The intracellular action of sphingosine 1-phosphate in GPVI-mediated Ca2+ mobilization in platelets

Thrombosis Research (2005) 115, 409 — 415

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The intracellular action of sphingosine 1-phosphate in GPVI-mediated Ca2+ mobilization in platelets T. Ohmoria, Y. Yatomib,*, M. Osadaa, Y. Ozakia a

Department of Laboratory Medicine, University of Yamanashi Faculty of Medicine, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan b Department of Laboratory Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Received 8 September 2004; received in revised form 15 October 2004; accepted 18 October 2004 Available online 10 November 2004

KEYWORDS GPVI; Sphingosine 1-phosphate; Sphingosine kinase; Ca2+ mobilization; Platelet

Abstract We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cg2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways. D 2004 Published by Elsevier Ltd.

Introduction Sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by Sph kinase, is a bioactive

Abbreviations: Sph-1-P, sphingosine 1-phosphate; Sph, sphingosine; TRAP, thrombin receptor-activating peptide; MoAb, monoclonal antibody; PLC, phospholipase C; DMS, N,N-dimethylsphingosine; TxA2, thromboxane A2. * Corresponding author. Tel.: +81 3 5800 8730; fax: +81 3 5689 0495. E-mail address: [email protected] (Y. Yatomi). 0049-3848/$ - see front matter D 2004 Published by Elsevier Ltd. doi:10.1016/j.thromres.2004.10.008

lysophospholipid mediator capable of inducing a wide spectrum of biological responses, including cell growth, differentiation, survival, and motility [1,2]. Although it was originally reported that Sph-1-P could serve as an intracellular second messenger, regulating intracellular Ca2+ mobilization and cell growth and survival, recent evidence has indicated that Sph-1-P also acts as an extracellular mediator, interacting with the S1P (also called endothelial differentiation gene (EDG)) family of G protein-coupled receptors [1,2]. Accordingly, Sph-1-P is now considered to

410 be a unique lipid mediator that has dual actions, signaling inside and outside of the cell [1,2]. Blood platelets are unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and to a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation [3,4]. In view of the diverse biological effects of Sph-1-P, Sph-1-P released from activated platelets may act as an intercellular messenger through S1Ps in a variety of (patho)physiological processes [1,2,5,6]. However, despite of the high Sph kinase activity in platelets, the role of Sph-1-P for intracellular signaling pathway in these anucleate cells has not been established. In this study, we examined the signaling role of Sph-1-P, especially the intracellular one in platelets. In contrast with agonists acting on G proteincoupled receptors, convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was found to induce intracellular Ca2+ mobilization through mediation of intracellular Sph-1-P, at least partly. Our results may be worthy of notice because collagen is one of the most important macromolecular constituents of the subendothelial layer, which becomes exposed and accessible to platelets when the integrity of the vascular endothelium is disrupted.

Materials and methods Materials Convulxin, a GPVI-selective agonist [7], was a gift from Dr. T. Morita (Meiji Pharmaceutical University, Tokyo, Japan). The following materials were obtained from the indicated suppliers: U46619 (Calbiochem—Novabiochem, CA); collagen (Hormon-Chemie, Munich, Germany); GRGDS peptide and thrombin receptoractivating peptide (TRAP; Peptide Institute, Osaka, Japan); antiphosphotyrosine monoclonal antibody (MoAb; 4G10; Upstate Biotechnology, Lake Placid, NY); anti-Syk MoAb (Wako, Tokyo, Japan); anti-phospholipase C (PLC) g2 polyclonal antibody (Santa Cruz Biotech., Santa Cruz, CA); antiphosphotyrosine MoAb (PY20; Transduction Laboratories, Lexington, KY); protein A-Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden); derythro-sphingosine (Sph) and N,N-dimethylsphingosine (DMS; Sigma, St. Louis, MO); Sph-1-P (Biomol, Plymouth Meeting, PA); d-erythro[3-3H]Sph (22.0 Ci/mmol; DuPont NEN, Boston, MA).

T. Ohmori et al.

Platelet preparation and aggregation monitoring Platelet-rich plasma obtained from healthy donors was incubated with 1 mM acetylsalicylic acid for 30 min to exclude secondary effects of thromboxane A2 (TxA2). Washed platelets were then prepared as previously described [8]. Platelet aggregation was monitored by measuring light transmission. To eliminate the involvement of integrin aIIbh3-mediated responses and Ca2+ influx, platelets were preincubated with 500 AM GRGDS (a competitive antagonist to inhibit fibrinogen—aIIbh3 interaction) plus 1 mM EGTA for 5 min; platelet aggregates were not formed under these conditions, even after stimulation (data not shown).

Measurement of the intracellular Ca2+ concentration ([Ca2+]i) The [Ca2+]i measurement was performed with the use of Ca2+-sensitive fluorophore fura2 as described previously [9].

Immunoprecipitation and immunoblottings Platelets were lysed in equal amounts of 2ice-cold lysis buffer (100 mM Tris (pH 7.4), 2% Triton X-100, 20 mM NaF, 100 mM NaCl, 6 mM EDTA, 1 mM Na3VO4, 0.5 mM PMSF, and 25 Ag/ml of leupeptin). A part of each lysate was used as a whole cell lysate. The subsequent immunoprecipitation and immunoblotting were performed as described previously [8].

Metabolism of [3H]Sph Washed platelets were incubated with 1 AM (0.2 ACi) [3-3H]Sph. Lipids were extracted, and then analyzed for [ 3 H]sphingolipid metabolism as described previously [10].

In vitro Sph kinase activity Washed platelets (109 cells), after being stimulated, were resuspended with A buffer (20 mM Tris (pH 7.5), 0.5 mM EGTA, 1 mM PMSF). All subsequent steps were carried out at 4 8C. After the extensive sonication (15 sec4), the membrane fraction was isolated by centrifugation at 100,000g for 1 h with HIMAC CS 100FX (Hitachi Koki, Tokyo, Japan). The pellets (the membrane fraction) were washed and resuspended with 1 ml of A buffer. The supernatant was used as the cytosol fraction. Each fraction containing 300 Ag of protein was used for the Sph kinase assay as previously described [11].

The intracellular action of sphingosine 1-phosphate in GPVI-mediated Ca2+ mobilization in platelets

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Statistics When indicated, statistical analysis was performed by Student’s t test, and pb0.05 was considered significant.

Results Sph kinase and its product Sph-1-P have been shown to play an important role in triggering Ca2+ release from intracellular stores [12,13]. We first analyzed whether Sph kinase is indeed involved in intra-

Figure 2 Effects of DMS on convulxin-induced tyrosine phosphorylation. Platelets pretreated without ( ) or with (+) 20 AM DMS were stimulated with 50 ng/ml of convulxin for the indicated durations. Whole cell tyrosine phosphorylation (A) and tyrosine phosphorylation of PLCg2 (B) and Syk (C) were examined as described in Materials and methods. In panel A, molecular weight markers are indicated on the right (in kDa). The data shown are representative of four separate experiments.

Figure 1 Inhibition by DMS of Ca2+ mobilization induced by convulxin, but not U46619, in platelets. (A) Fura2loaded platelets were preincubated with 20 AM DMS for 5 min, and stimulated with 50 ng/ml of convulxin or 1 AM U46619. Changes in [Ca2+]i were monitored by the ratio of fura2 fluorescence. Data are representative of three separate experiments. (B) The peak increases in [Ca2+]i upon stimulation with convulxin (.) or U46619 (o) were calculated and presented as a percentage of the control (without DMS pretreatment). The results are presented as the meanFS.D. (n=3). *Statistically significant ( pb0.05).

cellular calcium mobilization in platelets that possess high Sph kinase activity. We utilized a commonly used Sph kinase inhibitor, DMS [9,14] for this purpose. Sph kinase activity in intact platelets, as evaluated by [3H]Sph conversion into [3H]Sph-1-P [3,9], was abolished by preincubation of the cells with DMS (20 AM) for 5 min (data not shown). Under the conditions, DMS did not affect Ca2+ mobilization induced by U46619, a stable TxA2 analogue, which acts on its G protein-coupled receptor on platelets [15] (Fig. 1). Similar results were obtained by other G protein-stimulating agonists such as TRAP and ADP (data not shown), as we described previously [16]. On the other hand, this DMS treatment partially but significantly inhibited Ca2+ mobilization elicited by convulxin, an agonist of the collagen receptor GPVI [7]; about 30% of Ca2+ mobilization induced by convulxin was inhibited by DMS (Fig. 1). Similar results were obtained in collagen-stimulated platelets (data not shown). These data suggest that Sph kinase activity is at least partially linked to GPVIinduced Ca2+ mobilization.

412 GPVI, a member of the immunoglobulin superfamily, is critically involved in collagen-stimulated platelet activation and noncovalently associated with the FcR g-chain [17,18]. Convulxin activation of platelets through GPVI results from clustering of the receptor and subsequent tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif in FcR g-chain by Src family tyrosine kinases [17,18]. The resultant tyrosine phosphorylation of signaling molecules is essential for GPVImediated platelet activation [17,18]. Accordingly, we next examined whether the inhibitory effect of DMS on convulxin-induced Ca2+ mobilization is due to the inhibition of tyrosine phosphorylation. It was found that preincubation of platelets with DMS failed to inhibit tyrosine phosphorylation elicited by convulxin (Fig. 2A). Furthermore, tyrosine phosphorylations of Syk and PLCg2, which are important for GPVI-mediated Ca2+ mobilization [17,18], were not affected by DMS in immunoprecipitation studies (Fig. 2B,C). These phosphorylations were rather enhanced by preincubation with DMS (Fig. 2), by unknown reasons. These results

T. Ohmori et al. suggest that Sph kinase involvement in Ca 2+ mobilization is independent of tyrosine phosphorylation events in platelet GPVI-mediated signaling pathway. Several biological stimuli, among which growth and survival factors are well-known, activate Sph kinase and thereby increase the intracellular concentration of Sph-1-P [13,19]. We next examined Sph kinase activity in GPVI-mediated platelet activation. When 1 AM [3H]Sph was added exogenously to platelet suspensions, [3H]Sph incorporated into platelets was rapidly converted to [3H]Sph-1-P (Fig. 3A). The conversion of [3H]Sph into [3H]Sph-1-P was not affected by stimulation with collagen or convulxin (Fig. 3A), which is consistent with our previous observation that Sph kinase is highly active in resting platelets and that its total activity remains even after activation [3]. Targeting of signaling proteins to specific sites of the cell is important in the signal transduction mechanism(s), and previous studies demonstrated that translocation of Sph kinase to the membrane fraction is important for FceRI-mediated Ca2+

Figure 3 Sph kinase activity in activated platelets. (A) Platelets incubated with 1 AM (0.2 ACi) [3-3H]Sph were stimulated without (control) or with 50 ng/ml of convulxin or 50 Ag/ml of collagen for the indicated durations. Lipids were extracted from the cells, and then analyzed for [3H]sphingolipid metabolism. The data shown are representative of four separate experiments. (B) Platelets were stimulated without (control) or with 50 ng/ml of convulxin, 50 Ag/ml of collagen, 20 AM TRAP, or 1 AM U46619 for 1 min. The membrane fraction and cytosol fractions were isolated, and Sph kinase activity of each fraction is measured as described in Materials and methods. The membrane Sph kinase activity was calculated as a percentage of the total Sph kinase activity, and the results were expressed as a percentage of control platelets without stimulation (meanFS.D., n=3). *Statistically significant ( pb0.05).

The intracellular action of sphingosine 1-phosphate in GPVI-mediated Ca2+ mobilization in platelets mobilization in human mast cells upon antigen stimulation [20]. We hence investigated the intracellular localization of Sph kinase activity upon platelet activation. Sph kinase activity was present in the cytosol and membrane fractions in unstimulated platelets; about 40% of Sph kinase activity was present in the cytosol fraction (data not shown), as described previously [21]. When platelets were stimulated with TRAP or U46619, Sph kinase activity of each fraction was not altered (Fig. 3B). On the other hand, Sph kinase activity of the membrane fraction increased in convulxin- or collagen-stimulated platelets (Fig. 3B). These data support our idea that Sph kinase is specifically involved in the GPVI-mediated signaling pathway.

Discussion Collagen-induced platelet activation is probably one of the most important and integrated processes of platelet physiology [17,18]. When the integrity of the vascular endothelium is disrupted, a variety of macromolecular constituents of the subendothelial layer become exposed and accessible to platelets; collagen is the most important molecule in this context. It is widely accepted that the interaction between platelets and collagen involves firstly adhesion and subsequently activation, leading to the second-phase platelet activation, including firm adhesion, secretion, and irreversible aggregation [17,18]. Glycoprotein VI (GPVI) is the most important platelet collagen receptor and is critically involved in this first step of platelet activation [17,18]. GPVI-dependent signaling, which has been studied in detail, follows a pathway requiring the associated FcR g-chain and culminates after a series of tyrosine phosphorylation steps in activation of PLCg2 [17,18]. In this study, our data provide a new model of platelet activation via GPVI where Sph kinase might be involved in Ca2+ mobilization. In platelets, Ca2+ mobilization from the intracellular store, originating from phosphoinositide hydrolysis through PLC, has been believed to be essential. Seven PLC isoenzymes belonging to the PLCh, g, and u families have been identified in platelets, the major isoforms being PLCh2, PLCh3, and PLCg2 [22]. Most platelet agonists, which stimulate their own G protein-coupled receptors, including ADP, thrombin, and TxA2, activate PLCh isozymes through Gq [23], while GPVI reportedly tyrosine-phosphorylates and activates PLCg2 [17]. The results presented here add the information of Ca2+ mobilization other than PLC activation in

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platelet signal transduction; Sph kinase at least partially is involved in Ca2+ mobilization induced via GPVI, but not that by G protein-coupled receptor agonists. In platelets, Ca2+ mobilization induced by convulxin was moderately but specifically inhibited by the Sph kinase inhibitor DMS. The inhibition by specific Sph kinase inhibitors of the stimulated increases in intracellular Ca2+ concentration has been reported in other cells. Ca2+ mobilization triggered by FceRI is reportedly dependent on Sph kinase activation in bone marrow-derived mast cells [20] and the rat mast cell line RBL-2H [24]. An important role of Sph kinase for intracellular calcium mobilization has been also described in the high-affinity IgG receptor FcgRI of human myeloid cells [25]. It is worthy of notice that these receptors (including GPVI) all employ FcR g-chain to recruit cytosolic tyrosine kinases for further signal transduction. These facts support the importance of Sph kinase activation and the resultant Sph-1-P generation in mobilization of Ca2+ from internal sources in a PLC-independent—and hence inositol trisphosphate-independent—manner although intracellular targets that are responsible for this effect of Sph-1-P have not been identified yet [1]. As reported in several nucleate cells, Sph kinase activation upon stimulation results in a transient increase in intracellular Sph-1-P, thereby initiating a cell signaling cascade leading to diverse biological processes, including calcium mobilization, cell growth, differentiation, motility, and cytoskeletal reorganization [13,19]. The Sph-1-P-related metabolism in platelets is in contrast with the findings on other cells; Sph kinase is present in platelets as an active enzyme under resting conditions, and its total activity is not changed after cell activation by physiological agonists. However, it is possible that Sph-1-P acts as a pivotal messenger leading to Ca2+ mobilization as a result of translocation of Sph kinase activity, although whether the substrate for Sph kinase, Sph, is available at the plasma membrane after the translocation remains to be determined. Given that Sph formation upon stimulation, reported previously [26], occurred in or close to the plasma membrane concomitant with Sph kinase translocation, it might be possible to postulate that the GPVI activation results in an increase in actual Sph-1-P concentration close to the plasma membrane within platelets. Open questions to be addressed are the mechanisms by which GPVI activation translocates Sph kinase to the membrane fraction in platelets. Rosenfeldt et al. [27] demonstrated an accumulation of Sph kinase at membrane ruffles after PDGF stimulation, suggesting that localized Sph-1-P production aids in directional migration through bind-

414 ing to S1P1 (EDG-1). Other agonist activating Sph kinase could be mediated by PKC-induced translocation of Sph kinase [28]. Translocation of Sph kinase to the plasma membrane in response to Ca2+-mobilizing stimuli occurs in a calmodulindependent manner because it is blocked by the calmodulin inhibitor W-7 [29]. Furthermore, Erk1/ 2-mediated phosphorylation is necessary for translocation of Sph kinase from the cytosol to the plasma membrane [30]. However, these possibilities are remote in platelets because TRAP and U46619, which activate both PLC and Erk1/2, fail to translocate Sph kinase to the membrane fraction. The failure of PLCg2 / platelets to respond to GPVI agonists reveals an absolute need for PLCg2 in a pathway immediately downstream of GPVI [31]. Under the conditions where Sph kinase activity was completely inhibited by DMS, Syk and PLCg2 phosphorylation was not affected. Taken together, GPVI-mediated Sph kinase translocation might be a direct downstream target of PLCg, as is the case with PDGF-mediated Sph kinase activation [32]. Which signaling components are required for its translocation is yet to be elucidated. These studies are now under way in our laboratory. To date, two isoforms of sphingosine kinase (SPHK1 and SPHK2) have been cloned [33,34]. These kinases have homology to the diacylglycerol kinase catalytic site and calmodulin-binding motifs, and do not contain hydrophobic transmembrane sequences [33]. Furthermore, approximately 70% of SPHK1 activity is found in the cytosol. In platelets, multiple forms of Sph kinase activity are reportedly present, and the major part of Sph kinase is associated with the membrane fraction [21]. Although the cytosolic Sph kinase activity of Swiss 3T3 cells is stimulated with acidic phospholipids, including phophatidylserine [35], the platelet membrane-associated Sph kinase is less sensitive to these acidic phospholipids [21]. Furthermore, platelet Sph kinase activity is not changed after platelet activation, but translocates to the plasma membrane only by a selective agonist, as discussed above. These results imply that the difference in Sph kinase characteristics may reflect the existence of platelet-specific sphingosine kinases. Thorough identification of Sph kinases in platelets should lead to the better understanding of the intracellular role of Sph-1-P in activation of these anucleate cells.

Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Educa-

T. Ohmori et al. tion, Culture, Sports, Science and Technology, Japan. The authors are thankful to Prof. Takashi Morita (Meiji Pharmaceutical University, Tokyo, Japan) for the donation of convulxin.

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