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First published online April 26, 2007
Stem Cells Vol. 25 No. 7 July 2007, pp. 1713 -1719
doi:10.1634/stemcells.2006-0725; www.StemCells.com
© 2007 AlphaMed Press

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THE STEM CELL NICHE

Sphingosine 1-Phosphate Mediates Proliferation and Survival of Mesoangioblasts

Chiara Donatia, Francesca Cencettia, Paola Nincherib, Caterina Bernacchionib, Silvia Brunellic,d, Emilio Clementie,f, Giulio Cossuc,g, Paola Brunia

aDipartimento di Scienze Biochimiche and Instituto Interuniversitario di Miotogia, Università di Firenze, Firenze, Italy;
bDipartimento di Scienze Biochimiche, Università di Firenze, Firenze, Italy;
cStem Cell Research Institute, Istituto Scientifico H. San Raffaele, Milano, Italy;
dDipartimento di Medicina Sperimentale, Università di Milano-Bicocca, Monza, Italy;
eIstituto di Ricovero e Cura a Carattere Scientifico E. Medea, Bosisio Parini, Italy; Dipartimenti di
fScienze Precliniche Laboratorio Interdisciplinare Tecnologie Avanzate-Vialba and
gBiologia, Università di Milano, Milano, Italy

Key Words. Cellular proliferation • Apoptosis • Cell signaling • Mammalian stem cells • Sphingosine 1-phosphate

Correspondence: Paola Bruni, Ph.D., Dipartimento di Scienze Biochimiche Università di Firenze, Viale G.B. Morgagni 50, 50134 Firenze, Italy. Telephone: 39-0554-598328; Fax: 39-0554-598905; e-mail: paola.bruni{at}unifi.it

Received on November 8, 2006; accepted for publication on April 10, 2007.

First published online in STEM CELLS EXPRESS  April 26, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mesoangioblasts are stem cells capable of differentiating in various mesodermal tissues and are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases, as well as myocardial infarction. The enhancement of their proliferation and survival after injection in vivo could greatly improve their ability to repopulate damaged tissues. In this study, we show that the bioactive sphingolipid sphingosine 1-phosphate (S1P) regulates critical functions of mesoangioblast cell biology. S1P evoked a full mitogenic response in mesoangioblasts, measured by labeled thymidine incorporation and cell counting. Moreover, S1P strongly counteracted the apoptotic process triggered by stimuli as diverse as serum deprivation, C2-ceramide treatment, or staurosporine treatment, as assessed by cell counting, as well as histone-associated fragments and caspase-3 activity determinations. S1P acts both as an intracellular messenger and through specific membrane receptors. Real-time polymerase chain reaction analysis revealed that mesoangioblasts express the S1P-specific receptor S1P3 and, to a minor extent, S1P1 and S1P2. By using S1P receptor subtype-specific agonists and antagonists, we found that the proliferative response to S1P was mediated mainly by S1P2. By contrast, the antiapoptotic effect did not implicate S1P receptors. These findings demonstrate an important role of S1P in mesoangioblast proliferation and survival and indicate that targeting modulation of S1P-dependent signaling pathways may be used to improve the efficiency of muscle repair by these cells.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mesoangioblasts are mesodermal progenitors associated with vessels during the fetal stage of development and persist in postnatal life. They exhibit stem cell features, such as pluripotency and self-renewal ability, and can differentiate in vivo and in vitro into different mesoderm cell types, such as muscle, bone, and adipocytes, in response to specific extracellular cues [1, 2]. Upon injection into the arterial bloodstream, mesoangioblasts are capable of crossing the endothelium, migrate in the tissue interstitium, and are then incorporated into regenerating skeletal muscle fibers. Such an action opens therapeutic perspectives for degenerative diseases of heart and skeletal muscles. In particular, mesoangioblasts have been successfully used to correct morphologically and functionally the pathological alterations observed in the {alpha}-sarcoglycan-null mice, a model of severe muscular dystrophy [3]. Moreover, it has recently been shown that mesoangioblasts repair the infarcted heart, being as effective as bone marrow progenitor cells in reducing postinfarction left ventricular dysfunction [4]. Despite these promising results, problems related to in vitro expansion, efficient engrafting, and survival of mesoangioblasts in the toxic environment of the damaged muscle still hamper their therapeutic development [5]. The present knowledge of the extracellular agents capable of regulating key biological processes in mesoangioblasts is still limited. The characterization of the physiologically relevant agonists capable of regulating proliferation and survival of these stem cells becomes fundamental to enhancing their therapeutic efficacy. Studies of the last 15 years have clearly demonstrated that sphingosine 1-phosphate (S1P), a sphingolipid metabolite physiologically present in the serum, is endowed with powerful biological activities [6, 7]. The bioactive sphingolipid is produced from the metabolism of sphingomyelin, and although it was initially regarded as an intracellular mediator of growth factors and cytokines, it is presently recognized to elicit most of its effects as a ligand of at least five different specific seven-spanning membrane receptors named S1P1–5 [8]. A number of different studies have demonstrated that S1P regulates key biological processes, such as cell proliferation, motility, and survival, in many different cell types. An important aspect of this regulation is that S1P receptors (S1PRs) are differentially coupled to multiple G proteins upstream of distinct signaling pathways; thus, given that their expression pattern is highly cell-specific, S1P can exert different biological effects, depending on the cell type. For example, endothelial cells [9] and vascular smooth muscle cells [10] have been shown to proliferate in response to S1P; in contrast, in other cell types, such as T lymphocytes [11], hepatocytes [12], and skeletal myoblasts [13], the sphingolipid exerted an antiproliferative effect. Although in seminal pioneering studies the mitogenic action of S1P had been reported to be independent of S1PR engagement [14], it was subsequently found to implicate, in many instances, S1P1 and/or S1P3 [8], whereas S1P2 was identified as the receptor responsible for the antiproliferative properties of S1P [12, 13]. S1P also appears capable of increasing cell survival and inhibiting the apoptotic process in a number of different cell types [15, 16]. Notably, general attention to the dissection of the effects of S1P on cell proliferation and survival has been further increased by recent experimental work showing that a number of growth factors, cytokines, and hormones generate S1P and that modulation of S1P metabolism is integral to their mitogenic and/or antiapoptotic activity [6, 17, 18]. In this study, we report that S1P is a potent mitogenic factor for mesoangioblasts and protects these cells from apoptosis induced by various stimuli, such as serum deprivation, C2-ceramide treatment, and staurosporine treatment, supporting the view that S1P availability to these cells can be critical to improving their survival in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture and Treatment with Agonists, Antagonists, and Inhibitors
Murine mesoangioblasts [2, 3] were routinely grown in Dulbecco's modified Eagle's medium supplemented with heat-inactivated 20% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Human mesoangioblasts, isolated from adult muscle biopsies [19], were routinely grown in Mega Cell (Sigma-Aldrich) supplemented with heat-inactivated 5% FCS and 5 ng/ml basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, http://www.peprotech.com) [20]. Cells were challenged with 1 µM D-erythro-S1P (Calbiochem, San Diego, http://www.emdbiosciences.com; 2 mM stock solution in dimethyl sulfoxide). Specific antagonists of S1PR, VPC23019 [21] and JTE-013 [22], and inhibitors of p38 mitogen-activated protein kinase (MAPK) or p42/44 MAPK pathway, SB203580 and U0126 (Tocris Cookson Ltd., Bristol, U.K., http://www.tocris.com), respectively, were administered to the cells 30 minutes before agonist addition. The S1P1-specific agonist SEW2871 [23] was a kind gift of Dr. H. Rosen (The Scripps Research Institute, La Jolla, CA), and its efficacy was tested by evaluating its ability to promote p42/44 MAPK phosphorylation (data not shown).

Animals
{alpha}-Sarcoglycan ({alpha}-SG)-null C57BL/6 mice [24] were a kind gift of Dr. K. Campbell (Iowa University, Iowa City, IA, USA). Animals were housed in the pathogen-free facility at our institution and treated in accordance with the European Community guidelines and with the approval of the Institutional Ethical Committee.

Cell Proliferation Determination
To evaluate cell proliferation by [3H]thymidine incorporation, mesoangioblasts, seeded in 12-well plates and used when approximately 40% confluent, were serum-starved overnight and then challenged with 1 µM S1P for 24 hours. [3H]Thymidine, 1 or 2 µCi/well (Amersham Pharmacia Biotech, Uppsala, Sweden, http://www.ge.com), was added during the last 2 hours of incubation. Cells were washed twice in ice-cold phosphate-buffered saline (PBS) before the addition of 500 µl of 10% trichloroacetic acid for 5 minutes at 4°C. Cells were washed again in ice-cold PBS, and 250 µl of ethanol:ether (3:1, vol/vol) solution was added to the insoluble material. Samples were then lysed in 0.2 N NaOH for 1 hour at 37°C. Incorporation of [3H]thymidine was measured by scintillation counting.

Alternatively, proliferation was evaluated by cell counting. Briefly, mesoangioblasts, seeded in six-well plates at a density of approximately 1 x 105 cells per well, were serum-starved overnight and then challenged with 1 µM S1P for the indicated time intervals before being trypsinized and counted by a hemocytometer.

Reverse Transcription-Polymerase Chain Reaction
One µg of total RNA extracted with TriReagent (Sigma-Aldrich) from murine mesoangioblasts was reverse-transcribed into DNA as previously described [25] and subjected to polymerase chain reaction (PCR) using the following software-designed oligonucleotides (Amersham Pharmacia Biotech): S1P1 forward, 5'-GTGTCCACTAGCATCCCGGAGGTTAAAGCTCTCCGCAGCTCA-3'; S1P1 reverse, 5'-CCCAACAGGGGTAGCAGGAAGACCCC-3'; S1P2 forward, 5'-TCGCGAATGCTGATGCTCATCGGG-3'; S1P2 reverse, 5'-TCAGACCACCGTGTTGCCCTCCAG-3'; S1P3 forward, 5'-GCAACCACGCATGCGCAGGGCCAC-3'; S1P3 reverse, 5'-GCGGTTGTGAAATTTATTGTTTTTCCAG-3'; S1P4 forward, 5'-CTGCTGCCCCTCTACTCCAA-3'; S1P4 reverse, 5'-ATTAATGGCTGAGTTGAACAC-3'; S1P5 forward, 5'-GAGCGCCACCTTACCATG-3'; S1P5 reverse, 5'-GGAGCAGCTGGTGTCCAT-3'; ß-actin forward, 5'-GCGGGAAATCGTGCGTGACATT-3'; ß-actin reverse, 5'-GATGGAGTTGAAGGTAGTTTCGTG-3'. PCR amplification products were separated on a 1.2% agarose gel.

Real-Time PCR
To quantify mRNA expression of S1P1, S1P2, and S1P3 real-time quantitative reverse transcription-PCR (TaqMan PCR; Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) using the automated ABI Prism 7700 Sequence Detector System (Applied Biosystems) was performed essentially as previously described [26]. All samples were run in triplicate in Micro-Amp optical 96-well plates (Applied Biosystems) with a TaqMan Universal PCR Master Mix (Applied Biosystems). Simultaneous amplification of the target sequence (Assay on Demand; S1P1, Mm00514644_m1; S1P2, Mm01177794_m1; S1P3, Mm00515669_m1; Applied Biosystems) together with the housekeeping gene, 18S rRNA, was carried out with the following universal profile: initial denaturation for 10 minutes at 95°C was followed by denaturation for 15 seconds at 95°C and primer annealing and elongation at 60°C for 1 minute for 40–50 cycles. Results were analyzed by ABI Prism Sequence Detection System software (version 1.7; Applied Biosystems) and plotted by Microsoft Excel software (Microsoft, Redmond, WA, http://www.microsoft.com). The 2–{Delta}{Delta}CT method was applied as a comparative method of quantification [27].

Western Blot Analysis
Mesoangioblasts were lysed for 30 minutes at 4°C in a buffer containing 50 mM Tris, pH 7.5, 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 15 mM Na4P2O7, 20 mM NaF, 1% Nonidet P40, and protease inhibitor cocktail (1.04 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.08 µM aprotinin, 0.02 mM leupeptin, 0.04 mM bestatin, 15 µM pepstatin A, 14 µM L-trans-epoxy-succinyl-leucylamido(4-guanidino)butane) essentially as described by Donati et al. [13]. To prepare total cell lysates, cell extracts were centrifuged for 15 minutes at 10,000g at 4°C. Proteins (30 µg) from lysates were resuspended in Laemmli's sodium dodecyl sulfate (SDS) sample buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis and Western analysis as previously described [13]. Bound phospho-p42/p44 MAPK and phospho-p38 MAPK antibodies (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) were detected using ECL reagents (Amersham Pharmacia Biotech).

Apoptosis Measurement
Mesoangioblasts were seeded at a density of approximately 1 x 105 cells per well and used for experiments after 24 hours. For serum starvation-induced and C2-ceramide-induced apoptosis, cells were incubated in serum-free medium in the presence or absence of 10 µM C2-ceramide (Sigma-Aldrich) for 24 hours, and when requested, 1 µM S1P was administered 30 minutes and 18 hours after serum starvation. For staurosporine-induced apoptosis, 0.5 µM staurosporine (Sigma-Aldrich) was added for the last 4 hours of incubation to cells serum-starved for 24 hours, treated or not treated at 30 minutes and 18 hours of incubation with 1 µM S1P. To quantitate DNA fragmentation, the Cell Death Detection ELISAPLUS Kit (Roche Applied Science, Mannheim, Germany, http://www.roche-applied-science.com) was used to quantitatively determine in vitro cytoplasmic histone-associated DNA fragments characteristic of apoptotic cell death. The optical density was determined on an ELISA Reader, (Bio-Rad, Hercules, CA, http://www.bio-rad.com) using 405 and 490 nm (reference) filters. Alternatively, apoptosis was measured by caspase-3 activity assay. Briefly, cells were washed twice with PBS and then lysed for 20 minutes at 4°C in 20 mM Tris-HCl buffer, pH 7.4, containing 250 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 4 mM sodium vanadate, and 1 mM dithiothreitol (DTT) [28]. The lysis was completed by sonication, and total protein content was determined in the clarified lysates with the Coomassie Blue reagent (Bio-Rad). Aliquots of total proteins (50 µg) were diluted in 50 mM HEPES-KOH buffer, pH 7.0, containing 10% glycerol, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 2 mM EDTA, 10 mM DTT. Caspase-3 activity was determined by incubating the protein sample for 4 hours at 37°C in the presence of 30 µM Ac-DEVD-AFC (excitation, 400 nm; emission, 505 nm) (Biomol Research Laboratories Inc., Plymouth Meeting, PA, http://www.biomol.com). To determine nonspecific substrate degradation, the assays were also performed by preincubating total protein samples for 15 minutes at 37°C with or without the specific caspase inhibitor (200 nM Ac-DEVD-CHO) before substrate addition. The apoptotic response was also evaluated by counting the trypsinized surviving cells by a hemocytometer.

In Vivo Cell Survival Assay
D16-green fluorescent protein (D16-GFP) mesoangioblasts [3] were pretreated with 1 µM S1P for 12 hours, after which cells were suspended in culture medium. Delivery of mesoangioblasts was by injection of 5 x 105 cells directly into tibialis anterior muscles [29]. Muscles recovered from the mesoangioblasts-injected animals were dissected and frozen in liquid N2-cooled isopentane. Serial muscle sections were processed for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Apoptag; Chemicon, Temecula, CA, http://www.chemicon.com) to assess the presence of apoptotic nuclei and immunostained as previously described [3] with the anti-GFP antibody (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). The primary antibody was detected using appropriate secondary antibodies conjugated with Alexa 489 (Molecular Probes), and nuclei were visualized with the DNA dye 4',6-diamidino-2-phenylindole.

Statistical Analysis
Data reported are means ± SEM of triplicates of a representative experiment performed at least three times with analogous results. Statistical analysis was performed using Student's t test (*, p < .05).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
We initially evaluated the pattern of S1PR expression in murine mesoangioblasts. Amplified fragments of ~1,000, 600, and 225 base pairs, corresponding to mRNA transcripts encoding S1P1, S1P2, and S1P3, respectively, were detected by reverse transcription-PCR analysis (Fig. 1A), whereas S1P4 and S1P5 were not (data not shown). Real-time PCR analysis revealed that the relative mRNA abundance was S1P3 >> S1P1 > S1P2 (Fig. 1B).


Figure 1
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  		Figure 1. mRNA expression levels of S1P receptors in D16 mesoangioblasts. (A): Reverse transcription-polymerase chain reaction (PCR) analysis. (B): Real time PCR analysis. Quantitative mRNA analysis was performed by simultaneous amplification of the target S1P1, S1P2, and S1P3 genes together with the housekeeping gene 18S rRNA. Results are expressed as fold changes according to the 2–{Delta}{Delta}CT method, using S1P1 as a calibrator. Abbreviations: bp, base pairs; S1P, sphingosine 1-phosphate.

 
The effect of S1P on mesoangioblast proliferation was evaluated by labeled thymidine incorporation assay experiments. The sphingolipid action was concentration-dependent, with a maximal effect at 1 µM and a half-maximal effective concentration of 32.6 nM ± 7.1 (n = 3) (Fig. 2A). The mitogenic action of S1P was also confirmed by cell counting experiments, since the sphingolipid was still capable of increasing cell number population up to 96 hours of incubation (Fig. 2B). Analogous results were obtained when cell proliferation in response to S1P treatment was evaluated in other independent mesoangioblast clones (A2 and A6) (data not shown). These results demonstrate that S1P behaves in these cells as a full mitogen.


Figure 2
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  		Figure 2. S1P stimulates cell proliferation of D16 mesoangioblasts. (A): Incorporation of [3H]thymidine. Serum-starved D16 mesoangioblasts were stimulated with various concentrations of S1P for 24 h. [3H]thymidine (1 µCi/well) was added in the last 2 h of incubation. (B): Cell counting. Serum-starved mesoangioblasts were stimulated with 1 µM S1P for the indicated times before being counted by a hemocytometer. Abbreviations: h, hours; S1P, sphingosine 1-phosphate.

 
Since all the S1PRs that we found expressed by mesoangioblasts have been implicated in the proliferative response elicited by the sphingolipid in different cell types, we investigated the role of individual receptors by using selective agonists and antagonists. The selective S1P1 agonist SEW2871 in the concentration range 1–30 µM did not trigger cell proliferation, excluding the involvement of S1P1 in S1P-stimulated cell proliferation (Fig. 3A). Pharmacological inhibition of S1P2 using the selective antagonist JTE-013 significantly reduced the mitogenic effect of S1P, since the compound was effective starting from 30 nM (Fig. 3B). Instead, the S1P1/S1P3 antagonist VPC23019 (100 nM) enhanced basal [3H]thymidine incorporation and slightly reduced the proliferative action of S1P (Fig. 3 C), suggesting a minor role, if any, for S1P3. On the whole, these results indicate that the mitogenic action of S1P in mesoangioblasts occurs mostly via stimulation of S1P2 receptor. Since the mitogenic effect of S1P can be accompanied by a prosurvival action of the sphingolipid, the potential antiapoptotic effect of S1P in mesoangioblasts was also investigated. S1P fully prevented the formation of cytoplasmic histone-associated DNA fragments induced by 24 hours of culture under serum deprivation or in the presence of C2-ceramide (10 µM) and significantly reduced the amount of histone associated mono- and oligonucleosomes formed in response to staurosporine (0.5 µM) administration (Fig. 4A). Consistently, S1P significantly diminished the increase in caspase-3 activity and the reduction in total cell number induced by the different apoptogenic stimuli (Fig. 4B, 4C). S1P exhibited a similar protective effect against apoptosis in mesoangioblast clones A2 and A6 (data not shown).


Figure 3
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  		Figure 3. Role of S1P receptors in the proliferative action of S1P. Effect of SEW2871 (A), JTE-013 (B), and VPC23019 (C) on mesoangioblast proliferation. The experimental conditions were those described in the legend to Figure 2. Mesoangioblasts were incubated for 24 hours in the presence of SEW2871 at the indicated concentrations or preincubated for 30 minutes in the presence of different concentrations of JTE-013 or 100 nM VPC23019 before being stimulated with 1 µM S1P for 24 hours. Abbreviation: S1P, sphingosine 1-phosphate.

 


Figure 4
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  		Figure 4. S1P protects D16 mesoangioblasts from apoptosis. The apoptotic response induced by 24 hours of serum starvation, by treating serum-starved cells with 0.5 µM staurosporine for 4 hours or with 10 µM C2-ceramide for 24 hours in the presence or absence of S1P, was evaluated by quantitating DNA fragmentation (A), by measuring caspase-3 activity (B), or by counting the trypsinized surviving cells by a hemocytometer (C). (D): Approximately 5 x 105 GFP-expressing mesoangioblasts pretreated with 1 µM S1P for 12 hours were injected into the tibialis anterior muscles of 4-month-old {alpha}-sarcoglycan-null mice. After 12 hours, muscles were recovered. Apoptosis of GFP-expressing mesoangioblasts was assessed by the TUNEL technique (images from one out of four reproducible experiments). Also shown is the DAPI staining of the nuclei and its overlay with the GFP and TUNEL staining. Abbreviations: C, control; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; S1P, sphingosine 1-phosphate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

 
To assess the prosurvival effect of S1P in vivo, the D16-GFP mesoangioblasts, previously treated with 1 µM S1P for 12 hours, were injected directly into the right tibialis anterior muscle of {alpha}-SG-null mice. Twelve hours later, treated and controlateral (control) muscles were removed, and cell death was assessed by staining with the TUNEL technique. As shown in Figure 4D, the dystrophic muscle showed signs of apoptosis and provoked cell death of injected GFP-positive mesoangioblasts. Mesoangioblast treatment with S1P before their injection reduced apoptosis by 51% ± 4.3% (n = 4; p < .01 vs. not treated), also demonstrating a clear-cut in vivo prosurvival action of the sphingolipid. The involvement of S1PRs in the antiapoptotic effect of S1P was examined next. As shown in Figure 5, 1 µM SEW2871 did not prevent the staurosporine-induced formation of histone-associated DNA fragmentation, excluding the involvement of S1P1. Similarly, the protective effect of S1P was only slightly diminished by JTE-013 (1 µM) or VPC23019 (100 nM), also ruling out a role for S1P3 or S1P2. Thus, the antiapoptotic action of S1P appears to occur through S1PR-independent pathways. The possible molecular mechanisms involved in the biological effects exerted by S1P in mesoangioblasts were then investigated. In particular, we studied the involvement of the MAPK signaling pathways that have been shown to be activated by S1P and have an effect in both mitogenesis and apoptosis [9, 30–32]. To this end, we used U0126 (10 µM) and SB203580 (5 µM), specific inhibitors of the p42/44 and p38 MAPK pathways, respectively. In control experiments, the two inhibitors strongly reduced the S1P-induced activation of p42/44 MAPK and p38 MAPK, respectively (Fig. 6A). In Figure 6A, it is also shown that cell incubation with 2 µM JTE-013 reduced p38 MAPK and p42/44 MAPK phosphorylation induced by 10 minutes of treatment with 1 µM S1P. Cell treatment with U0126 (10 µM) significantly reduced basal [3H]thymidine incorporation and attenuated the stimulation of DNA synthesis brought about by S1P; conversely, SB203580 (5 µM) did not affect the cell response to S1P (Fig. 6B). Neither U0126 nor SB203580 influenced the protective action of S1P during cell death (Fig. 6C). These results indicate that S1P activates the p42/44 pathway, as well as the p38 MAPK pathway, and that p42/44 MAPK is required for the mitogenic effect of S1P, whereas protection from apoptosis is independent of MAPK activation. Finally, from the perspective of cell therapy in patients, it was evaluated whether the biological effects elicited by S1P in murine mesoangioblasts also take place in the same cell type isolated from human donors. As illustrated in Figure 7A, treatment with 1 µM S1P significantly stimulated [3H]thymidine incorporation into human mesoangioblasts. The sphingolipid was also found to strongly protect human mesoangioblasts from apoptosis. In particular, 1 µM S1P significantly reduced the formation of cytoplasmic mono- and oligonucleosomes in response to 24 hours of serum deprivation or 0.5 µM staurosporine administration (Fig. 7B). Notably, the mitogenic and antiapoptotic effect of the sphingolipid was comparable to that induced in murine cells.


Figure 5
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  		Figure 5. Role of S1P receptors in the antiapoptotic action of S1P. Effect of 1 µM SEW2871 (gray bar) and 1 µM JTE-013 or 100 nM VPC23019 in the absence (open bars) or presence (hatched bars) of 1 µM S1P on serum-starved mesoangioblast DNA fragmentation induced by 0.5 µM staurosporine treatment for 4 hours. Abbreviation: S1P, sphingosine 1-phosphate.

 


Figure 6
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  		Figure 6. Role of S1P-induced activation of p42/44 MAPK and p38 MAPK pathways (A) in the mitogenic (B) and antiapoptotic (C) action of the sphingolipid in D16 mesoangioblasts. (A): Western blot analysis of lysates (30 µg) of serum-starved mesoangioblasts incubated with or without 1 µM S1P for the indicated times in the presence or absence of 10 µM U0126, 5 µM SB203580, or 2 µM JTE-013. (B): Effect of 10 µM U0126 or 5 µM SB203580 on the proliferative action of S1P in D16 mesoangioblasts. (C): Effect of 10 µM U0126 or 5 µM SB203580 on the protective effect of S1P on staurosporine-induced DNA fragmentation. Abbreviations: MAPK, mitogen-activated protein kinase; min, minutes; S1P, sphingosine 1-phosphate.

 


Figure 7
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  		Figure 7. S1P exerts mitogenic (A) and antiapoptotic effect (B) in human mesoangioblasts. (A): Incorporation of [3H]thymidine. Serum-starved cells were stimulated with 1 µM S1P for 24 hours. [3H]Thymidine (2 µCi/well) was added to the cells in the last 2 hours of incubation. (B): The apoptotic response induced by 24 hours of serum starvation or by treating serum-starved cells with 0.5 µM staurosporine for 4 hours in the absence or presence of S1P was evaluated by quantitating DNA fragmentation. Abbreviation: S1P, sphingosine 1-phosphate.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This study represents the first experimental evidence for a key biological role of S1P in mesoangioblasts. In particular, S1P was found to promote cell proliferation and prevent programmed cell death.

The mitogenic effect exerted by S1P reported here is in agreement with a number of studies performed in different cell types in which the bioactive lipid is capable of stimulating cell proliferation and appears to depend on the action of S1P on its membrane receptors [8–10]. Murine mesoangioblasts were found to express S1P1, S1P2, and S1P3, in agreement with the pattern of S1PR expression recently identified in human embryonic stem cells [33]. Although S1P1 receptor is expressed in mesoangioblasts, it does not play a role in mitogenesis, since the specific S1P1 agonist SEW2871 was unable to reproduce the mitogenic action of the sphingolipid. This is a striking difference with the other cell types investigated thus far, in which this receptor plays a pivotal role in the sphingolipid-dependent cell proliferation [8–10]. Instead, the observed attenuation of the proliferative response to S1P by S1P2 antagonist JTE-013 is in favor of a mitogenic role of S1P2, whereas the modest inhibitory effect determined by S1P1/S1P3 antagonist VPC23019 rules out a major role for S1P3 in S1P-induced mesoangioblast proliferation. So far, S1P2 was found to be positively coupled to cell proliferation only in mesangial cells [32], an interesting observation in light of the common perithelial niche of both mesangial cells and mesoangioblasts [19]; in contrast, in other studies, the same receptor was implicated in the antiproliferative response elicited by the sphingolipid [12, 13]. This observation may have important implications in the in vitro expansion of these cells for therapeutic purposes, although selective S1P2 agonists have not yet been developed. The study of the signaling pathways involved in the mitogenic effect of S1P highlighted a key role for p42/44 MAPK, in agreement with a number of other studies performed in different cells [9, 34, 35].

Another important finding of this study is the powerful prosurvival effect exerted by S1P in mesoangioblast apoptosis induced by serum deprivation or treatment with C2-ceramide or staurosporine. S1P was found to fully prevent the DNA fragmentation caused by serum deprivation or C2-ceramide treatment, whereas chromatin damage induced by the more potent apoptogenic agent staurosporine was appreciably decreased by the sphingolipid. Moreover, S1P was capable of potently attenuating caspase-3 activation and counteracting the reduction in cell number induced by the apoptogenic stimuli. Furthermore, mesoangioblasts pretreated in vitro with S1P exhibited an increased survival when injected into tibialis anterior muscles of {alpha}-SG-null mice. On the whole, these results demonstrate that S1P plays a key role in the prevention of in vitro programmed cell death of mesoangioblasts.

The molecular mechanisms implicated in the antiapoptotic action of S1P appear complex and need further investigation. The prosurvival effect of the sphingolipid was not mimicked by the S1P1 agonist SEW2871 and was not significantly influenced by S1PR antagonists VPC23019 or JTE-013, thus excluding the involvement of S1PRs and suggesting the possibility of an intracellular action of S1P [14, 36, 37], since the exogenous sphingolipid may be transported across the plasma membrane, possibly via specific transporters [38]. However, at this stage it cannot be ruled out that the prosurvival effect of S1P in mesoangioblasts is due, alternatively, to G protein-coupled receptors different from S1PRs, as already demonstrated in other cell systems [8, 39].

Mesoangioblasts are presently regarded as very promising stem cells in the therapy of muscular dystrophy [3]. However, the reduced survival of these cells after injection in vivo is at least in part responsible for their partial ability to repopulate the damaged tissue, representing an obstacle to their use in cell therapy. The present identification of S1P as powerful mitogenic and antiapoptotic agent for mesoangioblasts suggests that their efficiency of muscle repair could be increased by the target delivery of exogenous S1P. In view of a future cell therapy, it is noteworthy that S1P was here found able to stimulate proliferation of human mesoangioblasts and protect these cells from apoptosis.

Moreover, it has to be considered that S1P is present in the plasma of mammals in a concentration range of 0.1–0.6 µM [40, 41], which is in principle sufficient to fully occupy the majority of S1PRs borne by circulating cells. However, since the sphingolipid is largely bound to the high-density lipoprotein fraction, its real bioavailability remains to be determined [42]. In agreement, it is of note that in vivo administration of S1P or S1P agonists is presently regarded as a valid therapeutical approach in a number of different pathologies ranging from renal failure to lung injury [43, 44], as well as to prevent transplant rejection and to treat autoimmune disorders [45].


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We are indebted to Drs. F. Torricelli and B. Minuti (Cytogenetics and Genetics Unit, Azienda Ospedaliera Careggi, Florence, Italy) for skillful assistance with real-time PCR experiments. This work was supported in part by funds from the University of Florence, Fondazione Cassa di Risparmio di Lucca, and Associazione Italiana Ricerca sul Cancro.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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