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Keywords:

  • cell proliferation;
  • cytokine;
  • ganglioside;
  • glycosyltransferase;
  • neural progenitor cell;
  • Ras-mitogen-activated protein kinase pathway

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

The CNS consists of neuronal and glial cells generated from common neural progenitor cells during development. Cellular events for neural progenitor cells, such as proliferation and differentiation, are regulated by multiple intrinsic and extrinsic cell signals. Although much is known on the importance of the proteinous factors in regulating the fate of neural progenitor cells, the involvement of other molecules such as gangliosides, sialic acid-containing glycosphingolipids, remains to be clarified. To elucidate the biological functions of gangliosides in neural progenitor cells, we transfected an immortalized neural progenitor cell line, C17.2, which does not express GD3 ganglioside, with a fusion protein of GD3-synthase (ST-II) and enhanced green fluorescent protein (ST-II-EGFP). Analysis of the ST-II transfectants revealed the ectopic expression of b- and c-series gangliosides. In the ST-II transfectants, proliferation induced by epidermal growth factor (EGF) was severely retarded. EGF-induced proliferation of C17.2 cells was dependent on the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, and the EGF-induced activation of this pathway was significantly repressed in the transfectants. Thus, ST-II overexpression retarded proliferation of C17.2 cells via repression of the Ras-MAPK pathway. The result supports the concept that gangliosides may play an important role in regulating the proliferation of neural progenitor cells.

Abbreviations used
bFGF

basic fibroblast growth factor

CTB

cholera toxin B subunit

DMEM

Dulbecco's modified Eagle's medium

EGF

epidermal growth factor

EGFP

enhanced green fluorescent protein

EGFR

EGF receptor

ERK

extracellular signal-regulated kinase

FCS

fetal calf serum

FGFR

fibroblast growth factor receptor

G3PDH

glyceraldehyde-3-phosphate dehydrogenase

GalNAcT

UDP-N-acetylgalactosamine GM3 N-acetylgalactosaminyltransferase

GSL

glycosphingolipids

HS

horse serum

IL

interleukin

LIF

leukemia inhibitory factor

LIFR

LIF receptor

Mab

monoclonal antibody

MAPK

mitogen-activated protein kinase

MBCD

methyl-β-cyclodextrin

RT-PCR

reverse transcription-polymerase chain reaction

STAT3

signal transducer and activator 3

ST-I

CMP-sialic acid : lactosylceramide sialyltransferase (GM3-synthase)

ST-II

CMP-sialic acid : GM3 sialyltransferase II (GD3-synthase)

ST-III

CMP-sialic acid: GD3 sialyltransferase II (GT3-synthase)

TLC

thin-layer chromatography

The central nervous system (CNS) is organized by neuronal and glial cells generated from common neural progenitor cells during development (Reynolds et al. 1992; McKay 1997; Gage 2000). The fate of neural progenitor cells, such as proliferation, differentiation, survival and death, is regulated by multiple intrinsic and extrinsic cell signals. Among intrinsic signals, basic helix-loop-helix transcription factors play central roles in neural development (Kageyama and Nakanishi 1997). As for cell extrinsic signals, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and interleukin 6 (IL-6)-type cytokines are known to promote proliferation and self renewal of neural precursor cells (Reynolds et al. 1992; Shimazaki et al. 2001). In addition, IL-6-type cytokines, for example leukemia inhibitory factor (LIF), induce astrocyte differentiation in co-operation with bone morphogenetic proteins (Nakashima et al. 1999a,b). Although the importance of these proteinous factors in regulation of neural precursor cell fate is clearly revealed, involvement of molecules other than proteins remains to be clarified.

Gangliosides are sialic acid-containing glycosphingolipids (GSLs) ubiquitously expressed in the plasma membrane of cells (Hakomori 1990). The expression of gangliosides is abundant in neural cells and drastically changed during development of the CNS (Yu 1994; Yu et al. 2004). Recently, GSLs including gangliosides were revealed to be enriched in cell-surface microdomains, lipid rafts, with sphingomyelin and/or cholesterol (Hakomori et al. 1998; Simons and Toomre 2000). Lipid rafts have been suggested to be important for modulation of signal transduction and cell adhesion. Furthermore, gangliosides are reported to be involved in ceramide-induced apoptosis in the developing brain (Bieberich et al. 2001). Therefore, it is expected that gangliosides may have functions in organizing nervous systems via regulation of cytokine signaling and/or cell adhesion in neural progenitor cells. However, the exact functions of gangliosides in neural progenitor cells are still unclear.

Previously, we characterized the expression of gangliosides in a C17.2 cell line (Suetake et al. 2003). This cell line is an immortalized neural progenitor cell line established from neonatal mouse cerebellar cortex by v-myc transformation (Ryder et al. 1990). Similar to endogenous neural stem cells, C17.2 cells are capable of self renewal, differentiating into neuronal and glial cells in vivo, and proliferating in response to EGF and bFGF stimulation in vitro (Snyder et al. 1992; Kitchens et al. 1994). Interestingly, these cells express only a-series gangliosides, such as GM3, GM2, GM1 and GD1a; they do not express b- and c-series complex gangliosides due to the lack of expression of CMP-sialic acid : GM3 sialyltransferase (GD3-synthase, ST-II, Fig. 1a) (Suetake et al. 2003). In this study, we induced ectopic overexpression of ST-II in C17.2 cells to clarify the functions of gangliosides in neural development and found that cytokine-induced mitogen-activated protein kinase (MAPK) activation and proliferation was repressed in ST-II transfectants.

image

Figure 1. Expression of ST-II-EGFP fusion protein in C17.2 cells. (a) Biosynthetic pathways of major gangliosides. C17.2 cells lack expression of ST-II (underline) and products such as b-series gangliosides (Suetake et al. 2003). (b) Phase views of control C17.2 cells (co) and the ST-II transfectants (ST-II). (c) Expression of sialyltransferases in control C17.2 cells and the ST-II transfectants. – RT indicates negative control without reverse transcription. (d) Expression of gangliosides in control C17.2 cells and ST-II transfectants. Gangliosides purified from the cells were subjected to TLC and detected by orcinol-H2SO4 reagent. Lanes: authentic human brain gangliosides (lane 1), GD3 (lane 2), GT3 (lane 3), gangliosides purified from control cells (lane 4), gangliosides purified from ST-II transfectants (lane 5).

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Cell culture

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

The C17.2 murine neural progenitor cell line was kindly provided by Dr Evan Snyder (Burnham Cancer Institute, La Jolla, CA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 5% horse serum (HS) at 37°C in a humidified 5% CO2 atmosphere. Transfection was performed using TransIT LT-1 (Mirus, Madison, WI, USA). To inhibit MEK MAPK kinase (MAPKK) or functions of lipid rafts, the cells were treated with U0126 (4 µm for 1 h at 37°C; Sigma, St Louis, MO, USA) or methyl-β-cyclodextrin (MBCD, 10 mm for 1 h at 37°C, Sigma), respectively.

Reverse transcription-polymerase chain reaction (RT-PCR)

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

Total RNAs were isolated from C17.2 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNAs were synthesized with 5 µg total RNAs as templates in a 20 µL reaction mixture using Superscript II reverse transcriptase (Invitrogen) at 50°C for 50 min. After reverse transcription, each reaction mixture was diluted 2.5-fold with H2O, and 1 µL of each mixture was subjected to PCR using Taq PCR core kit (Qiagen, Valencia, CA, USA) with the following settings: 94°C for 2 min; 28–32 cycles of 94°C for 20 s, 60°C for 20 s and 72°C for 0.5–1 min; and 72°C for 2 min. Primer sets used were as follows: 5′-TTTGGAGTCTGGCTCCTG-TAC-3′, 5′-CTCTCAAGTGTTCAGGAAAGTC-3′[for CMP-sialic acid : lactosylceramide sialyltransferase (GM3-synthase, ST-I)]; 5′-ATGCTAGCTCGGAAATTCCCG-3′, 5′-CAGGGTCACAGCA-GTCTTCC-3′ (for ST-II); 5′-TCTTCACCACTCCCAAGTA-CG-3′, 5′-CTGACTCCCTGTCAAGATTCC-3′[for CMP-sialic acid : GD3 sialyltransferase (GT3-synthase, ST-III)]; 5′-ACCACAGTCCATGCCATCAC-3′, 5′-TCCACCACCCTGTTGCTGTA-3′ (for glyceraldehyde-3-phosphate dehydrogenase; G3PDH); 5′-TTGCCCTCAACACCGTGGAG-3′, 5′-TCAATTTCTGGCAGTTCTCCTC-3′[for EGF receptor (EGFR)]; 5′-CTTGACGTCGTGGAACGATCT-3′, 5′-TTCCAGAACGGTCAACCATGCAGA-3′[for FGF receptor (FGFR) 1 IIIc]; 5′-GACAGACACACGGATGTGCTGGA-3′, 5′-AGCACCACCAGCCACGCAGAGTGA-3′ (for FGFR3 IIIc); 5′-TCCTGGAATCTCGGTCGATAG-3′, 5′-GAATCAAGAATTGCCTGGAATTG-3′[for LIF receptor (LIFR); Nakashima et al. 1999b]; 5′-TGTCAGCACCAAGGATTTGGC-3′, 5′-GTAGCTGACCATACATGAAGTG-3′ (for gp130; Nakashima et al. 1999b); 5′-ATCCAGCTGATCCAGAACCAC-3′, 5′-TGCCATAGCTGCGAGCAAGG-3′ (for H-Ras); 5′-CAGACAGCTCTTGCTGTTTCC-3′, 5′-GGTTGTTTGGGCTGCTGGAC-3′ (for Raf-1); 5′-TGCCCAAGAAGAAGCCGACG-3′, 5′-ACTCGTGCAGTACCTGCAGC-3′ (for MEK1); 5′-GCTCTGCTTATGATAATCTCAAC-3′, 5′-AAGGCTTGAGGTCACGGTGC-3′ (for ERK2).

Thin-layer chromatography (TLC) and TLC-immunostaining

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

Total lipids were extracted from C17.2 cells with chloroform/methanol (C/M; 2 : 1, v/v) and C/M/H2O (1 : 2 : 0.8, v/v/v). After the separation of neutral and acidic lipid fractions by diethylaminoethyl(DEAE)-Sephadex A-25 (acetate form) column chromatography, the acidic lipid fraction was treated with 0.2 m NaOH and then de-salted using a Sep-Pak C-18 column (Waters, Milford, MA, USA). Gangliosides were separated on a high-performance TLC plate (silica gel 60; Merck Chemicals, Darmstadt, Germany) in a solvent system of C/M/0.2% CaCl2 in H2O (55 : 45 : 10, v/v/v). Gangliosides were visualized with the orcinol-H2SO4 reagent (Svennerholm 1956) for the detection of hexoses.

For TLC-immunostaining, the TLC plate after development was immersed in n-hexane containing 0.4% polyisobutylmethacrylate (Sigma) for 30 s. The plate was incubated with A2B5 monoclonal anti-c-series gangliosides antibody (Saito et al. 2001) at 37°C for 6 h, and then anti-mouse IgM antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA) at 37°C for 2 h. The reactive bands were detected using the WesternLightning western blot chemiluminescence reagent plus (Perkin Elmer Life and Analytical Sciences, Boston, MA, USA).

Flow cytometry

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

C17.2 cells were stained with biotin-conjugated cholera toxin B subunit (CTB, for GM1 detection, Gibco BRL, Gaithersburg, MD, USA), monoclonal antibody (Mab) R24 anti-GD3 (Matreya Inc., Pleasant Gap, PA, USA), Mab D1.1 anti-9-O-acetyl GD3 (a generous gift from Dr Joel Levine, SUNY, Stony Brook, NY, USA) or Mab A2B5 anti-c-series gangliosides antibody, on ice, for 20–30 min. To detect CTB, R-phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch) was used. To detect antibodies, R-phycoerythrin-conjugated anti-mouse IgG/M antibody (Jackson ImmunoResearch) was used. Stained cells were analyzed by a flow cytometer, FACSCalibur (BD Biosciences, San Jose, CA, USA).

Western blot analysis

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

After starvation in serum-free DMEM for 3 h, C17.2 cells were stimulated with EGF (20 ng/mL), bFGF (20 ng/mL, Peprotech) or LIF (40 ng/mL, Invitrogen) for 10 min, then lysed in lysis buffer containing 1% Triton X-100, 10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA, 2 mm Na3VO4 and a protease inhibitor cocktail (Sigma). Protein concentration was measured by a Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The lysates containing the same amount of proteins were applied to SDS-PAGE (4–20% polyacrylamide gel) and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories). Western blot analysis was performed using antibodies to extracellular signal-regulated kinase (ERK) 1, ERK2, signal transducer and activator 3 (STAT3), EGFR, caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT3 or phospho-ERK (Cell Signaling Technology, Beverly, MA, USA), FLAG M2 (Sigma) or flotillin-1 (BD Bioscience).

Lipid rafts preparation

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

Preparation of lipid rafts of C17.2 cells was performed, according to a modification of the method by Iwabuchi et al. (1998), using lysis buffer containing Triton X-100. Briefly, cells were homogenized with 1 mL lysis buffer containing 1% Triton X-100, 10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA and 2 mm Na3VO4 using a loose-fitting homogenizer (20 strokes). Homogenates were mixed with an equal volume of 80% sucrose (w/v) in 10 mm Tris-HCl (pH 7.4), 150 mm NaCl and 5 mm EDTA (TBS), overlaid with 30% sucrose and 5% sucrose in TBS, and centrifuged at 195 000 gfor 18 h in a Beckman SW41 rotor (Beckman Coulter). Ten fractions were collected from the top of the gradient. Lipid raft-containing fraction just above the 5–30% sucrose interface was mainly collected in fraction 4. Gradient fractions were subjected to SDS-PAGE and western blot analysis.

Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

C17.2 cells express a-series gangliosides such as GM3, GM2, GM1 and GD1a, but do not express b- and c- series gangliosides due to the lack of ST-II expression (Fig. 1a) (Suetake et al. 2003). To induce ectopic overexpression of b-series gangliosides and elucidate the functions of ganglioside in neural progenitor cells, we transfected the cells with a ST-II-EGFP fusion protein (Bieberich et al. 2002) and sorted EGFP-positive cells by a fluorescence-activated cell sorter (Fig. 1b). These cells are positive not only for EGFP (Fig. 2) but also for ST-II (Fig. 1c). Examination of the extracted gangliosides by TLC confirmed that the ST-II transfectants express b-series gangliosides, such as GD3, GD1b and GT1b (Fig. 1d, italic with asterisk), in addition to a-series gangliosides.

image

Figure 2. Analysis of ganglioside expression in C17.2 cells using flow cytometry. C17.2 control cells (co, gray histograms) and ST-II transfectants (ST-II, black histograms) were analyzed by FACSCalibur. Expression of GM1, GD3 and c-series gangliosides was detected by CTB, Mab R24 and Mab A2B5, respectively.

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Next, we analyzed the expression of EGFP and gangliosides in C17.2 cells using flow cytometry. Although we sorted EGFP-positive cells only, 70–20% of ST-II transfectants was positive for EGFP. EGFP expression was decreased in accordance with the passage of the cells. It is likely due to the transient transfection of ST-II-EGFP fusion protein. In the case that 24.0% of ST-II transfectants were positive for EGFP, 32.3% of ST-II transfectants were positive for GD3, a b-series ganglioside (Fig. 2). In ST-II transfectants, GM1 expression was slightly decreased (co, 94.2% vs. ST-II, 83.7%). Although ST-II transfectants do not express ST-III GT3 synthase (Fig. 1d), 79.5% of the cells were positive for c-series gangliosides. The c-series gangliosides-positive population disappeared after rinsing the cells with ethanol (data not shown), suggesting that Mab A2B5-positive immunoreactivity is attributable to glycolipids, not to glycoproteins. To confirm expression of c-series gangliosides, we performed TLC-immunostaining using an A2B5 antibody. As shown in Fig. 3, expression of GT3 was detected in ST-II transfectants (arrow). This result indicates that ST-II transfectants lack ST-III but still express c-series ganglioside(s).

image

Figure 3. Detection of GT3 ganglioside using TLC-immunostaining. Gangliosides purified from C17.2 cells were subjected to TLC and detected by orcinol-H2SO4 reagent (left panel), or immunostained by A2B5 anti-c-series gangliosides antibody (right panel). Lanes: authentic GT3 (lane 1), gangliosides purified from control cells (lane 2), gangliosides purified from ST-II transfectants (lane 3). An arrow indicates GT3 in gangliosides purified from ST-II transfectants.

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Ras-MAPK pathway-dependent proliferation of C17.2 cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

EGF is known to promote proliferation and self renewal of neural stem/progenitor cells. Similar to primary neural stem/progenitor cells, C17.2 cells proliferate in response to EGF stimulation (Kitchens et al. 1994; Fig. 4a). To reveal the downstream pathway regulating proliferation of C17.2 cells, we treated the cells with an inhibitor of MEK MAPKK, U0126. U0126 (4 µm) significantly inhibited ERK MAPK activation (Fig. 4b). When C17.2 cells were cultured in the presence of U0126 (4 µm), EGF-induced proliferation of the cells was severely repressed (Fig. 4c). This inhibitory effect of U0126 was dose-dependent (Fig. 4d). These results clearly indicate that EGF-induced proliferation of C17.2 cells was dependent on the Ras-MAPK pathway.

image

Figure 4. Ras-MAPK pathway-dependent proliferation of C17.2 cells. (a) C17.2 cells were cultured in the presence (closed circles) or absence (open circles) of EGF (20 ng/mL). The number of the cells was counted by a cell counter, Vi-Cell. (b) C17.2 cells treated with or without a MEK inhibitor, U0126 (4 or 10 µm) were stimulated with or without EGF (20 ng/mL for 10 min). ERK activation was assessed by western blot analysis. (c) C17.2 cells treated with (closed circles) or without (open circles) U0126 (4 µm) were cultured in the presence EGF. (d) C17.2 cells treated with or without U0126 (0.1, 1 or 4 µm) were cultured in the presence of EGF for 3 days.

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Repression of proliferation and MAPK activation in ST-II transfectants

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

Since GD3 is significantly expressed in proliferating neural cells and suggested to be involved in melanoma proliferation (Yu 1994; Birkle et al. 2000; Zeng et al. 2000), we examined proliferation of C17.2 ST-II transfectants. Compared with control cells, proliferation of ST-II transfectants in the presence of EGF or FCS was severely retarded (Figs 5a and c). However, there is no significant difference in viability between these cells (Figs 5b and d). It was considered that ectopic expression of gangliosides induced by ST-II transfection specifically repressed proliferation of C17.2 cells.

image

Figure 5. Repression of proliferation and MAPK activation in ST-II transfectants. C17.2 control cells (open circles) or ST-II transfectants (closed circles) were cultured in the presence of 20 ng/mL of EGF (a and b) or 10% FCS HS (c and d). The number (a and c) or viability (b and d) of the cells were analyzed by a Vi-Cell. (e) C17.2 control cells (co) or ST-II transfectants (ST-II) were stimulated with EGF (20 ng/mL for 0, 10, 30, 60 or 120 min). (f) C17.2 control cells (co) or ST-II transfectants (ST-II) were stimulated with EGF (20 ng/mL), bFGF (20 ng/mL) or LIF (40 ng/mL) for 10 min. Activation of ERK and STAT3 was assessed by western blot analysis.

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As shown in Fig. 5, proliferation of C17.2 cells is dependent on the Ras-MAPK pathway. Therefore, we examined EGF-induced ERK MAPK activation in ST-II transfectants. In control C17.2 cells, ERK activation was induced by EGF for 10 min and rapidly decreased (Fig. 5e). However, in ST-II transfectants, this EGF-induced ERK activation was significantly repressed. It is known that other cytokines, bFGF and LIF, also have a mitotic effect on primary neural progenitor cells (Shimazaki et al. 2001). As well as EGF and bFGF, LIF, belonging to IL-6-type cytokines, activates the Ras-MAPK pathway in addition to the Janus kinase-STAT3 pathway (Taga and Kishimoto 1997). ERK activation induced by these mitogens was also repressed in ST-II transfectants (Fig. 5f). Therefore, it was suggested that proliferation of ST-II transfectants was retarded via repression of the Ras-MAPK pathway.

As described above, in accordance with the passage, the ST-II transfectant lost expression of an ST-II-EGFP fusion protein. In the late-passaged ST-II transfectants cultured for more than 1 month, ST-II-EGFP fusion protein expression almost disappeared, and the expression pattern of gangliosides was similar to control C17.2 cells (Fig. 6a). Furthermore, these late-passaged ST-II transfectants showed recovery of EGF-induced ERK activation and proliferation (Figs 6b and 6c, respectively). These results indicate that repression of ERK activation and proliferation in ST-II transfectants is reversible and due to the ectopically-expressed gangliosides, not to the transformation of the cells.

image

Figure 6. Recovery of ganglioside expression, EGF-induced ERK activation and proliferation in late-passaged ST-II transfectants. (a) C17.2 control cells (co, gray histograms) and late-passaged ST-II transfectants [ST-II(LP), blank histograms with black line] were analyzed by FACSCalibur. Expression of GM1, GD3 and c-series gangliosides was detected by CTB, R24 and A2B5 antibodies, respectively. (b) C17.2 control cells (co) or late-passaged ST-II transfectants [ST-II(LP)] were stimulated with EGF (20 ng/mL) for 10 min. Activation of ERK was assessed by western blot analysis. (c) C17.2 control cells (open circles) or late-passaged ST-II transfectants (closed circles) were cultured in the presence of 20 ng/mL of EGF. The number of the cells was analyzed by a Vi-Cell.

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Not only GD3 but also its O-acetylated derivative, 9-O-acetyl GD3, were suggested to be involved in proliferation of a melanoma cell line (Birkle et al. 2000). As shown by TLC analysis (Fig. 1e), GD3 expression was observed in ST-II transfectants but detection of 9-O-acetyl GD3 was impossible since it was converted to GD3 by base during purification of gangliosides. Therefore, we tried to detect 9-O-acetyl GD3 in ST-II transfectants using flow cytometry. In the case that 45.0% of ST-II transfectants were positive for EGFP, 84.4% and 62.6% of the cells were positive for 9-O-acetyl GD3 and GD3, respectively (Fig. 7). These results (Figs 1–7) suggest that ST-II transfection drastically induces ectopic expression of various kinds of gangliosides in C17.2 cells, and that these ectopic gangliosides induce the repression of the Ras-MAPK pathway and proliferation of ST-II transfectants. On the other hand, it was found that expression of GM3 was significantly decreased in ST-II transfectants (Fig. 1d). Because GM3 is a ganglioside expressed in proliferative neural cells (Yu 1994), it is possible that not only ectopic overexpression of b- and c-series gangliosides, but also decrease of GM3 might be involved in repression of the Ras-MAPK pathway and proliferation in ST-II transfectants. Further studies are required to dissect out the specific ganglioside(s) that are involved in the modulatory effects.

image

Figure 7. Expression of 9-O-acetyl GD3 in ST-II transfectants. C17.2 control cells (co, gray histograms) and ST-II transfectants (ST-II, black histograms) were analyzed by FACSCalibur. Expression of GD3 and 9-O-acetyl GD3 (OAcGD3) was detected by Mabs R24 and D1.1, respectively.

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Expression of signaling molecules in ST-II transfectants

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

Next, we tried to reveal the molecular mechanism of ERK MAPK repression in ST-II transfectants. First, we hypothesized down-regulation of components of cytokine receptors or the Ras-MAPK pathway in ST-II transfectants. As cytokine receptor components, EGFR (for EGF), FGFR1 IIIc and FGFR3 IIIc (for bFGF), and LIFR and gp130 (for LIF), were examined. As components of the Ras-MAPK pathway, H-Ras, Raf-1, MEK1 and ERK were examined. It was confirmed by RT-PCR that there is no significant reduction of mRNAs of these genes in ST-II transfectants (Figs 8a and b). Furthermore, not only mRNAs, but also proteins of some components (EGFR and ERKs), were expressed in ST-II transfectants (Fig. 8d). These results indicate that ERK MAPK repression in ST-II transfectants is not due to the down-regulation of components of cytokine receptors or the Ras-MAPK pathway.

image

Figure 8. Expression of components of cytokine receptors and the Ras-MAPK pathway. Expression of components of (a) receptors for EGF (EGFR), bFGF (FGFR1 IIIc or FGFR3 IIIc) and LIF (LIFR and gp130), and (b) the Ras-MAPK pathway (H-Ras, Raf-1, MEK1 and ERK2) in control C17.2 cells (co) or ST-II transfectants (ST-II) was examined by RT-PCR. – RT indicates negative control without reverse transcription. (c) Expression of EGFR, and ERK1 and ERK2 proteins, was confirmed by western blot analysis. (d) In ST-II transfectants, ERK activation was induced by constitutively active H-Ras (12 V).

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In order to examine whether the Ras-MAPK pathway is functionally intact, we transfected ST-II transfectants with H-Ras expression constructs. Although a control vector (vehicle) or wild-type H-Ras (WT) did not induce ERK activation, a constitutively active form of H-Ras (12V) induced ERK activation in ST-II transfectants (Fig. 8d). Therefore, it is suggested that the MAPK pathway is intact and defects are localized upstream of the MAPK pathway in ST-II transfectants.

Lipid rafts of C17.2 cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

GSLs including gangliosides were shown to be enriched in cell surface microdomains, lipid rafts (Hakomori et al. 1998; Simons and Toomre 2000). Lipid rafts have been suggested to be important for modulation of signal transduction and cell adhesion. Additionally, SOS-Grb2 complex and Ras, signaling molecules upstream of the MAPK pathway, are reported to be localized in lipid rafts (Wu et al. 1997). Therefore, we hypothesized that repression of proliferation and MAPK activation in ST-II transfectants are attributed to defects in lipid rafts.

First, we prepared lipid rafts from control C17.2 cells and ST-II transfectants by sucrose gradient ultracentrifugation (Iwabuchi et al. 1998) and performed western blot analysis for lipid raft markers, flotillin-1 and caveolon-1 (Bickel et al. 1997). Lipid rafts are known to concentrate at the interface between 5% and 30% sucrose solutions (fraction 4 in our protocol). Flotillin-1 was localized in fraction 4 of control cells and ST-II transfectants (Fig. 9a). Although it was detected in soluble fractions (fractions 9 and 10), caveolin-1 was localized in fraction 4 of both cells. This suggests that lipid rafts exist in ST-II transfectants as well as in control cells.

image

Figure 9. Involvement of lipid rafts in cytokine-induced ERK activation in C17.2 cells. (a) Lipid rafts were prepared from control C17.2 cells (co) or ST-II transfectants (ST-II) by sucrose gradient ultracentrifugation and then analyzed by western blot. Flotillin-1 and caveolin-1, marker proteins of lipid rafts, were mainly detected in fraction 4. (b) Viability of C17.2 cells treated with or without a lipid raft inhibitor, MBCD (1, 10 or 20 mm), for 1 h was evaluated by a Vi-Cell. (c) Localization of flotillin-1 and caveolin-1 in lipid raft fraction (fraction 4) and soluble fraction (fractions 9 and 10) of C17.2 cells treated with or without MBCD was assessed by western blot analysis. (d) C17.2 cells treated with MBCD (10 mm) for 1 h were stimulated with or without EGF or bFGF (20 ng/mL for 10 min) and activation of ERK was assessed by western blot analysis.

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Next, we examined the involvement of lipid rafts in cytokine-induced ERK activation. MBCD is an inhibitory molecule that binds to cholesterol and disrupts the functions of lipid rafts (Scheiffele et al. 1997). Since treatment by MBCD at a concentration of 10 mm for 1 h at 37°C is widely used for various cell types to disrupt lipid rafts, we treated C17.2 cells with these conditions. Although treatment with MBCD (10 mm for 1 h at 37°C) did not affect the viability of the cells (Fig. 9b), it induced a shift of lipid raft marker proteins (flotillin-1 and caveolin-1) to soluble fractions (Fig. 9c). These results suggest that MBCD specifically disrupts lipid rafts in C17.2 cells. In the cells treated with MBCD, ERK MAP activation induced by bFGF or EGF was repressed (Fig. 9d), suggesting that lipid rafts are involved in cytokine-induced ERK activation. Since lipid rafts were detected in ST-II transfectants (Fig. 9a), there might be functional defects in lipid rafts of ST-II transfectants.

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

The expression of gangliosides is abundant in neural cells and drastically changed during development of the CNS (Yu 1994; Yu et al. 2004). Therefore, gangliosides have been suggested to play an important role in the development of the nervous system. However, information regarding how gangliosides function in neural development is still scanty. Previously, we characterized the ganglioside composition in a C17.2 immortalized neural progenitor cell line that expresses a-series gangliosides but not b- and c-series gangliosides due to the lack of ST-II (GD3-synthase) transcripts (Suetake et al. 2003). In this study, we induced ectopic overexpression of b-series gangliosides in C17.2 cells by transfection with an ST-II-EGFP fusion protein. In ST-II transfectants, although a-series ganglioside expression was sustained, up-regulation of b-series gangliosides such as GD3, 9-O-acetyl GD3, GD1b and GT1b was detected. Surprisingly, GT3 ganglioside was also detected in ST-II transfectants, although ST-III (GT3-synthase) was not expressed in the cells. Nakayama et al. (1996) reported that ST-II synthesizes not only GD3 but also GT3, based on an overexpression experiment. This led them to conclude, mistakenly, that ST-II and ST-III are the same enzyme, whereas ST-II and ST-III are actually different enzymes (Yoshida et al. 1995; Zeng et al. 1996; Gao et al. 1997). Our data clearly indicated that overexpression of ST-II could induce c-series ganglioside(s), in addition to b-series gangliosides, in ST-II transfectants in the absence of ST-III transcripts.

Gangliosides are reported to modulate cell proliferation induced by cytokines such as EGF (review by Allende and Proia 2002). For example, GM3 ganglioside interacts with EGFR and inhibits signal transduction (Miljan et al. 2002). GD3 and its downstream derivative, 9-O-acetyl-GD3, are significantly expressed in proliferating neuroblast cells (Yu 1994) and were suggested to be involved in proliferation of a melanoma cell line (Zeng et al. 1999; Birkle et al. 2000). EGF-induced proliferation of ST-II transfectants ectopically expressing b- and c-series gangliosides was severely retarded. EGF-induced proliferation of C17.2 cells was dependent on the Ras-MAPK pathway, and the EGF-induced activation of this pathway was significantly repressed in the ST-II transfectants. Thus, it is considered that ST-II overexpression retarded proliferation of C17.2 cells via repression of activation of the Ras-MAPK pathway.

Neural stem/progenitor cells use EGF, bFGF and LIF as mitogens (Reynolds et al. 1992; Shimazaki et al. 2001). In ST-II transfectants, ERK MAPK activation induced by bFGF or LIF was also repressed. Furthermore, there was no defect in expression of cytokine receptors and signaling molecules of the Ras-MAPK pathway in ST-II transfectants. These results suggest that there are defects in a common signaling molecule(s) upstream of the MAPK pathway, such as a SOS-Grb2 complex or Ras molecule. Gangliosides are important components of lipid rafts on the cell surface, and SOS and Ras molecules are localized in lipid rafts (Wu et al. 1997). Additionally, bFGF- or EGF-induced ERK activation in C17.2 cells was dependent on the lipid rafts. Mitsuda et al. (2002) reported that overexpression of GM1 ganglioside repressed platelet-derived growth factor-induced ERK activation via dispersion of the receptor molecule in lipid rafts. Although the molecular mechanism underlying the repression of cell proliferation and ERK activation in ST-II transfectants is not clear, it may be via modification of lipid rafts by ectopic overexpression of gangliosides.

So far, some genetically-engineered mice lacking synthesis of major gangliosides such as UDP-N-acetylgalactosamine : GM3 N-acetylgalactosaminyltransferase (GalNAcT), ST-I, ST-II, or GalNAcT and ST-II, have been established (Kolter et al. 2002). Although these mutant mice all present certain phenotypic defects, for example lethal audiogenic seizures in GalNAcT/ST-II double-knockout mice and development of peripheral neuropathies in GalNAcT knockout mice, significant impairment of neural development was not reported. Because these mice do not completely lack ganglioside expression, the possibility that other gangliosides compensate for the lack of certain gangliosides cannot be eliminated. Instead of loss-of-function experiments, we used gain-of-function experiments in this study and found that ectopic expression of b- and c-series gangliosides repressed proliferation of immortalized neural progenitor cells via MAPK suppression. Therefore, this approach may be useful to reveal a bona fide function of gangliosides.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References

We thank Drs Evans Snyder for the C17.2 cell line, Erhard Bieberich for a ST-II-EGFP fusion protein expression vector, Joel Levine for a D1.1 antibody, and Shigekazu Nagata, Akihiko Yoshimura, Tetsuya Taga and Ikuo Nobuhisa for H-Ras-BOSE expression vectors. This work was supported by grants from National Institutes of Health (NS11853, NS26994) and a grant from the Children's Medical Research Foundation.

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  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Cell culture
  5. Reverse transcription-polymerase chain reaction (RT-PCR)
  6. Thin-layer chromatography (TLC) and TLC-immunostaining
  7. Flow cytometry
  8. Cell growth assay
  9. Western blot analysis
  10. Lipid rafts preparation
  11. Results
  12. Ectopic overexpression of b- and c-series gangliosides in ST-II transfectants
  13. Ras-MAPK pathway-dependent proliferation of C17.2 cells
  14. Repression of proliferation and MAPK activation in ST-II transfectants
  15. Expression of signaling molecules in ST-II transfectants
  16. Lipid rafts of C17.2 cells
  17. Discussion
  18. Acknowledgements
  19. References
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