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Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
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Abstract |
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Introduction |
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S1P functions as a specific ligand for a family of G protein-coupled receptors, termed S1P1–5 (Chun et al. 2002). These differentially expressed receptors couple to a different subset of heterotrimeric G-proteins, which in part determine their distinct roles in cellular responses. On the other hand, considerable evidence also indicates that S1P can act as an intracellular second messenger (Payne et al. 2002). An early clue that S1P may play a role as a second messenger surfaced with the finding that sphingosine derivatives generated inside cells stimulated the release of calcium (Ghosh et al. 1990). In fact, microinjection of S1P into fibroblasts increases DNA synthesis (van Brocklyn et al. 1998) and calcium mobilization from internal stores (Himmel et al. 1998). More recently, studies from our laboratory indicate that SphK2 possesses a nuclear import signal as well as nuclear export signals and shuttles between the nucleus and cytoplasm depending on cellular conditions, i.e. it enters the nucleus under the conditions of high cell density (Igarashi et al. 2003; Ding et al. 2007) or stress such as serum removal (Okada et al. 2005) and stops proliferation or induces apoptosis, suggesting intranuclear action of S1P. However, cytoplasmic or intranuclear targets for S1P remain undefined.
After the observation that platelet-derived growth factor (PDGF)-directed cell motility requires cross-talk from the PDGF receptor to S1P1 receptor via activation of SphK1 and formation of S1P (Hobson et al. 2001), it became clear that this new concept was valid for other systems including allergic reactions (Rosen & Goetzl 2005) and neurotransmitter secretion (Kajimoto et al. 2007) as well. This new paradigm is now accepted as an autocrine/paracrine action or an inside to outside signaling. However, since this paradigm was introduced, it has been unclear whether intracellular S1P has roles distinct from receptor-mediated S1P actions or it is just secreted and exerts S1P receptor-mediated actions in cell migration. Our recent observation that S1P-induced cell migration is strongly inhibited by SphK inhibitors in many cell lines prompted us to dissect S1P signaling into intracellular and receptor-mediated actions of S1P and to re-evaluate the ‘receptor-mediated S1P actions’. In the present studies we have demonstrated that intracellularly generated S1P functions as an intracellular mediator per se to enhance nondirectional cell movement whereas extracellular S1P facilitates the formation of cell polarity thus two pools of S1P enhance directional cell movement in a coordinated fashion.
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Results |
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S1P is known to induce cell migration in a variety of cells through S1P receptors. To dissect cellular signaling in S1P-induced cell migration, the effects of an SphK inhibitor were tested on S1P-induced cell migration in various cell types using a Boyden chamber method (Fig. 1). When L6 cells were treated with an SphK-specific inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (HACPT), S1P-induced cell migration was strongly inhibited. The inhibitory effects of HACPT were not cytotoxic ones because HACPT at the same concentration had no effect on hepatocyte growth factor (HGF)-induced cell migration. Similar inhibition of S1P-induced cell migration by HACPT was observed in COS7 and SH-SY5Y neuroblastoma cells. When 2 µM dimethylsphingosine, a nonspecific inhibitor of SphK, was used S1P-induced cell migration was similarly inhibited (data not shown). Estimation of S1P receptor expression analyzed by quantitative real-time PCR revealed that L6 cells expressed S1P2 receptor exclusively and no other types of the S1P receptors (Fig. 2). To enhance our understanding of mechanisms underlying S1P-mediated cell migration we further characterized S1P actions using L6 cells as a model system.
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Consistent with the observation that S1P2 is the only detectable S1P receptor subtype in L6 cells (Fig. 2), S1P-induced cell migration was almost completely blocked by an S1P2 receptor-specific antagonist JTE013 (Fig. 3). On the other hand, HGF-induced cell migration was insensitive to this blocker. As we already showed that exogenous S1P-induced cell migration was attenuated by HACPT in various cells including L6 cells (Fig. 1), the involvement of SphK in these phenomena was further assessed by studying the effect of SphK downregulation using a gene silencing technique. Because SphK1 is known to be involved in growth factor or chemokine-induced cell migration, e.g. cell migration induced by PDGF (Hobson et al. 2001) and that endogenous SphK2, another subtype of SphK, was localized mainly in the nucleus of L6 cells (data not shown), we focused on the effect of SphK1 down-regulation on S1P-induced cell migration. When SphK1 was down-regulated by SphK1-siRNA, expression of SphK1 mRNA was 40% inhibited compared with control siRNA whereas SphK2 mRNA levels were almost unchanged (Fig. 4A). Consistent with mRNA results SphK1 protein was about 40% down-regulated compared with that in control siRNA-treated cells (Fig. 4B). S1P-induced migration of L6 cells transfected with SphK1-siRNA showed 70% inhibition compared with cells treated with control siRNA (Fig. 4C). This inhibition caused by gene silencing was rescued by the simultaneous expression of mouse SphK1 but not by a catalytically inactive version SphK1G81D, suggesting the importance of catalytic activity of SphK1 and the subsequent generation of S1P. The expression levels of GFP-SphK1 and GFP-SphK1G81D were nearly identical (data not shown).
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Because both intracellular and extracellular S1P have the ability to enhance cell migration (Payne et al. 2002), it is important to clarify which S1P pool is involved in the enhancement of L6 cell migration. Requirement of SphK activity for S1P-induced cell motility (Figs 1,4) suggests that SphK product S1P must be generated after exogenous addition of S1P. To confirm this we measured S1P-induced S1P generation in L6 cells. As expected, exogenously added S1P caused intracellular accumulation of S1P in a dose-dependent manner with the highest intracellular accumulation of S1P being achieved by the concentration of around 10 µM exogenous S1P (Fig. 5A) in agreement with a previous report (Meyer zu Heringdorf et al. 2001). The dose-dependence curve of S1P necessary for cell migration was almost completely paralleled by that for S1P generation curve (Fig. 5B). These results strongly suggest that SphK-catalyzed formation of intracellular S1P may be important for cell migration.
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To address the importance of intracellular S1P in cell migration, S1P was introduced directly into the cells using a liposomal transfer technique. Extensively dialyzed S1P-embedded liposomes were added to the cells and cell migration was analyzed (Fig. 6). Addition of liposomal S1P induced cell migration with a potency similar to that of the exogenous addition of free S1P, although liposomes without S1P showed no effect. This liposomal S1P effect was insensitive to JTE013, whereas the effect of free S1P was completely blocked by the S1P2 antagonist, suggesting that through the liposomal S1P technique S1P indeed acted intracellularly and that this liposomal S1P effect was not mediated by a fractional free S1P present in the liposomal preparations acting through S1P2 receptor. Importantly, liposomal S1P effect was also insensitive to SphK inhibitor HACPT in sharp contrast with the observation that free S1P effect was almost abrogated by the inhibitor (Figs 1,6). These results indicate that intracellular, but not extracellular, S1P has the potency to promote cell migration. This enhancement of cell migration by liposomal S1P was observed in other cell types including COS7 and NIH3T3 cells (data not shown), suggesting that the ability of intracellular S1P to enhance cell migration is a common mechanism utilized by many cell types.
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We showed that exogenous addition of S1P caused enhanced cell migration in a manner inhibited by SphK inhibitors (Fig. 1) or by SphK1 down-regulation by SphK1-targetted gene silencing (Fig. 4) and that introduction of S1P using a liposomal transfer technique has the ability to enhance cell migration (Fig. 6). Next important issue to be clarified is whether intracellular S1P has roles distinct from the receptor-mediated S1P actions in cell migration. To answer these questions, we analyzed the effect of S1P in terms of cell polarity-based movement using a Boyden chamber assay. Cells may move across small pores toward lower chambers in a manner called nondirectional or chemokinetic when S1P is added in both chambers, whereas cells migrate in a directional or chemoattractant manner when S1P is added only in the lower chamber. S1P-induced cell migration was strongly inhibited when S1P was added in both chambers compared with the cell movement when S1P was added only in the lower chamber (Fig. 7), suggesting that extracellular S1P can enhance cell motility depending on S1P concentration gradient. It is reasonable to assume that such S1P gradient may facilitate the formation of cell polarity for movement. Interestingly, when S1P was delivered directly into the cells by a liposomal transfer, this S1P behaved like a chemokinetic factor and enhanced nondirectional cell movement, which was not influenced by the rout of administration (Fig. 7). These results strongly suggest that intracellular S1P per se enhances chemokinesis.
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We have demonstrated that S1P gradient may facilitate the directional cell movement (Fig. 7). To address the molecular mechanisms underlying S1P-induced enhancement of directional cell movement we next studied the effect of S1P on cell polarity formation using a wound-healing assay. Down-regulation of SphK1 in COS7 cells by transient transfection of SphK1-siRNA resulted in a marked reduction of the right cell polarity, whereas control siRNA-transfected cells showed a normal cell polarity with the Golgi facing toward the wounds as assessed by the microscopic examination of the nuclear-Golgi axis (Kupfer et al. 1982) (Fig. 8). To further assess the importance of SphK activity in the formation of cell polarity, the effect of SphK inhibitor HACPT on cell polarity formation after wound treatment was analyzed. HACPT dose-dependently inhibited the right-sided cell polarity formation in a variety of cell types tested so far.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In a Boyden chamber assay S1P enhanced cell movement toward lower chambers when S1P was included in the lower chamber, whereas S1P-induced cell movement was strongly attenuated when S1P was added in both chambers (Fig. 7), indicating that S1P has the ability to enhance directional cell movement depending on the S1P gradient. On the other hand, liposomal introduction of S1P into the cells caused enhancement of chemokinesis or nondirectional cell movement irrespective of the rout of administration (Fig. 7). Assuming that liposomal introduction of S1P into cells mimics intracellularly generated S1P, intracellular actions of S1P may induce nondirectional cell movement. Based on these observations along with the fact of high concentrations (several hundred nanomolar) of S1P in serum (Yatomi et al. 1995), it is quite possible that extracellular S1P may enhance cell migration through the concerted actions of extracellular and intracellular S1P, i.e. the former is involved in the formation of cell polarity, while the latter enhances chemokinetic activity.
What is the mechanism underlying cell polarity formation by SphK/S1P signaling? We and others (Maceyka et al. 2008) have observed that during cell migration SphK1 translocates from the cytoplasm to the membrane-ruffled regions in a variety of cells such as L6, NIH3T3 and HEK293 (see Supporting Fig. 1). This translocation of SphK1 may lead to the production of S1P and subsequent activation of S1P receptors proximal to the ruffled membranes. To support this S1P2-GFP and yellow fluorescent protein (YFP)-SphK1 were well colocalized at membrane-ruffled regions of L6 cells transiently expressing these proteins, suggesting that S1P receptor may be activated specifically at membrane ruffled regions, i.e. areas of leading edge for cell movement (see Supporting Fig. 1). We hypothesized that the activation of S1P receptors at localized membranes may contribute to the formation of cell polarity. This hypothesis was supported by the finding that SphK1 inhibition by HACPT or SphK1 down-regulation by SphK1-siRNA resulted in the reduction of right-sided cell polarity in a variety of cell types as assessed by wound-healing assay (Fig. 8). In addition, transient expression of catalytically inactive SphK1 in COS7 cells resulted in reduced directional persistence ratio using a random migration assay (see Supporting Fig. 2). These results indicate that translocation of SphK1 to some specialized areas of the cells, e.g. membrane ruffled regions, leads to the activation of S1P receptors at the localized areas of the cells, which may be important for the formation of cell polarity.
We have also demonstrated that S1P-induced directional cell movement was strongly inhibited by SphK inhibitor HACPT in a variety of cells as assessed by Boyden chamber method (Fig. 1), suggesting the importance of S1P generation in the cells. This intracellular S1P presumably enhances nondirectional cell movement as measured by Boyden chamber assay (Fig. 7). However we have also observed that liposomal S1P enhanced directional cell movement toward the wound as assessed by wound-healing assay (data not shown). The differences between the effects of liposomal S1P using Boyden chamber assay and wound-healing assay may be as follows: in Boyden chamber assay enhancement of cell motility by liposomal S1P is a cell-polarity-independent phenomenon and reflects increased chemokinetic activities, whereas in the case of wound-healing assay cell polarity may naturally be formed after wound treatment by mechanisms still unknown. Once cell polarity is formed presumably via localized S1P receptor activation, further enhancement of chemokinetic activity through intracellular S1P actions may enhance directional cell movement in total. The molecular mechanisms underlying intracellular actions of S1P in the regulation of cell motility need to be explored.
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Experimental procedures |
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JTE013 was purchased from Cayman Chemical (Ann Arbor, MI). S1P and dimethylsphingosine were obtained from Biomol (Plymouth Meeting, PA). Collagen type I was form BD Biosciences (Heidelberg, Germany). HACPT was from Calbiochem (La Jolla, CA). Anti-SphK1 antibody was prepared as described earlier (Kajimoto et al. 2007).
Plasmid construction
N-terminally green fluorescent protein (GFP)-tagged mouse SphK1 and its catalytically inactive mutant, mSphK1G81D were constructed as described (Igarashi et al. 2003).
Cell cultures
L6 myoblast cells were cultured in alpha-MEM containing 10% fetal calf serum, 100 units/mL penicillin, 100 µg/mL streptomycin, at 37 °C in a humidified 5% CO2 incubator. COS7, HEK293 and SH-SY5Y cells were similarly cultured in Dulbecco's modified Eagle's medium with serum and antibiotics using standard protocols.
siRNA
Small interfering RNAs (siRNAs) for rSphK1 (5'-GGGCAAGGCUCUGAAGCUCdTdT-3' and 5'-GAGCUUCAGAGCCUUGCCCdTdT-3') and control siRNA (5'-UUCUCCGAACGUGUCACGUdTdT-3' and 5'-ACGUGACACGUUCGGAGAAdTdT-3') were synthesized at Japan Bio Services (Saitama, Japan). Cells were transfected both with siRNAs and either empty vector or various expression vector constructs using Lipofectamine2000 (Invitrogen) 2 days before the assays. Transfection efficiency of siRNA was determined by using a commercially available kit (Block-iT Alexa Fluor Red Fluorescent Oligo; Invitrogen).
Cell migration assay
Cell migration was assessed using 24-well Transwell plates (Costar). The insert membranes (8.0 µm pore size) were coated with collagen type I (50 µg/mL in 5% acetic acid) for 2 h. L6 cells were trypsinized and resuspended in alpha-MEM containing 0.1% fatty acid-free bovine serum albumin. The cells were then added to Transwell plate at 5 x 104 cells per insert. The chemotactic agents were added to the lower wells. After 6 h at 37 °C the chamber was disassembled and the top of the filter was scraped to remove nonmigrated cells. The filter was fixed, and stained with DiffQuick (Sysmex, Kobe, Japan). The migratory cells were counted using a microscope with a 10x objective. Each data point is the average number of cells in sixteen random fields and is the mean ± SE of 16 individual wells.
Wound healing assay
COS7 cells were grown to confluence in multi-chamber slides (Nunc) and wounded with a P200 pipette tip. Wounded monolayers were washed three times with growth medium and returned to the incubator to recover from wounding. At 6 h after wounding, cells were treated with 5 µM BODIPY FL-ceramide (Invitrogen) for 30 min to stain Golgi apparatus and fixed with 4% paraformaldehyde. Orientation of the Golgi apparatus relative to the nucleus stained with DAPI was scored. Cells were scored as having a mislocalized Golgi apparatus if Golgi apparatus was outside of a 60° sector from a line bisecting the nucleus and perpendicular to the wound edge (Kupfer et al. 1982). All cells (>100) in four random fields of three separate wounding assays were examined.
Liposomal transfer of S1P
S1P-containing liposomes and control liposomes were prepared as described previously (Brailoiu et al. 2002). Briefly, 76 µL of 1 mM S1P in methanol solution was dissolved in 140 mM KCl (pH 6.9) to obtain 0.833 mL of S1P at a final concentration of 8.7 x 10–5 M. Separately, 50 mg of phosphatidylcholine (Sigma) was dissolved in 2.5 mL of diethylether. These two mixtures (phosphatidylcholine/diethylether and S1P/KCl) were mixed together. After additional vortexing of the emulsion for 5 min, the organic solvents (diethylether and methanol) were evaporated in vacuo using a rotary evaporator at 20 °C. Liposome batches were dialyzed against Ringer's solution (110 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 2.0 mM Tris-HCl (pH 7.2) and 5.6 mM glucose) to remove non-incorporated agent. Control liposomes that did not contain S1P were prepared similarly. Concentration of S1P in the liposome preparations was estimated by the ninhydrin staining on thin layer chromatography plate.
Real-time quantitative reverse transcription-PCR
Total RNA was extracted from L6 cells (2 x 106 cells) using ISOGEN (Nippon gene, Toyama, Japan) according to the manufacturer's instructions. First strand cDNA synthesis and real-time quantitative PCR were carried out as described previously. The primer sequences (sense and antisense) were as follows: rat S1P1, 5'-TTGTTGCAAATGCCCCAACG-3' and 5'-TTTGCTGCGGCTAAATTCCATG-3'; rat S1P2, 5'-TCGCCAAGGTCAAGCTCTACG-3' and 5'-AGACAATTCCAGCCCAGGATGG-3'; rat S1P3, 5'-CACCTGACCATGATCAAGATGAG-3' and 5'-ACCCAGCGAGAAGGCAATTAGC-3'; rat S1P4, 5'-TGTGTATGGCTGCATCGGTCTG-3' and 5'-GAGCACATAGCCCTTGGAGTAG-3'; rat S1P5, 5'-GCTCTACGCCAAGGCCTATGTG-3' and 5'-GCACCTGACAGTAAATCCTTGC-3'; rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5'-TGCCCCCATGTTTGTGATG-3' and 5'-TGTGGTCATGAGCCCTTCC-3'.
S1P determination
L6 cells were plated in 24-well culture plates and to each well was added 160 µL of assay mixture (150 mM NaCl, 100 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM Na3VO4, 10 mM NaF and 0.5 mM 4-deoxypyridoxine), 20 µL of 1 mM sphingosine conjugated with 0.2% fatty acid-free bovine serum albumin, 10 µL of ATP solution (20 mM ATP with [
-32P]ATP (1 µCi/well)), and 10 µL of different concentrations of S1P solution. Cells were incubated at 37 °C for 30 min. The radio labeled S1P was extracted and separated by thin layer chromatography as described previously (Siow & Wattenberg 2007). Radioactivity of S1P was quantitated using a Fujix Bio-imaging Analyzer BAS 2000 (Fuji Photo Film, Japan).
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work. 
aPresent address: Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt.
* Correspondence: snakamur{at}kobe-u.ac.jp |
References |
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Received: 13 January 2009
Accepted: 17 February 2009
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