Address correspondence and reprint requests to Carmen Sato-Bigbee, Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, VA 23298–0614, USA. E-mail: firstname.lastname@example.org
We had found previously that neurotrophin-3 (NT-3) is a potent stimulator of cAMP-response element binding protein (CREB) phosphorylation in cultured oligodendrocyte progenitors. Here, we show that CREB phosphorylation in these cells is also highly stimulated by sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is known to be a potent mediator of numerous biological processes. Moreover, CREB phosphorylation in response to NT-3 involves sphingosine kinase 1 (SphK1), the enzyme that synthesizes S1P. Immunocytochemistry and confocal microscopy indicated that NT-3 induces translocation of SphK1 from the cytoplasm to the plasma membrane of oligodendrocytes, a process accompanied by increased SphK1 activity in the membrane fraction where its substrate sphingosine resides. To examine the involvement of SphK1 in NT-3 function, SphK1 expression was down-regulated by treatment with SphK1 sequence-specific small interfering RNA. Remarkably, the capacity of NT-3 to protect oligodendrocyte progenitors from apoptotic cell death induced by growth factor deprivation was abolished by down-regulating the expression of SphK1, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Altogether, these results suggest that SphK1 plays a crucial role in the stimulation of oligodendrocyte progenitor survival by NT-3, and demonstrate a functional link between NT-3 and S1P signaling, adding to the complexity of mechanisms that modulate neurotrophin function and oligodendrocyte development.
Myelin is the highly specialized, multi-lamellar membrane sheath that surrounds axons, allowing the rapid saltatory conduction of nerve impulses. Multiple sclerosis (MS), a major cause of disability in young adults, is not only characterized by the loss of myelin but also by damage and death of oligodendrocytes, the cells that make the myelin membrane in the central nervous system (CNS). Thus, a major challenge in the treatment of this disease is the development of effective therapies to stimulate oligodendrocyte survival and the replenishment of lost oligodendrocyte pools. A major reason for the slow progress in this area is the fact that the molecular mechanisms that control oligodendrocyte biology are poorly understood. Oligodendrocyte development and survival involve the co-ordinated action of numerous factors, including cytokines, hormones and neurotrophins (Barres and Raff 1994; Barres et al. 1994; Casaccia-Bonnefil et al. 1996b; Cohen et al. 1996; Kumar et al. 1998; Kahn et al. 1999; Wilson et al. 2003).
Upon neurotrophin binding, Trks dimerize and become catalytically active, resulting in autophosphorylation of the receptor subunits. This process triggers the activation of several signaling cascades, including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase and phospholipase C-γ pathways that culminate in the activation of several transcription factors (Finkbeiner 2000; Kaplan and Miller 2000; Patapoutian and Reichardt 2001). In this regard, previous work from our laboratory demonstrated that NT-3 is a potent activator of the transcription factor CREB (Johnson et al. 2000; Saini et al. 2004).
CREB, a member of the leucine zipper family of transcription factors, binds to the consensus sequence known as Ca2+/cyclic AMP-response element (CRE) (Montminy et al. 1990; Sheng et al. 1991), and is activated by phosphorylation at ser133 (Gonzalez et al. 1989). We have previously shown that different neuroligands and signal transduction pathways stimulate CREB phosphorylation at specific stages of oligodendrocyte development (Sato-Bigbee et al. 1999). In committed oligodendrocytes, at a maturational stage that immediately precedes myelination, CREB is involved in the stimulation of myelin basic protein (MBP) expression in response to cAMP-mediated pathways (Sato-Bigbee and DeVries 1996; Afshari et al. 2001). At an earlier stage of development, when the cells are immature oligodendrocyte progenitors, CREB plays a crucial role in the NT-3-dependent stimulation of cell proliferation (Johnson et al. 2000). In addition, we recently demonstrated that NT-3 stimulates the expression of the anti-apoptotic protein Bcl-2 in oligodendrocyte progenitors and this process involves the direct regulation of the Bcl-2 gene by CREB (Saini et al. 2004).
The present report indicates that CREB phosphorylation in oligodendrocyte progenitors is also stimulated by sphingosine-1-phosphate (S1P), a lipid metabolite that has emerged in recent years as a potent mediator in numerous biological processes (Spiegel et al. 2002; Watterson et al. 2005). Interestingly, previous studies demonstrated a role for S1P as a mediator of NGF action in PC12 cells, and in hippocampal and DRG neurons (Edsall et al. 1997; Rius et al. 1997; Culmsee et al. 2002; Toman et al. 2004). These observations led us to investigate whether S1P could also play a role in NT-3 signaling. Our results indicate that CREB phosphorylation in response to NT-3 also involves sphingosine kinase 1 (SphK1), an enzyme that catalyzes S1P synthesis. Treatment of oligodendrocyte progenitors with NT-3 results in intracellular translocation and activation of SphK1. Our results demonstrate that SphK1 plays a crucial role in the stimulation of oligodendrocyte survival induced by NT-3, and indicate for the first time the presence of a cross-talk between NT-3 and S1P signaling.
Materials and methods
Isolation and culture of oligodendrocyte progenitors
Immature oligodendrocyte progenitors were isolated from 2- to 3-day-old Sprague–Dawley rat brain using a Percoll (Sigma, St Louis, MO, USA) gradient and differential adhesion, as described previously (Sato-Bigbee et al. 1999; Johnson et al. 2000). Immediately after isolation, the cells were plated in 24-well plates coated with 25 µL/well reduced growth factor-Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) and maintained overnight in chemically defined medium (CDM) [Dulbecco's modified Eagle's medium (DMEM)/F-12 medium (1 : 1; Invitrogen, Grand Island, NY, USA) supplemented with 1 mg/mL fatty acid-free bovine serum albumin, 50 µg/mL transferrin, 5 µg/mL insulin, 30 nm sodium selenite, 0.11 mg/mL sodium pyruvate, 10 nm biotin, 20 nm progesterone and 100 µm putrescine (Sigma)]. Cultures prepared from these cells are immature oligodendrocyte progenitors that are either bipolar or possess several simple processes and are stained with the A2B5/O4 antibodies (Sato-Bigbee et al. 1999). Astroglial contamination of these cultures, as assessed by glial fibrillary acid protein staining, was less than 5%. The identity of these cells as oligodendrocyte progenitors is further supported by the observation that when maintained for 7 days in CDM containing triiodothyronine, a hormone that stimulates oligodendrocyte differentiation, more than 80% of the cells become MBP-positive mature oligodendrocytes (Sato-Bigbee et al. 1999). For each set of experiments, the purity of the cultures was assessed by immunocytochemical staining with the antibodies mentioned above. Whenever indicated, NT-3 (PeproTech Inc., Rocky Hill, NJ, USA) was used at a concentration of 50 ng/mL. For S1P and dihydro-S1P treatments, both sphingolipids were dissolved in a solution containing 4 mg/mL fatty acid-free bovine serum albumin and added to the culture medium at a final concentration of 0.1 µm; control media were supplemented with vehicle alone. Animal use and isolation of oligodendrocytes were conducted in accordance with the guidelines from the National Institutes of Health and approved by the Virginia Commonwealth University Animal Care and Use Committee.
Western blot analysis
Equal amounts of protein were subjected to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis in 12% acrylamide gels and electrotransferred to nitrocellulose membranes. The membranes were then subjected to immunoblot analysis as previously described (Johnson et al. 2000; Afshari et al. 2001) using the following antibodies: anti-CREB (dilution 1 : 1000; Cell Signaling, Beverly, MA, USA), anti-phosphorylated CREB (1 : 1000; Upstate, Charlottesville, VA, USA), anti-beta-actin (1: 2000; Sigma), anti-SphK1 (1 : 1000; kindly provided by Drs T. Murate and Y. Banno, Gifu University School of Medicine, Gifu, Japan) and anti-SphK2 (1 : 1000; Jolly et al. 2004). The immunoreactive bands were detected by chemiluminescence with Super Signal West Dura reagent (Pierce, Rockford, IL, USA). The relative amount of immunoreactive protein in each band was determined by scanning densitometric analysis of the X-ray films using the NIH Image J program.
Inhibition of SphK1 expression by small interfering RNA transfection
SphK1 expression was down-regulated with sequence-specific small interfering RNA (siRNA) (Toman et al. 2004). Chemically-synthesized double-stranded siRNA for rat SphK1, 5′-CUGGCCUACCUUCCUGUAGdTT-3′ and 5′-CUACAGGAAGGUAGGCCAGdTT-3′, and a scrambled control siRNA, 5′-UUCUCCGAACGUGUCACGUdTT-3′ and 5′-AAGAGGCUUGCACAGUGCAdTT-3′, were purchased from Xeragon-Qiagen (Bethesda, MD, USA). Oligodendrocyte progenitors were transfected for 6 h with the siRNA duplexes using GeneJammer transfection reagent (Stratagene, La Jolla, CA, USA) following the manufacturer's recommendations. To assess the effectiveness of treatment, 24 h after transfection, total RNA was isolated from the cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The levels of endogenous SphK1 and SphK2 mRNA were determined after reverse transcription and polymerase chain reaction amplification (RT-PCR) of the RNA using sequence-specific primers (Ambion, Austin, TX, USA) to each enzyme. SphK1 and SphK2 mRNA levels were also determined by quantitative real-time PCR (QPCR) and normalized to the 18S ribosomal RNA. SphK1 and SphK2 protein levels were determined by western blot analysis with their respective antibodies.
To examine SphK1 intracellular localization, oligodendrocyte progenitors were incubated for different times in DMEM/F-12 without or with 50 ng/mL NT-3. At the end of the incubation, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and then permeabilized with 0.1–0.2% Triton X-100 for 5–10 min at room temperature (22°C). The cells were then incubated in 3% H2O2 in PBS for 60 min to quench endogenous peroxidase activity, washed three times with PBS, blocked for 60 min with PBS containing 2% bovine serum albumin (BSA), 0.05% Tween-20 and 5% normal goat serum, and incubated overnight at 4°C with a rabbit polyclonal antibody against rat SphK1 (1: 500) in PBS containing 0.05% Tween 20 (Murate et al. 2001). After extensive washing followed by a 30 min incubation in blocking buffer, the cells were incubated for 2 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS containing 0.05% Tween-20. The antibody signal was then detected using a Tyramide Signal Amplification Kit Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA), followed by analysis with a Leica TCS-SP2 AOBS confocal microscope.
Sphingosine kinase activity assay
SphK1 activity was determined as previously described (Jolly et al. 2004) with minor modifications. Oligodendrocyte progenitors were incubated for different times in medium without or with 50 ng/mL NT-3, and then harvested and lysed by freeze–thawing in SphK1 buffer (0.02 m Tris, pH 7.4, 1 mm EDTA, 0.5 mm deoxypyridoxine, 15 mm NaF, 0.1 mmβ-mercaptoethanol, 10 µg/mL each leupeptin, aprotinin and trypsin inhibitor, 40 mm glycerol phosphate, 1 mm phenylmethylsulfonyl fluoride and 10% glycerol). Lysates were centrifuged at 700 g for 10 min to remove unbroken cells, and then at 100 000 g for 60 min to obtain the cytosolic and membrane fractions. Aliquots from each fraction (5–10 µg protein) were incubated for 30 min at 37°C in SphK1 buffer in a total volume of 200 µL containing 50 µm sphingosine (previously dissolved in 5% Triton X-100), 30 µCi γ-[32P]ATP (specific activity, 100 mCi/mm) and 10 mm MgCl2. Reactions were terminated by addition of 800 µL chloroform/methanol/HCl (100 : 200 : 1, v/v). After vigorous mixing, 250 µL chloroform and 250 µL 2 m KCl were added, and the phases were separated by centrifugation. Lipids in the organic phase were separated by thin layer chromatography on silica gel G60 using as solvent chloroform/acetone/methanol/acetic acid/water (10 : 4 : 3 : 2:1, v/v). [32P]S1P was identified by its RF value compared with S1P standard, and quantified with an FX Molecular Imager (Bio-Rad Laboratories, Hercules, CA, USA).
Detection of apoptosis by TUNEL labeling
SphK1 expression was down-regulated by treatment of the oligodendrocyte progenitors with siRNA as described above. At the end of the transfection procedure, the medium was replaced with DMEM/F-12, alone or supplemented with 50 ng/mL NT-3. After 18 h, the cells were fixed with 4% paraformaldehyde for 1 h at room temperature. DNA fragmentation was then assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using a cell death detection kit (in situ Kit, Roche Diagnostics Corp., Indianapolis, IN, USA), as previously reported (Saini et al. 2004). Fluorescein-labeled nucleotides incorporated at 3′-OH ends were detected by incubation with HRP-conjugated anti-fluorescein antibody, followed by detection of peroxidase activity with diaminobenzidine (DAB) (metal enhanced DAB substrate kit, Roche Diagnostics Corp.) and microscopic examination. For each condition, at least 20 visual fields containing approximately 200 cells were analyzed.
Statistical analysis was performed by one-way anova and the ad hoc Tukey–Kramer test. Differences were considered statistically significant when p-values were < 0.05.
S1P is a potent stimulator of CREB phosphorylation in oligodendrocyte progenitors
The observation that S1P stimulates CREB phosphorylation in vascular smooth muscle cells (Coussin et al. 2003; Han et al. 2003) prompted us to investigate whether this also occurs in oligodendrocyte progenitors. As shown in Fig. 1(a), treatment of the cells with S1P did not alter the levels of total CREB but indeed, resulted in a significant increase in CREB phosphorylation. The most important biological responses to S1P are dependent on the activation of plasma membrane S1P receptors (Watterson et al. 2005). Sphinganine-1-phosphate (dihydro-S1P), an S1P mimetic which binds to and activates S1P receptors but has no direct intracellular effects (Spiegel and Milstien 2003), also stimulated CREB phosphorylation (Fig. 1b), suggesting the involvement of S1P receptors in this stimulation.
Stimulation of CREB phosphorylation by S1P involves ERK and PKC activities
We next investigated the signaling pathways by which S1P stimulates CREB phosphorylation in the oligodendrocyte progenitors. In agreement with other reports (Hida et al. 1999; Yu et al. 2004), treatment of oligodendrocytes with S1P markedly stimulated extracellular regulated protein kinase (ERK)1/2 phosphorylation (Fig. 2a). We had previously found that ERK1/2 was involved in CREB phosphorylation in oligodendrocyte progenitors (Johnson et al. 2000) and thus, these observations raise the possibility that the S1P-dependent phosphorylation of CREB could be downstream of ERK1/2 activation. To investigate this, oligodendrocyte progenitors were treated with the MEK (ERK kinase) inhibitor, PD98059 (MEKi), and then stimulated with S1P. Incubation with MEKi effectively blocked the S1P-dependent ERK1/2 phosphorylation (Fig. 2b). Moreover, the MEKi abolished the capacity of S1P to stimulate CREB phosphorylation (Fig. 2c), supporting a role of ERK1/2 in this stimulation.
Our previous studies had suggested that CREB phosphorylation in oligodendrocyte progenitors also involves protein kinase C (PKC) (Sato-Bigbee et al. 1999; Johnson et al. 2000). As shown in Fig. 3(a), incubation of the oligodendrocytes with the PKC inhibitor, bisindolylmaleimide (PKCi), effectively blocked the S1P-dependent increase in ERK1/2 phosphorylation. This inhibition was also accompanied by a lack of increased CREB phosphorylation in response to S1P stimulation (Fig. 3b). Thus, S1P-induced ERK1/2 activation and CREB phosphorylation are also dependent on PKC.
SphK1 plays an important role in the NT-3-dependent stimulation of CREB phosphorylation
We previously found that treatment of oligodendrocyte progenitors with NT-3 results in a robust increase in CREB phosphorylation (Johnson et al. 2000; Saini et al. 2004). The observation that CREB phosphorylation in these cells is also stimulated by S1P led us to investigate whether this lipid molecule plays a role in NT-3 signaling, as previous findings had indicated a cross-talk between nerve growth factor (NGF) and S1P in PC12 cells and neurons (Edsall et al. 1997; Rius et al. 1997; Culmsee et al. 2002; Toman et al. 2004). To investigate this possibility, we examined the effects of N-N-dimethylsphingosine (DMS) (Edsall et al. 1998) and 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (HPCT) (French et al. 2003), two inhibitors of SphK, the enzyme that forms S1P. Interestingly, the NT-3-dependent phosphorylation of CREB is significantly decreased by DMS (Fig. 4a) or HPCT (Fig. 4b), suggesting that SphK may also function as a downstream mediator of NT-3 signaling.
SphK exists in two different isoforms, SphK1 and SphK2 (Watterson et al. 2005), and while SphK1 activity has been associated with protection from cell death (Olivera et al. 1999; Edsall et al. 2001), SphK2 has been linked to the induction of apoptosis (Liu et al. 2003). Since NT-3 stimulates oligodendrocyte survival (Barres et al. 1994; Kumar et al. 1998; Saini et al. 2004), SphK1 may be the form involved in NT-3 signaling. To test this possibility, we next investigated the ability of NT-3 to stimulate CREB phosphorylation in oligodendrocyte progenitors in which endogenous SphK1 expression was down-regulated by transfection with small interfering RNA duplexes (siRNA) targeted to SphK1 mRNA. As shown in Fig. 5(a and b), transfection with SphK1 siRNA decreased SphK1 mRNA levels by 55%, in comparison with control cells treated with scrambled siRNA. Western blot analysis showed that this decrease in SphK1 mRNA levels was accompanied by a 60% reduction in SphK1 protein levels (Fig. 5c). It is important to note that the siRNA treatment specifically targeted SphK1, as neither SphK2 mRNA (Figs 5a and b) nor SphK2 protein (Fig. 5c) levels were affected.
Figure 5(d) shows that incubation of the control cells with NT-3 elicits a robust increase in CREB phosphorylation. However, this stimulation is significantly reduced in the oligodendrocytes transfected with the SphK1 siRNA, further indicating that SphK1 plays a crucial role in NT-3 signaling.
Treatment of oligodendrocyte progenitors with NT-3 induces translocation of SphK1 accompanied by increased membrane-associated SphK1 activity
If, as our results suggest, SphK1 is an important mediator of NT-3 signaling, then NT-3 should stimulate SphK1. Studies in other cell types have shown that SphK1 is primarily a cytosolic enzyme, which, upon cell stimulation, is rapidly translocated to the plasma membrane where its sphingosine substrate resides (Spiegel and Milstien 2003). Immunofluorescent staining and confocal microscopy indicated that in unstimulated cells (Figs 6a and b), SphK1 is diffusely distributed in the cytoplasm. In contrast, treatment with NT-3 (Figs 6c and d) results in SphK1 localization to the plasma membrane. This NT-3-dependent translocation of SphK1 was clearly observed in 62% of the cells (data not shown).
To substantiate these observations, SphK1 activity was then measured in the cytosolic and membrane fractions of the cells. Treatment with NT-3 results in a robust increase in SphK1 activity in the membrane fraction (Fig. 7). On the other hand, NT-3 did not induce detectable changes in cytosolic SphK1 activity. Altogether, these observations support the idea that NT-3 induces translocation and activation of SphK1.
Inhibition of SphK1 expression blocks the survival effect of NT-3
Previous results indicated that NT-3 prevents oligodendrocyte apoptotic cell death induced by growth factor deprivation (Saini et al. 2004). Therefore, we next examined whether SphK1 was important for this NT-3 survival effect. SphK1 expression was down-regulated by transfection of the oligodendrocyte progenitors with SphK1 siRNA as indicated above, and apoptotic cell death following growth factor withdrawal was assessed by detection of DNA fragmentation with the TUNEL assay. As shown in Fig. 8, 60% of the cells exhibited TUNEL-positive nuclei following overnight incubation in DMEM/F-12 medium alone. As expected, addition of NT-3 was accompanied by reduced apoptosis, with only 30% of the cells exhibiting TUNEL-positive nuclei. However, this protective effect of NT-3 was abrogated by down-regulation of SphK1 expression, indicating that this enzyme plays a crucial role in the oligodendrocyte progenitor survival induced by NT-3.
The molecular mechanisms underlying NT-3 action in oligodendrocytes are still poorly understood. We have previously shown that treatment of oligodendrocyte progenitors with NT-3 results in rapid activation of the transcription factor CREB (Johnson et al. 2000; Saini et al. 2004). The present results demonstrate that this stimulation involves a novel and complex interplay with SphK1, the enzyme that catalyzes sphingosine phosphorylation generating S1P. S1P by itself induces CREB phosphorylation in the oligodendrocyte progenitors by a mechanism that involves both ERK1/2- and PKC-dependent pathways. CREB phosphorylation was similarly stimulated by S1P and dihydro-S1P, which activates S1P receptors but has no direct intracellular actions, suggesting that this response is a S1P receptor-mediated effect. Initial studies on the identification of S1P receptors in the brain found that white matter tracts exhibit particularly high levels of S1P2, suggesting the presence of this receptor in the mature myelin-producing oligodendrocytes (Im et al. 2001). Later reports indicated the presence of S1P5 receptors in oligodendrocyte progenitors, and of mRNAs encoding S1P5, S1P1, S1P2 and S1P3 in differentiated oligodendrocytes (Terai et al. 2003; Yu et al. 2004). Recently, S1P5 expression in the brain has been determined to be restricted to cells of the oligodendrocyte lineage, where it appears to play a role in regulating survival of mature oligodendrocytes and process retraction in the immature cells (Jaillard et al. 2005). Thus, it will be interesting to establish the identity of the S1P receptor(s) that are activated in the oligodendrocyte progenitors and mediate the S1P-induced CREB phosphorylation.
The present report indicates that CREB phosphorylation in response to NT-3 involves SphK1, the enzyme that synthesizes S1P. Immunocytochemical localization studies and analysis of enzymatic activity indicated that NT-3 elicits translocation of SphK1 from the cytosol to the plasma membrane. The functional significance of these observations is further supported by results indicating that the NT-3-dependent stimulation of oligodendrocyte survival requires SphK1 expression.
Cross-talk between neurotrophin and sphingolipid signaling was first reported for NGF effects in PC12 cells (Edsall et al. 1997; Rius et al. 1997). Subsequently, SphK has been implicated in the neuroprotective effect of NGF in hippocampal neurons (Culmsee et al. 2002), and in NGF-induced neurite extension in PC12 cells and dorsal root ganglion neurons (Toman et al. 2004). Our results suggest that a cross-talk with SphK signaling may be a common feature of neurotrophin actions. Moreover, the observation that the NT-3-dependent phosphorylation of CREB involves SphK1 raises the possibility that this may also be the case for NGF and BDNF, two neurotrophins that are also known to stimulate CREB phosphorylation (Ginty et al. 1994; Bonni et al. 1995; Xing et al. 1996; Finkbeiner 2000).
We have previously shown that CREB phosphorylation in response to NT-3 involves the concerted action of ERK and PKC (Johnson et al. 2000). We have now found that these two enzymes are also required for S1P to stimulate CREB phosphorylation. However, NT-3 and S1P induce CREB phosphorylation by mechanisms that are differently affected by MEK/ERK and PKC inhibition. CREB phosphorylation in response to NT-3 was only reduced by about 50% by inhibition of either ERK or PKC activities, and simultaneous inhibition of both was required to completely block the NT-3-dependent activation of CREB (Johnson et al. 2000). In contrast, CREB phosphorylation in response to exogenously added S1P was completely blocked by inhibition of either ERK or PKC. Based on these observations, we speculate that NT-3 may stimulate CREB phosphorylation by two complementary pathways, one that is mainly dependent on ERK and another that requires SphK1 activation and S1P formation, and involves both PKC and ERK activities (see Fig. 9). Speculation on a signaling model is complicated by the fact that ERK and PKC activities could play a role both upstream and downstream of SphK1. ERK and PKC may, in part, be required for SphK1 activation and intracellular translocation. It has been shown that treatment of HEK293T cells with TNF-α results in SphK1 activation accompanied by SphK1 phosphorylation at ser122 by an ERK-dependent mechanism (Pitson et al. 2003), and experiments in NIH3T3 cells indicated that this phosphorylation is essential for SphK1 translocation to the plasma membrane (Pitson et al. 2005). In addition, experiments in human embryonic kidney cells suggested that the translocation and activation of SphK1 could also be mediated by PKC (Johnson et al. 2002). Moreover, activation of SphK1 and S1P production could, in turn, cause ERK and PKC activation. S1P is known to induce ERK (Hida et al. 1999; Yu et al. 2004) and PKC activation (Meacci et al. 2003; Rabano et al. 2003; Watterson et al. 2003, 2005) in a variety of cells, and we have now found that both ERK and PKC activities are downstream effectors in the S1P-dependent phosphorylation of CREB.
The role of SphK1/S1P signaling in oligodendrocytes is still poorly understood. Experiments in CEINGE cl3-transformed oligodendrocytes concluded that S1P may modulate Ca2+ signaling triggered by platelet-derived growth factor (PDGF) receptor stimulation (Fatatis and Miller 1997). In addition, SphK1 has been implicated in the PDGF-dependent up-regulation of mRNAs encoding the Kv1.5 and Kv1.6 K+ channels, suggesting that this stimulation could play a role in regulating oligodendrocyte proliferation in response to PDGF (Soliven et al. 2003).
A very important finding of the present study is that SphK1 plays a crucial role in the stimulation of oligodendrocyte survival by NT-3 and, therefore, the bases for this stimulation need to be determined. Our previous results showed that treatment of oligodendrocyte progenitors with NT-3 results in increased expression of the anti-apoptotic protein Bcl-2, by a process that involves the direct regulation of the Bcl-2 gene by CREB (Saini et al. 2004). In this regard, up-regulation of Bcl-2 expression together with down-regulation of the pro-apoptotic protein, Bim, has been shown to be part of the mechanism by which SphK1 stimulates the survival of endothelial cells (Limaye et al. 2005). Thus, it is possible that SphK1 may mediate NT-3-dependent survival by affecting the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins. In addition to CREB, SphK1 may also trigger the activation of other transcription factors, which, like AP-1 and NF-κB, are involved in survival and have been shown to be stimulated by S1P (Su et al. 1994; Wang et al. 1996; Shatrov et al. 1997; Xia et al. 1998; Takeshita et al. 2000).
It is also possible that the survival effect of SphK1 is due to reduction of the levels of ceramide and sphingosine. It has been proposed that SphK1 is a critical regulator of the ‘sphingolipid rheostat’, since this enzyme not only produces anti-apoptotic S1P but concomitantly decreases the levels of the pro-apoptotic S1P precursors, sphingosine and ceramide, in a variety of cells, including T-cell lymphocytes, basophilic leukemia cells and fibroblasts (Olivera and Spiegel 2001). This is particularly important because several lines of evidence have shown induction of oligodendrocyte death by sphingosine and its derivative, psychosine (d-galactosyl-β-1,1′ sphingosine), a lipid that accumulates in globoid cell leukodystrophy, a disease characterized by oligodendrocyte apoptosis and progressive demyelination (Hida et al. 1999; Jatana et al. 2002). Oligodendrocyte cell death is also induced by ceramide (Casaccia-Bonnefil et al. 1996a; Larocca et al. 1997; Hida et al. 1998; Craighead et al. 2000; Jatana et al. 2002), and levels of this lipid were shown to correlate with DNA fragmentation in brains from patients with neuroinflammatory diseases like MS (Singh et al. 1998). Moreover, ceramide levels can be increased by TNF-α (Dobrowsky et al. 1994), a cytokine whose production by CD4(+) T cells predicts long-term increase lesions in MS (Lucchinetti et al. 2004). Based on these observations, SphK1 and formation of S1P may influence oligodendrocyte survival by the concerted action of multiple signaling pathways and downstream effectors.
Discovery of cross-talk between NT-3 and SphK1 signaling, which is implicated in regulating oligodendrocyte survival, is particularly relevant in view of recent reports indicating that FTY720, an immunosuppressant drug that is an analog of sphingosine and is phosphorylated by SphKs (Brinkmann et al. 2004), protects animals against experimental autoimmune encephalomyelitis (EAE) (Fujino et al. 2003; Rausch et al. 2004; Webb et al. 2004), an experimental model for MS. Hence, an interesting possibility is that FTY720 may not only block the immune response but may also stimulate the survival of oligodendrocytes. Further investigation into these signaling pathways may provide important clues to stimulate remyelination after demyelinating lesions as occur in MS.
The authors wish to thank Dr Scott Henderson, Department of Anatomy and Neurobiology, VCU School of Medicine, for his help and advice in the analysis of SphK1 by confocal microscopy. This project was supported by Jeffress Trust J-639 and National Multiple Sclerosis Society RG3432A2/2 grants to CS-B, NIH fellowship F31 NS 48 733–01A1 to HSS, NIH grant GM43880 to SS and NIH grant 1C06RR17393 to VCU.