Sphingosine-1-phosphate Lyase Is Involved in the Differentiation of F9 Embryonal Carcinoma Cells to Primitive Endoderm*

Akio KiharaDagger , Mika IkedaDagger , Yuki KariyaDagger , Eun-Young Lee§, Yong-Moon Lee§, and Yasuyuki IgarashiDagger

From the Dagger  Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan and the § College of Pharmacy, Chungbuk National University, Chongju 361-763, South Korea

Received for publication, November 8, 2002, and in revised form, February 3, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased ~2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sphingosine 1-phosphate (S1P)1 is a sphingolipid metabolite that functions as both an extracellular and intracellular signaling mediator in regulating diverse biological processes such as proliferation, differentiation, apoptosis, and cell motility (1-3). Extracellular effects of S1P are mediated via members of the endothelial differentiation gene (Edg)/S1P receptor family (Edg1/S1P1, Edg3/S1P3, Edg5/S1P2, Edg6/S1P4, and Edg8/S1P5). These receptors are coupled distinctly (via Gq-, Gi-, G12/13-, and Rho-dependent routes) to multiple downstream signaling pathways including those associated with adenylate cyclase, MAP kinase, phospholipases C and D, c-Jun N-terminal kinase, and nonreceptor tyrosine kinase (2, 4, 5). Intracellularly, S1P has been implicated in inositol trisphosphate-independent calcium mobilization, inhibition of caspase activity, and activation of nonreceptor tyrosine kinases (2, 3).

S1P is formed through the phosphorylation of sphingosine (Sph), catalyzed by Sph kinase. Once formed, S1P is rapidly cleaved by S1P lyase to hexadecenal and phosphoethanolamine or dephosphorylated by S1P phosphohydrolase (Fig. 1). Hence, intracellular S1P levels are determined by the balance of Sph kinase-mediated synthesis and its degradation by S1P lyase or S1P phosphohydrolase. Platelets, which possess high Sph kinase activity and lack S1P lyase activity, accumulate S1P abundantly (6); consequently, S1P lyase is thought to play a central role in keeping intracellular S1P levels low. Identical to S1P but lacking the 4,5-trans double bond, another sphingolipid biosynthesis intermediate dihydrosphingosine (dihydro-Sph) can also be phosphorylated by Sph kinase to dihydrosphingosine-1-phosphate (dihydro-S1P). Dihydro-S1P binds to Edg receptors and activates them, yet does not mimic other effects of S1P such as cell survival (7).


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Fig. 1.   Pathway of sphingolipid metabolism. Shown are the pathways for de novo sphingolipid biosynthesis as well as the sphingolipid-to-glycerolipid conversion. In the sphingolipid biosynthetic pathway, ceramide is converted to sphingomyelin or one of hundreds of glycosphingolipids. Dihydro-Sph, a sphingolipid biosynthesis intermediate, and Sph, a sphingomyelin metabolite, are phosphorylated to dihydro-S1P and S1P, respectively, by Sph kinase. S1P lyase converts dihydro-S1P and S1P to fatty aldehydes (hexadecanal and hexadecenal, respectively) and phosphoethanolamine, all of which then enter the glycerolipid pathways of metabolism.

S1P lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with a conserved pyridoxal-dependent decarboxylase domain positioned at the middle of the protein (Fig. 2A). Recently, S1P lyase has been identified in several organisms including Saccharomyces cerevisiae, Dictyostelium discoideum, and Caenorhabditis elegans, and in mammalian cells (8-11). Mutant analyses demonstrated that yeast strains lacking S1P lyase (Bst1p/Dpl1p) exhibit resistance to heat stress and unregulated proliferation upon approaching the stationary phase (12, 13). The disruption in the S1P lyase gene (sglA) of D. discoideum resulted in a mutant strain with an increased viability during the stationary phase (10). The sglA null mutant also had a strong developmental phenotype and produced aberrant fruiting bodies, with short thick stalks and no obvious apical spore mass (10, 14).


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Fig. 2.   Targeted disruption of the SPL gene. A, structure of the SPL protein and schematic representation of the SPL targeting strategy. TM, PDCD, and IRES, a putative transmembrane segment, a pyridoxal-dependent decarboxylase conserved domain, and an internal ribosome entry site, respectively. Exons 7-14, the primers for genomic PCR (A-D), the loxP sites (closed triangle), the neomycin-resistant gene (Neor), the puromycin-resistant gene (Puror), and internal ribosome entry site (IRES) are indicated. B, PCR amplification of genomic DNA. Genomic DNA isolated from F9 (SPL+/+; lane 1), F9-1 (SPL+/-; lane 2), and F9-2 (SPL-/-; lane 3) cells was subjected to PCR using primers C and D. C, RT-PCR analysis. The levels of SPL transcripts were monitored in F9 (lane 1) and F9-2 (lane 2) cells by RT-PCR as described under "Experimental Procedures." D, immunoblotting analysis. F9 cells were transfected with pCE-puro (vector; lanes 1 and 3) or pCE-puro HA-SPL (lanes 2 and 4), and 24 h later, total lysates were prepared. Fixed amounts of proteins (15 µg) were subjected to immunoblotting using anti-SPL antiserum (lanes 1 and 2) or anti-HA antibodies (lanes 3 and 4). Total proteins (20 µg) prepared from F9 (lane 5), F9-1 (lane 6), and F9-2 (lane 7) cells were separated by SDS-PAGE and detected by immunoblotting with anti-SPL antiserum. The asterisk indicates nonspecific background. E, in vitro S1P lyase assay. Total cell lysates (135 µg of protein) prepared from F9-4 (SPL+/+; lanes 1 and 2) and F9-2 (lanes 3 and 4) cells were incubated with 0.45 µCi of [3H]dihydro-S1P at 37 °C for 0.5 h (lanes 1 and 3) and 2 h (lanes 2 and 4). Lipids were extracted and separated by TLC. HD, hexadecanal. F and G, metabolism of exogenously added [3H]Sph. F9-4 (lane 1) and F9-2 (lane 2) cells in 1 ml of culture medium were treated with 0.8 µCi of [3H]Sph plus cold Sph (total 100 pmol) at 37 °C for 1 h. Lipids were extracted and separated by TLC. In G, quantitative results for the amounts of S1P, ceramide, and sphingomyelin in F9-2 cells (open columns) relative to those in F9-4 cells (closed columns) are shown. Values represent the mean ± S.D. from three independent experiments. CER, ceramide; SM, sphingomyelin. H, measurement of intracellular amounts of S1P (closed columns) and dihydro-S1P (open columns) in F9-2 and F9-4 cells using HPLC. Values represent the mean ± S.D. from three independent experiments.

Mouse F9 embryonal carcinoma (EC) cells are a useful model system for studying the mechanism of endoderm differentiation in mouse early embryogenesis. F9 cells can be induced to differentiate to primitive endoderm (PrE) by the addition of retinoic acid (RA) (15). PrE cells express several specific markers such as tissue plasminogen activator, Type IV collagen, c-jun, cytokeratin ENDO A, and Disabled-2 (Dab2)/DOC-2 (15-19). The subsequent addition of dibutyryl cyclic AMP (bt2cAMP) induces further differentiation of PrE cells to parietal endoderm (PE) (20). Differentiation to PE induces expression of thrombomodulin (21), and the levels of tissue type plasminogen activator, Type IV collagen, and laminin are increased (16, 20).

To investigate the role of mouse S1P lyase (SPL) as well as that of intracellular S1P in the differentiation processes, we have generated SPL knockout F9 cells by homologous recombination. The SPL-/- cells possess no S1P lyase activity and accumulate intracellular S1P and dihydro-S1P. RA-induced PrE differentiation of the SPL-/-cells was accelerated compared with the wild-type cells. Moreover, expression of SPL as well as Sph kinase activity was up-regulated by RA treatment. These results suggest that SPL and intracellular S1P play roles in PrE differentiation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Mouse F9 EC cells were grown in Dulbecco's modified Eagle's medium (D6429; Sigma) containing 10% fetal calf serum supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin in 0.1% gelatin-coated dishes. For differentiation experiments, 1 µM all-trans-RA (Sigma) and 250 µM bt2cAMP (Sigma) were added to the medium.

Plasmids-- The SPL genomic fragments were obtained by PCR using F9 genomic DNA as follows. The SPL genomic regions corresponding to exons 7-9 (2.1 kb), exons 9-11 (2.4 kb), exons 11-12 (0.6 kb), and exons 12-14 (1.8 kb) were amplified using the primers (5'-GGCGTTAGAGAAGGGGATCAAAACTCC-3' and 5'-GCATAGCTGTGTTCCTGGAGATGGC-3', 5'-GCCATCTCCAGGAACACAGCTATGC-3' and 5'-CCAGGCCGTGAGCCAGCTATGCTTGG-3', 5'-CCAAGCATAGCTGGCTCACGGCCTGG-3' and 5'-CGGTAAATGTCAAAATCGTTGGATCCC-3', and 5'-GGGATCCAACGATTTTGACATTTACCG-3' and 5'-CCTGTCAATGGTTGCCTGGGCCATGCC-3', respectively), and these were then connected. The targeting vectors (targeting vector Neo and targeting vector Puro) were constructed by replacing a 105-bp fragment corresponding to the middle of exon 9 to the ScaI site in intron 9 with loxP site-flanked neomycin-resistant gene and puromycin-resistant gene, respectively (Fig. 2A). The internal ribosome entry site of the encephalomyocarditis virus, which permits the translation of two open reading frames from one mRNA (22), was inserted upstream of the loxP site-flanked puromycin-resistant gene in the targeting vector Puro.

pcDNA3-HA1, a derivative of pcDNA3 (Invitrogen), was constructed to create an N-terminal hemagglutinin (HA)-tagged gene. pCE-puro, a derivative of pCI-neo (Promega), was designed for protein expression under the control of the human elongation factor 1alpha promoter. For this construction, the cytomegalovirus immediate early promoter of pCI-neo was first replaced by the human elongation factor 1alpha promoter from pCE-FL (a gift from S. Sugano; Tokyo University) by N. Mizushima (National Institute for Basic Biology, Okazaki, Japan) to generate pCE-neo. Then the neomycin-resistant gene was replaced by the puromycin-resistant gene from pPGKpurobpA to generate pCE-puro.

The SPL cDNA was amplified by PCR using primers 5'-AGATCTCCCGGAACCGACCTCCTCAAGC-3' and 5'-GTGTGCAGTCTGTTCCAAACGCC-3' and F9 total cDNA as a template. The amplified fragments were cloned into pGEM-T Easy (Promega) to generate pGEM-SPL plasmid. The pcDNA3-HA-SPL plasmid, which encodes N-terminally HA-tagged SPL, was constructed by cloning the 1.8 kb of the BglII-NotI fragment of pGEM-SPL into the BamHI-NotI site of pcDNA3-HA1. pCE-puro HA-SPL was then constructed by cloning of the HA-SPL region of pcDNA3-HA-SPL into the pCE-puro plasmid.

The pcDNA3-HA-SPHK1a, which encodes N-terminally HA-tagged mouse Sph kinase 1a (SPHK1a), was constructed by cloning of the 1.2 kb of the BamHI-EcoRI fragment of pcDNA3-FLAG-SPHK1a (23) into the BamHI-EcoRI site of pcDNA3-HA1. pCE-puro HA-SPHK1a was then constructed by cloning of the HA-SPHK1a region of pcDNA3 HA-SPHK1a into the pCE-puro plasmid.

Production of SPL-/- F9 Cells and Stable Transformants-- The linearized targeting vector Neo (1 µg) was transfected into 4 × 105 F9 cells using LipofectAMINETM 2000 reagent (Invitrogen). Cells were subjected to G418 selection at 900 µg/ml for 1 week. Homologous recombination was examined by PCR amplification of genomic DNA using primer A (5'-GTGACTTCTGGGGGAACGGAAAGC-3'), located in exon 7 but outside the genomic sequences present in the targeting vector, and primer B (5'-ATCGGAATTCCTCGAGTCTAGAGCG-3'), located upstream of the loxP site (Fig. 2A). For isolation of F9 SPL-/- cells, the heterozygous clone (F9-1) was transfected with the linearized targeting vector Puro (1 µg). Cells were then cultured in the presence of 0.5 µg/ml puromycin for 8 days. Genomic DNAs were prepared from resistant clones, and homologous recombination was examined by PCR using primer C (5'-GCCATCTCCAGGAACACAGCTATGC-3'), located in exon 9, and primer D (5'-CCAGGCCGTGAGCCAGCTATGCTTGG-3'), located in exon 11. One of the puromycin-resistant clones, F9-2, exhibited an SPL-/- genotype. F9-4 (SPL+/+, neomycin- and puromycin-resistant) cells were obtained in the course of this targeting procedure.

To obtain stable transformants of the HA-SPHK1a gene, the pCE-puro HA-SPHK1a plasmid (1 µg) was transfected into 4 × 105 F9 cells using LipofectAMINETM 2000 reagent. Cells were subjected to puromycin selection at 0.5 µg/ml for 1 week. One of the stable transformants, F9-9, expressed the highest level of HA-SPHK1a among the isolated clones and was used for further analyses.

Reverse Transcription (RT) PCR-- F9 total RNA, isolated using Trizol reagent (Invitrogen), was converted to cDNA using oligo(dT) primer and ProSTARTM first strand RT-PCR kit (Stratagene). The SPL cDNA was amplified by PCR using primer E (5'-CCCGGAACCGACCTCCTCAAGCTGAAGG-3'), primer F (5'-GTGTGCAGTCTGTTCCAAACGCC-3'), and F9 total cDNA as a template.

Antibodies-- Anti-SPL antiserum was raised against recombinant full-length SPL proteins expressed as hexahistidine-tagged fusion proteins. Anti-Dab2, anti-HA Y-11, and anti-actin (A-2066) antibodies were purchased from Transduction Laboratories (Lexington, KY), Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA), and Sigma, respectively.

Measurement of S1P Lyase Activity-- Cells suspended in buffer A (phosphate-buffered saline (PBS), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor mixture (CompleteTM; Roche Molecular Biochemicals), 1 mM dithiothreitol) were lysed by sonication and subjected to in vitro S1P lyase assay as described previously (24). For use as a substrate, [4,5-3H]dihydro-S1P was prepared by phosphorylation of [4,5-3H]dihydro-Sph (50 Ci/mmol; American Radiolabeled Chemical Inc.) using recombinant maltose-binding protein-fused mouse SPHK1a. After the reaction, lipids were extracted by successive additions of a 3.75-fold volume of chloroform/methanol/HCl (100:200:1, v/v/v), a 1.25-fold volume of chloroform, and a 1.25-fold volume of 1% KCl, with mixing. Phases were then separated by centrifugation, and the organic phase was recovered, dried, and suspended in chloroform/methanol (2:1, v/v). The labeled lipids were resolved by TLC on Silica Gel 60 high performance TLC plates (Merck) with 1-butanol/acetic acid/water (3:1:1, v/v/v).

Sph Kinase Assay-- The Sph kinase assay was performed as described previously (25).

Immunoblotting-- Cells were washed with PBS twice, suspended in buffer A, and sonicated. After centrifugation at 300 × g for 5 min at 4 °C, the supernatant was treated with an equal volume of 10% (w/v) trichloroacetic acid and incubated for 20 min at 0 °C. Protein precipitates were washed with acetone and suspended in buffer B (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS). After quantification of protein concentrations using a BCA protein assay kit (Pierce), samples were diluted with equal volumes of 2× SDS sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and a trace amount of bromphenol blue). Proteins were separated by SDS-PAGE and transferred to ImmobilonTM polyvinylidene difluoride membrane (Millipore Corp.). The resulting membrane was incubated with primary antibody (anti-SPL antiserum, anti-Dab2 antibodies, or anti-HA Y-11 antibodies, each diluted 1:1000; or anti-actin antibodies, diluted 1:200) for 1 h and then with secondary antibody (peroxidase-conjugated donkey anti-rabbit IgG F(ab')2 fragment or sheep anti-mouse IgG F(ab')2 fragment, both from Amersham Biosciences, and diluted 1:7500) for 1 h. Labeling was detected by the ECL detection method (Amersham Biosciences).

[3-3H]S1P and [3-3H]Sph Labeling Experiments-- [3-3H]S1P was prepared by phosphorylation of [3-3H]Sph (23.5 Ci/mmol; PerkinElmer Life Sciences) using recombinant maltose-binding protein-fused mouse SPHK1a. Cells at ~70% confluence in six-well dishes were incubated with 1 ml/well medium containing 0.8 µCi of [3-3H]Sph plus cold Sph (total 100 pmol of Sph) or 0.8 µCi of [3-3H]S1P plus cold S1P (total 100 pmol), which had been complexed with 1 mg/ml fatty acid-free bovine serum albumin (Sigma catalog no. A-6003), for 1 h at 37 °C. Lipids were extracted and separated by TLC as described above.

Measurement of Intracellular S1P Level by HPLC-- Intracellular amounts of S1P and dihydro-S1P were measured by HPLC as described previously (26).

Phalloidin Staining-- F9 cells grown on coverslips were fixed with 3.7% formaldehyde in PBS at 37 °C for 10 min, permeabilized with 0.1% Triton X-100 in PBS, blocked with blocking solution (10 mg/ml bovine serum albumin in PBS), and incubated at room temperature for 30 min with Alexa FluorTM 488 phalloidin (Molecular Probes, Inc., Eugene, OR). Cells were washed, mounted with Slow Fade Light Antifade Kit (Molecular Probes), and observed under a fluorescence microscope (Axiophot 2 Plus; Carl Zeiss) with a plane-APOCHROMAT lens (×63) (Carl Zeiss).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of F9 SPL-/- Cells-- To inactivate the SPL gene, we constructed targeting vectors for homologous recombination. To obtain genomic information about the SPL gene, the mouse genome data base MGSCV3 was searched using the BLAST program. We found that the Mus musculus WGS supercontig Mm10_WIFeb01_211 (accession number NW_000027) contains the SPL gene. This sequence data revealed that the SPL gene is located on chromosome 10, and the open reading frame consists of 14 exons. Based on this sequence information, we obtained a SPL genomic fragment that corresponds to exons 7-14. We constructed two targeting vectors in which exon 9 was replaced by a loxP site-flanked neomycin-resistant gene (targeting vector Neo) or a puromycin-resistant gene (targeting vector Puro) (Fig. 2A). We utilized a promoter selection strategy (i.e. selection markers lacking their promoters and transcribed under the control of the SPL promoter only when properly targeted). To facilitate internal translation of the puromycin-resistant gene, we introduced an internal ribosome entry site upstream of the puromycin-resistant gene in the targeting vector Puro. The SPL protein is a PLP-dependent enzyme and contains a conserved pyridoxal-dependent decarboxylase domain positioned from amino acid 167 to amino acid 452 (Fig. 2A). In a PLP-dependent enzyme, PLP exists as a Schiff base with its aldehyde group forming an imine linkage with the epsilon -amino group of a lysine residue. In SPL, Lys-353 is predicted to be the Schiff base-forming residue. Since Lys-353 is encoded within exon 10, the disruption of exon 9 was expected to render the C-terminally truncated SPL completely inactive.

To target the first allele of the SPL gene, F9 cells were transfected with the targeting vector Neo. One of the neomycin-resistant clones, named F9-1, showed a SPL+/- genotype confirmed by PCR (data not shown) using its genomic DNA and primers A and B shown in Fig. 2A. F9-1 cells were then transfected with the targeting vector Puro, and a clone containing the second targeted SPL allele (named F9-2) was isolated. Fig. 2B shows the result of PCR analysis using the genomic DNA, primer C located in exon 9, and primer D located in exon 11. Although only a 2.4-kb fragment was amplified from genomic DNA prepared from F9 cells (Fig. 2B, lane 1), both a 2.4-kb and a 3.8-kb DNA fragment were detected in PCR products from F9-1 cells (Fig. 2B, lane 2). On the other hand, a 3.8-kb fragment and a faint 4.8-kb fragment were amplified from genomic DNA of F9-2 cells, but the 2.4-kb fragment was not detected (Fig. 2B, lane 3). RT-PCR analysis demonstrated the loss of SPL mRNA in F9-2 cells (Fig. 2C).

Next, we prepared an antibody against recombinant full-length mouse SPL to detect the SPL protein. Fig. 2D shows the immunoblotting analysis of the total lysates prepared from F9 cells transfected with HA-SPL cDNA. The anti-SPL antiserum, as well as anti-HA antibodies, revealed that the HA-SPL protein migrated slightly faster (59 kDa) than the predicted molecular mass (65.0 kDa) (Fig. 2D, lanes 2 and 4). The lysates of the vector transfectant did not produce this band (Fig. 2D, lanes 1 and 3). The anti-SPL antiserum also detected the endogenous SPL protein in the lysates of the vector- and HA-SPL cDNA-transfected and untransfected F9 cells as a 58-kDa band (Fig. 2D, lanes 1, 2, and 5). However, the SPL protein was reduced in F9-1 (mSPL+/-) cells and was absent in F9-2 (mSPL-/-) cells (Fig. 2D, lanes 6 and 7). Above all, these results confirmed the proper targeting in F9-2 cells. In the course of the targeting experiments, we also obtained F9 cells resistant to both neomycin and puromycin but carrying an intact SPL gene. We used these cells (F9-4), which always exhibited the same phenotype as original F9 cells, as a wild-type control for further analyses as indicated.

To investigate the S1P lyase activity in the SPL-/- cells, we performed an in vitro S1P lyase assay using total cell lysates and [4,5-3H]dihydro-S1P. Wild-type cell lysates converted dihydro-S1P to hexadecanal in a time-dependent manner (Fig. 2E, lanes 1 and 2), also generating dihydro-Sph by the action of the phosphohydrolase. In contrast, lysates from SPL-/- cells converted dihydro-S1P only to dihydro-Sph and displayed no S1P lyase activity (Fig. 2E, lanes 3 and 4), indicating that SPL is the sole S1P lyase in F9 cells.

We next examined the intracellular accumulation of S1P using exogenously added [3-3H]Sph. After a 1-h incubation with [3-3H]Sph, lipids were extracted and separated by TLC. Wild-type cells accumulated only a small amount of S1P and converted most of the Sph to ceramide and sphingomyelin (Fig. 2F, lane 1). The SPL-/- cells showed an increased accumulation of S1P by 3.4-fold compared with wild-type cells, whereas conversion to ceramide and sphingomyelin in the SPL-/- cells was indistinguishable from that in wild-type cells (Figs. 2, F and G). Next, we measured steady-state levels of S1P and dihydro-S1P using HPLC. The SPL-/- cells showed an increase in intracellular S1P (about 2-fold) and dihydro-S1P (about 2.5-fold) compared with the SPL+/+ cells (Fig. 2H; see also Fig. 7E).

Disrupting the SPL Gene Accelerates the Differentiation of F9 Cells-- We examined the effect of SPL gene disruption on cell growth and morphology. The growth rate of SPL-/- cells was only slightly reduced compared with that of wild-type cells (data not shown). The morphology of SPL-/- cells was indistinguishable from that of wild-type cells (data not shown). Recently, it was reported that S1P lyase has a central role in the development of D. discoideum (10). With this in mind, we investigated the role of SPL in the differentiation of F9 cells to PrE and, subsequently, PE in the presence of 1 µM RA and 250 µM bt2cAMP. Fig. 3A shows phase-contrast images and phalloidin-staining patterns of EC, PrE, and PE cells. PrE cells manifest an enlarged and flattened morphology, whereas PE cells exhibit rounded shapes with long cell processes (15, 20) (Fig. 3A). By day 3 of treatment, all of the wild-type cells had differentiated to a cell type with typical PrE morphology (Fig. 3B), whereas almost no cells (<1%) had differentiated to PE. PE morphology was observed within small areas of the culture at day 4 (5%), and about 78% of the cells had differentiated to PE at day 5 (Fig. 3B). In contrast, SPL-/- cells that had differentiated to exhibit PE morphology could be detected even at day 3 (21%) (Fig. 3B). Moreover, most of the SPL-/- cells by day 4 (83%) and day 5 (95%) had differentiated to PE (Fig. 3B), further establishing that differentiation to PE was accelerated by the disruption of the SPL gene.


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Fig. 3.   Effects of SPL knockout on differentiation. A, morphology of F9 EC, PrE, and PE cells. F9 EC cells were differentiated to PrE cells and PE cells by incubation with 1 µM RA and by incubation with 1 µM RA and 250 µM bt2cAMP, respectively, for 5 days. Cells were then fixed, permeabilized, and stained with phalloidin to visualize F-actin. Left panels, phase-contrast images; right panels, phalloidin-staining. B, kinetics of differentiation of F9-4 (SPL+/+) and F9-2 (SPL-/-) cells. Cells at a density of 400 cells/cm2 were incubated with 1 µM RA and 250 µM bt2cAMP for the indicated time periods. Cells were photographed under a phase-contrast microscope at ×200 magnification. The arrows indicate PE-differentiated cells. C, expression of Dab2 is up-regulated in SPL-/- cells. F9-2 (lanes 1, 3, 5, 7, 9, 11, and 13) and F9-4 (lanes 2, 4, 6, 8, 10, 12, and 14) cells were cultured in the presence of 1 µM RA and 250 µM bt2cAMP for the indicated time periods. Total proteins (10 µg) were separated by SDS-PAGE, followed by immunoblotting with anti-Dab2 antibodies or, to demonstrate uniform protein loading, anti-actin antibodies.

It is possible that the accelerated PE differentiation observed in the SPL-/- cells was due to acceleration from EC cells to PrE cells, acceleration from PrE cells to PE cells, or both. To distinguish between these possibilities, we next examined whether differentiation from EC cells to PrE cells was accelerated in the SPL-/- cells, using the differentiation-specific marker Dab2, a candidate tumor suppressor of breast and ovarian tumors (27, 28). A previous study demonstrated that PrE differentiation is accompanied by the expression of two spliced isoforms of Dab2, p96 and p67 (19, 29). p96 binds to the Src homology 3 domains of Grb2 and may function to modulate Ras pathways by competing with Sos for binding to Grb2 (30). Unlike p96, p67 largely resides in nuclei and may function as a transcriptional co-factor (31). We prepared total cell lysates from the wild-type cells and the SPL-/- cells treated with RA and bt2cAMP for 0-6 days and examined the expression levels of Dab2 (Fig. 3C). Although both p96 and p67 appeared only at day 3 in the wild-type cells (Fig. 3C, lane 8), they could both be detected even at day 2 in the SPL-/- cells (Fig. 3C, lane 5). Moreover, expression of Dab2 was up-regulated in SPL-/- cells, compared with that in the wild-type cells, at day 3 (Fig. 3C, lanes 7 and 8). We also obtained similar results when these cells were incubated with only RA, which induces differentiation of F9 EC cells to PrE cells but not further into PE cells (data not shown). These results confirmed that the differentiation of EC cells to PrE cells was accelerated in SPL-/- cells.

Next, we investigated whether differentiation of PrE cells to PE is also enhanced by disruption of the SPL gene. For this purpose, we first cultured both the wild-type and the SPL-/- cells in medium containing only RA for 5 days to allow them to differentiate to PrE, and then bt2cAMP was added to the cultures. Within 1 day after the addition of bt2cAMP, 50% of the wild-type cells had differentiated to PE (Fig. 4, left upper panel). More importantly, the SPL-/- cells had also differentiated to PE at a frequency similar to the wild-type cells (48%) (Fig. 4, right upper panel), indicating that SPL is not involved in the differentiation of PrE cells to PE.


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Fig. 4.   Disruption of SPL has no effect on differentiation of PrE cells to PE cells. F9-4 (SPL+/+; left panels) and F9-2 (SPL-/-; right panels) cells were first differentiated to PrE cells by incubation with 1 µM RA for 5 days. These cells were then treated with 1 µM RA and 250 µM bt2cAMP for 1 day in the absence (upper panels) or presence of 1.5 µM DMS (lower panels). Cells were photographed under a phase-contrast microscope at ×200 magnification. The arrows indicate PE-differentiated cells.

Disruption of the SPL gene has two effects: the accumulation of S1P (and dihydro-S1P) and the cessation of the sphingolipid-to-glycerolipid pathway. To examine which of these is the cause of the accelerated differentiation, we utilized a Sph kinase inhibitor, N,N-dimethylsphingosine (DMS). Given that S1P exhibited a positive effect on the differentiation, it would be expected that DMS would inhibit the differentiation. In contrast, if one of the S1P metabolites such as hexadecanal or phosphoethanolamine exerts a negative effect on the differentiation, DMS may stimulate the differentiation in wild-type cells and may have no effect on the differentiation of the SPL-/- cells. We cultured the wild-type and the SPL-/- cells in the presence of RA and bt2cAMP with or without DMS. Since high concentrations of DMS can inhibit other protein kinases (32), we used DMS at low concentrations (1-2 µM). Within this range, DMS cannot inhibit Sph kinase activity completely (33), yet even 1.5 µM DMS had an inhibitory effect on the differentiation (Fig. 5A). Although in the absence of DMS 80% of the SPL-/- cells differentiated to PE by day 4, those incubated with DMS differentiated at a very low frequency (3%) (Fig. 5A, upper panels). Similar results were obtained from experiments using wild-type cells. Although the wild-type cells differentiated to PE efficiently by day 5 (82%) in the absence of DMS, most still exhibited PrE morphology in the presence of DMS at day 5 (PE cells, 1%) (Fig. 4A, lower panels). We also investigated the effect of DMS on the expression of Dab2. Both the p96 and p67 forms of Dab2 were reduced by treatment with DMS in a dose-dependent manner (Fig. 5B) In contrast, DMS had no effect on differentiation of already PrE-differentiated wild-type and SPL-/- cells to PE (Fig. 4, lower panels).


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Fig. 5.   S1P plays a role in the PrE differentiation. A, F9-2 (SPL-/-; upper panels) and F9-4 (SPL+/+; lower panels) cells, each at a density of 400 cells/cm2, were incubated with 1 µM RA and 250 µM bt2cAMP for the indicated time periods in the absence (left panels) or presence of 1.5 µM DMS (right panels). Cells were photographed under a phase-contrast microscope at ×200 magnification. The arrows indicate PE-differentiated cells. B, expression of Dab2 is inhibited by DMS. F9-2 (lanes 1-4) and F9-4 (lanes 5-8) cells at concentrations of 12,000 cells/cm2 and 3,000 cells/cm2, respectively, were treated with 1 µM RA, 250 µM bt2cAMP, and 0 µM (lanes 1 and 5), 1 µM (lanes 2 and 6), 1.5 µM (lanes 3 and 7), or 2 µM DMS (lanes 4 and 8) and cultured for the indicated time periods. Total proteins (15 µg) were separated by SDS-PAGE, followed by immunoblotting with anti-Dab2 antibodies. C, PrE differentiation is accelerated by overexpression of Sph kinase. F9 (lanes 1 and 3) and F9-9 (HA-SPHK1a stable transformants; lanes 2 and 4) cells, each at a concentration of 12,000 cells/cm2, were incubated with 1 µM RA and 250 µM bt2cAMP for 2 days, and lysates were prepared. Total proteins (15 µg) were separated by SDS-PAGE, followed by immunoblotting with anti-HA (lanes 1 and 2) or anti-Dab2 (lanes 3 and 4) antibodies.

The above results suggested that S1P (dihydro-S1P), but not its metabolites, plays a role in the EC-to-PrE differentiation. To confirm this, we investigated the effects of overproduction of Sph kinase on the differentiation. Previous study has revealed that overexpression of Sph kinase results in an increase in intracellular amounts of S1P (34). We established F9 clones (F9-9) stably expressing HA-tagged mouse SPHK1a. We detected HA-SPHK1a in the lysates of F9-9 cells by immunoblotting with anti-HA antibodies (Fig. 5C, lane 2). Then we investigated the differentiation status of the cells by immunoblotting with anti-Dab2 antibodies. F9-9 cells treated with RA and bt2cAMP for 2 days expressed an increased amount of Dab2 compared with the control F9 cells (Fig. 5C, lanes 3 and 4). Thus, PrE differentiation was accelerated in the presence of higher levels of S1P, whether they resulted from SPL disruption or overexpression of Sph kinase.

S1P can act both extracellularly, via Edg/S1P family receptors, and intracellularly. Therefore, the possibility that S1P accumulated in the SPL-/- cells is released into the medium and is acting extracellularly in an autocrine or paracrine fashion cannot be excluded. To distinguish whether S1P stimulates the differentiation intracellularly or extracellularly, we investigated the effects of exogenously added S1P or dihydro-S1P on the F9 differentiation. The wild-type and SPL-/- cells were incubated with S1P or dihydro-S1P at 0.1 or 1 µM for 2 days. Then total cell lysates were prepared and subjected to immunoblotting using anti-Dab2 antibodies. As shown in Fig. 6A, neither S1P nor dihydro-S1P treatment enhanced the expression of Dab2 in either cells; rather, these compounds were slightly inhibitory at 1 µM. Moreover, the appearance of PE morphology was also unchanged by S1P or dihydro-S1P (data not shown). Thus, exogenous S1P or dihydro-S1P did not induce differentiation, indicating that the lipids act intracellularly to stimulate the differentiation.


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Fig. 6.   Effects of exogenous S1P and dihydro-S1P on differentiation. A, F9-4 (SPL+/+; lanes 1-5) and F9-2 (SPL-/-; lanes 6-10) cells, each at a concentration of 12,000 cells/cm2, were treated with 1 µM RA, 250 µM bt2cAMP, and 0 µM S1P (lanes 1 and 6), 0.1 µM S1P (lanes 2 and 7), 1 µM S1P (lanes 3 and 8), 0.1 µM dihydro-S1P (lanes 4 and 9), or 1 µM dihydro-S1P (lanes 5 and 10) and incubated for 2 days. Total proteins (15 µg) were separated by SDS-PAGE and detected by immunoblotting with anti-Dab2 antibodies. B, F9 cells in 1 ml of culture medium were treated with 100 pmol of Sph containing 0.8 µCi of [3H]Sph (lane 1) or with 100 pmol of S1P containing 0.8 µCi of [3H] S1P (lanes 2 and 3) at 37 °C for 1 h. Lipids were extracted, separated by TLC, and visualized by autoradiography. Lane 3 represents a longer autoradiography exposure of lane 2. CER, ceramide; SM, sphingomyelin.

To examine whether extracellular S1P can be imported into the cell, thereby causing an accumulation of intracellular S1P, we next performed a [3H]S1P labeling experiment. Although [3H]Sph was rapidly imported into the cells and converted to ceramide and sphingomyelin (Fig. 6B, lane 1), the labeling by [3H]S1P was very inefficient, and S1P was not detected (Fig. 6B, lane 2). A longer autoradiography exposure (Fig. 6B, lane 3) revealed a pattern of metabolism for S1P similar to that for Sph.

Up-regulation of SPL and Sph Kinase Activities during PrE Differentiation of F9 Cells-- We next examined whether SPL expression is regulated during differentiation. F9 cells were cultured in the presence of RA and bt2cAMP for 0-6 days, and SPL protein levels were measured in the cell lysates by immunoblotting. We found that SPL increased in a time-dependent manner and reached maximal level at 5 days (Fig. 7A). Cells incubated with RA alone for 5 days (PrE cells) accumulated the same amount of SPL compared with cells incubated with RA and bt2cAMP together (PE cells) (Fig. 7B), indicating that SPL expression is up-regulated during differentiation of EC cells to PrE. Consistent with these observed increases in SPL levels, an in vitro S1P lyase assay using total cell lysates further demonstrated that the S1P lyase activity of PrE cells was about 4-fold higher than that of EC cells (data not shown).


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Fig. 7.   Expression of the SPL protein and Sph kinase activity are up-regulated during differentiation to PrE cells. A, kinetics of intracellular SPL accumulation during differentiation. F9-4 (SPL+/+) cells were cultured in the presence of 1 µM RA and 250 µM bt2cAMP for 0 (lane 1), 1 (lane 2), 2 (lane 3), 3 (lane 4), 4 (lane 5), 5 (lane 6), and 6 (lane 7) days. Total proteins (10 µg) were separated by SDS-PAGE, followed by immunoblotting with anti-SPL antiserum or, to demonstrate uniform protein loading, anti-actin antibodies. B, expression levels of the SPL protein in different stages of differentiation. F9 cells were incubated with no agents (lane 1) or with 1 µM RA (lane 2) or 1 µM RA and 250 µM bt2cAMP (lane 3) for 5 days. Total proteins (15 µg) were separated by SDS-PAGE and detected by immunoblotting with anti-SPL antiserum. C, Sph kinase assay. F9-4 (SPL+/+) and F9-2 (SPL-/-) cells were differentiated to PrE cells by incubation with 1 µM RA for 6 days. Total cell lysates were prepared from undifferentiated (EC) and PrE-differentiated cells, and 50 µg of protein were subjected to an in vitro Sph kinase assay using 50 µM D-erythro-Sph, [gamma -32P]ATP, and 1 mM cold ATP for 15 min at 37 °C. Lipids were separated by TLC, and radioactivities associated with S1P were quantified using a PhosphorImager BAS2000 (Fuji Film). Values are illustrated relative to the Sph kinase activity associated with F9-4 EC cells (0.625 ± 0.076 pmol/mg/min) and represent the mean ± S.D. from three independent experiments. SK, Sph kinase. D, metabolism of exogenous Sph in EC and PrE cells. F9-4 (lanes 1 and 2) and F9-2 (lanes 3 and 4) cells incubated with mock agent (lanes 1 and 3) or 1 µM RA for 6 days were treated with 0.8 µCi of [3H]Sph plus cold Sph (total 100 pmol) at 37 °C for 1 h. Lipids were extracted and separated by TLC. CER, ceramide; SM, sphingomyelin. E, measurement of intracellular amounts of S1P using HPLC. F9-4 and F9-2 cells incubated with mock agent (closed columns) or 1 µM RA (open columns) for 6 days were subjected to HPLC analysis. Values represent the mean ± S.D. from three independent experiments.

Although intracellular S1P levels were only about 2-fold higher in the SPL-/- cells than in wild-type cells (Fig. 2H), significant acceleration was observed in the differentiation of the SPL-/- cells (Fig. 3). This observation led us to consider the possibility that differences in intracellular S1P levels between the wild-type and SPL-/- cells may be even greater after differentiation. To investigate this possibility, we first measured Sph kinase activity in F9 EC and RA-induced PrE cells. We found that PrE cells possessed 2.7-fold higher Sph kinase activity than EC cells (Fig. 7C). A similar increase in Sph kinase activity was observed in the SPL-/- cells (Fig. 7C). Thus, Sph kinase activity is also up-regulated during PrE differentiation. Next, we investigated intracellular S1P accumulation using [3H]Sph labeling. The wild-type and SPL-/- cells were incubated with [3H]Sph for 1 h, and labeled membrane was separated by TLC. The PrE-differentiated wild-type cells accumulated only low amounts of S1P, nearly the same levels as in the undifferentiated cells (Fig. 7D, lanes 1 and 2). In contrast, the S1P levels increased 3.9-fold during the differentiation of the SPL-/- cells to PrE cells (Fig. 7D, lanes 3 and 4). Next, we also investigated intracellular S1P levels using HPLC. Again, the S1P levels were unchanged in the wild-type cells during the differentiation, whereas a significant increase in the S1P levels was observed in the SPL-/- cells (Fig. 7E). These results indicated that the intracellular amounts of S1P are regulated by not only synthesis but also degradation during PrE differentiation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we constructed a SPL-deficient F9 cell line. Until now, disruptants of the S1P lyase gene were generated only in S. cerevisiae and D. discoideum (10, 12-14). Thus, this is the first report of gene disruption of mammalian S1P lyase. The SPL-/- F9 cells constructed in this study had no S1P lyase activity (Fig. 2E), indicating that the SPL protein is the sole S1P lyase, at least in F9 cells and, most likely, in mammalian cells. Since S1P is stored in high concentrations in platelets, which lack S1P lyase activity but have S1P phosphohydrolase activity, it is likely that S1P lyase has a central role in clearing intracellular S1P. A previous study demonstrated that a 500-fold overproduction of the Sph kinase SPHK1a resulted in only a 4-8-fold increase in S1P levels (34). One possibility for these moderate increases is that degradation by SPL keeps intracellular S1P levels low. Consistent with this theory, a 2.6-fold increase in Sph kinase activity during PrE differentiation resulted in a 1.7-fold increase in S1P levels in the SPL-/- cells but not in the wild-type cells (Fig. 7E).

Disruption of the sglA gene, which encodes S1P lyase in D. discoideum, affects multiple stages throughout development, including the ability to form migrating slugs, the control of cell type-specific gene expression, and terminal spore differentiation (10). Here we demonstrated that the mammalian S1P lyase, SPL, is also involved in the differentiation of F9 cells. Disruption of the SPL gene resulted in accelerated differentiation to PrE cells (Fig. 3). Expression of the PrE-specific marker Dab2 was observed earlier and was enhanced in the SPL-/- cells (Fig. 3C). This situation is similar to that observed in sglA mutant cells, which proceed through early development slightly faster than wild-type cells (10). Moreover, expression of contact site A, which is expressed at the onset of development, occurred slightly earlier and was extended longer in the mutant (10). Thus, it seems that the function of S1P lyase in early development is conserved.

There are two plausible accounts for the effect of SPL knockout on promoting differentiation. Since S1P lyase catalyzes the conversion of S1P (dihydro-S1P) to fatty aldehyde and phosphoethanolamine, both of which are used as glycerolipid precursors, inactivation of S1P lyase leads to both the accumulation of S1P and the loss of its products (i.e. the shutdown of the cross-talk between sphingolipids and glycerolipids). To examine which of these is the cause of the accelerated differentiation, we utilized an inhibitor of Sph kinase (DMS), which diminishes both intracellular S1P and its degradation products. Treatment of cells with low concentrations of DMS efficiently inhibited the PrE differentiation (Fig. 5, A and B). Moreover, overexpression of Sph kinase also resulted in an acceleration of the differentiation (Fig. 5C). These results support the conclusion that accumulation of S1P rather than shutdown of the sphingolipid-to-glycerolipid pathway is responsible for the effect of SPL disruption.

S1P can act extracellularly by binding to members of the Edg/S1P family of G protein-coupled receptors. Our RT-PCR results showed that F9 EC cells express Edg1/S1P1, Edg5/S1P2, and Edg6/S1P4 but not Edg3/S1P3.2 A previous study demonstrated that Edg5/S1P2 is down-regulated during RA- and bt2cAMP-induced differentiation (35). The authors proposed a role for Edg5/S1P2 in maintaining the stem cell phenotype of undifferentiated F9 cells. Our results presented here revealed that exogenously added S1P or dihydro-S1P does not induce the differentiation but rather slightly inhibits it. Therefore, we suppose that S1P signaling mediated by Edg receptors may be inhibitory to the differentiation. We also demonstrated that exogenously added Sph did not accelerate the differentiation either (data not shown). As shown in Figs. 2F and 6B, conversion of exogenous Sph to S1P is only <1%. Most Sph was converted to other sphingolipids. Taking into account the fact that other sphingolipids such as ceramide and Sph are also signaling molecules, the effects of exogenous Sph cannot be attributed only to the effects of S1P. Our results using 3H-labeled S1P demonstrated that exogenously added S1P did not result in an increase in intracellular amounts of S1P (Fig. 6B). The pattern of metabolism of S1P was similar to that of Sph, and the signals of metabolites of S1P were weaker than those of Sph (Fig. 6B). Therefore, we suppose that F9 can import S1P only through conversion to Sph by the phosphatase on the cell surface.

There is abundant evidence that S1P can also function intracellularly in calcium homeostasis, inhibition of apoptosis, and cell growth (36-38). The level of S1P is very low in a steady-state system and is rapidly increased by the activation of Sph kinase induced by numerous external stimuli including platelet-derived growth factor (39), tumor necrosis factor alpha  (40), nerve growth factor (41), activation of protein kinase C (42), and cross-linking of the immunoglobulin receptors Fcepsilon RI (43) and Fcgamma R1 (44). Thus, S1P appears to function as a second messenger. However, molecular mechanisms of intracellular S1P actions are largely unknown. Therefore, it is also unclear how the accumulated S1P causes the accelerated differentiation of F9 cells. Previous studies demonstrated that RA-induced differentiation of F9 cells to PrE cells causes activation of Ras and a decline in Gialpha 2 levels, both of which lead to activation of ERK (21, 45). Moreover, transfection of the constitutively active form of mitogen-activated protein kinase kinase, MEK, induced PrE differentiation of F9 cells without RA treatment (21). Although an ERK-independent pathway in RA-induced PrE differentiation has been reported (21), antisense disruption of p42 ERK abolished PrE differentiation (45). Thus, ERK seems to play an important role in RA-induced progression of F9 EC cells to PrE. We investigated the activation of ERK in wild-type and SPL-/- cells treated with RA and found no difference between them (data not shown), indicating that ERK is not involved in the promotional effect of S1P on differentiation. Therefore, further work will be needed to determine the precise molecular mechanism of the role of S1P in this differentiation as well as any intracellular S1P signaling pathway. The SPL-/- cells constructed here can be used as a new tool in investigating the role of intracellular S1P.

    ACKNOWLEDGEMENTS

We thank N. Mizushima for the pCE-neo plasmid and useful advice, S. Sugano for the pCE-FL plasmid, A. Wada (this laboratory) for pcDNA3-HA1 and pcDNA3-HA-SPHK1a plasmids, and Chie Ogawa (this laboratory) for technical support.

    FOOTNOTES

* This work was supported in part by a Grant-in-aid for Scientific Research on Priority Areas (B) 12140201 from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Ono Pharmaceutical Co., Inc.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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan. Tel.: 81-11-706-3970; Fax: 81-11-706-4986; E-mail: yigarash@pharm.hokudai.ac.jp.

Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M211416200

2 A. Kihara, C. Ogawa, and Y. Igarashi, unpublished results.

    ABBREVIATIONS

The abbreviations used are: S1P, sphingosine-1-phosphate; Edg, endothelial differentiation gene; Sph, sphingosine; PLP, pyridoxal 5'-phosphate; EC, embryonal carcinoma; PrE, primitive endoderm; RA, retinoic acid; Dab2, Disabled-2; bt2cAMP, dibutyryl cyclic AMP; PE, parietal endoderm; SPL, S1P lyase; HA, hemagglutinin; SPHK1a, sphingosine kinase 1a; RT, reverse transcription; PBS, phosphate-buffered saline; DMS, N,N-dimethylsphingosine; ERK, extracellular signal-regulated kinase; HPLC, high-performance liquid chromatography; contig, group of overlapping clones.

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