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Published as doi: 10.1096/fj.06-7420com.
 (The FASEB Journal. 2007;21:1503-1514.)
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Activation of sphingosine-1-phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration

Alexander S. Novgorodov{dagger}, Mazen El-Alwani{ddagger}, Jacek Bielawski§, Lina M. Obeid*,{ddagger} and Tatyana I. Gudz*,{dagger},1

* Ralph H. Johnson Veterans Affairs Medical Center, and the Departments of

{dagger} Neuroscience,

{ddagger} Medicine and

§ Biochemistry of Medical University of South Carolina, Charleston, South Carolina, USA

1Correspondence: Department of Neuroscience, Medical University of South Carolina, 114 Doughty St. Charleston, SC 29425, USA. E-mail: gudz{at}musc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sphingosine-1-phosphate (S1P) acts as an extracellular ligand for a family of G-protein coupled receptors that are crucial in cell migration. S1P5 is exclusively expressed in oligodendrocytes and oligodendrocyte precursor cells (OPCs), which migrate considerable distances during brain development. The current studies suggest a physiological role for S1P and S1P5 in regulation of OPC migration. mRNA expression levels of S1P2 and S1P5 are comparable in OPCs, but S1P binding specifically to the S1P5 receptor blocked OPC migration (IC50=29 nM). Thus, knocking down S1P5 using siRNA prevented the S1P-induced decrease in OPC migration, whereas knocking down S1P2 did not have any effect. S1P-induced modulation of OPC migration was insensitive to pertussis toxin, suggesting that S1P5-initiated signaling is not mediated by the G{alpha}i-protein coupled pathway. Furthermore, S1P5 appears to engage the G{alpha}12/13 protein coupled Rho/ROCK signaling pathway to impede OPC migration. To modulate OPC motility, extracellular S1P could be derived from the export of intracellular S1P generated in response to glutamate treatment of OPCs. These studies suggest that S1P could be a part of the neuron-oligodendroglial communication network regulating OPC migration and may provide directional guidance cues for migrating OPCs in the developing brain.—Novgorodov, A. S., El-Alwani, M., Bielawski, J., Obeid, L. M., Gudz, T. I. Activation of sphingosine-1-phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration.


Key Words: brain development • S1P2 receptor • FTY720 • directional guidance cue


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SPHINGOSINE-1-PHOSPHATE (S1P) is a pleiotropic sphingolipid mediator that has been implicated in regulation of cell proliferation, differentiation, survival, and migration (1) . S1P acts as an extracellular ligand for receptors belonging to S1P family. These receptors, referred to as S1P1–5 (originally termed EDG-1, 3, 5, 6, and 8), couple to multiple G proteins and regulate intracellular signaling pathways. Generally, S1P1, S1P2, and S1P3 receptors are widely expressed in humans and rodents, whereas S1P4 is restricted to lymphoid compartments (2) . S1P5 is predominantly expressed in the white matter tracts and oligodendrocytes and is particularly abundant in the anterior commissure, corpus collosum, and optic tract (3) . S1P5 expression in the brain is regulated throughout development: S1P5 mRNA was detected on 7 and 17 day embryos but not in 11 or 15 day embryos (4 , 5) . At the cellular level, the S1P5 receptor is already detectable in oligodendrocyte precursor cells (OPCs) and remains expressed throughout the development of the oligodendrocyte lineage, including mature myelinating oligodendrocytes (6) .

The S1P5 receptor is encoded within a single exon as are the other members of the S1P receptor family. Human and mouse S1P5 share >95% amino acids with rat S1P5 and have an apparent molecular mass of 43 kDa (7) . Similar to the other members of the family, S1P5 is a high affinity S1P receptor (Kd=2–6 nM) (8) . S1P binding to each of the S1P receptors activates different intracellular signaling pathways depending on which G{alpha} protein they couple to intracellularly (9) . S1P5 is linked to G{alpha}i and G{alpha}12/13 but apparently not to G{alpha}s or G{alpha}q. Coupling of S1P5 to G{alpha}i is reflected in pertussis toxin (PT)-sensitive inhibition of adenylyl cyclase activity by S1P in cells overexpressing the receptor (10) . Direct S1P-induced activation of endogenous G{alpha}i and also of G{alpha}12, but not G{alpha}s and G{alpha}q, was observed in cells overexpressing the S1P5 receptor using a GDP/[35S]GTP{gamma}S exchange assay (8) .

Functionally, S1P5 has been found to repress serum-induced proliferation in a manner that is insensitive to PT but sensitive to orthovanadate, suggesting that phosphatases may be downstream of S1P5 (8) . S1P5 appears to mediate S1P-induced survival of mature oligodendrocytes through a PT-sensitive, Akt-dependent pathway (6) . In contrast, S1P activation of the S1P5 receptor on immature oligodendrocytes induced process retraction via a Rho/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from S1P5-deficient mice (6) . It is noteworthy that S1P1, S1P2, and S1P3 receptors have been shown to play critical roles in cytoskeletal reorganization and cell migration (11 12 13) , but the role of S1P5 in cell migration remains to be elucidated. The Rho family of GTPases, particularly, Rho, Rac, and Cdc42, has emerged as a central coordinator of cell migration. Activation of S1P1 or S1P3 increases directional or chemotactic migration (13 , 14) , and both receptors mediate activation of Rac via G{alpha}i (12) . In contrast, stimulation of S1P2 decreases chemotaxis (15) and membrane ruffling partly due to suppression of Rac activation (16) . Unlike S1P1 that only couples to Rac via G{alpha}i, the repellent receptor S1P2 also couples and stimulate Rho via G{alpha}12/13 (17 , 18) . Thus, the ability of S1P to stimulate multiple S1P receptors, which in turn activate multiple G{alpha}{alpha} protein-coupled pathways, creates a complex signaling system downstream of S1P receptors. The balance of counteracting signals from the G{alpha}i and G{alpha}12/13-Rho pathways has been proposed to direct either positive or negative regulation of cell migration (16) .

Although it has long been established that oligodendrocytes express the S1P5 receptor along with S1P1, S1P3, and S1P2 (3 , 6 , 10 , 19) , little is yet known of S1P involvement in regulation of OPC migration. We hypothesized that S1P/S1P5 receptor signaling could modulate OPC migration. In the mammalian central nervous system (CNS), OPCs arise in restricted germinal areas of the developing CNS, ventricular, or subventricular domains and migrate to the developing white matter, where they differentiate and elaborate myelin sheaths around axons. The cellular and molecular mechanisms that guide this migration have begun to be clarified. Mounting data demonstrate that OPCs express both ligands and receptors able to respond to classical guidance cues that have been identified to direct axons through the complex terrain of the developing CNS (20 , 21) . Axons are guided along specific pathways by attractive and repulsive cues in the extracellular environment. Genetic and biochemical studies have identified highly conserved families of guidance molecules, including netrins, slits, semaphorins, and ephrins (22) . These are not the only known guidance molecules, but they are by far the best understood, and substantial evidence indicates a key role of netrins, semaphorins, and ephrins in guiding OPC migration (23) . It is noteworthy that the expression of the chemokine CXCL1 by astrocytes in the developing white matter appears to instruct migrating OPCs to stop (24) . CXCL1 signals via the chemokine receptor CXCR2, which is expressed by OPCs. In addition, a recent study provides evidence that the neurotransmitter glutamate is a powerful regulator of {alpha}v ß3 integrin-mediated OPC migration (25) . Herein, we uncovered an important role for S1P/S1P5 receptor signaling pathway as a negative regulator of OPC motility. The current studies suggest that S1P could be an essential modulator of OPC migration in the developing brain and may provide directional guidance cues for migrating OPCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
D-Erythro-sphingosine-1-phosphate, D-erythro-C18-ceramide, and D-erythro-sphingosine were provided by the MUSC Lipidomics Core (Department of Biochemistry, MUSC, Charleston, SC, USA). FTY720 (2-amino-2[2-(4-octylphenyl) ethyl]-1,3-propanediol) was purchased from Cayman Chemical (Ann Arbor, MI, USA), and HA1077 [1-(5-isoquinilinesulfonyl) homoperazine] was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Pertussis toxin, glutamate, fatty acid free BSA, and C3 exoenzyme were from Sigma (St. Louis, MO, USA).

Cell culture
Dissociated rat neonatal cortices were cultured on PLL-coated flasks as described (26) . Briefly, the cerebra of rat pups were dissected and minced to generate a single cell suspension. Cells were plated into 75 cm2 flasks and grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) medium with 10% FBS at 37°C and 5% CO2. By day 10, mixed glial cultures were obtained, consisting of OPCs and microglia growing on astrocyte monolayer. OPCs were purified from mixed glial cell cultures using a shake-off procedure. Cells were shaken initially for 1 h at 100 rpm to remove microglia, refed, and shaken for 22–24 h at 37°C at 200 rpm. OPCs were collected by centrifugation at 1200 g for 5 min. Cells were resuspended in DMEM medium supplemented with N2 (Sigma) and 2% fatty acid free BSA and centrifuged again to remove FBS. Cells were immediately used for migration assays or for transfection with siRNA.

Reverse transcription
RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. Isolated RNA (2 µg) was added to 1 µl of 10 mM dNTP and 1 µl of 0.5 mg/ml oligo(dT) and brought to a volume of 10 µl with ddH2O. The mixture was incubated at 65°C for 5 min. Then, 2 µl of 10x first strand buffer, 2 µl of 0.1 M dithiothreitol, 4 µl of 25 mM MgCl2, and 1 µl of RNase Out (Invitrogen) were added, and the contents were incubated at 42°C for 2 min. Next, 1 µl of Superscript II reverse transcriptase (Invitrogen) was added and the mixture was kept at 42°C for 50 min. The enzyme was then inactivated at 70°C for 15 min.

Real time reverse transcriptase-polymerase chain reaction
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on a PE Applied Biosystems Gene Amp 5700 Sequence detection System (Foster City, CA, USA). QuantiTest SYBER Green PCR kit was purchased from Qiagen, whereas the AmpErase UNG enzyme was purchased from PE Applied Biosystems. The standard reaction volume was 10 µl and contained 4.2 µl of QuantiTest SYBER Green PCR reagents, 0.1 µl of AmpErase UNG enzyme, 0.7 µl of cDNA template, 0.8 µl of the forward primer, 0.8 µl of the reverse primer(both the forward and reverse primers at 3.125 µM concentration), and 3.4 µl of water. Primers were designed using the PerkinElmer Primer Express software. The following primers were used for PCR amplification: S1P1 (accession number U10303; forward, 5'-CTGACCTTCCGCAAGAACATCT-3', reverse, 5'-CTTCAGCAAGGCCAGAGACTTC-3'), S1P2 (accession number AF022138; forward, 5'-GCCTTGTACGTCCGAATCTACTTC-3', reverse, 5'-AGCGTCTGAGGACCAGCAA-3'), S1P3 (accession number AF184914; forward, 5' -ACGCGCGCATCTACTTCCT-3', reverse, 5'-TGGATCTCTCGGAGTTGTGGTT-3'), S1P5 (accession number AF233649; forward, 5'- CTCTAGAGCGCCACCTTACCAT-3', reverse, 5'-CCCAGCAGCAGCGACAA-3'), ß-actin (accession number VO1217; forward, 5'-ATGCCCCGAGGCTCTCTT-3', reverse, 5'-CTTCATGATGGAATTGAATGTAGTTTC-3'). Initial steps of RT-PCR were 2 min at 50°C for AmpErase UNG enzyme activation, followed by a 10 min hold at 95°C for its deactivation. Cycles (n=40) consisted of a 15 s melt at 95°C, followed by 1 min annealing/extension at 60°C for 1 min. All reactions were performed in triplicate. The threshold cycle (CT) analysis for all samples was set at 0.15 relative fluorescence units.

Data analysis
The ß-actin gene was used as an internal reference control to normalize relative levels of gene expression. Real-time RT-PCR results were analyzed using Q-Gene software (27) , which expresses data as mean normalized expression, which is directly proportional to the amount of RNA of the target gene relative to the amount of RNA of the reference gene.

Silencing RNA transfection
Silencing RNA (siRNA) targeting the S1P5 receptor or the S1P2 receptor was purchased from Qiagen High Performance GenomeWide siRNA bank (Qiagen, Valencia, CA,USA). The following target sequences were used: S1P5, 5'-CAGAATCTATTCATTCATCTA-3' or S1P2, 5'-ACCGACATTTCTGGA GGGTAA-3'. OPCs were transfected with siRNA targeting S1P5 or S1P2 receptor using the Nucleofector electroporation system according to manufacturer’s instructions with efficiencies of >70% (Amaxa Biosystems, Gaithersburg, MD, USA). Cells (5–7x106) are mixed with 100 µl of Nucleofector reagent and 2 µl siRNA in the cuvette of the Amaxa electroporation device. Scrambled siRNA was used as a control. After transfection, OPCs were plated on top of confluent astrocyte cultures and then cultured for 48 h. OPCs were then shaken off the mixed cultures for 24 h. Total time of siRNA treatment was 72 h.

Migration assay
To quantify OPC migration, we used a transwell (TW)-based assay in 24-well plates (Corning, Acton, MA, USA). TW chamber membranes (8.0 µm pore size) were coated with 10 µg/ml fibronectin (Chemicon). Cells were plated at 60,000 cells/TW in DMEM/N2 medium and allowed to migrate for 4 h. Cells were labeled with 5 µM calcein-AM (Molecular Probes, Carlsbad, CA, USA) 1 h before the end of assay. Cells that had not migrated were removed from the upper chamber of the TW with a cotton swab. Calcein fluorescence of cells on the bottom of the TW was measured in a plate reader (Wallac Victor 1420, PerkinElmer, Wellesley, MA, USA) at 530 nm emission (480 nm excitation).

Analysis of sphingolipids by mass spectroscopy
Cells were lysed in buffer containing 10 mM Tris, 1% TritonX-100, pH 7.4 for electrospray ionization/MS/MS analysis of ceramides and sphingomyelins which were performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode according to methodology described by Novgorodov et al. (28) . Cell lysate samples, fortified with internal standards, were extracted with ethyl/acetate/isopropyl alcohol/water (60:30:10, v/v/v), evaporated to dryness, and reconstituted in 100 µl of methanol. The samples were injected on the Surveyor/TSQ 7000 liquid chromatography/MS system and gradient-eluted from the BDS Hypersil C8, 150 x 3.2-mm, 3 µm particle size column, with a 1.0 mM methanolic ammonium formate, 2 mM aqueous ammonium formate mobile phase system. The peaks for the target analytes and internal standards were collected and processed using the Xcalibur software system. Calibration curves were constructed by plotting peak area ratios of synthetic standards, representing each target analyte and the corresponding internal standard. The target analyte peak area ratios from the samples were similarly normalized to their respective internal standard and compared with the calibration curves using a linear regression model.

Rho pull-down assay
Measurements of activation of Rho were performed using a commercially available kit manufactured by Upstate Biotechnology (Lake Placid, NY, USA). Briefly, cells were plated in 60 mm plates coated with 10 µg/ml fibronectin and cultured for 4 h in DMEM/N2 medium in the presence of 0.1 µM S1P. Cells were washed twice with ice-cold Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 8.0). Cell lysates were made in Mg2+ lysis buffer, pH 7.5 (provided by the manufacturer) supplemented with 25 mM NaF, 1 mM Na3VO4, and Complete Mini Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN, USA). After 1 h on ice, samples were centrifuged at 15,000 g for 10 min to remove insoluble material. Protein concentration was determined using the bicinchoninic acid method (Sigma) with BSA as a standard. Cell lysates (1 mg protein/ml) were incubated for 1 h at 4°C with glutathione S-transferase (GST)-Rhotekin already bound to glutathione-agarose beads to precipitate GTP-bound RhoA. Precipitated complexes were washed three times in lysis buffer and boiled in reducing sample buffer.

Immunoblotting
Total lysates and precipitates were resolved on 10% SDS-polyacrylamide gel and transferred to the polyvinylene difluoride membrane. The membrane was blocked overnight in 5% nonfat dry milk (Bio-Rad, Hercules, CA, USA) in TBS-T buffer (10 mM Tris, 150 mM NaCl, and 0.2% Tween-20, pH 8.0). The proteins were detected by blotting with monoclonal anti-Rho antibody (clone 55, Upstate Biotechnology) or polyclonal anti-S1P5 and anti-S1P2 antibody (Exalpha Biologicals, Watertown, MA, USA) or monoclonal anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive bands were visualized using an enhanced luminescence kit (ECL-Plus; Amersham Biosciences, Piscataway, NJ, USA) or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA).

Cell viability
OPCs were plated at a concentration of 10,000 cells/well in 96-well plates in 100 µl of DMEM/N2 media. Cells were treated with vehicle, S1P, sphingosine (SPH), glutamate, or glutamate plus cyclothiazide (CZ) for 4 or 24 h. Cell viability was determined using a 3-[4,5-dimethylthizaol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)-based kit (Sigma) following the manufacturer’s instructions. The absorbance of the samples was measured at 560 nm in a microplate reader (FLUOstar Optima, BMG LABTECH Inc., Durham, NC, USA).

Apoptosis
Apoptotic cell death was determined using FLICA apoptosis detection kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. OPCs were plated at a concentration of 10 000 cells/well in 96-well plates in 100 µl of DMEM/N2 media. Cells were treated with vehicle, S1P, sphingosine, glutamate, or glutamate plus cyclothiazide for 24 h. Before the end of the assay, cells were labeled for 1 h with fluorescent inhibitor of activated caspase-3. Plate was centrifuged, and fluorescence was determined in the microplate reader (FLUOstar Optima) at 530 nm emission with 485 nm excitation.

Statistical analysis
All assays were performed three or more times. Typically, there were four to six replicates of each treatment in each assay. Data were collected, and the mean value of the treatment groups and SE was calculated. Data were analyzed for statistically significant differences between groups by one-way ANOVA with a post hoc Bonferroni’s test, which adjusts for multiple simultaneous comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of S1P5 inhibits OPC migration
OPC migration was quantified in TW-based assays. In these studies, TWs were precoated with fibronectin, the ligand of several integrin receptors expressed in oligodendrocytes, including {alpha}v ß1, {alpha}v ß3, and {alpha}v ß5. Recently, we demonstrated that OPC migration on fibronectin is predominantly mediated by the {alpha}v ß3 integrin receptor (25) , which is the most abundant integrin of the {alpha}v family expressed in oligodendrocytes at this stage of differentiation (29) . First, we examined whether S1P regulates integrin-mediated OPC migration (Fig. 1 ). In the presence of increasing concentrations of S1P (10–30 nM), the number of OPCs migrating through the TW was significantly decreased, and these S1P concentrations are within the range of S1P receptors activation (7) . S1P reduced OPC migration with IC50 = 29 nM. To determine the specificity of S1P-induced inhibition of OPC migration, we measured OPC migration in the presence of biologically active analogs of S1P, sphingosine, and C18-ceramide. We showed that sphingosine is a less potent inhibitor of OPC motility (IC50=600 nM). This is consistent with possible conversion of sphingosine into S1P, which, in turn, would block OPC migration. In contrast, C18-ceramide had no effect on OPC migration. To rule out cytotoxicity of sphingosine or S1P, cell viability was evaluated after 4 or 24 h treatment with 1 µM sphingosine or 1 µM S1P. Stimulation of glutamate receptor with 0.8 mM glutamate in the presence of the receptor desensitization blocker, cyclothiazide was used as a positive control. There was no change in cell viability following the treatment with sphingosine or S1P (Fig. 2 A). As expected, almost 60% of the cells treated with glutamate and cyclothiazide lost their viability at 24 h. In contrast, glutamate alone did not cause any toxicity to OPCs after 24 h treatment. This indicates that glutamate receptor desensitization prevents glutamate from causing receptor-mediated OPC toxicity. The data are consistent with the previous findings that OPCs are resistant to relatively high concentrations of glutamate due to desensitization of glutamate receptors (30) and the robust glutamate uptake and utilization by OPCs (31) . There was no change in OPC morphology after the treatment with S1P or sphingosine or glutamate (not shown). The lack of S1P-mediated cytotoxicity raises the issue of whether the S1P-induced inhibition of OPC migration could be an early manifestation of the induction of apoptosis. As shown in Fig. 2B , treatment with S1P caused no apparent apoptotic cell death at 24 h as compared to OPCs treated with glutamate and cyclothiazide. Together, these results suggest that S1P acts as a specific and potent inhibitor of integrin-mediated OPC migration.


Figure 1
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  		Figure 1. S1P reduces OPC migration. OPCs were allowed to migrate in the presence of various concentrations of S1P (closed diamonds), sphingosine (closed squares), and C18-ceramide (closed triangles) for 4 h. TWs were precoated with 10 µg fibronectin. Calcein-AM (10 µM) was added 1 h before end of assay. Nonmigrated cells were removed from top compartment of TW with a cotton swab, and calcein fluorescence of migrated cells on the bottom of the TW was measured. Data are mean ± SE (12 replicates from at least 3 independent experiments). *P < 0.01, **P < 0.05 compared to vehicle control.


Figure 2
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  		Figure 2. S1P is nontoxic and do not cause apoptotic death of OPCs. OPCs were treated with 1 µM S1P, 1 µM sphingosine (SPH), 0.8 mM glutamate, or 0.8 mM glutamate with 50 µM cyclothiazide (CZ). A) Viability of cells was measured at 4 or 24 h. B) Apoptotic cell death was determined at 24 h. Data are mean ± SE (36 replicates from 3 independent experiments). *P < 0.01 compared to vehicle control.

S1P has been identified as a ligand of four S1P receptors expressed in differentiated oligodendrocytes with a relative mRNA abundance of S1P5>S1P1 = S1P2>S1P3 (19) . To quantify the levels of expression of S1P receptors in OPCs, we employed real-time RT-PCR using ß-actin as the reference gene for normalization purposes. As shown in Fig. 3 , in three independent OPC preparations, four S1P receptors were found with the following rank order of gene expression: S1P1 = S1P2 = S1P5>S1P3. Among four S1Ps tested, S1P3 was the least abundant. In contrast to differentiated oligodendrocytes, the levels of expression of S1P5 in OPCs were similar to S1P2 or S1P1. Activation of the S1P1 or S1P3 receptor could result in stimulation of directional or chemotactic cell migration (11 12 13 14) , and ligation of S1P2 inhibits chemotaxis of cells overexpressing the receptor (15 , 16) . Although the biological functions of S1P5 are not well understood, a recent study suggests that activation of S1P5 elicited process retraction in OPCs, indicating the involvement of S1P5 in modulation of the cell adhesion (6) .


Figure 3
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  		Figure 3. S1P receptor mRNA expression profile in OPCs. mRNA expression was measured by real-time RT-PCR. Data represent expression of individual S1P mRNA relative to expression of ß-actin mRNA. Data are mean ± SE (9 replicates from 3 independent experiments). *P < 0.01 compared to S1P1, S1P2, or S1P5.

To determine which S1P receptor governs OPC migration, we knocked down S1P2 or S1P5 in these cells using small interfering RNAs. A dose-response experiment with S1P5-specific siRNA showed that mRNA and protein levels were maximally reduced with 50 nM siRNA at 72 h after OPC transfection (Fig. 4 A). The protein level was ~20% of that in scrambled siRNA-treated cells. This effect was highly specific for S1P5: no reduction in S1P2 mRNA or protein was observed. Similarly, S1P2-specific siRNA (50 nM) knocked down mRNA up to 70% within 72 h after OPC transfection (Fig. 4B ). S1P2 protein levels were significantly reduced. We next examined the effect of S1P on cell migration. Cells depleted of the S1P5 receptor did not respond to S1P suggesting that S1P-mediated inhibition of OPC migration occurs via S1P5-initiated signaling pathways (Fig. 5 A). In contrast, knockdown of S1P2 had no effect on OPC migration in the presence of S1P. The data clearly demonstrate that S1P binding to S1P5 initiates the intracellular signaling to reduce the integrin-mediated OPC motility. To confirm these findings, we employed a close analog of S1P, FTY720, which is a high-affinity agonist for all S1P receptors except S1P2 (32 33 34) . If S1P-induced inhibition of OPC migration is mediated by the S1P2 receptor, FTY720 will not affect OPC migration. However, FTY720 had a profound inhibitory effect on OPC migration as shown in Fig. 5B . Moreover, knockdown of the S1P5 prevented FTY720-induced reduction in OPC motility, whereas knockdown of the S1P2 did not have any effect. These results suggest that S1P is a negative regulator of OPC migration, and its binding to S1P5 governs integrin-mediated OPC migration.


Figure 4
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  		Figure 4. Knocking down S1P2 or S1P5 receptor in OPCs. OPCs were treated with receptor-specific siRNA or scrambled (SCR) for 72 h. Upper panel) Decrease in S1P5 (A) or S1P2 (B) protein expression level on treatment with siRNA. Lower panel) Real-time RT-PCR S1P5 (A) or S1P2 (B) mRNA knockdown. Mean normalized expression is directly proportional to the amount of S1P receptor mRNA relative to amount of ß-actin mRNA. Data are mean ± SE (9 replicates from 3 independent experiments). **P < 0.01 compared to SCR.


Figure 5
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  		Figure 5. S1P5 receptor is required for S1P- and FTY720-induced inhibition of OPC migration. OPCs were treated with 50 nM scrambled siRNA (closed triangles), 50 nM S1P5-specific siRNA (closed squares), and 50 nM S1P2-specific siRNA (closed circles), for 72 h. OPCs were allowed to migrate in the presence of various concentrations of S1P (A), FTY720 (B), for 4 h. TWs were precoated with 10 µg/ml fibronectin. Calcein-AM (10 µM) was added 1 h before end of assay. Nonmigrated cells were removed from top compartment of TW with a cotton swab, and calcein fluorescence of migrated cells on the bottom of TW was measured. Data are mean ± SE (12 replicates from at least 3 independent experiments). *P < 0.01, **P < 0.05 compared to scrambled siRNA.

Rho activation mediates S1P5-induced inhibition of OPC migration
To elucidate the S1P5-initiated signaling pathways that control OPC migration, we first determined whether the G{alpha}i protein coupled to the S1P5 receptor is involved. In this study, an inhibitor of G{alpha}i protein, pertussis toxin (PT), had no effect on S1P-induced inhibition of OPC motility indicating a lack of G{alpha}i involvement (Fig. 6 ). These results are in agreement with the previously identified role of the G{alpha}i protein coupled to the S1P5 receptor in promoting the survival of mature oligodendrocytes (6) . To further examine S1P5-induced signaling pathways that modulate OPC motility, we focused on another G{alpha} protein coupled to the S1P5 receptor, G{alpha}12/13, and its downstream target Rho GTPase. Rho proteins, including Rac, Cdc42, and Rho, are pivotal regulators of the actin cytoskeleton and act as molecular switches in various signal transduction pathways. Furthermore, it appears that integrins regulate Rho proteins, and Rho family GTPases regulate integrin functions (35) . Thus, engagement of integrin receptors with fibronectin has been shown to trigger a decrease in Rho activity that was essential for cell spreading on fibronectin (36) . We therefore investigated the role of Rho signaling in S1P5–induced decreases in integrin-mediated OPC migration using specific inhibitors of the Rho signaling pathway (Fig. 7 ). C3 exotoxin from Clostridium botulinum ADP ribosylates Rho at an asparagine residue and inhibits its activity (37) by interfering with its interaction with downstream targets. HA1077 is a potent inhibitor of Rho kinase (ROCK), which is a downstream target of Rho. Both C3 exotoxin and HA1077 abolished S1P-induced decreases in OPC motility, thus supporting the notion that S1P reduces integrin-mediated cell migration by engaging the Rho signaling pathway. To examine whether Rho is activated by S1P in OPCs, we used a Rho activation assay (Fig. 8 ). S1P treatment of OPCs significantly stimulated Rho activity, but total Rho expression did not increase. Rho activation was S1P5 dependent: knocking down S1P5 with siRNA abolished the effect. In contrast, knocking down S1P2 did not have any effect. Our data suggest that S1P activates Rho via the S1P5 receptor and that Rho activity is required for the inhibition of OPC migration.


Figure 6
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  		Figure 6. S1P effect on OPC migration is G{alpha}i-independent. OPCs were allowed to migrate in the presence of various concentrations of S1P (closed triangles) or in the presence of S1P with 50 µM pertussis toxin (closed circles) for 4 h. TWs were precoated with 10 µg/ml fibronectin. Calcein-AM (10 µM) was added 1 h before end of assay. Nonmigrated cells were removed from the top compartment of the TW with a cotton swab, and the calcein fluorescence of migrated cells on the bottom of TW was measured. Data are mean ± SE (12 replicates from at least 3 independent experiments).


Figure 7
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  		Figure 7. S1P5 receptor engages Rho/ROCK signaling pathway to inhibit OPC migration. OPCs were allowed to migrate in the presence of various concentrations of S1P (closed diamonds); or in the presence of S1P and Rho inhibitor, 250 µg/ml C3 exozyme (closed circles); or in the presence of S1P and ROCK inhibitor, 3 µM HA1077 (closed triangles) for 4 h. TWs were precoated with 10 µg/ml fibronectin. Calcein-AM (10 µM) was added 1 h prior to the end of the assay. Nonmigrated cells were removed from the top compartment of TW with a cotton swab, and the calcein fluorescence of migrated cells on bottom of TW was measured. Data are mean ± SE (12 replicates from at least 3 independent experiments). *P < 0.01 compared to cells treated with S1P alone.


Figure 8
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  		Figure 8. S1P stimulation of S1P5 receptor activates Rho. OPCs were treated with 50 nM scrambled siRNA or 50 nM S1P5-specific siRNA or 50 nM S1P2-specific siRNA for 72 h. Cells were plated on fibronectin-coated dishes and treated with or without 100 nM S1P for 30 min. Active Rho was precipitated using GST-Rhotekin which bears a consensus GTP-Rho-binding sequence, as described under Methods. Activation of Rho was detected by immunoblotting with antibody against Rho and visualized using SuperSignal West Femto Maximum Sensitivity Substrate. S1P binding specifically to the S1P5 receptor resulted in activation of Rho, and effect was abolished by knocking down S1P5. Knocking down S1P2 had no effect on S1P-induced activation of Rho.

S1P and S1P5 receptor mediate glutamate-induced decrease in OPC motility
In the immune system, it has been well documented that S1P is produced intracellularly in the organelles and in the plasma membrane of platelets or macrophages, and subsequently secreted (38) . Extracellular S1P has both paracrine and autocrine functions and elicits many responses in immune cells, including the control of cell migration. In the CNS, recent studies show that S1P could be released from astrocytes treated with phorbol ester (39) or in response to basic fibroblast growth factor (bFGF; ref 40 ). This suggests that extracellular S1P may participate in a paracrine network regulating OPC migration in the CNS. Next, we examined a possible role of S1P in autocrine regulation of OPC migration. It has been shown that integrin-mediated OPC migration is under control of glutamate receptor signaling (25) . To elucidate the role of S1P in glutamate modulation of OPC migration, we measured S1P synthesis and release from OPCs in response to glutamate treatment. OPCs were plated on fibronectin and treated with 0.8 mM glutamate. Sphingolipid analysis revealed that glutamate enhanced S1P generation in cells up to 15-fold, and this enhancement peaked at 1 h (Fig. 9 A). S1P production seems to result from the stimulation of de novo ceramide synthesis because it was completely abolished by the ceramide synthase inhibitor fumonisin B1. Ceramide is readily converted into sphingosine, which is then phosphorylated by sphingosine kinase to produce S1P (41) . The intracellular accumulation of S1P was accompanied by S1P secretion into the extracellular space. Approximately 30% of newly produced S1P was released from the OPCs into the medium (Fig. 9B ). To elucidate S1P involvement in glutamate modulation of OPC migration, we measured OPC migration in the presence of glutamate (Fig. 10 ). Consistent with our findings that glutamate induced S1P release from OPCs, which, in turn, inhibits OPC migration, glutamate inhibited integrin-mediated OPC migration in a dose-dependent manner. Knocking down the S1P5 completely abolished the effect of glutamate on OPC migration whereas knocking down the S1P2 did not have any effect. Taken together, the results of these studies suggest that S1P could be a part of an autocrine cascade induced by glutamate that controls integrin-mediated OPC migration, and S1P acts via binding to the S1P5 receptor (Fig. 11 ).


Figure 9
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  		Figure 9. Glutamate stimulates S1P synthesis and its extracellular export in OPCs. Cells were plated on fibronectin-coated dishes and treated with 0.8 mM glutamate with and without 50 µM fumonisin B1 (FB1). Cells and media were harvested, and S1P content was analyzed by mass spectroscopy. Glutamate treatment of OPCs resulted in robust increase in S1P accumulation in the cells and its release into the surrounding medium. S1P accumulation was prevented by blocking de novo ceramide synthesis with FB1. Data are mean ± SE (12 replicates from at least 3 independent experiments).


Figure 10
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  		Figure 10. Glutamate-induced inhibition of OPC migration is mediated by S1P5 receptor. OPCs were treated with 50 nM scrambled siRNA (closed diamonds); 50 nM S1P5-specific siRNA (closed triangles); or 50 nM S1P2-specific siRNA (closed squares), for 72 h. OPCs were allowed to migrate in the presence of various concentrations of S1P for 4 h. TWs were precoated with 10 µg/ml fibronectin. Calcein-AM (10 µM) was added 1 h before end of assay. Nonmigrated cells were removed from top compartment of TW with a cotton swab, and calcein fluorescence of migrated cells on bottom of TW was measured. Data are mean ± SE (12 replicates from at least 3 independent experiments). *P < 0.01, **P < 0.05 compared to scrambled siRNA.


Figure 11
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  		Figure 11. Scheme for S1P regulation of {alpha}v ß3 integrin-mediated cell migration. Previous studies have demonstrated that fibronectin binding to {alpha}v ß3 integrin results in a decrease of Rho activity, and it stimulates cell motility and cell spreading (36) . S1P activates the S1P5 receptor coupled with G{alpha}12/13, which enhances Rho activity leading to the reduction of {alpha}v ß3 integrin-driven OPC migration. Glutamate is a powerful regulator of OPC migration. At low concentrations of glutamate, OPC migration is increased (25) . In contrast, higher concentrations of glutamate boost the intracellular production of S1P and facilitate its release into the medium where it binds to the S1P5 receptor, which reduces OPC migration.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this study was to investigate the role of the S1P5 receptor in regulation of cell migration. We have shown that S1P binding to the S1P5 receptor results in reduction of integrin-mediated OPC migration. This is the first demonstration of the involvement of the S1P5 receptor in the negative regulation of cell migration. Fundamental differences in signaling through S1P receptors relate primarily to variations in G-protein coupling. Typically, activation of S1P1 or S1P3 increases directional or chemotactic migration (13 , 14) , and stimulation of S1P2 decreases chemotaxis (15) . S1P1 couples to G{alpha}i, whereas S1P3 and S1P2 couple to G{alpha}i as well as G{alpha}q and G{alpha}12/13 (42 , 43) . S1P5 has been reported to couple to G{alpha}i and G{alpha}12/13 (10) . Our data showing that S1P5 inhibits cell migration are consistent with the notion that G{alpha}i-linked S1P1 and S1P3 are chemoattractant receptors and G{alpha}12/13-linked S1P2 and S1P5 are chemorepellant receptors.

S1P5 is predominantly expressed in primary oligodendrocytes throughout their life span (6) , including OPCs, which migrate from the site of their emergence toward their final destination, mainly the future white matter tracts in the developing brain. Along with the S1P5 receptor, these cells also express S1P1, S1P2, and S1P3 receptors. Remarkably, S1P selectively engages the S1P5 receptor signaling pathway to inhibit OPC migration despite the presence of S1P2, another S1P receptor capable of inhibiting cell migration. Our results highlight the strong specificity of the effect of S1P on OPC migration. The data provide further evidence that S1P receptors are not entirely redundant despite some commonalities in G-protein coupling (44) .

The inhibitory effect of S1P on OPC migration was mimicked by its analog FTY720, which is a novel immunomodulator that acts as a high-affinity agonist at the S1P receptor where it internalizes the receptor and causes alterations to the normal circulation of lymphocytes between blood and lymphoid tissue (33 , 45) . FTY720 is potent immunosuppressant in animal models of transplantation and autoimmunity, especially in acute experimental encephalomyelitis (46) , a widely used as a model of multiple sclerosis. The etiology of the disease remains uncertain but is believed to involve autoimmune destruction of myelin in the CNS. The protective antiinflammatory effect of treatment with FTY720 was, to a large extent, due to inhibition of the T cell migration into the CNS (46) . It is noteworthy that the hallmarks of multiple sclerosis lesions in the brain include axon demyelination and dysfunctional oligodendrocytes, which are incapable of remyelinating axons (47) . Our data showing that FTY720 reduces OPC motility suggest that this compound could negatively affect the remyelination process, and this warrants further investigation in in vivo disease models.

In the present study, we explored the mechanism of S1P5-mediated inhibition of OPC migration by examining downstream effectors of S1P5. Whereas it has been shown that S1P5 engages G{alpha}i to stimulate mature oligodendrocyte survival (6) , S1P5-mediated inhibition of OPC migration was insensitive to the G{alpha}i inhibitor PT. Functional data for S1P5 indicate that this receptor couples primarily to G{alpha}12/13, which activate small GTP-ase Rho signaling pathway in order to reduce cell motility. The results of Rho pull-down studies and inhibitory analysis suggest that activation of Rho is required for S1P-induced reduction of OPC migration. Activated GTP-bound Rho could stimulate several downstream signaling pathways, among which ROCK is a prominent player. ROCK is a serine/threonine kinase, and it phosphorylates a number of substrates involved in actin-filament assembly, which, in turn, governs cell motility (48) . Blocking ROCK activity completely abolished the S1P effect on OPC migration. These results highlight novel signaling determinants of OPC migration and provide new evidence suggesting that the S1P5-initiated Rho kinase-dependent signaling regulates OPC motility.

Cell migration is a multistep process involving dynamic and spatially regulated changes to the cytoskeleton and cell adhesion. The Rho GTPases play key roles in coordinating the cellular responses required for cell migration (48) . Recently, it has become clear that integrins regulate Rho GTPases and the Rho GTPases and their effectors regulate the adhesive functions of the integrins (35) . The speed of cell migration is dependent on substrate composition, and indeed, the relative levels of Rho vary with matrix composition (49) . It is well established that Rho stimulates contractile actin-myosin filaments, resulting in formation of stress fibers and focal adhesions, which inhibit cell locomotion. As well as being involved in strengthening the attachment to extracellular matrix (ECM), Rho promotes the migration by stimulating cell body contraction (48) . Rho acts via ROCK to affect myosin light chain phosphorylation (MLC), both by inhibiting MLC phosphatase (50) and by phosphorylating MLC (51 , 52) . Based on the fact that integrins and Rho GTPases are intimately connected at the multiple levels, it has been hypothesized that integrins and Rho GTPases could be organized in the complex signaling cascade regulating cytoskeletal organization, cell adhesion, migration, and polarity (35) . The results of the present studies showing S1P5 receptor-dependent modulation of Rho activity add more complexity to the integrin signaling cascade regulating OPC migration. This suggests that a crosstalk between integrins and S1P5 receptor, involving modulation of Rho activity, contributes to the regulation of OPC migration.

Altogether, the results of these studies indicate a possible role of S1P as a guidance cue for OPC migration in the developing brain. Four major families of axon guidance molecules—netrins, semaphorins, slits, and ephrins—direct axons to specific sites by providing attractive or repulsive cues during the formation of neural networks (22) . Recent studies demonstrated that the migration of OPCs is guided by directional cues used in axonal migration (20 , 23) . These cues may be soluble, matrix bound, or displayed on the cell surface and may exist in gradients. For instance, semaphorin 3A, which is a secreted protein, exerts a repulsive chemotactic effect by binding to its receptor neuropilin expressed on OPCs (53) . Elegant studies by Sugimoto’s group demonstrated that semaphorin 3A and netrin-1 are chemorepellents distributed unevenly along the trajectory of OPCs migration, and the gradient of these proteins provides guidance cues for the cell migration (54) . There are obvious similarities in the effects of semaphorin 3A and S1P on OPCs. It has been pointed out that semaphorin 3A and S1P induce the retraction of processes in oligodendrocytes acting via receptors (neuropilin for semaphorin 3A and S1P5 for S1P) expressed by oligodendrocytes and common downstream elements, Rho kinase and CRMP2 (6) . Studies indicating that S1P can modulate OPC migration via the Rho kinase signaling pathway support the hypothesis that S1P could be involved in directional guidance of OPC migration. In the immune system, accumulating data suggest that S1P and its receptors are required for the emigration of thymocytes from the thymus, the trafficking of lymphocytes in secondary lymphoid organs, and the migration of B cells into splenic follicles (38) . Coordinated activities of biosynthetic and biodegradative enzymes stringently maintain the tissue concentration of S1P in the ranges that are required for optimal physiological activities (38 , 55 , 56) . The brain tissue levels of S1P are around 5–20 nM, which is within the range of affinities of S1P receptors (Kd=2–63 nM; ref 42 ). Emerging evidence indicates that S1P could be secreted by neural cells such as astrocytes and cerebellar granule cells, and this is under control of growth factor receptors (39 , 40) . In OPCs, glutamate treatment resulted in robust secretion of S1P from the cells leading to reduction of OPC motility via S1P5 receptor-initiated signaling. These data suggest that, in vivo, the S1P and S1P5 receptor could be a part of autocrine/paracrine network regulating chemotactic responses of migrating OPCs in the developing brain. Mounting evidence suggests that neurotransmitters released locally form axons control proliferation, differentiation, and migration of OPCs (57 , 58) . The inhibition of OPC migration by relatively high concentrations of glutamate is further support for the hypothesis of the widespread role for glutamate as chemoattractant that provides positional cues for neurons and glial cells in the developing nervous system (25 , 59 60 61) . Typically, chemoattractant mediates both start and stop motility signals in various cells (62 63 64) . Although very low concentrations of attractant stimulate migration, excessive levels of attractant hinder further motility. In the developing brain, low concentrations of glutamate would stimulate OPCs to begin migrating whereas cells approaching their targets (axons) will encounter increasing levels of glutamate that could signal the cells to stop moving. It appears that glutamate stimulation of OPC migration is mediated by glutamate receptor-mediated Ca2+-dependent intracellular signaling (25) . Activation of glutamate receptors could also inhibit OPC proliferation and prevent their differentiation in a way that is not attributable to a cytotoxic action of glutamate (65) . Antiproliferative effects of glutamate are Ca2+ independent and arise from an increase in intracellular Na+ and subsequent block of outward K+ currents (65) . Glutamate also modulates OPC responses via glutamate receptor-independent mechanism (66) . High concentrations of glutamate are known to induce depletion of intracellular cystine by acting on glutamate/cystine exchanger on the plasma membrane (30 , 66) . Cystine is a precursor of glutathione, which is a major cellular antioxidant, glutamate may activate free radical-dependent intracellular signaling and stress response kinases signaling cascades (66) . How glutamate slows down OPC migration is not clear yet, but our studies demonstrate that S1P plays a critical role. More investigation in developmental and disease models should provide new knowledge into the role of S1P as an instructional guidance cue for neural cell migration.


   ACKNOWLEDGMENTS
 
We thank Drs. Sergei Novgorodov and Mark Kindy for many stimulating discussions regarding the manuscript. We thank Ting Ting Hsieh Kinser and Bryan McElveen for the expert technical assistance in preparation of cell cultures. This work is supported by the NIH/NCCR COBRE in Lipidomics and Pathobiology P20 RR 17677–04 (T. I.G.), VA Merit Awards (T. I.G., L. M.O.), and GM062887 (L. M.O.).

Received for publication October 5, 2006. Accepted for publication December 14, 2006.


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