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Cell Growth & Differentiation Vol. 12, 39-50, January 2001
© 2001 American Association for Cancer Research

Articles

Differences in Early and Late Responses between Neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y Neuroblastoma Cells1

Anders Edsjö, Bengt Hallberg, Sofia Fagerström, Christer Larsson, Håkan Axelson and Sven Påhlman2

Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, S-205 02 Malmö [A. E., S. F., C. L., H. A., S. P.], and Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå [B. H.], Sweden

Abstract

Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.

Introduction

Neuroblastoma is a malignancy of infancy and childhood with embryonic characteristics and is derived from the sympathetic nervous system (1) . Phenotypically, neuroblastoma cells resemble immature sympathetic neuroblasts, but some tumors also contain cells with sympathetic extra-adrenal chromaffin characteristics (1, 2, 3) . The NT3 receptor gene trkA is frequently expressed in tumors with favorable outcome but not in highly malignant neuroblastomas (Refs. 4 and 5 ) and reviewed in Ref. 6 . Expression of full-length trkC and the gene coding for the low-affinity NT receptor p75NTR also correlates with favorable outcome (5 , 7 , 8) , although it has been noted that tumors with detectable trkC expression are less frequent than tumors expressing trkA (8 , 9) . On the basis of these findings, an impaired NT-driven differentiation in unfavorable neuroblastomas has been suggested (4 , 10) . Introduction of exogenous trkA into established neuroblastoma cell lines, which invariably are derived from highly malignant tumors, demonstrates that such cells retain the capacity to differentiate in response to NGF (10, 11, 12) . Furthermore, xenotransplanted trkA-transfected neuroblastoma cells in mice form tumors, which become growth arrested and differentiated upon treatment with NGF (11) . This could suggest that unfavorable neuroblastomas are immature because the sympathetic differentiation is blocked at an early developmental stage when trkA under normal conditions is not yet expressed.

In mammals, the NT family consists of NGF, brain-derived neurotrophic factor, NT-3, and NT-4/5 (13, 14, 15) . The NTs are ligands for receptor protein tyrosine kinases of the Trk family, i.e., NGF binds p140trkA, brain-derived neurotrophic factor and NT-4/5 bind to p145trkB, and NT-3 is the ligand for p145trkC (16, 17, 18) .

In vitro studies of the growth factor dependence of the developing sympathetic nervous system cells have identified bFGF, IGF-I, and CNTF, among other growth factors, as important mitogenic, trophic, and differentiation-inducing factors for sympathetic neuroblasts before they become dependent on NTs (19, 20, 21, 22) . Under the influence of these growth factors, sympathetic neuroblasts begin to express trkC. During this phase, NT-3 serves as a trophic and survival factor, resulting in growth arrest, trkA expression, and NGF dependency (23, 24, 25, 26) . However, studies on knockout mice have shown that trkA is expressed at normal levels in sympathetic ganglia in trkC-/- mice, suggesting either redundant mechanisms or that the switch in trk expression observed in vitro may not require NT-3-induced p145trkC signaling in vivo (27) .

SH-SY5Y neuroblastoma cells (SH-SY5Y/wt) resemble sympathetic neuroblasts in that they differentiate toward a neuronal phenotype when treated with a combination of bFGF and IGF-I (28) . Similar to a number of other neuroblastoma cell lines, SH-SY5Y/wt cells express low levels of trkA, and limited differentiation is seen in response to NGF alone (10 , 12) . When SH-SY5Y/wt cells are stably transfected with exogenous trkA, NGF induces morphological differentiation observed as neurites with growth cones and varicosities and expression of neuronal sympathetic differentiation marker genes (10 , 12) . Very low trkC expression in SH-SY5Y/wt cells at the mRNA level has been reported (7) , but as shown in this report, p145trkC cannot be detected, and the weak response to NT-3 in wild-type cells appears to be mediated via p140trkA. The present study aims to investigate whether trkC-transfected SH-SY5Y/wt cells differentiate with NT-3, how this phenotype compares with that of NGF-treated SH-SY5Y/trkA cells (10 , 12) , and whether the NT-3 treatment results in growth arrest and up-regulation of the expression of trkA, as seen in normal sympathetic neuroblasts.

Results

Growth Factor-induced Differentiation of SH-SY5Y/wt Cells.
To test whether NT-3 alone or in combination with NGF could induce differentiation of SH-SY5Y/wt cells, cells were cultured in the presence of 100 ng/ml NT-3 and/or NGF. In the presence of 10% FCS, a clear effect on morphology of NGF, but not of NT-3, was noted, whereas neither factor alone or in combination induced morphological differentiation under serum-free conditions (Fig. 1ACitation and as quantified below). Treatment of SH-SY5Y/wt cells with phorbol esters in the presence of serum or with a combination of bFGF and IGF-I leads to phenotypic alterations indicative of a neuronal sympathetic phenotype (28 , 29) . These alterations include increased synthesis of neurotransmitters, e.g., norepinephrine, NPY, and increased expression of proteins involved in axonal growth, e.g., GAP-43 (Fig. 1BCitation ; Refs. 28 and 29 ). Expression of two of these differentiation markers, GAP-43 and NPY, did not increase in NT-3- or NGF-treated SH-SY5Y/wt cells (Fig. 1B)Citation . As mentioned, bFGF and IGF-I serve as mitogens and trophic factors for sympathetic neuroblasts and induce differentiation at a time when NT receptors are not yet expressed. The combination of bFGF and IGF-1 also induces differentiation of SH-SY5Y/wt cells (30) . To test whether pretreatment of SH-SY5Y/wt cells with bFGF and IGF-I would make these cells susceptible to NT-3 and/or NGF treatment, the cells were treated under serum-free conditions with NTs and a combination of 3 nM bFGF and 5 nM IGF-I. In summary, no additional effects on the morphological differentiation induced by bFGF and IGF-I were seen when NT-3 (100 ng/ml) alone, NGF (100 ng/ml) alone, or a combination of both were included (Fig. 1Citation A, panels c and d, and not shown). This suggests that bFGF/IGF-I treatment did not induce a functional NT-3/p145trkC or NGF/p140trkA response. Furthermore, addition of CNTF to these protocols did not result in further morphological differentiation (not shown).



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  		Fig. 1. Growth factor-induced differentiation of SH-SY5Y/wt cells. A, phase-contrast micrographs of SH-SY5Y/wt cells grown in the presence of bFGF + IGF-I and/or NT-3 + NGF. Cells were grown for 4 days in serum-free medium alone (a), in serum-free medium supplemented with 100 ng/ml NT-3 + 100 ng/ml NGF (b), 3 nM bFGF + 5 nM IGF-I (c), or 3 nM bFGF + 5 nM IGF-I + 100 ng/ml NT-3 + 100 ng/ml NGF (d). Bar in a, 25 µm. B, Northern blot analysis of the expression of the neuronal differentiation marker genes, NPY (0.8 kb) and GAP-43 (1.4 kb) in SH-SY5Y/wt cells. Cells were grown for 4 days in serum-containing medium alone or in serum-containing medium supplemented with 100 ng/ml NT-3, 100 ng/ml NGF, 100 ng/ml NT-3 + 100 ng/ml NGF, or the differentiation inducer TPA (16 nM). Fifteen µg of total RNA of each sample were electrophoretically separated and blotted onto the same filter. GAPDH (1.5 kb) mRNA levels were used as a reference for the amount of loaded RNA.

 
Generation of SH-SY5Y Cells Stably Expressing Functional p145trkC.
To investigate whether the introduction of trkC could confer an NT-3 response, SH-SY5Y/wt cells were stably transfected with a full-length trkC cDNA. As demonstrated in Fig. 2ACitation ,trkC expression could not be detected in wild-type cells, in cells transfected with empty vector (clone 1:9), or in previously generated (10) , stably trkA-transfected cells (SH-SY5Y/trkA, clone 6:2). In contrast, expression of exogenous trkC could readily be detected as one major 2.8-kb and one minor 1.2-kb mRNA in the trkC-transfected (SH-SY5Y/trkC) 3:1 and 3:2 clones (Fig. 2A)Citation . The SH-SY5Y/trkC clones resembled SH-SY5Y/wt cells in terms of morphology and growth properties. Furthermore, the capacity of SH-SY5Y/trkC and SH-SY5Y/trkA cells to differentiate with established differentiation protocols for wild-type cells (28) was comparable, as shown by the induction of differentiation markers NPY and GAP-43 in response to either TPA or bFGF + IGF-I in combination (Fig. 2B)Citation .



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  		Fig. 2. Characterization of stably transfected SH-SY5Y cell clones. A, Northern blot analysis of trkC expression in SH-SY5Y/trkC cells. Fifteen µg of total RNA from SH-SY5Y/wt, SH-SY5Y cells transfected with trkC (clones 3:1 and 3:2), with trkA (clone 6:2), or with empty pMEXneo vector (clone 1:9) were transferred to the same filter and probed for trkC and GAPDH expression, the latter as an mRNA loading control. Bars at left, approximate sizes of trkC transcripts (2.8 and 1.2 kb). B, Northern blot analysis of the expression of the neuronal marker genes, NPY (0.8 kb) and GAP-43 (1.4 and 1.6 kb) in differentiated SH-SY5Y/trkC (clone 3:1) and SH-SY5Y/trkA cells. Cells were grown for 4 days in serum-containing medium alone or in serum-containing medium supplemented with 3 nM bFGF and 5 nM IGF-I or with 16 nM TPA. Fifteen µg of total RNA of each sample were electrophoretically separated and blotted onto the same filter. GAPDH (1.5 kb) mRNA levels were used as a reference for the amount of loaded RNA.

 
To test whether the SH-SY5Y/trkC clones expressed a functional p145trkC tyrosine kinase, these and SH-SY5Y/wt cells were stimulated with 100 ng/ml of NT-3 for 5 min. Cell lysates were subjected to immunoprecipitation using an anti-Pan-Trk antiserum, followed by immunoblotting with first an anti-phosphotyrosine antibody and then the same anti-Pan-Trk antiserum used for immunoprecipitation (Fig. 3A)Citation . A tyrosine phosphorylated protein in the Mr 145,000 molecular weight range (arrow, top panel of Fig. 3ACitation ) was detected with the anti-phosphotyrosine antibody in lysates from NT-3-stimulated SH-SY5/trkC cells. Reprobing with the anti-Pan-Trk antiserum detected a major protein of corresponding size in the SH-SY5Y/trkC cells. Similar results were obtained with both SHSY5Y/trkC clones tested (Fig. 3, A–C)Citation . Taken together, our data showed that these cells express a functional p145trkC. In both SH-SY5Y/trkC and SH-SY5Y/wt cells, a minor immunoreactive protein of slightly lower molecular weight was detected by the anti-Pan-Trk antiserum. This antiserum also detected a major protein doublet of similar size in the SH-SY5Y/trkA cells (Fig. 3C)Citation . As shown by anti-phosphotyrosine blotting of the same filter, the protein represented by the doublet became phosphorylated after NGF stimulation. On the basis of these data, we conclude that this anti-Pan-Trk immunoreactive protein represents p140trkA, that p140trkA and p145trkC can be discriminated, and that their phosphorylation status can be assessed by these analyses.



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  		Fig. 3. Trk and Erk 1/2 phosphorylation in trk-transfected SH-SY5Y cells. A, Western blot analysis of Trk phosphorylation status in NT-3-treated wild-type and trkC-transfected SH-SY5Y cells (clone 3:1). Serum-starved cells were incubated with 100 ng/ml NT-3 at 37°C for 10 min. After immunoprecipitation with anti-Pan-Trk antiserum and SDS-PAGE, the filter was incubated with an anti-phosphotyrosine ({alpha}-PY) antibody (upper panel) and then reprobed with anti-Pan-Trk antiserum (lower panel). The approximate positions of p145trkC and p140trkA are indicated by an arrow and *, respectively. B, Western blot analysis of Trk receptor levels in SH-SY5Y/trkC cells. Serum-starved cells (clones 3:1 and 3:2) were incubated at 37°C for 10 min with 100 ng/ml NT-3 or NGF present where indicated. Cells were lysed, and cell lysates where analyzed as in A. The approximate positions of p145trkC and p140trkA are indicated by an arrow and *, respectively. C, Western blot analysis of NT-induced Trk receptor phosphorylation in trk-transfected SH-SY5Y cells. SH-SY5Y/trkC and SH-SY5Y/trkA cell lysates were analyzed as in A, with 100 ng/ml NT-3 or NGF added, where indicated. The approximate position of p145trkC is indicated by an arrow, and the p140trkA doublet is indicated by *. D, serum-starved SH-SY5Y/trkC cells (clone 3:1) were stimulated with 100 ng/ml NT-3 or NGF for 10 min and then lysed. Whole-cell lysate proteins were separated by SDS-PAGE, followed by immunoblotting with anti-phospho-Erk 1/2 antiserum and reprobing with an anti-Pan-Erk antibody. IP, immunoprecipitation; IB, immunoblot.

 
In SH-SY5Y/trkC cells, NGF-induced phosphorylation of the endogenous p140trkA, and the phosphorylation level was in parity to the NT-3-induced phosphorylation of p145trkC in these cells (Fig. 3C)Citation . To test the signal transduction capacity of phosphorylated p145trkC and p140trkA in the SH-SY5Y/trkC cells, they were stimulated with either NGF or NT-3, and phosphorylation of two major downstream targets of p140/145trk activation, Erk 1 and 2, was analyzed. Despite similar receptor phosphorylation status, NT-3 induced a considerably stronger Erk 1/2 phosphorylation in SH-SY5Y/trkC cells than did NGF (Fig. 3D)Citation , showing that both receptors could convey signals into the cell and that p145trkC appeared to be a more efficient inducer of Erk 1/2 phosphorylation than the endogenous p140trkA phosphorylated to a similar extent as p145trkC. As reported previously (10) , NGF induced a robust phosphorylation of p140trkA in SH-SY5Y/trkA cells (Fig. 3C)Citation . NT-3 was also capable of inducing phosphorylation of a Trk protein in SH-SY5Y/trkA cells. It is known that NT-3 is able to bind p140trkA and to induce receptor phosphorylation (30) . Given the molecular weight of the phosphorylated receptor observed here and the lack of detectable trkC expression in the SH-SY5Y/trkA cells, we conclude that the phosphorylated protein is p140trkA (Fig. 3Citation C). Finally, when comparing the p145trkC and p140trkA levels in the two clones, p140trkA was slightly more abundant in SH-SY5Y/trkA than p145trkC in SH-SY5Y/trkC cells. More importantly, p140trkA became considerably more phosphorylated than did p145trkC upon NT stimulation (Fig. 3Citation C).

NT-3 Induces Morphological Differentiation of SH-SY5Y/trkC Cells.
SH-SY5Y/wt and trk-transfected cells were treated for up to 10 days with 100 ng/ml NT-3 and/or NGF. Under serum as well as serum-free (not shown) conditions, NT-3-treated SH-SY5Y/trkC cells differentiated morphologically, as manifested by neurite outgrowth and growth cone formation (Fig. 4Citation A, panel b). NGF alone induced a morphological differentiation of these cells (Fig. 4Citation A, panel c), and the morphological differentiation was enhanced in cells treated with a combination of NT-3 and NGF (Fig. 4Citation A, panel d). To quantify these differences, the number of cells with long neurites was counted. As an aid to distinguish individual cells and neurites, cells were transiently transfected with an expression vector containing green fluorescent protein cDNA. This quantification confirmed the morphological effects of NT-3 and NGF on both tested SH-SY5Y/trkC cell clones (Fig. 4, ACitation and C). NT-3 had no effect on neurite outgrowth in wild-type cells (Fig. 4B)Citation , suggesting that the morphological differentiation induced by NT-3 in SH-SY5Y/trkC cells is mediated via p145trkC. Because no cross-reactivity between NGF and p145trkC has been reported to date, the effect of NGF on morphology in SH-SY5Y/trkC cells most likely reflects the activation of endogenous p140trkA (Fig. 3C)Citation . Nontreated SH-SY5Y/trkA cultures displayed a higher basal number of cells with long neurites (27%), and NGF treatment of these cells leads to a considerably higher number of cells with long neurites (63%) than observed in SH-SY5Y/trkC cultures upon NT-3 addition (18 and 22% in clones 3:1 and 3:2, respectively). The differences in basal neurite outgrowth between the analyzed clones might reflect clonal variability. Despite the observation that NT-3 induced a pronounced phosphorylation of p140trkA in SH-SY5Y/trkA cells, NT-3 consistently failed to induce neurite outgrowth in SH-SY5Y/wt and SH-SY5Y/trkA cells, nor did it enhance the effect of NGF (Fig. 4Citation , B and D).



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  		Fig. 4. NT-induced morphological differentiation and neurite outgrowth in SH-SY5Y/wt, SH-SY5Y/trkC, and SH-SY5Y/trkA cells. A, phase-contrast micrographs of SH-SY5Y/trkC cells grown in the presence of NT-3 and/or NGF. Cells were grown for 10 days in serum-containing medium alone (a), in serum-containing medium supplemented with 100 ng/ml NT-3 (b), 100 ng/ml NGF (c), or 100 ng/ml NT-3 + 100 ng/ml NGF (d). Bar in a, 25 µm. B, percentage of cells with long neurites in SH-SY5Y/wt cells. Cells were grown for 8 days in serum-containing medium alone or with supplementation of 100 ng/ml NT-3, 100 ng/ml NGF, or 100 ng/ml NT-3 + 100 ng/ml NGF. C, SH-SY5Y/trkC cells (clones 3:1 and 3:2) treated as in B, supplemented with 100 ng/ml NT-3 or 100 ng/ml NGF and analyzed as in B. D, SH-SY5Y/trkA cells treated and analyzed as in C.

 
NT-3 Does Not Induce Growth Arrest or trkA Expression in SH-SY5Y/trkC Cells.
Because NT-3 can induce growth arrest and trkA expression in cultured sympathetic neuroblasts, we investigated whether NT-3 stimulation of SH-SY5Y/trkC cells could induce similar responses. SH-SY5Y/trkC clones were grown for 3 days in serum-containing medium with 100 ng/ml of NT-3 or NGF. The amount of viable cells was assessed using an MTT cell viability assay. As a control for the potential contribution of NT-3/p140trkA-driven proliferation, wild-type cells were also tested. In the presence of NT-3, a slight increase in SH-SY5Y/trkC cell numbers was seen relative to nontreated cultures (Table 1)Citation . A small increase was also seen in wild-type cells. NGF induced a similar increase in the wild-type and trkC clones, and no additive effect was seen when cells were grown in the presence of both factors (Table 1)Citation . Long-term cultures of SH-SY5Y/trkC cells in the presence of NT-3 became confluent. However, analysis of the number of cells with a capacity to synthesize DNA and incorporate thymidine at day 8 revealed that most cells, control as well as NT-3-treated SH-SY5Y/trkC cells, did synthesize DNA (79–90% cells with labeled nuclei; Table 1Citation ). Thus, after 8 days in culture, NT-3 had not induced growth arrest in SH-SY5Y/trkC cells.


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  		Table 1 Effect of NTs on the number of viable and proliferating SH-SY5Y/wt and SH-SY5Y/trkC cells grown in serum-containing medium, as measured by MTT and thymidine incorporation assays

 
The trk mRNA levels were assessed using Northern blot analyses. None of the NT combinations had any detectable effect on endogenous trkA mRNA expression (Fig. 5A)Citation ; endogenous trkA mRNA was slightly smaller in size (3.2 kb), allowing discrimination between endogenous and exogenous (3.5-kb) trkA mRNAs (10) . However, NT-3 stimulation resulted in higher exogenous trkC mRNA levels in SH-SY5Y/trkC cells, and NGF had a similar effect on exogenous trkA mRNA levels in SH-SY5Y/trkA cells (Fig. 5A)Citation . The mechanism(s) behind these changes in trk mRNA levels is not known to us and has not been analyzed further.



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  		Fig. 5. Northern blot analysis of expression of trkA, trkC, and of the neuronal marker genes NPY and GAP-43 in SH-SY5Y/trkC and SY5Y/trkA cells treated with NTs. A, cells were grown for 4 days in serum-free medium alone or in medium supplemented with 100 ng/ml NT-3, 100 ng/ml NGF, or 100 ng/ml NT-3 + 100 ng/ml NGF. Total RNA (15 µg) was analyzed for expression of NPY (0.8 kb), trkA (3.5 kb), and trkC (bars at left, 2.8 and 1.2 kb). B, SH-SY5Y/trkC (3:1) and SY5Y/trkA cells were grown for 4 days in serum-containing medium supplemented with increasing concentrations of NT-3 or NGF. Fifteen µg of total RNA were analyzed for expression of NPY and GAP-43. C, SH-SY5Y/trkC (3:1 and 3:2) and SY5Y/trkA cells were grown for 4 days in serum-containing medium supplemented with 100 ng/ml NT-3 or NGF. In all panels, GAPDH (1.5 kb) mRNA levels were used as a reference for the amount of loaded RNA.

 
NT-3 Enhances Expression of the Neuronal Differentiation Marker Genes GAP-43 and NPY in SH-SY5Y/trkC Cells.
In SH-SY5Y/trkC cells treated with NT-3, a dose-dependent elevation of mRNA levels of both NPY and GAP-43 was seen (Fig. 5B)Citation . The induction of differentiation markers was similar in both tested SH-SY5Y/trkC clones, as demonstrated for NPY (Fig. 5C)Citation . Stimulation of SH-SY5Y/trkC cells with NGF alone did not lead to an appreciable elevation of NPY levels (Fig. 5A)Citation , despite the p140trkA autophosphorylation and morphological differentiation induced in these cells (Figs. 3CCitation and 4C)Citation . In accordance with the morphology data, NT-3 had no apparent effect on the expression of the differentiation marker NPY in SH-SY5Y/trkA cells (Figs. 5ACitation and 4D)Citation . Furthermore, a stimulation of either SH-SY5Y/trkA or SH-SY5Y/trkC cells with a combination of NGF and NT-3 did not alter the NPY mRNA levels in comparison to stimulation with the corresponding NT alone (Fig. 5A)Citation .

NT-3-treated SH-SY5Y/trkC Cells Lack Important Functional Sympathetic Neuronal Characteristics.
Norepinephrine is the major neurotransmitter synthesized by sympathetic neurons. To further characterize the phenotypes induced upon NT treatment, the norepinephrine concentration and expression of synaptophysin, both related to a functional sympathetic phenotype, were investigated. In SH-SY5Y/trkA cells, the norepinephrine concentration increased ~6-fold over basal level with NGF treatment. In contrast, NT-3 treatment did not significantly increase the level of norepinephrine in SH-SY5Y/trkC cells (Fig. 6A)Citation .



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  		Fig. 6. Norepinephrine and synaptophysin levels in NT-treated SH-SY5Y/trkC and SH-SY5Y/trkA cells. A, norepinephrine was determined by high-performance liquid chromatography analysis in cells grown for 4 days in serum-containing medium ± 100 ng/ml of the indicated NT. Norepinephrine levels were adjusted for sample protein content and expressed as means (n = 4–5); bars, SE. B, distribution of synaptophysin in growth cones and cell bodies from NT-treated SH-SY5Y/trkC and SH-SY5Y/trkA cells, as determined by Western blot analysis. Cells were grown for 4 days in serum-containing medium supplemented with 100 ng/ml of NT-3 and NGF, respectively. Growth cones were prepared as described in "Materials and Methods," and 100 µg of protein from each of the cell body (CB) and growth cone (GC) preparations were separated by SDS-PAGE and blotted onto a nitrocellulose filter. The filter was incubated with an anti-synaptophysin antibody. Overexposure reveals a band (*) of the expected size for synaptophysin (Mr 42,000), also in the SH-SY5Y/trkC growth cone lane.

 
Synaptophysin is a synaptic vesicle and dense core granule protein (31 , 32) expressed in neuronal sympathetic cells (1) . When SH-SY5Y/wt cells differentiate in response to phorbol esters, they send out growth cone-terminated neurites with varicosities. These structures contain the neurosecretory granules, as determined by electron microscopy and synaptophysin immunofluorescence (33) . The growth cones of the differentiated cells can be isolated from the residual cell bodies, allowing for biochemical analyses of these preparations, revealing that synaptophysin is highly enriched in growth cones (33) . Using this methodology, growth cones and cell bodies were prepared from NGF-treated SH-SY5Y/trkA- and NT-3-treated SH-SY5Y/trkC cells, and the synaptophysin content was determined by immunoblot analysis (Fig. 6B)Citation . Growth cones isolated from differentiated SH-SY5Y/trkA cells contained severalfold more synaptophysin than the corresponding cell body preparation. In contrast, growth cones from differentiated SH-SY5Y/trkC cells contained only small amounts of synaptophysin. Thus, both norepinephrine and synaptophysin data indicate that NT-3-treated SH-SY5Y/trkC cells were not fully developed in terms of a sympathetic phenotype, although these cells were clearly more differentiated toward a neuronal phenotype than nontreated SH-SY5Y/trkC or wild-type cells, as judged by morphology, and the NPY and GAP-43 expression data.

NT-3/p145trkC- and NGF/p140trkA-induced Signaling Differ in Early Responses.
Comparison of the effects of NT-3 on SH-SY5Y/trkC cells with those of NGF on SH-SY5Y/trkA cells revealed distinct differences with respect to induced phenotype. Principally, this could be explained by quantitative and/or qualitative differences between p145trkC- and p140trkA-mediated signaling. Although the receptor levels appeared to be in the same concentration range in the tested trkC- and trkA-transfected clones, the induced receptor autophosphorylation differed considerably, as shown in Fig. 3Citation . To test whether this difference could explain the differences in the downstream effects induced by the cognate ligand, the transfected cells where stimulated with increasing concentrations of ligands to reach an experimental condition where the receptor autophosphorylation levels are similar in the NT-3-treated SH-SY5Y/trkC and NGF-treated SH-SY5Y/trkA cells. To this end, serum-starved SH-SY5Y/trkC and SH-SY5Y/trkA cells were stimulated for 10 min with NT-3 or NGF (Fig. 7)Citation . In both cell systems a dose-dependent increase in autophosphorylation was observed. However, when comparing NT-3/p145trkC- and NGF/p140trkA-induced receptor phosphorylation, 100 ng/ml NT-3 seemed to result in a tyrosine phosphorylation of a magnitude between that induced by 10 and 30 ng/ml NGF (Fig. 7Citation , top panel, arrow and *, respectively).



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  		Fig. 7. Receptor phosphorylation at increasing NT concentration. Western blot analysis of Trk phosphorylation status in NT-3-treated SH-SY5/trkC (clone 3:1) and NGF-treated SH-SY5/trkA cells. Serum-starved cells were incubated at 37°C for 10 min with increasing concentrations of NT-3 or NGF. After immunoprecipitation with anti-Pan-Trk antiserum and SDS-PAGE, the filter was incubated first with an anti-phosphotyrosine ({alpha}-PY) antibody and then reprobed with anti-Pan-Trk antiserum. The positions of p145trkC and p140trkA are indicated by an arrow and *, respectively. IP, immunoprecipitation; IB, immunoblot.

 
To investigate early NT-3/p145trkC- and NGF/p140trkAinduced transcriptional events at the ligand concentrations tested above, the expression profiles of a set of immediate-early genes, known to be regulated by NTs (34) , were analyzed. The temporal expression of the genes was established. As demonstrated in Fig. 8ACitation ,c-fos expression was strongly induced by 100 ng/ml NGF in SH-SY5Y/trkA cells, whereas p145trkC activation induced a barely detectable increase in c-fos expression. In contrast, c-jun and NGFI-A mRNA levels increased in both cell systems after NT stimulation, although this response was stronger in NGF-stimulated SH-SY5Y/trkA cells (Fig. 8A)Citation . Thus, early transcriptional events induced by NT-3/p145trkC and NGF/p140trkA differed in both activation pattern and intensity. The NT dose dependency of these events was then tested at 60 min of stimulation, the approximate time of maximum immediate-early gene activation (Fig. 8B)Citation . As for Erk 1/2 phosphorylation (Fig. 3DCitation and below), 10 ng/ml of ligand proved sufficient to elicit a maximum response. Again, the clear-cut c-fos induction seen in SH-SY5Y/trkA cells was contrasted by a virtual absence of c-fos induction in the tested trkC-transfected clones (Fig. 8B)Citation .



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  		Fig. 8. Northern blot analysis of the expression of immediate-early genes in NT-3-stimulated SH-SY5Y/trkC and NGF-stimulated SH-SY5Y/trkA cells. A, SH-SY5Y/trkC (clone 3:1) and SH-SY5Y/trkA cells, grown in serum-containing medium, were stimulated with 100 ng/ml of NT-3 and NGF, respectively, for the indicated time periods. Total RNA (15 µg) was analyzed for expression of c-jun (2.7 and 3.2 kb), c-fos (2.2 kb), and NGFI-A (3.0 kb). B, SH-SY5Y/trkC (clones 3:1 and 3:2) and SH-SY5Y/trkA cells, grown in serum-containing medium, were stimulated for 60 min with increasing concentrations of NT-3 or NGF. Total RNA (15 µg) was analyzed for expression of c-fos (2.2 kb) and NGFI-A (3.0 kb). In both panels, GAPDH (1.5 kb) mRNA levels were used as a reference for the amount of loaded RNA.

 
To analyze whether the magnitude of receptor-induced activation of specific downstream target proteins would reflect the difference in receptor phosphorylation and activation of immediate-early genes, activation of Ras and phosphorylation of Erk 1/2 and Akt/PKB was analyzed. For these experiments, cells were stimulated for 5 min by their cognate ligands. In both SH-SY5Y/trkC and SH-SY5Y/trkA cells, Ras could be activated by the respective NT to a similar extent (Fig. 9A)Citation , as monitored using the specific interaction between Ras-GTP and the RBD of Raf-1 (35 , 36) . Also, the phosphorylation of Erk 1/2 and Akt/PKB (appearance of a protein doublet; Fig. 9Citation A, arrows) was induced in these cell clones after NT stimulation. However, the Erk 1/2 phosphorylation level in NT-3-stimulated SH-SY5Y/trkC cells was substantially higher than in the NGF-stimulated SH-SY5Y/trkA cells, and Akt/PKB phosphorylation was also more pronounced in the NT-3-stimulated SH-SY5Y/trkC cells. To assess Erk activation at different ligand concentrations, cell lysates from the experiment shown in Fig. 7Citation were analyzed for Erk 1/2 phosphorylation status (Fig. 9B)Citation . Erk 1/2 were phosphorylated already at 10 ng/ml ligand, with a higher degree of phosphorylation upon NT-3-p145trkC-mediated than upon NGF-p140trkA-mediated signaling. These results are in line with the p140trkA- and p145trkC-induced Erk 1/2 phosphorylation data in SH-SY5Y/trkC cells shown above in Fig. 3DCitation , where p145trkC appeared more potent than endogenous p140trkA at similar levels of receptor phosphorylation (Fig. 3C)Citation .



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  		Fig. 9. NT-induced activation of Ras and phosphorylation of Erk 1/2 and Akt/PKB in SH-SY5Y/trkC and SH-SY5Y/trkA cells. A, cells were stimulated with 100 ng/ml NT-3 or 100 ng/ml NGF for 5 min. Lysates were subjected to affinity precipitation with GST-Raf-RBD. The resulting precipitates and corresponding whole-cell lysates were separated on a 10% SDS-PAGE gel. The filter with precipitated material was immunoblotted with an anti-Ras antibody. The filter with whole-cell lysate was immunoblotted with the same anti-Ras antibody, an anti-Akt/PKB, and an anti-phospho-Erk 1/2 antiserum. Arrows, an Akt/PKB protein doublet; the top band is phosporylated Akt/PKB. B, Erk 1/2 phosphorylation at increasing NT concentration. Serum-starved SH-SY5Y/trkC (clone 3:1) and SH-SY5Y/trkA cells were stimulated with increasing concentrations of NT-3 or NGF for 10 min. Whole-cell lysate proteins were separated on a 10% SDS-PAGE gel, followed by immunoblotting with anti-phospho-Erk 1/2 antiserum and reprobing with an anti-Pan-Erk antibody. IP, immunoprecipitation; IB, immunoblot.

 
Discussion

Here we report that stable expression of trkC in SH-SY5Y neuroblastoma cells conferred responsiveness to NT-3, resulting in neurite outgrowth and increased expression of neuronal marker genes such as NPY and GAP-43. However, both early and late responses differed considerably compared with NGF-induced events in trkA-transfected SH-SY5Y cells. Despite a modest NT-3-induced autophosphorylation of p145trkC, activation of specific downstream events (particularly Erk 1/2) was more pronounced than in NGF-treated SH-SY5Y/trkA cells. In contrast, c-fos expression was not induced in NT-3-stimulated trkC-transfected cells, whereas NGF activation of p140trkA resulted in a prominent c-fos induction. Finally, activation of either NT receptor led to morphological differentiation and increased expression of some neuronal differentiation markers, but an increase in norepinephrine and enrichment of synaptophysin occurred only in the NGF-treated SH-SY5Y/trkA cells. On the basis of our data, we conclude that activation of p145trkC and p140trkA, respectively, results in the transduction of slightly different signals and that the NT-3-treated SH-SY5Y/trkC cells become less differentiated than the NGF-treated SH-SY5Y/trkA cells. In addition, stimulation of SH-SY5Y/trkC cells with NT-3 did not induce growth arrest or increased expression of p140trkA, which contrasts with what has been observed in normal sympathetic neuroblasts in vitro (23, 24, 25, 26) .

Analysis of the receptor levels in the two different cell lines with anti-Pan-Trk antiserum suggested that the expression of p140trkA in SH-SY5Y/trkA cells is somewhat higher than the p145trkC expression in SH-SY5Y/trkC cells. A more obvious difference was observed when the induced autophosphorylation levels were compared. One might therefore argue that low p145trkC expression and/or receptor autophosphorylation could explain the difference in sympathetic neuronal phenotype between differentiated SH-SY5Y/trkC and SH-SY5Y/trkA cells. However, several of our observations speak against this interpretation. Even at an NGF concentration of 10 ng/ml, a concentration where tyrosine phosphorylation of p140trkA in SH-SY5Y/trkA cells equals or is surpassed by that of NT-3-induced p145trkC phosphorylation in SH-SY5Y/trkC cells, c-fos is clearly induced by NGF. This was in striking contrast to the virtual absence of c-fos induction in SH-SY5Y/trkC cells simulated by 10–100 ng/ml NT-3. There are also examples of activation more readily detected after NT-3/p145trkC activation. For instance, over the entire ligand concentration range tested, Erk 1/2 phosphorylation was consistently more prominent upon NT-3 stimulation of SH-SY5Y/trkC cells than upon NGF stimulation of SH-SY5Y/trkA cells. Taken together, the differences between differentiating SH-SY5Y/trkA and SH-SY5Y/trkC cells appear not explainable simply by differences in receptor and/or Trk phosphorylation levels. In concordance with the recent results showing that activation of p145trkB and p140trkA, respectively, induce different phenotypes in neuroblastoma cells (37) , we conclude that there are qualitative differences in the signaling mechanisms induced by p145trkC and p140trkA, presumably explaining the phenotypic differences observed.

Progenitor cells of the sympathetic nervous system give rise to three major cell types: neurons, chromaffin cells of the adrenal gland and paraganglia, and small intensely fluorescent cells. Both trkA and trkC are expressed in all of these cell types during development (1 , 9) . Neuroblastoma cells have sympathetic progenitor cell characteristics. Consequently, introduction of either trkC or trkA into neuroblastoma cells might principally generate cells differentiating along a sympathetic chromaffin or small intensely fluorescent lineage, as well as a neuronal lineage, upon NT treatment. The major neurotransmitter produced by cells of the sympathetic neuronal lineage is norepinephrine. On the basis of norepinephrine levels as well as on morphological changes and NPY and GAP-43 expression, we conclude that stimulation of SH-SY5Y/trkC cells with NT-3 results in cells with neuronal characteristics (morphology and increased GAP-43 expression) and retained sympathetic phenotype (increased NPY expression and maintained synthesis of norepinephrine). In comparison with the corresponding SH-SY5Y/trkA cells stimulated with NGF, the differentiated SH-SY5Y/trkC cells appeared less mature. The morphology was not as well developed, the NPY and GAP-43 mRNA levels were slightly lower, and the levels of norepinephrine and synaptophysin did not increase upon NT-3 stimulation. This lack of increase in norepinephrine might suggest that these cells differentiate toward a noncatecholaminergic phenotype. Indeed, in chicken sympathetic neurons, NT-3 promotes cholinergic differentiation (38) . However, expression of the vesicular acetylcholine transferase gene, a cholinergic marker gene, did not increase in NT-3-treated SH-SY5Y/trkC cells (data not shown).

Highly differentiated, low-stage neuroblastomas regularly express trkA and/or trkC. Neuroblastoma cell lines are typically established from high-stage tumors that do not express these receptors or express them at levels where complementary treatment is needed to elicit a NT response. There are, however, ways to induce p140/145trk receptor expression; SH-SY5Y/wt cells have been demonstrated to express functional levels of p140trkA when treated with mitogenic inhibitors (12 , 39) , and previous transfection experiments have shown that the capacity to differentiate in response to NGF is partially restored after introduction of exogenous trkA (10, 11, 12) . The SH-SY5Y/trkC cells described here differentiated in response to NT-3, but the resulting phenotype was lacking a number of important functional characteristics, and expression of trkA was not induced. Thus, despite the forced expression of a relevant NT receptor, a fully mature neuronal phenotype could not be reached. Together with the absence of experimental evidence of structural alterations in the trkA and trkC loci in highly malignant neuroblastomas, this suggests that low trkA and trkC expression is a consequence rather than the cause of the immature phenotype of these tumors.

Upon stimulation with NGF, a small effect on neurite outgrowth and p140trkA phosphorylation could be observed in SH-SY5Y/wt cells cultured in serum-containing medium (40 , 41) . A similar effect was also detected in the NGF-stimulated SY5Y/trkC cells. In these cells, NGF induced a modest neurite outgrowth, but we were unable to detect a corresponding increase in expression of markers of neuronal differentiation. This could suggest that neurite outgrowth requires activation of fewer receptor molecules and/or phosphorylation of few but specific tyrosine residues than needed for a complete differentiation response. Studies using PC-12 cells transfected with mutant trkA could support this conclusion, because they suggest that only activation of the Suc1-associated neurotrophic factor target pathway, thus far, is necessary for initiation of p140trkA-induced neurite outgrowth (42 , 43) . Other studies have further shown that neurite outgrowth can be induced separately from induction of a full differentiation program (44) . The fact that the marked NT-3-induced p140trkA phosphorylation in SH-SY5Y/trkA cells did not lead to neurite outgrowth is difficult to explain. However, it has been shown in sympathetic neurons that NT-3 stimulation of p140trkA, even when comparing conditions resulting in similar p140trkA phosphorylation levels, elicits a weaker response than that induced by NGF (30) . Observations like these leave many unanswered questions regarding the roles of NT-3, trkC, and trkA during normal sympathetic development.

trkC expression in mouse sympathetic neuroblasts of the superior cervical ganglion during normal development is detectable at embryonic day 11.5 (E11.5) and remains high at E15.5 (27) . In these neuroblasts, trkA expression is first detected at E13.5 and becomes robust at E15.5. Although the trkA expression stays high throughout postnatal development, the trkC expression diminishes significantly between E15.5 and birth (27) . Also, expression of trkC precedes trkA expression in cultured sympathetic neuroblasts. Whatever function trkC has in the development of the sympathetic nervous system, it is most likely exerted at an embryonal stage when the neuroblasts still have a capacity to proliferate and are not fully mature (23, 24, 25, 26) . The data presented here are in accordance with these findings, because NT-3-treated SH-SY5Y/trkC cells do not become fully differentiated. One can further speculate about NT-3/p145trkC function in sympathetic neuroblasts that is not fulfilled by NGF/p140trkA in vivo. In mice lacking trkC, the number of sympathetic neurons is normal, which could suggest that the receptor is not essential for survival of these neurons or their progenitor cells (27) . However, there are probably mechanisms compensating for the lack of p145trkC signaling that explain this finding, and we assume that trkC also has a role in sympathetic differentiation. The data presented here suggest that the signaling from p140trkA and p145trkC in a neuroblastoma cell context differs slightly and results in cells with different phenotypes. The NT-3-induced Akt/PKB phosphorylation in the SH-SY5Y/trkC cells might serve as a clue to one role of p145trkC during sympathetic development. The Akt/PKB kinase is dependent on phosphatidylinositol 3,4-biphosphate, a lipid generated in response to activation of phosphatidylinositol 3-kinase (45) . Activation of this pathway is sufficient for the survival of PC-12 cells, and cultured rat cerebellar neurons survive when Akt/PKB is overexpressed (46 , 47) . Thus, one function for p145trkC could be to transduce cell survival signals, and NT-3 has indeed been shown to support proliferation of cultured sympathetic precursor cells by promoting their survival (26 , 48) .

The exact mechanism by which NT-3 exerts its effects on cells expressing both trkA and trkC will not be trivial to establish. For instance, in primary cultured postnatal rat sympathetic neuroblasts, it is well-documented that NT-3 binds p140trkA and induces neurite outgrowth via this receptor and not via p145trkC, which is expressed at low levels (30) . The SH-SY5Y cell clones described in this report offer a system of human cells in which p140trkA- and p145trkC-evoked signal transduction pathways and phenotypic outcome can be studied in a sympathetic neuronal context. A better understanding of these signaling events has the potential to unravel defects causing differentiation arrest in neuroblastoma cells.

Materials and Methods

Cell Culture.
SH-SY5Y/wt cells, a subclone of the non-N-myc-amplified human neuroblastoma cell line SK-N-SH (49) kindly provided by Dr. June Biedler (Sloan Kettering Institute, New York, NY), and stably transfected cell clones based on SH-SY5Y/wt were used. These were trkA-transfected SH-SY5Y/trkA cells, clone 6:2 (10) , and trkC-transfected SH-SY5Y/trkC cells, clones 3:1 and 3:2 (see below). All cells were cultured in Eagle’s Minimum Essential Medium with the following additives: 10% FCS, 100 IU/ml penicillin V, and 100 µg/ml streptomycin (Eagle’s/FCS). To induce differentiation, the following growth factors and substances were used: bFGF (Promega), IGF-I (kind gift from Pharmacia), NT-3 (kind gift from Regeneron Pharmaceuticals, purchased from PeproTech), NGF (Promega), and TPA (Sigma). For serum-free conditions, the cells were plated and grown for 1 day in serum-containing medium and washed twice with RPMI 1640 before addition of serum-free medium (RPMI 1640 containing 30 nM sodium selenite, 10 nM hydrocortisone, 30 µg/ml transferrin, and 10 nM ß-estradiol with added antibiotics as described above). Media, serum, and antibiotics were from Life Technologies, Inc.

Generation of Stably trkC-expressing SH-SY5Y Cells.
One day before transfection, cells were plated in 100-mm tissue culture dishes (2 x 106 cells/dish). Three h prior to transfection, fresh Eagle’s/FCS was added to the dishes. Fifteen µg of pFL20, i.e., the pMEXneo plasmid with an 2500-bp insert of full-length porcine trkC cDNA (18) , a kind gift from Dr. Mariano Barbacid (Centro Nacional de Investigaciones Oncológicas Carlos III, Madrid, Spain), were diluted with water to a volume of 440 µl, mixed with 62 µl of 2 M CaCl2, and precipitated by adding it dropwise into 500 µl of a solution containing 21 mM HEPES (pH 7.1), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM dextrose. The DNA was allowed to precipitate for 30 min at room temperature before the dropwise addition to the dishes. Sixteen h after transfection, the cells were washed once with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.3), and fresh complete medium was added. Selection with 600 µg/ml G-418 (Geneticin; Life Technologies) was initiated 48 h after transfection. A few surviving cells formed clonal colonies that, when grown to a size of ~100 cells, were transferred to separate dishes with the use of a small pipette. Fresh G-418 containing Eagle’s/FCS was added twice a week until the stable clones had been identified, collected, and expanded. G-418 selection was subsequently performed every fourth week. As a control, SH-SY5Y/wt cells were stably transfected with the empty pMEXneo vector using the same protocol (clone 1:9).

Quantification of Morphological Changes.
Cells were seeded on coverslips in 35-mm dishes (75,000 cells/dish). The following day, cells were transiently transfected with 2 µg of the pEGFP-N1 vector (Clontech Laboratories, Inc.) using Lipofectamine (Life Technologies) as described by Zeidman et al. (44) . After 8 days, cells were fixed in 4% paraformaldehyde in PBS for 4 min, mounted on microscopy slides using PVA-DABCO solution (9.6% polyvinyl alcohol, 24% glycerol, and 2.5% 1,4-diazabicyclo[2.2.2]octane in 67 mM Tris-HCl, pH 8.0), and used for morphological evaluation. The cells were scored positive for long neurites if these extended more than two cell body diameters. Two hundred cells were counted on each coverslip.

Assessment of Cell Number and Thymidine Incorporation.
Cells were plated in Eagle’s/FCS at a density of 7500 cells/well in 96-well culture dishes. Factors used were added in 50 µl of medium prior to the addition of cells in the same volume. Cells were grown for 72 h, and the amount of viable cells was analyzed measuring the conversion of the tetrazolium salt MTT to formazan (CellTiter 96; Promega). To determine whether long-term NT-3-treated SH-SY5Y/trkC cells still have the capability to synthesize DNA and divide, cells were grown in 35-mm dishes in serum-containing medium for 8 days with or without 100 ng/ml of NT-3, with medium changed day 4. In the last 24 h, the cells were exposed to [3 H]thymidine (1.48 kBq/ml). The cells were then washed in PBS and fixed in methanol:acetic acid (3:1), and film emulsion was added to the dishes. Labeled nuclei were counted after 7 days of exposure.

Catecholamine Analysis.
Cells were plated in Eagle’s/FCS at a density of 106 cells/10-cm dish, and after 24 h, the cells received fresh medium with or without NTs. Four days later, the cells were washed and detached in ice-cold PBS and pelleted at 500 x g for 5 min at 4°C. The pellet was resuspended in 0.5 M trichloroacetic acid and subjected to centrifugation at 21,000 x g for 5 min at 4°C. The protein content of the pellets was, after resuspension in 1 M NaOH, determined according to Bradford (50) . Prior to further analysis, the pH of the supernatants was adjusted to pH 4 with NaOH, and internal standard (3,4-dihydroxynorepinephrine) was added. The samples were extracted and derivatized with 1,2-diphenylethylenediamine as described (51) . Samples were thereafter analyzed with high-performance liquid chromatography using a 25 x 2-mm precolumn (Perisorb RP 18; 30–40 µm particle diameter) and a 250 x 4.6-mm column (Hypersil ODS; 5 µm particle diameter) using 0.05 M sodium acetate (pH 7.0):acetonitrile:methanol (5:4:1) as mobile phase. The fluorescence was detected with either a Shimadzu RF-10A XL or a Jasco Fluorescence Detector 821, and the signal was integrated with a Shimadzu C-R3A integrator.

Isolation of RNA and Northern Blot Analysis.
The guanidine-isothiocyanate/phenol-chloroform extraction method described (52) was used for isolation of total cellular RNA. Fifteen µg of RNA were electrophoretically separated on an 1% agarose-formaldehyde gel and blotted onto a nylon membrane (Hybond-N; Amersham Pharmacia Biotech) using the capillary blot technique. Probes were labeled with [32P]dCTP using an oligonucleotide labeling kit (Amersham Pharmacia Biotech). Filters were prehybridized for 1 h in 5x SSC, 5x Denhardt’s, 0.5% SDS. Denatured probe was added, and hybridization was performed overnight. Hybridized filters were washed, 15 min in 1x SSC, 0.1% SDS and then 15 min in 0.1x SSC, 0.1% SDS. All hybridization and washing steps were performed at 65°C. Washed filters were exposed to X-ray film (Agfa-Gevaert) at -70°C in the presence of intensifier screens. Hybridized RNA was, in some cases, visualized with the help of a Molecular Dynamics PhosphorImager. The following probes of human origin were used: trkA (18) , GAPDH (53) , NPY (54) , GAP-43 (55) , c-fos (56) , c-jun (ATCC 63026), and NGFI-A (57) . A porcine trkC probe (17) was also used.

Trk Protein Phosphorylation Studies.
Cells were plated overnight in Eagle’s/FCS at a density of 5 x 106 cells/10-cm cell culture dish and then serum-starved in serum-free medium for 24 h prior to stimulation by NTs. To synchronize receptor autophosphorylation and to reduce unspecific phosphorylation, cells were, in the initial experiments, kept on ice with or without NT added for 30 min and thereafter incubated at 37°C for 10 min. Similar results were, however, obtained when the incubation on ice was excluded; and in later experiments, the ice incubation step was omitted. The cells were washed twice with ice-cold PBS in the presence of protease inhibitors (Complete Protease Inhibitor; Roche Molecular Biochemicals) and lysed in 10 mM Tris-HCl (pH 7.2), 160 mM NaCl, 1% Triton X-100, 1% sodium desoxycholate, 0.1% SDS, 1 mM EGTA, 1 mM EDTA, 1 mM Na3VO4 in the presence of the same protease inhibitors. For immunoprecipitation, equal amounts of protein from two dishes [1–3 mg, determined according to Bradford (50) ] was incubated for 1 h at 4°C with 2 µg of anti-Pan-Trk antiserum (Santa Cruz Biotechnology, Inc.; sc-139) and for another hour at 4°C with protein G-Sepharose (Amersham Pharmacia Biotech). After washing with ice-cold lysis buffer, bound proteins were eluted and separated by 5% SDS-PAGE and blotted onto Hybond C Extra filters (Amersham Pharmacia Biotech). The anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology) was used as a primary antibody, followed by horseradish peroxidase-coupled secondary antibodies (Amersham Pharmacia Biotech). Filters with immunoprecipitated material were, after a brief wash, reprobed with the sc-139 anti-Pan-Trk antiserum. Immunoreactivity was detected using the enhanced chemiluminescent method.

Erk 1/2 and Akt/PKB Phosphorylation and Ras Pull-Down Assays.
Approximately 10 x 106 cells plated in a 10-cm cell culture dish were serum-starved for 24 h prior to stimulation by NTs. Cells were incubated in a cell incubator for 5 min, with or without added NTs, washed once with ice-cold PBS, and lysed in 50 mM Tris (pH 7.5), 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 15 mM MgCl2, 1 mM DTT, 10 mM benzamidine, and 10 µg/ml of aprotinin, leupeptin, and pepstatin A. Protein content was determined with Bio-Rad Protein Assay 500-0006 (Bio-Rad, Hercules, CA). Equal amounts of protein were incubated for 30 min at 4°C with a glutathione S-transferase-RBD fusion protein (36) immobilized on glutathione-Sepharose. After washing with ice-cold PBS with 0.1% Triton X-100 and 10 mM MgCl2, bound proteins were eluted, separated by 12.5% SDS-PAGE, and blotted onto an Immobilon-P filter (Millipore, Bedford, MA). The filter was then incubated with a monoclonal anti-Ras antibody (Transduction Labs; R02120). In parallel, total cell lysate was analyzed for Akt/PKB and phosphorylated Erk immunoreactivities, using anti-Akt/PKB and anti-phospho-Erk 1/2 (Thr202/Tyr204) antiserum (New England Biolab; nos. 9272 and 9101S). For separate analysis of Erk 1/2 phosphorylation, serum-starved cells were incubated in a cell incubator for 10 min, with or without added NTs. Cells were lysed, protein content was determined, SDS-PAGE was run, and transfer was performed as described for Trk protein phosphorylation studies. The filter was analyzed using the anti-phospho-Erk 1/2 antiserum detailed above. After a brief wash, the filter was reprobed with an anti-Pan-Erk antibody (Transduction Labs; E17120).

Isolation of Growth Cones.
SH-SY5Y/trkA and SH-SY5Y/trkC cells were harvested after 4 days of treatment with NT-3 and NGF under serum-free conditions. After gentle homogenization, the cell homogenate was fractionated, and the growth cone and cell body fractions were saved as described previously (33) . Equal amounts of protein from each fraction were separated by 10% SDS-PAGE, and an anti-synaptophysin antibody (Dako) was used as primary antibody.

Acknowledgments

We thank June Ljungberg and Åsa Lindeheim for technical assistance, Ulf Rosén and May-Lill Svensson for catecholamine analysis, Ruth Palmer for valuable discussions, Mariano Barbacid for the kind gift of the pFL20 plasmid, Regeneron Pharmaceuticals for kindly providing NT-3, and Pharmacia for the kind gift of IGF-I.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Swedish Cancer Society, the Children Cancer Foundation of Sweden, HKH kronprinsessan Lovisas förening för barnasjukvård, Hans von Kantzows stiftelse, Crafoordska stiftelsen, Inga och John Hains stiftelse, the Swedish Society for Medical Research, and Malmö University Hospital and its research funds. Back

2 To whom requests for reprints should be addressed, at Department of Laboratory Medicine, Lund University, University Hospital MAS, Entrance 78, S-205 02 Malmö, Sweden. Phone: 46-40337403. Fax: 46-40337322; E-mail: sven.pahlman{at}molmed.mas.lu.se Back

3 The abbreviations used are: NT, neurotrophin; NGF, nerve growth factor; bFGF, basic fibroblast growth factor; IGF-I, insulin-like growth factor I; NPY, neuropeptide Y (tyrosine); GAP, growth-associated protein; CNTF, ciliary neurotrophic factor; Erk, extracellular signal-regulated kinase; RBD, Ras binding domain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back

Received for publication 1/24/00. Revision received 11/ 8/00. Accepted for publication 11/27/00.

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