Phosphorylated cis-4-Methylsphingosine Mimics the Mitogenic Effect of Sphingosine-1-phosphate in Swiss 3T3 Fibroblasts

Abstract

The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155–167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analoguecis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitorl-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate.The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound. The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155–167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analoguecis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitorl-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate. The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound. sphingolipid bovine serum albumin Dulbecco's modified Eagle's medium sphingosine-1-phosphate l-threo-C12-sphinganine (the term sphinganine is used for dihydrosphingosine). Sphingolipids (SL),1 i.e. glycosphingolipids and sphingomyelin, are structural molecules of the plasma membrane of eukaryotic organisms. Glycosphingolipids are involved in intercellular and cell-matrix interactions and membrane binding of antibodies, bacteria, and viruses (1Svennerholm L. Asbury A.K. Reisfeld R.A. Sandhoff K. Suzuki K. Tettamanti G. Toffano G. Biological Functions of Gangliosides, Progress in Brain Research. Vol. 101. Elsevier Science Publishers B.V., Amsterdam1994: 17-29Google Scholar). Sphingolipid catabolism converges on ceramide, which is consecutively degraded to sphingosine and sphingosine-1-phosphate (SPP). Evidence emerged that these three compounds function as intracellular second messengers with wide spectrum activity in signal transduction and cell growth regulation (2Spiegel S. Merrill Jr., A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (650) Google Scholar, 3Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Crossref PubMed Scopus (474) Google Scholar, 4Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1496) Google Scholar). Although sphingosine is widely known to be growth inhibitory and cytotoxic, it was shown to stimulate growth in Swiss 3T3 fibroblasts (5Zhang H. Buckley N.E. Gibson K. Spiegel S. J. Biol. Chem. 1990; 265: 76-81Abstract Full Text PDF PubMed Google Scholar). However, several lines of evidence suggest that the mitogenic effect of sphingosine is mediated, at least to a substantial part, by SPP. SPP was shown to stimulate proliferation in quiescent Swiss 3T3 fibroblasts by an activation of the mitogen-activated protein kinase (Raf-1/MKK/MAP kinase) pathway (6Wu J. Spiegel S. Sturgill T.W. J. Biol. Chem. 1995; 270: 11484-11488Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In these cells, exogenous SPP triggers mobilization of calcium from intracellular stores as well as an activation of phospholipase D, and both events are thought to be involved in mediating the proliferative response of Swiss 3T3 cells (7Gosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Crossref PubMed Scopus (328) Google Scholar, 8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar, 9Desai N.N. Zhang H. Olivera A. Mattie M.E. Spiegel S. J. Biol. Chem. 1992; 267: 23122-23128Abstract Full Text PDF PubMed Google Scholar). Furthermore, activation of sphingosine kinase in response to growth factors resulted in a rapid and transient increase of endogenous SPP in Swiss 3T3 fibroblasts (10Su Y. Rosenthal D. Smulson M. Spiegel S. J. Biol. Chem. 1994; 269: 16512-16517Abstract Full Text PDF PubMed Google Scholar, 11Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (816) Google Scholar), suggesting that SPP production mediates the proliferative stimulus. The intracellular concentration of SPP appears to be relatively low (8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar) and is efficiently regulated by phosphorylation of sphingosine by sphingosine kinase (12Buehrer B.M. Bell R.M. Adv. Lipid Res. 1993; 26: 59-67PubMed Google Scholar, 13Buehrer B.M. Bell R.M. J. Biol. Chem. 1992; 267: 3154-3159Abstract Full Text PDF PubMed Google Scholar) and rapid ultimate cleavage into ethanolamine and the corresponding aldehyde by sphingosine-phosphate lyase (14van Veldhoven P.P. Mannaerts G.P. Adv. Lipid Res. 1993; 26: 69-98PubMed Google Scholar). The rapid turnover of SPP provides an appropriate requirement for a signaling second messenger (10Su Y. Rosenthal D. Smulson M. Spiegel S. J. Biol. Chem. 1994; 269: 16512-16517Abstract Full Text PDF PubMed Google Scholar); however, it makes SPP-mediated signaling difficult to examine. Recent results from our laboratory suggest that the phosphorylated derivative of the sphingosine analoguecis-4-methylsphingosine might act as a metabolically stable mimetic of SPP (15van Echten-Deckert G. Zschoche A. Bär T. Schmidt R.R. Raths A. Heinemann T. Sandhoff K. J. Biol. Chem. 1997; 272: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Like sphingosine,cis-4-methylsphingosine is efficiently phosphorylated in primary cultured mouse cerebellar neurons. In contrast to SPP, the corresponding phosphorylated derivative is, however, not subject to rapid degradation but was demonstrated to accumulate intracellularly. To further determine the potential role of cis-4-methylsphingosine phosphate as a mimetic of SPP, the effect of cis-4-methylsphingosine on cell growth regulation was examined in Swiss 3T3 fibroblasts. As previously shown in primary cultured neurons (15van Echten-Deckert G. Zschoche A. Bär T. Schmidt R.R. Raths A. Heinemann T. Sandhoff K. J. Biol. Chem. 1997; 272: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we demonstrate here a substantial accumulation of cis-4-methylsphingosine phosphate in Swiss 3T3 cells.cis-4-Methylsphingosine treatment mimicked the mitogenic effect of SPP, but resulted in a much stronger proliferative response. The decrease of both proliferation and phosphorylation by inhibition of sphingosine kinase indicates that cis-4-methylsphingosine phosphate mediates the proliferative stimulus. cis-4-Methylsphingosine was synthesized and purified as described (15van Echten-Deckert G. Zschoche A. Bär T. Schmidt R.R. Raths A. Heinemann T. Sandhoff K. J. Biol. Chem. 1997; 272: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).l-threo-C12-sphinganine was synthesized in our laboratory according to Clasen (16Clasen K. Synthese von Sphingosin und SphingosinderivatenPh.D. thesis. Institut für Organische Chemie und Biochemie, Universität Bonn, Germany1994Google Scholar). SPP was obtained from Biomol (Hamburg, Germany). All culture media were obtained from Life Technologies, Inc. (Karlsruhe, Germany). All other chemicals and supplies were purchased as described previously (15van Echten-Deckert G. Zschoche A. Bär T. Schmidt R.R. Raths A. Heinemann T. Sandhoff K. J. Biol. Chem. 1997; 272: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Swiss 3T3 fibroblasts, obtained from Prof. A. H. Merrill Jr. (Emory University, Atlanta, GA), were cultured in DMEM, supplemented with 2 mm glutamine, 10% heat-inactivated fetal calf serum as well as penicillin (100 units/liter) and streptomycin (100 mg/liter). For experiments, cells were subcultured in 8-cm2 Petri dishes. Medium was renewed every 48 h until confluency was reached. Swiss 3T3 fibroblasts were kept 5 days confluent and quiescent before use. Cells were washed with phosphate-free DMEM and subsequently incubated with this medium containing 32Pi (40 μCi/ml) for 24 h as described (8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar). The cells were treated with 10 μm of the respective sphingoid for either 1 or 24 h. For inhibition studies, l-threo-sphinganine (20 μm) was added simultaneously. Sphingosine phosphates were extracted following the method of Yatomi et al. (17Yatomi Y. Ruan F. Ohta H. Welch R.J. Hakomori S. Igarashi Y. Anal. Biochem. 1995; 230: 315-320Crossref PubMed Scopus (120) Google Scholar), as described in detail previously (15van Echten-Deckert G. Zschoche A. Bär T. Schmidt R.R. Raths A. Heinemann T. Sandhoff K. J. Biol. Chem. 1997; 272: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Phosphorylated sphingosines were resolved by thin layer chromatography in 1-butanol/methanol/acetic acid/water (80:20:10:20, by volume), visualized by autoradiography, and identified by their RF value. DNA synthesis was estimated by incorporation of tritiated thymidine into trichloroacetic acid-precipitable cellular material as described previously (8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar). Quiescent cultures were washed three times with DMEM to remove residual serum and incubated for 12 h with 1 ml of DMEM/Waymouth medium (1:1) supplemented with 20 μg/ml bovine serum albumin (BSA) and 5 μg/ml transferrin. Cells were treated with the respective sphingoid added as a complex with BSA. After 18 h, cells were pulsed with 1 μCi of [3H]thymidine for 6 h. The incorporation of radioactivity into trichloroacetic acid-insoluble material was measured as described by Spiegel and Panagiotopoulos (18Spiegel S. Panagiotopoulos C. Exp. Cell Res. 1988; 177: 414-427Crossref PubMed Scopus (50) Google Scholar). Cytoplasmic free calcium concentrations [Ca2+]i were determined with the fluorescent calcium indicator dye Fura-2 in a Perkin-Elmer LS-5B spectrofluorimeter equipped with a fast filter device as described previously (19Meyer zu Heringdorf D. van Koppen C.J. Windorfer B. Himmel H.M. Jakobs K.H. Naunyn-Schmiedeberg's Arch. Pharmacol. 1996; 354: 397-403Crossref PubMed Google Scholar). Briefly, confluent and quiescent Swiss 3T3 cells were harvested by trypsination, and the cell pellet was resuspended in a buffer (pH 7.4) containing 1 μm Fura-2/AM, 137 mm NaCl, 2.7 mm KCl, 0.9 mmCaCl2, 0.5 mm MgCl2, 6.5 mm Na2HPO4, 1.5 mmKH2PO4, 1 mg/ml BSA, and 1 mg/ml glucose (0.5–1.0 × 106 cells/ml). After 1 h at 37 °C, cells were washed twice, resuspended in fresh buffer and immediately used for fluorescence measurements. To remove dye leaked into the medium, aliquots were pelleted in a microfuge, resuspended in prewarmed buffer without BSA, and transferred to a thermostatted cuvette (37 °C) in the spectrofluorimeter. 10 μm of the respective sphingosine derivatives were added as complexes with BSA in phosphate-buffered saline. Excitation was alternating at 340 and 380 nm, and emission read at 495 nm. Fluorescence data were converted into calcium concentration using software supplied by the manufacturer. DNA fragmentation was determined by using “Cell Death Detection ELISAPLUS” test kit according to the instructions given by the manufacturer (Boehringer Mannheim, Mannheim, Germany). This photometric enzyme immunoassay allows for the quantitative in vitro measurement of histone-associated DNA fragments (mono and oligonucleosomes) in the cytoplasmic fraction of cell lysates. Cells were harvested and lysed 24 h after addition of the respective sphingosine derivative. The cytoplasmic nucleosome enrichment factor was calculated from the ratio of absorbance measured in treated cells versus untreated control cells, thus illustrating the specific enrichment of mono- and oligonucleosomes released into the cytoplasm of treated cells. In addition, chromatin fragmentation was determined by quantitative nuclear fluorescence morphology assay using bisbenzimide (Hoechst stain H 33258, obtained from Sigma, Deisenhofen, Germany). Cells were incubated with 10 μm of the respective sphingosine derivative for 24 h, trypsinized, washed with phosphate-buffered saline, and fixed in phosphate-buffered saline containing 3% paraformaldehyde. Cells were stained with bisbenzimide for 15 min at room temperature, transferred to glass slides, and examined by fluorescence microscopy. 300 cells per experiment were scored on chromatin fragmentation. The presence of at least three chromatin fragments per cell was considered as apoptosis, and the ratio to cells with intact nuclei was calculated. To examine the effect of cis-4-methylsphingosine on proliferation of quiescent Swiss 3T3 fibroblasts, [3H]thymidine incorporation into DNA was assayed and compared with the effect of SPP and sphingosine, respectively, as shown in Fig. 1. Relative to untreated control cells, incubation of cells withcis-4-methylsphingosine resulted in an ∼10-fold increase of [3H]thymidine incorporation, whereas incorporation was 3-fold and nearly 2-fold after treatment of cells with SPP and sphingosine, respectively. To further differentiate whether cis-4-methylsphingosine or its endogenously phosphorylated derivative caused the marked effect on proliferation in Swiss 3T3 fibroblasts, sphingosine kinase was blocked with the competitive inhibitorl-threosphinganine (12Buehrer B.M. Bell R.M. Adv. Lipid Res. 1993; 26: 59-67PubMed Google Scholar). As shown in Fig. 1, when l-threo-sphinganine was combined withcis-4-methylsphingosine, the proliferative effect of this compound was significantly reduced by about 50%.l-threo-sphinganine also reduced the mitogenic effect of sphingosine, however, to a lesser extent. These results suggest that the induction of the proliferative response relied on the phosphorylated derivative of cis-4-methylsphingosine. Fig. 2 depicts the in vivophosphorylation of cis-4-methylsphingosine in Swiss 3T3 fibroblasts prelabeled with 32Pi. Relative to the amount of endogenously labeled SPP of control cells (100%), the amount of phosphorylated cis-4-methylsphingosine was found to be elevated about 13-fold after 1 h of treatment, rising to about 45-fold after 24 h of treatment. When sphingosine kinase was inhibited withl-threo-sphinganine (20 μm), the elevated intracellular amount of phosphorylatedcis-4-methylsphingosine was reduced by about 15 and 50% after 1 and 24 h, respectively (Fig. 2). Thus the decrease of cis-4-methylsphingosine-induced cell proliferation observed upon simultaneous treatment withl-threo-sphinganine is paralleled by its inhibitory effect on phosphorylation. The SPP-induced stimulation of DNA synthesis was shown to correlate well with an SPP-triggered mobilization of calcium from intracellular stores, suggesting that the mitogenic response is mediated, at least in part, by intracellular calcium release (8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar). The effect of 10 μm cis-4-methylsphingosine, SPP, and sphingosine on mobilization of intracellular calcium is shown in Fig. 3. The release of free calcium was similar when cells were incubated with SPP and cis-4-methylsphingosine, respectively. Aftercis-4-methylsphingosine treatment, the amplitude of free calcium was less pronounced compared with SPP, and the decrease of free calcium to baseline levels was somewhat delayed. These results, however, suggest that both compounds trigger analogous mechanisms in cellular metabolism. In contrast, less calcium release was induced by sphingosine, which correlates with the reduced effect of this compound on cell proliferation. Sphingoid bases have been demonstrated to induce apoptosis in a variety of cell types (20Merrill Jr., A.H. Schmelz E-M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Crossref PubMed Scopus (567) Google Scholar). Also, SPP (20 μm) was demonstrated to induce apoptosis in human hepatoma cells Biochem. Res. 1996; PubMed Scopus Google Scholar). To or in withl-threo-sphinganine in Swiss 3T3 cells, apoptosis was by of histone-associated DNA fragments and oligonucleosomes) in the cytoplasmic fraction of the cell in vitro by a photometric enzyme immunoassay (Boehringer In addition, apoptosis was by nuclear fluorescence was used as As given in treatment with 10 μm caused cell of the other compounds or in as with both of apoptosis in Swiss 3T3 enrichment of mono- and oligonucleosomes of cells fragments was determined in quiescent Swiss 3T3 cells by of DNA fragmentation after 24 h of treatment with the used were 10 μm 20 μm and 10 μm other respectively. The the cytoplasmic nucleosome enrichment factor obtained from the Cell Death Detection test kit (Boehringer which was calculated from the ratio of absorbance measured in treated versus untreated control cells. The the of cells at least three chromatin as determined by a quantitative nuclear fluorescence morphology assay from a of 300 cells. are of at least three not determined as the of this with the absorbance in a was determined in quiescent Swiss 3T3 cells by of DNA fragmentation after 24 h of treatment with the used were 10 μm 20 μm and 10 μm other respectively. The the cytoplasmic nucleosome enrichment factor obtained from the Cell Death Detection test kit (Boehringer which was calculated from the ratio of absorbance measured in treated versus untreated control cells. The the of cells at least three chromatin as determined by a quantitative nuclear fluorescence morphology assay from a of 300 cells. are of at least three not determined as the of this with the absorbance SPP, the phosphorylated degradation of sphingosine, was shown previously to function as a second messenger in inducing cell proliferation in Swiss 3T3 cells (6Wu J. Spiegel S. Sturgill T.W. J. Biol. Chem. 1995; 270: 11484-11488Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar, A. Zhang H. Mattie M.E. Schmidt R.R. Spiegel S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In the present study, treatment of Swiss 3T3 fibroblasts with cis-4-methylsphingosine resulted in a much stronger proliferative effect compared with SPP. The of sphingosine degradation SPP is to cis-4-methylsphingosine as As demonstrated in the present study, the compound is efficiently however, whereas the of SPP is relatively of rapid ultimate degradation G. Chem. PubMed Scopus Google Scholar, K. Chem. 354: PubMed Scopus Google Scholar), phosphorylatedcis-4-methylsphingosine in the cells, as was also shown previously in primary cultured cerebellar neurons (15van Echten-Deckert G. Zschoche A. Bär T. Schmidt R.R. Raths A. Heinemann T. Sandhoff K. J. Biol. Chem. 1997; 272: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). as both cell types have the degradation of this compound appears to be to the which the of either sphingosine degradation or or the of the of cis-4-methylsphingosine and SPP treatment on proliferation in Swiss 3T3 cells, evidence for the phosphorylated derivative the mitogenic was obtained in the present by sphingosine kinase with the competitive inhibitorl-threo-sphinganine B.M. Bell R.M. J. Biol. Chem. 1992; 267: 3154-3159Abstract Full Text PDF PubMed Google Scholar). This compound was shown to decrease DNA synthesis in Swiss 3T3 cells induced by growth factor or serum by and respectively F. Buckley N.E. Olivera A. K.A. Y. Spiegel S. J. 1996; PubMed Scopus Google Scholar). l-threo-sphinganine the proliferative effect of cis-4-methylsphingosine to a similar extent. for the of phosphorylation of cis-4-methylsphingosine on the proliferative effect from of phosphorylation in cultured cells in the and presence of When sphingosine kinase was of cis-4-methylsphingosine was reduced by about 50% after 24 h compared with cells. This caused was not of an effect of this when in with cis-4-methylsphingosine The proliferative effect of SPP in Swiss 3T3 cells was demonstrated to be associated with mobilization of calcium from intracellular stores (8Zhang H. Desai N.N. Olivera A. Seki T. Brooker G. Spiegel S. J. Cell Biol. 1991; 114: 155-167Crossref PubMed Scopus (564) Google Scholar). This is by our data that SPP and cis-4-methylsphingosine, both compounds inducing substantial calcium with sphingosine, which less proliferative resulted in an calcium In cultured cells, the calcium mobilization by exogenous SPP was demonstrated to be mediated by with membrane whereas sphingosine and other to be to these as by their calcium effect Koppen C.J. zu Heringdorf D. Zhang C. Jakobs K.H. M. L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). However, binding of SPP to cell membrane in human S. T. 1997; PubMed Scopus Google Scholar) as well as M. van S. C. R. Spiegel S. T. Science. PubMed Scopus Google and S. T. 1997; PubMed Scopus Google Scholar) mediates a of the cellular of SPP but not calcium mobilization van H. Biochem. J. PubMed Scopus Google Scholar). however, that the proliferative effect of endogenous SPP, which is thought to as mitogenic signaling molecule, on the activation of on the cell substantial of the SPP-induced cell proliferation might be mediated by intracellular (7Gosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Crossref PubMed Scopus (328) Google Scholar, M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In this calcium mobilization phosphorylation of the respective sphingosine derivative which the calcium release induced by exogenous sphingosine compared with SPP, and the much effect of accumulated to these The results of this demonstrate a of the of SPP and phosphorylated cis-4-methylsphingosine on cell growth regulation in quiescent Swiss 3T3 fibroblasts. The stronger effect of cis-4-methylsphingosine on cell proliferation compared with SPP most likely corresponds to the unusual intracellular accumulation of this which might in stimulation of signaling Prof. Sandhoff Bonn, for and also Prof. R. Schmidt for and zu Heringdorf for in the of intracellular The of and is