Essential Roles of Sphingosine 1-Phosphate/S1P1 Receptor Axis in the Migration o - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Atsushi Kimuraa,b,c, Tsukasa Ohmorib, Ryunosuke Ohkawad, Seiji Madoiwab, Jun Mimurob, Takashi Murakamic, Eiji Kobayashic, Yuichi Hoshinoa, Yutaka Yatomid, Yoichi Sakatab作者单位：aDepartment of Orthopedic Surgery, Jichi Medical University School of Medicine, Tochigi, Japan;bResearch Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan;cDivision of Organ Replacement Research, Center for Molecular M + }# v3 l, d6 ~9 K! ]0 d+ E  B" c
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      【摘要】) t/ W2 T6 S9 H) @6 A8 q
   Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell-based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that sphingosine 1-phosphate (Sph-1-P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph-1-P receptors, S1P1 was abundantly expressed. Sph-1-P-induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y-27632 (a Rho kinase inhibitor), and VPC23019 (a competitive inhibitor of S1P1 and S1P3). Sph-1-P does not act as intracellular mediator or in an autocrine manner, because sphingosine, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation-dependent formation and extracellular release of Sph-1-P were not observed. Further, Sph-1-P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph-1-P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S1P1, given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S1P1 abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph-1-P/S1P1 pathway may have therapeutic potential for CNS injuries.
      【关键词】 Migration Neural stem cell Transplantation Lysophospholipid7 w6 T+ j2 n! t) A1 {
   INTRODUCTION

Neural stem/progenitor cell (NSPC) migration is an essential process for the development of the central nervous system (CNS) as well as the ongoing neurogenesis that occurs in the mature CNS . Because this migratory property of NSPCs could theoretically be exploited for cell-based therapeutics of CNS disease, identification of NSPC chemoattractant factors would not only aid a better understanding of the mechanisms responsible for injury-mediated NSPC migration but also have clinical implications for cell-based therapeutic strategies for injury repair.( Y4 R6 `, `* y* D, R0 t

Although little is known of the mechanisms responsible for the injury-mediated NSPC migratory responses, a variety of protein and peptide factors have been shown to influence NSPC migration. Cytokines and growth factors with known important functions in CNS development, such as stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), fibroblast growth factor (FGF), and epidermal growth factor (EGF), are capable of enhancing injury-induced NSPC proliferation and migration .
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Another type of candidate molecule for regulating NSPC migration may be lipid mediators. The physiological roles of lipid mediators produced during membrane phospholipid degradation are currently receiving much attention in biomedical research fields. In particular, lysophospholipids, which are exemplified by lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P), are known to be important mediators of normal cellular proliferation, survival, and motility in various cells through the LPA and S1P families of G protein-coupled receptors ). Here, we examined the physiological roles of lysophospholipids for the migration of NSPCs.
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MATERIALS AND METHODS& P  x" y) J" K. t6 O; y' l

Antibodies, J7 ?; H# p) u( q
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Anti-Sph-1-P mouse monoclonal antibody (clone NHS1P) was a gift from Alfresa Pharma Corporation (Tokyo, http://www.alfresa-pharma.co.jp/english). This antibody is specific for Sph-1-P (Japan Patent Office: number P2002-243737A; August 28, 2002). It was confirmed that clone NHS1P did not react with sphingosine, sphingomyeline, lysosphingomyeline, ceramide, ceramide 1-phosphate, phosphatidylserine, lysophosphatidylcholine, or lysophosphatidylethanolamine.
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The following materials were obtained from the indicated suppliers: anti-S1P1 polyclonal antibody, 1-oleoyl-LPA, lysophosphatidylcholine, D-erythro-sphingosine (Sph), C2-ceramide, anandamide, and recombinant human EGF (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com); Sph-1-P (BIOMOL International, L.P., Plymouth Meeting, PA, http://www.biomol.com/1); VPC23019 (Avanti Polar Lipids, Inc., Alabaster, AL, http://www.avantilipids.com); recombinant human SCF, recombinant human SDF-1, and recombinant human basic FGF (PeproTech EC Ltd., London, http://www.peprotechec.com); Y-27632 (Calbiochem, San Diego, http://www.emdbiosciences.com); anti-nestin mouse monoclonal antibody, anti-microtubule associated protein-2 (MAP2) mouse monoclonal antibody, and anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (Chemicon International, Temecula, CA, http://www.chemicon.com); and anti-CD11b mouse monoclonal antibody (Serotec Ltd., Oxford, U.K., http://www.serotec.com).6 Z/ V3 h4 r' [- l( q5 Z
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NSPC Isolation and Culture

All animal procedures were approved by the institutional Animal Care and Concern Committee at Jichi Medical University, and animal care was performed in accordance with the committee's guidelines. Rat NSPCs were isolated and cultured as described previously . Briefly, rat forebrains at embryonic day 14.5 of gestation were isolated and mechanically dissociated into a suspension of single cells. The dissociated cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with B27 supplement (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 20 ng/ml FGF, and 20 ng/ml EGF. The cells were grown in flasks as neurospheres and prevented from attaching to the plates by periodic gentle agitation of the plates each day. EGF and FGF were added every other day, and the culture medium was passaged every 3¨C4 days. The cells were used for experiments during 2¨C4 passages. The NSPCs used in our study expressed neural stem cell markers nestin (95.1% ¡À 1.21%) and Sox2 (96.3% ¡À 0.62%) but lacked markers of lineage-specific differentiation. When NSPCs were cultured in the presence of 1 µM retinoic acid and 1% serum, mixed numbers of Tuj1  neuron (36.7% ¡À 2.60%), GFAP  astrocytes (42.2% ¡À 3.67%), and O4  oligodendrocytes (18.8% ¡À 1.99%) were observed. These results indicated that the cells used in our study have stem cell properties.

Reverse Transcription-Polymerase Chain Reaction

Total RNA was isolated from the indicated tissues using an RNA isolation kit (RNeasy Protect kit; Qiagen Inc., Valencia, CA, http://www1.qiagen.com). The RNA samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers and an RT-PCR kit (SuperScript One-Step RT-PCR System; Invitrogen). A primer pair for mouse/rat GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) was used for control RT-PCR experiments. The oligonucleotide primer pairs used in this study were shown in supplemental online Table 1. The RT-PCR amplification was initiated by incubations at 55¡ãC for 30 minutes and then 95¡ãC for 5 minutes. The thermal cycling profile consisted of 40 cycles of 94¡ãC for 15 seconds, 60¡ãC for 30 seconds, and 68¡ãC for 30 seconds.# {1 z0 l; T2 |2 o& @) K3 e$ ^
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Quantification of mRNA expression was performed by real-time quantitative RT-PCR using a SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen). The mouse GAPDH control reagents described above were used to estimate the amount of RNA analyzed. During the extension phase of the PCR, the fluorescent dye from a specific primer was detected using an ABI PRISM 7700 Sequence Detector System (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com). A standard curve was established by analyzing double aliquots of serial dilutions of each PCR product-containing plasmid. Aliquots of 100 ng of purified RNA were used as the templates. For each sample, the amount of the target sequence was estimated by reference to the standard curve, and the quantity was determined by dividing the copy number of the sequence by that of the rat GAPDH sequence.
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Migration

NSPC migration was assessed by a modified Boyden's chamber assay (i.e., in Transwell cell culture chambers ). Polycarbonate filters with 8-µm pores, used to separate the upper and lower chambers, were coated with 10 µg/ml collagen type IV (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). NSPCs were added to the upper chamber at a density of 1 x 105 per 100 µl of medium containing 1% fatty acid-free bovine serum albumin (BSA) and incubated for 12 hours at 37¡ãC. The NSPCs were allowed to migrate toward the indicated chemoattractant in the lower chamber. After the reaction, the filters were fixed and subjected to Giemsa staining. After removal of the nonmigrated cells by wiping with cotton swabs, cells that had migrated through the filter to the lower surface were counted manually under a microscope in five predetermined fields at a magnification of x100. When cells transduced with a short hairpin RNA interference (shRNAi) lentiviral vector were used, migrated cells that expressed green fluorescent protein (GFP) (considered to be an expressing shRNAi) were counted using fluorescence microscopy under the same conditions.
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Metabolism of Sph

NSPCs (2 x 105 cells) were incubated with 1 µM (0.2 µCi) . Finally, portions of the extracted lipids were applied to silica gel high-performance thin-layer chromatography plates (Merck, Darmstadt, Germany, http://www.merck.com), and the plates were then developed in butanol/acetic acid/water (3:1:1), followed by autoradiography.

Spinal Cord Injury and Transplantation of NSPCs8 m: `3 V( F  R2 W0 E
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Contusion spinal cord injury was induced using an Infinite Horizon Impactor (Precision Systems and Instrumentation, LLC, Fairfax, VA, http://www.presysin.com) .3 z" J) h$ C4 R) x

When indicated, rats with a spinal cord injury received transplantation of NSPCs to examine their in vivo migration. Briefly, at 7 days after the injury, an additional laminectomy was performed at the T10-11 level to expose the caudal lesion of the injured spinal cord. A total volume of 5 µl of cell suspension containing 5 x 105 NSPCs transduced with LentiLox lentiviral vector was injected into the region 8-mm caudal and 0.8-mm deep relative to the epicenter of the spinal cord injury using a 10-µl Hamilton syringe (Hamilton Company, Reno, NV, http://www.hamiltoncompany.com) held in a micromanipulator. All rats received tacrolimus immunosuppression (0.64 mg/kg intramuscularly) daily to reduce rejection reactions. At 3 days after the transplantation, the animals were sacrificed for histological analysis. Serial transverse sections of the spinal cord from ¨C10 mm to  10 mm relative to the NSPC injection site were prepared. In our transplantation experiments, the GFP-positive area in serial transverse sections of injured spinal cord was thought of as transplanted NSPCs transduced with a lentiviral vector. First, the sections were stained with 4,6-diamidino-2-phenylindole to confirm the nuclear location and were then photographed at intervals of 0.5 mm using a camera with a fluorescent microscope. After digitalization using Photoshop 7.0 software (Adobe Systems Incorporated, San Jose, CA, http://www.adobe.com), image analysis was performed using Scion Image for Windows (Scion Corporation, Frederick, MD, http://www.scioncorp.com). Briefly, GFP-positive areas were extracted and transformed into binary images, and the pixel numbers of GFP-positive areas were quantified.

Measurement of Sph-1-P

After excision of a 12-mm length of each spinal cord, 3 ml of ice-cold chloroform/methanol (1:2) was added, and the samples were extensively sonicated. Lipids were extracted from the samples, and the Sph-1-P concentrations were measured by reaction with o-phthalaldehyde as described previously . Hence, the actual Sph-1-P content in the spinal cord was estimated by multiplying the amount after lipid extraction by 1/0.37.
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Immunohistochemistry/ p3 Z- q- D- k. G

The spinal cord was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2 hours at 4¡ãC, incubated with PBS containing sucrose (10%¨C30%), and then frozen in the presence of OCT compound in dry ice/ethanol. Sections were prepared from frozen tissues at ¨C25¡ãC and attached to poly(lysine)-coated glass slides. For detection of Sph-1-P localization by immunohistochemistry, tissue sections were blocked with 1% BSA in PBS containing 0.2% Triton X-100 and incubated with a mouse monoclonal anti-Sph-1-P antibody (1:200) at 4¡ãC for 12 hours. After washing with PBS, the sections were incubated with a biotin-conjugated rabbit anti-mouse immunoglobulin M (IgM) antibody followed by streptavidin-conjugated horseradish peroxidase and were visualized with VECTOR SG (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Counterstaining was performed with nuclear Fast Red solution (Sigma-Aldrich). To detect the expression of Sph-1-P in specific cell types, sections probed with the anti-Sph-1-P antibody were further incubated with the following specific antibodies for neural cell markers: anti-nestin for NSPCs (1:200); anti-MAP2 for neurons (1:200); anti-GFAP for astrocytes (1:500); and anti-CD11b for microglia (1:100). The sections were then incubated with a species-specific secondary antibody conjugated with cyanine 3 (Chemicon International) and a biotin-conjugated rabbit anti-mouse IgM antibody and then with Alexa 488-conjugated streptavidin (Invitrogen). Immunofluorescence staining was observed and photographed using a confocal microscope.3 a6 j! G8 j7 d- x, R
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Construction of Lentiviral shRNAi Vectors. n- l/ d- y$ }' ]. {

A gene transfer vector, pLL3.7, for constructing replication-defective self-inactivating HIV shRNAi vectors was purchased from American Type Culture Collection (Manassas, VA, http://www.atcc.org) . Putative short interfering RNA (siRNA) target sequences were designed against the rat S1P1 gene using the Web software provided by Dharmacon RNA Technologies (Dharmacon, Inc., Chicago, http://www.dharmacon.com). Sequences were determined to be unique to the rat gene by BLAST (Basic Local Alignment Search Tool) searches of the GenBank database. Oligonucleotide sequences were designed corresponding to the sense and antisense sequences of the siRNA target sites of interest separated by a hairpin loop sequence (see Fig. 5A). The oligonucleotide pairs were then annealed and cloned into the HpaI/XhoI site of pLL3.7.5 q0 k% e* ]" @; E
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The pLL3.7 shRNAi plasmid was transfected into HEK293T cells, together with three packaging plasmids encoding gag-pol (PLP-1) (Invitrogen), rev (PLP-2) (Invitrogen), and VSV-G env (Clontech, Mountain View, CA, http://www.clontech.com), using the Lipofectamine Plus reagent (Invitrogen). After 12 hours, the culture medium was renewed to initiate harvesting of virus particles. At 48 hours, harvesting was performed, and the virus particles were concentrated by ultracentrifugation. Transduction units of the lentiviral vector were measured as described previously . After the transduction of NSPCs, enhanced GFP (eGFP) expressions were routinely examined using fluorescence microscopy or fluorescence-activated cell sorting analysis. This was because it is possible that lower transduction efficiency affects the interpretation of shRNAi experiments. We consistently confirmed GFP expression of 65%¨C90% in transduced NSPCs with each lentiviral vector. Further, the high proportion of nestin-positive cells and the negativity for lineage-specific markers did not change after transduction with lentiviral vectors expressing shRNAi., b  Q5 S! A( z

Statistical Analysis3 R0 G) v; A3 i9 E( e0 N- l' K
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Statistical significance was assessed using Student's paired t test where indicated in the figure legends.

RESULTS
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Expression of Lysophospholipid Receptors in NSPCs6 P, F* H% l' ]
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First, we examined the expressions of lysophospholipid receptors in NSPCs, adult brain, and adult spinal cord. Specifically, we investigated the Sph-1-P receptors S1P1, S1P2, S1P3, S1P4, and S1P5 and the LPA receptors LPA1, LPA2, and LPA3 . The expressions of S1P1 and LPA2 were predominantly found together with trace expressions of S1P2, S1P3, S1P4, LPA1, and LPA3 (Fig. 1A). S1P5 expression was predominantly found in the spinal cord and marginally in NSPCs (Fig. 1A). Real-time quantitative RT-PCR analysis of NSPCs revealed abundant expression of S1P1 and a lower level of S1P4 expression (Fig. 1B). Regarding the LPA receptors, NSPCs expressed a low level of LPA2 (Fig. 1B). Confocal microscopy analysis confirmed that neurospheres simultaneously expressed nestin, an NSPC marker, and S1P1 (Fig. 1C). Accordingly, we concluded that NSPCs mainly express S1P1 as their lysophospholipid receptor.

Figure 1. Expression of lysophospholipid receptors in neural stem/progenitor cells (NSPCs). (A): Reverse transcription-polymerase chain reaction (RT-PCR) analyses of transcripts derived from the genes for S1P1¨C5 and LPA1¨C3 in NSPCs, adult rat brain (Brain), and adult rat spinal cord (Spine) are shown. As a control, RT-PCR analysis for the rat GAPDH transcript was performed simultaneously. The data are representative of four experiments. (B): The mRNA expression levels of the S1P1¨C5 and LPA1¨C3 genes in NSPCs were quantified by real-time quantitative RT-PCR. Columns and error bars represent the mean ¡À SD (n = 4 per group). (C): Confocal microscopic images of NSPCs as neurospheres dual-immunostained with antibodies against S1P1 and nestin. The data are representative of four experiments. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Migration of NSPC Toward Sph-1-P

We next evaluated the effects of various lipid mediators, FGF, EGF, SCF, and SDF-1, on NSPC migration. We examined the migration induced by several concentrations of all reagents (1¨C1,000 ng/ml cytokines, 1 nM to 10 µM lipid mediators) (data not shown) and then compared the results of the concentration that had the maximal migratory response (Fig. 2A). Among the lipid mediators examined, Sph-1-P, a sphingosine-based lysophospholipid, was the most powerful chemoattractant for NSPCs (Fig. 2A). The Sph-1-P-induced response was concentration-dependent and reached a maximum response at 100 nM (Fig. 2B). Another important lysophospholipid mediator, LPA, which is reportedly important for NSPC viability , but their effects were much weaker than those of Sph-1-P (Fig. 2A). To clarify whether the NSPC migration was dependent on the presence of a concentration gradient of Sph-1-P between the lower and upper chambers, a checkerboard analysis was conducted. The maximal migration induction occurred in the presence of a positive Sph-1-P concentration gradient (Table 1), showing that Sph-1-P induces NSPC chemotaxis. In addition, the presence of equal concentrations of Sph-1-P above and below the membrane produced a slight but significant enhancement of NSPC migration, indicating that Sph-1-P has a chemokinetic effect on NSPCs (Table 1).1 R7 M6 C) e* t' i3 x

Figure 2. Stimulation of neural stem/progenitor cell (NSPC) migration. (A): NSPC migration was assessed using a modified Boyden's chamber assay. A solution containing 100 nM Sph-1-P, lysophosphatidic acid (LPA), or sphingosine (Sph), 1 µM of C2-ceramide (Cer), anandamide (AEA), or lysophosphatidylcholine (LPC), 100 ng/ml of SDF-1, SCF, EGF, or FGF, or no additive (as a control) was placed in each lower chamber, and the NSPCs were allowed to migrate for 12 hours (n = 4). (B): The indicated concentrations of Sph-1-P and LPA were placed in each lower chamber, and the NSPCs were allowed to migrate for 12 hours (n = 4). (C): NSPCs preincubated without (control) or with 300 ng/ml pertussis toxin (PTX) or 20 µM Y-27632 for 2 hours were allowed to migrate for 12 hours toward the lower chamber that contained 100 nM Sph-1-P or no Sph-1-P (n = 4). (D): NSPCs were pretreated without or with various concentrations of VPC23019 for 30 minutes. NSPC migration toward the indicated concentrations of VPC23019 or 100 nM Sph-1-P was examined (n = 4). Columns and error bars represent the mean ¡À SD. *p * t, N9 E9 _8 I' n; M

Table 1. Checkerboard analysis of neural stem/progenitor cells

S1Ps are seven-pass transmembrane receptors coupled with Gi, Gq, and G12/13 .
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Sph Metabolism in NSPCs- D7 Y' ^2 a2 I
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Sph-1-P is a bioactive sphingolipid, acting as an intracellular second messenger in some cells and as an extracellular mediator in others Sph metabolism was not affected by the established NSPC agonists such as FGF, EGF, SCF, and SDF-1 (Fig. 3B). Accordingly, it is unlikely that Sph-1-P acts as an intracellular second messenger in NSPCs. Further, 3H-labeled Sph-1-P was not detected in the medium under these conditions, and stimulation-dependent extracellular Sph-1-P release did not occur (Fig. 3), indicating that Sph-1-P does not act in an autocrine manner.
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Figure 3. Metabolism of Sph for 1 minute and then challenged without (Control) or with 100 ng/ml of EGF, FGF, SCF, or SDF-1 for 20 minutes. The results shown are representative of three independent experiments. Abbreviations: Cer, Ceramide; Cont, control; EGF, epidermal growth factor; FGF, fibroblast growth factor; O, origin; SCF, stem cell factor; SDF-1, stromal cell-derived factor-1; SM, sphingomyelin; Sph, sphingosine; Sph-1-P, sphingosine 1-phosphate.4 w& k: @# f/ P/ g  B
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Enhancement of Sph-1-P Content at the Spinal Cord Injury$ R& b4 h9 J, a7 A: V, y

To investigate the physiological role of Sph-1-P in the injured CNS, lipids were extracted from spinal cords with or without a contusion injury, and the Sph-1-P concentrations were measured. The Sph-1-P concentrations in the spinal cord were significantly enhanced at 7 days after the spinal cord injury (Fig. 4A, 4B). Next, we used immunohistochemistry to examine which site(s) were responsible for the Sph-1-P elevation after the spinal cord injury. In the normal spinal cord, Sph-1-P was expressed mainly in neurons in the gray matter (Fig. 4C). At 7 days after the spinal cord injury, destruction of the normal structure of the CNS and accumulation of microglia, immunoreactive cells of non-neural lineages, were observed (Fig. 4C). Microglia reportedly release an unidentified chemoattractant(s) for NSPCs after CNS injury and thus play an important role in directing the replacement of damaged or lost cells in the CNS . Interestingly, Sph-1-P was highly expressed in the region of microglia accumulation in the injury site (Fig. 4C). Confocal microscopy of individual cells confirmed the preferred expression of Sph-1-P in neurons of the normal spinal cord (data not shown). In injured spinal cords, reactive astrocytes around the injury and CD11b-positive microglia highly coexpressed Sph-1-P at the site of the injury (Fig. 4D), suggesting that the accumulation of reactive astrocytes and microglia after spinal cord injury may be responsible for the increased level of Sph-1-P. Further, although Sph-1-P was abundantly expressed in injured areas, few MAP2-positive cells were seen to exist (Fig. 4D). This result strengthened our assertion that the accumulations of microglia and astrocytes at an injured site are responsible for Sph-1-P elevation.9 Z8 W$ P) B3 n. u

Figure 4. Enhancement of the Sph-1-P concentration at the site of a spinal cord injury. (A): The Sph-1-P content in the spinal cord at 0 (Control) or 7 days after a contusion injury (Injured Spinal Cord) was measured by high-performance liquid chromatography. Representative chromatographic patterns are shown. (B): Sph-1-P contents in the spinal cord at various times after the injury are shown. Values represent the mean ¡À SD (n = 3 per group). **p = .002, two-tailed Student t test. (C): Distribution of Sph-1-P in the spinal cord at 0 (Normal) or 7 days after the injury (Injury). Sections were immunostained with an antibody against Sph-1-P and visualized with VECTOR SG (black). Counterstaining was carried out with Nuclear Fast Red (red). A section of normal spinal cord stained with an isotype-matched control antibody (Negative Control) is also shown. In the normal spinal cord (Normal), Sph-1-P-positive cells consist mainly of neurons in the gray matter. Abundant Sph-1-P-positive cells are present in the injured area (arrow; Injury). Higher magnifications of the numbered boxed regions are shown on the right. (D): Characterization of Sph-1-P-positive cells in the injured spinal cord. Sections were double-immunostained for MAP2 and Sph-1-P, GFAP and Sph-1-P, or CD11b and Sph-1-P. The merged images show colocalization (yellow) of MAP2 and Sph-1-P, GFAP and Sph-1-P, or CD11b and Sph-1-P. Abbreviations: GFAP, glial fibrillary acidic protein; MAP2, microtubule associated protein-2; Sph-1-P, sphingosine 1-phosphate.

Involvement of Sph-1-P/S1P1 Pathway in the Migration of NSPCs

Next, we used the HIV lentiviral system LentiLox, which simultaneously expresses shRNAi and eGFP ; however, NSPC migration induced by FGF and EGF was not inhibited by transduction with LentiLox-S1P1D (Fig. 5E).
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Figure 5. Effects of S1P1 silencing using LentiLox lentiviral vectors on neural stem/progenitor cell (NSPC) migration. (A): Schematic representation of the LentiLox lentiviral vector. The expected structures of the shRNAi for S1P1 and the random sequence are shown on the right. (B): Cultured NSPCs were transfected with LentiLox (MOI = 30), and GFP-positive cells were photographed using a fluorescent microscope at 48 hours after the transduction. (C): NSPCs were transfected with increasing concentrations of LentiLox for 48 hours. Expression of GFP in the cells was analyzed by flow cytometry. The percentages of transduced cells expressing GFP are shown. Columns and error bars represent the mean ¡À SD (n = 3). (D): NSPCs were transduced with the empty vector (Emp), LentiLox-random (Rand), or LentiLox-S1P1D (S1P1D). At 3 days after the transduction, silencing of S1P1 was quantified by real-time quantitative reverse transcription-polymerase chain reaction. Columns and error bars represent the mean ¡À SD (n = 4). (E): Migration of transduced NSPCs in the presence of the empty vector (Emp), LentiLox-random (Rand), or LentiLox-S1P1D (S1P1D) toward 100 nM Sph-1-P. Columns and error bars represent the mean ¡À SD (n = 4). Abbreviations: GFP, green fluorescent protein; MOI, multiplicity of infection; shRNAi, short hairpin RNA interference.  l2 F0 f) o. \

Finally, we examined whether an Sph-1-P gradient/S1P1 axis is associated with the in vivo migration of NSPCs toward a spinal cord injury. We induced a thoracic spinal cord injury in female rats as described in Materials and Methods. At 7 days after the injury, NSPCs transduced with LentiLox-random or LentiLox-S1P1D were transplanted into the region 8 mm caudal to the injury site. The migrated GFP-positive cells were evaluated histologically at 3 days after the transplantation. NSPCs transduced with LentiLox-random migrated toward the spinal cord injury (Fig. 6). Notably, LentiLox-S1P1D abolished the injury-mediated NSPC migration (Fig. 6), indicating that the locally elevated Sph-1-P concentration in the injured area plays an important role in NSPC migration through its receptor S1P1 in vivo.
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Figure 6. Inhibition of transplanted neural stem/progenitor cell (NSPC) migration toward spinal cord injury by S1P1 silencing. (A): NSPCs transduced with LentiLox-random or LentiLox-S1P1D were injected into the region 8 mm caudal and 0.8 mm deep relative to the epicenter of the spinal cord injury. At 3 days after the transplantation, the animals were sacrificed for histological analysis. Transverse sections of the spinal cord at the indicated distances from the NSPC injection site are shown. The sections were stained with DAPI to confirm the nuclear location (blue). GFP-positive cells (green) indicate transplanted NSPCs transduced with the LentiLox lentiviral vector. (B): Representative data of higher magnification at 6 mm from the injection site are shown. (C): Quantification of the GFP-positive area in each 0.5-mm segment in transverse sections of the spinal cord (triangles: LentiLox-random; circles: LentiLox-S1P1D). The data represent the mean ¡À SEM (n = 5 and 7 per group, respectively). *p
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DISCUSSION

Sph-1-P currently attracts much attention as a bioactive sphingolipid with various cellular functions, although its precise physiological and pathophysiological roles are yet to be defined. Although originally postulated to function as an intracellular second messenger . In the present study, we examined the involvement of Sph-1-P in the migration of NSPCs and explored its physiological role in the CNS.. L% s" |* g1 p* e% n; ^
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Of the Sph-1-P receptor actions, Sph-1-P receptors have been shown to have especially critical roles in cell migration. Activation of S1P1 or S1P3 by Sph-1-P in many cell types increases directional or chemotactic migration, whereas S1P2 abolishes migration and membrane ruffling . Although further studies are clearly needed to more precisely define the role of the intracellular signaling pathways in NSPC migration induced by Sph-1-P, our data clearly demonstrated that the Sph-1-P/S1P1 pathway is activated within the spinal cord after a contusion injury and that it contributes to NSPC migration toward the sites of injury.
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An invariant feature of damage to the CNS is the migration of microglia to the site of injury and their subsequent activation; in addition, astrocytes are also activated. The activation of microglia can result in either neuroprotective or neurotoxic effects, or both . In our study, Sph-1-P concentration was enhanced at the site of spinal cord injury, and Sph-1-P was highly expressed at the site of microglia accumulation. Sph-1-P enhances the viability, migration, and differentiation of NSPCs, which suggests that Sph-1-P may be an important neuroprotective factor released from microglia.
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Questions remaining to be addressed are the mechanisms by which Sph-1-P concentration in the spinal cord is enhanced after spinal cord injury. Our data suggest that accumulation of microglia and reactive astrocytes may contribute to the elevation of Sph-1-P at the injured site. Sph kinase is known as a key enzyme in the regulation of cellular Sph-1-P, and microglia that express macrophage marker CD11b probably contains a great deal of Sph-1-P, as is the case with white blood cells . Sph-1-P content in spinal cord may depend on various factors, including Sph kinase expression and activation in microglia and astrocytes, and the lipid phosphate phosphatase and Sph lyase activities of the cells. Investigations into the precise mechanisms of injury-mediated Sph-1-P elevation and Sph metabolism at injury sites are now under way in our laboratory.2 h! S2 g' N) e- {9 e. x  {
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As there are so many chemoattractant factors in an injured area, a critical question is how silencing of one lysophospholipid receptor, S1P1, could have such dramatic effects on NSPC migration toward CNS injury in vivo. There are several proposed mechanisms to explain the importance of Sph-1-P/S1P1 to other signaling pathways. First, some growth factors stimulate and translocate Sph kinase to the plasma membrane, resulting in local formation of Sph-1-P and activation of Sph-1-P receptors . Thus, it is possible that the silencing of S1P1 blocks not only direct action of the Sph-1-P/S1P1 axis, but also the amplification loops for cell migration via the functions of a number of other unknown receptors.

Although our data add important information concerning the role of Sph-1-P in the CNS, its precise role for embryonic brain development remains unknown. Because NSPC migration is an essential process for the development of the CNS . It will be important to investigate whether these structurally related lysophospholipids elicit overlapping and/or distinct signaling pathways and thereby induce different functional responses in embryonic brain development.

CONCLUSION' C0 o+ `1 m( s& [
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Sph-1-P through its receptor S1P1 is an important factor involved in NSPC migration toward injured areas of the CNS. Other mediators that regulate NSPC reactivity are also present in injured areas of the CNS: growth factors (FGF and EGF), cytokines and chemokines (SDF-1 and SCF), and neurotrophins such as brain-derived neurotrophic factor. However, we postulate that Sph-1-P is the potent mediator that enhances NSPC migration and might be the central controller of several amplification loops of cell migration. Sph-1-P receptor agonists, such as FTY720, may be useful not only for promoting immunomodification and angiogenesis  but also for treating CNS diseases, including spinal cord injury, cerebral infarction, and degenerative diseases. Further, strategies for delivering specific S1P1 agonists into injured areas may lead to new therapeutic approaches for mobilizing endogenous or transplanted NSPCs to repair various types of CNS lesions.

DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS+ _0 ]# n/ J7 y1 s

We thank Drs. K. Takeuchi, Y. Hakamata, and K. Shimazaki (Jichi Medical University) and J. Francis (University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School) for technical advice. This work was partly supported by Grants-in-aid for Scientific Research from the Ministry of Education and Science; Jichi Medical University Young Investigator Award; and Grants for "High-Tech Center Research" Projects for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2002¨C2006. A.K. and T.O. contributed equally to this work.
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