![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan (A.D., H.T., M.K., M.T., K.S., C.M., F.O.); and Department of Microbiology, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, Japan (H.N., K.T.)
Received June 28, 2004; accepted December 28, 2004
Abstract
Sphingosine 1-phosphate (S1P) has been shown to exert a variety of biological responses through extracellular specific receptors or intracellular mechanisms. In the present study, we characterized a signaling pathway of S1P-induced cAMP accumulation in human coronary artery smooth muscle cells (CASMCs). S1P induced biphasic cAMP accumulation composed of a short-term and transient response (a peak at 2.5 min) and a late and sustained response (
4-6 h). The late phase of cAMP accumulation was parallel to the increment of cyclooxygenase-2 protein expression and was inhibited by N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS398), a cyclooxygenase-2-specific inhibitor. We were surprised to find that the cyclooxygenase-2 inhibitor also inhibited short-term cAMP accumulation even when cyclooxygenase-2 protein expression was not yet increased. More interestingly, the short-term cAMP accumulation was also completely inhibited by pertussis toxin, an inhibitor of Gi/o proteins. JTE-013, a specific antagonist for S1P2 receptors, inhibited the S1P-induced cAMP accumulation. Furthermore, small interfering RNAs targeted for S1P2 receptors significantly inhibited the S1P-induced cAMP accumulation. The cAMP response was also inhibited by specific inhibitors for phospholipase C, extracellular signal-regulated kinase pathways, and cytosolic phospholipase A2. S1P actually activated these enzyme activities and stimulated prostaglandin I2 (PGI2) synthesis. Finally, exogenously applied arachidonic acid and PGI2 induced cAMP accumulation to a similar extent as S1P. In conclusion, S1P induced cAMP accumulation through S1P receptors, including S1P2 receptor and Gi/o protein-mediated stimulation of intracellular signaling pathways involving cyclooxygenase-2-dependent PGI2 synthesis.
; Tamama and Okajima, 2002; Ishii et al., 2004). In vascular smooth muscle cells as well, S1P induces pleiotropic actions (i.e., contraction of the cells, stimulation of DNA synthesis, and inhibition of migration). These physiological actions are associated with the stimulation or inhibition of intracellular signaling pathways, including Ca2+ mobilization, cAMP accumulation, regulation of small molecular weight G proteins, and regulation of several protein kinases (Saba and Hla, 2004). Although most of the regulatory mechanisms of these signaling pathways are well characterized, there are still uncertainties with respect to the primary action sites for S1P. S1P was first recognized as a second messenger of some kinds of cytokines, including platelet-derived growth factor and tumor necrosis factor-
(Spiegel and Milstien, 2000). Later, S1P-specific cell-surface GPCRs, which are coupled to several intracellular signaling pathways, were identified; five subtypes of S1P receptors, i.e., EDG-1/S1P1, EDG-5/S1P2, EDG-3/S1P3, EDG-6/S1P4, and EDG-8/S1P5, have been identified so far (Hla et al., 2001; Tamama and Okajima, 2002; Ishii et al., 2004). We have previously shown that S1P-induced Ca2+ mobilization and inhibition of migration are mediated by cell surface S1P receptors, especially EDG-5/S1P2 in rat aortic smooth muscle cells (Tamama et al., 2001).
Bornfeldt et al. (1995) first reported that S1P increased cAMP accumulation, which seems to play a role in the inhibition of platelet-derived growth factor-induced migration of the cells (Bornfeldt et al., 1995). Cyclic AMP also acts as a second messenger for some vasodilators, such as prostaglandin I2, and prostaglandin E2, and may also be involved in the inhibition of contraction and proliferation (Weber et al., 1998). The mechanism by which S1P increased cAMP accumulation in vascular smooth muscle cells, however, remains uncharacterized. Proliferation or migration of vascular smooth muscle cells plays a pivotal role in the formation and progression of atherosclerotic lesions and restenotic lesions after angioplasty (Ross, 1999; Tamama and Okajima, 2002). Thus, the elucidation of the mechanism of cAMP accumulation as an inhibitory signal for cell proliferation and migration may be important for not only understanding the intracellular signaling networks but also developing therapeutic drugs for cardiovascular diseases, although cAMP signaling itself is a scientifically old subject. The regulation of adenylyl cyclase by GPCRs is well established; GPCRs couples to stimulatory (Gs) or inhibitory (Gi) G proteins and thereby stimulate or inhibit enzyme activity (Dessauer et al., 1996; Patel et al., 2001). In Chinese hamster ovary cells overexpressing the respective S1P receptor subtype, we demonstrated that S1P2 and S1P3 have the potential ability to couple to Gs proteins, resulting in cAMP accumulation (Kon et al., 1999). On the other hand, S1P may indirectly stimulate cAMP accumulation through prostaglandin synthesis (Davaille et al., 2000).
In the present study, we characterized S1P-induced cAMP accumulation in coronary artery smooth muscle cells (CASMCs) and found that there are several unique regulatory mechanisms of cAMP accumulation. For example, S1P induced early phase (2.5 min) and late phase (4-6 h) biphasic cAMP accumulation; both phases of cAMP accumulation involve cyclooxygenase-2. Furthermore, the early phase of cAMP accumulation was inhibited by pertussis toxin, suggesting Gi/o protein-mediated cAMP accumulation. Our results indicate that the early phase of cAMP accumulation is mediated through S1P receptors/Gi/o proteins/cytosolic phospholipase A2 (cPLA2)/cyclooxygenase-2/prostaglandin I2 pathways and the late phase is mediated by cyclooxygenase-2 induction.
Materials and Methods
Materials. Human CASMCs were purchased from Cambrex Bio Science Rockland (Rockland, ME); S1P, AACOF3, and NS398 were from Cayman Chemical (Ann Arbor, MI); arachidonic acid, prostaglandin D2, prostaglandin I2, prostaglandin E2, and SW2871 were from Sigma-Aldrich (St. Louis, MO); fatty acid-free bovine serum albumin was from Calbiochem (San Diego, CA); pertussis toxin was from List Biological Laboratories Inc. (Campbell, CA); fura 2/acetoxymethl ester was from Dojindo (Tokyo, Japan); U73122
[GenBank]
and U73343
[GenBank]
were generously provided by Upjohn Co. (Kalamazoo, MI); cAMP radioimmunoassay kit was from Yamasa (Chosi, Japan); [3H]arachidonic acid was from American Radiolabeled Chemicals (St. Louis, MO); an ERK-specific antibody (K-23, against amino acids 305-327 of rat ERK1, which recognizes both ERK1 and ERK2) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and an antibody specific to phosphorylated forms of ERK (phospho-p44/42MAPK E10) was from Cell Signaling Technology Inc. (Beverly, MA). Rabbit antibody against synthetic carboxyl-terminal peptide (ASSSRSGLDDINPT, 581-594) of cyclooxygenase-2 was prepared by immunizing animals with peptide-KLH conjugate and specifically purified from antisera. JTE-013 was a gift from the Central Pharmaceutical Research Institute, Japan Tobacco Incorporation (Osaka, Japan). The sources of all other reagents were the same as described previously (Kon et al., 1999; Sato et al., 1999).
Cell Culture. CASMCs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA), 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor-2, and 5 µg/ml insulin in a humidified air/CO2 (19:1) atmosphere. For cAMP assay, CASMCs were plated on rat-tail collagen (400 µg/ml)-coated 12-multiplates or 10-cm dishes. Twenty-four hours before experiments, the medium was replaced with serum-free medium containing 0.1% (w/v) bovine serum albumin (fraction V). Where indicated, 100 ng/ml pertussis toxin was added to the culture medium 24 h before experiments.
cAMP Accumulation. CASMCs (passages 7-9) were washed once and preincubated for 20 min (PD98059, U0126, AACOF3, indomethacin, and NS398) or for 10 min (JTE-013) at 37°C in HEPES-buffered medium. The HEPES-buffered medium was composed of 20 mM HEPES, pH 7.4, 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 2.5 mM NaHCO3, 5 mM glucose, and 0.1% (w/v) bovine serum albumin (fraction V). After the preincubation, the cells were again washed once in the HEPES-buffered medium and then stimulated with S1P or other agonists for 2.5 min unless otherwise specified. In the experiments using U73122 [GenBank] and U73343 [GenBank] , the cells were harvested from 10-cm dishes with trypsin (0.05% in phosphate-buffered saline containing 0.53 mM EDTA) and washed by sedimentation (250g for 5 min). The washing procedure was repeated, and the cells were finally resuspended in the same medium. The cells (about 5 x 106 cells) were preincubated for 2 min with 5 µM U73122 [GenBank] or U73343 [GenBank] and further incubated for 2.5 min with S1P. The reaction was terminated by adding 100 µl of 1 N HCl. The cAMP in the acid extract was measured using cAMP radioimmunoassay kit.
Measurement of [Ca2+]i. The cells were harvested from 10-cm dishes with trypsin as described above, and then [Ca2+]i was measured based on the change in fura-2 fluorescence as described previously (Kon et al., 1999).
Western Blot Analysis. The cells were washed once, preincubated for 10 min at 37°C in the HEPES-buffered medium and incubated with test agents for 2.5 min at 37°C. The incubation was terminated by washing twice with ice-cold phosphate-buffered saline and adding 0.5 ml of a lysis buffer composed of 50 mM HEPES, pH 7.0, 250 mM NaCl, 0.1% Nonidet P-40, 100 mM NaF, 0.2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. The cells were then harvested from the dishes with a rubber policeman. The recovered lysate was incubated for 30 min on ice and was centrifuged at 14,000g for 20 min. The supernatant was analyzed by Western blotting with the cyclooxygenase-2-specific or the ERK-specific antibodies. Protein extracts were subjected to 10% SDS-polyacrylamide gel electrophoresis, and proteins in the gel were transferred to a polyvinylidene difluoride membrane (ProBlott; Applied Biosystems, Foster City, CA) by electroblot. The membranes were blocked with 5% dry milk for 2 h and incubated with primary antibodies (1:1300 dilution for cyclooxygenase-2, 1:1000 dilution for ERK) for 2 h. The membranes were then incubated with a second antibody conjugated with alkaline phosphatase for 1 h and were visualized using the nitro blue teterazolium/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt system (Sato et al., 1999).
Quantitative RT-PCR Analysis. Total RNA was isolated using Tri Reagent (Sigma-Aldrich) according to the instructions from the manufacturer. After DNase I (Promega, Madison, WI) treatment to remove possible traces of genomic DNA contaminating in the RNA preparations, 5 µg of the total RNA was reverse transcribed using random priming and Multiscribe reverse transcriptase according to the instructions from the manufacturer (Applied Biosystems). To evaluate the expression level of the cyclooxygenase-2, S1P1, S1P2, S1P3, S1P4, and S1P5 mRNAs, quantitative RT-PCR was performed using real-time TaqMan technology with a sequence detection system model 7700 (Applied Biosystems). The human cyclooxygenase-2, S1P1-, S1P2-, S1P3-, S1P4-, and S1P5-specific probes were obtained from TaqMan gene expression assays (Applied Biosystems). The identification number of the products is Hs00153133 for cyclooxygenase-2, Hs00173499 for S1P1, Hs00244677 for S1P2, Hs00245464 for S1P3, Hs00269446 for S1P4, Hs00258220 for S1P5, and Hs99999905 for GAPDH. The expression level of the target mRNA was normalized to the relative ratio of the expression of GAPDH mRNA. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ± S.E.
Transfection of siRNA. CASMCs were plated on 12-multiplates at 2.0 x 105 cells/well. Sixteen hours later, siRNAs (100 nM) were introduced into cells using RNAiFect reagent (QIAGEN, K.K.) according to the manufacturer's instructions. The cells were further cultured for 24 h. The S1P receptor mRNA level was measured using real-time TaqMan technology. Cyclic AMP response was performed 24 h after serum starvation as described above. The nonsilencing siRNA was obtained from QIAGEN, K.K. The siRNA targeted for S1P2 was obtained from Dharmacon (Lafayette, CO). The identification number of S1P2 is M-003952-00.
Release of Arachidonic Acid and Its Metabolites. The cells were cultured for 2 days in 12 multiplates, and the medium was replaced with serum-free medium containing 0.1% bovine serum albumin 24 h before experiments. [3H]Arachidonic acid (0.1 µCi/well) was then added to the medium 10 h before experiments. The cells were washed three times with HEPES-buffered medium and incubated with appropriate agents for 2.5 min. The supernatant was then collected, and its radioactivity ([3H]arachidonic acid and its metabolites) was measured.
Prostaglandin I2 Measurement. Prostaglandin I2 levels were determined using an enzyme immunoassay kit, according to the manufacturer's instructions (Cayman Chemical). The amounts of prostaglandin I2 released in the supernatant were estimated from the levels of its stable metabolite 6-keto-prostaglandin F1 (6-keto-PGF1).
Statistical Analysis. All experiments were performed in duplicate or triplicate, and the results of multiple observations were presented as means ± S.E. of at least three independent experiments, unless otherwise stated. Statistical significance was assessed by Student's t test, and values were considered significant at p < 0.05 (*).
Results
Biphasic Accumulation of cAMP by S1P in CASMCs. The time-dependent effect of S1P on cAMP accumulation in CASMCs is shown in Fig. 1A. S1P rapidly increased the cAMP level 10- to 20-fold 2.5 min after the treatment, and its level then decreased. The decrease in the cAMP level may not be caused by a loss of S1P during incubation because an additional supply of the same amount of S1P during the 30-min incubation did not change the magnitude and pattern of cAMP response (data not shown). The cAMP level, however, started to gradually increase again from 60 min to at least 6 h after the S1P treatment. Thus, S1P induced biphasic cAMP accumulation, (i.e., a short-term and transient response and a late and sustained response). As shown in Fig. 1B, we measured the expression of cyclooxygenase-2 protein by Western blotting and found that S1P markedly induced cyclooxygenase-2 protein expression. The time course of cyclooxygenase-2 expression was parallel to the late and sustained cAMP response to S1P. As expected, cAMP accumulation in the late phase (at 240 min) was attenuated by NS398, a cyclooxygenase-2-specific inhibitor (Fig. 1C). We were surprised to find, however, that cAMP accumulation at 2.5 min was also inhibited by NS398 (Fig. 1C). A significant cyclooxygenase-2 expression was not observed with our detection method at such an early phase (Fig. 1B). Thenceforth, we focused on the acute phase of cAMP accumulation and analyzed its signaling mechanisms.
|
Involvement of Pertussis Toxin-Sensitive Gi/o Proteins in the S1P-Induced Acute Phase of cAMP Accumulation. As shown in Fig. 2, S1P induced the acute phase of cAMP accumulation with a half-maximal effective concentration of around 10 nM, in agreement with the reported Kd value of S1P receptors (Lee et al., 1998). If cyclooxygenase-2 is involved in S1P-induced action, prostaglandins may mediate cAMP accumulation. We examined the effects of prostaglandin I2, prostaglandin E2, and prostaglandin D2 on cAMP accumulation and found that prostaglandin I2 was the most potent stimulator (Fig. 2B). Treatment of the cells with pertussis toxin markedly inhibited the stimulatory effect of S1P (Fig. 2A) but not prostaglandin I2 (Fig. 2B) on cAMP accumulation. Thus, the pertussis toxin effect was specific, and we postulated that S1P receptors that coupled to pertussis toxin-sensitive Gi/o proteins may mediate the S1P-induced cAMP response.
|
S1P2 Receptors Is Involved in S1P-Induced cAMP Accumulation. Fig. 3A shows the mRNA expression pattern of S1P receptor subtypes measured by the real-time TaqMan PCR method. All the receptor subtypes except for S1P4 seemed to be expressed in CASMCs; the rank order of expression of mRNA was S1P2 > S1P1 > S1P3 > S1P5. We therefore examined the possible involvement of S1P2 receptors, which are expressed at the highest level. In Fig. 3B, we examined the effect of JTE-013, a specific antagonist of S1P2 receptor, on the S1P-induced action (Arikawa et al., 2003; Ohmori et al., 2003). JTE-013 inhibited the S1P-induced cAMP accumulation in a competitive manner without any significant effect on the prostaglandin I2-induced cAMP accumulation, suggesting that the S1P2 receptor is involved in the S1P action.
|
In the second line of experiments, we performed siRNA experiments. As shown in Fig. 3C, the siRNA specific to S1P2 receptors decreased the S1P2 receptor mRNA expression to 30 to 40% of the initial expression without any significant effect on S1P1 receptor mRNA expression. Under these conditions, the siRNA inhibited about 40% of the S1P-induced cAMP accumulation (Fig. 3D, left) but hardly affected prostaglandin I2-induced cAMP accumulation (Fig. 3D, right). These results indicate that the S1P2 receptors, at least, may mediate S1P-induced cAMP accumulation in CASMCs.
Intracellular Signaling Pathways of the S1P-Induced Acute Phase of cAMP Accumulation. Because cAMP formation through GPCR systems that were not thought to stimulate it has often been reported (Brown and Rietow, 1981; Pyne et al., 1997), we next characterized the mechanism underlying the Gi/o protein-mediated cAMP formation in CASMCs. An involvement of cyclooxygenase-2 was postulated from the finding that the enzyme-specific inhibitor NS398 almost completely inhibited S1P-induced cAMP accumulation (Fig. 1). We examined the specificity of this observation. S1P-induced cAMP accumulation was also inhibited by indomethacin, a potent inhibitor for both cyclooxygenase-1 and cyclooxygenase-2 (Fig. 4A), but not by 100 nM mofezolac, an inhibitor of cyclooxygenase-1 (Goto et al., 1998) (data not shown). The inhibition of the cAMP response by NS398 and indomethacin was not caused by the nonspecific action of the drugs, as evidenced by the finding that prostaglandin I2-induced cAMP accumulation was not affected by these inhibitors (Fig. 4A).
|
Because cyclooxygenase-2 protein expression was not yet increased by S1P under such an acute phase (Fig. 1), the supply of arachidonic acid, the substrate for the enzyme, was expected to be increased. We postulated an involvement of cPLA2 as an enzyme for arachidonic acid production. We also assumed the participation of ERK and phospholipase C/[Ca2+]i system because ERK has been reported to regulate cPLA2 (Lin et al., 1993; Pyne et al., 1997) and phospholipase C/[Ca2+]i signaling pathway has been shown to be involved in ERK activation (Agell et al., 2002). As shown in Fig. 4A, PD98059 and U0126, ERK kinase inhibitors, and AACOF3, a cPLA2 inhibitor, abolished S1P-induced cAMP accumulation. U73122
[GenBank]
, a phospholipase C inhibitor, but not U73343
[GenBank]
, an inactive form of U73122
[GenBank]
, also abolished S1P-induced cAMP accumulation (Fig. 4B). These results suggest the involvement of phospholipase C, ERK, and cPLA2 for the S1P action.
The involvement of these signaling pathways was confirmed by the association of their activation by S1P. S1P increased [Ca2+]i levels, which were inhibited by phospholipase C inhibitor U73122
[GenBank]
but not by U73343
[GenBank]
, reflecting phospholipase C activation (Fig. 5A). The increase in [Ca2+]i was inhibited by pertussis toxin pretreatment, indicating Gi/o proteins are also involved in this pathway like S1P-induced cAMP accumulation (Fig. 5B). S1P also induced ERK1/2 phosphorylation, reflecting the activation of the enzyme, in a manner sensitive to pertussis toxin and the ERK kinase inhibitor PD98059, whereas prostaglandin I2 failed to activate the enzyme (Fig. 6A). The phospholipase C inhibitor U73122
[GenBank]
but not U73343
[GenBank]
also inhibited S1P-induced ERK1/2 phosphorylation, suggesting that phospholipase C/[Ca2+]i signaling pathway may be located upstream in the ERK activation (Fig. 6B). Furthermore, S1P stimulated arachidonic acid release in a manner sensitive to U0126, another ERK kinase inhibitor (Fig. 7A), and exogenous arachidonic acid induced cAMP accumulation to the same extent as S1P (Fig. 7B). Finally, we examined whether S1P actually induced prostaglandin synthesis in CASMCs. We measured 6-keto-prostaglandin F1, a stable metabolite of prostaglandin I2, in the culture medium in the presence or absence of various inhibitors and found S1P markedly induced 6-keto-prostaglandin F1 production (Fig. 8A). As expected, S1P-induced 6-keto-prostaglandin F1 production was almost completely inhibited by several inhibitors that were effective for the cAMP response (Fig. 8B).
|
|
|
|
Discussion
Numerous studies have shown that cyclooxygenase-2 is involved in a variety of biological functions induced by extracellular signaling molecules, including G protein-coupled receptor ligands and cytokines, through prostaglandin synthesis (Robida et al., 2000; Martinez-Gonzalez et al., 2004). In many cases, the onset of cyclooxygenase-2-dependent extracellular signaling molecule-induced actions is rather slow because time for the induction of the cyclooxygenase-2 protein is necessary. In CASMCs as well, S1P induced the expression of cyclooxygenase-2 1 to 2 h after S1P treatment, accompanied by an increase in cAMP accumulation. A similar late phase of S1P-induced actions on prostaglandin synthesis depending on cyclooxygenase-2 induction has been shown in other cell types (Davaille et al., 2000; Kim et al., 2003; Pettus et al., 2003). The mechanisms by which S1P induces cyclooxygenase-2 expression, however, are controversial, especially with respect to its primary action site. S1P-induced prostaglandin synthesis seems to be mediated by the S1P receptors S1P1 and S1P3 in amnion-derived WISH cells (Kim et al., 2003); however, it seems to be mediated by intracellular mechanisms in hepatic myofibroblasts (Davaille et al., 2000), cultured fibroblasts, and lung adenocarcinoma cells (Pettus et al., 2003), although intracellular S1P targets still remain unidentified. Our preliminary experiments showed that S1P-induced cyclooxygenase-2 mRNA expression was attenuated by pertussis toxin pretreatment in CASMCs, suggesting an involvement of S1P receptors in the induction (data not shown).
In addition to the late response, S1P also exerted short-term and transient cAMP accumulation in CASMCs; a 10- to 20-fold increase in cAMP accumulation was observed at 2.5 min after S1P treatment. We were surprised to find that this short-term and transient cAMP response was also completely inhibited by the cyclooxygenase-2-specific inhibitor NS398. At such an early time (2.5 min), of course, cyclooxygenase-2 protein induction was not stimulated by S1P. Thus, S1P induced biphasic cAMP accumulation composed of a short-term and transient response and a late and sustained response; both responses seem to depend on cyclooxygenase-2. Although cyclooxygenase-2 protein expression was too low to be detected with our detection method, the endogenous enzyme must have high enough activity to synthesize prostaglandins when its substrate arachidonic acid is supplied.
Four types of S1P receptors (i.e., S1P1, S1P2, S1P3, and S1P5) seem to be expressed in CASMCs (Fig. 3A). Pharmacological (JTE-013) and molecular biological (siRNA) experiments suggested that S1P-induced stimulation of cAMP accumulation may be mediated by at least S1P2 receptors and pertussis toxin-sensitive Gi/o proteins (Fig. 3, B-D). Although S1P2 receptor siRNA showed the specificity to the receptors, the inhibition of mRNA expression was partial. We cannot therefore exclude the possible involvement of S1P receptors other than S1P2 receptors. In relation to this, our preliminary results showed that suramin, an antagonist for S1P3 receptors (Ancellin and Hla, 1999), did not attenuate the S1P-induced cAMP accumulation. Furthermore, SW2871, a specific agonist for S1P1 receptors (Sanna et al., 2004), alone did not increase cAMP accumulation but enhanced the S1P-induced cAMP accumulation (data not shown). These results suggest that S1P2 receptors may play a major role in the S1P-induced cAMP accumulation; however, it would be possible that there is a cross-talk between S1P receptor subtype signalings. Further studies are necessary to evaluate the role of the respective S1P receptor subtype in the S1P actions. Nevertheless, our present results strongly suggest that the S1P-induced cAMP accumulation is mediated by Gi/o protein-coupled S1P receptors, at least S1P2 receptors, but not by intracellular mechanisms.
These results were not necessarily expected because Gi/o proteins usually mediate the inhibition of cAMP accumulation and therefore may show that the S1P action is a secondary response through intracellular and/or intercellular signaling pathways but not a primary action through a typical adenylyl cyclase system in plasma membranes (Dessauer et al., 1996; Patel et al., 2001). The intracellular pathways involve cyclooxygenase-2 and prostaglandins, especially prostaglandin I2 synthesis. Prostaglandin I2 then activates adenylyl cyclase, in an autocrine manner, through a typical enzyme system composed of a stimulatory receptor (in this case, prostaglandin I2 receptor) and Gs proteins.
Because the acute phase of prostaglandin I2 synthesis was not associated with an increase in cyclooxygenase-2 protein expression, an increase in the supply of arachidonic acid, a substrate of the enzyme, was expected. Indeed, S1P induced an increase in arachidonic acid release, and the cPLA2 inhibitor AACOF3 inhibited S1P-induced prostaglandin I2 synthesis and cAMP accumulation. The arachidonic acid release was completely inhibited by the ERK kinase inhibitor U0126, suggesting that cPLA2 is regulated by ERK. Indeed, cPLA2 has been reported to be activated by phosphorylation by ERK (Lin et al., 1993; Pyne et al., 1997). Phospholipase C inhibitor U73122
[GenBank]
inhibited the S1P-induced phosphorylation of ERK, suggesting that phospholipase C/[Ca2+]i signaling pathway may be located upstream in the ERK activation. Participation of phospholipase C/[Ca2+]i signaling pathway in ERK activation has been shown (Agell et al., 2002). The S1P-induced [Ca2+]i increase was inhibited by pertussis toxin, suggesting that Gi/o proteins mediate stimulation of phospholipase C/[Ca2+]i signaling pathway. The intracellular signaling pathways of S1P receptor stimulation leading to prostaglandin I2 synthesis and cAMP accumulation in CASMCs are illustrated in Fig. 9. This scheme may explain the acute phase of cAMP accumulation without a net increase in cyclooxygenase-2 induction. Although the late phase of cAMP accumulation was not analyzed in detail in the present study, prostaglandin I2 synthesis may be synergistically stimulated by cyclooxygenase-2 induction (Fig. 1) and increase in the supply of arachidonic acid by cPLA2 activation (Hamilton et al., 1999).
|
S1P has been shown to exert pleiotropic actions in a variety of cells and organs. In vascular cell systems, S1P induced both antiatherogenic and proatherogenic actions (Okajima, 2002; Tamama and Okajima, 2002). Thus, S1P stimulates proliferation, survival, migration, barrier integrity, nitric oxide synthesis, and other functions in endothelial cells and inhibits the migration of smooth muscle cells (Kimura et al., 2001, 2003; Tamama et al., 2001; Okajima, 2002; Saba and Hla, 2004). These actions seem to be antiatherogenic. On the other hand, S1P has been shown to increase the expression of adhesion molecules, such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and thereby to accelerate monocyte interaction with endothelial cells and monocyte penetration into subendothelial space or the intima of arterial walls (Xia et al., 1998; Auge et al., 2000). This proatherogenic action of S1P has been reported to be mediated by intracellular targets, although the molecular mechanisms are not fully characterized (Xia et al., 1998; Auge et al., 2000). The stimulation of the prostaglandin I2/cAMP system by S1P, shown in the present study, may be considered an antiatherogenic aspect of S1P. In vascular smooth muscle cells, an increase in intracellular cAMP seems to be inhibitory for proliferation, migration, and contraction (Klemm et al., 2001). Moreover, prostaglandin I2 has been shown to be a potent inhibitor for platelet aggregation (Fitzgerald et al., 1987). In the S1P-induced cAMP accumulation, phospholipase C/[Ca2+]i signaling pathway seems to be involved. On the other hand, S1P has been shown to induce contraction of CASMCs through S1P2 receptors (Ohmori et al., 2003). This contraction might be partly explained by the phospholipase C/[Ca2+]i signaling pathway. Thus, S1P-induced phospholipase C/[Ca2+]i signaling may be functioning in the CASMC contraction not only as a positive regulator but also as a negative regulator through cAMP accumulation, which might make possible a fine control of the contraction.
We have previously shown that S1P is accumulated in lipoprotein fractions, especially high-density lipoproteins (HDLs) (Murata et al., 2000; Kimura et al., 2001, 2003). The HDL-associated S1P mediates HDL-induced antiatherogenic actions, such as the stimulation of survival and the migration of endothelial cells (Murata et al., 2000; Kimura et al., 2001, 2003). HDL has also been shown to increase prostaglandin I2 synthesis and cAMP accumulation in vascular smooth muscle cells (Vinals et al., 1999; Kothapalli et al., 2004) and endothelial cells (Fleisher et al., 1982). These results suggest that HDL-induced prostaglandin I2 synthesis and cAMP accumulation may be partly mediated through S1P and S1P receptors. This is our next important subject of investigation.
Acknowledgements
We are grateful to Japan Tobacco Inc. Research Laboratory for providing JTE-013. We thank Masayo Yanagita for technical assistance.
Footnotes
This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science and by a research grant from ONO Medical Research Foundation. This work was also partly supported by Japan-Korea Basic Scientific Cooperation Program.
ABBREVIATIONS: S1P, sphingosine 1-phosphate; GPCR, G protein-coupled receptor; EDG, endothelial differentiation gene; CASMC, coronary artery smooth muscle cell; cPLA2, cytosolic phospholipase A2; AACOF3, arachidonyltrifluoromethyl ketone; ERK, extracellular signal-regulated kinase; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; 6-keto-PGF1, 6-keto-prostaglandin F1; NS398, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; U-73122, 1-[6-[[17
-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; U-73343, 1-[6-[[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione; SW2871, 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluorometryl)phenyl]-1,2,4-oxadiazole.
Address correspondence to: Dr. Hideaki Tomura, Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan. E-mail: tomurah{at}showa.gunma-u.ac.jp
References
Agell N, Bachs O, Rocamora N, and Villalonga P (2002) Modulation of the Ras/Raf/MEK/ERK pathway by Ca2+ and calmodulin. Cell Signal 14: 649-654.[CrossRef][Medline]
Ancellin N and Hla T (1999) Differential pharmacological properties and signal transduction of the sphingosine 1-phosphate receptors EDG-1, EDG-3 and EDG-5. J Biol Chem 274: 18997-189002.
Arikawa K, Takuwa N, Yamaguchi H, Sugimoto N, Kitayama J, Nagawa H, Takehara K, and Takuwa Y (2003) Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor. Requirement of inhibition of cellular RAC activity. J Biol Chem 278: 32841-32851.
Auge N, Negre-Salvayre A, Salvayre R, and Levade T (2000) Sphingomyelin metabolites in vascular cell signaling and atherogenesis. Prog Lipid Res 39: 207-229.[CrossRef][Medline]
Bornfeldt KE, Graves LM, Raines EW, Igarashi Y, Wayman G, Yamamura S, Yatomi Y, Sidhu JS, Krebs EG, Hakomori S, et al. (1995) Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of human arterial smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction. J Cell Biol 130: 193-206.
Brown JH and Rietow M (1981) Muscarinic-dopaminergic synergism on retinal cyclic AMP formation. Brain Res 215: 388-392.[CrossRef][Medline]
Davaille J, Gallois C, Habib A, Li L, Mallat A, Tao J, Levade T, and Lotersztajn S (2000) Antiproliferative properties of sphingosine 1-phosphate in human hepatic myofibroblasts. A cyclooxygenase-2 mediated pathway. J Biol Chem 275: 34628-34633.
Dessauer CW, Posner BA, and Gilman AG (1996) Visualizing signal transduction: receptors, G-proteins and adenylate cyclases. Clin Sci (Lond) 91: 527-537.[Medline]
Fitzgerald GA, Catella F, and Oates JA (1987) Eicosanoid biosynthesis in human cardiovascular disease. Hum Pathol 18: 248-252.[Medline]
Fleisher LN, Tall AR, Witte LD, Miller RW, and Cannon PJ (1982) Stimulation of arterial endothelial cell prostacyclin synthesis by high density lipoproteins. J Biol Chem 257: 6653-6655.
Goto K, Ochi H, Yasunaga Y, Matsuyuki H, Imayoshi T, Kusuhara H, and Okumoto T (1998) Analgesic effect of mofezolac, a non-steroidal anti-inflammatory drug, against phenylquinone-induced acute pain in mice. Prostaglandins Other Lipid Mediat 56: 245-254.[Medline]
Hamilton LC, Mitchell JA, Tomlinson AM, and Warner TD (1999) Synergy between cyclooxygenase-2 induction and arachidonic acid supply in vivo: consequences for nonsteroidal antiinflammatory drug efficacy. FASEB J 13: 245-251.
Hla T, Lee MJ, Ancellin N, Paik JH, and Kluk MJ (2001) Lysophospholipids-receptor revelations. Science (Wash DC) 294: 1875-1878.
Ishii I, Fukushima N, Ye X, and Chun J (2004) Lysophospholipid receptors: signaling and biology. Annu Rev Biochem 73: 321-354.[CrossRef][Medline]
Kim JI, Jo EJ, Lee HY, Cha MS, Min JK, Choi CH, Lee YM, Choi YA, Baek SH, Ryu SH, et al. (2003) Sphingosine 1-phosphate in amniotic fluid modulates cyclooxygenase-2 expression in human amnion-derived WISH cells. J Biol Chem 278: 31731-31736.
Kimura T, Sato K, Kuwabara A, Tomura H, Ishiwara M, Kobayashi I, Ui M, and Okajima F (2001) Sphingosine 1-phosphate may be a major component of plasma lipoproteins responsible for the cytoprotective actions in human umbilical vein endothelial cells. J Biol Chem 276: 31780-31785.
Kimura T, Sato K, Malchinkhuu E, Tomura H, Tamama K, Kuwabara A, Murakami M, and Okajima F (2003) High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors. Arterioscler Thromb Vasc Biol 23: 1283-1288.
Klemm DJ, Watson PA, Frid MG, Dempsey EC, Schaack J, Colton LA, Nesterova A, Stenmark KR, and Reusch JE (2001) cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration. J Biol Chem 276: 46132-46141.
Kon J, Sato K, Watanabe T, Tomura H, Kuwabara A, Kimura T, Tamama K, Ishizuka T, Murata N, Kanda T, et al. (1999) Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells. J Biol Chem 274: 23940-23947.
Kothapalli D, Fuki I, Ali K, Stewart SA, Zhao L, Yahil R, Kwiatkowski D, Hawthorne EA, FitzGerald GA, Phillips MC, et al. (2004) Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation. J Clin Investig 113: 609-618.[CrossRef][Medline]
Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, and Hla T (1998) Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science (Wash DC) 279: 1552-1555.
Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, and Davis RJ (1993) cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278.[CrossRef][Medline]
Martinez-Gonzalez J, Escudero I, and Badimon L (2004) Simvastatin potentiates PGI2 release induced by HDL in human VSMC: effect on Cox-2 up-regulation and MAPK signalling pathways activated by HDL. Atherosclerosis 174: 305-313.[CrossRef][Medline]
Murata N, Sato K, Kon J, Tomura H, Yanagita M, Kuwabara A, Ui M, and Okajima F (2000) Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions. Biochem J 352: 809-815.
Ohmori T, Yatomi Y, Osada M, Kazama F, Takafuta T, Ikeda H, and Ozaki Y (2003) Sphingosine 1-phosphate induces contraction of coronary artery smooth muscle cells via S1P2. Cardiovasc Res 58: 170-177.
Okajima F (2002) Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator? Biochim Biophys Acta 1582: 132-137.[Medline]
Patel TB, Du Z, Pierre S, Cartin L, and Scholich K (2001) Molecular biological approaches to unravel adenylyl cyclase signaling and function. Gene 269: 13-25.[CrossRef][Medline]
Pettus BJ, Bielawski J, Porcelli AM, Reames DL, Johnson KR, Morrow J, Chalfant CE, Obeid LM, and Hannun YA (2003) The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha. FASEB J 17: 1411-1421.
Pyne NJ, Tolan D, and Pyne S (1997) Bradykinin stimulates cAMP synthesis via mitogen-activated protein kinase-dependent regulation of cytosolic phospholipase A2 and prostaglandin E2 release in airway smooth muscle. Biochem J 328: 689-694.
Robida AM, Xu K, Ellington ML, and Murphy TJ (2000) Cyclosporin A selectively inhibits mitogen-induced cyclooxygenase-2 gene transcription in vascular smooth muscle cells. Mol Pharmacol 58: 701-708.
Ross R (1999) Atherosclerosis-an inflammatory disease. N Engl J Med 340: 115-126.
Saba JD and Hla T (2004) Point-counterpoint of sphingosine 1-phosphate metabolism. Circ Res 94: 724-734.
Sanna MG, Liao J, Jo E, Alfonso C, Ahn MY, Peterson MS, Webb B, Lefebvre S, Chun J, Gray N, et al. (2004) Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate. J Biol Chem 279: 13839-13848.
Sato K, Tomura H, Igarashi Y, Ui M, and Okajima F (1999) Possible involvement of cell surface receptors in sphingosine 1-phosphate-induced activation of extracellular signal-regulated kinase in C6 glioma cells. Mol Pharmacol 55: 126-133.
Spiegel S and Milstien S (2000) Sphingosine-1-phosphate: signaling inside and out. FEBS Lett 476: 55-57.[CrossRef][Medline]
Tamama K, Kon J, Sato K, Tomura H, Kuwabara A, Kimura T, Kanda T, Ohta H, Ui M, Kobayashi I, et al. (2001) Extracellular mechanism through the Edg family of receptors might be responsible for sphingosine-1-phosphate-induced regulation of DNA synthesis and migration of rat aortic smooth-muscle cells. Biochem J 353: 139-146.[CrossRef][Medline]
Tamama K and Okajima F (2002) Sphingosine 1-phosphate signaling in atherosclerosis and vascular biology. Curr Opin Lipidol 13: 489-495.[CrossRef][Medline]
Vinals M, Martinez-Gonzalez J, and Badimon L (1999) Regulatory effects of HDL on smooth muscle cell prostacyclin release. Arterioscler Thromb Vasc Biol 19: 2405-2411.
Weber AA, Zucker TP, Hasse A, Bonisch D, Wittpoth M, and Schror K (1998) Antimitogenic effects of vasodilatory prostaglandins in coronary artery smooth muscle cells. Basic Res Cardiol 93: 54-57.
Xia P, Gamble JR, Rye KA, Wang L, Hii CS, Cockerill P, Khew-Goodall Y, Bert AG, Barter PJ, and Vadas MA (1998) Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway. Proc Natl Acad Sci USA 95: 14196-14201.
This article has been cited by other articles:
|
|
 |
|
  L. Brizuela, M. Rabano, P. Gangoiti, N. Narbona, J. M. Macarulla, M. Trueba, and A. Gomez-Munoz Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways J. Lipid Res., October 1, 2007; 48(10): 2264 - 2274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Damirin, H. Tomura, M. Komachi, J.-P. Liu, C. Mogi, M. Tobo, J.-Q. Wang, T. Kimura, A. Kuwabara, Y. Yamazaki, et al. Role of lipoprotein-associated lysophospholipids in migratory activity of coronary artery smooth muscle cells Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2513 - H2522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Dragusin, S. Wehner, S. Kelly, E. Wang, A. H. Merrill Jr., J. C. Kalff, and G. van Echten-Deckert Effects of sphingosine-1-phosphate and ceramide-1-phosphate on rat intestinal smooth muscle cells: implications for postoperative ileus FASEB J, September 1, 2006; 20(11): 1930 - 1932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. H. Zhang, M. R. Vasko, and G. D. Nicol Intracellular sphingosine 1-phosphate mediates the increased excitability produced by nerve growth factor in rat sensory neurons J. Physiol., August 15, 2006; 575(1): 101 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Skaznik-Wikiel, T. Kaneko-Tarui, A. Kashiwagi, and J. K. Pru Sphingosine-1-Phosphate Receptor Expression and Signaling Correlate with Uterine Prostaglandin-Endoperoxide Synthase 2 Expression and Angiogenesis During Early Pregnancy Biol Reprod, March 1, 2006; 74(3): 569 - 576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tomura, J.-Q. Wang, M. Komachi, A. Damirin, C. Mogi, M. Tobo, J. Kon, N. Misawa, K. Sato, and F. Okajima Prostaglandin I2 Production and cAMP Accumulation in Response to Acidic Extracellular pH through OGR1 in Human Aortic Smooth Muscle Cells J. Biol. Chem., October 14, 2005; 280(41): 34458 - 34464. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
or closed column) or not treated (
or open column) with PTX (100 ng/ml) for 24 h, were incubated for 2.5 min with the indicated concentrations of S1P in A or 1 µM each of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), or prostaglandin I2 (PGI2) in B. The asterisk (*) indicates that the effect of PTX was significant.
, 10 µM) or absence (
