34.
Wang, Y.,
and
J. P. Durkin.
-Amino-3-hydroxy-5-methyl-4-isoxazplepropionic acid, but not N-methyl-D-aspartate, activates mitogen-activated protein kinase through G-protein
subunits in rat cortical neurons.
J. Biol. Chem.
270:
22783-22787,
1995
35.
Wurgler-Murphy, S. M.,
and
H. Saito.
Two-component signal transducers and MAPK cascades.
Trends Biochem. Sci.
22:
172-176,
1997[Medline].