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Role of Sphingosine 1-Phosphate in the Mitogenesis Induced by Oxidized Low Density Lipoprotein in Smooth Muscle Cells via Activation of Sphingomyelinase, Ceramidase, and Sphingosine Kinase

Role of Sphingosine 1-Phosphate in the Mitogenesis Induced by Oxidized Low Density Lipoprotein in... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 31, Issue of July 30, pp. 21533–21538, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of Sphingosine 1-Phosphate in the Mitogenesis Induced by Oxidized Low Density Lipoprotein in Smooth Muscle Cells via Activation of Sphingomyelinase, Ceramidase, and Sphingosine Kinase* (Received for publication, November 16, 1998, and in revised form, April 23, 1999) Nathalie Auge ´‡§¶, Mariana Nikolova-Karakashiani, Ste ´ phane Carpentier‡, Sampath Parthasarathy§, Anne Ne ` gre-Salvayre‡, Robert Salvayre‡, Alfred H. Merrill, Jr.i, and Thierry Levade‡** From the ‡Laboratoire de Biochimie, INSERM U. 466, Universite ´ Paul Sabatier, CHU Rangueil, 31403 Toulouse, France and the Departments of §Gynecology and Obstetrics and iBiochemistry, Emory University, Atlanta, Georgia 30322-3050 Oxidized LDL (oxLDL) have been implicated in di- Oxidized low density lipoproteins (LDL) are believed to play a critical role in atherosclerosis (1, 2). Oxidized LDL exert verse biological events leading to the development of atherosclerotic lesions. We previously demonstrated diverse biological effects on the different cell types, including that the proliferation of cultured vascular smooth mus- smooth muscle cells (SMC), which are present in the athero- cle cells (SMC) induced by oxLDL is preceded by an sclerotic lesions (3). The responses of cultured SMC depend on increase in neutral sphingomyelinase activity, sphingo- the degree of oxidation and on the extracellular concentration myelin turnover to ceramide, and stimulation of mito- of oxidized LDL, and include production of growth factors (3, 4), gen-activated protein kinases (Auge ´ , N., Escargueil- chemotaxis (5), and cell proliferation (6 – 8) as well as induction Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., of cytotoxicity (7, 9), all of which are considered to be key events Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffre ´ zou, in the development of atherosclerosis (3, 10). J. P., Laurent, G., Levade, T., Ne ` gre-Salvayre, A., and Oxidized LDL (oxLDL) induce the proliferation of cultured Salvayre, R. (1998) J. Biol. Chem. 273, 12893–12900). SMC (11, 12), which has recently been shown to be accompa- Since ceramide can be converted to other bioactive me- nied by the activation of a neutral, magnesium-independent tabolites, such as the well established mitogen sphingo- sphingomyelinase that induces sphingomyelin (SM) hydrolysis sine 1-phosphate (S1P), we investigated whether addi- and ceramide generation (13). Ceramide (N-acylsphingosine) tional ceramide metabolites are involved in the oxLDL- belongs to the family of sphingolipids (14), and has recently induced SMC proliferation. We report here that emerged as an important signaling molecule that is involved in incubation of SMC with oxLDL increased the activities the regulation of cell growth, differentiation, or most notably of both acidic and alkaline ceramidases as well as sphin- apoptotic cell death (15–20). gosine kinase, and elevated cellular sphingosine and In previous studies, the induction of SM turnover was shown S1P. Furthermore, the mitogenic effect of oxLDL was D-erythro-2-(N-myristoylamino)-1-phenyl-1- to be mitogenic for SMC through the activation of the mitogen- inhibited by activated protein kinases p42 and p44 (13); however, the exact propanol and N,N-dimethylsphingosine which are in- hibitors of ceramidase and sphingosine kinase, respec- nature of the SM metabolite(s) that were responsible for this tively. These findings suggest that S1P is a key mediator response was not elucidated. The ceramide formed from SM of the mitogenic effect of oxLDL. In agreement with this turnover might be hydrolyzed by ceramidases to liberate the conclusion, exogenous addition of sphingosine stimu- sphingoid base backbone (sphingosine), which can be re-acy- lated the proliferation of cultured SMC, and this effect lated to ceramide or phosphorylated to sphingosine 1-phos- was abrogated by dimethylsphingosine but not by fumo- phate (S1P) by sphingosine kinase (14, 20) (see Fig. 1). While nisin B1, an inhibitor of the acylation of sphingosine to ceramide and sphingosine have most frequently been described ceramide. Exogenous S1P also promoted SMC prolifera- as potent inhibitors of cell growth or as cytotoxic agents (20), tion. Altogether, these results strongly suggest that the S1P has been found to be growth stimulatory for fibroblasts mitogenic effect of oxLDL in SMC involves the combined (21) and SMC (22, 23). The mitogenic effects of S1P have been activation of sphingomyelinase(s), ceramidase(s), and attributed to calcium mobilization, activation of phospholipase sphingosine kinase, resulting in the turnover of sphin- D, and generation of the second messenger phosphatidic acid, gomyelin to a number of sphingolipid metabolites, of engagement of the MAPK pathway, and activation of the which at least S1P is critical for mitogenesis. transcription factor AP-1 (24); furthermore, S1P can inhibit apoptosis (25). * This work was supported in part by the Universite ´ Paul Sabatier (Toulouse), INSERM, European Community (Biomed-2 BMH4-CT98- Sphingosine kinase is activated by PDGF, phorbol esters, 3191), and National Institutes of Health Grant GM 46368 (to A. H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked The abbreviations used are: LDL, low density lipoproteins; SMC, “advertisement” in accordance with 18 U.S.C. Section 1734 solely to smooth muscle cells; oxLDL, mildly oxidized LDL; SM, sphingomyelin; indicate this fact. S1P, sphingosine-1-phosphate; DMS, N,N-dimethylsphingosine; ¶ Recipient of fellowships from the American Heart Association D-MAPP, D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; FB1, fu- (Georgia Affiliate) and Association pour la Recherche sur le Cancer. monisin B1; MAPK, mitogen-activated protein kinases; TPCK, L-1- ** To whom correspondence should be addressed: INSERM U. 466, tosylamido-2-phenylethyl chloromethyl ketone; C -NBD-ceramide, Laboratoire de Biochimie, “Maladies Me ´ taboliques”, Institut Louis Bug- 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl sphingosine; nard, Ba ˆ t. L3, C.H.U. Rangueil, 1 Avenue Jean Poulhe ` s, F-31403 Tou- PDGF, platelet-derived growth factor; FCS, fetal calf serum; DMEM, louse Cedex 4, France. Tel.: 33-561-32-20-60; Fax: 33-561-32-20-84 (or Dulbecco’s modified Eagle’s medium; HPLC, high performance liquid 29-53); E-mail: [email protected]. chromatography. This paper is available on line at http://www.jbc.org 21533 This is an Open Access article under the CC BY license. 21534 OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin Metabolic Labeling of Cellular Lipids—For SM determination, SMC were metabolically labeled to apparent equilibrium with [methyl- H]choline (0.5 mCi/ml) in MEM medium containing 1% FCS for 48 h. The cells were washed once with PBS and incubated for2hin fresh medium containing 1% FCS (to allow them to adjust to the medium change), then incubated with or without oxLDL (10 mg of apoB/ml). At the indicated times, cells were washed with ice-cold PBS, harvested, and sedimented by centrifugation (300 3 g for 5 min). Cell pellets were immediately frozen at 220 °C. Alternatively, SM and ceramide con- tents were determined by [9,10- H]palmitic acid labeling using the previously reported experimental conditions (12). For the estimation of cellular S1P concentrations, subconfluent cells were incubated for 24 h with phosphate-free DMEM medium containing [ P]phosphoric acid (40 mCi/ml). After1hof chase in phosphate-free medium, cells were treated as indicated above. Lipid Extraction and Analyses—Cell pellets were suspended in 0.6 ml of distilled water and homogenized by sonication (2 3 10 s, using a FIG.1. Metabolic pathways of S1P formation. Abbreviations: MSE probe sonicator). An aliquot was saved for protein determination Cer, ceramide; Sph, sphingosine. (29). Lipids from 0.5 ml of the cell lysate were extracted by 2.5 ml of chloroform/methanol (30). The lipid phase was evaporated under nitro- and a number of other growth stimulatory factors (24). Rela- gen. [ H]Choline-labeled SM was quantified as described (31). The [9,10- H]palmitic acid-labeled lipids were separated by TLC on Silica tively little is known about the modulation of ceramide hydrol- Gel G-60 analytical plates, using 3 successive runs, one with chloro- ysis by various agonists, however, PDGF (26) and interleu- form/methanol/water (100:42:6, by volume) up to 14 cm, a second run kin-1b (27) have been shown to activate ceramidase activities with chloroform, methanol, 16 N ammonia (90:10:1, by volume) up to 18 in mesenchymal cells and hepatocytes, respectively. cm, and a last run with petroleum ether/diethyl ether (80:20, by vol- The present study investigated whether the mitogenic sig- ume) up to 19.5 cm. Radioactive lipids were localized using a Berthold naling triggered by oxLDL in cultured SMC is mediated by the radiochromatoscan and after exposure to iodine vapors. For S1P quan- tification, lipids were extracted exactly as described for the experiments hydrolysis of SM to ceramide combined with the activation of with radioactive choline, that is after mild alkaline hydrolysis. P- ceramidase and sphingosine kinase to form S1P. The results, Labeled phospholipids were separated by TLC using chloroform/meth- indeed, establish that oxLDL induce activation of this panel of anol/water (60:35:8, by volume) as developing solvent. Radioactive S1P enzymes, and strongly implicate S1P as a key mediator of the was localized by autoradiography (Kodak BioMax film) and by iodine mitogenic effect of oxLDL. vapors. All radioactive spots were scraped off and counted by liquid scintillation. Sphingosine mass was determined as described previously EXPERIMENTAL PROCEDURES (27). Chemicals—Minimal essential medium (MEM), phosphate-free In Vitro Ceramidase Assay—After stimulation with or without ox- DMEM medium, penicillin, streptomycin, trypsin-EDTA, L-glutamine, LDL, SMC were washed with ice-cold PBS, scraped from two 60-mm and fetal calf serum (FCS) were from Life Technologies, Inc. (Cergy- dishes, pooled and sedimented, and ceramidase assays were performed 3 3 Pontoise, France). [ H]Thymidine (5 Ci/mmol) and [9,10- H]palmitic as described previously (27). After incubation for1hat37 °Cin10mM acid (53 Ci/mmol) were obtained from Amersham (Les Ulis, France); Tris, pH 7.2 (for neutral ceramidase assay), 0.5 M acetate buffer, pH 5 [methyl- H]choline chloride (86 Ci/mmol) was from DuPont NEN (for acidic ceramidase), or 10 mM Hepes-Tris, pH 8 (for alkaline cerami- (Courtaboeuf, France), and [ P]phosphoric acid (500 mCi/ml) and dase), the reaction was stopped and the products were analyzed by [g- P]ATP (5 Ci/mmol) were from ICN (Orsay, France). D-Erythro- HPLC as described below. Alternatively, the enzymatic reaction (after MAPP, DMS, S1P, and N-acetylsphingosine (C -ceramide) were from 2 h incubation at 37 °C) was terminated by adding chloroform/metha- TEBU-Biomol (Le Perray en Yvelines, France). Bacillus cereus sphin- nol, and the lipids were extracted, separated on aluminum TLC plates gomyelinase, D-erythro-sphingosine, TPCK, and FB1 were obtained using chloroform/methanol (95:5, by volume), and then petroleum from Sigma. C -NBD-ceramide was from Molecular Probes (Eugene, ether/diethyl ether (80:20, by volume). The fluorescent bands were cut, OR). Other reagents and solvents (Merck, Darmstadt, Germany) were eluted in chloroform/methanol (2:1, by volume), and quantified using a of analytical grade. Jobin-Yvon 3D spectrofluorometer (at 466 and 536 nm, for the excita- Cell Culture—Rabbit femoral SMC (obtained from ATCC, Manassas, tion and emission wavelengths, respectively). VA) were routinely grown in MEM supplemented with 10% FCS, L- In Situ Ceramidase Assay—SMC were incubated with C -NBD-cer- glutamine (2 mmol/liter), penicillin (100 units/ml), and streptomycin amide (6 mM) for 6 h before addition of oxLDL. After varying incubation (100 mg/ml) at 37 °C in humidified CO (5%) atmosphere. When indi- times, the cells were washed with ice-cold PBS and 1.5 ml of methanol/ cated, C -ceramide, sphingosine, D-MAPP, FB1, and DMS were added water/phosphoric acid (850:150:1.5, by volume) was added, and the to the cells in ethanolic solution (final concentration of ethanol, 0.1%), suspension was incubated for1hat37 °C with gentle shaking. The while S1P was introduced as an aqueous solution prepared in MEM insoluble material was removed by centrifugation and the supernatant containing 10% FCS. was analyzed by HPLC as described in the following section. Lipoprotein Isolation and Oxidation—Human LDL (d 1.019 –1.063) HPLC Analysis of NBD Lipids—NBD lipids were injected into a were isolated from pooled fresh sera as described previously (6). OxLDL reverse-phase column (Nova-Pak, C , Bio-Rad) and eluted with meth- were obtained by copper oxidation: LDL in 0.15 M NaCl were oxidized by anol/water/phosphoric acid (850:150:1.5, by volume) as described (27). incubation for 30 min at 37 °C with copper sulfate (2 mM). Oxidation was Under these conditions, NBD fatty acid appears as a single peak at 1.5 stopped by adding EDTA (2 mM) to the solution. This procedure was set min and C -NBD-ceramide at 10.3 min. The NBD fluorescence was up to obtain mildly oxidized LDL, i.e. LDL characterized by a moderate analyzed with excitation at 455 nm and emission at 530 nm. amount of lipid peroxidation products (8.5 6 1.2 nmol of thiobarbituric Sphingosine 1-Kinase Assay—Sphingosine kinase activity was deter- acid-reactive substances/mg of apolipoprotein B), and by slight modifi- mined as described previously (32) with minor modifications. After cation of the relative electrophoretic mobility as compared with native incubation with or without oxLDL, cells were washed with ice-cold PBS, LDL (data not shown). harvested, and sedimented by centrifugation. Cell pellets were imme- Cell Proliferation and Cytotoxicity Measurements—SMC were seeded diately solubilized in the kinase buffer (see Ref. 32) and frozen in liquid at a density of 50,000 cells/ml in 24-multiwell Nunc culture plaques, nitrogen. The kinase assay was performed by mixing protein samples and grown for 24 h in medium containing 10% FCS. Then the medium (50 mg) with 10 mlof1mM sphingosine (dissolved in 5% Triton X-100) was removed, cells were washed once with phosphate-buffered saline, and [ P]ATP (1 mCi, 20 mM) containing 200 mM MgCl . After incuba- and further grown for 24 h in MEM containing 1% FCS. Cells were then tion for 30 min at 37 °C, the reaction was stopped by addition of 20 ml incubated with the indicated concentration of mitogenic agent for 24 or of 1 N HCl, and the [ P]ATP-labeled S1P was extracted, isolated by 48 h, and labeled for the last 12 h of the experiment with [ H]thymidine TLC, and quantified as described (32). Alternatively, a nonradioactive (0.5 mCi/ml). Incorporation of [ H]thymidine was determined as de- method was used. Cells were extracted in the kinase buffer as described scribed previously (13). The cytotoxicity was evaluated by using the above and the kinase reaction was performed by incubating 50 mgof 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide test (28). protein sample with 50 mM octyl-b-D-glucopyranoside, 20 mM sphingo- OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin 21535 TABLE I SMC proliferation induced by sphingomyelinase (SMase), C -ceramide, sphingosine, and S1P [ H]Thymidine incorporation was evaluated after 24 or 48 h incubation in the presence of the indicated additive. The values (mean 6 S.D.; n 5 3 to 7) are expressed as percentage of those measured in untreated cells (grown in 1% FCS). During this time period, no cytotoxicity (as measured using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide test or by cell counting) was observed with any inhibitor. Statistical differences to the values obtained in cells treated in the absence of the indicated inhibitor are given. Very similar results were obtained by cell counting (data not shown). [ H]Thymidine incorporation (% of control) Inhibitor Time OxLDL SMase C -ceramide Sphingosine S1P mM h10 mg/ml 0.1 units/ml 5 mM 5 mM 5 mM None 24 134 6 12 211 6 13 129 6 6 145 6 16 145 6 7 48 152 6 19 143 6 12 134 6 12 126 6 6 151 6 16 a a a D-MAPP (20) 24 88 6 14 58 6 6 81 6 20 137 6 12 138 6 10 a a a 48 88 6 16 60 6 17 86 6 11 120 6 7 138 6 10 a a a a DMS (1) 24 100 6 5 109 6 11 112 6 14 108 6 7 150 6 16 a a a a 48 103 6 16 93 6 13 75 6 26 62 6 10 140 6 5 *, p , 0.001; according to Student t test. sine, and 1 mM ATP in a final volume of 100 ml during 30 min at 37 °C. The reaction was stopped by adding 750 ml of methanol. Then 100 mlof 10 mM EDTA and 50 mlof5% ortho-phthalaldehyde were added to the mixture. After 15 min of derivatization at room temperature in the dark, the samples were injected into HPLC (C reverse-phase column) using methanol/potassium phosphate, pH 7.2, tetrabutylammonium dihydrogen phosphate (83:16:1, by volume) as mobile phase. S1P was analyzed with excitation at 340 nm and emission at 455 nm. RESULTS Previous studies have shown that oxLDL induce SMC pro- liferation as reflected in small, but significant, increases in 3 FIG.2. Activation of SM turnover in cultured SMC by oxLDL. [ H]thymidine incorporation (6, 8, 11–13), increases in cell A, SMC were metabolically labeled with [methyl- H]choline and incu- number (6, 11), and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl bated in the presence of oxLDL (10 mg of apoB/ml). At the indicated tetrazolium bromide assays, and this growth stimulation by times, incubations were stopped and SM levels determined as described under “Experimental Procedures.” Results are expressed as % of the SM oxLDL is thought to be significant as a contributor to athero- amounts measured in untreated cells (about 400,000 dpm/mg of cell genesis (3, 10). We have shown in bovine aortic SMC that protein) at the corresponding time. B, cells metabolically labeled for oxLDL induced SM hydrolysis to ceramide, and that this was 3 48 h with [9,10- H]palmitic acid (1 mCi/ml) were treated as above. At strongly associated with the mitogenesis induced by oxLDL the indicated times, lipids were extracted and separated by TLC; SM and ceramide (Cer) spots were scraped off and counted by liquid scin- (12, 13). Therefore, this study investigated the role of sphingo- tillation. Radioactive lipids (mean 6 S.E. of three independent experi- lipid metabolites in greater detail using the related model, ments) are expressed as % of the amounts measured at time 0 (about rabbit femoral SMC, which also display oxLDL-induced mito- 200,000 and 80,000 dpm/mg of cell protein for SM and ceramide, genesis (see Table I). respectively). OxLDL Activate SM Turnover in Rabbit SMC—As shown in Fig. 2, oxLDL at a concentration that is mitogenic for SMC (10 mg of apoB/ml; see Table I and Fig. 3) induced a time-dependent decrease in SM. Maximal hydrolysis of [ H]choline-labeled SM (38 6 11.5%) was observed within 40 –70 min after oxLDL addition, then, [ H]SM returned progressively toward the orig- inal level (Fig. 2A). Similar results were obtained when using cells labeled with [ H]palmitic acid (Fig. 2B). OxLDL also transiently increased the amounts of labeled ceramide (Fig. 2B), but the amount of radiolabel in ceramide was about 60% compared with the loss in SM, supporting the conclusions that not only oxLDL stimulate a sphingomyelinase activity but also that some of the ceramide is being hydrolyzed FIG.3. OxLDL-induced SMC proliferation is inhibited by both to other products. ceramidase and sphingosine kinase inhibitors. Rabbit SMC were Both [ H]palmitate-labeled SM and ceramide levels recov- incubated for 24 h with (solid bars) or without (empty bars) oxLDL (10 ered to baseline within 3 h. These results are similar to the mg of apoB/ml) in the presence or absence of the indicated concentration of D-MAPP, DMS, or FB1. In the case of D-MAPP, cells were pretreated responses seen previously with UV-oxidized LDL (12), and for 12 h with the inhibitor. [ H]Thymidine incorporation was evaluated provide further evidence that oxidized LDL stimulates tran- during the last 12 h of incubation. The values are expressed as % of the sient ceramide release from SM via sphingomyelinase radioactivity measured in cells grown in medium containing 1% FCS activation. (mean 6 S.E., n 5 three separate experiments, each being performed in triplicate). OxLDL-induced SMC Proliferation is Abrogated by Inhibi- tors of Ceramidase and Sphingosine Kinase—The increase in ceramide might mean that this metabolite is responsible for the sible that subsequent metabolite(s) are involved. To explore mitogenic response of SMC to oxLDL, however, it is also pos- this possibility, the cells were treated with oxLDL in the pres- ence of D-MAPP, a ceramidase inhibitor (33) or DMS, an inhib- B. Caligan and A. H. Merrill, Jr., manuscript in preparation. 3 itor of sphingosine kinase (34, 35). The proliferative effect of N. Auge ´ , A. Ne ` gre-Salvayre, R. Salvayre, and T. Levade, unpub- lished data. oxLDL was completely abolished by co-incubation with 20 mM 21536 OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin FIG.5. Ceramidase activity in untreated and oxLDL-stimu- lated SMC cell extracts. SMC were treated for the indicated times with or without oxLDL (10 mg/ml). After which, ceramidase activities FIG.4. In situ ceramidase activity in control and oxLDL-stim- were assayed on cell extracts at pH 5 or 8 using C -NBD-ceramide as ulated SMC. Cells were incubated for 6 h with C -NBD-ceramide and substrate as described under “Experimental Procedures.” The data then treated with (f) or without (M) oxLDL (10 mg/ml) for the indicated (derived from the determinations of fluorescence intensities after TLC times. Afterward, ceramidase activity was evaluated as described under separation) are expressed as percentage of the values on cells incubated “Experimental Procedures.” The data, expressed as the ratio of C - in the absence of oxLDL, and correspond to three separate experiments NBD-fatty acid produced to the intracellular C -NBD-ceramide, repre- (means 6 S.E.) performed in duplicate. Similar results were obtained sent the average of two separate experiments, each performed in trip- using HPLC analysis (two independent experiments performed in licate. Inset, cellular sphingosine mass was quantified after 120 min triplicate). incubation as described under “Experimental Procedures.” D-MAPP or 1–2 mM DMS (Fig. 3). In contrast, the mitogenic effect was not blocked by an inhibitor of ceramide synthase, i.e. fumonisin B1 (except at the concentration of 50 mM which also affected SMC proliferation in the absence of oxLDL). These data suggested the involvement of both ceramidase and sphin- gosine kinase in the mitogenic effect induced by oxLDL, there- fore, these activities were next assayed. OxLDL Activate in Situ and in Vitro Ceramidase Activi- ties—To test whether oxLDL stimulate ceramide turnover, cells were incubated with the fluorescent ceramide analog C - NBD-ceramide and then exposed to oxLDL for varying times. The ratio of fluorescent fatty acid to ceramide, which is a FIG.6. In vitro sphingosine kinase activity in oxLDL-treated reflection of in situ ceramidase activity (27), was higher at all SMC. Cells were treated for the indicated times with or without oxLDL (10 mg/ml), and sphingosine kinase activity was determined in cell time points for the cells treated with oxLDL (Fig. 4). Consistent extracts using either exogenous D-erythro-sphingosine as substrate and with this increased turnover of ceramide, there was an increase [ P]ATP (A) or a nonradioactive, HPLC method (B) as described under in the amount of sphingosine in the cells treated with oxLDL “Experimental Procedures.” In A, the results are expressed as % of the versus the untreated control (Fig. 4, inset). The effect of oxLDL values in untreated cells at the corresponding time. The data corre- spond to the mean 6 S.E. of five independent experiments performed in was also determined by measuring in vitro ceramidase activi- duplicate (A) or two experiments in triplicate (B). ties under acidic (pH 5) and alkaline (pH 8) conditions (Fig. 5). Under basal conditions, the specific activity of ceramidase at pH 5 (6.0 6 0.3 nmol/mg of protein/h) was somewhat higher 120 min. This increase in S1P in response to oxLDL was abro- than the activity at pH 8 (3.8 6 0.4 nmol/mg of protein per h) gated by the serpin TPCK (Fig. 7B), which was previously (assays were also conducted at pH 7.2, but no activity was shown to inhibit SM hydrolysis (13, 36, 37). detected at neutral pH, data not shown). Fig. 5 shows that The amount of labeled S1P was also elevated when cells were incubation of SMC with oxLDL resulted in enhanced cerami- treated for 1 h (data not shown) or 2 h with exogenous bacterial dase activity at both acidic and alkaline pH. This increase was sphingomyelinase, C -ceramide, or sphingosine (Fig. 7C). The detected by 60 min and appeared to be maximal between 120 increases with exogenous sphingomyelinase and C -ceramide and 150 min. demonstrate, again, that SMC can metabolize endogenously OxLDL Induce Sphingosine Kinase Activation and S1P Pro- generated and exogenously added ceramide(s) to S1P. In all duction in SMC—The possibility that sphingosine kinase is cases, including in the presence of oxLDL, the production of activated in response to oxLDL was examined using the in vitro S1P was considerably inhibited by co-administration of the assay described by Spiegel et al. (32) (Fig. 6A) as well as by an sphingosine kinase inhibitor DMS or the ceramidase inhibitor assay that analyzes the product mass by HPLC (Fig. 6B). D-MAPP (Fig. 7C) at concentrations that abrogated SMC pro- OxLDL induced a 40% increase in sphingosine kinase activity liferation (see Fig. 3). Note also that D-MAPP did not inhibit when cell homogenates were assayed 90 –120 min after addi- sphingosine induction of proliferation (see Table I). Thus, these tion of the oxLDL (Fig. 6). As shown in Fig. 7A, cells treated results strongly implicate the involvement of ceramidase and with oxLDL also exhibited higher amounts of P-labeled S1P, sphingosine kinase, as well as sphingomyelinase, in the re- with an apparent maximum (about 2-fold over the control) at sponse of SMC to oxLDL, thereby resulting in the formation of S1P. SMC Proliferation Is Induced by Exogenous Sphingomyeli- It should be noted that a very low concentration of DMS was nase, C -ceramide, Sphingosine, and S1P—To further define employed because this compound exhibited a strong cytotoxicity for 2 SMC above 5 mM. the role of downstream metabolites of ceramide in the mito- OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin 21537 gosine 1-phosphate, and sphingosylphosphocholine, are highly bioactive in a growing number of species (20, 24 38). In general, the biological effects of ceramides range from induction of cell differentiation to apoptosis, with ceramide acting as a mediator of stress (16). In contrast, S1P is usually a positive modulator of cell growth stimulation (24, 39 – 41), and can protect cells from apoptosis (25, 42, 43). In previous studies on SMC, oxLDL induced sphingomyeli- nase activation, SM hydrolysis, and ceramide generation, which was followed by MAPK stimulation and mitogenesis (12, 13), suggesting that ceramide might mediate the oxLDL-in- duced SMC proliferation. However, since S1P is a more likely candidate in mitogenic signaling (24), we investigated whether this sphingolipid could be responsible for the cell proliferation triggered by oxLDL. Regarding SMC, this hypothesis was sup- ported by previous observations showing a mitogenic role for S1P in guinea pig airway SMC (23) and human arterial SMC (22). The present study demonstrates that oxLDL stimulate an enzymatic cascade leading to the production of S1P from SM. The oxLDL-stimulated enzymes include neutral sphingomyeli- nase (13), ceramidase(s), and sphingosine kinase, as shown in Fig. 1. This conclusion is supported by our observations that FIG.7. Sphingosine 1-phosphate amounts in SMC treated with not only are each of these activities increased in a relevant time oxLDL, exogenous sphingomyelinase, C -ceramide, or sphingo- scale after exposure of SMC to oxLDL, but also, by the expected sine. Cells were grown for 24 h in a phosphate-free medium containing variations in the intracellular amounts of the corresponding [ P]phosphate. After a 1-h chase, cells were incubated with oxLDL (10 products. Furthermore, the notion that S1P is a critical medi- mg/ml) for various times (A), or for 2 h with oxLDL (10 mg/ml) in the presence or absence of 5 mM TPCK (B). In C, cells were incubated for 2 h ator was strengthened by the inhibition of S1P formation, and with oxLDL (10 mg/ml), exogenous sphingomyelinase (SMase; 0.1 units/ mitogenesis, by D-MAPP and DMS. ml), C -ceramide (C2-cer;5 mM), or sphingosine (Sph;5 mM), in the This metabolic scheme has been corroborated by previous presence or absence of D-MAPP or DMS. D-MAPP was incubated with reports that have, typically, focused on one or two of the enzy- the cells for 12 h before other additions, while TPCK and DMS were added 1 h before. Cellular S1P was quantified as described under matic steps of the cascade. Spiegel and associates (21) demon- “Experimental Procedures.” Each experiment was performed at least 3 strated that PDGF induces formation of sphingosine, activa- times (except for C where the data are from two separate experiments tion of sphingosine kinase, and the subsequent production of with a variation averaging 10%). S1P in Swiss 3T3 cells. A PDGF-stimulated increase in S1P levels has been reproduced by others on airway SMC (23). In genic response of SMC, we investigated the ability of cellular embryonic rat thoracic aorta SMC, PDGF was shown to pro- ceramide produced through membrane SM hydrolysis and of duce an increase in sphingosine levels (44), and later studies exogenously added cell-permeant ceramide, sphingosine, and using primary rat mesangial cells reported the activation of a S1P to mimic the mitogenic effect of oxLDL. As shown in Table 5 neutral sphingomyelinase and an alkaline ceramidase in re- I, treatment of SMC with bacterial sphingomyelinase in- 3 sponse to PDGF (26). Besides PDGF and other mitogens, nerve creased the incorporation of [ H]thymidine into DNA; signifi- growth factor, a neurotrophin described to trigger SM break- cant increases in thymidine labeling both after 24 and 48 h down and ceramide generation (45), has also been reported to incubation were induced by all of the exogenous sphingolipids activate sphingosine kinase (42). The same is true for vitamin (Table I). In each case, the effects of the inhibitors were con- (43). In addition, recent studies of the regulation of sistent with the mitogenesis involving formation of S1P. For CYP2C11 in rat hepatocytes have shown that interleukin-1b example, 20 mMD-MAPP suppressed the effects of C -ceramide activates SM hydrolysis (46, 47), ceramide breakdown (via an and sphingomyelinase (and oxLDL), but did not affect sphin- increase in ceramidase activity), and S1P formation, although gosine or S1P-induced growth. A mitogenic effect was also the latter was based only on the use of sphingosine kinase observed after treatment with both sphingosine and S1P (Table inhibitors (27). Finally, the tumor necrosis factor-induced ex- I), and the sphingosine kinase inhibitor DMS (1 mM) blocked pression of adhesion molecules by endothelial cells has recently only the thymidine incorporation induced by sphingosine. Last, been reported to be preceded by sphingomyelinase and sphin- sphingosine-induced SMC proliferation was not blocked by FB1 gosine kinase activation (48). Thus, as far as we are aware, the (155 6 7%) (data not shown), indicating that re-acylation of current study of the mitogenic effects of oxLDL on SMC pro- sphingosine to ceramide is not required for the effect of sphin- vides one of the only analyses of all three metabolites and the gosine. All these data favor the hypothesis that S1P is primar- activities of enzymes that form them. The subcellular location ily responsible for the mitogenic effect of oxLDL. of the metabolic cascade initiated by oxLDL that leads to S1P DISCUSSION still remains to be elucidated. The backbones of sphingolipids are now recognized as impor- The mechanism(s) by which the oxLDL-stimulated S1P trig- tant players in the regulation of both normal and abnormal cell gers SMC proliferation is(are) not yet clarified. However, sev- function (16, 20, 38). Among these molecules, not only ceramide eral lines of evidence indicate that the MAPK pathway is (15, 16, 18), but also ceramide 1-phosphate, sphingosine, sphin- activated downstream of S1P. First, it should be borne in mind that S1P has both extracellular and intracellular receptors, which complicates interpretation of its site(s) of action (49). Under these conditions, sphingomyelinase was not toxic but re- Nonetheless, it is clear that formation of S1P intracellularly sulted in hydrolysis of about 70% of cellular radiolabeled SM within 15 min, with a concomitant production of ceramide (12). plays a role in the mitogenicity of oxLDL because inhibition of 21538 OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin 16. Hannun, Y. A. (1996) Science 274, 1855–1859 sphingosine kinase by DMS blocked the growth stimulation by 17. Mathias, S., Pena, L. A., and Kolesnick, R. N. (1998) Biochem. J. 335, 465– 480 oxLDL. Second, several reports suggest that the Raf/MEK/ 18. Kolesnick, R. N., and Kro ¨ nke, M. (1998) Annu. Rev. Physiol. 60, 643– 665 19. Riboni, L., Viani, P., Bassi, R., Prinetti, A., and Tettamanti, G. (1997) Prog. MAPK pathway is stimulated by S1P (24, 50). We (13) and Lipid Res. 36, 153–195 others (51) have previously shown that mildly oxidized LDL are 20. Spiegel, S., and Merrill, A. H., Jr. (1996) FASEB J. 10, 1388 –1397 able to stimulate MAPK in cultured SMC, and that the oxLDL- 21. Olivera, A., and Spiegel, S. (1993) Nature 365, 557–560 22. Bornfeldt, K. E., Graves, L. M., Raines, E. W., Igarashi, Y., Wayman, G., induced DNA synthesis is blocked by a MAPK kinase inhibitor. Yamamura, S., Yatomi, Y., Sidhu, J. S., Krebs, E. G., Hakomori, S. I., and In addition, incubation of SMC with the serpin TPCK which Ross, R. (1995) J. Cell Biol. 130, 193–206 blocked SM hydrolysis was found to abolish the activation of 23. Pyne, S., Chapman, J., Steele, L., and Pyne, N. J. (1996) Eur. J. Biochem. 237, 819 – 826 MAPK and mitogenesis (13). Finally, S1P is known to enhance 24. van Brocklyn, J. R., Cuvillier, O., Olivera, A., and Spiegel, S. (1998) J. Lipo- the DNA binding activity of the transcription factor AP-1 (52), some Res. 8, 135–145 25. Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, a molecular event believed to take place downstream of MAPK, J. S., and Spiegel, S. (1996) Nature 381, 800 – 803 and which has also been described following exposure to oxLDL 26. Coroneos, E., Martinez, M., McKenna, S., and Kester, M. (1995) J. Biol. Chem. (53). Therefore, although other potential mechanisms (24) can- 270, 23305–23309 27. Nikolova-Karakashian, M., Morgan, E. T., Alexander, C., Liotta, D. C., and not be excluded, the activation of the MAPK pathway repre- Merrill, A. H., Jr. (1997) J. Biol. Chem. 272, 18718 –18724 sents a likely candidate to transduce the oxLDL-induced mito- 28. Denizot, F., and Lang, R. (1986) J. Immunol. Methods 89, 371–377 genic signaling of S1P. 29. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, M. N., Olson, B. J., and Klenk, In conclusion, this study has emphasized the concept that a D. C. (1985) Anal. Biochem. 150, 76–85 given agonist can activate an entire cascade of sphingolipid 30. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497–509 metabolism, as exemplified by the triggering of SM hydrolysis 31. Andrieu, N., Salvayre, R., and Levade, T. (1994) Biochem. J. 303, 341–345 in SMC by oxLDL leading to the formation of S1P and mito- 32. Olivera, A., and Spiegel, S. (1997) in Methods in Molecular Biology (Bird, I. M., genesis. However, this entire pathway might not be activated ed) Vol. 105, pp. 233–242, Humana Press, Totowa, NJ 33. Bielawska, A., Greenberg, M. S., Perry, D., Jayadev, S., Shayman, J. A., in other cell types in response to the same stimulus. As a McKay, C., and Hannun, Y. A. (1996) J. Biol. Chem. 271, 12646 –12654 possible example, oxLDL have recently been reported to induce 34. Yatomi, Y., Ruan, F., Megidish, T., Toyokuni, T., Hakomori, S. I., and Igarashi, Y. 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Mazie ` re, C., Djavaheri-Mergny, M., Frey-Fressart, V., Delattre, J., and 14. Merrill, A. H., Jr., and Sweeley, C. C. (1996) in New Comprehensive Biochem- istry: Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D. E., Mazie ` re, J. C. (1997) FEBS Lett. 409, 351–356 54. Harada-Shiba, M., Kinoshita, M., Kamido, H., and Shimokado, K. (1998) and Vance, J. E., eds) Vol. 31, pp. 309 –338, Elsevier, Amsterdam 15. Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125–3128 J. Biol. Chem. 273, 9681–9687 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Role of Sphingosine 1-Phosphate in the Mitogenesis Induced by Oxidized Low Density Lipoprotein in Smooth Muscle Cells via Activation of Sphingomyelinase, Ceramidase, and Sphingosine Kinase

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 31, Issue of July 30, pp. 21533–21538, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of Sphingosine 1-Phosphate in the Mitogenesis Induced by Oxidized Low Density Lipoprotein in Smooth Muscle Cells via Activation of Sphingomyelinase, Ceramidase, and Sphingosine Kinase* (Received for publication, November 16, 1998, and in revised form, April 23, 1999) Nathalie Auge ´‡§¶, Mariana Nikolova-Karakashiani, Ste ´ phane Carpentier‡, Sampath Parthasarathy§, Anne Ne ` gre-Salvayre‡, Robert Salvayre‡, Alfred H. Merrill, Jr.i, and Thierry Levade‡** From the ‡Laboratoire de Biochimie, INSERM U. 466, Universite ´ Paul Sabatier, CHU Rangueil, 31403 Toulouse, France and the Departments of §Gynecology and Obstetrics and iBiochemistry, Emory University, Atlanta, Georgia 30322-3050 Oxidized LDL (oxLDL) have been implicated in di- Oxidized low density lipoproteins (LDL) are believed to play a critical role in atherosclerosis (1, 2). Oxidized LDL exert verse biological events leading to the development of atherosclerotic lesions. We previously demonstrated diverse biological effects on the different cell types, including that the proliferation of cultured vascular smooth mus- smooth muscle cells (SMC), which are present in the athero- cle cells (SMC) induced by oxLDL is preceded by an sclerotic lesions (3). The responses of cultured SMC depend on increase in neutral sphingomyelinase activity, sphingo- the degree of oxidation and on the extracellular concentration myelin turnover to ceramide, and stimulation of mito- of oxidized LDL, and include production of growth factors (3, 4), gen-activated protein kinases (Auge ´ , N., Escargueil- chemotaxis (5), and cell proliferation (6 – 8) as well as induction Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., of cytotoxicity (7, 9), all of which are considered to be key events Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffre ´ zou, in the development of atherosclerosis (3, 10). J. P., Laurent, G., Levade, T., Ne ` gre-Salvayre, A., and Oxidized LDL (oxLDL) induce the proliferation of cultured Salvayre, R. (1998) J. Biol. Chem. 273, 12893–12900). SMC (11, 12), which has recently been shown to be accompa- Since ceramide can be converted to other bioactive me- nied by the activation of a neutral, magnesium-independent tabolites, such as the well established mitogen sphingo- sphingomyelinase that induces sphingomyelin (SM) hydrolysis sine 1-phosphate (S1P), we investigated whether addi- and ceramide generation (13). Ceramide (N-acylsphingosine) tional ceramide metabolites are involved in the oxLDL- belongs to the family of sphingolipids (14), and has recently induced SMC proliferation. We report here that emerged as an important signaling molecule that is involved in incubation of SMC with oxLDL increased the activities the regulation of cell growth, differentiation, or most notably of both acidic and alkaline ceramidases as well as sphin- apoptotic cell death (15–20). gosine kinase, and elevated cellular sphingosine and In previous studies, the induction of SM turnover was shown S1P. Furthermore, the mitogenic effect of oxLDL was D-erythro-2-(N-myristoylamino)-1-phenyl-1- to be mitogenic for SMC through the activation of the mitogen- inhibited by activated protein kinases p42 and p44 (13); however, the exact propanol and N,N-dimethylsphingosine which are in- hibitors of ceramidase and sphingosine kinase, respec- nature of the SM metabolite(s) that were responsible for this tively. These findings suggest that S1P is a key mediator response was not elucidated. The ceramide formed from SM of the mitogenic effect of oxLDL. In agreement with this turnover might be hydrolyzed by ceramidases to liberate the conclusion, exogenous addition of sphingosine stimu- sphingoid base backbone (sphingosine), which can be re-acy- lated the proliferation of cultured SMC, and this effect lated to ceramide or phosphorylated to sphingosine 1-phos- was abrogated by dimethylsphingosine but not by fumo- phate (S1P) by sphingosine kinase (14, 20) (see Fig. 1). While nisin B1, an inhibitor of the acylation of sphingosine to ceramide and sphingosine have most frequently been described ceramide. Exogenous S1P also promoted SMC prolifera- as potent inhibitors of cell growth or as cytotoxic agents (20), tion. Altogether, these results strongly suggest that the S1P has been found to be growth stimulatory for fibroblasts mitogenic effect of oxLDL in SMC involves the combined (21) and SMC (22, 23). The mitogenic effects of S1P have been activation of sphingomyelinase(s), ceramidase(s), and attributed to calcium mobilization, activation of phospholipase sphingosine kinase, resulting in the turnover of sphin- D, and generation of the second messenger phosphatidic acid, gomyelin to a number of sphingolipid metabolites, of engagement of the MAPK pathway, and activation of the which at least S1P is critical for mitogenesis. transcription factor AP-1 (24); furthermore, S1P can inhibit apoptosis (25). * This work was supported in part by the Universite ´ Paul Sabatier (Toulouse), INSERM, European Community (Biomed-2 BMH4-CT98- Sphingosine kinase is activated by PDGF, phorbol esters, 3191), and National Institutes of Health Grant GM 46368 (to A. H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked The abbreviations used are: LDL, low density lipoproteins; SMC, “advertisement” in accordance with 18 U.S.C. Section 1734 solely to smooth muscle cells; oxLDL, mildly oxidized LDL; SM, sphingomyelin; indicate this fact. S1P, sphingosine-1-phosphate; DMS, N,N-dimethylsphingosine; ¶ Recipient of fellowships from the American Heart Association D-MAPP, D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; FB1, fu- (Georgia Affiliate) and Association pour la Recherche sur le Cancer. monisin B1; MAPK, mitogen-activated protein kinases; TPCK, L-1- ** To whom correspondence should be addressed: INSERM U. 466, tosylamido-2-phenylethyl chloromethyl ketone; C -NBD-ceramide, Laboratoire de Biochimie, “Maladies Me ´ taboliques”, Institut Louis Bug- 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl sphingosine; nard, Ba ˆ t. L3, C.H.U. Rangueil, 1 Avenue Jean Poulhe ` s, F-31403 Tou- PDGF, platelet-derived growth factor; FCS, fetal calf serum; DMEM, louse Cedex 4, France. Tel.: 33-561-32-20-60; Fax: 33-561-32-20-84 (or Dulbecco’s modified Eagle’s medium; HPLC, high performance liquid 29-53); E-mail: [email protected]. chromatography. This paper is available on line at http://www.jbc.org 21533 This is an Open Access article under the CC BY license. 21534 OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin Metabolic Labeling of Cellular Lipids—For SM determination, SMC were metabolically labeled to apparent equilibrium with [methyl- H]choline (0.5 mCi/ml) in MEM medium containing 1% FCS for 48 h. The cells were washed once with PBS and incubated for2hin fresh medium containing 1% FCS (to allow them to adjust to the medium change), then incubated with or without oxLDL (10 mg of apoB/ml). At the indicated times, cells were washed with ice-cold PBS, harvested, and sedimented by centrifugation (300 3 g for 5 min). Cell pellets were immediately frozen at 220 °C. Alternatively, SM and ceramide con- tents were determined by [9,10- H]palmitic acid labeling using the previously reported experimental conditions (12). For the estimation of cellular S1P concentrations, subconfluent cells were incubated for 24 h with phosphate-free DMEM medium containing [ P]phosphoric acid (40 mCi/ml). After1hof chase in phosphate-free medium, cells were treated as indicated above. Lipid Extraction and Analyses—Cell pellets were suspended in 0.6 ml of distilled water and homogenized by sonication (2 3 10 s, using a FIG.1. Metabolic pathways of S1P formation. Abbreviations: MSE probe sonicator). An aliquot was saved for protein determination Cer, ceramide; Sph, sphingosine. (29). Lipids from 0.5 ml of the cell lysate were extracted by 2.5 ml of chloroform/methanol (30). The lipid phase was evaporated under nitro- and a number of other growth stimulatory factors (24). Rela- gen. [ H]Choline-labeled SM was quantified as described (31). The [9,10- H]palmitic acid-labeled lipids were separated by TLC on Silica tively little is known about the modulation of ceramide hydrol- Gel G-60 analytical plates, using 3 successive runs, one with chloro- ysis by various agonists, however, PDGF (26) and interleu- form/methanol/water (100:42:6, by volume) up to 14 cm, a second run kin-1b (27) have been shown to activate ceramidase activities with chloroform, methanol, 16 N ammonia (90:10:1, by volume) up to 18 in mesenchymal cells and hepatocytes, respectively. cm, and a last run with petroleum ether/diethyl ether (80:20, by vol- The present study investigated whether the mitogenic sig- ume) up to 19.5 cm. Radioactive lipids were localized using a Berthold naling triggered by oxLDL in cultured SMC is mediated by the radiochromatoscan and after exposure to iodine vapors. For S1P quan- tification, lipids were extracted exactly as described for the experiments hydrolysis of SM to ceramide combined with the activation of with radioactive choline, that is after mild alkaline hydrolysis. P- ceramidase and sphingosine kinase to form S1P. The results, Labeled phospholipids were separated by TLC using chloroform/meth- indeed, establish that oxLDL induce activation of this panel of anol/water (60:35:8, by volume) as developing solvent. Radioactive S1P enzymes, and strongly implicate S1P as a key mediator of the was localized by autoradiography (Kodak BioMax film) and by iodine mitogenic effect of oxLDL. vapors. All radioactive spots were scraped off and counted by liquid scintillation. Sphingosine mass was determined as described previously EXPERIMENTAL PROCEDURES (27). Chemicals—Minimal essential medium (MEM), phosphate-free In Vitro Ceramidase Assay—After stimulation with or without ox- DMEM medium, penicillin, streptomycin, trypsin-EDTA, L-glutamine, LDL, SMC were washed with ice-cold PBS, scraped from two 60-mm and fetal calf serum (FCS) were from Life Technologies, Inc. (Cergy- dishes, pooled and sedimented, and ceramidase assays were performed 3 3 Pontoise, France). [ H]Thymidine (5 Ci/mmol) and [9,10- H]palmitic as described previously (27). After incubation for1hat37 °Cin10mM acid (53 Ci/mmol) were obtained from Amersham (Les Ulis, France); Tris, pH 7.2 (for neutral ceramidase assay), 0.5 M acetate buffer, pH 5 [methyl- H]choline chloride (86 Ci/mmol) was from DuPont NEN (for acidic ceramidase), or 10 mM Hepes-Tris, pH 8 (for alkaline cerami- (Courtaboeuf, France), and [ P]phosphoric acid (500 mCi/ml) and dase), the reaction was stopped and the products were analyzed by [g- P]ATP (5 Ci/mmol) were from ICN (Orsay, France). D-Erythro- HPLC as described below. Alternatively, the enzymatic reaction (after MAPP, DMS, S1P, and N-acetylsphingosine (C -ceramide) were from 2 h incubation at 37 °C) was terminated by adding chloroform/metha- TEBU-Biomol (Le Perray en Yvelines, France). Bacillus cereus sphin- nol, and the lipids were extracted, separated on aluminum TLC plates gomyelinase, D-erythro-sphingosine, TPCK, and FB1 were obtained using chloroform/methanol (95:5, by volume), and then petroleum from Sigma. C -NBD-ceramide was from Molecular Probes (Eugene, ether/diethyl ether (80:20, by volume). The fluorescent bands were cut, OR). Other reagents and solvents (Merck, Darmstadt, Germany) were eluted in chloroform/methanol (2:1, by volume), and quantified using a of analytical grade. Jobin-Yvon 3D spectrofluorometer (at 466 and 536 nm, for the excita- Cell Culture—Rabbit femoral SMC (obtained from ATCC, Manassas, tion and emission wavelengths, respectively). VA) were routinely grown in MEM supplemented with 10% FCS, L- In Situ Ceramidase Assay—SMC were incubated with C -NBD-cer- glutamine (2 mmol/liter), penicillin (100 units/ml), and streptomycin amide (6 mM) for 6 h before addition of oxLDL. After varying incubation (100 mg/ml) at 37 °C in humidified CO (5%) atmosphere. When indi- times, the cells were washed with ice-cold PBS and 1.5 ml of methanol/ cated, C -ceramide, sphingosine, D-MAPP, FB1, and DMS were added water/phosphoric acid (850:150:1.5, by volume) was added, and the to the cells in ethanolic solution (final concentration of ethanol, 0.1%), suspension was incubated for1hat37 °C with gentle shaking. The while S1P was introduced as an aqueous solution prepared in MEM insoluble material was removed by centrifugation and the supernatant containing 10% FCS. was analyzed by HPLC as described in the following section. Lipoprotein Isolation and Oxidation—Human LDL (d 1.019 –1.063) HPLC Analysis of NBD Lipids—NBD lipids were injected into a were isolated from pooled fresh sera as described previously (6). OxLDL reverse-phase column (Nova-Pak, C , Bio-Rad) and eluted with meth- were obtained by copper oxidation: LDL in 0.15 M NaCl were oxidized by anol/water/phosphoric acid (850:150:1.5, by volume) as described (27). incubation for 30 min at 37 °C with copper sulfate (2 mM). Oxidation was Under these conditions, NBD fatty acid appears as a single peak at 1.5 stopped by adding EDTA (2 mM) to the solution. This procedure was set min and C -NBD-ceramide at 10.3 min. The NBD fluorescence was up to obtain mildly oxidized LDL, i.e. LDL characterized by a moderate analyzed with excitation at 455 nm and emission at 530 nm. amount of lipid peroxidation products (8.5 6 1.2 nmol of thiobarbituric Sphingosine 1-Kinase Assay—Sphingosine kinase activity was deter- acid-reactive substances/mg of apolipoprotein B), and by slight modifi- mined as described previously (32) with minor modifications. After cation of the relative electrophoretic mobility as compared with native incubation with or without oxLDL, cells were washed with ice-cold PBS, LDL (data not shown). harvested, and sedimented by centrifugation. Cell pellets were imme- Cell Proliferation and Cytotoxicity Measurements—SMC were seeded diately solubilized in the kinase buffer (see Ref. 32) and frozen in liquid at a density of 50,000 cells/ml in 24-multiwell Nunc culture plaques, nitrogen. The kinase assay was performed by mixing protein samples and grown for 24 h in medium containing 10% FCS. Then the medium (50 mg) with 10 mlof1mM sphingosine (dissolved in 5% Triton X-100) was removed, cells were washed once with phosphate-buffered saline, and [ P]ATP (1 mCi, 20 mM) containing 200 mM MgCl . After incuba- and further grown for 24 h in MEM containing 1% FCS. Cells were then tion for 30 min at 37 °C, the reaction was stopped by addition of 20 ml incubated with the indicated concentration of mitogenic agent for 24 or of 1 N HCl, and the [ P]ATP-labeled S1P was extracted, isolated by 48 h, and labeled for the last 12 h of the experiment with [ H]thymidine TLC, and quantified as described (32). Alternatively, a nonradioactive (0.5 mCi/ml). Incorporation of [ H]thymidine was determined as de- method was used. Cells were extracted in the kinase buffer as described scribed previously (13). The cytotoxicity was evaluated by using the above and the kinase reaction was performed by incubating 50 mgof 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide test (28). protein sample with 50 mM octyl-b-D-glucopyranoside, 20 mM sphingo- OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin 21535 TABLE I SMC proliferation induced by sphingomyelinase (SMase), C -ceramide, sphingosine, and S1P [ H]Thymidine incorporation was evaluated after 24 or 48 h incubation in the presence of the indicated additive. The values (mean 6 S.D.; n 5 3 to 7) are expressed as percentage of those measured in untreated cells (grown in 1% FCS). During this time period, no cytotoxicity (as measured using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide test or by cell counting) was observed with any inhibitor. Statistical differences to the values obtained in cells treated in the absence of the indicated inhibitor are given. Very similar results were obtained by cell counting (data not shown). [ H]Thymidine incorporation (% of control) Inhibitor Time OxLDL SMase C -ceramide Sphingosine S1P mM h10 mg/ml 0.1 units/ml 5 mM 5 mM 5 mM None 24 134 6 12 211 6 13 129 6 6 145 6 16 145 6 7 48 152 6 19 143 6 12 134 6 12 126 6 6 151 6 16 a a a D-MAPP (20) 24 88 6 14 58 6 6 81 6 20 137 6 12 138 6 10 a a a 48 88 6 16 60 6 17 86 6 11 120 6 7 138 6 10 a a a a DMS (1) 24 100 6 5 109 6 11 112 6 14 108 6 7 150 6 16 a a a a 48 103 6 16 93 6 13 75 6 26 62 6 10 140 6 5 *, p , 0.001; according to Student t test. sine, and 1 mM ATP in a final volume of 100 ml during 30 min at 37 °C. The reaction was stopped by adding 750 ml of methanol. Then 100 mlof 10 mM EDTA and 50 mlof5% ortho-phthalaldehyde were added to the mixture. After 15 min of derivatization at room temperature in the dark, the samples were injected into HPLC (C reverse-phase column) using methanol/potassium phosphate, pH 7.2, tetrabutylammonium dihydrogen phosphate (83:16:1, by volume) as mobile phase. S1P was analyzed with excitation at 340 nm and emission at 455 nm. RESULTS Previous studies have shown that oxLDL induce SMC pro- liferation as reflected in small, but significant, increases in 3 FIG.2. Activation of SM turnover in cultured SMC by oxLDL. [ H]thymidine incorporation (6, 8, 11–13), increases in cell A, SMC were metabolically labeled with [methyl- H]choline and incu- number (6, 11), and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl bated in the presence of oxLDL (10 mg of apoB/ml). At the indicated tetrazolium bromide assays, and this growth stimulation by times, incubations were stopped and SM levels determined as described under “Experimental Procedures.” Results are expressed as % of the SM oxLDL is thought to be significant as a contributor to athero- amounts measured in untreated cells (about 400,000 dpm/mg of cell genesis (3, 10). We have shown in bovine aortic SMC that protein) at the corresponding time. B, cells metabolically labeled for oxLDL induced SM hydrolysis to ceramide, and that this was 3 48 h with [9,10- H]palmitic acid (1 mCi/ml) were treated as above. At strongly associated with the mitogenesis induced by oxLDL the indicated times, lipids were extracted and separated by TLC; SM and ceramide (Cer) spots were scraped off and counted by liquid scin- (12, 13). Therefore, this study investigated the role of sphingo- tillation. Radioactive lipids (mean 6 S.E. of three independent experi- lipid metabolites in greater detail using the related model, ments) are expressed as % of the amounts measured at time 0 (about rabbit femoral SMC, which also display oxLDL-induced mito- 200,000 and 80,000 dpm/mg of cell protein for SM and ceramide, genesis (see Table I). respectively). OxLDL Activate SM Turnover in Rabbit SMC—As shown in Fig. 2, oxLDL at a concentration that is mitogenic for SMC (10 mg of apoB/ml; see Table I and Fig. 3) induced a time-dependent decrease in SM. Maximal hydrolysis of [ H]choline-labeled SM (38 6 11.5%) was observed within 40 –70 min after oxLDL addition, then, [ H]SM returned progressively toward the orig- inal level (Fig. 2A). Similar results were obtained when using cells labeled with [ H]palmitic acid (Fig. 2B). OxLDL also transiently increased the amounts of labeled ceramide (Fig. 2B), but the amount of radiolabel in ceramide was about 60% compared with the loss in SM, supporting the conclusions that not only oxLDL stimulate a sphingomyelinase activity but also that some of the ceramide is being hydrolyzed FIG.3. OxLDL-induced SMC proliferation is inhibited by both to other products. ceramidase and sphingosine kinase inhibitors. Rabbit SMC were Both [ H]palmitate-labeled SM and ceramide levels recov- incubated for 24 h with (solid bars) or without (empty bars) oxLDL (10 ered to baseline within 3 h. These results are similar to the mg of apoB/ml) in the presence or absence of the indicated concentration of D-MAPP, DMS, or FB1. In the case of D-MAPP, cells were pretreated responses seen previously with UV-oxidized LDL (12), and for 12 h with the inhibitor. [ H]Thymidine incorporation was evaluated provide further evidence that oxidized LDL stimulates tran- during the last 12 h of incubation. The values are expressed as % of the sient ceramide release from SM via sphingomyelinase radioactivity measured in cells grown in medium containing 1% FCS activation. (mean 6 S.E., n 5 three separate experiments, each being performed in triplicate). OxLDL-induced SMC Proliferation is Abrogated by Inhibi- tors of Ceramidase and Sphingosine Kinase—The increase in ceramide might mean that this metabolite is responsible for the sible that subsequent metabolite(s) are involved. To explore mitogenic response of SMC to oxLDL, however, it is also pos- this possibility, the cells were treated with oxLDL in the pres- ence of D-MAPP, a ceramidase inhibitor (33) or DMS, an inhib- B. Caligan and A. H. Merrill, Jr., manuscript in preparation. 3 itor of sphingosine kinase (34, 35). The proliferative effect of N. Auge ´ , A. Ne ` gre-Salvayre, R. Salvayre, and T. Levade, unpub- lished data. oxLDL was completely abolished by co-incubation with 20 mM 21536 OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin FIG.5. Ceramidase activity in untreated and oxLDL-stimu- lated SMC cell extracts. SMC were treated for the indicated times with or without oxLDL (10 mg/ml). After which, ceramidase activities FIG.4. In situ ceramidase activity in control and oxLDL-stim- were assayed on cell extracts at pH 5 or 8 using C -NBD-ceramide as ulated SMC. Cells were incubated for 6 h with C -NBD-ceramide and substrate as described under “Experimental Procedures.” The data then treated with (f) or without (M) oxLDL (10 mg/ml) for the indicated (derived from the determinations of fluorescence intensities after TLC times. Afterward, ceramidase activity was evaluated as described under separation) are expressed as percentage of the values on cells incubated “Experimental Procedures.” The data, expressed as the ratio of C - in the absence of oxLDL, and correspond to three separate experiments NBD-fatty acid produced to the intracellular C -NBD-ceramide, repre- (means 6 S.E.) performed in duplicate. Similar results were obtained sent the average of two separate experiments, each performed in trip- using HPLC analysis (two independent experiments performed in licate. Inset, cellular sphingosine mass was quantified after 120 min triplicate). incubation as described under “Experimental Procedures.” D-MAPP or 1–2 mM DMS (Fig. 3). In contrast, the mitogenic effect was not blocked by an inhibitor of ceramide synthase, i.e. fumonisin B1 (except at the concentration of 50 mM which also affected SMC proliferation in the absence of oxLDL). These data suggested the involvement of both ceramidase and sphin- gosine kinase in the mitogenic effect induced by oxLDL, there- fore, these activities were next assayed. OxLDL Activate in Situ and in Vitro Ceramidase Activi- ties—To test whether oxLDL stimulate ceramide turnover, cells were incubated with the fluorescent ceramide analog C - NBD-ceramide and then exposed to oxLDL for varying times. The ratio of fluorescent fatty acid to ceramide, which is a FIG.6. In vitro sphingosine kinase activity in oxLDL-treated reflection of in situ ceramidase activity (27), was higher at all SMC. Cells were treated for the indicated times with or without oxLDL (10 mg/ml), and sphingosine kinase activity was determined in cell time points for the cells treated with oxLDL (Fig. 4). Consistent extracts using either exogenous D-erythro-sphingosine as substrate and with this increased turnover of ceramide, there was an increase [ P]ATP (A) or a nonradioactive, HPLC method (B) as described under in the amount of sphingosine in the cells treated with oxLDL “Experimental Procedures.” In A, the results are expressed as % of the versus the untreated control (Fig. 4, inset). The effect of oxLDL values in untreated cells at the corresponding time. The data corre- spond to the mean 6 S.E. of five independent experiments performed in was also determined by measuring in vitro ceramidase activi- duplicate (A) or two experiments in triplicate (B). ties under acidic (pH 5) and alkaline (pH 8) conditions (Fig. 5). Under basal conditions, the specific activity of ceramidase at pH 5 (6.0 6 0.3 nmol/mg of protein/h) was somewhat higher 120 min. This increase in S1P in response to oxLDL was abro- than the activity at pH 8 (3.8 6 0.4 nmol/mg of protein per h) gated by the serpin TPCK (Fig. 7B), which was previously (assays were also conducted at pH 7.2, but no activity was shown to inhibit SM hydrolysis (13, 36, 37). detected at neutral pH, data not shown). Fig. 5 shows that The amount of labeled S1P was also elevated when cells were incubation of SMC with oxLDL resulted in enhanced cerami- treated for 1 h (data not shown) or 2 h with exogenous bacterial dase activity at both acidic and alkaline pH. This increase was sphingomyelinase, C -ceramide, or sphingosine (Fig. 7C). The detected by 60 min and appeared to be maximal between 120 increases with exogenous sphingomyelinase and C -ceramide and 150 min. demonstrate, again, that SMC can metabolize endogenously OxLDL Induce Sphingosine Kinase Activation and S1P Pro- generated and exogenously added ceramide(s) to S1P. In all duction in SMC—The possibility that sphingosine kinase is cases, including in the presence of oxLDL, the production of activated in response to oxLDL was examined using the in vitro S1P was considerably inhibited by co-administration of the assay described by Spiegel et al. (32) (Fig. 6A) as well as by an sphingosine kinase inhibitor DMS or the ceramidase inhibitor assay that analyzes the product mass by HPLC (Fig. 6B). D-MAPP (Fig. 7C) at concentrations that abrogated SMC pro- OxLDL induced a 40% increase in sphingosine kinase activity liferation (see Fig. 3). Note also that D-MAPP did not inhibit when cell homogenates were assayed 90 –120 min after addi- sphingosine induction of proliferation (see Table I). Thus, these tion of the oxLDL (Fig. 6). As shown in Fig. 7A, cells treated results strongly implicate the involvement of ceramidase and with oxLDL also exhibited higher amounts of P-labeled S1P, sphingosine kinase, as well as sphingomyelinase, in the re- with an apparent maximum (about 2-fold over the control) at sponse of SMC to oxLDL, thereby resulting in the formation of S1P. SMC Proliferation Is Induced by Exogenous Sphingomyeli- It should be noted that a very low concentration of DMS was nase, C -ceramide, Sphingosine, and S1P—To further define employed because this compound exhibited a strong cytotoxicity for 2 SMC above 5 mM. the role of downstream metabolites of ceramide in the mito- OxLDL-induced Formation of Sphingosine 1-Phosphate from Sphingomyelin 21537 gosine 1-phosphate, and sphingosylphosphocholine, are highly bioactive in a growing number of species (20, 24 38). In general, the biological effects of ceramides range from induction of cell differentiation to apoptosis, with ceramide acting as a mediator of stress (16). In contrast, S1P is usually a positive modulator of cell growth stimulation (24, 39 – 41), and can protect cells from apoptosis (25, 42, 43). In previous studies on SMC, oxLDL induced sphingomyeli- nase activation, SM hydrolysis, and ceramide generation, which was followed by MAPK stimulation and mitogenesis (12, 13), suggesting that ceramide might mediate the oxLDL-in- duced SMC proliferation. However, since S1P is a more likely candidate in mitogenic signaling (24), we investigated whether this sphingolipid could be responsible for the cell proliferation triggered by oxLDL. Regarding SMC, this hypothesis was sup- ported by previous observations showing a mitogenic role for S1P in guinea pig airway SMC (23) and human arterial SMC (22). The present study demonstrates that oxLDL stimulate an enzymatic cascade leading to the production of S1P from SM. The oxLDL-stimulated enzymes include neutral sphingomyeli- nase (13), ceramidase(s), and sphingosine kinase, as shown in Fig. 1. This conclusion is supported by our observations that FIG.7. Sphingosine 1-phosphate amounts in SMC treated with not only are each of these activities increased in a relevant time oxLDL, exogenous sphingomyelinase, C -ceramide, or sphingo- scale after exposure of SMC to oxLDL, but also, by the expected sine. Cells were grown for 24 h in a phosphate-free medium containing variations in the intracellular amounts of the corresponding [ P]phosphate. After a 1-h chase, cells were incubated with oxLDL (10 products. Furthermore, the notion that S1P is a critical medi- mg/ml) for various times (A), or for 2 h with oxLDL (10 mg/ml) in the presence or absence of 5 mM TPCK (B). In C, cells were incubated for 2 h ator was strengthened by the inhibition of S1P formation, and with oxLDL (10 mg/ml), exogenous sphingomyelinase (SMase; 0.1 units/ mitogenesis, by D-MAPP and DMS. ml), C -ceramide (C2-cer;5 mM), or sphingosine (Sph;5 mM), in the This metabolic scheme has been corroborated by previous presence or absence of D-MAPP or DMS. D-MAPP was incubated with reports that have, typically, focused on one or two of the enzy- the cells for 12 h before other additions, while TPCK and DMS were added 1 h before. Cellular S1P was quantified as described under matic steps of the cascade. Spiegel and associates (21) demon- “Experimental Procedures.” Each experiment was performed at least 3 strated that PDGF induces formation of sphingosine, activa- times (except for C where the data are from two separate experiments tion of sphingosine kinase, and the subsequent production of with a variation averaging 10%). S1P in Swiss 3T3 cells. A PDGF-stimulated increase in S1P levels has been reproduced by others on airway SMC (23). In genic response of SMC, we investigated the ability of cellular embryonic rat thoracic aorta SMC, PDGF was shown to pro- ceramide produced through membrane SM hydrolysis and of duce an increase in sphingosine levels (44), and later studies exogenously added cell-permeant ceramide, sphingosine, and using primary rat mesangial cells reported the activation of a S1P to mimic the mitogenic effect of oxLDL. As shown in Table 5 neutral sphingomyelinase and an alkaline ceramidase in re- I, treatment of SMC with bacterial sphingomyelinase in- 3 sponse to PDGF (26). Besides PDGF and other mitogens, nerve creased the incorporation of [ H]thymidine into DNA; signifi- growth factor, a neurotrophin described to trigger SM break- cant increases in thymidine labeling both after 24 and 48 h down and ceramide generation (45), has also been reported to incubation were induced by all of the exogenous sphingolipids activate sphingosine kinase (42). The same is true for vitamin (Table I). In each case, the effects of the inhibitors were con- (43). In addition, recent studies of the regulation of sistent with the mitogenesis involving formation of S1P. For CYP2C11 in rat hepatocytes have shown that interleukin-1b example, 20 mMD-MAPP suppressed the effects of C -ceramide activates SM hydrolysis (46, 47), ceramide breakdown (via an and sphingomyelinase (and oxLDL), but did not affect sphin- increase in ceramidase activity), and S1P formation, although gosine or S1P-induced growth. A mitogenic effect was also the latter was based only on the use of sphingosine kinase observed after treatment with both sphingosine and S1P (Table inhibitors (27). Finally, the tumor necrosis factor-induced ex- I), and the sphingosine kinase inhibitor DMS (1 mM) blocked pression of adhesion molecules by endothelial cells has recently only the thymidine incorporation induced by sphingosine. Last, been reported to be preceded by sphingomyelinase and sphin- sphingosine-induced SMC proliferation was not blocked by FB1 gosine kinase activation (48). Thus, as far as we are aware, the (155 6 7%) (data not shown), indicating that re-acylation of current study of the mitogenic effects of oxLDL on SMC pro- sphingosine to ceramide is not required for the effect of sphin- vides one of the only analyses of all three metabolites and the gosine. All these data favor the hypothesis that S1P is primar- activities of enzymes that form them. The subcellular location ily responsible for the mitogenic effect of oxLDL. of the metabolic cascade initiated by oxLDL that leads to S1P DISCUSSION still remains to be elucidated. The backbones of sphingolipids are now recognized as impor- The mechanism(s) by which the oxLDL-stimulated S1P trig- tant players in the regulation of both normal and abnormal cell gers SMC proliferation is(are) not yet clarified. However, sev- function (16, 20, 38). Among these molecules, not only ceramide eral lines of evidence indicate that the MAPK pathway is (15, 16, 18), but also ceramide 1-phosphate, sphingosine, sphin- activated downstream of S1P. First, it should be borne in mind that S1P has both extracellular and intracellular receptors, which complicates interpretation of its site(s) of action (49). 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Published: Jul 1, 1999

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