Mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets

• DOI: 10.1194/jlr.m500268-jlr200
• Publication Date: 2005-08-02T00:26:43Z
• Authors: Motohiro Tani; Takamitsu Sano; Makoto Ito; Yasuyuki Igarashi
• Keywords: Sphingolipid; Sphingosine Kinase

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high (Sph) kinase activity, lack the de novo sphingolipid biosynthesis necessary to provide substrates. Here, we reveal a generation pathway for Sph, precursor of S1P, human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than megakaryoblastic cells rapidly converted it S1P. Furthermore, formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) neutral ceramidase (CDase) was into suggesting that use as source express SM, possibly supplied lipoproteins, at cell surface. Treating with SMase resulted surface, conceivably action membrane-bound CDase. Simultaneously, time-dependent increase S1P levels observed. Finally, demonstrated secretory acid also induces increases platelets.In conclusion, our results suggest platelets, least two sources: followed incorporation, outer leaflet membrane, initiated surface SM degradation. In Sphingolipid metabolites been implicated modulators membrane signal transduction systems shown involved diverse cellular processes (1Merrill Jr, A.H. De biosynthesis: necessary, but dangerous, pathway.J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text PDF PubMed Scopus (496) Google Scholar, 2Pettus B.J. Chalfant C.E. Hannun Y.A. Ceramide apoptosis: an overview current perspectives.Biochim. Biophys. Acta. 1585: 114-125Crossref (675) 3Cuvillier O. Sphingosine apoptosis signaling.Biochim. 153-162Crossref (290) 4Hla T. Signaling biological actions 1-phosphate.Pharmacol. Res. 2003; 47: 401-407Crossref (241) Scholar). Generally, (SM), which distributed mainly on exoplasmic side broken down signal-activated sphingomyelinases (SMases), leading formation ceramide (Cer), important signaling functions (2Pettus Subsequently, Cer deacylated generate (Sph); because not produced synthesis, its restricted this (5Wang E. Norred W.P. Bacon C.W. Riley T.R. H. Merrill A. Inhibition fumonisins. Implications diseases associated Fusarium moniliforme.J. 1991; 266: 14486-14490Abstract Scholar), referred here "SM degradation pathway." final step, occurs cytoplasmic region cells, phosphorylated (S1P). has attracted great deal attention key molecule, functioning both intracellular second messenger ligand five G protein-coupled receptors, endothelial differentiation gene now named S1P1 S1P5 (4Hla With such multiple pleiotropic targets, influences numerous functions, including vascular maturation during development (6Liu Y. Wada R. Yamashita Mi Deng C-X. Hobson J.P. Rosenfeldt H.M. Nava V.E. Chae S-S. Lee M-J. et al.Edg-1, receptor sphingosine-1-phosphate, essential maturation.J. Clin. Invest. 2000; 106: 951-961Crossref (993) heart rate (7Sanna M.G. Liao J. Jo Alfonso C. Ahn M-Y. Peterson M.S. Webb B. Lefebvre S. Chun Gray N. al.Sphingosine subtypes S1P3, respectively, regulate lymphocyte recirculation rate.J. 2004; 279: 13839-13848Abstract (538) (8Matloubian M. Lo G.G. Cinamon G. Lesneski M.J. Xu Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Lymphocyte egress thymus peripheral lymphoid organs dependent 1.Nature. 427: 355-360Crossref (2075) S1Ps are present serum (9Yatomi Igarashi Yang L. Hisano Qi Asazuma Satoh K. Ozaki Kume 1-phosphate, normal constituent serum.J. Biochem. (Tokyo). 1997; 121: 969-973Crossref (412) This thought generated through pathways: phosphorylation (10Ancellin Colmont Su Li Q. Mittereder S.S. Stefansson Liau Hla Extracellular export kinase-1 enzyme. induction angiogenic 6667-6675Abstract (256) release certain types (11Hobson Barak L.S. Olivera Poulton Caron Milstien Spiegel Role sphingosine-1-phosphate EDG-1 PDGF-induced motility.Science. 2001; 291: 1800-1803Crossref (384) 12Yang Yatomi Miura Metabolism functional effects sphingolipids blood cells.Br. Haematol. 1999; 107: 282-293Crossref (112) At present, widely accepted most store intracellularly extracellularly after stimulation protein C activators (13Yatomi Ruan F. Hakomori Sphingosine-1-phosphate: platelet-activating agonist-stimulated platelets.Blood. 1995; 86: 193-202Crossref 14Yatomi Yamamura platelet activation shares lysophosphatidic acid.J. 272: 5291-5297Abstract (204) low lyase (15Zhou Saba J.D. Identification first mammalian phosphate expression yeast.Biochem. Commun. 1998; 242: 502-507Crossref (152) activity highly active kinases, so accumulate concentrations (14Yatomi 16Stoffel W. Heimann Hellenbroich platelets.Hoppe Seylers Z. Physiol. 1973; 354: 562-566Crossref (62) just how remains unclear. study, determined or within Although biosynthetic found very weak, Additionally, their expressed supply lipoproteins. Exogenously added triggered increase, subsequent (conceivably CDase), and, finally, summary, propose depends sources, membrane. CHOP were kindly provided Dr. Nara (Mitsubishi Kagaku Institute Life Sciences, Tokyo, Japan). Apyrase purchased Nacalai Tesque (Tokyo, BSA (essentially fatty acid-free), palmitoyl-CoA, fumonisin B1 (FB1), UDP-glucose, prostaglandin E1 obtained Sigma (St. Louis, MO). Bacillus cereus Funakoshi Precoated Silica Gel 60 TLC plates Merck (Whitestation, NJ). [3H]Sph, [14C]serine, [γ-32P]ATP Perkin-Elmer Sciences (Boston, MA), [14C] palmitoyl-CoA American Radiolabeled Chemical, Inc. C6-4-nitrobenzo-2-oxa-1,3-diazole (NBD)-SM C6-NBD-Cer Matreya, (Pleasant Gap, PA), C12-NBD-Cer prepared described elsewhere (17Tani Kita Nakagawa Ito Enzymatic synthesis ω-amino-ceramide: preparation sensitive fluorescent substrate ceramidase.Anal. 263: 183-188Crossref (40) All other reagents highest purity available. isolated healthy adult volunteers. collected 0.2 volume anticoagulant ACD (0.8% citric acid, 2.2% sodium citrate, 2.45% glucose) centrifuged 120 g 15 min room temperature obtain platelet-rich plasma. fraction incubated 3 units apyrase 37°C 10 min. Then, 1.0 buffer A [138 mM NaCl, 3.3 NaH2PO4, 2.9 KCl, 1 MgCl2, 20 HEPES-NaOH (pH 7.4), mg/ml glucose] containing 1% BSA, μM E1, fraction, centrifugation 1,107 temperature. then washed ACD. resuspended 0.1% used suspensions. Plasma anticoagulated 12,000 buffered (20 mM, pH 7.4). lines CMK MEG-O1 grown RPMI 1640 medium supplemented 10% FBS, 100 μg/ml streptomycin, U/ml penicillin 5% CO2 incubator. Chinese hamster ovary polyoma large T antigen support replication eukaryotic vectors (18Heffernan Dennis W.J. Polyoma papovavirus antigen-mediated shuttle cells.Nucleic Acids 19: 85-92Crossref (94) HEK293T embryonic kidney α-MEM DMEM, each penicillin. Transfection performed using LipofectAMINE Plus™ Reagent (Invitrogen Co., Carlsbad, CA). To prepare lysates, (1 × 106), (2 (3.5 108) ml PBS, suspended Tris-HCl 7.5) 1× protease inhibitor mixture (Roche Diagnostics Indianapolis, IN) phenylmethylsulfonyl fluoride, sonicated (three times 30 s 4°C), −80°C before use. CDase measured (19Tani Okino Mitsutake Tanigawa Izu Purification characterization mouse liver: single catalyzes reversible reaction hydrolyzed synthesized.J. 275: 3462-3468Abstract (75) different conditions GTA [3,3-dimethyl-glutaric Tris(hydroxymethyl) aminomethane, 2-amino-2-methyl-1,3-propanediol] assay according method al. (20Olivera Kohama Tu rat kinase.J. 273: 12576-12583Abstract (202) Scholar) slight modifications. brief, appropriate amount lysate μCi [γ-32P] ATP 50 μl mix [20 0.25% Triton X-100, 5 12 β-glycerophosphate, 0.25 EDTA, pyrophosphate]. terminated addition 375 chloroform-methanol-concentrated HCl (100:200:1), lipids extracted Bligh Dyer (21Bligh E.G. rapid total lipid extraction purification.Can. 1959; 37: 911-917Crossref (42879) Portions extract applied plate, developed butanol-acetic acid-water (3:1:1, v/v). Radioactive bands visualized quantified imaging analyzer (FLA5000; Fuji Film). 5.0 C6-NBD-SM substrate. Briefly, (550 pmol) [100 acetate 5.0) X-100]. Where indicated, concentration 0.1 ZnCl2 mix. stopped adding chloroform-methanol (2:1, v/v), resulting lower layer chloroform-methanol-25% ammonia (90:20:0.5, remaining analyzer. glucosyltransferase synthase activities Lipsky Pagano (22Lipsky N.G. R.E. Intracellular translocation cultured fibroblasts: endogenously synthesized glucocerebroside analogues pass Golgi apparatus en route membrane.J. Cell 1985; 100: 27-34Crossref (205) (10 nmol ethanol) mixed 200 distilled water 0.01% form complex. An complex 32°C h [25 7.5), 500 UDP-glucose]. 150 chloroform-methanol-12 MgCl2 (65:25:4, C6-NBD-glucosylCer Serine palmitoyltransferase (23Merrill Characterization serine cells.Biochim. 1983; 754: 284-291Crossref (118) Specifically, 0.5 [14C]serine 300 8.0), DTT, pyridoxal 5-phosphate]. 600 radiolabeled 3-ketosphinganine Determination sphinganine N-acyltransferase Venkataraman (24Venkataraman Riebeling Bodennec Riezman Allegood J.C. Sullards M.C. Jr., Futerman Upstream growth factor (uog1), homolog yeast longevity assurance (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) B1-independent manner cells.J. 35642-35649Abstract (235) [14C]palmitoyl-CoA (d18:0) 25 250 sucrose, 2 MgCl2]. (90:10:1, dihydroCer amounts HPLC (Agilent 1100 series; Agilent Technologies) derivatization o-phthalaldehyde essentially (25Min J.K. Yoo H.S. E.Y. Y.M. Simultaneous quantitative analysis sphingoid base 1-phosphates samples precolumn dephosphorylation alkaline phosphatase.Anal. 303: 167-175Crossref (140) suspension 750 (1:1, Water (160 μl) added, phases separated centrifugation. organic phase dried, sn-1,2-diacylglycerol (26Perry D.K. Bielawska Quantitative determination diglyceride kinase.Methods Enzymol. 312: 22-31Crossref vector, cDNA encoding enzyme subcloned pcDNA3.1/Myc-His(+) vector Co.) PCR 5′ primer KpnI restriction site (5′-AGGGTACCGCAATGCCCCACCACAGAGCA-3′) 3′ XbaI disrupted stop codon (5′-AGTCTAGAGCACAACAGGGGGCGTGACCAC-3′). mutant (secretory possesses sequence Vh chain instead signal/anchor sequence, follows. fragment lacking Myc-His epitope end amplified PstI (5′-CGAAGTCGACAAAGATTCAGGGAATCACTGGGT-3′) NotI (5′-GGGCGGCCGCTCAATGGTGATGATGATGATGACC-3′) cDNA-containing (27Mitsutake Tani Mori Ichinose Omori Iida Nakamura Purification, characterization, molecular cloning, subcellular distribution kidney.J. 276: 26249-26259Abstract (99) template. digested pCMV/Myc/ER (Invitrogen). sequences these constructs verified DNA sequencer (model 377; Applied Biosystems transiently transfected plasmid SMase. more efficiently wild-type CDase, ∼10-fold (data shown). Eighteen hours transfection, media replaced serum-free DMEM ml/well six-well plates) additional 24 h. culture subjected supernatants conditioned media. examine metabolic pathways, labeled analyzed TLC. Human controls. Cer, well glycerophospholipids, detected (Fig. 1A). despite efficient labeling little detected. Reportedly, inhibition FB1 causes sphinganine, intermediate biosynthesis, consequently, (28Riley R.T. Enongene Voss K.A. Meredith F.I. Sharma R.P. Spitsbergen Williams D.E. Carlson D.B. perturbations mechanisms fumonisin.Environ. Health Perspect. 109: 301-308Crossref (216) Using inhibitor, examined effect confirm effectiveness [3H]Sph presence 1, 3, 8 As result, strongly inhibited conversion [3H]Cer no inhibitory observed [3H]S1P expected, marked treatment 1B). contrast, accumulation even 18 FB1. Together, indicate synthesize few novo. inability suggested initial step condensation 3-ketosphinganine, defective. We enzymes metabolism lysates CHOP, CMK, cells. Table exhibited (<10% control cells). Similarly, glucosyltransferase, synthase, all compared lines. higher had activity. It appears loss megakaryocyte leads novo.TABLE 1Activity synthesizing metabolizing enzymesEnzymeCHOPCMKMEG-O1Plateletspmol/min/mg proteinSerine palmitoyltransferase5.09 ± 0.326.11 0.504.67 0.330.48 0.07Sphinganine N-acyltransferase17.86 1.7231.68 1.7428.19 0.3611.75 0.26Ceramide glucosyltransferase53.58 1.4242.09 0.9834.83 1.041.95 0.64Sphingomyelin synthase7.32 0.798.47 0.045.58 0.342.50 0.12Ceramidase 7.5)0.62 0.020.61 0.060.79 0.051.17 0.05Sphingosine kinase4.46 0.190.14 0.0020.11 0.00729.06 4.28Total line used. Results means SD three independent experiments. Open table new tab Total Because possible supply. Thus, investigated ability incorporate phosphorylate 2). incorporation notably completely disappeared supernatant still detectable physiologic conditions, would major available recently reported rate-limiting (29Tani O-Glycosylation mucin-like domain retains membranes type II integral protein.J. 278: 10523-10530Abstract (56) substrate, optimum near 7.0 3A). dependence similar purified CDases investigate whether hydrolysis plasma, treated markedly increased 3B), simultaneously, increased, presumably result 3C). greatly enhanced upon indicating plays role when 37°C, 3D). significant only whereas untreated These produce SM. phospholipids, lipoproteins via endocytosis-independent transfer (30Engelmann Kogl Kulschar Schaipp Transfer phosphatidylcholine, phosphatidylethanolamine low- high-density lipoprotein platelets.Biochem. 1996; 315: 781-789Crossref (49) content 4A). majority exogenous decrease 4B). compound generating possibility consequence treating b