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J. Biol. Chem., Vol. 277, Issue 15, 12724-12734, April 12, 2002

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Apoptotic Effect of Sphingosine 1-Phosphate and Increased Sphingosine 1-Phosphate Hydrolysis on Mesangial Cells Cultured at Low Cell Density^*

Isabelle Gennero, Josette Fauvel, Michèle Niéto, Clotilde Cariven, Frédérique Gaits, Fabienne Briand-Mésange, Hugues Chap, and Jean Pierre Salles

From INSERM Unité 326, Institut Claude de Préval (Institut Fédératif de Recherche 30), Hôpital Purpan, Place du Dr. Baylac, 31059 Toulouse Cedex, France

Received for publication, September 17, 2001, and in revised form, January 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The lipid mediator sphingosine 1-phosphate (S1P) may alter the proliferation of mesangial cells during pathophysiological processes. Here, S1P stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at subconfluent cell density. Both effects were inhibited by pertussis toxin treatment. Mesangial cells expressed several S1P receptors of the endothelial differentiation gene family: EDG-1, -3, -5, and -8. Conversely, S1P induced apoptosis at low cell density (2 × 10⁴ cells/cm²), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells and was not reverted by lysophosphatidic acid or platelet-derived growth factor. S1P enhanced phosphorylation of SAPKs. Incubation with [³³P]S1P, [³H]S1P, and [³H]sphingosine demonstrated increased S1P hydrolysis, resulting in enhanced intracellular sphingosine levels and decreased S1P levels. A rise in total ceramide levels was also observed; however, ceramide did not originate from [³H]sphingosine, and S1P-induced apoptosis was not inhibited by fumonisin B, precluding involvement of de novo ceramide synthesis in apoptosis. Therefore, we suggest that sphingosine accumulation and decreased S1P are primarily responsible for S1P-induced apoptosis. In conclusion, incubation of low-density mesangial cells with S1P results in apoptosis, presumably due to increased S1P hydrolysis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glomerular diseases are often characterized by the increasing proliferation rate of mesangial cells, also responsible for altered composition of the extracellular matrix, with both mechanisms leading to progressive fibrosis (1-4). Because of this, the identification of agents regulating the growth of mesangial cells has been the topic of numerous studies. The prominent members of the family of lysophospholipids (LPLs),¹ the structurally related molecules sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA; 1-acyl-2-hydroxy-sn-glycerol 3-phosphate), have attracted attention in the last decade as potent growth factors (see reviews in Refs. 5-9). Activated platelets or blood cells secrete S1P and LPA, and so mesangial cell may be a target for LPLs during inflammatory and/or degenerative processes. Several studies have documented the proliferative effect of LPLs on these cells (10-16). LPLs act through homologous G protein-coupled receptors of the endothelial differentiation gene (EDG) family, several members of which are known today. S1P preferentially activates EDG-1, -3, -5, -6, and -8, whereas LPA activates EDG-2, -4, and -7 (7). LPLs promote cell growth by activating several EDG-evoked signaling pathways. G_i/o induces mitosis via the mitogen-activated protein kinase (MAPK) pathway; G_q mediates mobilization of intracellular calcium; and G_12/13 activates Rho-related pathways. In addition, S1P may act as an intracellular second messenger (17).

S1P activates multiple responses in various cells. For example, S1P induces migration of endothelial cells, and it is regarded, together with the expression of its receptor EDG-1, as a factor promoting angiogenesis (18). S1P is also considered as a factor of cell survival. This is the case for both exogenous S1P (9, 19-21) as well as S1P generated by activation of sphingosine kinase by growth factors or by overexpression of the enzyme (22, 23). Contrary to this, cell proliferation is negatively influenced by other sphingolipids such as ceramides and sphingosine (24-27). Thus, the balance between S1P and ceramide/sphingosine cellular levels is probably crucial to control the proliferation of mesangial cells (28, 29).

We previously observed that the effect of S1P on mesangial cell proliferation is variable and depends upon the density of the cell culture (15). In the present study, we documented that an increased hydrolysis of S1P to sphingosine occurred in mesangial cells cultured at low cell density, leading to increased sphingosine and decreased S1P intracellular levels. This phenomenon, in association with the increased production of ceramide, seems a key factor in inducing the apoptosis of mesangial cells. To our knowledge, this is the first time that increased S1P hydrolysis as a mechanism leading to apoptosis has been observed in cells derived from a primary culture. It might also be relevant under pathophysiological conditions, where excessive mesangial cell proliferation is present.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- S1P, LPA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue), collagenase, pertussis toxin, propidium iodide, and Hoechst 33342 were obtained from Sigma. RPMI 1640 medium with Glutamax I, trypsin, fetal calf serum (FCS), penicillin, streptomycin, the SuperScript pre-amplification system, and TRIzol were from Invitrogen. RPMI 1640 medium with D-valine was purchased from BioMedia (Boussens, France). N-Hexanoyl[3-³H]sphingosine (C₆-[³H]ceramide; 10-20 Ci/mmol) was from PerkinElmer Life Sciences. [methyl-³H]Thymidine (25 Ci/mmol) and [-³³P]ATP (2500 Ci/mmol) were from Amersham Biosciences. Culture flasks and multiwells were from Falcon Plastic (Paisley, Scotland). Polyvinylidene difluoride membrane (Immobilon-P) was from Millipore Corp. (Bedford, MA). Anti-phospho-Thr¹⁸³/Thr¹⁸⁵ ERK1/2 antibody, anti-phospho-SAPK antibody, and CDP star chemiluminescent reagent were from New England Biolabs Inc. (Beverly, MA). Anti-rabbit secondary antibody and the lactate dehydrogenase activity kit were from Promega (Madison, WI). Escherichia coli sn-1,2-diacylglycerol kinase, N-octanoyl-D-erythro-sphingosine (C₈-ceramide) and N-palmitoyl-D-erythro-sphingosine (C₁₆-ceramide) were from Calbiochem.

Isolation of Rat Renal Glomeruli and Culture of Mesangial Cells-- Renal glomeruli were isolated from kidneys from 2-month-old male Wistar rats weighing 150-200 g. Most of the procedure was adapted from Striker and Striker (30) as previously described (31). Briefly, the kidneys were removed from their capsule, and the cortex fragments (3-4 mm³) were submitted to four repeated enzymatic digestions with collagenase at 37 °C in PBS solution. The glomeruli purified by successive mechanical sieving were retained on the 100-, 80-, and 60-µm pore stainless steel sieves. Glomerular mesangial cells were obtained from glomerular preparations and cultured under standard conditions at 37 °C in a humidified 5% (v/v) CO₂ incubator in RPMI 1640 medium supplemented with 10% (v/v) heat inactivated FCS plus 100 units/ml penicillin and 100 µg/ml streptomycin. Passages of primary cultures were done after 3-4 weeks. To eliminate contamination by either epithelial or endothelial cells, the culture was performed in RPMI 1640 medium with D-valine until passage 5. Thereafter, experiments were performed between passages 5 and 20. Mesangial cells were characterized by their morphological appearance in phase contrast and their biological and biochemical properties as previously described (31): 1) positive staining with antibodies against myosin or vimentin and negative staining with antibodies against factor VIII or cytokeratins by standard immunofluorescence; 2) ability to contract in response to 10⁵ M angiotensin; and 3) ability to grow in selective D-valine-containing medium. In all experiments, cells were made quiescent by serum starvation in RPMI 1640 medium with 0.5% (v/v) FCS for 48 h. When testing the effect of cell density, equivalent numbers of cells were seeded on variable culture areas. Consequently, for analysis, we used equal cell material originating from the cell at different densities. Cell density varied between 1 × 10⁴ and 1.7 × 10⁵ cells/cm² as indicated below and in the figure legends.

[³H]Thymidine Incorporation-- Mesangial cells were seeded with RPMI 1640 medium containing 10% (v/v) FCS in tissue culture dishes at different densities as indicated. After 24 h, the medium was changed for 48 h to RPMI 1640 medium containing 0.5% (v/v) FCS to make the cells quiescent. The agents to be tested were then added for 24 h. At this time, [³H]thymidine (0.5 µCi/ml) was added, and the incubation was continued for 4 h. Cells were washed with PBS, and the DNA was precipitated with 10% (w/v) trichloroacetic acid. The trichloroacetic acid-precipitable material was dissolved in 0.5 M NaOH, and the radioactivity was measured with a liquid scintillation counter. Data from triplicate values are expressed as a percentage of the average control value.

MTT Cell Proliferation Assay-- Cells were cultured as described above for thymidine incorporation, except that the agents to be tested were added for 36 h. Cells were incubated with 5 µg/ml MTT for 2 h at 37 °C. The medium was aspirated, the formazan crystals formed were dissolved in isopropyl alcohol with 60 mM HCl, and the absorbance at 570 nm was determined with a spectrophotometer (Beckman Instruments). Data from triplicate values are expressed as a percentage of the average control value.

Western Blotting-- Mesangial cells were cultured at high (1 × 10⁵ cells/cm²) or low (2 × 10⁴ cells/cm²) cell density in RPMI 1640 medium with 10% (v/v) FCS. They were rendered quiescent by treatment in RPMI 1640 medium with 0.5% FCS for 48 h. 2 h before the stimulation, the medium was changed to RPMI 1640 medium alone. The cells were then stimulated, washed twice with PBS, and lysed with SDS sample buffer containing 62 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 50 mM dithiothreitol, and 0.1% (w/v) bromphenol blue. Homogenates were sonicated for 10 s, boiled for 5 min, clarified for 2 min at 13,000 rpm, subjected to SDS-PAGE, and then electrotransferred to a polyvinylidene difluoride membrane. The membrane was blocked for 1 h in Tris-buffered saline/Tween 20 with 5% (w/v) nonfat dry milk, incubated overnight with primary antibody (1:1000, v/v), washed twice, and finally incubated with anti-rabbit secondary antibody (1:7500, v/v) for 30 min. Detection was performed with the CDP star chemiluminescent system.

RNA Isolation, Reverse Transcription, and PCR-- Total cellular RNA was extracted by the TRIzol method according to the manufacturer's instructions. The cDNAs encoding parts of the open reading frame of EDG receptors were amplified with the SuperScript pre-amplification system. The primers (MWG Biotech) used for cDNA PCR amplifications were as follows: for rat sequences: EDG-1 (5'-GCTATGGTGTCCTCCACCAG and 5'-CCGGTTTTAAGAAGAAGAATTGACG), EDG-2 (5'-TCATGGCAGCTGCCTCTACT and 5'-CCTTCTAAACCACAGAGTGGTCA), EDG-3 (5'-CATTGGCAACTTGGCTCTCT and 5'-ATCACTACGGTCCGCAGAAG), EDG-5 (5'-CCCCACCATGGGCG and 5'-ACATTTCCCCTCAGACCACTG), EDG-8 (5'-CGAGGTCATCGTCCTTCACT and 5'-GTTGGGAAGCGTCAGTCTGT), and glyceraldehyde-3-phosphate dehydrogenase (5'-ATGGTGAAGGTCGGTGTGAA and 5'-GGGCCCTCGGCCG); and for human sequence: EDG-6 (5'-ACATGCGGTCGCGACG and 5'-ATGCTCCGCACGCTGG). PCRs were performed for 30 cycles of denaturation for 30 s at 95 °C; annealing for 1 min at 58 °C for EDG-1 sequence, at 50 °C for EDG-2 sequence, and at 55 °C for the others; and elongation for 1 min at 72 °C. PCR products were analyzed by agarose gel electrophoresis after staining with ethidium bromide. We precluded contamination with DNA by checking the absence of amplification of glyceraldehyde-3-phosphate dehydrogenase from mRNA extracts not previously submitted to reverse transcription.

Lactate Dehydrogenase Assay-- Lactate dehydrogenase release from damaged cells was used to assess cytotoxicity. Quiescent mesangial cells were cultured in 12-well dishes and treated with the stimulus as indicated. Lactate dehydrogenase activity was determined in both the supernatant and cells according to the manufacturer's instructions as previously described (11). Lactate dehydrogenase activity in the supernatant is expressed as percent of the total release (lactate dehydrogenase released from the supernatant and cells).

Flow Cytometry Analysis-- Apoptotic cells were quantitatively evaluated by flow cytometry. Quiescent cells (5 × 10⁵) were cultured at high or low density and treated with the agonist or vehicle. At the indicated times, both adherent and non-adherent cells were harvested, washed twice with PBS, and fixed in 70% cold ethanol. Briefly, cells were labeled with propidium iodide as described previously (32) and analyzed in a flow cytometer (Coulter ELITE).

Hoechst Staining Assay-- Intact cells were stained with Hoechst 33342 (5 µg/ml) and propidium iodide (10 µg/ml) for 10 min and analyzed in a fluorescence microscope (Zeiss Axioskop) with excitation at 360 nm. Under these conditions, the Hoechst dye stained all nuclei, whereas propidium iodide stained the nuclei of only necrotic cells with disrupted plasma membranes. With Hoechst staining, viable cells were observed with intact nuclei, and apoptotic cells with fragmented or condensed nuclei.

Synthesis of [³³P]S1P and [³H]S1P-- [³³P]S1P was synthesized as described previously (33). The reaction uses the ability of E. coli diacylglycerol kinase to phosphorylate ceramides. C₈-ceramide was solubilized in 5 mM cardiolipin, 7.5% octyl -glucopyranoside, and 1 mM diethylenetriaminepentaacetic acid and resuspended in buffer containing 50 mM imidazole (pH 6.6), 50 mM NaCl, 10 mM MgCl₂, 1 mM EGTA, 10 mM dithiothreitol, and 0.4 international units/ml diacylglycerol kinase. The reaction was started with 10 mM [-³³P]ATP (25 Ci/mmol) overnight. C₈-ceramide 1-[³³P]phosphate was treated with 6 M HCl/butanol-1 (1:1, v/v) for 60 min at 100 °C to release [³³P]S1P. This compound was purified by thin-layer chromatography using butanol-1/acetic acid/water (3:1:1, v/v). [³³P]S1P migrated like S1P in the solvent system described above. [³H]S1P was synthesized from C₆-[³H]ceramide using the same method with nonradioactive ATP.

Measurement of S1P Lipid Phosphate Phosphohydrolase Activity in Mesangial Cells-- Quiescent cells were seeded, cultured at low or high density, and treated with 10 µM [³³P]S1P (10 Ci/mol) in 0.1% (w/v) BSA. At the indicated times, 0.5 ml of the incubation medium was transferred into 0.5 ml of 1 M HClO₄ to precipitate proteins and the labeled lipid. The ³³P_i present in the medium was measured as described previously (34). The radioactivity was counted in a scintillation counter. The initial rate was deduced from experimental data obtained between 0 and 10 min in the linear part of the activity curve. Activity is expressed as nanomoles of inorganic phosphate released per 10⁶ cells.

Metabolism of [³H]S1P in Mesangial Cells-- Mesangial cells were seeded, cultured at low or high density, and treated with the agonists or vehicle in RPMI 1640 medium plus 0.01% (w/v) BSA as indicated. Lipids were then extracted from the extracellular medium by the Bligh and Dyer method (35). Lipids were extracted from cells as follows. Cells were washed twice with PBS and scraped in 0.75 ml of methanol. The plates were washed with an additional 0.75 ml of methanol, and 0.75 ml of chloroform was added to the pooled methanol extracts. The samples were stored overnight at 20 °C, and the precipitated proteins were removed by centrifugation. S1P, sphingosine, and ceramides were analyzed by thin-layer chromatography with butanol-1/acetic acid/water (3:1:1, v/v) as solvent. Radioactivity was detected with an automatic TLC linear analyzer (Bertold, Wildbad, Germany). Spots corresponding to the different lipids were scraped, and the radioactivity was counted in a scintillation counter. A control incubation with radiolabeled substrates without cells demonstrated that ~5% of the compounds remained bound to the plate, with no clear time dependence. This value was considered as the zero value and was subtracted from all experimental data.

Measurement of Cellular Ceramides by the Diacylglycerol Kinase Assay-- The cellular ceramide level was measured according to a method described previously (36) with slight modifications. Quiescent cells were seeded, cultured at low or high density, and treated with S1P as indicated. Cells were washed twice with PBS and scraped in 2 ml of PBS. Lipids were extracted according to the Bligh and Dyer method (35). Samples from the lower chloroform phase were dried and incubated with 0.08 N NaOH in chloroform/methanol (2:0.5, v/v) at 37 °C for 30 min for alkaline hydrolysis of diacylglycerol. The reaction was stopped by lipid extraction, and ceramide was converted to ceramide 1-[³³P]phosphate by E. coli diacylglycerol kinase in the presence of [-³³P]ATP (25 Ci/mmol) as described above, except that the reaction was carried out for 30 min with 1 IU/ml diacylglycerol kinase. The labeled lipids were extracted by the Bligh and Dyer method (35) and separated by thin-layer chromatography in chloroform/methanol/acetone/acetic acid/water (10:2:4:2:1,v/v). The R_F value for ceramide phosphate was 0.20 in this system. Radioactivity was measured with a PhosphorImager 445 SI (Molecular Dynamics, Inc.). Spots corresponding to ceramide phosphate were scraped, and the radioactivity was counted in a scintillation counter. Addition of C₁₆-ceramide to the initial medium as a control testified for ~70% recovery during the overall procedure. This rate of recovery was taken into account to estimate the quantity of ceramide initially present in the assay medium.

Statistics-- Results (expressed as means ± S.E.) are representative of at least three experiments as indicated in the figure legends. Comparisons were done using Student's t test. Results were considered to be significant when p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Density and Opposite Effects of S1P on Mesangial Cell Proliferation-- Although S1P acts as a mitogen toward diverse cell types, few reports have described an antiproliferative effect on cells derived from a primary culture (37, 38). In a preliminary study, we observed that S1P exerts opposite effects on the proliferation rate of mesangial cells, depending upon the density of the cell culture (15). Therefore, our first aim was to assess precisely the effect of cell density on mesangial cell proliferation. We tested a range of cell densities varying between 1 × 10⁴ and 1.7 × 10⁵ cells/cm² (Fig. 1). The effect of S1P on [³H]thymidine incorporation strongly depended upon the cell density, whereas under the same conditions, PDGF was constantly proliferative (data not shown). At subconfluent cell density, 10 µM S1P increased DNA synthesis by up to 6-fold. The effect of 10 µM S1P or 10 µM LPA tested under the same conditions was similar (Fig. 2C). Above 1.5 × 10⁵ cells/cm², the decrease in DNA synthesis was interpreted logically as a confluence-dependent inhibition of cell growth. When decreasing the cell density, the effect of S1P progressively reverted toward an unexpected antiproliferative one at the lowest densities. Because of these first results, we tested the effect of cell density more extensively. In the following experiments, low density corresponded to distinct cells with rare membrane contacts (1-2 × 10⁴ cells/mm²), and high density corresponded to subconfluent cells (1-1.2 × 10⁵ cells/mm²).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effect of S1P on mesangial cell proliferation dependent on cell density. Quiescent cells were seeded at variable density (1 × 104 to 1.7 × 105 cells/cm2) and incubated with 10 µM S1P or vehicle (0.01% (w/v) BSA in PBS) as a control for 24 h. Cells were then incubated with [3H]thymidine for 4 h. Absolute values for [3H]thymidine incorporation in control cells were between 12,000 and 70,000 cpm ± 10% at the lowest and highest cell densities, respectively. Results expressed as percent of the control value are means ± S.E. from triplicate determinations, representative of three independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effects of S1P on mesangial cell proliferation at high cell density. A and B, various concentrations of S1P were tested on quiescent cells cultured at high cell density (1 × 105 cells/cm2). In A, cells were tested for [3H]thymidine incorporation. Quiescent cells were incubated for 24 h with S1P or vehicle (0.01% (w/v) BSA in PBS). Cells were then incubated with [3H]thymidine for 4 h. In B, cells were tested with the MTT technique. C, [3H]thymidine incorporation was evaluated in quiescent cells treated with 10 µM S1P or 10 µM LPA, with or without pretreatment with 100 ng/ml pertussis toxin (PTX) for 16 h. In A-C, results are means ± S.E. from triplicate determinations, representative of three independent experiments. The absolute value for [3H]thymidine incorporation in control cells was 50,000 cpm ± 20%. *, p < 0.05; **, p < 0.01. D, quiescent cells were stimulated with 10 µM S1P for various periods of time (5-120 min) (upper panel) or for 10 and 120 min with or without pertussis toxin (100 ng/ml) pretreatment for 16 h (lower panel). C indicates the control without S1P. MAPK phosphorylation was analyzed by Western blotting with an antibody against the phosphorylated fraction of the protein. Results are representative of two independent experiments. E, shown is the expression of EDG receptors in mesangial cells. Reverse transcription-PCR analysis of EDG-1, -2, -3, -5, and -8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was carried out with RNA isolated from rat mesangial cells as described under "Experimental Procedures."

S1P-induced Proliferation at High Cell Density-- At high cell density (1 × 10⁵ cells/cm²), the treatment of mesangial cells with S1P increased DNA synthesis as demonstrated by [³H]thymidine incorporation (Fig. 2A). Dose dependence was demonstrated by a significant difference between the effect of 1 and 10 µM S1P (p < 0.05). The proliferative effect of S1P was confirmed by the MTT technique. Fig. 2B shows a 2.5-fold increase with 10 µM S1P, with a 3-fold increase being induced by 10 µM LPA as a positive control. Pretreatment of mesangial cells with pertussis toxin almost completely inhibited the S1P-induced mitogenic response, as it did for 10 µM LPA (Fig. 2C), suggesting the involvement of a G_i-coupled pathway for the proliferative response to S1P. Phosphorylation of p42 and p44 MAPKs (ERK1/2) showed a two-wave phosphorylation profile induced by 10 µM S1P that persisted for >2 h, as tested by Western blot analysis using anti-phospho-p42/44 antibody (Fig. 2D). This is in agreement with the results of the thymidine incorporation assay. In addition, activation of ERK1/2 was partly inhibited by pertussis toxin treatment (Fig. 2D).

Extracellular S1P is known to act through receptors of the EDG family. mRNA expression of EDG receptors by mesangial cells was examined using reverse transcription-PCR. As shown in Fig. 2E, most of the identified receptors for S1P were well expressed by mesangial cells, i.e. EDG-1, -3, -5, and -8, with EDG-1, -3, and -5 being expressed the most. As the sequence of the rat EDG-6 receptor was unknown, we amplified it using primers for human EDG-6. We could amplify a homologous form of human EDG-6 from genomic rat DNA. However, we were unable to amplify EDG-6 from either rat or human mesangial cell mRNAs. Altogether, these results suggest that mesangial cells do not express EDG-6. The ubiquitous LPA receptor EDG-2 was also well expressed by mesangial cells.

S1P-induced Apoptosis at Low Cell Density-- As mentioned above, S1P inhibited the proliferation of rat mesangial cells at the lowest cell densities (1-2 × 10⁴ cells/cm²) (Fig. 1). This effect started at 1 µM, was significant at 5 µM S1P, and was at maximum at >10 µM S1P (Fig. 3A). Moreover, S1P significantly decreased the [³H]thymidine incorporation at 24 h when incubated simultaneously with mitogenic agents such as PDGF (40 ng/ml) and FCS (10%, v/v) (Fig. 3, B and C). The hypothesis of DNA synthesis being simply delayed was proved to be wrong in the case of longer S1P incubation (up to 30 h) (data not shown). To clarify the mechanism involved in the deleterious effect of S1P, we first excluded the toxic effect of S1P. Lactate dehydrogenase measured in the supernatant of cells stimulated with 10 µM S1P precluded cell lysis even after 15 h, with the values being similar to those obtained in the control (7.64 ± 1.07% versus 6.87 ± 1.02% of lysed cells, respectively). Apoptosis was studied with Hoechst 33342 staining. Condensed chromatin fragments were observed in cells treated with S1P for 24 h (Fig. 4A). Under the same conditions, propidium iodide remained excluded from the cells, precluding significant necrosis. After 24 h, the percentage of apoptotic cells was 24.9% in cells treated with S1P versus 4.3% in control cells. Another piece of evidence that S1P promoted DNA fragmentation was obtained by flow cytometry analysis after propidium iodide staining (Fig. 4B, upper panel). After 24 h, hypodiploid DNA content increased by up to 18% in S1P-processed cells versus 7% in control cells. The discrepancy between Hoechst staining and cytometry analysis is probably due to the fact that part of the non-adherent apoptotic cells are lost during harvesting before cytometry analysis. In addition, condensed (but not fragmented) nuclei were probably not detected by cytometry. S1P was also tested in cells maintaining a growing mode in the presence of 5% FCS. Although a percentage of the cells in G₂-S phase were detected under these conditions, the apoptotic effect of S1P remained significant, with the same being valid in the presence of PDGF (Fig. 4B, lower panel). S1P was also apoptotic in quiescent cells in the presence of 10 µM LPA (Fig. 4C, upper panel), which acts as a growth factor for mesangial cells (11). LPA decreased the percentage of apoptosis in quiescent cells, but did not prevent apoptosis in the presence of S1P. Apoptosis persisted in growing cells in the presence of PDGF (Fig. 4C, lower panel). These data demonstrate that the apoptotic effect of S1P at low cell density at concentrations >1 µM is not dependent on the privation of growth factors and is not reverted by them.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Antiproliferative effect of S1P at low cell density: dose response to S1P and effect of growth factors. [3H]Thymidine incorporation was evaluated in quiescent cells cultured at low density (2 × 104 cells/cm2). Cells were incubated for 24 h with S1P or vehicle (0.01% (w/v) BSA in PBS). Cells were then incubated with [3H]thymidine for 4 h. A, cells were treated with increasing doses of S1P or vehicle. *, p < 0.05; **, p < 0.01. B and C, cells were treated with a vehicle control (C) or with 10 µM S1P with or without growth factors: 10% (v/v) FCS (in B) or 40 ng/ml PDGF (in C). In B, p < 0.01 (**) compared with vehicle for S1P and with FCS for FCS + S1P. In C, p < 0.01 (**) compared with vehicle for S1P and with PDGF for PDGF + S1P. Results are means ± S.E. from triplicate determinations, representative of three independent experiments. The absolute value for [3H]thymidine incorporation in control cells was 15,000 cpm ± 20%.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Mesangial cell apoptosis induced by S1P at low cell density in quiescent cells and in growth factor-treated cells. A, shown is the Hoechst staining of quiescent mesangial cells after treatment for 24 h with 10 µM S1P or vehicle (0.01% (w/v) BSA in PBS). Arrows show condensed or fragmented nuclei in S1P-treated cells. B, the DNA content from rat mesangial cells treated with 10 µM S1P for 24 h was analyzed by single label flow cytometry with propidium iodide. Upper panel, cells were maintained quiescent in 0.5% (v/v) FCS for 48 h and stimulated for 24 h with vehicle or 10 µM S1P. Lower panel, cells were maintained growing in 10% (v/v) FCS and stimulated for 24 h with 10 µM S1P in the presence of 2% (v/v) FCS. C, the percentage of hypodiploid cells was measured by single label flow cytometry with propidium iodide. Upper panel, quiescent cells as described for B were subjected for 24 h to vehicle or 10 µM S1P with or without 10 µM LPA. Lower panel, non-quiescent cells maintained growing in 10% (v/v) FCS as described for B were subjected to 2% (v/v) FCS ± 10 µM S1P or to 40 ng/ml PDGF ± 10 µM S1P for 24 h. **, p < 0.01. Results are means ± S.E. from triplicate determinations, representative of three independent experiments. D, quiescent cells cultured at variable density were stimulated with 10 µM S1P for various periods of time (5-120 min), and SAPK phosphorylation was analyzed by Western blotting with an antibody against the phosphorylated (SAPK-P) or whole (SAPK) fraction of the protein. Results are representative of two independent experiments.

Phosphorylation and activation of SAPKs are important signaling pathways for programmed cell death. SAPK phosphorylation was detected in S1P-treated cells only when cultured at low density, with activation lasting for >2 h (Fig. 4C).

Apoptotic Effect of Sphingosine at Low Cell Density-- As the proliferative effect of S1P reverted to apoptosis in cells cultured at low density, we supposed that the sphingolipid rheostat would be modified toward its apoptotic components under these conditions. We hypothesized that the conversion of S1P to sphingosine could be responsible for the apoptotic effect of S1P at low cell density (S1P possibly being dephosphorylated to sphingosine). Therefore, we tested the effect of sphingosine, which also appeared to be density-dependent, resulting in stimulated thymidine incorporation at high cell density and inhibition at low cell density (Fig. 5A). The effect of sphingosine on low-density cells was significant in the concentration range of 1-10 µM (Fig. 5B). 1 µM sphingosine inhibited the proliferation induced by PDGF (40 ng/ml), and significant apoptosis occurred with 10 µM sphingosine, as demonstrated by flow cytometry analysis. (Note that the vehicle used in the sphingosine assay (Me₂SO, 1:1000, v/v) was responsible for increased apoptosis in control cells.)

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Density dependence of the effect of sphingosine on mesangial cells and apoptotic effect at low cell density. A, shown is the density dependence of the sphingosine effect on [3H]thymidine incorporation. Quiescent cells were seeded at variable density (3 × 104 to 2 × 105 cells/cm2) and incubated for 24 h with 10 µM sphingosine or vehicle (0.5% (v/v) Me2SO) as a control. Cells were then incubated with [3H]thymidine for 4 h. Absolute values for [3H]thymidine incorporation in control cells were between 8000 and 35,000 cpm ± 10% at the lowest and highest cell densities, respectively. B, [3H]thymidine incorporation was evaluated in quiescent cells cultured at low density (2 × 104 cells/cm2) and treated for 24 h with vehicle with or without 40 ng/ml PDGF in the presence of variable concentrations of S1P as indicated. *, p < 0.05; **, p < 0.01. Results are means ± S.E. from triplicate determinations, representative of three independent experiments. C, the DNA content from quiescent rat mesangial cells treated with vehicle or 10 µM sphingosine for 24 h was analyzed by single label flow cytometry. The percent of hypodiploid cells is indicated. Results are representative of three independent experiments.

S1P Phosphohydrolase Activity Involved in Apoptosis of Mesangial Cells-- To test the hypothesis of increased conversion of S1P to sphingosine at low cell density, S1P hydrolysis was measured. [³³P]S1P was synthesized as described under "Experimental Procedures." The amount of ³³P_i released in the medium over 1 h was significantly higher at low compared with high cell density (Fig. 6A), suggesting increased S1P hydrolysis. The initial kinetic rate was increased by 2-fold, being 21 ± 2 nmol of P_i/min/10⁶ cells at high cell density versus 42 ± 6 nmol at low cell density (means ± S.E.). When co-incubated with S1P, LPA induced a significant dose-dependent decrease in S1P hydrolysis exclusively in cells cultured at low density (Fig. 6B).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of cell density on S1P hydrolysis. A, time course of S1P hydrolysis at low (2 × 104 cells/cm2) or high (1 × 105 cells/cm2) cell density. Activity was measured as the amount of 33Pi radioactivity released in the medium from mesangial cells treated with 10 µM [33P]S1P (10 nmol, 220,000 cpm). B, effect of LPA on S1P hydrolysis. S1P hydrolysis was measured as described for A at low (2 × 104 cells/cm2) or high (1 × 105 cells/cm2) cell density in the presence of variable concentrations of LPA or vehicle (0.01% (w/v) BSA in PBS) in the assay. *, p < 0.05. Results are means ± S.E. from triplicate determinations, representative of three (A) or two (B) independent experiments.

Metabolism of S1P in Mesangial Cells at Low and High Density-- To clarify the metabolic fate of S1P as a function of cell density, mesangial cells were incubated with [³H]S1P. Cellular [³H]S1P and [³H]sphingosine levels increased progressively. At 120 min, the [³H]S1P level was higher in high-density cells. Conversely, the [³H]sphingosine level increased more in low- than in high-density cells (Fig. 7, A and B). Over 120 min, the [³H]S1P level decreased by ~25%/10⁶ cells in the extracellular medium of low-density cells versus 18%/10⁶ cells in the medium of high-density cells. Under the same conditions, [³H]sphingosine remained nearly undetectable (data not shown). Incubation with [³H]sphingosine provided essentially similar data. The initial uptake of [³H]sphingosine was higher in cells cultured at low density (Fig. 7D). At 120 min, the [³H]S1P level was significantly higher in high- than in low-density cells, whereas the [³H]sphingosine level increased more in the low- than in the high-density cells (Fig. 7, E and F). Altogether, these data suggest that increased conversion of [³H]S1P to [³H]sphingosine occurs in cells cultured at low density and correlates with increased cellular uptake of [³H]sphingosine and decreased intracellular conversion of [³H]sphingosine to [³H]S1P. Consequently, the ratio of [³H]S1P to [³H]sphingosine decreased in low-density cells. Parallel to this, the [³H]ceramide levels increased similarly in cells at low and high density in the presence of [³H]S1P (Fig. 7C). This suggests that there is no difference regarding the conversion of [³H]S1P or [³H]sphingosine to [³H]ceramide.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Metabolism of [3H]S1P and [3H]sphingosine in mesangial cells. 10 µM [3H]S1P (A-C) or 10 µM [3H]sphingosine (D-F) (10 nmol, 220,000 cpm) was added to mesangial cells cultured at low (2 × 104 cells/cm2) or high (1 × 105 cells/cm2) cell density. Quantification of lipids, S1P, sphingosine, and ceramides was performed at various incubation times in the extracellular medium (D) or in cell extracts (A-C, E, and F) after separation by thin-layer chromatography as described under "Experimental Procedures." *, p < 0.05. Results are means ± S.E. from triplicate determinations, representative of two independent experiments.

Generation of Ceramides in Mesangial Cells at Low Cell Density-- Increased conversion of [³H]sphingosine to [³H]ceramide at low cell density was not suggested by the above experiments (Fig. 7C). However, as ceramides are usually involved in the apoptosis process, a hypothesis could be made that ceramide can be independently generated. To clarify this point, the total ceramides were quantified by the diacylglycerol kinase technique during S1P-induced apoptosis. Upon S1P stimulation, a significantly higher increase in the ceramide level was observed in low- compared with high-density cells (Fig. 8). We then tested the hypothesis that an increased de novo synthesis of ceramide is involved in S1P-induced apoptosis. Low-density cells were subjected for 24 h to 10 µM S1P in the presence of 15 µM fumonisin B, used as a ceramide synthase inhibitor (20). Apoptosis induced by etoposide was used as a control for the effect of fumonisin B. Fumonisin B (15 µM) reduced the apoptosis of low-density cells treated for 24 h with 30 µM etoposide (15.1 ± 1.2% versus 10.2 ± 1.1% apoptotic cells, n = 3) by 27%. On the contrary, under the same conditions, fumonisin B slightly increased the apoptosis induced by 10 µM S1P (17.8 ± 2.5% versus 21.1 ± 1.8% apoptotic cells in the presence of fumonisin B, n = 3; not significant).

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Production of endogenous ceramide in mesangial cells depending on cell density. Shown is the time course of the production of endogenous ceramide in mesangial cells after treatment with 10 µM S1P or vehicle (0.01% (w/v) BSA in PBS). Ceramide production was assessed in mesangial cells cultured at low (2 × 104 cells/cm2) or high (1 × 105 cells/cm2) cell density in the presence of 5% (v/v) FCS. Quantification of ceramide was performed with the diacylglycerol kinase method as described under "Experimental Procedures." C8-ceramide was used as a standard for quantification, and C8-ceramide phosphate was used as a standard for migration. Results are means ± S.E. from triplicate determinations, representative of two independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LPLs (S1P and LPA) are potent mitogenic mediators supposed to influence cell responsiveness in various pathophysiological situations (5-9). Regarding sphingolipids, the balance between S1P and sphingosine is considered as a rheostat for cells deciding between death and survival (17, 20). We have previously observed that the effect of S1P on the proliferation of mesangial cells is variable (15). In that study, it was suggested that the role of the density of the cell culture is an important factor, something that we have tested further here.

We have confirmed in this study that the effect of S1P is highly dependent upon the density of the cell culture, as demonstrated by [³H]thymidine incorporation assays. At subconfluent cell density, S1P stimulated the proliferation of rat mesangial cells in a dose-dependent fashion. S1P-induced proliferation involved the long-term phosphorylation of the MAPKs ERK1 and ERK2. This result is in agreement with other studies in mesangial cells where sustained activation of ERK1/2 has been related to mitogenesis induced by growth factors such as PDGF, LPA, and angiotensin II (11, 39, 40). The known S1P receptors of the EDG family are coupled with heterotrimeric G proteins. In this case, the steps between the receptor and Ras-ERK1/2 activation may involve various processes such as G_i/-linked pathways and transactivation (41-44). In our experiments, [³H]thymidine incorporation and ERK1/2 phosphorylation were pertussis toxin-dependent, suggesting that G_i-dependent pathways are involved in S1P-induced mesangial cell proliferation. S1P is a ligand of several receptors of the EDG family. We found that several of these receptors were well expressed by rat mesangial cells (i.e. EDG-1, -3, -5, and -8), some of them being potentially coupled to G_i (7-9). As recently reviewed, the biological properties of S1P may be due to both intracellular and extracellular signaling (17). However, ERK1/2 activation by S1P is frequently pertussis toxin-dependent (8), as in our model; and in many cases, S1P-induced proliferation can be related to EDG-dependent signaling.

The unexpected result of this study was that S1P became antiproliferative when the density of the cell culture was decreased. Under these conditions, S1P even inhibited the proliferation induced by PDGF, LPA, or serum. As analysis of the release of lactate dehydrogenase precluded any cell toxicity, S1P was supposed to induce apoptosis in that case. DNA fragmentation was evidenced in S1P-treated low-density cells by Hoechst staining and flow cytometry. Moreover, SAPK phosphorylation suggested that S1P elicited signaling pathways leading to a cell death program in low-density cells.

Our results have suggested a relationship between the apoptosis of mesangial cells cultured at low cell density and the formation of sphingosine. In a number of cell types, sphingosine induces either apoptosis or proliferation, specifically depending on the activity of sphingosine kinase (17). For instance, sphingosine and its methylated derivative N,N-dimethylsphingosine induce apoptosis of cancer cells and transformed mesangial cells (28). Conversely, activation of sphingosine kinase, allowing conversion of sphingosine to S1P, prevents apoptosis (22, 23). In our study, we observed that sphingosine, like S1P, induced apoptosis of low-density mesangial cells in a dose-dependent manner. Therefore, we tested whether or not S1P hydrolysis was increased in cells cultured at low density. Enhanced S1P hydrolysis was demonstrated by incubation with [³³P]S1P. Incubation with [³H]S1P also demonstrated that low-density cells accumulated [³H]sphingosine, with [³H]sphingosine being rapidly incorporated into the cell and poorly converted to [³H]S1P. As a result, the ratio of [³H]sphingosine to [³H]S1P rose significantly in low-density cells at 120 min. Incubation with [³H]sphingosine provided concordant results. It is interesting to note that [³H]sphingosine was more actively taken up by low-density cells (Fig. 7, D and E). Moreover, a low conversion rate of [³H]sphingosine to [³H]S1P was observed, which was in favor of decreased sphingosine kinase activity in low-density cells. The down-regulation of sphingosine kinase at low cell density is in agreement with the increase in thymidine incorporation observed with sphingosine only in high-density cells.

In our opinion, the increased S1P hydrolysis as documented by [³³P]S1P incubation in Fig. 6 could account for the accumulation of sphingosine observed in low-density cells. We suggest that S1P hydrolysis and sphingosine uptake are crucial steps leading to the accumulation of [³H]sphingosine in low-density cells. Moreover, this could be enhanced by the decrease in sphingosine kinase activity. Hence, sphingosine accumulation seems pertinent to explain the S1P-induced apoptosis of cells at low density. In this regard, it is worth noting that the dose responses of sphingosine and S1P were at a similar level because either 5 µM S1P or 1 µM sphingosine was sufficient to induce apoptosis.

A similar concentration has been used in another study of sphingosine-induced apoptosis (28). In that study, it was suggested that apoptosis of serum-starved cells may result from the mixed effect of treatment and starvation. Specially, the intracellular level of sphingosine may be modified by the down-regulation of sphingosine kinase activity due to the starvation of growth factors like PDGF (22). However, it was demonstrated in the study of Sweeney et al. (28) that sphingosine-induced apoptosis may occur in various cell lines even in the presence of growth factors. This was the case in our study, where S1P-induced apoptosis persisted in growing cells and was reverted neither by 10 µM LPA in quiescent cells nor by PDGF in growing cells (Fig. 4). Starvation of growth factors, which can be responsible for a modification of the "apoptosis threshold" (for instance, by modifying sphingosine kinase activity), was not necessary for apoptosis at low density. Therefore, one must suppose an additional effect of low density, such as increased S1P hydrolysis, to explain the S1P-induced apoptosis.

The role of intracellular sphingolipids in cell death is dependent upon two major interrelated components: ceramide and sphingosine. Both compounds may play an active part in apoptosis. Although the role of ceramide has been largely investigated in the past, more recent studies have pointed to the accumulation of sphingosine as being directly responsible for cell death (26, 45-47). Sphingosine may be directly involved in mitochondrion-dependent apoptosis, as recently suggested (26). In this regard, the relevance of sphingosine as an inductor of apoptosis is also related to a modification of the balance between sphingosine and S1P as discussed above. Alternatively, increased sphingosine levels may also enhance the synthesis of ceramide by ceramide synthase. Ceramide possesses apoptotic properties that are also intimately related to mitochondrial function, as recently reviewed (27).

In this study, we observed generation of ceramide, but no increased conversion of [³H]sphingosine to [³H]ceramide, during S1P-induced apoptosis. This suggests that de novo synthesis of ceramide from sphingosine, under the control of sphingosine N-acetyltransferase (ceramide synthase), is not significantly involved in this process. On the other hand, apoptosis was not inhibited in this study by incubation with fumonisin B, an inhibitor of ceramide synthase. On the contrary, fumonisin B inhibited etoposide-induced apoptosis, but slightly increased S1P-induced apoptosis. Logically, the blockade of ceramide synthase by fumonisin B would result in decreased ceramide levels and increased intracellular sphingosine and sphinganine levels. As discussed above, increased levels of sphingosine and sphinganine may be responsible for cell death. Hence, apoptosis seems more related to the accumulation of sphingosine than of ceramide in our model. Similarly, in the study of Sweeney et al. (28), N,N-dimethylsphingosine and sphingosine induced apoptosis in the absence of ceramide generation. Moreover, in a recent study, the generation of ceramide protected mesangial cells against sphingosine-induced cell death (29).

Another pathway to ceramide production must be hypothesized to explain the progressive accumulation of ceramide in our study. Activation of acid sphingomyelinase is a frequent event during apoptosis following the recruitment of the adaptor proteins TRADD and FADD and activation of the early caspases (27, 48). In this regard, SAPK phosphorylation as observed here is frequently associated with activation of acid sphingomyelinase (49). Therefore, the generation of ceramide by sphingomyelinase could explain the ceramide increase observed here, which could, in turn, be involved in later phases of the apoptosis process.

Thus, these data do not allow unambiguous interpretation of the respective roles of sphingosine and ceramide during S1P-induced apoptosis of low-density mesangial cells. Nevertheless, they do not suggest a major role for ceramide generation in the induction of apoptosis. Ceramides are not generated from the accumulated sphingosine. As sphingosine may alter mitochondrial function, sphingosine alone can start apoptosis. However, later production of ceramide by sphingomyelinase may be involved as an additional factor.

Alternatively, other mechanisms not related to the altered metabolism of sphingolipids have been proposed in a few cases where S1P induces cell growth arrest. Apoptosis or proliferation arrest has been observed in cells derived from primary cultures such as hippocampal neurons and hepatic myofibroblasts (37, 38). It has also been described in several cell lines such as hepatoma cells, ovarian cancer cells, and HEK293 cells transfected with EDG receptors (50-52). In hepatic myofibroblasts, the phenotype of which is similar to that of mesangial cells, S1P-induced growth arrest is related to cyclooxygenase-2 induction and prostaglandin E₂ production (38). However, in our work with mesangial cells, we ruled out such a mechanism, as incubation with ibuprofen (a cyclooxygenase-2 inhibitor) was of no benefit during S1P-induced apoptosis (data not shown). In other cases, S1P-induced apoptosis may be related to receptor-dependent events. Overexpression of EDG-3 or EDG-5 receptors is responsible for apoptosis induced by 0.1 µM S1P (52). S1P-induced apoptosis in hippocampal neurons is associated with activation of calcium-dependent phosphatases (37), a mechanism possibly related to activation of EDG-3/EDG-5 G_q-coupled receptors. EDG receptors may also be involved in the expression of Bax protein observed during S1P-induced apoptosis of hepatoma cells (50). The hypothesis that EDG receptors are involved in the process has not been precisely tested here. However, we performed semiquantitative evaluations of the expression of EDG receptors and did not detect a clear difference between mesangial cells cultured at low and high density (data not shown). In addition, the similar effect observed with S1P and sphingosine did not favor a mechanism dependent on S1P receptors in our model.

Finally, S1P-induced apoptosis of mesangial cells may be closely compared with the modulated growth and adhesion of ovarian cancer cells (51). In that study, S1P induced cell death of cells in suspension, but stimulated growth of attached cells. This suggests that the presence of homologous cell-cell contacts may drastically modify the responsiveness to S1P. This allows a comparison with our model; however, the effect of sphingosine was not tested in the study of Hong et al. (51). Similarly, it has been demonstrated that up-regulation of the cadherin-catenin complex decreases sphingosine-induced cell death (46), and a relationship between sphingosine and -catenin-regulated pathways is supported by another recent study (47).

Hydrolysis of LPLs is dependent on lipid phosphate phosphohydrolases (53). Three isoforms of the type 2 lipid phosphate phosphohydrolase (lipid phosphate phosphohydrolase-1, -2, and -3) have been cloned from mammalian cells, with these enzymes having broad substrate specificity with similar efficiencies against LPA, S1P, phosphatidic acid, ceramide 1-phosphate, and diacylglycerol pyrophosphate. However, genes encoding for phosphatases with a remarkable specificity for phosphorylated sphingoid bases have been identified in yeast; and recently, murine S1P phosphohydrolase-1, a homolog of the yeast phosphohydrolases, was cloned (54, 55). Murine S1P phosphohydrolase-1 differs from the other mammalian lipid phosphohydrolases by sequence particularities and by its high specificity for S1P. In NIH 3T3 cells, overexpression of murine S1P phosphohydrolase-1 leads to a 4-fold increase in its activity, which is responsible for S1P-induced apoptosis. A 2-fold increase in S1P hydrolysis was observed at low cell density, which could explain the apoptotic effect of S1P. The nature of the enzyme involved has not been documented in this study. However, the lipid phosphohydrolase activity that increased at low density was probably not specific for S1P because LPA partly inhibited S1P hydrolysis in the in vitro assay (Fig. 6B). Even so, this would not preclude the involvement of S1P phosphohydrolase-1 in that case. We detected both lipid phosphate phosphohydrolase-1 and the rat homologous sequence of S1P phosphohydrolase-1 using semiquantitative reverse transcription-PCR. We observed no significant difference between cells cultured at low and high cell density (data not shown), but increased lipid phosphate phosphohydrolase activity may be due to post-translation regulation (53). Note that even if LPA phosphohydrolase activity is increased, it is probably not relevant in explaining the apoptosis occurring at low cell density. LPA did not increase S1P-induced apoptosis (Fig. 4). Moreover, it is worth noting that S1P-induced apoptosis occurred in a serum-free medium in the absence of any other factor such as LPA contained in FCS.

Finally, the physiological relevance of these results may be considered. First, the notion of cell density is not easily transferable in vivo. However, a number of cell functions seem to be regulated by the density of cell cultures. For instance, the up- or down-regulation of the tyrosine phosphorylation status of focal adhesion kinase or paxillin has been observed as a function of cell density (56). It is worth noting that the cadherin-catenin complex, which is highly regulated by the level of homologous cell-cell contacts, modulates the apoptosis induced by sphingosine (46). This suggests that the effect of cell density observed here may be valid for other cells. The response of human osteoblasts to sphingolipids is also density-dependent, as determined by preliminary data.² Another point is related to the relevance of the concentrations able to induce apoptosis. The actual concentration of S1P in renal glomeruli is unknown. However, the S1P concentration can rise to micromolar levels in plasma (57). Platelet activation might account for significantly increased local S1P levels in several renal diseases. In addition, the hypothesis of paracrine production of S1P in vascular systems is strongly supported by the recent observation of the extracellular export of sphingosine kinase (58). Altogether, these data suggest the possibility of increased S1P concentrations in the vicinity of mesangial cells. Resident mesangial cells are normally maintained in a quiescent phenotype and probably develop poor cell-cell contacts. In pathology, enhanced production of S1P may occur. Then S1P hydrolysis might act as one of the glomerular "self-defense mechanisms" (59), allowing apoptosis to be a component of the glomerular remodeling after injury.

In conclusion, it was demonstrated that S1P stimulated the proliferation of subconfluent mesangial cells, whereas at low density, increased S1P hydrolysis was responsible for phosphorylation of SAPKs and apoptosis. Apoptosis seemed to be mainly determined by the accumulation of sphingosine, together with decreased conversion to S1P. Ceramide that did not originate from the accumulated sphingosine was also generated. Thus, it was suggested that the sphingolipid rheostat was readjusted at low cell density, with up-regulation of S1P hydrolysis and down-regulation of sphingosine kinase. As S1P may be produced actively in glomerular diseases, it may be of interest in the future to assess the level of S1P hydrolysis in circumstances involving excessive proliferation of mesangial cells.

	ACKNOWLEDGEMENTS

We thank Yvette Jonquières and Christiane Muss for editorial help in the preparation of this manuscript.

	FOOTNOTES

^* This work was supported by grants from the Association pour la Recherche contre le Cancer and from the Ligue contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-5-61779400; Fax: 33-5-61779401; E-mail: jpsalles@toulouse.inserm.fr.

Published, JBC Papers in Press, January 30, 2002, DOI 10.1074/jbc.M108933200

² I. Gennero, J. Fauvel, M. Niéto, C. Cariven, F. Gaits, F. Briand-Mésange, H. Chap, and J. P. Salles, unpublished data.

	ABBREVIATIONS

The abbreviations used are: LPLs, lysophospholipids; S1P, sphingosine 1-phosphate; LPA, lysophosphatidic acid; EDG, endothelial differentiation gene; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FCS, fetal calf serum; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PDGF, platelet-derived growth factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES