Engagement of endogenous ganglioside GM1a induces tyrosine phosphorylation involved in neuron-like differentiation of PC12 cells

Masafumi Kimura1, Kazuya I.-P. Jwa Hidari1, Takashi  Suzuki, Daisei Miyamoto and Yasuo Suzuki2

Department of Biochemistry, University of Shizuoka Pharmaceutical Sciences, 52-1 Yada, Shizuoka-shi, Shizuoka 422-8526, Japan

Received on October 4, 2000; revised on December 6, 2000; accepted on December 15, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Using the cholera toxin B subunit (CTB) that specifically binds to ganglioside GM1a on the plasma membrane, we investigated intracellular signaling mediated by endogenous GM1a involved in neuronal differentiation of PC12 cells. The treatment with CTB induced morphological alternations of PC12 cells, such as augmentation of the cell body, neurite extension, and branched spikes of tips of neurites. The neurite extension induced with CTB was strongly suppressed by the pretreatment of tyrosine kinase inhibitors in a dose-dependent manner. Western blotting analysis showed that CTB induced tyrosine phosphorylation of several cellular proteins with molecular masses around 120, 70, and 45–40 kDa in PC12 cells. Some of the proteins identified were extracellular-signal regulated kinase (ERKs) (ERK1 and ERK2). The peak activation of ERKs lasted for 60–90 min and gradually decreased thereafter.

Immunoprecipitation analysis demonstrated that the intracellular events induced with CTB are not related with the activation of Trk proteins, suggesting that signals evoked by ligation of endogenous GM1a are unique and distinct from those induced with exogenous GM1a.

Although the presence of a tyrosine kinase inhibitor, genistein, at a concentration of 10 µM diminished the neurite extension of PC12 cells induced with CTB, ERK activation was still observed. However, pretreatment with a MEK inhibitor, PD98059, abolished the activation of ERKs induced with CTB in a dose-dependent manner and only attenuated the morphological alternations of PC12 cells.

Considered together, we concluded that tyrosine phosphorylation induced with CTB was responsible for neuron-like differentiation of PC12 cells and that the MEK–ERK cascade is part of the biological signals mediated by endogenous ganglioside GM1a on PC12 cells.

Key words: ERK/ganglioside GM1a/PC12 cells/tyrosine phosphorylation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycosphingolipids consisting of sugar moieties and ceramide are ubiquitously distributed in eukaryotic cells (Hakomori, 1990Go). Gangliosides, sialic acid–containing glycosphingolipids, exist in the nervous system relatively more than in other systems, such as the immune system. They are localized in various types of neurons as well as glial cells (Yu and Iqbal, 1979Go).

In ontogenesis, the distribution and composition of gangliosides was markedly altered in neural cells (Seybold and Rahmann, 1985Go). Based on these findings, gangliosides are thought not only to be structural constituents of the plasma membrane of the cells but also to have important functions in the nervous system (Sheikh et al., 1999Go). Indeed, both protective effects and curative effects were observed when gangliosides were administrated into animals whose nervous systems were injured (Ala et al., 1990Go; Geisler et al., 1991Go). Recently, mice lacking of complex gangliosides, including GM1a, were generated (Takamiya et al., 1996Go). Those mice were normally developed and born. Phenotypes of the mutant mice were apparently similar to those of wild type, indicating that endogenous complex gangliosides are not indispensable for normal development of mice. The biological (physiological) activities of endogenous gangliosides are still controversial.

In the past decade, many studies have reported that exogenous gangliosides exert trophic effects on neuronal cells or induce differentiation to cultured cell lines, such as neuroblastoma and glioma cells. Especially in terms of the effects on primary-cultured neurons, exogenous gangliosides demonstrated a modulation of the axon potential. Recently, it was reported that exogenous gangliosides enhanced tyrosine phosphorylation induced with nerve growth factor (NGF) and that the treatment with ganglioside GM1a augmented NGF-mediated differentiation of PC12 cells (Mutoh et al., 1993Go). The effect of exogenous GM1a was associated with the activation of Trks, receptors for NGFs. In pharmacological studies, administration of gangliosides promoted regeneration of neurofilaments of mice whose nerve codes were surgically dissected (Itoh et al., 1999Go). Other groups reported that a treatment of ganglioside GM1 recovered nonhuman primates in which Parkinsonism was experimentally induced with MPTP (Schneider et al., 1992Go) and that administration of GM1a exerted therapeutic effects on Alzheimer’s disease (Ala et al., 1990Go).

However, clinical applications of gangliosides into animals, especially humans, are still very difficult because of their unique properties, such as insolubility and immunogenicity. Ligation of endogenous gangliosides, which mimic the effect of exogenous gangliosides, is probably an alternative and useful approach to remedy injury and disorders in animals. Several studies addressing the functions of endogenous gangliosides, especially GM1a, were performed using specific probes for gangliosides, such as the cholera toxin B subunit (CTB) for ganglioside GM1a (Fishman, 1982Go; Sixma et al., 1991Go). Although CTB is not a physiological substance, it exerted biological effects against lymphocytes (Dixon et al., 1987Go) and various types of cultured cell lines, such as fibroblast, epithelial, neuroblastoma, and glioma cells (Spiegel and Fishman, 1987Go; Buckley et al., 1990Go; Masco et al., 1991Go). In fibroblast and neuroblastoma cells, CTB induced intracellular calcium mobilization that may be required for trophic effects and neuronal differentiation (Carlson et al., 1994Go; Buckley et al., 1995Go). These findings suggested that intracellular signals mediated by endogenous ganglioside GM1a on the plasma membranes are responsible for some cellular functions. However, characterizations of biological signals transduced through endogenous GM1a on the plasma membrane are not well defined.

In the present study, we newly identified that tyrosine phosphorylation and MEK–extracellular-signal regulated kinase (MEK–ERK) activation induced by engagement of ganglioside GM1a on the cell surface was involved in neuron-like differentiation of PC12 cells.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CTB altered the morphology of PC12 cells
Figure 1 shows that the microscopic images of morphology of PC12 cells at 48 h after stimulation with CTB in the presence or absence of the tyrosine kinase inhibitor genistein. When PC12 cells were stimulated with CTB, we observed morphological alternations like NGF-differentiated PC12 cells, such as protuberance of a cyton, neurite extension, and branched spikes of tips of neurites (Greene and Tischler, 1976Go).



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Fig. 1. Tyrosine kinase inhibitor (genistein) blocks morphological alterations of PC12 cells induced with CTB. PC12 cells were treated with genistein for 30 min at the indicated concentrations, followed by the incubation with CTB or NGF for 48 h. ctrl. (control), not stimulated with either CTB or NGF.

 
Next, we investigated whether the neuron-like differentiated PC12 cells expressed neuron-associated proteins. We determined the expression of MAP2B, one of the microtubule-associated proteins (MAPs) and expressed in neuronal cells (Kindler et al., 1990Go). The cellular expression of this protein was strongly enhanced by stimulation with CTB, as compared with nonstimulated cells (data not shown). This response was very similar to that of PC12 cells differentiated with NGF.

The neurite extension induced with CTB was suppressed by the pretreatment of genistein in a dose-dependent manner (Figure 1). We observed a similar effect with the erbstatin analog, another tyrosine kinase inhibitor on neurite extension induced with CTB (data not shown). We also observed that both genistein and the erbstatin analog have cell toxicity to some extent. The number of surviving PC12 cells in the presence of CTB increased significantly more than that of untreated cells, suggesting that CTB appeared to protect the cells from the toxic effect of the tyrosine kinase inhibitors.

Taken together, it was suggested that CTB, like NGF, possesses a trophic activity in the survival of neurons or neuron-like differentiation and that the tyrosine phosphorylation was involved in neuron-like differentiation induced with CTB, in a similar manner to NGF.

Detection of CTB-binding gangliosides in PC12 cells
The above findings indicated that the interaction of CTB with molecules on the plasma membrane of PC12 cells triggered signaling events, such as tyrosine phosphorylation, followed by biological responses (morphological changes). Many studies have shown that CTB specifically bound to gangliosides, such as GM1a and GM1a-related gangliosides (Hidari et al., 1993Go), but not glycoproteins or proteoglycans. We next investigated the reactive substances with CTB in PC12 cells.

Lipids extracted from PC12 cells were applied onto thin-layer chromatography (TLC) plates and subjected to binding-assay using CTB. CTB clearly bound to a component with a TLC mobility similar to GM1a from bovine brain (Figure 2A), indicating that the major lipid that reacted with CTB in PC12 cells had an identical carbohydrate structure to GM1a. Using a different solvent system for TLC development, we observed a minor component that reacted with CTB (Figure 2B, asterisk). In a previous study using the same solvent system, it was reported that a novel ganglioside with a slower mobility on a TLC plate was a GM1a-related ganglioside termed de-N-acetylated GM1a (Hidari et al., 1993Go). It was suggested that the minor lipid was de-N-acetylated GM1a. Both the reactive lipids in PC12 cells with CTB were resistant against alkaline hydrolysis. On the other hand, there were not other reactive lipids labile to the alkaline reagents (data not shown). We did not find any reactive substances with CTB in protein extracts from PC12 cells (data not shown).



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Fig. 2. Detection of reactive glycolipids in PC12 cells with CTB. Lane 1: ganglioside mixture from bovine brain (5 µg), lane 2: ganglioside GM1a (1 nmol), lane 3: total lipids from PC12 cells (5 x 105 cells). TLC-binding assay with CTB were performed as described in Materials and methods. (A) The TLC plate developed with CHCl3/MeOH/12 mM MgCl2 (5:4:1, v/v/v). (B) The TLC plate developed with CHCl3/MeOH/12 mM MgCl2/25% NH3 (50:40:7:3, v/v/v/v). *, unknown glycolipid(s).

 
These findings showed that the reactive substances in PC12 cells with CTB were GM1a ganglioside (including GM1a-related ganglioside, de-N-acetylated GM1a), but not other lipids or proteins.

Therefore, these findings suggested that the effect of CTB on morphological changes of PC12 cells resulted from some biological signals triggered by the interaction between CTB and GM1a on the plasma membrane.

CTB-induced protein tyrosine phosphorylation in PC12 cells
As mentioned above, we found that neurite extension in PC12 cells induced with CTB was strongly suppressed with tyrosine kinase inhibitors in a dose-dependent manner. Next, we investigated tyrosine phosphorylation events in PC12 cells stimulated with CTB by western blotting analysis.

Several cellular proteins in PC12 cells were detected with anti-phosphotyrosine antibody when the cells were treated with CTB (Figure 3). We observed that tyrosine-phosphorylated proteins appeared within 10 min and lasted up to 120 min post–CTB treatment of the cells. The protein(s) with molecular masses around 120 kDa had been phosphorylated before the stimulation. The level of phosphorylation of the 120 kDa protein(s) increased from 60 min posttreatment with CTB (Figure 3, upper arrow). CTB markedly induced tyrosine phosphorylation of protein(s) with molecular masses of 70 kDa within 10 min and still enhanced the phosphorylation level at 120 min poststimulation (Figure 3, middle arrow). It was also observed that tyrosine phosphorylation of protein(s) around 45 kDa increased to a much lesser extent and peaked at 90 min poststimulation with CTB (Figure 3, lower arrow). Considered with microscopic analysis using tyrosine kinase inhibitors, we concluded that tyrosine phosphorylation induced with CTB was involved in neuron-like differentiation of PC12 cells.



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Fig. 3. CTB induces tyrosine phosphorylation in PC12 cells. PC12 cells were incubated with CTB for the indicated times and then lysed with 1% SDS-containing Tris-buffered saline. The lysates were subjected to SDS–PAGE and western blotting with anti-phosphotyrosine antibody. Arrows indicated tyrosine-phosphorylated proteins induced with CTB.

 
Detection of phosphorylation of Trks induced with CTB in PC12 cells
It was reported that ganglioside GM1a bound to an NGF receptor, TrkB, and modulated the function (Ferrari et al., 1995Go). We examined whether ligation of endogenous ganglioside GM1a with CTB-induced tyrosine phosphorylation of Trks.

Figure 4A showed detection of tyrosine phosphorylated proteins immunoprecipitated with anti-Trk antibody. CTB significantly induced tyrosine phosphorylation of proteins from PC12 cells as described above. However, any signals in anti-Trk immunoprecipitates were not apparently detected with antiphosphotyrosine antibody even when the blotted membrane was exposed for long time (3 h). Anti-Trk antibody certainly immunoprecipitated Trk proteins from PC12 cell lysates. Those findings suggest that the signaling events evoked by engagement of endogenous GM1a with CTB is not dependent on tyrosine phosphorylation of Trks in PC12 cells.



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Fig. 4. CTB does not induce phosphorylation of Trk proteins in PC12 cells. Immunoprecipitation analysis was carried out as described in Materials and methods. (A) PC12 cells were incubated with (+) or without (–) CTB for 90 min and then lysed. The lysates were immunoprecipitated with anti-Trk antibody or normal mouse IgG. The detection of signals was carried out by chemiluminescence method. The X-ray film was exposed for 3 h. (B) blotted membrane was stripped and then reprobed with anti-Trk (A/B/C) antibody to confirm that Trk proteins were immunoprecipitated. Arrowhead, a signal derived from primary antibody.

 
Activation of ERK induced by CTB treatment
It was reported that NGF activated ERKs in differentiated PC12 cells. ERK classified into mitogen-activated protein kinase (MAPK) is well known as tyrosine-phosphorylated proteins when cells were exposed to various stimuli. In this study, although we detected subtle enhancement of tyrosine phosphorylation of the proteins around 45 kDa on SDS–PAGE gel, we examined whether CTB activates ERKs in PC12 cells using an antibody specific for phosphorylated ERKs.

As shown in Figure 5A, CTB, like NGF, induced ERK phosphorylation in PC12 cells. Double bands with molecular weights 44 and 42 kDa represented ERK1 and ERK2, respectively. The activation of ERKs reached to a peak at 60–90 min and gradually decreased thereafter. The time course of activation of ERKs was concomitant with that of the tyrosine phosphorylation of proteins with molecular masses of around 45 kDa (Figure 3). We confirmed a similar amount of ERK proteins blotted on the membrane with anti-ERK antibody (Figure 5B).



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Fig. 5. CTB induces phosphorylation of ERK in PC12 cells. (A) PC12 cells were incubated with CTB for the indicated times and then lysed. The lysates were subjected to SDS–PAGE and western blotting with anti-phospho-ERK1/2 antibody. (B) blotted membrane was stripped and then reprobed with anti-MAP kinase (ERK1/2) antibody to confirm similar amounts of ERK proteins were blotted on the membrane.

 
These findings indicated that at least two tyrosine-phosphorylated proteins, observed around 45 kDa, are active forms of ERK1 and ERK2, respectively.

Effect of tyrosine kinase inhibitor genistein on the activation of ERKs
By western blotting analysis, we examined the activation of ERK induced with CTB in the presence of the tyrosine kinase inhibitor genistein. As shown in Figure 6A, the activation of ERK1 and ERK2 induced with CTB was partially suppressed by pretreatment with genistein. Figure 6B showed that the same amounts of ERKs were blotted on the membrane.



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Fig. 6. Tyrosine kinase inhibitor (genistein) attenuates phosphorylation of ERK induced with CTB. (A) PC12 cells were pretreated with genistein for 30 min, followed by the incubation with CTB for 60 min and then lysed. The lysates were subjected to SDS–PAGE and western blotting with anti-phospho-ERK1/2 antibody. ctrl. (control), no treatment of cells with either CTB or genistein. CTB, incubation of cells with CTB for 60 min. (B) blotted membrane was stripped and then reprobed with anti-MAP kinase (ERK1/2) antibody.

 
The pretreatment of PC12 cells with 10 µM of genistein, which completely blocked neuron-like differentiation, was inadequate for abolishment of ERK activation.

Effect of PD98059 on neuron-like differentiation induced with CTB
We investigated the involvement of activation of the ERK cascade in neuron-like differentiation of PC12 cells induced with CTB. For this purpose, we used an inhibitor, PD98059, against MEK (MAPK/ERK kinase), which is located upstream of ERKs.

Figure 7 shows the microscopic images of PC12 cells by the treatment with CTB for 48 h in the presence or absence of PD98059.



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Fig. 7. MEK inhibitor (PD98059) attenuates morphological alterations of PC12 cells induced with CTB. PC12 cells were treated with PD98059 for 30 min at the indicated concentrations, followed by incubation with CTB or NGF for 48 h. ctrl. (control), no stimulation with either CTB or NGF.

 
Although the reduction of morphological alteration with PD98059 was observed in a dose-dependent manner, it only attenuated neuron-like differentiation of PC12 cells induced with CTB, but did not abolish the effect of CTB when the cells were pretreated with 10 µM of PD98059.

MEK inhibitor–abolished activation of ERKs induced with CTB
The activation of ERKs, which are physiological substrates of MEK, was determined. We examined the activation of ERKs induced with CTB in the presence or absence of PD98059. As shown in Figure 8, the activation of ERK1 and ERK2 induced with CTB was blocked by pretreatment with PD98059 in a dose-dependent manner. The treatment of cells with 10 µM of PD98059 decreased the MEK activation with CTB to the basal level.



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Fig. 8. MEK inhibitor (PD98059) blocks phosphorylation of ERK induced with CTB. PC12 cells were treated with PD98059 for 30 min, followed by the incubation with CTB for 60 min and then lysed. The lysates were subjected to SDS–PAGE and western blotting with anti-phospho-ERK1/2 antibody. ctrl. (control), no treatment of cells with MEK inhibitor.

 
These findings indicated that a cascade, MEK–ERK, is part of the signaling events involved in CTB-induced neuron-like differentiations and that other signaling molecules, including tyrosine-phosphorylated proteins, may exert additional biological effects on the morphological alterations of PC12 cells induced with CTB.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this study, we found that the interaction of CTB with GM1a on the cell surface caused tyrosine phosphorylation of several cellular proteins and activation of the MEK–ERK cascade. The resultant signals were associated with neuron-like differentiation of PC12 cells.

It is well known that Raf, one of the MAPKKKs, which is located upstream of MEK in a canonical ERK pathway, is activated by growth factor–mediated signaling complexes, including Shc, Grab-2, and Sos-1 (Seger and Krebs, 1995Go). In the canonical pathway, tyrosine phosphorylation caused by occupation of growth factor receptors with their ligands is sufficient for the activation of the MEK–ERK cascade. In our case, however, MEK–ERK activation induced with CTB was not entirely dependent on tyrosine phosphorylation, suggesting that other types of signaling molecules are involved. The MEK inhibitor PD98059 abolished the activation of ERK, but did not completely inhibit morphological alternations of PC12 cells induced with CTB (Figures 7 and 8). These findings suggested that the MEK–ERK pathway (MAP kinase cascade) is a part of the configuration of the neuron-like differentiation of PC12 cells induced with CTB. Possible candidates involved in CTB-induced signals in PC12 cells may be calcium signaling pathways. Indeed, CTB induced calcium mobilization that was related to cellular proliferation (Buckley et al., 1995Go). In addition, endogenous ganglioside GM1 modulates L-type calcium channel activity in neuroblastoma cells and release of Ca2+ from intracellular storage (Spiegel and Panagiotopoulos, 1988Go; Carlson et al., 1994Go). Further studies are required to elucidate the mechanisms of MEK–ERK activation as well as characterization of tyrosine-phosphorylated proteins induced with CTB.

A previous study showed that CTB (1 µg/ml) primed PC12 cells in the presence of lower concentrations of NGF (1 ng/ml) but did not induce any effects by itself (Li et al., 1998Go), suggesting that putative signals mediated by GM1a possibly cross-talk with NGF-mediated signals but are insufficient for induction of neuron-like differentiation of PC12 cells. In contrast, the present findings clearly demonstrated that CTB at a similar concentration is capable of differentiating the cells. We also found that treatment of the cells with NGF at lower concentrations (1 ng/ml) still induced morphological alterations in PC12 cells, indicating that PC12 cells, we used in the present study, may be a different sub-clone from previously used clones and more sensitive to either NGF or CTB.

Signaling pathways of NGF were well characterized (Ohmichi et al., 1993Go). Occupation of the NGF receptor TrkA triggers multiple signals inside cells, which finally attain activation of Raf, one of the MAPKKKs in the canonical MAP kinase cascade (Cobb and Goldsmith, 1995Go; Schaeffer and Weber, 1999Go). Our biochemical analyses of the CTB signals showed that gel profiles of tyrosine-phosphorylated proteins induced by CTB are similar to those by induced NGF and that CTB causes MEK–ERK activation like NGF signals (Powers et al., 1999Go). However, the biological nature of the CTB signals also appears to be similar to NGF, compared with NGF signals, in which most tyrosine phosphorylation of cellular proteins peaked within 10 min poststimulation (Maher, 1988Go; Vetter et al., 1991Go). The kinetics of tyrosine phosphorylation of proteins induced with CTB was much slower and longer than that with NGF. For example, the level of phosphorylation of the 120-kDa protein(s) increased from 60 min poststimulation, and phosphorylation of 70-kDa protein(s) lasted until 120 min. It was reported that exogenous ganglioside GM1a executed a neurotrophic effect through the activation of TrkA (Ferrari and Greene, 1996Go). In this study, we did not detect that stimulation with CTB induced the phosphorylation of either TrkA or TrkB (Figure 4). None of the Src family protein kinases, such as Src, Yes, Lyn, or Fyn, were apparently phosphorylated with CTB (data not shown). Taken together, we suppose that the biochemical properties of the CTB signals may differ from those of NGF signals, and that the CTB signals are unique and distinct from exogenous ganglioside GM1a signals in PC12 cells.

CTB bound to GM1a and/or GM1a-related gangliosides on the cell surface of PC12 cells, but not to any of the glycoproteins tested in the present study. Glycosphingolipids, including gangliosides, are predominantly distributed at the outer leaflet of the plasma membrane (Harder et al., 1998Go), meaning that gangliosides do not have any signaling domain in the cytoplasm or even any portions capable of interacting with cytosolic molecules. It is not clear how the interaction of surface GM1a with CTB transduces signals inside cells. Recently, several studies demonstrated that glycosphingolipids are indispensable components that form domains on the plasma membrane associated with outside–in signal transduction (Kotani et al., 2000Go). Some gangliosides are localized to a specific domain termed the caveolae on the plasma membrane (Wolf et al., 1998Go; Prinetti et al., 1999Go) and are associated with nonreceptor types of tyrosine kinases in a caveolae-like domain of the rat brain (Kasahara et al., 1997Go). In addition, incorporated ganglioside GM1a bound to TrkB and regulated its receptor function (Mutoh et al., 1995Go). The above findings suggest the possibility that the binding of their ligands to gangliosides, including GM1a, changes the potential of the microdomains on the plasma membrane and the resultant changes activate signaling molecules, such as receptor and/or nonreceptor types of tyrosine kinases.

In the present study, using CTB as a specific probe of ganglioside GM1a, we demonstrated that ligation of endogenous GM1a on the cell surface mimics the trophic effect on PC12 cells, like exogenous gangliosides (Ferrari et al., 1993Go, 1995) and biochemically identified unique signaling pathways responsible for the effect. We suppose that the unique signals by occupation of endogenous gangliosides with their specific ligands or probes may be applicable to remedy neuronal degeneration, such as Alzheimer’s and Parkinson’s disease. CTB is not a physiological substance. The most critical issue in future studies is to identify physiological counterparts to gangliosides, especially GM1a. Very recently, it was reported that galectin-1 was able to bind to ganglioside GM1a (Kopitz et al., 1998Go). In addition, administration of galectin-1 regenerated surgical amputated neurofilaments in rats (Horie et al., 1999Go). These findings suggest that galectin-1 is a physiological ligand to GM1a and help elucidates the physiological function of GM1a-mediated signals in the nervous system.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Culture of PC12 cells
PC12 cells (RCB0009) were obtained from Riken Cell Bank (Japan). The cells were maintained in Dulbecco’s modified Eagles medium (DMEM; Nissui, Japan) with 10% fetal calf serum (FCS; Sigma), 10% horse serum (Life Technologies, USA), 0.1% N-2 supplement (Life Technologies), 1000 U/ml penicillin (Life Technologies), 1000 µg/ml streptomycin (Life Technologies). The cells were cultured at 37°C in 5% CO2.

Preparation of the assay plates and PC12 cells
Collagen (Cellmatrix Type I-C, Nitta gelatin, Japan) was diluted with HCl solution (pH 3.0) to 0.3 mg/ml. Plates were coated with the collagen solution at room temperature for 12 h. After coated, the plates were washed three times with the culture medium. PC12 cells were then inoculated and cultured in at 37°C in 5% CO2 for 18 h.

The plates were rinsed with an assay medium consisting of DMEM/F12 mixture (1:1, v/v) (Life Technologies) containing 0.1% N-2 supplement, insulin-transferrin-selenium–X supplement (Life Technologies), and 20 µM progesterone (Wako Chemicals, Japan).

Pretreatment of PC12 cells with tyrosine kinase inhibitors or MEK inhibitor, followed by stimulation of CTB or NGF
Genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; 4', 5, 6-trihydroxy-isoflavone, RBI) and erbstatin analog (methyl 2, 5-dihydroxycinnamate, RBI) as the tyrosine kinase inhibitors and PD98059 (Calbiochem, USA) as the MEK inhibitor were dissolved in dimethyl sulfoxide (DMSO) at concentrations of 100 mM (Akiyama et al., 1987Go; Umezawa et al., 1990Go; Pang et al., 1995Go).

The tyrosine kinase inhibitors or MEK kinase inhibitor were added to the assay medium at final concentrations of 0.1, 1.0, or 10 µM. The pretreatment of the PC12 cells with inhibitors were performed at 37°C in 5% CO2 for 30 min. As a negative control, cells were treated with DMSO only.

Subsequently, the cells were cultured in the assay medium supplemented with CTB (RBI) (1 µg/ml at the final concentration) or NGF (Life Technologies) (50 ng/ml at the final concentration) at 37°C for 48 h.

Immunofluorescein staining of PC12 cells
PC12 cells (5 x 102 cells/well) were seeded onto collagen-coated eight-chamber slides (Nunc). After incubated for 48 h with 1 µg/ml of CTB or 50 ng/ml of NGF at the final concentration, PC12 cells were fixed at room temperature for 10 min with phosphate-buffered saline (PBS) containing 3.7% formaldehyde and 0.1% Triton X-100 (Nacali Tesque, Japan). Fixed cells were gently rinsed with Hanks’s balanced salt solution (Life Technologies). The cells were blocked at room temperature for 10 min with PBS containing 1% bovine serum albumin (BSA; Roche) to minimize nonspecific binding of the antibodies.

The cells were incubated with anti-MAP2B antibody (Transduction Laboratories, USA) at room temperature for 45 min. After washing three times with PBS, FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, USA) were added at room temperature for 45 min to the cells. After washing three times with PBS, the cells were mounted. The mounted cells were observed using a confocal laser scanning microscope system (LSM510, Carl Zeiss Co., Ltd.).

Extraction of total lipids from PC12 cells
PC12 cells (1 x 107 cells) were harvested and sedimented at 4°C for 5 min. The cells were washed twice with 1 ml of PBS. The cell pellet was then suspended in 1 ml of CHCl3/MeOH (1:1, v/v). The suspension was sonicated for 5 min. After brief sedimentation, the supernatant was collected. The pellet was resuspended in 1 ml of CHCl3/MeOH (1:1,v/v.), sonicated, and sedimented again. The combined supernatant (about 2 ml) was evaporated under an N2 stream. Dried total extracts were hydrolyzed at room temperature overnight in a solution containing 0.5 N NaOH (Hidari et al., 1991Go).

After hydrolysis, the solution was applied to the YMC Dispo SPE (C18) column (YMC, USA) preactivated by MeOH. The column was thoroughly washed with 40 ml of water. Bound materials were eluted with 1 ml of MeOH and 5 ml of CHCl3/MeOH (1:1, v/v) sequentially. The eluate was dried under an N2 stream and dissolved in 100 µl of CHCl3/MeOH (1:1, v/v). This was then subjected to TLC analysis.

TLC-binding assay
Five microliters of lipid extracts (equivalent to 5 x 105 of PC12 cells) was spotted onto TLC plates (Polygram Sil G, Macherey-Nagel). One micromole of ganglioside GM1a and 5 µg of total ganglioside from bovine brain were used as standard materials. The TLC plates were developed using either solution system, acetone and CHCl3/MeOH/12 mM MgCl2 (5:4:1, v/v/v), or CHCl3/MeOH/12 mM MgCl2/25% NH3 (50:40:7:3, v/v/v/v) (Hidari et al., 1993Go).

After drying thoroughly, the plates were blocked at 4°C overnight with PBS containing 1% BSA and 1% polyvinyl pyrrolidone (Wako Chemicals). The plates were washed five times for 3 min with PBS containing 0.05% Tween 20. The plates were then incubated at room temperature for 1 h in a blocking solution containing biotinylated CTB (List Biological Laboratories, USA). After washing five times for 3 min with PBS, the plates were incubated at room temperature for 1 h in a blocking solution containing horseradish peroxidase–conjugated avidin.

After washing five times for 3 min with PBS, the plates were visualized with a substrate solution (2 µM 4-chloro-1-naphthol, 60 µM N,N-diethylphenylenediamine monohydrochloride, 0.15% H2O2, 100 mM citrate buffer, pH 6.0).

Western blotting analysis of tyrosine-phosphorylated proteins in PC12 cells induced with CTB
PC12 cells (1.6 x 106 cells) were seeded onto collagen-coated 60-mm dishes (Falcon). The cells were stimulated with CTB (1 µg/ml at the final concentration) following pretreatment with tyrosine kinase inhibitors, as described previously. The stimulated cells were incubated at 37°C for the indicated times.

After the indicated periods, the cells were rinsed once with ice-cold PBS, lysed in 100 µl buffer (150 mM NaCl, 1% SDS, 1 mM PMSF [Wako Chemicals], 10 µg/ml aprotinin [Wako Chemicals], 10 µg/ml leupeptin [Wako Chemicals], 10 µg/ml pepstatin A [Wako Chemicals], 1 mM Na3VO4 [Wako Chemicals], and 20 mM Tris–HCl [pH 7.5]) and immediately boiled for 10 min. The lysates were sonicated and boiled again. The lysates were sedimented, and the supernatants were subjected to SDS–PAGE under reducing conditions (Laemmli, 1970Go).

The proteins were electrotransferred to polyvinylidene fluoride membrane (Bio-Rad) and incubated with primary antibodies after blocking of the membrane with 2% BSA for antiphosphotyrosine antibody (Transduction Laboratories) or 2% nonfat milk for other primary antibodies (BioLabs). The immune complexes were visualized using an HRP-Luminol system (Pierce).

Immunoprecipitation analysis of Trk proteins in PC12 cells
PC12 cells were seeded onto collagen-coated 100-mm dishes (Falcon). The cells were incubated with or without CTB (1 µg/ml at the final concentration). The stimulated cells were incubated at 37°C for 90 min. The cells were lysed as described above. The lysates were diluted 10-fold by the addition of a dilution buffer (150 mM NaCl, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM Na3VO4, and 20 mM Tris–HCl [pH 7.5]).

Five micrograms of goat anti-mouse IgG antibody (Jacson ImmunoResearch) and 20 µl of Protein A Sepharose (Pharmacia Biotech) were incubated for 6 h at 4°C. After conjugation, the resin was thoroughly washed with TTBS. Subsequently, 5 µg of anti-Trk antibody (Santa Cruz Biotechnology) or 5 µg of normal mouse IgG (Jacson ImmunoResearch) were conjugated to the goat anti-mouse IgG–preadsobed protein A Sepharose for 6 h at 4°C.

Anti-Trk– or normal mouse IgG–preadsobed Protein A Sepharose and diluted total lysates of PC12 cells were incubated at 4°C for 12 h. Immunoprecipitants were resolved by SDS–PAGE, followed by Western blotting with antiphosphotyrosine antibody.

The blotted membrane was striped and reprobed with anti-Trk antibody. The signals were visualized using on alkaline phosphatase substrate (Western Blue, Promega).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This study was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (No. 11780429, K.I.P.J.H) from the Japan Society for the Promotion of Science and a Grant-in-Aid for Scientific Research (B) (No. 11694303 and 10470081, Y.S.) from the Ministry of Education, Science, Sports, and Culture.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; CTB, cholera toxin B subunit; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; MAP2B, microtubule-associated protein 2B; MEK, MAPK/ERK kinase; NGF, nerve growth factor; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TLC, thin-layer chromatography.


    Footnotes
 
1 These authors equally contributed to this work. Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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