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• W2005244540 abstract "Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. TheSPL 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, sinceN,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. Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. TheSPL 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, sinceN,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. Sphingosine 1-phosphate (S1P) 1The abbreviations used are: S1Psphingosine-1-phosphateEdgendothelial differentiation geneSphsphingosinePLPpyridoxal 5′-phosphateECembryonal carcinomaPrEprimitive endodermRAretinoic acidDab2Disabled-2bt2cAMPdibutyryl cyclic AMPPEparietal endodermSPLS1P lyaseHAhemagglutininSPHK1asphingosine kinase 1aRTreverse transcriptionPBSphosphate-buffered salineDMSN,N-dimethylsphingosineERKextracellular signal-regulated kinaseHPLChigh-performance liquid chromatographycontiggroup of overlapping clones1The abbreviations used are: S1Psphingosine-1-phosphateEdgendothelial differentiation geneSphsphingosinePLPpyridoxal 5′-phosphateECembryonal carcinomaPrEprimitive endodermRAretinoic acidDab2Disabled-2bt2cAMPdibutyryl cyclic AMPPEparietal endodermSPLS1P lyaseHAhemagglutininSPHK1asphingosine kinase 1aRTreverse transcriptionPBSphosphate-buffered salineDMSN,N-dimethylsphingosineERKextracellular signal-regulated kinaseHPLChigh-performance liquid chromatographycontiggroup of overlapping clones 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 (1Igarashi Y. J. Biochem. (Tokyo). 1997; 122: 1080-1087Crossref PubMed Scopus (131) Google Scholar, 2Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 3Spiegel S. Milstien S. FEBS Lett. 2000; 476: 55-57Crossref PubMed Scopus (241) Google Scholar). 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 (2Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 4Spiegel S. Milstien S. Biochim. Biophys. Acta. 2000; 1484: 107-116Crossref PubMed Scopus (186) Google Scholar, 5Hla T. Prostaglandins Other Lipid Mediators. 2001; 64: 135-142Crossref PubMed Scopus (118) Google Scholar). Intracellularly, S1P has been implicated in inositol trisphosphate-independent calcium mobilization, inhibition of caspase activity, and activation of nonreceptor tyrosine kinases (2Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 3Spiegel S. Milstien S. FEBS Lett. 2000; 476: 55-57Crossref PubMed Scopus (241) Google Scholar). sphingosine-1-phosphate endothelial differentiation gene sphingosine pyridoxal 5′-phosphate embryonal carcinoma primitive endoderm retinoic acid Disabled-2 dibutyryl cyclic AMP parietal endoderm S1P lyase hemagglutinin sphingosine kinase 1a reverse transcription phosphate-buffered saline N,N-dimethylsphingosine extracellular signal-regulated kinase high-performance liquid chromatography group of overlapping clones sphingosine-1-phosphate endothelial differentiation gene sphingosine pyridoxal 5′-phosphate embryonal carcinoma primitive endoderm retinoic acid Disabled-2 dibutyryl cyclic AMP parietal endoderm S1P lyase hemagglutinin sphingosine kinase 1a reverse transcription phosphate-buffered saline N,N-dimethylsphingosine extracellular signal-regulated kinase high-performance liquid chromatography group of overlapping clones 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 (6Yatomi Y. Igarashi Y. Yang L. Hisano N. Qi R. Asazuma N. Satoh K. Ozaki Y. Kume S. J. Biochem. (Tokyo). 1997; 121: 969-973Crossref PubMed Scopus (412) Google Scholar); consequently, S1P lyase is thought to play a central role in keeping intracellular S1P levels low. Identical to S1P but lacking the 4,5-transdouble 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 (7Van Brocklyn J.R. Lee M.J. Menzeleev R. Olivera A. Edsall L. Cuvillier O. Thomas D.M. Coopman P.J.P. Thangada S. Liu C.H. Hla T. Spiegel S. J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (446) Google Scholar). 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, andCaenorhabditis elegans, and in mammalian cells (8Saba J.D. Nara F. Bielawska A. Garrett S. Hannun Y.A. J. Biol. Chem. 1997; 272: 26087-26090Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 9Zhou J. Saba J.D. Biochem. Biophys. Res. Commun. 1998; 242: 502-507Crossref PubMed Scopus (152) Google Scholar, 10Li G. Foote C. Alexander S. Alexander H. Development. 2001; 128: 3473-3483PubMed Google Scholar, 11Van Veldhoven P.P. Gijsbers S. Mannaerts G.P. Vermeesch J.R. Brys V. Biochim. Biophys. Acta. 2000; 1487: 128-134Crossref PubMed Scopus (99) Google Scholar). Mutant analyses demonstrated that yeast strains lacking S1P lyase (Bst1p/Dpl1p) exhibit resistance to heat stress and unregulated proliferation upon approaching the stationary phase (12Skrzypek M.S. Nagiec M.M. Lester R.L. Dickson R.C. J. Bacteriol. 1999; 181: 1134-1140Crossref PubMed Google Scholar, 13Gottlieb D. Heideman W. Saba J.D. Mol. Cell. Biol. Res. Commun. 1999; 1: 66-71Crossref PubMed Scopus (63) Google Scholar). The disruption in the S1P lyase gene (sglA) of D. discoideum resulted in a mutant strain with an increased viability during the stationary phase (10Li G. Foote C. Alexander S. Alexander H. Development. 2001; 128: 3473-3483PubMed Google Scholar). 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 (10Li G. Foote C. Alexander S. Alexander H. Development. 2001; 128: 3473-3483PubMed Google Scholar,14Li G. Alexander H. Schneider N. Alexander S. Microbiology. 2000; 146: 2219-2227Crossref PubMed Scopus (72) Google Scholar). 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) (15Strickland S. Mahdavi V. Cell. 1978; 15: 393-403Abstract Full Text PDF PubMed Scopus (1132) Google Scholar). 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 (15Strickland S. Mahdavi V. Cell. 1978; 15: 393-403Abstract Full Text PDF PubMed Scopus (1132) Google Scholar, 16Rickles R.J. Darrow A.L. Strickland S. J. Biol. Chem. 1988; 263: 1563-1569Abstract Full Text PDF PubMed Google Scholar, 17Bjersing J.L. Brorsson A. Heby O. J. Cell. Biochem. 1997; 67: 378-385Crossref PubMed Scopus (8) Google Scholar, 18Duprey P. Morello D. Vasseur M. Babinet C. Condamine H. Brûlet P. Jacob F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8535-8539Crossref PubMed Scopus (86) Google Scholar, 19Cho S.Y. Cho S.Y. Lee S.H. Park S.S. Mol. Cells. 1999; 9: 179-184PubMed Google Scholar). The subsequent addition of dibutyryl cyclic AMP (bt2cAMP) induces further differentiation of PrE cells to parietal endoderm (PE) (20Strickland S. Smith K.K. Marotti K.R. Cell. 1980; 21: 347-355Abstract Full Text PDF PubMed Scopus (592) Google Scholar). Differentiation to PE induces expression of thrombomodulin (21Verheijen M.H. Wolthuis R.M. Bos J.L. Defize L.H. J. Biol. Chem. 1999; 274: 1487-1494Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and the levels of tissue type plasminogen activator, Type IV collagen, and laminin are increased (16Rickles R.J. Darrow A.L. Strickland S. J. Biol. Chem. 1988; 263: 1563-1569Abstract Full Text PDF PubMed Google Scholar,20Strickland S. Smith K.K. Marotti K.R. Cell. 1980; 21: 347-355Abstract Full Text PDF PubMed Scopus (592) Google Scholar). To investigate the role of mouse S1P lyase (SPL) as well as that of intracellular S1P in the differentiation processes, we have generatedSPL knockout F9 cells by homologous recombination. TheSPL−/− 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. 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. 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 (22Jackson R.J. Howell M.T. Kaminski A. Trends Biochem. Sci. 1990; 15: 477-483Abstract Full Text PDF PubMed Scopus (280) Google Scholar), 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 1α promoter. For this construction, the cytomegalovirus immediate early promoter of pCI-neo was first replaced by the human elongation factor 1α 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 theHA-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 theBamHI-EcoRI fragment of pcDNA3-FLAG-SPHK1a (23Murate T. Banno Y., T- Koizumi K. Watanabe K. Mori N. Wada A. Igarashi Y. Takagi A. Kojima T. Asano H. Akao Y. Yoshida S. Saito H. Nozawa Y. J. Histochem. Cytochem. 2001; 49: 845-855Crossref PubMed Scopus (53) Google Scholar) into the BamHI-EcoRI site of pcDNA3-HA1. pCE-puro HA-SPHK1a was then constructed by cloning of theHA-SPHK1a region of pcDNA3 HA-SPHK1a into the pCE-puro plasmid. 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 theloxP site (Fig. 2A). For isolation of F9SPL−/− 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 anSPL−/− 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. 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. 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. 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 (24Van Veldhoven P.P. Methods Enzymol. 2000; 311: 244-254Crossref PubMed Scopus (78) Google Scholar). 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). The Sph kinase assay was performed as described previously (25Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). 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 mmTris-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′)2fragment 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 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. Intracellular amounts of S1P and dihydro-S1P were measured by HPLC as described previously (26Min J.K. Yoo H.S. Lee E.Y. Lee W.J. Lee Y.M. Anal. Biochem. 2002; 303: 167-175Crossref PubMed Scopus (140) Google Scholar). 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). To inactivate theSPL gene, we constructed targeting vectors for homologous recombination. To obtain genomic information about the SPLgene, 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 theSPL 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 ε-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 aSPL+/− 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 SPLallele (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 ofSPL 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 withHA-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 theSPL−/− 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. TheSPL−/− 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). We examined the effect of SPL gene disruption on cell growth and morphology. The growth rate ofSPL−/− 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 (10Li G. Foote C. Alexander S. Alexander H. Development. 2001; 128: 3473-3483PubMed Google Scholar). 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 μmbt2cAMP. Fig. 3Ashows 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 (15Strickland S. Mahdavi V. Cell. 1978; 15: 393-403Abstract Full Text PDF PubMed Scopus (1132) Google Scholar, 20Strickland S. Smith K.K. Marotti K.R. Cell. 1980; 21: 347-355Abstract Full Text PDF PubMed Scopus (592) Google Scholar) (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. It is possible that the accelerated PE differentiation observed in theSPL−/− 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 theSPL−/− cells, using the differentiation-specific marker Dab2, a candidate tumor suppressor of breast and ovarian tumors (27Mok S.C. Wong K.K. Chan R.K. Lau C.C. Tsao S.W. Knapp R.C. Berkowitz R.S. Gynecol. Oncol. 1994; 52: 247-252Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 28Tseng C.P. Ely B.D. Li Y. Pong R.C. Hsieh J.T. Endocrinology. 1998; 139: 3542-3553Crossref PubMed Scopus (82) Google Scholar). A previous study demonstrated that PrE differentiation is accompanied by the expression of two spliced isoforms of Dab2, p96 and p67 (19Cho S.Y. Cho S.Y. Lee S.H. Park S.S. Mol. Cells. 1999; 9: 179-184PubMed Google Scholar, 29Smith E.R. Capo-chichi C.D. He J. Smedberg J.L. Yang D.H. Prowse A.H. Godwin A.K. Hamilton T.C. Xu X.X. J. Biol. Chem. 2001; 276: 47303-47310Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 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 (30Xu X.X. Yi T. Tang B. Lambeth J.D. Oncogene. 1998; 16: 1561-1569Crossref PubMed Scopus (107) Google Scholar). Unlike p96, p67 largely resides in nuclei and may function as a transcriptional co-factor (31Cho S.Y. Jeon J.W. Lee S.H. Park S.S. Biochem. J. 2000; 352: 645-650Crossref PubMed Scopus (23) Google Scholar). We prepared total cell lysates from the" @default.
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• W2005244540 cites W1986898174 @default.
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• W2005244540 cites W1996192399 @default.
• W2005244540 cites W1996200711 @default.
• W2005244540 cites W2008161863 @default.
• W2005244540 cites W2014457420 @default.
• W2005244540 cites W2020555116 @default.
• W2005244540 cites W2020874843 @default.
• W2005244540 cites W2022589671 @default.
• W2005244540 cites W2023166258 @default.
• W2005244540 cites W2023384909 @default.
• W2005244540 cites W2034349845 @default.
• W2005244540 cites W2034441887 @default.
• W2005244540 cites W2055041485 @default.
• W2005244540 cites W2056899647 @default.
• W2005244540 cites W2057365786 @default.
• W2005244540 cites W2066321464 @default.
• W2005244540 cites W2070369144 @default.
• W2005244540 cites W2070547356 @default.
• W2005244540 cites W2071202837 @default.
• W2005244540 cites W2073829786 @default.
• W2005244540 cites W2075468442 @default.
• W2005244540 cites W2077080612 @default.
• W2005244540 cites W2079930884 @default.
• W2005244540 cites W2087008002 @default.
• W2005244540 cites W2110797507 @default.
• W2005244540 cites W2141921458 @default.
• W2005244540 cites W2144506623 @default.
• W2005244540 cites W2150592919 @default.
• W2005244540 cites W2157395014 @default.
• W2005244540 cites W2160917745 @default.
• W2005244540 cites W2163438453 @default.
• W2005244540 cites W2414496502 @default.
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