Sphingosine-phosphate Lyase Enhances Stress-induced Ceramide Generation and Apoptosis

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer. Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer. Sphingosine 1-phosphate (S1P) 1The abbreviations used are: S1P, sphingosine 1-phosphate; AFC, 7-amino-4-trifluoromethylcoumarin; CHAPS, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate; DHS1P, dihydrosphingosine 1-phosphate; DPL1, dihydrosphingosine-phosphate lyase; DTT, dithiothreitol; ER, endoplasmic reticulum; EST, expressed sequence tag; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PLP, pyridoxal 5′-phosphate; RT, reverse transcriptase; S1PP, sphingosine-1-phosphate phosphatase; SPHK, sphingosine kinase; SPL, sphingosine-phosphate lyase. is a sphingolipid metabolite that regulates cell migration, survival, differentiation, angiogenesis, and development. S1P has been shown to activate an extracellular signaling pathway mediated through a family of specific G protein-coupled receptors (1.Kluk M. Hla T. Biochim. Biophys. Acta. 2002; 1582: 72-80Crossref PubMed Scopus (280) Google Scholar, 2.Pyne S. Pyne N. Biochim. Biophys. Acta. 2002; 1582: 121-131Crossref PubMed Scopus (81) Google Scholar). However, evidence indicates that S1P also mediates effects through a receptor-independent mechanism by functioning as a second messenger within cells (3.Payne S. Milstien S. Spiegel S. FEBS Lett. 2002; 531: 54-57Crossref PubMed Scopus (185) Google Scholar). Intracellular S1P levels are regulated primarily by three highly conserved enzymes: sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine-producing S1P, S1P phosphatase (S1PP), which reverses the former reaction, and S1P lyase (SPL), a PLP-dependent enzyme that catalyzes the essentially irreversible cleavage of S1P at the C2-3 carboncarbon bond, yielding ethanolamine phosphate and a long-chain aldehyde (4.Maceyka M. Payne S. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (508) Google Scholar, 5.Mandala S.M. Prostaglandins Other Lipid Mediat. 2001; 64: 143-156Crossref PubMed Scopus (86) Google Scholar, 6.Van Veldhoven P.P. Merrill Jr., A.H. Hannun Y.A. Sphingolipid Metabolism and Cell Signaling Part A. 311. Academic Press, New York2000: 244-254Google Scholar). One approach to uncovering roles of S1P signaling in biology has been to manipulate expression of the recently cloned genes of S1P metabolism. Toward that end, altered SPL expression in a variety of cell lines and mutant model systems has yielded pronounced effects and severe phenotypes. For example, Saccharomyces cerevisiae dpl1 null mutants, which lack SPL activity, are highly resistant to heat stress and nutrient deprivation, whereas overexpression of DPL1 can correct certain defects of endocytosis (7.Mao C. Saba J. Obeid L. Prostaglandins Other Lipid Mediat. 1999; 342: 667-675Google Scholar, 8.Skrzypek M. Nagiec M. Lester R. Dickson R. J. Bacteriol. 1999; 181: 1134-1140Crossref PubMed Google Scholar, 9.Gottlieb D. Heideman W. Zhou J. Oskouian B. Saba J. Mol. Cell. Biol. Res. Commun. 1999; 1: 66-71Crossref PubMed Scopus (64) Google Scholar, 10.Birchwood C.J. Saba J.D. Dickson R.C. Cunningham K.W. J. Biol. Chem. 2001; 276: 11712-11718Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11.Grote E. Vlacich G. Pypaert M. Novick P. Mol. Biol. Cell. 2000; 11: 4051-4065Crossref PubMed Scopus (38) Google Scholar). In mouse embryonal carcinoma cells, disruption of the SPL gene enhances cellular differentiation in response to retinoic acid (12.Kihara A. Ikeda M. Kariya Y. Lee E. Lee Y. Igarashi Y. J. Biol. Chem. 2003; 278: 14578-14585Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In Drosophila melanogaster, Caenorhabditis elegans, and Dictyostelium discoideum SPL expression is required for global functions, including embryogenesis, reproduction, survival, and movement (13.Herr D. Fyrst H. Phan V. Heinecke K. Georges R. Harris G. Saba J. Development. 2003; 130: 2443-2453Crossref PubMed Scopus (104) Google Scholar, 14.Mendel J. Heinecke K. Fyrst H. Saba J. J. Biol. Chem. 2003; 278: 22341-22349Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15.Li G. Foote C. Alexander S. Alexander H. Development. 2001; 128: 3473-3483PubMed Google Scholar). These observations indicate that SPL has important and, in some cases, essential functions in cells and organisms. The effects of manipulating SPL expression are presumed, and in some cases have been demonstrated, to be secondary to alterations in intracellular pools of S1P and its availability to carry out downstream signaling functions. However, the specific intracellular mechanisms by which SPL exerts its effects are not well understood. In this study, a recombinant human SPL-GFP fusion protein was expressed in HEK293 cells to elevate SPL activity and diminish cellular S1P. Somewhat unexpectedly, stable expression also enhanced stress-induced increases in ceramide. Overexpression of catalytically active SPL also increased apoptosis, which was reversed by the addition of exogenous S1P or the ceramide synthase inhibitor fumonisin B1. In contrast, the products of the reaction catalyzed by SPL, ethanolamine phosphate, and long-chain aldehydes, did not promote apoptosis. These studies show that SPL can affect cell fate and influence sphingolipid metabolism beyond the regulation of intracellular S1P. Materials—d-erythro-Sphingosine, fumonisin B1, ethanolamine phosphate, and MTT reagent were obtained from Sigma Chemical Co. (St. Louis, MO), 4,5-3Hd-erythro-dihydrosphingosine 1-phosphate (DHS1P) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO), S1P was from Avanti Polar Lipids (Alabaster, AL), ISP-1 was from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA), Sequenase 2.0 and l-14Cserine were from Amersham Pharmacia Biotech (Piscataway, NJ), and Taq polymerase and PCR reagents were from Promega Corp. (Madison, WI). Hexadecenal and hexadecanal were synthesized essentially as described previously (16.Baumann W. Schmid H. Mangold H. J. Lipid Res. 1969; 10: 132-133Abstract Full Text PDF PubMed Google Scholar). cDNA Cloning—BLAST search using the mouse SPL cDNA sequence against GenBank™ dbEST identified two homologous human EST sequences, GenBank™ accession numbers T86263 and AA338781 (17.Zhou J. Saba J. Biochem. Biophys. Res. Commun. 1998; 242: 502-507Crossref PubMed Scopus (154) Google Scholar). T86263 was homologous to the 3′-end and AA338781 to the 5′ region of mouse SPL. To clone the overlapping region of these two ESTs, primers H070F2 (Table I) and H967R2 were used for PCR amplification of the target cDNA from a human cDNA library constructed in the yeast shuttle J. C. T. K. M. M. S. A. PubMed Scopus Google Scholar). This PCR was cloned and was using the S. A. S. A. PubMed Scopus Google Scholar). To clone the of the of the a on the and a on EST sequence were used for PCR amplification using the cDNA The region of the cDNA was by amplification of from human using and This was cloned at to of primers used for PCR and of in a sequence was at with an to the for expression in mammalian cells M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This is to as of the sequence from to G the the for a to that of an The fusion was by of the region from G. Research with the of in Cell and cells American were in and For HEK293 cells were at cells in in cells were with the using reagent was by by the of cells. For cells were at To cell cells were to at at of cells were and for to the green protein expression of recombinant S1P in to was at fumonisin B1 in was to phosphate was in and were in and in a with of mammalian SPL an was in against a sequence of acid at the of the protein HEK293 cells were by in were at for The were in the For of protein from the was by to with the were with using For cells were at and deprivation, by in DTT, and and and with were by at in a for to the and cell The was at for to and this was and at for of from were on a by to The were in a and in for at mouse or mouse were in the and were with the for by of in were in and to with the for by three of in and a in The were using a from the SPL were by disruption and for in Veldhoven P.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were obtained by at for were by for with at was by the using Laboratories, SPL activity was using as of protein was and were out at for SPL-GFP and of recombinant SPL to the endoplasmic was by the of from the fusion protein that of a fluorescent protein the sequence of and the sequence in HEK293 cells expressing and with the mammalian expression were on and using a and The of was by the of was to two was The was by a and was through an The was by a and was through a and was using on a Sphingolipid sphingolipid in cell were by and as described previously Merrill A.H. 2001; Scholar, M. 2000; PubMed Google Scholar). The for of the were obtained from Avanti Polar Lipids (Alabaster, and of and of was by of synthesized as previously described G. A. T. R. A. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were at cells in and under in with for was with activity, cells were and from cell by with for at cell was by and of the was for To the was with at for with by on to and with were to and in as previously described V. R. M. Biochem. J. PubMed Scopus Google Scholar). Lipids were by and identified by and was by and Sphingolipid was by cellular as well as and was to the of cells and to cellular B. P. M. J. J. Biochem. 1999; PubMed Scopus Google Scholar). was by the the cDNA was to a of primers target sequence on and which the for The reaction was out to the The to as was to the Cell of cells was in under the for to the MTT reagent was to a of and cells were at for was by of was to and were with and for at Cell was by at were in the of of by the cell the cells. were by in CHAPS, for on by at for at and of the activity was by the of the fluorescent from the of the Research Meeting, of protein was in reaction CHAPS, at activity was at and activity was by as of of and of previously the of SPL cDNA from (17.Zhou J. Saba J. Biochem. Biophys. Res. Commun. 1998; 242: 502-507Crossref PubMed Scopus (154) Google Scholar). the search the mouse SPL cDNA sequence against the EST and identified two homologous human EST this EST sequence and a PCR as described under cloned human SPL cDNA from a human cDNA The region of was by from human and on This of cDNA accession with a of This sequence is with that recently by Veldhoven P.P. S. V. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar). To that the cloned sequence human SPL, was expressed in the S. cerevisiae mutant which is of SPL enzyme activity and is to the effects of exogenous of in this using the yeast expression in of sphingosine and of SPL enzyme activity not Overexpression of in HEK293 HEK293 cells were with using the expression with an mouse and human SPL a increase in protein in HEK293 cells. The SPL was not under these shown in the of the and recombinant activity in the SPL activity in the of from HEK293 cells was cells, that the recombinant protein is fusion protein constructed to and GFP, localized to the and activity to recombinant as shown in activity in and of and human HEK293 cells or HEK293 cells were for Cell and were by and SPL activity in of protein was using as in a of human SPL by overexpressing and expressing the were by as described under activity of cells or expressing three human SPL SPL enzyme activity was in the of HEK293 cells or with or SPL activity in of protein was using shown three Stable Overexpression of to under Stress has been shown to affect and mammalian cell To whether human SPL expression cell fate decisions, cell lines expressing the fusion protein were In stable were to potential or with the and of from cells. The cells a increase in SPL activity with cells with a cells overexpressing appeared to the as by cell and MTT were However, of the two behaved the cells a of the the cells a in cell at and through of The of SPL on to an in metabolism has been shown to affect survival, and apoptosis in cell including HEK293 cells J. C. Obeid J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. R. S. C. A. J. Spiegel S. S. A. 2000; PubMed Scopus Google Scholar, A. T. L. V. S. Spiegel S. J. Cell Biol. 1999; PubMed Scopus Google Scholar). To investigate whether overexpression of cell through an of of the was activity was in cells overexpressing in response to for In contrast, was in cells under SPL to whether the effects in with overexpression SPL enzyme activity, a mutant SPL with no enzymatic activity was The SPL acid of S. C. elegans, M. and D. of including a acid SPL within this region are a protein kinase phosphorylation and a on to the of PLP-dependent mutant with a acid at the was by of the the wild type mutant is highly expressed and to the ER, as by not However, and stable expression of that the mutant protein is of SPL activity stable expressing cells were under to cells, that SPL activity is required to promote apoptosis S1P and Sphingosine in were in whether the cellular sphingolipid is by SPL Toward that end, and long-chain and were using in HEK293 cells under shown in and S1P, and sphingosine levels were diminished under baseline in cells overexpressing wild type but not mutant SPL, whereas the of was not of and in under Stress the effects of SPL overexpression on sphingolipid and cellular were by these were in these the amounts of S1P and sphingosine were not diminished in the cells overexpressing SPL and some of ceramide and were In cases, this was and was the In contrast, the amounts of and not were not by SPL The of increased amounts of ceramide in cells with cells under these was and that a availability sphingosine and ceramide not SPL is under stress in cells and cells overexpressing wild type or mutant human SPL. In HEK293 cells expressing or the mutant were as described in and for sphingolipid by as described under are as of are in cells. S1P, sphingosine 1-phosphate; ceramide. are ceramide and ceramide of the ceramides yielded not In acid and of ceramide and from HEK293 cells with the the active and the inactive for in The are identified the region of the by the by the of in the with a are and in in in were the at are as were at not These show that cells have well as and a of the acid The not to be to SPL activity, because is also in the cells with mutant that SPL some effects on cells through that are of its The ceramide of also the in ceramides in wild type and mutant cells and the cells a of the as These show that overexpression of SPL to in ceramide that in of the as well as some in acid these are that with the and of ceramides and in some cases such the these of The of in were in the for ceramide in cells overexpressing SPL. that S1P not be well by the cell and a cellular response to increase sphingolipid In sphingolipid was by the of a increase in and a increase in were in cells overexpressing wild type but not mutant SPL. These that sphingolipid be enhanced in response to expression of an active SPL. To investigate whether sphingolipid was required for apoptosis, was by cells with which of cells with ISP-1 led to a in stress-induced apoptosis not ISP-1 apoptosis in cells at by of has been shown to against apoptosis by ceramide and Y. Y. M. D. T. Y. S. H. J. 2001; PubMed Scopus Google Scholar, Lee J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, E. Y. B. J. 1999; Google Scholar, J. R. Biochim. Biophys. Acta. 2002; 1585: PubMed Scopus (83) Google Scholar). To whether apoptosis be by S1P, cells with amounts of exogenous S1P shown in and apoptosis was reversed by addition of S1P in a dose-dependent of the SPL to in HEK293 whether the effects of SPL on apoptosis be to the increased of the products of the reaction catalyzed by this cells expressing a were with ethanolamine phosphate, or the products of the cleavage of S1P or DHS1P, shown in of cells with of to of these three for did not an as by in SPL the of whether the in long- and very long-chain ceramides to the enhanced apoptosis by cells overexpressing SPL, cells were with the ceramide synthase inhibitor fumonisin B1 fumonisin B1 of cells overexpressing SPL reduced stress-induced apoptosis to the of cells. This that the increase in apoptosis in this cell the generation of ceramide. in or SPL ceramide with in the the of the of cells expressing wild type or mutant SPL shown in and the was in cells overexpressing catalytically active SPL. In contrast, wild type cells did not amounts of the was in the of cells overexpressing mutant SPL. expression of mutant SPL did not or ceramide these that ceramide to the increased apoptosis in cells, but that is not or be to promote apoptosis. The of S1P in the regulation of cell survival, differentiation, and movement and its to the of are The of S1P by SPHK, which is by and through has in the as a potential target in the of S. R. 2002; PubMed Scopus Google Scholar, D. R. Res. 2003; PubMed Google Scholar). has also been to that S1P S.M. Prostaglandins Other Lipid Mediat. 2001; 64: 143-156Crossref PubMed Scopus (86) Google Scholar, H. C. H. Milstien S. S. Biochim. Biophys. Acta. 2002; 1582: PubMed Scopus Google SPL has been as a the enzyme that S1P. This is because studies in and in mammalian cells that SPL a in biology previously as a of differentiation and (12.Kihara A. Ikeda M. Kariya Y. Lee E. Lee Y. Igarashi Y. J. Biol. Chem. 2003; 278: 14578-14585Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, D. Fyrst H. Phan V. Heinecke K. Georges R. Harris G. Saba J. Development. 2003; 130: 2443-2453Crossref PubMed Scopus (104) Google Scholar, 14.Mendel J. Heinecke K. Fyrst H. Saba J. J. Biol. Chem. 2003; 278: 22341-22349Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15.Li G. Foote C. Alexander S. Alexander H. Development. 2001; 128: 3473-3483PubMed Google Scholar). Thus, is not to in addition to and S1PP, SPL can human cell fate SPL expression not influences the metabolism of S1P and ceramide but also cell under certain observations that in cells is by cells with exogenous S1P or by ceramide generation that these affect apoptosis by SPL. The of S1P by SPL and, the addition of exogenous S1P to cells of this affect cell through extracellular or intracellular To SPL apoptosis in cells, the of from the the an important of in the The long- and very long-chain ceramide that in cells have been shown recently to to and amplification of the response S. H. Hannun Y.A. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). ceramide S1P in cells overexpressing catalytically active SPL to the for apoptosis to that overexpression of a catalytically inactive SPL mutant to a of is not enhanced in this cell the that functions of SPL was that ceramide amounts were not reduced by SPL because lyase activity be to cellular This be to an increase in sphingolipid an with and with studies by G. A. T. R. A. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. R. G. R. K. J. Biol. Chem. Full Text PDF PubMed Google that have that long-chain activity and sphingolipid was also that cells overexpressing SPL in ceramide of which indicate that of this pathway the that to are also The in ceramide in the of was not to SPL activity, because the was in cells overexpressing the mutant the that the lyase influence the of ceramides that are or not these in ceramide have indicate a regulation of this pathway example, through of some of the in a These are important on levels. SPL expression regulates not intracellular S1P and sphingosine but also to increase ceramide generation under stress This the that sphingolipid metabolism is a highly and that of a enzyme in this pathway can to and through inhibition and mechanisms of studies have shown that SPL is in regulation of protein through its influence on metabolism A. M. J. R. 2002; PubMed Scopus Google Scholar). Thus, SPL exerts global effects on metabolism in cells. the of SPL to cell fate decisions its in and is with observations of apoptosis in Drosophila SPL (13.Herr D. Fyrst H. Phan V. Heinecke K. Georges R. Harris G. Saba J. Development. 2003; 130: 2443-2453Crossref PubMed Scopus (104) Google Scholar). the of SPL to affect cell fate to the that of SPL expression to cell In of the region the human SPL have been in a variety of including and of and S. G. K. M. J. 1998; PubMed Scopus Google Scholar, S. M. K. K. M. J. 2000; PubMed Scopus Google Scholar, A. S. K. C. M. A. B. T. J. A. 2000; 10: PubMed Scopus Google Scholar, Y. N. Y. J. J. Res. 2000; PubMed Scopus Google Scholar, S. S. 2000; PubMed Scopus Google Scholar, J. M. B. M. M. M. R. P. P. S. S. 1998; PubMed Scopus Google Scholar, L. G. C. J. PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar, A.H. P. PubMed Scopus Google Scholar, R. S. J. K. Y. Res. Google Scholar, PubMed Scopus (86) Google Scholar, M. J. Full Text PDF PubMed Scopus Google Scholar, K. E. A. H. V. 1998; PubMed Scopus Google Scholar). Thus, in the SPL of cells have and indicate that SPL be a target for in the of cancer. has been that the of apoptosis as a the that increased ceramide also to increased S1P the effect on the cell Chem. 2003; 11: PubMed Scopus Google Scholar). The generation of ceramide that not to S1P or the generation of ceramide and inhibition of are to this However, SPL ceramide and S1P this in The of SPL for not be because and regulation of SPL expression has recently been in and mammalian cells. The of specific in SPL gene regulation to manipulate SPL for in the for