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
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Abstract |
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Key words: EGF receptor / GnT-III / integrin / MAPK / neurite outgrowth
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Introduction |
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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., 1989; Gregoriou, 1993
; Hakomori, 2002
; Taniguchi et al., 2001
; Zheng et al., 1994
). 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., 1992
). 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., 1997
). On the other hand, the binding of lectins to epidermal growth factor (EGF) modulates the receptor functions (Zeng et al., 1995
). Concanavalin A inhibits EGF binding to its receptor, receptor autophosphorylation, and cell proliferation (Hazan et al., 1995
). 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., 2000
). 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 factorinduced 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, 1999; Wang et al., 1998
; Yamada and Even-Ram, 2002
). Changes in the N-glycan structures of integrins can also affect cellcell and cellECM interactions, thereby affecting cell adhesion, migration, and tumor malignancy (Chakraborty et al., 2001
; Demetriou et al., 1995
; Dennis et al., 2002
; Miyoshi et al., 1999
). 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., 2002
). In addition, B16 melanoma cells that overexpress GnT-III have been shown to reduce invasive ability and lung colonization (Yoshimura et al., 1995
). 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., 1996
). 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., 2001
).
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.
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Results |
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Discussion |
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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, 1997). 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., 1996
). 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., 1996
). Our present studies show that collagen I or laminin has the ability to rescue PC12 cells from serum-depletioninduced 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., 2002
). 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, 1995). The PC12 cells are wildly used as a cell system for the study of growth factorstimulated 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., 2001
). 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)
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., 1997). 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., 2001
). 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., 1998; Kao et al., 2001
; Kaplan et al., 1991
). 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., 1997
). 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 functionsincluding cell spreading, migration (unpublishedn data), metastasis (Yoshimura et al., 1995), and cell differentiation (Ihara et al., 1997
; 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., 1993
). Furthermore Stanley's group recently reported that mice lacking GnT-III are still viable and fertile (Bhaumik et al., 1998
; Priatel et al., 1997
), but mice carrying a truncated GnT-III gene that encodes inactive enzyme represents a subtle neurological phenotype (Bhattacharyya et al., 2002
). 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.
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Materials and methods |
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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 5-containing laminin, laminin-10 (
5ß1
1), and laminin-11 (
5ß2
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), as described previously (Ihara et al., 2002
). 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., 1989).
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 TrisHCl, 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 sulfatepolyacrylamide 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 0.0050.15 ng and unlabeled EGF over a concentration range of 01.2 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 -counter.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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