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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).
The neurotrophin family consists of six highly homologous peptides (Barbacid 1994): nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, NT-6 and NT-7 (Gotz et al. 1994; Fagan et al. 1996; Nilsson et al. 1998). Neurotrophins play crucial roles during CNS development (Confort et al. 1991; de la Rosa et al. 1994; Segal et al. 1995) and NT-3, in particular, is known to induce both survival and proliferation of oligodendrocytes (Barres et al. 1992, 1993; Cohen et al. 1996; Johnson et al. 2000; Wilson et al. 2003; Saini et al. 2004; Fressinaud 2005). Neurotrophin actions are transduced by interaction with two different classes of cell surface receptors. All neurotrophins bind to the low affinity receptor, p75 (Casaccia-Bonnefil et al. 1999). However, functional specificity of each neurotrophin is provided by binding to different members of the Trk family of tyrosine protein kinase receptors (Barbacid 1994; McDonald and Chao 1995). The Trks include TrkA (a receptor for NGF), TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Developing oligodendrocytes express TrkC (Cohen et al. 1996; Kumar et al. 1998; Kahn et al. 1999), and a role for NT-3 and TrkC in oligodendrocyte development in vivo is supported by the observations that the number of oligodendrocytes in the optic nerve is reduced by delivery of neutralizing antibodies against NT-3 (Barres et al. 1994), and that mutant mice lacking NT-3 and TrkC expression have decreased brain and spinal cord oligodendrocyte numbers (Kahn et al. 1999). NT-3 has been shown to diminish the susceptibility of cultured oligodendrocytes to tumor necrosis factor-α (TNF-α) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-glutamate receptor-mediated excitotoxicity (Kavanaugh et al. 2000), and studies with animal models have suggested that NT-3 may play an important role in regulating oligodendrocyte number and myelin regeneration following CNS injury and demyelination (McTigue et al. 1998; Jean et al. 2003). However, the molecular mechanisms of NT-3 action in oligodendrocytes are not fully understood.
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.
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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.
Figure 9. Proposed mechanism of NT-3-dependent CREB phosphorylation in oligodendrocyte progenitors. Upon NT-3 binding, TrkC receptor subunits autophosphorylate and initiate a signaling cascade that results in CREB activation through the concerted action of ERK1/2- and PKC-dependent pathways. This cascade involves a traditional MEK/ERK1/2 pathway as well as translocation of SphK1 to the plasma membrane of the cells, resulting in S1P synthesis. S1P may act as an autocrine factor that binds to S1P receptors, further stimulating CREB phosphorylation by means of ERK and PKC activities.
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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.