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(The FASEB Journal. 1998;12:307-314.)
© 1998 FASEB

RESEARCH COMMUNICATION

Prosaptide activates the MAPK pathway by a G-protein-dependent mechanism essential for enhanced sulfatide synthesis by Schwann cells

W. Marie Campanaa, Masao Hiraiwaa, and John S. O'Briena,1

a Department of Neurosciences, University of California, San Diego, School of Medicine, La Jolla, California 92093, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prosaposin, the precursor of saposins A, B, C, and D, was recently reported to be a neurotrophic factor in vivo and in vitro. The neurotrophic region of prosaposin has been localized to a 12-amino acid sequence within the saposin C domain and has been used to derive biologically active synthetic peptides (14–22 residues), called prosaptides. Treatment of primary Schwann cells and an immortalized Schwann cell line, iSC, with a 14-mer prosaptide, TX14(A) (10 nM), enhanced phosphorylation of mitogen-activated kinases ERK1 (p44 MAPK) and ERK2 (p42 MAPK) within 5 min, which was blocked by 4 h pretreatment with pertussis toxin. Furthermore, incubation of Schwann cells with the nonhydrolyzable GDP analog GDP-ßS inhibited TX14(A)-induced ERK phosphorylation. TX14(A) enhanced the sulfatide content of primary Schwann cells by 2.5-fold, which was inhibited by pretreatment with pertussis toxin or the synthetic MAP kinase kinase inhibitor PD098059. In addition, TX14(A) increased the tyrosine phosphorylation of all three isoforms of the adapter molecule, Shc, which coincided with the association of p60Src and PI(3)K. Inhibition of PI3(K) by wortmannin blocked TX14(A)-induced ERK phosphorylation. These data demonstrate that TX14(A) uses a pertussis toxin-sensitive G-protein pathway to activate ERKs, which is essential for enhanced sulfatide synthesis in Schwann cells.—Campana, W. M., Hiraiwa, M., O'Brien, J. S. Prosaptide activates the MAPK pathway by a G-protein-dependent mechanism essential for enhanced sulfatide synthesis by Schwann cells. FASEB J. 12, 307–314 (1998)


Key Words: prosaposin • saposin C • myelin • MAP kinase • PI3 kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROSAPOSIN IS A neurotrophic factor (1) that was originally identified as the precursor of four small lysosomal glycoproteins called saposins A, B, C, and D (2). Saposins are found in lysosomes and activate the hydrolysis of sphingolipids by lysosomal hydrolases. Within prosaposin, the neurotrophic sequence is localized to a 12-amino acid stretch at the amino-terminal end of the saposin C domain (3). Several synthetic peptides (14–22 residues) derived from this region are equally as bioactive as prosaposin and are called prosaptides (3, 4). Prosaptides induce differentiation and prevent cell death in a variety of neuronal cells (3–7); recently, prosaposin and prosaptides have been shown to increase sulfatide synthesis, a myelin glycolipid essential for myelin stability (8) in Schwann cells and oligodendrocytes (9). It has been suggested that hypomyelination in prosaposin-deficient humans (10) and prosaposin-deficient transgenic mice (11) is a consequence of the absence of the myelinotrophic action of prosaposin (9).

Mitogen-activated kinase is a general name for a family of serine/threonine kinases that play an important role in cell signaling by a variety of ligands and receptors including receptor tyrosine kinases (12) and G-protein-coupled receptors (13–15). The extracellular signal-regulated protein kinases ERK1 and ERK2 make up a part of the extracellular signal-regulated kinase (MAPK)2 family: p44 MAPK, ERK1; and p42 MAPK, ERK 2. Activation of ERKs have traditionally been thought to be a result of a linear signaling cascade from growth factor receptors, SH2/SH3 adapter proteins, guanine nucleotide exchange factors, p21 Ras, Raf-1, and MAPK-activating kinases such as MAP kinase kinase (MEK). However, there is emerging data indicating that signaling proteins such as phosphatidylinositol-3-kinase [PI(3)K] and protein kinase C (PKC) can also phosphorylate MEK and ERKs independent of the p21 Ras pathway (16, 17).

In many cellular systems, activation of the MAPK pathway by trophic factors has been implicated in the regulation of gene transcription (18) associated with proliferation and differentiation (19). In oligodendrocytes, ERKs appear to play an important role in process extension (20). However, signal transduction cascades involved in myelin lipid synthesis by myelinating cells have not been identified. We previously reported that prosaptides bind to a cell surface protein with high affinity and activated ERK1 and ERK2 phosphorylation in PC12 cells (21), Schwann cells, and oligodendrocytes (9). We demonstrate that prosaptides activate ERK activity via a pertussis toxin-sensitive mechanism involving the adapter proteins, Shc, the nonreceptor tyrosine kinase, p60Src, and PI(3)K. Furthermore, G-protein-stimulated ERK activity appears to be essential for enhanced synthesis of sulfatide in Schwann cells after prosaptide treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
TX14(A), a prosaptide (TX14(A)=TXLIDNNATEEILY, where X equals D-alanine) derived from the neurotrophic region of saposin C, was synthesized commercially to 98% purity (AnaSpec, San Jose, Calif.). Platelet-derived growth factor (PDGF) was purchased from Genzyme (Cambridge, Mass.). GDP-ßS, PD098059, wortmannin (WT), and pertussis toxin (PT) were purchased from CalBiochem (San Diego, Calif.). Anti-phosphotyrosine monoclonal Ab, anti-Src monoclonal Ab, anti-PI(3)K polyclonal Ab, and anti-Shc polyclonal Ab were purchased from Upstate Biotechnology Incorporated (Lake Placid, N.Y.). Anti-sulfatide antisera was the generous gift of Dr. Y. Hirabayashi (Riken Frontier, Tokyo).

Cell culture
Two Schwann cell cultures were used: a spontaneously transformed cell line, iSC, from rat primary Schwann cells (22); and primary Schwann cells prepared from neonatal rats, as described previously (23). At the first passage, Schwann cells were further selected from fibroblasts by using an anti-fibronectin antibody and rabbit complement. This resulted in approximately 99% pure Schwann cell cultures as assessed by S100 and fibronectin immunoflourescence. iSC cells were maintained in DME/F12 containing 10% horse serum and P/S (100 U/ml penicillin and 100 µg/ml streptomycin). Primary Schwann cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), P/S, 21 µg/ml bovine pituitary extract, and 4 mM forskolin. All cells were incubated at 37°C under humidified 7.5% CO2.

Phosphorylation assays
Primary Schwann cells and iSC cells were grown to 85% confluency in maintenance media and changed to serum-free media (SFM) 6 h (primary Schwann cells) or 16–18 h (iSC cells) before experimentation. Experiments involving the nonhydrolyzable GDP analog, GDP-ßS, were performed by permeabilizing serum-starved cells with saponin (20 µg/ml) for 3 min in the presence of GDP-ßS. Cells were then rinsed twice with SFM and reincubated at 37°C with GDP-ßS for 20 min prior to the addition of effectors. Cells were pretreated with either PT, PD098059, or WT at the times and concentrations described in the figure legends. In all experiments, cells were stimulated with effectors for 5 min, washed three times with ice-cold phosphate-buffered saline (PBS) containing 1 mM sodium vanadate, and lysed on ice in lysis buffer as previously described (21). The protein content of each sample was determined by the bicinchonic acid method (Sigma Chemical Co., St. Louis, Mo.). Western immunoblotting and densitometry were performed as described elsewhere (21) except that nitrocellulose membranes were used instead of polyvinylidene difluoride membranes. Differences in treatments were analyzed by analysis of variance (ANOVA) and treatment means were analyzed by the Student's-Newman-Keuls multiple comparisons test.

Kinase activity
ERK activity was assessed by using a MAP kinase activity kit (New England Biolabs, Cambridge, Mass.) with minor modifications. Briefly, Schwann cells were prepared as described above, stimulated with effectors for 5 min, and lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM sodium vanadate, 1 µg/ml leupeptin, and 1 mM PMSF. The protein content of each sample was determined as described above. Primary Schwann cell lysates (100 µg) and iSC cell lysates (200 µg) were incubated with 1:200 phospho-MAP kinase antibody overnight at 4°C. Immunoprecipitates were obtained by adding 20 µl (50% slurry) protein A sepharose CL-4B (Sigma) and incubating at 4°C for 4 h or overnight. Beads were washed twice in lysis buffer and then washed in kinase buffer containing 25 mM Tris (pH 7.5), 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM sodium vanadate, and 10 mM MgCl2. Immunoprecipitates were incubated at 30°C for 30 min in kinase buffer containing 1 µg ELK-1 fusion protein and 100 µM ATP. Reactions were terminated by the addition of 25 µl 3x sodium dodecyl sulfate (SDS) sample buffer. Samples were boiled for 5 min and proteins were resolved by sodium SDS-polyacrylamide gel electrophoresis (-PAGE) (24). Proteins were electroblotted onto nitrocellulose membrane and ERK activity was identified by immunoblotting with a phospho-ELK-1 antibody, followed by detection with ECL (Amersham, Arlington Heights, Ill.).

Immunoprecipitations
iSC cells (approximately 2.0x107) were incubated in DMEM/F12 without serum 18 h before stimulation with TX14(A) for 5 min at 37°C. Cells were then lysed and immunoprecipitated as previously described (25). Protein concentrations were determined by the bicinchoninic acid method (Sigma Chemical Co.). Immunoprecipitates containing equal amounts of protein were resolved by SDS-PAGE and electrotransferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.). After blocking with 3% bovine serum albumin (BSA) and 0.05% Tween 20, membranes were probed with specific antibodies at 4°C overnight in 1% BSA diluted in T-TBS (20 mM Tris-HCL pH 7.6 150 mM NACl and 0.05% Tween 20). After extensive washing, proteins from the immunocomplexes were detected by horseradish peroxidase-conjugated, species-specific secondary antibodies (Bio-Rad, Hercules, Calif.), followed by ECL (Amersham).

Immunoassay for sulfatide
Primary Schwann cells were incubated in DMEM containing 0.5% FBS with and without effectors for 48 h. Cells treated with PT (50 ng/ml) were preincubated for 4 h in 0.5% FBS containing media before the addition of effectors. Cells treated with the recently described synthetic inhibitor of MEK, PD098059 (26), were preincubated at 37°C for 30 min before the addition of effectors. Cells were rinsed with PBS, harvested, and sonicated in 100 µl distilled water. An aliquot of cell lysate was removed for protein analysis and the remainder was extracted with 5 ml of chloroform/methanol, 2:1 (v/v). Schwann cell lipid extracts were chromatographed and immunostained, as described, with an anti-sulfatide monoclonal antibody that is highly specific for sulfatide (9). The effect of treatment changes in sulfatide synthesis was tested by comparing the differences by ANOVA and treatment means were analyzed by the Student's-Newman-Keuls multiple comparisons test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In this study, TX14(A) increased both ERK1 and ERK2 phosphorylation in Schwann cells ( Fig. 1A). There was a larger increase in the ratio of ERK1 phosphorylation to total ERK1 protein (18-fold that of controls) than that of ERK2 (3-fold greater than controls) ( Fig. 1C). When iSC cells were preincubated with PT, which catalyzes the ADP ribosylation of Gi/Go {alpha} subunits of G-proteins, TX14(A)-induced ERK phosphorylation was inhibited ( Fig. 1A–C). Similar results were also observed in primary Schwann cells (data not shown). In contrast, PDGF, which binds to a tyrosine kinase receptor and stimulates proliferation of Schwann cells, stimulated ERK1 (four- to sixfold) and ERK2 (twofold) phosphorylation, but was not inhibited by PT pretreatment ( Fig. 1C). To further confirm that ERK phosphorylation by TX14(A) involved G-proteins, we incubated the iSC cells with GDP-ßS; this treatment also blocked TX14(A)-induced ERK phosphorylation ( Fig. 2).




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  		Figure 1. Inhibition of TX14(A)-induced ERK phosphorylation by pertussis toxin (PT) in iSC cells. A) Western blot of ERK phosphorylation using a polyclonal antibody that recognizes phosphorylated ERK1 and ERK2. Lanes: 1, control; 2, TX14(A) (10 nM) for 5 min; 3, pretreated with PT (100 ng/ml) for 4 h; 4, pretreated with PT (100 ng/ml) for 4 h prior to TX14(A) at 10 nM for 5 min. B) Western blot of total ERK proteins using a polyclonal antibody that recognizes both ERK1 and ERK2 independent of phosphorylation state. Lanes 1–4, same as above. C) The ratio of phosphorylated ERKs to total ERK protein quantified by densitometry. The data were normalized to controls for each experiment and expressed as mean ±SEM of three to five independent experiments. *Differences from controls with P < 0.05.



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  		Figure 2. Inhibition of TX14(A)-induced ERK phosphorylation by GDP-ßS in iSC cells. After permeabilization with saponin, iSC cells were treated with GDP-ßS and stimulated with TX14(A) for 5 min. A) Western blot of phosphorylated ERK as in Fig. 1. Lanes: 1, control; 2, GDP-ßS (100 µM); 3, TX14(A) 10 nM for 5 min; 4, TX14(A) for 5 min + GDP-ßS (100 µM). B) Western blot of total ERK proteins as in Fig. 1.

ERK protein kinases are activated by phosphorylation of tyrosine and threonine residues, and both are required for full protein kinase activity. Since the antibody we used only recognized the phosphorylated tyrosine residue on ERKs, we investigated whether TX14(A)-induced phosphorylation of ERK was correlated with ERK catalytic activity. As shown in Fig. 3, kinase activity was also increased in both primary Schwann cells and iSC cells after treatment with PDGF and TX14(A).



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  		Figure 3. Stimulation of ERK activity by TX14(A) and PDGF in iSC and primary Schwann cells. After in vitro kinase assays were performed, proteins were resolved by 12% SDS-PAGE and Western blotted for phosphorylated ELK-1. Lanes: 1, primary Schwann cell control; 2, primary Schwann cells stimulated by TX14(A) 10 nM for 5 min; 3, iSC cell control; 4, iSC cells stimulated by PDGF 1 nM for 5 min; 5, iSC cells stimulated by TX14(A) 10 nM for 5 min.

We also examined the activation of the adapter protein Shc in TX14(A) signaling. iSC cells expressed all three isoforms (p46, p52, and p66) of the Shc proteins. As shown in Fig. 4A, immunoprecipitation of iSC cell lysates with a polyclonal antibody to all three isoforms of Shc, followed by Western blotting with an anti-phosphotyrosine antibody, demonstrated that TX14(A) greatly enhanced tyrosine phosphorylation of all three Shc isoforms. Furthermore, two unidentified tyrosine phosphorylated proteins of approximately 60 and 85 kDa in size were observed in the Shc immunoprecipitates. Western blotting of Shc immunoprecipitates with an antibody to p60Src revealed that the 60 kDa tyrosin- phosphorylated protein was p60Src ( Fig. 4B); an antibody to p85 PI(3)K revealed that the 85 kDa phosphorylated protein was indeed the p85 subunit of PI(3)K ( Fig. 4C). Moreover, after TX14(A) treatment, there was more PI(3)K associated with Shc immunoprecipitates than controls. Blots were reprobed with anti-Shc to demonstrate that equal amounts of unphosphorylated Shc proteins were loaded onto the gel ( Fig. 4D). We then immunoprecipitated iSC cell lysates with an antibody to p60Src and Western blotted them with an antibody to phosphotyrosine; this experiment showed enhanced tyrosine phosphorylation of PI(3)K after treatment with TX14(A) (data not shown). Furthermore, preincubation of iSC cells with wortmannin completely blocked TX14(A)-induced ERK phosphorylation ( Fig. 5); in unstimulated cells, wortmannin treatment reduced ERK phosphorylation below control levels.



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  		Figure 4. Tyrosine phosphorylation of Shc and association of Shc with p60Src and PI(3)K in iSC cells. iSC cells were treated with TX14(A) at 10 nM for 5 min. A) Tyrosine phosphorylation of Shc by TX14(A). Cell lysates were immunoprecipitated with anti-Shc, Western blotted, and probed with an antibody to phosphorylated tyrosines. B) Association of Shc with p60Src. Cell lysates were immunoprecipitated with anti-Shc and probed with anti-p60Src. C) Association of Shc with PI(3)K. Cell lysates were immunoprecipitated with anti-Shc, Western blotted, and probed with anti-p85 PI(3)K. D) Western blot of Shc immunoprecipitates reprobed with anti-Shc.



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  		Figure 5. Inhibition of TX14(A)-induced ERK phosphorylation by wortmannin (WT) in iSC cells. Prior to TX14(A) stimulation for 5 min, iSC cells were treated with WT (50 nM) for 15 min. Proteins from cell lysates were resolved by 10% SDS-PAGE, immunoblotted for phosphorylated ERK1 and ERK2, and reprobed for total ERK1 and ERK2 protein as in Fig. 1. Data were normalized to controls and expressed as mean ±SEM of two independent experiments performed in duplicate.

TX14(A) was previously reported to stimulate synthesis of sulfatide in Schwann cells (9). To determine whether G-protein-mediated ERK phosphorylation was involved in the synthesis of sulfatide, we preincubated primary Schwann cells with either PT or the synthetic inhibitor of MEK, PD098059, before TX14(A) stimulation. Similar to our previous results (9) the anti-sulfatide monoclonal antibody identified only sulfatide that had the same mobility as purified sulfatide in all samples. In addition, TX14(A) treatment increased the sulfatide content 2.5-fold over controls ( Fig. 6). Pretreatment with either PT or PD098059 inhibited TX14(A)-induced sulfatide synthesis. The viability of Schwann cells treated with either PD098059 or PT after 48 h did not differ from controls, as determined by trypan blue exclusion. To confirm that the dose of PD098059 used to inhibit sulfatide synthesis also inhibited ERK phosphorylation in primary Schwann cells, we performed ERK phosphorylation experiments in cells pretreated with PD098059. As shown in Fig. 7, TX14(A) increased the phosphorylation of ERKs; however, the magnitude of the increase was less than that observed in iSC cells. The same dose of PD098059 (50 µM) used in the sulfatide experiments blocked TX14(A)-induced phosphorylation of ERK in primary Schwann cells. In addition, PD098059 decreased ERK1 and ERK2 phosphorylation below control levels. Time course experiments of TX14(A)-induced phosphorylation of ERKs in iSC cells demonstrated that TX14(A) rapidly activates ERK1 and ERK2 within 5 min, which returned to baseline levels by 30 min ( Fig. 8 A–C).




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  		Figure 6. Inhibition of TX14(A)-induced sulfatide content in primary Schwann cells by pertussis toxin (PT) and PD098059 (PD). Schwann cells were pretreated with PT (50 ng/ml) for 4 h before the addition of TX14(A) (10 nM) or with PD (50 µM) for 30 min before the addition of TX14(A) (10 nM). A) TLC immunostaining of sulfatide after treatment with TX14(A) or TX14(A) + PT for 48 h. B) TLC immunostaining of sulfatide after treatment with TX14(A) or TX14(A)+PD after 48 h. C) Sulfatide content after quantification by densitometry. Lanes: 1, control; 2, TX14(A) (10 nM); 3, PD098059 (50 µM); 4, TX14(A) 10 nM + PD098059 (50 µM); 5, PT (50 ng/ml); 6, TX14(A) 10 nM + PT (50 ng/ml). Data are expressed as mean ±SEM; **differences from controls with P < 0.01.



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  		Figure 7. PD098059 (50 µM) blocks TX14(A)-induced ERK phosphorylation in primary Schwann cells. Proteins from cell lysates were resolved by 10% SDS-PAGE, immnoblotted for phosphorylated ERK1 and ERK2, and reprobed for total ERK proteins as in Fig. 1. Data were normalized to controls and expressed as mean SEM for three independent experiments *Differences from controls with P < 0.05.




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  		Figure 8. Time course of ERK phosphorylation in iSC cells. A) Western blot of ERK phosphorylation as in Fig. 1. Lanes: 1, control; 2, TX14(A) 10 nM for 5 min; 3, TX14(A) 10 nM for 15 min; 4, TX14(A) 10 nM for 30 min. B) Western blot of total ERK proteins as in Fig. 1. Lanes 1–4, same as above. C) The ratio of phosphorylated ERKs to total ERK protein quantified by densitometry. The data were normalized to controls for each experiment and expressed as mean ±SEM of three or four independent experiments *Differences from controls with P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Identification of a G-protein-dependent mechanism for TX14(A) signaling
We previously reported that TX14(A) dose-dependently stimulated ERK phosphorylation in both iSC and primary Schwann cells (9). After quantification and expression of the data as a ratio of phosphorylated ERKs to total ERK proteins, TX14(A) preferentially phosphorylated ERK1, although Schwann cells contained a greater amount of immunoreactive ERK2 protein. We observed the same phenomenon in PC12 cells (21), and others have reported that ERK1 is preferentially activated in oligodendrocytes (20). In the present study, TX14(A)-stimulated ERK phosphorylation was blocked by PT treatment, which indicated that the primary mechanism of activation involved one or more PT-sensitive G-proteins such as Gi or G0, both of which are abundantly expressed in Schwann cells (27). It was reported recently that ERK activation was associated with PT-sensitive G-protein signaling in COS-7 cells (28), CHO cells (29), and Swiss 3T3 cells (30), and that the mechanism of MAP kinase activation by G-coupled receptors involves Gß{gamma} subunits (28, 30–31). We have demonstrated that prosaptides and prosaposin specifically bind to PC12 cells in a dose-dependent, saturatable manner with high affinity (Kd=2.5 nM and 18 nM, respectively) (21). Similarly, cell surface binding assays using radiolabeled TX14(A) gave a single high-affinity constant for binding to iSC cells, with a Kd of 10 nM (data not shown). These findings suggest that prosaposin and TX14(A) bind to a putative receptor that associates with PT-sensitive G-protein to mediate signal transduction. Pertussis toxin-sensitive ERK signaling has been reported for the insulin-like growth factor receptor tyrosine kinase (15) as well as the more common seven-transmembrane, G-protein-coupled receptors. Experiments are under way to clone and elucidate the molecular structure of the prosaposin receptor to determine which subtype it represents.

Enhanced sulfatide synthesis is dependent on TX14(A)-induced ERK activation
The pathways of signal transduction that underlie myelination have not been clearly defined. In oligodendrocytes, the initial stages of myelination appeared to involve nonreceptor tyrosine kinases of the Src family (32), and ERK activation has been shown to play an important role in process extension (20). In the peripheral nerve, tissue concentrations of ERKs have been shown to increase after peripheral nerve injury (day 3); ERKs were localized to activated Schwann cells and increased concomitant with remyelination (33). However, no evidence has been reported to indicate that pathways resulting in ERK activation lead to enhanced synthesis of myelin constituents. We demonstrate here for the first time that inhibition of MEK by PD098059 completely blocked TX14(A)-enhanced synthesis in Schwann cells of sulfatide, an essential myelin lipid component of both central and peripheral nervous system myelin. This concentration of PD098059 (50 µM) has been shown to specifically inhibit MEK and not other kinases such as PKC, PI(3)K, or p38 MAP kinase (26). PD098059 did decrease ERK phosphorylation below controls, suggesting that primary Schwann cells in culture contain autocrine-regulated ERKs.

In addition, the time course of ERK activation by TX14(A) indicates that only 5 min of stimulation is sufficient for the enhanced sulfatide synthesis observed 48 h later. Transient activation of ERKs in PC12 cells with growth factors such as epidermal growth factor does not lead to pronounced nuclear translocation (34), suggesting that in Schwann cells, TX14(A)-induced ERK may act in the cytosol to contribute to myelin lipid synthesis. Our present hypothesis is that signal transduction through the ERK pathway is an essential signaling pathway responsible for myelination by Schwann cells.

TX14(A) induces signaling through multiple pathways
We have demonstrated that TX14(A) signaling involved the adapter protein, Shc, and the nonreceptor tyrosine kinase, p60Src. Our results demonstrated that Shc associated with p60Src after TX14(A) stimulation, which coincided with increased tyrosine phosphorylation of Shc. The association of p60Src and Shc has previously been observed in COS-7 cells after lysophosphatidate stimulation (35), and is proposed to be involved in early activation of ERKs via PT-sensitive G-protein-coupled receptors (14, 36, 37).

Our results with the MEK inhibitor PD098059 suggested that ERK activation by TX14(A) was a result of the p21 Ras-mediated signaling cascade in Schwann cells. However, our studies also demonstrated that PI(3)K plays a role in ERK activation in response to TX14(A), based on the ability of WT to block TX14(A)-induced ERK phosphorylation and the observation that TX14(A) induced a larger amount of p85 PI(3)K in Shc immunoprecipitates coincident with Shc tyrosine phosphorylation. The concentration of WT we used has previously been shown to specifically inhibit PI(3)K activity (38) in Swiss 3T3 fibroblasts (16) and L6 rat myoblasts (39). PI(3)K has been shown to activate ERKs via a p21 Ras-independent mechanism (16) and by linkage with G-protein-coupled receptors (14), suggesting that TX14(A) signaling involves multiple and perhaps novel pathways leading to ERK activation.

TX14(A) role in myelination
Prosaposin appears not only to be a neurotrophic factor (1), but an essential factor for events involved in myelination, including prevention of Schwann cell and oligodendrocyte death and synthesis of sulfatide, a myelin lipid (9). Moreover, prosaposin-deficient transgenic mice have severe hypomyelination (11) in both the central and peripheral nervous systems, apparently due to the failure of myelin synthesis rather than to demyelination. We have proposed that the deficiency of myelin in these animals and in prosaposin-deficient humans is due to the lack of a myelinotropic effect of prosaposin during development (9). We report here that TX14(A), encompassing the neurotrophic region of prosaposin, appeared to exert its trophic effect by binding to a high-affinity receptor, which activated a PT-sensitive G-protein (40) and signaled through ERKs to up-regulate the synthesis of sulfatide in Schwann cells. We demonstrated that inhibition of ERK activation blocked enhanced synthesis of sulfatide, implicating ERKs as a key signaling component in myelin lipid synthesis. Current experiments are aimed to define other key components involved in prosaposi-induced myelination and to assess the efficacy of TX14(A) in the treatment of demyelinating diseases.


   ACKNOWLEDGMENTS
 
We thank Drs. Lyn Bolin and Richard Quarles for iSC cells. This project was supported by a grant from Myelos Neurosciences Corp. (J.S.O.) and by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant DK07318–18 (W.M.C.).


   FOOTNOTES
 
1 Correspondence: Department of Neurosciences, Center for Molecular Genetics-115, School of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093–0634, USA. E-mail: jsobrien{at}ucsd.edu

2 Abbreviations: ANOVA, analysis of variance; TX14(A), 14-mer prosaptide; PDGF, platelet-derived growth factor; PT, pertussis toxin; WT, wortmannin; GDP-ßS, guanosine-5'-(3–0-thio) diphosphate; p44 MAPK, extracellular signal-regulated kinase, ERK1; p42MAPK, extracellular signal-regulated kinase, ERK2; PI(3)K, phosphatidylinositol-3-kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SFM, serum-free medium; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; MEK, MAP kinase kinase; PKC, protein kinase C; PBS, phosphate-buffered saline.

Received for publication September 1, 1997. Accepted for publication November 20, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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