The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

Introduction

The archetypal neurotrophin NGF has long been recognized for its roles in the survival, differentiation, and target innervation by sympathetic and sensory neurons, as well as the elaboration of neurites in rat pheochromocytoma PC12 cells, a model for sympathetic neurons. Signaling through the TrkA receptor tyrosine kinase is central to NGF action. Dimerization and autophosphorylation of TrkA couples this receptor to intracellular signaling cascades, including the Ras/ERK, phosphoinositide 3-kinase (PI3K)/Akt, and phospholipase Cγ pathways (for review see Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001).

In addition to well-established TrkA signaling systems, we previously found that TrkA activates sphingosine kinase type 1 (SphK1; Edsall et al., 1997), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P, an important lipid mediator (Spiegel and Milstien, 2002), is the ligand for five specific G protein–coupled receptors (GPCRs), designated S1P1–S1P5. These receptors are enriched in the nervous system and couple to different G proteins to regulate diverse signaling cascades. In particular, S1P1, S1P2, and S1P5 are highly expressed and temporally regulated in the brain (Lado et al., 1994; Glickman et al., 1999). S1P2 expression is highest in neuronal cell bodies during early stages of differentiation and in axons during branching (MacLennan et al., 1997). Moreover, during NGF-induced differentiation of PC12 cells, which endogenously express S1P1 (Lado et al., 1994), S1P2 (MacLennan et al., 2000), S1P3 (Molderings et al., 2002), and S1P5 (Glickman et al., 1999), expression of S1P2 and S1P5, but not S1P1, has been reported to be down-regulated (Glickman et al., 1999). Although the biological functions of these S1P receptors in the nervous system are largely unknown, they all have been implicated in directed cell movement and cytoskeleton rearrangements (Spiegel and Milstien, 2002).

Neurite extension and retraction, important processes in the establishment of neuronal networks during development, are largely orchestrated by the organization of the actin cytoskeleton controlled by the balance between the opposing actions of the small GTPases, Rho and Rac (Li et al., 2002). Although Rho induces collapse of growth cones and inhibition of neurite outgrowth (Nakamura et al., 2002), Rac is required for neurite outgrowth (Estrach et al., 2002). Of interest, in diverse cell types, S1P receptors differentially regulate Rac and Rho. Binding of S1P to S1P1 enhances Rac-coupled cortical actin formation (Rosenfeldt et al., 2001), whereas S1P2 stimulates Rho and suppresses activation of Rac, thereby inhibiting cell migration (Okamoto et al., 2000).

In this work, we examined the role of S1P receptors in NGF-induced neurite extension. We found that NGF, in a TrkA-dependent manner, transactivated S1P receptors in PC12 cells by stimulation of SphK1. Neurite elongation of cultured dorsal root ganglion (DRG) neurons induced by NGF was similarly regulated via SphK1 and activation of S1P receptors. Our results reveal that cross-communication between TrkA and S1P receptors, and the consequent regulation of opposing signaling pathways, such as Rac and Rho, may play an important role in neurite extension.

Results

NGF activates S1P receptors in PC12 cells

Similar to other GPCRs, GFP-tagged S1P receptors have been extensively used to study receptor internalization after activation and have been shown to transduce intracellular signals in a manner indistinguishable from the wild-type receptors (Liu et al., 1999). As expected, S1P1-GFP and S1P5-GFP were expressed mainly on the plasma membrane of serum-starved PC12 cells while GFP-vector was cytoplasmic (Fig. 1 A). Interestingly, internalization of S1P1 and S1P5 was significant within 2 min after addition of NGF (Fig. 1 A). The internalization occurred in the absence of exogenous S1P and was clearly evident within 10 min (Fig. 1, A and B). This time course is similar to that of S1P-induced trafficking of S1P1 in HEK 293 cells (Liu et al., 1999). On the other hand, NGF did not induce internalization of S1P1 or S1P5 in nnr5 cells, a TrkA-deficient PC12 mutant that is unresponsive to NGF-induced neuritogenesis (Loeb et al., 1991) (Fig. 1, A and B). These results suggest that NGF transactivates S1P1 and S1P5 in a TrkA-dependent manner.

SphK1 is required for NGF-induced transactivation of S1P receptors

Previously, we have shown that binding of NGF to TrkA stimulates SphK1, leading to a rapid increase in S1P levels in PC12 cells (Edsall et al., 1997). Although SphK1 is diffusely distributed in the cytosol of unstimulated PC12 cells, as it is in other cell types (Olivera et al., 1999), NGF induced its rapid translocation to the plasma membrane where its substrate sphingosine resides (Fig. 2, A and B). In sharp contrast, SphK1 appeared to be entirely cytoplasmic in nnr5 cells and translocation to the plasma membrane by NGF was not evident (Fig. 2, A and B), even after much longer exposure (unpublished data).

To examine whether SphK1 activity is required for NGF-induced translocation of SphK1 and S1P receptors, we used a catalytically inactive, dominant-negative mutant SphK1 prepared by site-directed mutagenesis of the second conserved glycine residue in the ATP-binding site (SGDGX17–21K(R)) present within the conserved catalytic domain of all SphK isoforms. In untreated PC12 cells, the catalytically inactive G82D mutant (SphK1-G82D) had a similar diffuse cytosplasmic distribution as wild-type SphK1. NGF treatment also translocated SphK1-G82D to the plasma membrane in a TrkA-dependent manner (Fig. 2, A and B), and the catalytically inactive mutant prevented NGF-induced internalization of S1P1 (Fig. 2 C).

In PC12 cells, NGF initiates time-dependent cellular differentiation and neurite outgrowth (Greene and Tischler, 1976). Because NGF translocates (Fig. 2 A) and stimulates SphK1 (Edsall et al., 1997), it was of interest to examine the role of SphK1 in neuritogenesis. In both naive and vector-transfected PC12 cells, there was no detectable neurite extension 1 d after NGF treatment and only minimal extension by 36 h (Fig. 2 D). In contrast, SphK1 overexpression enhanced the initiation of NGF-induced PC12 cell differentiation, resulting in a significant increase in PC12 cells bearing neurites twice the length of their cell bodies that was evident within 1 d and marked at 36 h. However, at 48 h, vector- and SphK1-transfected cells exhibited similarly extended neurites. Moreover, expression of SphK1-G82D significantly inhibited the rate of neurite extension induced by NGF (Fig. 2 D). These results suggest that SphK1 might play a role in NGF-mediated neurite elaboration.

Transactivation of S1P receptors by NGF differentially affects neurite extension

Untreated naive PC12 cells endogenously express mRNA for S1P1 (Lado et al., 1994), S1P2 (MacLennan et al., 2000), S1P3 (Molderings et al., 2002), and S1P5 (Glickman et al., 1999). A recent analysis established the successful use of anti-S1P receptor antibodies for immunocytochemical analysis in diverse cell types (Jin et al., 2003). Immunohistochemistry with specific S1P receptor antibodies also revealed the expression of S1P1, S1P2, and S1P5 in PC12 cells (Fig. 3 A). In agreement with previous reports (Lado et al., 1994; Glickman et al., 1999; MacLennan et al., 2000), treatment with NGF down-regulated all of the S1P receptors except S1P1 as demonstrated by RT-PCR (unpublished data) and by the nearly complete disappearance of immunohistochemical labeling with anti-S1P2 and anti-S1P5, but not with anti-S1P1 (Fig. 3 A).

Next, we examined the involvement of S1P receptors in neurite extension. Overexpression of S1P1 remarkably accelerated and strongly potentiated NGF-induced neurite extension in these cells, again in the absence of exogenous S1P. Within 1 d, nearly 20% of these cells had neurites twice the length of their cell bodies (Fig. 3 B), whereas barely detectable neurite extension was observed in naive or vector-transfected PC12 cells, and no neurites were detectable in S1P2- or S1P5-expressing cells (Fig. 3 B). This dramatic stimulation of NGF-induced neurite formation at 24 h was also clearly evident in the morphology of S1P1-transfected PC12 cells (Fig. 1 A, bottom). Moreover, overexpression of S1P1 enhanced NGF-induced differentiation even after prolonged exposure. By contrast, overexpression of either S1P2 or S1P5 strongly inhibited NGF-induced neurite extension (Fig. 3 B). Similar results were obtained with PC12 cells that were transfected with wild-type S1P1, S1P2, or S1P5, together with GFP at a 5:1 ratio to identify transfected cells (unpublished data), further supporting the notion that the GFP tag does not interfere with functions of the S1P receptors (Liu et al., 1999). When S1P was added to PC12 cells overexpressing S1P2 or S1P5 for only the final 3 h of a 120-h culture period in the presence of NGF, it initiated retraction of existing neurites (Fig. 3 C). Similarly, wild-type S1P2 and S1P3, but not S1P1, have previously been associated with S1P-induced rounding of NGF-differentiated PC12 cells (Van Brocklyn et al., 1999).

The opposing roles of Rac and Rho in neurite extension modulated by S1P receptors

The Rho family of GTPases, Rho, Rac, and Cdc42, has been shown to play an important role in neuronal morphogenesis and differentiation (Li et al., 2002; Nusser et al., 2002). In particular, Rho mediates retraction of neurites and inhibits their outgrowth (Yamaguchi et al., 2001; Nakamura et al., 2002; Nusser et al., 2002). Thus, we first examined the role of Rho in S1P2- and S1P5-mediated suppression of neuritogenesis by expressing a dominant-negative mutant Rho (N19RhoA). This mutant Rho inhibited the effect of S1P on neurite retraction mediated by both S1P2 and S1P5 (Fig. 3 C), in agreement with previous reports showing that S1P-induced neurite retraction and soma rounding of N1E-115 neuronal cells was blocked by Clostridium botulinum C3 transferase, which ADP-ribosylates and inactivates Rho (Postma et al., 1996). Expression of dominant-negative N17Rac resulted in nearly complete suppression of neuritogenesis in both vector and S1P receptor transfected cells, highlighting the critical importance of Rac in the process of neurite outgrowth.

Because it is well established that S1P1 and S1P2 can differentially regulate Rho and Rac, it was of interest to assess the roles of the S1P receptors in modulation of activation of Rho and Rac induced by NGF in PC12 cells. To this end, we measured the levels of active, GTP-bound forms of Rac and Rho by GST pull-down assays with the GST-fused CRIB-containing NH2 terminus binding domain of PAK and the GST-fused binding domain of rhotekin, respectively. To increase the sensitivity, we used PC12 cells stably expressing S1P1 or S1P2 as described previously (Van Brocklyn et al., 1999). Nonclonal pools were used to avoid potential phenotypic changes due to selection and propagation of clones derived from single individual cells. Overexpression of S1P1 or S1P2 resulted in marked increases in specific S1P binding as expected (Fig. 3 D). Similar to previous reports (Yamaguchi et al., 2001; Nusser et al., 2002), NGF rapidly and transiently increased GTP-Rac and reduced GTP-Rho (Fig. 3 E). In agreement with the opposing effects of different S1P receptors on neurite extension (Fig. 3 B), overexpression of S1P1 enhanced NGF-induced Rac activation, whereas NGF treatment of S1P2-expressing PC12 cells inhibited Rac activation and stimulated Rho (Fig. 3 E). To further examine the involvement of S1P1 in NGF-induced Rac activation, PC12 cells were pretreated with pertussis toxin (PTX) to inactivate Gi, as S1P1, in contrast to the other S1P receptors, is coupled solely to Gi. PTX reduced NGF-induced Rac activation (Fig. 3 F). Similarly, NGF-induced differentiation of PC12 cells is also sensitive to PTX (Rakhit et al., 2001).

S1P modulates NGF-induced differentiation of rat DRG neurons

To investigate the role of S1P and S1P receptors in regulation of neurite extension in primary neurons, we used DRG neurons isolated from embryonic rats. Not only do these neurons express TrkA, they have also been shown to respond to NGF (Markus et al., 2002a). To eliminate unintended lipid effects, the dissociated DRG neurons were grown in the absence of serum with NGF acting as the sole trophic factor. Although only 30% of the freshly dissociated neurons were TrkA positive, in agreement with some reports (Mu et al., 1993; Kashiba et al., 1995), other analyses using stained tissue sections or RNA hybridization assays (Wright and Snider, 1995; Molliver and Snider, 1997) have found higher proportions of TrkA-expressing cells. This variability could result from the trypsinization treatment necessary to dissociate the ganglia. Nevertheless, culturing the DRG neurons in the presence of NGF resulted in the death of most of the neurons not expressing TrkA, producing nearly homogenous TrkA-expressing cultures by 16 h (Fig. 4 A). Freshly dissociated DRG neurons express mRNA for S1P1, S1P2, and S1P5 (Fig. 4 B), indicating the potential for endogenous S1P signaling. Similar to PC12 cells, culturing these neurons in NGF-containing media resulted in the down-regulation of both S1P2 and S1P5, but not S1P1 (Fig. 4 B).

Because NGF induced translocation of SphK1 in PC12 cells, we next examined its effect on localization of endogenous SphK1 in DRG neurons. We used a specific antibody that recognizes SphK1 in rat brain and shows a single band of the expected size of 46 kD on immunoblots (Murate et al., 2001). Confocal immunohistochemistry revealed that similar to SphK1 overexpressing PC12 cells, endogenous SphK1 was mainly cytosolic in unstimulated DRG neurons (Fig. 4 C, d). NGF treatment induced translocation of SphK1 to the plasma membrane within 10 min (Fig. 4 C, f). This is consistent with movement of SphK1 to the plasma membrane by other agonists in different cell types (Rosenfeldt et al., 2001; Johnson et al., 2002).

The majority of freshly dissociated DRGs were rounded and had no processes before treatment with NGF. NGF induced a time-dependent increase in the number of neurons extending processes, and within 16 h, >70% of DRG neurons had extensive neurites of at least four times the cell body length (Fig. 5). Addition of 100 nM S1P for the last 1.5 h markedly potentiated neurite extension, increasing the number of neurons with neurites to nearly 100% (Fig. 5 B). Moreover, S1P-treated neurons had significantly elongated neurites that tended to have greater distal branching (Fig. 5, A and C). This was a specific response to S1P, as lysophosphatidic acid, a structurally related lysophospholipid that binds to a related yet distinct GPCR family, caused neurite retraction rather than extension at similar concentrations (Fig. 5).

Surprisingly, during the course of this investigation we noticed that when dissociated DRG neurons were exposed to both NGF and S1P at the time of plating, S1P inhibited neurite extension in a concentration-dependent manner, with a significant effect at a concentration as low as 10 nM and maximal inhibition at 100 nM (Fig. 6, A and B). S1P abrogated NGF-stimulated axonal growth without killing the cells. The striking contrast in responses to S1P at different stages of neurite development is reminiscent of its effects on PC12 cells and suggests that S1P and its receptors might exhibit temporally specific functions. Given that the opposing effect of S1P on neurite extension and retraction in PC12 cells was dependent on relative expression of the various S1P receptors, it was important to further substantiate that their levels in DRG neurons varied with the extent of neurite formation. In agreement with mRNA analyses (Fig. 4 B), immediately after dissociation, E15 DRG neurons expressed both S1P1 and S1P2 as determined by immunocytochemistry (Fig. 6 C). After culturing for 4 h in media containing NGF, DRG neurons expressed only S1P1, which was expressed on the cell surface (Fig. 6 C). After 16 h of NGF treatment, S1P1 was expressed in the soma as well as along the extended neurites (Fig. 6 D). These results suggest that similar to PC12 cells, S1P2 protein and mRNA expression is down-regulated in DRG neurons during neurite elongation.

Role of S1P1 in neurite extension of DRG neurons

Because S1P1 is prominently expressed on DRG neurons during neurite extension and is coupled to Gi, we used PTX to examine the role of S1P1 in this process. When NGF-treated, neurite-possessing DRG neurons were pretreated with PTX 3 h before adding S1P, PTX completely suppressed neurite extension induced by S1P (Fig. 7 A). To further confirm the role of S1P1 in S1P-induced DRG neurite extension, S1P1 expression was down-regulated by transfection with S1P1 antisense RNA, which was designed to bind to the translation initiation site on the S1P1 mRNA and thus block its expression. Previously, we have shown that S1P1 mRNA expression was almost completely eliminated in aortic smooth muscle cells by S1P1 antisense oligonucleotides without affecting S1P2 or S1P3 expression (Hobson et al., 2001). Antisense oligonucleotides for S1P1 specifically inhibited endogenous neurite extension, as sense and scrambled oligonucleotides had no effects (Fig. 7, B and C). To confirm that the neurons whose S1P receptors are repressed by antisense are those that have shorter neurites, fluorescent oligonucleotides were used in these experiments and effects on neuritogenesis of fluorescent neurons compared in the same cultures (Fig. 7 C) to the nonfluorescent (Fig. 7 D) and therefore nontransfected neurons. Importantly, nontransfected neurons in the same cultures behaved similarly as neurons transfected with the sense and scrambled oligonucleotides (Fig. 7, C and D). Not only did antisense S1P1 oligonucleotide significantly decrease the number of neurons with extensive neurites, nearly 31% of these cells failed to exhibit any neurite outgrowth at all (Fig. 7 C). These results further support the notion that activation of S1P1 by S1P may be required for NGF-induced neurite extension.

SphK1 plays an important role in neurite extension

As SphK1 activity was required for NGF-induced neurite extension in PC12 cells, it was of interest to determine whether it also played such a role in primary DRG neurons. We used d,l-threo-dihydrosphingosine and N,N-dimethylsphingosine (DMS), competitive inhibitors of SphK1 that block formation of S1P in PC12 cells (Edsall et al., 1997). Both inhibited neurite extension of DRG neurons in a dose-dependent manner (Fig. 8 A). DMS was somewhat more potent and was thus used in further experiments to define the importance of SphK1 in DRG neurite formation. If the effect of DMS is mediated specifically by inhibiting production of S1P, which in turn is required for stimulation of S1P1, then exogenous S1P should bypass this block and reverse the effects of DMS. Importantly, suppression of neurite extension by DMS was completely rescued by exogenous S1P (Fig. 8, A and B), suggesting that the S1P produced by SphK1 is essential for neurite extension in these neurons. Moreover, pretreatment with PTX to block S1P1 Gi-coupled downstream signaling (Fig. 8 A) completely prevented the ability of exogenous S1P to reverse the inhibition of neurite extension induced by DMS, pointing to an important role of S1P1 activation as a consequence of activation of SphK1 and formation of S1P.

To further substantiate the role of SphK1, we also used small interfering RNA (siRNA) targeted to SphK1. In agreement with the SphK inhibitor results (Fig. 8 A), transfection of dissociated DRG neurons with siRNA targeted to SphK1 decreased neurite extension by nearly 50%, whereas control siRNA had no significant effects (Fig. 8 C). This reduction correlated with the down-regulation of SphK1 mRNA as determined by RT-PCR (Fig. 8 D).

Discussion

The importance of the local signals that promote axonal growth of peripheral neurons, particularly the ERK and PI3K pathways, coordinated by TrkA in response to NGF, are now beginning to be elucidated (Kaplan and Miller, 2000; Markus et al., 2002a). In cultures of sympathetic neurons, it was shown that the PI3K pathway is required for NGF-induced distal axon growth (Ming et al., 1999) and plays an important role in neuritogenesis of adult rat DRG sensory neurons (Kimpinski and Mearow, 2001). PI3K may regulate axonal growth cone actin cytoskeleton through its effectors Rac and Akt (Markus et al., 2002a). Although the involvement of ERK in NGF-induced neurite outgrowth in PC12 cells is well established (Vaudry et al., 2002), its role in nervous system development is much more complex. NGF-stimulated ERK is not necessary for neurite outgrowth of chick DRG neurons (Klinz et al., 1996) or mouse DRG neurons (Klesse and Parada, 1998). However, a recent report (Markus et al., 2002b) with sensory neurons lacking the proapoptotic protein Bax enabled the examination of individual signaling mediators on axonal growth without affecting survival. Although Ras was both necessary and sufficient for NGF-stimulated axon growth, the Ras effector Raf, an activator of the ERK pathway, and Akt, known to be activated by PI3K, mediated distinct morphological features of developing neurons (Markus et al., 2002b). TrkB, which stimulates signaling proteins in a manner similar to TrkA, mediates both neuronal survival and axonal outgrowth in rat sympathetic neurons by activating the PI3K and ERK signaling pathways (Atwal et al., 2000). Inhibitors of MEK–ERK and PI3K blocked axon growth from naive and NGF-stimulated E13 sensory neurons without effecting elongation of adult sensory neurons after a conditioning lesion, suggesting that the signaling mediators that underlie regenerative axon growth are distinct from those used during development (Liu and Snider, 2001).

In this report, we describe an extended paradigm for TrkA signaling in regulation of neurite extension. According to this model, activation of TrkA by NGF stimulates and translocates SphK1 from the cytosol to the plasma membrane, where its substrate sphingosine resides, to generate S1P in a spatially and temporally restricted manner. This S1P binds to and activates S1P1 on the cell surface, leading to activation of signaling pathways important for neurite extension. Our results also suggest that differential cross-communication of TrkA with S1P receptors, a family of GPCRs that regulates opposing downstream signals important for cytoskeletal changes, modulates neurite extension or retraction.

In support of this notion, it was recently shown that TrkA is tethered to the classical GPCR signaling molecules, GPCR kinase 2 and β-arrestin, providing a platform for integrative signaling by these two receptors to promote differentiation in PC12 cells (Rakhit et al., 2001). In addition, GAIP, a regulator of G protein–signaling protein, forms a complex with TrkA and is a putative link between G protein and receptor tyrosine kinase pathways (Lou et al., 2001).

Previously, we suggested that PDGF-induced cell migration required S1P1 (Hobson et al., 2001; Rosenfeldt et al., 2001), representing a similar mechanism of crosstalk between receptor tyrosine kinases and GPCRs. However, others have questioned the generality of the concept that SphK1 and S1P1 are downstream elements of PDGF-induced chemotaxis (Kluk et al., 2003). Recently, several different mechanisms for S1P1 and receptor tyrosine kinase cross-communication have been suggested (Lee et al., 2001; Waters et al., 2003). In the integrative signaling model, the PDGF receptor and S1P1 form complexes that are cointernalized together by PDGF as a functional signaling unit to regulate ERK1/2 (Waters et al., 2003). A second proposal suggests that tyrosine kinase receptors, such as the IGF-1 receptor, could transactivate S1P1 through its Akt-dependent phosphorylation in a manner that does not require the SphK pathway (Lee et al., 2001). Both of these models suggest that activation of SphK1 and intracellular generation of S1P do not play any role and introduce the concept of ligand-independent activation of S1P receptors.

In contrast, several lines of evidence support a central role for SphK1 and generation of S1P in NGF-induced neurite extension. Previously, we found that NGF rapidly stimulated SphK1 in PC12 cells that was followed by a prolonged increase in SphK activity and a concomitant increase in S1P levels (Edsall et al., 1997). The activation of SphK1 by NGF, but not by FGF, was blocked by K252a, an inhibitor of TrkA (Edsall et al., 1997). Here, we demonstrated that NGF induced translocation of SphK1 from the cytosol to the plasma membrane in PC12 cells and in primary DRG neurons. Moreover, overexpression of SphK1 in PC12 cells enhanced initiation of NGF-induced neuritogenesis. Conversely, a catalytically inactive mutant SphK1, which was also translocated by NGF to the plasma membrane, markedly reduced both initiation and extension of neurites. Interestingly, active SphK1 was required for NGF-induced activation and internalization of S1P receptors in PC12 cells, supporting the notion that formation of S1P in the vicinity of its receptors induces their activation. In agreement, it was recently reported that activation of PKC leads to translocation of SphK to the plasma membrane (Johnson et al., 2002). Importantly, SphK1 also plays a significant role in axonal growth of primary neurons. Inhibition of SphK1 in dissociated DRG neurons by pharmacological or molecular approaches drastically reduced NGF-induced neurite extension. However, most notably, addition of exogenous S1P to neurons in which SphK1 was inhibited reversed the inhibitory effects on neurite extension. Collectively, these results suggest that activation of SphK1 and formation of S1P plays an important role during NGF-induced neurite extension of sympathetic neurons.

Neurite extension and retraction are complex processes important in formation of neural networks. Our findings suggest that transactivation of distinct S1P receptors by NGF via TrkA regulates neuronal development in a reciprocal manner whereby S1P1 acts in opposition to S1P2 and S1P5 to coordinate neurite extension and morphological differentiation. Because the different S1P receptors are associated with a variety of opposing downstream signaling pathways, their temporally selective expression on the cell surface could consequently regulate neurite extension or retraction. In agreement, in PC12 cells, NGF-induced neuronal differentiation down-regulates expression of S1P2 and S1P5 (Glickman et al., 1999). Similarly, we have found that S1P has opposite effects on neuritogenesis of dissociated DRG neurons, depending on which S1P receptors are expressed. S1P inhibited neurite extension of freshly isolated DRG neurons, which express S1P1 and S1P2. In striking contrast, when S1P was added to DRG neurons that had already initiated neurite outgrowth and then only expressed S1P1, it dramatically enhanced neurite extension.

In concordance with the opposite actions of S1P1 compared with S1P2 and S1P5, down-regulation of S1P2 by antisense oligonucleotides has been shown to enhance neurite extension and prevent S1P-induced rounding in PC12 cells (MacLennan et al., 2000). Moreover, addition of S1P to PC12 cells transiently expressing S1P2 caused cell rounding (Van Brocklyn et al., 1999). In agreement, we found that overexpression of either S1P2 or S1P5 in PC12 cells inhibited NGF-induced neuritogenesis, whereas overexpression of S1P1 enhanced it, even in the absence of exogenous S1P. Similarly, in DRG neurons, inhibition of neurite extension by S1P is primarily regulated through S1P2, whereas S1P signaling through S1P1 enhances neurite extension. Thus, down-regulation of S1P1 expression by antisense RNA, or inactivation of its Gi-coupled signaling, completely abolished neurite extension induced by S1P. Additionally, PTX blocked the S1P “rescue” of SphK1-inhibited DRG neurons, suggesting once again that transactivation of S1P1 through SphK1 activation is critical for neurite elaboration.

Remodeling of the actin cytoskeleton provides the basis for both the motility of growth cones, the highly dynamic structures at the tips of axons, and neurite extension in response to neurotrophins. Recent analyses suggest that NGF signals through TrkA to stimulate Rac1 (Nusser et al., 2002), which in turn induces transient Rho inactivation during the initial phase of neuritogenesis (Nusser et al., 2002). Conversely, RhoA, acting through Rho-associated kinase, down-regulates NGF-induced Rac1 activation to inhibit neurite formation (Yamaguchi et al., 2001). In agreement, we found that the antagonistic actions of S1P1 and S1P2 on neurite extension correlate with their opposing effects on Rho and Rac. Activation of S1P2 elicits Rho-coupled stress fiber assembly and negatively regulates Rac activity (Okamoto et al., 2000). Although NGF activates Rac and represses Rho in S1P1-overexpressing PC12 cells, it represses Rac and activates Rho in S1P2-overexpressing PC12 cells. Moreover, inhibition of S1P1 signaling by PTX decreased NGF-induced Rac activation. These results agree with previous reports implicating S1P1 in Rac activation (Hobson et al., 2001; Lee et al., 2001). This diversity of signaling may be particularly important for remodeling neurons, as it provides a fine-tuning mechanism for the processes of neurite retraction and extension.

Materials And Methods

Cell culture

PC12 cells and DRG neurons were cultured and transfected as described in the online supplemental materials (available at http://www.jcb.org/cgi/content/full/jcb.200402016/DC1).

Plasmid constructs and site-directed mutagenesis

The QuikChange® Site-Directed Mutagenesis Kit (Stratagene) was used to prepare catalytically inactive SphK1 (G82D mutation primers: forward, 5′-GTCCGGTGATGATCTGATGCATGAAGTGG-3′ and reverse 5′-CCACTTCATGCATCAGATCATCACCGGAC-3′). The mutated sequence was verified by DNA sequencing. Texas red–tagged oligonucleotides were obtained from Invitrogen (sense 5′-XATGGGGCCCACCAGCGTC-3′; antisense 5′-XGACGCTGGTGGGCCCCAT-3′; and scrambled 5′-XTGATCCTTGGCGGGGCCG-3′). siRNA for rat SphK1 (5′-CUGGCCUACCUUCCUGUAGdTT-3′, 5′-CUACAGGAAGGUAGGCCAGdTT-3′), and the control sequence (5′-UUCUCCGAACGUGUCACGUdTT-3′, 5′-ACGUGACACGUUCGGAGAAdTT-3′) were synthesized by Xeragon.

Quantification of neurite extension

PC12 cells transfected with the various GFP constructs were grown in the presence of 100 ng/ml NGF in RPMI 1640 supplemented with 2% heat-inactivated horse serum for 72 h. Cells were visualized by fluorescence microscopy (Eclipse TE300; Nikon). Images were collected with a CoolSNAP Camera (Roper Scientific) using MetaMorph® software (Universal Imaging Corp.). Extended neurites were defined as having a length equal to or greater than twice the cell body.

Dissociated DRG neurons were stained with rhodamine-conjugated phalloidin (Molecular Probes, Inc.) and neurite extension was quantified by fluorescence microscopy, assessing the percentage of neurons bearing at least one neurite four times the length of their cell bodies. Unless otherwise indicated, a minimum of 300 PC12 cells or DRG neurons was examined in a blinded manner and results are reported as averages (± SD). Similar results were obtained in at least three independent experiments.

Immunocytochemistry

PC12 cells and DRG neurons were fixed with 4% PFA/4% sucrose in PBS. Cells were permeabilized with cold acetone before immunostaining and were incubated in PBS containing 5% goat serum, 2% BSA, and 0.05% Tween 20 for 1 h to block nonspecific staining. Cultures were incubated overnight at 4°C with purified polyclonal rabbit IgG antibodies directed against unique S1P receptor COOH-terminal peptides (S1P1 (X1093P), S1P2 (X1091P), and S1P5 (X1094P)) obtained from Exalpha and diluted 1:500 in PBS containing 1% goat serum and 0.05% Tween 20. Cells were washed with PBS and incubated for 1 h with rhodamine-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) diluted 1:150 in PBS containing 1% goat serum and 0.05% Tween 20 and then were mounted on slides using the ProLong® Antifade Kit (Molecular Probes, Inc.). Cultures were examined with a laser-scanning confocal microscope (FluoView 300; Olympus). No fluorescence was detected when the primary antibody was omitted or preincubated with the peptide antigen. Moreover, in agreement with previous characterization of S1P receptor antibodies (Jin et al., 2003), fluorescence was not observed in other cell types not expressing the corresponding S1P receptor. Conversely, fluorescence was enhanced by overexpression of the S1P receptor.

To examine expression of TrkA on DRG, fixed cells were permeabilized with cold acetone before immunostaining and incubated in PBS containing 5% goat serum, 2% BSA, and 0.05% Tween 20 for 1 h to block nonspecific staining. Cultures were incubated overnight at 4°C with TrkA-specific antibodies (sc-118; Santa Cruz Biotechnology, Inc.) diluted 1:250 in PBS containing 1% goat serum and 0.05% Tween 20. Cells were washed with PBS and incubated for 1 h with FITC-conjugated secondary antibody (1:150), rhodamine-conjugated phalloidin (1:500; Santa Cruz Biotechnology, Inc.), and Alexa Fluor® 647–conjugated cholera toxin subunit B (1:500, c-34778; Molecular Probes, Inc.) diluted in PBS containing 1% goat serum and 0.05% Tween 20, and were then mounted on slides. Cultures were examined by confocal fluorescence microscopy (LSM model 510; Carl Zeiss MicroImaging, Inc.) with a 63× oil immersion objective. Quantitative image analysis was performed using LSM 510 image-processing software. Control immunohistochemistry experiments were performed by using normal serum or by omitting the primary antibodies.

To examine translocation of SphK1, DRG neurons were grown on coverslips in medium containing both 10% FBS and 100 ng/ml NGF for 4 h and then in their absence for 6 h. After NGF treatment in serum-free medium for the indicated times, neurons were fixed with 4% PFA in 50 mM Tris-HCl, pH 7.3, for 4 h. Coverslips were washed three times with 10 mM Tris-HCl, pH 7.3, containing 150 mM sodium chloride (TBS), blocked for 30 min with TBS containing 2% BSA, 0.05% Tween 20, and 5% normal donkey serum, and incubated overnight at 4°C with a rabbit pAb to mouse SphK1 (2 μg/ml; provided by Dr. T. Murate and Dr. Y. Banno, Gifa University School of Medicine, Gifa, Japan) in TBS containing 0.05% Tween 20 (Murate et al., 2001). After washing and 30 min incubation in blocking solution, cells were incubated for 2 h at RT with Cy2-conjugated donkey anti–rabbit secondary antibody (7.5 μg/ml; Jackson ImmunoResearch Laboratories) with TBS containing 0.05% Tween 20, and were visualized by confocal microscopy as described above.

Online supplemental material

Details about cell culture and transfection, statistical analysis, and activation of Rho and Rac (provided by Dr. J. Silvio Gutkind, NIH, Bethesda, MD) can be found as supplemental material.

Acknowledgments

We are grateful to Dr. Lisa Edsal for help with the S1P binding assays.

This work was supported by National Institutes of Health (NIH) grant CA61774 (to S. Spiegel). R.E. Toman was supported by an NIH pre-doctoral fellowship (NS41706). Confocal microscopy was supported in part by NIH grant P30 CA16059.

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J.W. Bigbee and S. Spiegel contributed equally to this paper.

Abbreviations used in this paper: DMS, N,N-dimethylsphingosine; DRG, dorsal root ganglion; GPCR, G protein–coupled receptor; PI3K, phosphoinositide 3-kinase; PTX, pertussis toxin; S1P, sphingosine-1-phosphate; siRNA, small interfering RNA; SphK, sphingosine kinase.