ß1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells

Jianguo Gu1,2, Yanyang Zhao2, Tomoya Isaji2, Yukinao Shibukawa2, Hideyuki Ihara2, Motoko Takahashi2, Yoshitaka Ikeda2, Eiji Miyoshi2, Koichi Honke3 and Naoyuki Taniguchi2

2 Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka Suita, Osaka 565-0871, Japan; and 3 Department of Molecular Genetics, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan

Received on August 13, 2003; revised on October 1, 2003; accepted on October 3, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
A rat pheochromocytoma cell line (PC12), when transfected with ß1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)–mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.

Key words: EGF receptor / GnT-III / integrin / MAPK / neurite outgrowth


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
ß1,4-N-Acetylglucosaminyltransferase III (GnT-III), a pivotal glycosyltransferase, participates in the branching of N-glycans, catalyzing the formation of a unique sugar chain structure, a bisecting GlcNAc (Narasimhan, 1982Go). The introduction of the bisecting GlcNAc results in the suppression of further processing and the elongation of N-glycans catalyzed by other glycosyltransferases, because they are not able to use the bisected oligosaccharide as a substrate (Schachter, 1986Go). Thus GnT-III is generally regarded to be a key glycosyltransferase in N-glycan biosynthetic pathways. Interestingly, the tissue distribution of its mRNA showed that the GnT-III transcript is present in particularly high levels in the brain and kidney of the mouse (Nishikawa et al., 1990Go). Consistent with this, the bisecting GlcNAc containing sugar chain is more abundant in neural tissues than in other tissues (Shimizu et al., 1993Go). Due to the relative difficulty of studying signaling in neurons, neurotrophin signaling has been primarily studied using a rat pheochromocytoma tumor cell line (PC12) as a model system (Greene and Tischler, 1976Go; Tischler and Greene, 1975Go). PC12 cells respond to exposure to nerve growth factor (NGF) by differentiating to sympathetic neuron-like cells. On NGF stimulation, PC12 cells cease their division; neurites are extended and become electrically excitable and express neuronal markers (Greene and Tischler, 1976Go; Tischler and Greene, 1975Go).

The remodeling of cell surface growth factor receptors and extracellular matrix (ECM) receptors by modification of their oligosaccharide structures is associated with the function and biological behavior of tumor cells (Akiyama et al., 1989Go; Gregoriou, 1993Go; Hakomori, 2002Go; Taniguchi et al., 2001Go; Zheng et al., 1994Go). NGF has been shown to bind to its receptor, TrkA, on the surface of PC12 cells, resulting in TrkA dimerization and phosphorylation (Jing et al., 1992Go). TrkA-mediated neurite outgrowth and its tyrosine phosphorylation are blocked as the result of the transfection of GnT-III to PC12 cells, suggesting that bisecting structures may participate in the regulation of TrkA functions (Ihara et al., 1997Go). On the other hand, the binding of lectins to epidermal growth factor (EGF) modulates the receptor functions (Zeng et al., 1995Go). Concanavalin A inhibits EGF binding to its receptor, receptor autophosphorylation, and cell proliferation (Hazan et al., 1995Go). Furthermore, the sugar chain linked to Asn-420 of EGF receptor (EGFR) plays a crucial role in EGF binding and prevents spontaneous receptor oligomerization (Tsuda et al., 2000Go). Collectively, these results suggest that the oligosaccharide moieties of this receptor may be involved in receptor activation.

Integrins are receptors for ECM proteins that engage in reciprocal crosstalk with growth factor receptors. Recent studies have shown that growth factor–induced proliferation, cell-cycle progression, and differentiation require the adhesion of cells to the ECM, a process that is mediated by the integrin family of cell-surface receptors (Schwartz and Baron, 1999Go; Wang et al., 1998Go; Yamada and Even-Ram, 2002Go). Changes in the N-glycan structures of integrins can also affect cell–cell and cell–ECM interactions, thereby affecting cell adhesion, migration, and tumor malignancy (Chakraborty et al., 2001Go; Demetriou et al., 1995Go; Dennis et al., 2002Go; Miyoshi et al., 1999Go). In epithelial cells, a shift in integrin N-glycans to highly ß1,6 GlcNAc branched types leads to a decreased cell adhesion, resulting in an increase in both cell motility and tumorigenicity (Guo et al., 2002Go). In addition, B16 melanoma cells that overexpress GnT-III have been shown to reduce invasive ability and lung colonization (Yoshimura et al., 1995Go). One mechanism by which GnT-III could cause these effects appears to be through an increased cell surface expression of E-cadherin (Yoshimura et al., 1996Go). E-cadherin, with bisecting GlcNAc, is correlated with a suppressed tyrosine phosphorylation of beta-catenin, which may lead to a reduction in invasion and metastasis (Kitada et al., 2001Go).

The present study reports on an examination of the potential role of GnT-III on EGFR-mediated cellular signaling in neuron cells, PC12 cells that overexpress GnT-III were investigated under conditions where the cells were spread on ECM. Effects of oligosaccharides of the EGFR on EGF binding, receptor autophosphorylation, receptor-mediated mitogen-activated protein kinase (MAP) activation, and neurite formation in a serum-free differentiation system were compared between mock, wild-type, and mutant GnT-III transfectants after stimulation with EGF.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Establishment of GnT-III transfectants
The glycosylation of EGFR appears to be tissue-specific. For example, in A431 cells, a human epidermoid carcinoma cell line, EGFR was found to contain mainly a high mannose oligosaccharide structure (Soderquist and Carpenter, 1984Go). In contrast, in U373 MG cells, a human glioma cell line, EGFR contained a bisecting GlcNAc structure (Rebbaa et al., 1996Go). To investigate the functional role of the bisecting GlcNAc structure on EGFR in neuronal cells, stable GnT-III transfectants were generated in PC12 cells. Transfection was verified by the detection of GnT-III protein expression by immunoblot analysis. The bands migrated at 66 kDa and 46 kDa as seen in wild-type and D321A mutant transfectants are assumed to be derived from GnT-III, because these bands were not observed in mock transfectants (Figure 1A) and expression of recombinant GnT-III tagged with poly-His also showed two bands as previously described (Ikeda et al., 2000Go). The D321A mutant expression level was found to be similar to that of wild-type GnT-III. GnT-III enzyme activity was measured using a fluorescence-labeled agalacto-biantennary sugar chain as acceptor, and product formation was analyzed by high-performance liquid chromatography. In wild-type transfectants, an additional product peak identified as bisected biantennary glycan was demonstrated (Figure 1B). In contrast, no detectable activity was detected in mock and the D321A mutant transfectants. Interestingly, the minimal activity could be detected under a longer incubation time (4 h) in the mock transfectant but not in D321A transfectants (data not shown), suggesting that the D321A, similar to the D323A mutant described previously (Ihara et al., 2002Go), serves as a dominant negative molecule involved in competition with the corresponding endogenous wild-type GnT-III.



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Fig. 1. Expression and activities of the wild-type and mutant GnT-IIIs in PC12 cells. (A) Cell homogenates from PC12 cells stably transfected with mock, wild-type (Wt), or DN GnT-III (D321A), were separated on 10% SDS–PAGE. Expression of GnT-III was analyzed by immunoblot using a specific antibody to GnT-III. (B) GnT-III activities were examined with high-performance liquid chromatography using a fluorescence-labeled agalactobiantennary sugar chain as a substrate. S, substrate; P, product; ND, not detectable.

 
Effect of EGF on cell differentiation in PC12 cells
PC12 cells are frequently used as a cell model to study neuronal differentiation on stimulation with NGF (Greene and Tischler, 1976Go; Tischler and Greene, 1975Go). Our previous study showed that the overexpression of GnT-III disrupts the phosphorylation of TrkA with resultant NGF-induced neurite formation in PC12 cells (Ihara et al., 1997Go). To study the role of GnT-III and its product, bisecting GlcNAc, on neurite formation induced by costimulation with EGF and integrins, we established a serum-free differentiation assay using PC12 cells. The cells were suspended in serum-free medium and plated on culture dishes coated with collagen I, laminin, or fibronectin in the absence or presence of growth factors. Plating the cells on collagen I or laminin, but not fibronectin, led to their rapid attachment, because PC12 cells express higher levels of {alpha}1, {alpha}3, and {alpha}6 integrins (Vogelezang et al., 2001Go), the receptors for collagen I and laminin (Kikkawa et al., 2000Go), respectively, but lower levels of {alpha}5 integrin (Vogelezang et al., 2001Go), a receptor for fibronectin. Although it has been reported that alteration of the N-glycans in integrins may affect initial cell adhesion and spreading on ECM (Demetriou et al., 1995Go; Guo et al., 2002Go; Miyoshi et al., 1999Go; unpublished data), in this case, no apparent difference in cell adhesion between mock and GnT-III transfectants was detected after a 12-h incubation (data not shown). PC12 cells failed to undergo neuronal differentiation without treatment with growth factors, such as NGF, which is regarded as a positive control for the induction of PC12 cells. Conversely, PC12 cells plated on plastic dishes without an ECM coating, such as bovine serum albumin (BSA) and poly-L-lysine, began to die, even in the presence of NGF after 3 days of incubation, suggesting that integrin-mediated extracellular signals are required to prevent serum-depletion–induced apoptosis. After 7 days of incubation on collagen I, neurite formation was observed in PC12 cells transfected with mock or the dominant negative D321A mutant in the presence of EGF as well as NGF. However, the stimulation of neuronal differentiation was completely blocked in PC12 cells overexpressing wild-type GnT-III (Figure 2). These data indicate that the costimulation of EGFR and integrins are required for the induction of neurite outgrowth and that GnT-III disrupts not only TrkA- but EGFR-mediated neurite outgrowth in PC12 cells as well.



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Fig. 2. Neurite outgrowth in PC12 cells on EGF stimulation. Cells were replated on culture dishes coated with collagen I (A), laminin (B), or BSA (C) and then incubated with serum-free medium with or without EGF (5 ng/ml) or NGF (50 ng/ml). During the incubation, the culture medium was replaced with fresh medium at 2-day intervals. After 7 days of incubation, cells were photographed by phase-contrast microscopy. The results were reproducible in three independent experiments. The arrowheads point to neurites. (D) The data show the percentage of cells with neurite outgrowth spread on collagen I and expressed as the mean of 300 cells from 3 independent experiments. Those cells with neurite which length is longer than two times of cell body diameter are defined as positive cells.

 
Inhibition of ERK activation induced by EGF in GnT-III transfectants
It is well established that growth factors activate the Ras/MAPK cascade to control the neuronal differentiation of PC12 cells. To determine the effects of GnT-III transfection on EGF signaling, ERK activity was measured by immunoblotting with an antiphospho-ERK antibody. Unexpectedly, the basal levels of ERK phosphorylation were somewhat higher in the wild-type GnT-III transfectants than in mock and D321A GnT-III transfectants. However, ERK phosphorylation, as induced by EGF or NGF, was greatly enhanced in cells transfected with the mock or D321A mutant, whereas marginal activation of ERK was detected in the wild-type GnT-III transfectants after 5 min of incubation with EGF or NGF (Figure 3A). Furthermore, although the EGF-induced ERK activation began to decline at 30 min, significant activation of ERK persisted for 9 h in the mock and D321A transfectants. By contrast, in the wild-type GnT-III transfectants, the levels of activated ERK rapidly declined to the basal levels of the unstimulated control (Figure 3B). In fact, it has been suggested that persistent ERK activation is important for neuronal differentiation (Kao et al., 2001Go).



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Fig. 3. Activation of ERK in PC12 cells on EGF treatment. Serum-starved PC12 cells were detached and maintained in suspension for 30 min and then replated on culture dishes coated with collagen I. After incubation for 1 h, cells were treated with or without EGF (5 ng/ml) or NGF (50 ng/ml) for the indicated times. Cell lysates were subjected to 10% SDS–PAGE. After electroblotting, blots were analyzed for phosphorylated ERK (P-ERK, upper panel) and total ERK (T-ERK, lower panel). (A) A representative example of an assay for phospho-ERK treated with growth factors for 5 min. (B) Densitometric quantification of the levels of phosphorylated ERK (P-ERK). The data are shown as fold increases relative to the levels of the unstimulated control after normalization against the levels of total ERK (T-ERK). Data shown are the mean ± SD in three separate experiments.

 
Inhibition of MEK-1/ERK pathways blocks neurite formation in PC12 cells on collagen 1
To determine whether ERK activation is necessary for the EGF-induced differentiation of PC12 cells on collagen I, we examined the effects of PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). The blockage of ERK activation by PD98059 was found to abolish the differentiation of PC12 cells in the presence of EGF, as evidenced by neurite formation with phase-contrast microscopy, but it had no significant affect on cell death (Figure 4). On the other hand, we overexpressed dominant negative mutant of MEK-1 (DN-MEK-1) and constitutively activated mutant of MEK-1 (CA-MEK-1) to test whether the DN mutant could mimic the effects of PD98059 and if CA-MEK-1 could rescue the neurite formation that had been inhibited by the overexpression of GnT-III in PC12 cells, respectively. We cotransfected a puromycin-resistant plasmid with DN-MEK-1 or CA-MEK-1 and then selected transfectants for 3 days in medium containing puromycin. This puromycin selection procedure routinely yields ~90% positive populations of transfectants as previously described (Gu et al., 1999Go). The surviving cells were cultured in medium containing 10% horse serum (HS) and 5% fetal calf serum (FCS) without puromycin overnight and replated on tissue culture dishes coated with collage I, following by incubation for the indicated days in the serum-free medium with or without EGF. Consistent with the results shown in Figure 4, the overexpression of DN-MEK-1 significantly inhibited neurite formation in PC12 cells transfected with both mock and D321A mutant. Conversely, transfection with CA-MEK-1 significantly potentiated neurite formation in cells transfected with wild-type GnT-III, further confirming that ERK activation is necessary and sufficient for PC12 differentiation.



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Fig. 4. Effects of PD98059 and MEK-1 mutants on neurite formation. (A) PC12 cells were replated on culture dishes coated with collagen I and incubated with serum-free medium containing EGF (5 ng/ml) with or without PD98059 (20 nM) for 7 days, and then photographed by phase-contrast microscopy. (B) PC12 cells were cotransfected with pHA262pur with or without DN-MEK-1 or CA-MEK-1. After selection with puromycin, cells were detached and replated on culture dishes coated with collagen I and incubated with serum-free medium containing EGF (5 ng/ml) for 7 days, and then photographed by phase-contrast microscopy. (C) The data show the percentage of cells with neurite outgrowth spread on collagen I and expressed as the mean of 200 cells from 3 independent experiments. (D) The expression levels of DN-MEK-1 and CA-MEK-1 were detected by immunoblotting with anti-MEK-1. The results were reproducible in three independent experiments. The arrowheads point to neurites.

 
Glycosylation of EGFR
To determine whether the transfection affected the cell surface expression of EGFR, we carried out cell surface biotinylation and precipitation according to manufacturer's instructions. The finding showed that the three cell types expressed nearly the same amount of EGFR on the cell surface (Figure 5A). Glycosylation of the EGF receptor on the immunoprecipitated receptor was analyzed. The blot was probed with biotinylated E4-PHA lectin, which preferentially recognizes the bisecting GlcNAc structure of N-glycan. The EGFR from the wild-type GnT-III transfectants showed a markedly increased reactivity to E4-PHA compared with those from mock and D321A mutant GnT-III transfectants (Figure 5B). This result also indicates that the molecular mass of EGFR from wild-type GnT-III transfectants was lower than that of the mock and mutant GnT-III transfectants, supporting the hypothesis that bisecting oligosaccharide structures suppress the elongation of N-glycans (Koyota et al., 2001Go; Schachter, 1986Go).



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Fig. 5. Analysis of EGFR of GnT-III transfectants. (A) PC12 cells were biotinylated and immunoprecipitated with antibody against EGFR from 300 µg proteins of a whole cell lysate. The samples were subjected to 7.5% SDS–PAGE. After electroblotting, the biotinylated proteins were detected following manufacturer's instruction. (B) EGFR was immunoprecipitated from a whole cell lysate from mock or GnT-III transfectants and subjected on 7.5% SDS–PAGE. After electroblotting, blots were probed by E4-PHA (upper panel) and anti-EGFR antibody (lower panel).

 
Tyrosine phosphorylation and EGF binding of EGFR
It is well known that the activation of growth factor receptors (such as EGFR) stimulate nucleotide exchange on the Ras protein, which participates in the activation of the Ras family and then sequentially phosphorylates and activates the downstream components MEK-1/2 and ERK. To elucidate the mechanism by which GnT-III transfection affects EGFR-mediated ERK activation, tyrosine phosphorylation of EGFR was investigated. Tyrosine phosphorylation of the EGFR was greatly enhanced by EGF treatment in both mock and D321A mutant transfectants. By contrast, although the basal levels of tyrosyl-phosphorylated EGFR in the absence of EGF were higher in GnT-III transfectants than those in mock and D321A transfectants, EGF treatment did not lead to an increase in the levels of tyrosyl-phosphorylated EGFR from wild-type GnT-III transfectants (Figure 6). The diminution in EGFR activation is in agreement with ERK activation as described in Figure 3, suggesting that an overexpression of GnT-III inhibits EGFR function in PC12 cells. On the other hand, the binding of EGF to its cell surface receptor on mock- and GnT-III-transfected PC12 cells was examined. As shown in Figure 7A, the binding of 125I-EGF to EGFR on cell surface was significantly reduced in the wild-type GnT-III transfectants, compared with mock and D321A mutant transfectants. A Scatchard analysis revealed that high- and low-affinity EGFR were present in mock and D321A mutant transfectants. However, only low-affinity EGFR was detected in wild-type transfectants (Figure 7B). In fact, it has been reported that high-affinity population of EGFR is required and sufficient for EGF-induced responses (Bellot et al., 1990Go; Defize et al., 1989Go). Taken together, our results suggest that modulation of N-glycans with bisecting GlcNAc on EGFR disturbs the capacity of binding and affinity with its ligand, thereby inhibiting subsequent signaling.



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Fig. 6. Tyrosine phosphorylation of EGFR from mock and GnT-III transfectants on EGF stimulation. Serum-starved PC12 cells were replated on culture dishes coated with collagen I. After 1 h, cells were treated with or without EGF (5 ng/ml) for 5 min, and solubilized in lysis buffer as described under Materials and methods. The immunoprecipitates of the anti-EGFR antibody were detected by immunoblotting of antiphosphotyrosine antibody (upper panel) and anti-EGFR antibody (lower panel). P-EGFR, phosphorylated EGFR.

 


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Fig. 7. 125I-EGF binding to mock and GnT-III transfectants. (A) Representative competition binding curves of 125I-EGF to mock and GnT-III transfectants. The cells suspended in 1.5-ml tubes were incubated with 125I-EGF in the presence of unlabeled EGF. After a 2-h incubation at 4°C with agitation of 20-min intervals, unbound EGF was eliminated by washing the cells with PBS containing 0.1% BSA. The cells were then solubilized in 500 µl 1 N NaOH, and the binding of the radioactive EGF was measured by {gamma}-counter. Data shown are the means of triplicate experiments. The results were reproducible in two independent experiments. (B) Scatchard analysis of the binding experiments. The Kd value are the means of triplicate experiments.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Certain N-glycan structures of many glycoproteins, particularly adhesion molecules and growth factor receptors, appear to contribute to the folding, stability, and biological function of the molecules (Dwek, 1995Go). In the present study, the effects of GnT-III expression and its product, bisecting GlcNAc, on EGF signaling in rat PC12 cells were investigated. These cells were found to be a most useful and convenient model for the study of neuronal differentiation on stimulation with growth factors. We found the overexpression of GnT-III blocked the EGFR/ERK activation pathway, thereby inhibiting cell differentiation and neurite outgrowth on EGF stimulation. The inhibition of the EGFR/ERK/neurite outgrowth pathway was confirmed by treatment with the chemical inhibitor PD98059 and the overexpression of DN-MEK-1, which compromises the ability of EGF to transduce ERK activation. Our results provide clear evidence that GnT-III may play an important role in neural cell differentiation.

Costimulation with EGF and integrins induces neurite outgrowth in PC12 cells but not GnT-III transfectants
Interactions of cells with the ECM through integrins are known to suppress apoptosis in many cell types (Frisch and Ruoslahti, 1997Go). Mammary epithelial cells cultured on collagen I show extensive apoptosis over periods of several days, whereas the same cells do not when they are in contact with laminin-1 or Matrigel, a basement membrane-like gel containing laminin-1, collagen IV, nidogen, and perlecan (Pullan et al., 1996Go). However, laminin-1 may not be a survival ligand for other cells, because endothelial cells undergo apoptosis on a laminin-1 substrate but are protected from apoptosis on when grown on fibronectin or vitronectin substrates (Wary et al., 1996Go). Our present studies show that collagen I or laminin has the ability to rescue PC12 cells from serum-depletion–induced apoptosis, whereas fibronectin does not. Consistent with these data, laminin-10/11 is more potent than fibronectin in preventing apoptosis induced by serum depletion in A549 cells (Gu et al., 2002Go). Thus different cell types may have their own favored ECM for protection from apoptosis, depending on the repertoire of integrins expressed on their cell surface, which in turn may define the types of ECM ligands that are the most potent for protection from apoptosis.

On the other hand, it is well known that integrin-mediated cell adhesion cooperates with growth factor receptors in control of cell proliferation, cell differentiation, cell survival, and cell migration in epithelia cells and fibroblasts. To examine whether these synergistic effects are also needed for differentiation, PC12 cells in the serum-free medium were plated on plastic dishes without an ECM coating. Treatment with EGF or NGF alone failed to induce neurite formation in PC12 cells, suggesting that the integration of signaling pathways triggered by receptor tyrosine kinases and integrins are required for the regulation of PC12 cell differentiation. Interestingly, EGF-induced neurite outgrowth was completely blocked in PC12 cells transfected with wild-type GnT-III. In addition, GnT-III activity is essential for the inhibition. The overexpression of D321A mutant, a DN mutant of GnT-III, had no effect on neurite formation. Collectively, these results clearly indicate that GnT-III could be considered one of the negative regulators for cell differentiation in PC12 cells.

Constitutively activated MEK-1 restores neurite outgrowth suppressed by the overexpression of GnT-III
Proliferation and differentiation of cells in response to extracellular signals is influenced by the differential regulation of MAPKs (Marshall, 1995Go). The PC12 cells are wildly used as a cell system for the study of growth factor–stimulated cell functions, in which the intensity and duration of activation the ERK have been proposed to govern a distinct switch between cell proliferation and differentiation (Kao et al., 2001Go). It has been reported that EGF induces rapid and transient Ras- and Rap1-dependent ERK activation, whereas NGF treatment results in a sustained activation of this signaling pathway. However, our data showed that treatment with EGF in the absence of serum also promotes neurite formation in PC12 cells transfected with mock or DN GnT-III but not wild type GnT-III, presumably correlated with sustained ERK activation. Recently, Ho et al. (2001)Go demonstrated that in addition to NGF, EGF is also involved in determining the threshold level of ERK activation required for the directional migration of PC12 cells. The precise mechanisms responsible for the sustained ERK activation stimulated by EGF remain to be elucidated in PC12 cells. This result may support the postulated threshold theory, in which differentiation is determined by the duration of ERK activation. ERK activation was found to be specifically mediated by the activation of the EGFR channeled via the Ras/MEK/ERK cascade, as it was abolished by treatment with a specific MEK-1 inhibitor (data not shown), thereby inhibiting neurite outgrowth. Experiments on the overexpression of dominant negative MEK-1 are consistent with these results because it completely inhibited neurite outgrowth from cells that had been transfected with either mock or DN GnT-III. Conversely, the overexpression of constitutively activated MEK-1 absolutely restored neurite outgrowth that was suppressed by the introduction of wild-type GnT-III in PC12 cells, indicating that the overexpression of GnT-III down-regulates neurite outgrowth via the EGFR/MAPK pathway.

GnT-III overexpression down-regulates EGFR-mediated signaling in PC12 cells
Although several lines of evidence showed that the oligosaccharide portion of the EGFR is important for its functions, the effects of oligosaccharide may vary between cells lines. For example, U373 MG cells overexpressing GnT-III exhibit the inhibition of EGF binding to the cell surface and EGFR autophosphorylation (Rebbaa et al., 1997Go). Contrary to U373 MG cells, the expression of GnT-III in HeLa S3 cells results in an enhancement in EGFR-induced ERK activation via the up-regulation of the rate of internalization of the receptor but no decrease in EGFR autophosphorylation (Sato et al., 2001Go). However, the effects of oligosaccharides on cell biology have not been extensively investigated in those studies. In the present study, we clearly showed that the overexpression of wild-type GnT-III in PC12 cells down-regulates neurite outgrowth through inhibition of EGF binding, receptor autophosphorylation and receptor-mediated ERK activation. Furthermore GnT-III activity is required for such suppression, since these changes could not be observed in D321A mutant transfectants. In fact, EGFRs from wild-type GnT-III-transfected cells showed more E4-PHA staining than those from mock or DN GnT-III-transfected cells, whereas the expression levels of EGFR protein at the cell surface were not influenced by GnT-III overexpression. Thus the overexpression of the bisecting GlcNAc structure on EGFR appears to be responsible for the reduction in EGFR-mediated signaling.

The responses to EGF and NGF both require ERK activation for cell differentiation in PC12 cells. The NGF signaling pathway is initiated by the direct binding of NGF to TrkA, and tyrosyl-phosphorylated TrkA then recruits docking proteins Grb2, Crk, and FRS2 and a tyrosine phosphatase SHP-2 to its receptor complex to activate ERK (Hadari et al., 1998Go; Kao et al., 2001Go; Kaplan et al., 1991Go). We previously reported that the N-glycan of TrkA, when modified by a bisecting GlcNAc, causes its functional changes by disrupting the dimerization of TrkA, whereas there is no significant difference in the capacity of NGF binding to its receptor between mock and GnT-III transfectants (Ihara et al., 1997Go). By contrast, EGF binding to its receptor is down-regulated by introducing bisecting GlcNAc into the EGF receptor in PC12 cells, indicating that GnT-III inhibits these receptor-mediated signalings through different mechanism.

The precise reason why the bisecting GlcNAc usually negatively regulates cell biological functions—including cell spreading, migration (unpublishedn data), metastasis (Yoshimura et al., 1995Go), and cell differentiation (Ihara et al., 1997Go; present study)—remains to be elucidated. Interestingly, it has been reported that the bisecting GlcNAc structure of N-glycans are more abundant in neural tissues, such as the cerebrum, the cerebellum, and the brain stem, than in other tissues (Shimizu et al., 1993Go). Furthermore Stanley's group recently reported that mice lacking GnT-III are still viable and fertile (Bhaumik et al., 1998Go; Priatel et al., 1997Go), but mice carrying a truncated GnT-III gene that encodes inactive enzyme represents a subtle neurological phenotype (Bhattacharyya et al., 2002Go). Although still speculative, our data and those of others suggest that GnT-III may play an important role in the regulation of neural differentiation.

In conclusion, we have shown that GnT-III down-regulates neurite outgrowth induced by costimulation of integrins and EGFR through MAPK activation pathway in PC12 cells. The present study further supports the notion that sugar chains decorate glycoproteins on the cell surface, where the sugars can have critical functions, and provides insight into the molecular mechanisms of bisecting GlcNAc-regulated neural cell differentiation.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Cell culture
Rat pheochromocytoma PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% HS, 5% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml) under a humidified atmosphere containing 5% CO2.

Reagents and antibodies
PD98059 (a specific MEK-1 inhibitor) was purchased from Sigma (St. Louis, MO). Collagen I and laminin, which has been considered to be {alpha}5-containing laminin, laminin-10 ({alpha}5ß1{gamma}1), and laminin-11 ({alpha}5ß2{gamma}1), were obtained from Chemicon (Temecula, CA). Monoclonal antibodies against phospho-ERK and phosphotyrosine (4G10) were purchased from New England BioLabs (Beverly, MA) and Upstate Biotechnology, respectively. Monoclonal anti-MEK-1 was obtained from Transduction Laboratories (San Diego, CA). Polyclonal anti-ERK and EGFR were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antihemagglutinin (HA) was purchased from BAbCO (Richmond, CA), and the monoclonal antibody against GnT-III was from Fujirebio (Tokyo).

Plasmids
The wild-type cDNA encoding rat GnT-III was subcloned into the EcoRI sites of the pCXNII expression vector, which contained a neomycin-resistant gene. The D321A DN mutant was constructed by site-directed mutagenesis experiments according to Kunkel (1985)Go, as described previously (Ihara et al., 2002Go). The fidelity of each construct was confirmed by DNA sequencing. The expression plasmids for the DN HA-tagged MEK-1 (HA-MEK-1) and CA HA-MEK-1 were kindly provided by Dr. Natalie G. Ahn (Department of Chemistry and Biochemistry, University of Colorado). The puromycin-resistance plasmid pHA262pur was provided by Dr. Hein te Riele (The Netherlands Cancer Institute, Amsterdam).

Transfection and cell selection
To get colonies to overexpress GnT-III, PC12 cells were transfected with pCXNII (mock), pCXNII/GnT-III (wild-type), or pCXNII/D321AGnT-III using the LipofectAMINE reagent, following the manufacturer's instructions. Selection was performed in 10% HS and 5% FCS DMEM containing 1 mg/ml G418. After a 2-week incubation, G418-resistant colonies were isolated and recloned by serial dilution to ensure clonality, and the expression levels of GnT-III were finally confirmed by western blotting. On the other hand, in transient experiments, 5 µg of each expression plasmid (CA or DN HA-MEK-1) was cotransfected with 1 µg pHA262Puro into 2 x 106 PC12 cells using the Lipofectamine reagent. Cells were subcultured at a 1:3 dilution 12 h after transfection and maintained for 72 h in 1 µg/ml puromycin-containing medium. Before use, cells were cultured overnight in the absence of puromycin.

Cell differentiation assay
PC12 cells or those selected with puromycin were detached with trypsin-ethylenediamine tetra-acetic acid (EDTA), and kept in suspension in DMEM containing 0.1% BSA for 30 min. Cells were plated on dishes coated with either collagen I or laminin in serum-free DMEM with or without chemical inhibitors as indicated. The cells were photographed by phase-contrast microscopy at suitable intervals.

GnT-III activity assay
Cell lysates were homogenized in phosphate buffered saline (PBS) containing protease inhibitors, and the supernate, after removal of the nucleus fraction by centrifugation for 15 min at 900 x g was used for the assays by high-performance liquid chromatography methods using a pyridylaminated biantennary sugar chain as an acceptor substrate, as described previously (Taniguchi et al., 1989Go).

Immunoprecipitation and lectin blotting
Samples of mock, wild-type, and DN GnT-III transfectant cells on 100-mm tissue culture dishes were solubilized in 600 µl of 1% Triton lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol bis [2-aminoethyl ether]-tetra acetic acid, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM sodium vanadate, 10 µg/ml leupepetin, 10 µg/ml aprotinin, 1 mM phenymethylsulfonyl floride). The cell lysates were clarified by centrifugation at 20,000 x g for 15 min at 4°C. Proteins were then immunoprecipitated from the lysates using a combination of 2 µg of anti-EGF receptor and Protein G-Sepharose beads. Immunoprecitates were suspended in reducing sample buffer, heated to 100°C for 3 min, resolved on 7.5% sodium dodecyl sulfate–polyacrylamide gels, and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 3% BSA and then incubated with biotin-conjugated lectin E4-PHA (Vector Labs, Burlingame, CA). Lectin reactive proteins were detected by strepavidin-peroxidase and ECL reagent (Amersham, Buckinghamshire, England).

Western blotting
To characterize phospho-ERK and the tyrosine phosphorylation of EGFR in cultured cells treated with EGF, 24 h after serum starvation the transfectants were detached from culture dishes by treatment with trypsin-EDTA, washed with serum-free DMEM containing 1% BSA, and resuspended in the same medium. Cells (2 x 106) were allowed to spread on 100-mm tissue culture dishes coated with 10 µg/ml of collagen I for 1 h. After 5 min of EGF treatment, cells were washed in ice-cold PBS, and solubilized in the lysis buffer as described, and total lysates and immunoprecipitates of anti-EGF receptor antibody were then detected by the immunoblotting of antiphospho-ERK and antiphosphotyrosine antibody (4G10), respectively.

EGF binding assays
Because PC12 cells are very easy to detach from culture dishes when cells were kept on ice, it is difficult to use culture plates for assaying cell surface EGF binding. The cells were suspended in 50 µl PBS containing 0.1% BSA (2 x 105/tube), and then incubated with 200 µl PBS containing different amount of 125I-EGF over a concentration range of 0.005–0.15 ng and unlabeled EGF over a concentration range of 0–1.2 ng. Nonspecific binding was determined by adding 100 ng unlabeled EGF. After incubation for 2 h at 4°C with agitation of 20-min intervals, the cells were washed three times with ice-cold PBS containing 0.1% BSA and then solubilized in 500 µl 1 N NaOH. The radioactivity of the cell lysates was counted with a {gamma}-counter.


    Acknowledgements
 
This work was partly supported by a Grant-in-Aid for the 21st Century COE Program by the Ministry of Education, Science, Culture, Sports and Technology in Japan.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: jgu{at}biochem.med.osaka-u.ac.jp Back


    Abbreviations
 
CA, constitutively activated; DMEM, Dulbecco's modified Eagle's medium; DN, dominant negative; ECM, extracellular matrix; EDTA, ethylenediamine tetra-acetic acid; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; HA, hemagglutinin; HS, horse serum; MAPK, mitogen-activated protein kinase; MEK-1, mitogen- or extracellular signal-regulated kinase kinase-1; NGF, nerve growth factor; PBS, phosphate buffered saline


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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