Sphingosine 1-phosphate stimulates rat mesangial cell proliferation from outside the cells

Norio Hanafusa, Yutaka Yatomi1, Koei Yamada, Yuichi Hori, Masaomi Nangaku, Toshihiro Okuda, Toshiro Fujita, Kiyoshi Kurokawa2 and Masafumi Fukagawa3,

Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, 1 Department of Laboratory Medicine, Yamanashi Medical University, Yamanashi, 2 Division of Nephrology, Tokai University School of Medicine, Kanagawa, and 3 Division of Nephrology and Dialysis Center, Kobe University School of Medicine, Kobe, Japan



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Proliferation of mesangial cells (MCs) is the initial step in glomerulonephritis, and platelet-derived mediators have been shown to play a significant role in this proliferation. Sphingosine 1-phosphate (S1P), one of the sphingolipids, is abundantly stored in platelets and is released upon stimulation. We examined the effects of S1P and related sphingolipids on the cell fate of cultured MCs in order to elucidate potential roles of these lipid mediators in glomerulonephritis.

Methods. Cell proliferation was evaluated by bromodeoxy uridine (BrdU) incorporation together with MTS assay. Apoptosis of MCs was evaluated by examining annexin V staining and typical morphological changes in nuclei. We also examined the metabolism of [3H]sphingosine in MCs in either the presence or absence of platelet-derived growth factor (PDGF). The expression of endothelial differentiation genes (edg), which are the cell surface receptors for S1P in MCs, was examined by RT-PCR.

Results. S1P, but not the other sphingolipids, stimulated MC proliferation. In contrast, dimethylsphingosine (DMS) induced apoptosis in the MCs. The amount of sphingosine (Sph) converted into S1P was small and was not affected by PDGF. This observation suggested that Sph kinase activity producing S1P from Sph was low in the MCs. Furthermore, expression of edg-1, -2 and -5 in MCs was confirmed by RT-PCR.

Conclusions. Our observations suggest that S1P stimulates MC proliferation from outside the cells, and not as a second messenger for PDGF. The modulation of MC fate with sphingolipids may provide possible strategies for the treatment of glomerulonephritis.

Keywords: apoptosis; dimethylsphingosine; endothelial differentiation genes; mesangial cell; proliferation; sphingosine 1-phosphate



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Sphingolipids have recently emerged as a class of lipids involved in signal transduction [1,2]. The sphingolipids have been distinguished by their apparent ability to participate in pro- or anti-proliferative pathways [1,2]. For example, it has been shown that ceramide (Cer) and dimethylsphingosine (DMS) induce apoptosis [2], whereas sphingosine 1-phosphate (S1P) stimulates mitosis [1,3] and works as a second messenger in the proliferation induced by platelet-derived growth factor (PDGF) and by serum [3]. It has also been proposed that the balance between intracellular levels of Cer and S1P, as well as regulatory effects of these sphingolipids on members of mitogen-activated protein kinases, may determine cell fate [4].

Platelets demonstrate a unique metabolism of S1P. These anucleate cells exhibit high sphingosine (Sph) kinase activity, which phosphorylates Sph into S1P; however, they lack lyase activity, which degrades S1P into ethanolamine phosphate and fatty aldehyde [5]. As a result, platelets abundantly accumulate S1P [6] and release it upon stimulation [5]. Furthermore, S1P itself has been shown to stimulate platelets [5] as well as to protect endothelial cells from apoptosis [7]. These observations suggest that S1P acts as an autocrine or paracrine mediator released from activated platelets.

Proliferation of mesangial cells (MCs) is an important and initial step in the pathogenesis of glomerulonephritis. This early step is usually followed by the accumulation of extracellular matrix within the glomeruli [8]. Platelets have been shown to play a significant role in mediating MC proliferation in vivo. For example, platelets were found together with neutrophils in damaged or inflamed glomeruli [9]. In anti-Thy-1 nephritis, an experimental model of mesangioproliferative glomerulonephritis, platelet depletion by anti-platelet antibody decreased the extent of MC proliferation [10]. These actions have been mainly attributed to factors released by platelets, such as PDGF.

To elucidate the role of S1P in MC proliferation, we investigated the effects of S1P and related sphingolipids on the fate of MCs. We found that S1P, but not other Sph derivatives, stimulates MC proliferation, whereas DMS induced MC apoptosis. These findings suggest that S1P stimulates proliferation of MCs from outside the cells.



   Materials and methods
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 Materials and methods
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Preparation of rat MCs
Primary culture of rat MCs was prepared by a standard sieving method [11]. The cells were maintained in Dulbecco's modified Eagle's Medium (DMEM) (Nissui Seiyaku, Tokyo, Japan) containing 17% foetal bovine serum (JRH Biosciences, Lenexa, KS, USA) at 37°C under a humidified atmosphere of 5% CO2/95% air. In order to avoid contamination of epithelial cells or endothelial cells, we used cells between the 5th and 15th passage number. The cells were confirmed as MCs by positive staining for vimentin, alfa-smooth muscle actin and Thy-1 antigen (data not shown).

MC proliferation
MC proliferation was examined by two methods. The first method measured the amount of bromodeoxy uridine (BrdU) incorporated into chromosomal DNA during synthesis. The second method used a colorimetric assay of MTS, one of the tetrazolium compounds (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) that changes colour due to the intracellular reductive products in proportion to the number of cells.

MCs were seeded in 96-well plates (Becton Dickinson, Franklin Lakes, NJ, USA). When cells reached ~60% confluency, the medium was changed to DMEM without serum. After a 48 h serum starvation period, specific concentrations of S1P or 10 µM of other sphingolipids were added to the wells and the cells were incubated for an additional 24 h. S1P and DMS were obtained from BIOMOL (Plymouth Meeting, PA, USA). Sph and Cer (C2-Cer, cell membrane permeable type) were purchased from Sigma (St Louis, MO, USA). S1P was resolved in 50% (v/v) ethanol (EtOH) in phosphate-buffered saline (PBS). The other sphingolipids were resolved in 100% EtOH. Concentrations of EtOH in the media, including samples without sphingolipids, were adjusted to 0.5%, a concentration that did not have effects on MC proliferation or apoptosis.

For the BrdU incorporation assay, performed 6 h prior to the assays, 10 µM BrdU was added to the medium. The amount of incorporated BrdU was then determined by Cell Proliferation ELISA system, version 2 (Amersham LIFE SCIENCE, Amersham Place, Buckinghamshire, UK). This non-RI system detects the amount of BrdU within chromosomal DNA by peroxidase-labelled anti-BrdU antibody-coloured 3,3',5,5'-tetramethylbenzidine. The optic absorption at 450 nm was measured to detect BrdU using DIGISCAN (ASYS Hitech GmbH, Eugendorf, Austria), according to the supplied protocols. The results were expressed in relative data. The mean values of control wells without mitogen were defined as zero and wells containing 17% FCS were defined as 100.

CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) was employed for MTS assays. We followed the manufacturer's protocol. During the final 15 min of incubation with various stimulants, 20 µl of MTS solution was added to each well. At the end of the incubation period, optic absorbance at 550 nm was measured. Various numbers of MCs, counted using a counting chamber, were seeded and subjected to MTS assays on the calibration plate. The results were expressed as relative values. The mean number of MCs in the wells without mitogen was defined as 100.

Both BrdU assays and MTS assays were performed with an n value of 8 and were duplicated.

Detection of apoptosis
MCs were cultured on 6 cm plates (Corning, Corining, NY, USA) with up to 60% confluency. After 48 h of serum starvation, the cells were challenged with 10 µM of S1P or DMS for 24 h. The cells were then trypsinized (Trypsin-EDTA, Life Technologies, Rockville, MD, USA) and scraped from the plates. After centrifugation, the cells were stained with 1 mM bisbenzimide hydrochloride (Hoechst 33258) (Sigma, St Louis, MO, USA) in PBS for 2 min. Morphological changes to the nuclei were studied under a fluorescent microscope with FITC/DAPI/Tex Red filter (Olympus, Tokyo, Japan), and photographs were taken using Kodak Gold 100 film (Eastman Kodak, Rochester, NY, USA).

In addition to the morphological studies, the percentages of apoptotic cells were determined by flow cytometry. Cells were stained with both fluorescein (FITC)-labelled annexin V and propidium iodide (PI).

The cultured cells were submitted to the same procedures described in the morphological study. They underwent apoptosis with each of the 10 µm sphingolipids on the 6 cm plates. Cells were removed from the plates, trypsinized and subjected to the assays described below. For annexin V binding assays, we used Annexin V-FITC Apoptosis Detection Kit (Medical & Biological Laboratories, Nagoya, Japan). Cells were incubated at room temperature in binding buffer for 10 min with annexin V-FITC and PI. The cells were then analysed by FACScan and LYSYS II software for analysis (both from Becton Dickinson, Franklin Lakes, NJ, USA). The fraction of cells that were annexin positive and PI negative were defined as apoptotic.

Metabolism of [3H]Sph in MCs
MCs, cultured in 12-well plates (Becton Dickinson) to 90% confluency, were incubated with 1 µM Sph containing [3H]Sph (0.1 µCi) (NEN Life Science Products, Boston, MA, USA) in the presence or absence of rat recombinant PDGF-BB (5 ng/ml) (Sigma, St Louis, MO, USA). At the indicated time points, the reaction was terminated by removing the medium and by adding ice-cold methanol to the cells. Lipids were extracted separately from the cells and medium by the method of Bligh and Dyer [12], and were then analysed using tritium-labelled sphingolipids as described previously [5]. Portions of lipids obtained from the chloroform phase were applied to silica gel high-performance thin layer chromatography (TLC) plates (Merck, Darmstadt, Germany), and the plates were then developed in butanol/ acetic acid/water (3:1:1). The TLC plates were scanned with a BAS-2000 (Fujifilm, Tokyo, Japan).

Detection of edg expression
The expression of the edg family of receptors was detected by RT-PCR. Total RNA was extracted by a standard acid guanidinium thiocyanate-phenol-chloroform (AGPC) method from 90% confluent MCs cultured on 10-cm plates (Becton Dickinson). Complementary DNA was synthesized from 2 µg of total RNA with SuperScript II (Life Technologies, Rockville, MD, USA) according to the manufacturer's instructions. PCR reactions were performed with synthetic primers (Nippon Flour Mills, Tokyo, Japan), ExTaq and the appropriate buffer (TaKaRa, Tokyo, Japan). Amplification was conducted during 30 cycles of 30 s at 94°C, 30 s at 54°C, and 30 s at 72°C. We also performed PCR on nucleic acid samples without reverse transcription in order to exclude the possibility of contamination by chromosomal DNA. The following oligonucleotide primer pairs were used: 5'-CCTCCTTGCTATCGCCATTGAG-3' (667–688 bp) (sense) and 5'-TGAGTTCAGCACAGCCAGAACC-3' (1156–1177 bp) (antisense) for edg-1 [13]; 5'-ACGAGTTGCTTCTTGTGCCACC-3' (84–105 bp) (sense) and 5'-ACCACACGTCGGTTGCTCATTC-3' (624–645 bp) (antisense) for edg-2 [14]; and 5'-CATGTACCTGTTCCTCGGCAAC-3' (201–222 bp) (sense) and 5'-GCGAAGGCAAAGAAATAATGGG-3' (809–830 bp) (antisense) for h218/agr16, the rat homologue of edg-5 [15].

Statistical analyses
Stat View Version 5.0, (SAS Institute, Cary, NC, USA) was used for statistical analysis. Bonferroni/Dunn tests were used for comparisons between three or more groups. P values of <0.05 were defined as statistically significant. The data were expressed as means±SD (n=8 and duplicated for BrdU incorporation and MTS assays; n=3 for annexin V binding assay).



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DNA synthesis in MCs stimulated by S1P
The incorporation of BrdU into chromosomal DNA revealed that S1P stimulates DNA synthesis in MCs (Figure 1AGo). A mitogenic effect was observed at 0.1–3 µM. Regression analysis confirmed a dose relationship at doses between 0.1 and 1 µM (r=0.494, P=0.0004). For unknown reasons, the stimulatory effect of S1P became less evident at 10 µM. The MTS assays also revealed that 3 µM of S1P significantly increased the number of MCs (Figure 1BGo). In contrast, DMS at 10 µM decreased MC numbers and totally abolished MC viability (Figure 1CGo). We additionally counted actual numbers of MCs per 1 mm2 on 96-well plates using inverted microscopy (Olympus, Tokyo, Japan) and found that MC numbers were increased to values similar to the values in the MTS assay (data not shown).



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Fig. 1.  Stimulation of MC proliferation by S1P. (A) BrdU incorporation into MCs treated with the indicated concentrations of S1P was measured as described in Materials and methods. BrdU incorporation was significantly increased by S1P at concentrations between 0.1 and 3 µM compared with control cells. *P<0.05. Dose dependency was also observed between 0.1 and 1 µM (r=0.494, P<0.05). (B) The number of MCs was also increased by S1P. The maximum effect was observed at 3 µM. At that concentration, the increase in cell number was statistically significant (P<0.05). (C) DMS, another sphingolipid, decreased the number of MCs and totally abolished the reaction of MTS at 10 µM (#). Sph at 10 µM also decreased MC numbers compared with control (P<0.05). Open bars represent 1 µM, shaded bars are for 3 µM, and filled bars are for 10 µM of sphingolipids.

 

Effects of sphingolipids on MC apoptosis
Sphingolipid mediators, such as Cer, Sph and DMS, have been shown to induce apoptosis in other cell types [2]. In the present study, nuclei from either serum-deprived or S1P-added cells stained with Hoechst 33258 showed normal appearances (Figure 2AGo and BGo). However, a significant number of cells treated with DMS manifested characteristic appearances of apoptosis, including nuclear and cytoplasmic condensation and fragmentation (Figure 2CGo).



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Fig. 2.  Detection of apoptosis in MCs treated with various sphingolipids. MCs were challenged with 10 µM S1P (A), serum starvation only (B), or with 10 µM DMS (C). Morphological changes of MCs were detected using Hoechst 33258 staining and examined under a fluorescence microscope. DMS markedly induced apoptotic morphological changes in nuclei, such as apoptotic bodies (C, arrowheads). These changes were also induced by serum starvation in a limited number of cells (not shown).

 
Quantitative analyses of the proportion of apoptotic cells measured by annexin V binding revealed that DMS significantly increased the number of apoptotic MCs (Figure 3AGo and CGo). Conversely, MCs challenged with S1P did not show a significant difference compared with the vehicle-treated group (Figure 3AGo). These results indicated that DMS, unlike S1P, induces apoptosis in MCs.



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Fig. 3.  MC apoptosis induced by various sphingolipids determined by annexin V binding to the cells. (A) Percentage of apoptotic MCs determined by annexin V binding. Annexin V-positive and PI-negative cells were detected by flow cytometry. Ten micromoles of DMS significantly increased the population of apoptotic MCs compared with control. *P<0.05. FITC-labelled annexin V (horizontal axis) and PI (vertical axis) fluorescence profiles were plotted on a logarithmic scale (B and C). The results of cytograms show the population of apoptotic cells (lower right quadrant) induced by either serum starvation (B) or DMS (C).

 

An extracellular action of S1P in MC proliferation
It has been reported that Sph kinase activity is elevated and that S1P levels are increased by PDGF in Swiss 3T3 fibroblasts [3]. We examined the amount of S1P converted from Sph in order to determine the activity of Sph kinase in MCs. In these cells, incorporated [3H]Sph was mainly converted to [3H]Cer and then further to [3H]sphingomyelin in a time-dependent manner, whereas only a small amount of [3H]S1P was transiently produced from [3H]Sph (Figure 4AGo). Moreover, the conversion of Sph into S1P was not affected by PDGF (Figure 4BGo). This observation argues against a role of S1P as a second messenger for PDGF in MCs. [3H]S1P was not detected in the medium and this did not change with PDGF stimulation. This finding indicates that MCs do not release S1P into the extracellular environment.



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Fig. 4.  Metabolism of [3H]Sph in MCs. MCs incubated with [3H]Sph (0.1 µCi) were challenged with (B) or without (A) 5 ng/ml PDGF for various durations. Lipids were then extracted from the cells or the media and analysed for tritium-labelled sphingolipids by TLC autoradiography. Locations of standard lipids are indicated on the right. SM, sphingomyelin; Ori, origin.

 
In order to examine the possibility that S1P acts from outside the MCs, we measured the expression of S1P receptors, edgs [16] by RT-PCR. Both edg-1 and -5 were identified as receptors for S1P, and edg-2 was identified for both S1P and lysophosphatidic acid (LPA). RNA from MCs was prepared for reverse transcription, followed by PCR amplification of specific transcripts. As shown in Figure 5Go, MCs expressed mRNA for edg-1, -2 and -5.



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Fig. 5.  Detection of mRNA expression by RT-PCR for S1P receptors in the MCs. Amplified products for edg-1, -2 and -5 were electrophoresed in 2% agarose gel. The size of each product was consistent with the predicted values from the published sequences: 511, 562 and 630 bp, respectively.

 



   Discussion
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 Materials and methods
 Results
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In agreement with mitogenic properties of S1P in several cell lines [1,3,4,7], S1P stimulated MC proliferation and activated DNA synthesis. Similar findings were reported in a recent short communication [17]. Although S1P has been proposed as an intracellular second messenger in certain cell types [1,3,4], this possibility is unlikely in MCs. This is because Sph, the precursor of S1P, was not mitogenic for MCs. In addition, the level of S1P converted from Sph was not affected by PDGF even though Sph is mitogenic only when it is converted into S1P intracellularly. It is more likely that edgs, cell surface receptors for lysophospholipids including S1P [18], mediate the extracellular actions of S1P. The observation that S1P receptors were expressed in MCs supports an extracellular action of S1P on MCs.

Recently, it has been shown that intracellular Ca2+ signalling was induced by S1P in MCs [18]. This novel sphingolipid mediator is probably an important agonist for MCs. Furthermore, modulation of MCs by sphingolipids may have pathophysiological importance since S1P is released from platelets [5], and platelets are found in damaged or inflamed glomeruli [9]. S1P may be supplied as a survival factor for MCs from activated platelets in vivo. Supporting this, results of [3H]Sph metabolism indicated that S1P was not released from PDGF-activated MCs.

Of the various sphingolipids examined, DMS most potently induced apoptosis in the MCs. Although Cer had been reported to induce apoptosis in MCs [19], its effect in the present study was much smaller than DMS and not significant. DMS is a potent inhibitor of Sph kinase. However, the activity of Sph kinase appeared to be low in MCs because only a limited amount of S1P was produced from Sph. Thus, the action of DMS to inhibit Sph kinase may not be responsible for the apoptotic effect of DMS. Although it has been suggested that DMS induces apoptosis by inhibiting protein kinase C, recent reports have revealed that DMS induces apoptosis through complex mechanisms involving more than protein kinase C inhibition [2]. Further studies will elucidate the mechanisms of DMS-induced apoptosis in MCs.

Even though DMS exerts a variety of cellular actions (including induction of apoptosis) and Sph N-methyltransferase, the enzyme that synthesizes DMS, exhibits activity in certain tissues or cells [2], DMS was formed in neither resting nor PDGF-stimulated [3H]Sph-labelled MCs (Figure 4Go), nor in the media. Thus, the physiological and pathophysiological implications of apoptosis induced by DMS remain to be solved. Nevertheless, the effects of DMS may be therapeutically important, as discussed below.

Various glomerular diseases, including glomerulonephritis, are characterized by MC proliferation. This may be associated with MC activation and lead to mesangial matrix expansion [8] and glomerulosclerosis. Alternatively, apoptosis of MCs may be the major mechanism for resolution of glomerular hypercellularity in mesangioproliferative glomerulonephritis [20].

Accordingly, mechanisms that initiate, maintain and limit the proliferative response of MCs in glomerular diseases may provide useful insights into future therapeutic strategies for these diseases. In this context, modulation of MC fate by sphingolipids may prove to have important implications. Treatment of glomerulonephritis is currently limited to supportive therapy with or without non-specific immunosuppressive drugs. Further knowledge about the involvement of platelets or related sphingolipids in MC fate may identify new possibilities for treatment of kidney diseases in the future [20].



   Acknowledgments
 
The authors are grateful to Dr Libo Yang for her technical assistance. This work was supported in part by a Program Project Grant from the Ministry of Health (to M.F.), a Special Grant for Medical Research from Ministry of Post and Telecommunications (to M.F.), a grant from Ministry of Science and Education (to Y.Y.), grants in Aid for Scientific Research from the Ministry of Education, Science and Culture (Nos 11671030 and 13671100 to M.N.), and a grant from the Ministry of Health, Labour and Welfare (No. H13-21st century(Seikatu)-17 to M.N.).



   Notes
 
Correspondence and offprint requests to: Masafumi Fukagawa, Associate Professor and Chief, Division of Nephrology and Dialysis Center, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Email: fukagawa{at}med.kobe\|[hyphen]\|u.ac.jp Back



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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received for publication: 20. 2.01
Accepted in revised form: 9.10.01