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First published online 25 November 2008
doi: 10.1242/jcs.027995

Journal of Cell Science 121, 4089-4097 (2008)
Published by The Company of Biologists 2008
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EGFR-dependent migration of glial cells is mediated by reorganisation of N-cadherin

Anne Rappl1, Guido Piontek2 and Jürgen Schlegel1,2,*

1 Division of Neuropathology, Institute of Pathology, Technische Universität München, Ismaninger Str. 22, 81675 München, Germany
2 Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany


Figure 1
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  		Fig. 1. Diagram of the different HER2 constructs used in this study. The human HER2 receptor consists of an extracellular ligand-binding domain, a transmembrane (TM) domain and an intracellular domain containing the kinase domain and the region of the phosphorylation sites. HER2N represents the wild-type receptor. The constitutively active variant HER2VE was created by inserting the oncogenic mutation of the rat Neu gene into the transmembrane domain of the human ERBB2 (HER2) cDNA at amino acid position 659, substituting glutamate (E) for valin (V). The kinase-inactive form of the HER2 receptor HER2VEKA was created by mutating the ATP-binding site of the kinase domain of HER2VE at amino acid position 753, substituting alanin (A) for lysine (K).

 

Figure 2
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  		Fig. 2. Protein expression and activation of EGFR and HER2, and activation of EGFR- and HER2-dependent signalling pathways in human LN18 glial tumor cells after transfection with different HER2-receptor mutants. Parental (Ctrl) and LN18 cells stably expressing different HER2 constructs were serum-starved for 24 hours and then treated with EGF (100 ng/ml) for 5 minutes. (A) Expression of total HER2 or EGFR and levels of phosphorylated HER2 or EGFR were determined by western blot analysis using anti-HER2 or anti-EGFR antibodies, and antibodies specifically recognising P-HER2 or P-EGFR [pEGFR (pY1086), pHER2 (pY1248)]. {alpha}-tubulin was used as a loading control. (B) Western blot analysis of total and P-Akt [Akt and pAkt (Ser473), respectively] and total and P-MAPK [MAPK and pMAPK p42/p44 (Thr202/Thr204), respectively]. Total Akt and total MAPK were used as loading controls. Ctrl, wild-type control cells; N, HER2N cells; VE, HER2VE cells; VEKA, HER2VEKA cells.

 

Figure 3
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  		Fig. 3. Cell motility assays. (A) The migratory behaviour of parental LN18 cells (Ctrl), and LN18 cells transfected with wild-type HER2N, constitutively active HER2VE or dominant-negative HER2VEKA were serum starved and wound-healing assays were carried out as described in Materials and Methods. (Left panels) Initial wound edges. (Right panels) Phase-contrast pictures taken after 48 hours. Solid lines indicate initial scratches; dashed lines indicate healed area. Bar, 100 µm. (B) Wound closures of the different HER2-mutant-expressing cell clones were measured and are given as the percentage of the initial distance. Values are the means ± s.d. from at least three independent experiments. (C) Morphology of parental LN18 cells (Ctrl) and the HER2N-(N), HER2VE-(VE) and HER2VEKA cells (VEKA). Bar, 100 µm.

 

Figure 4
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  		Fig. 4. Expression and localisation of N-cadherin. (A) Western blot analysis of lysates of LN18 parental cells and the different HER2 cell clones cultured under standard growth conditions. Samples were immunoblotted for N-cadherin. {alpha}-tubulin was used as a loading control. (B) Different HER2 cell clones, immunostained for N-cadherin. Ctrl, wild-type control cells; N, HER2N cells; VE, HER2VE cells; VEKA, HER2VEKA cells. Bar, 20 µm.

 

Figure 5
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  		Fig. 5. Downregulation of N-cadherin results in enhanced motility of HER2VEKA cells. (A) Wound-healing assays of HER2VEKA-expressing cells treated with siRNA targeting N-cadherin (siNcad). Lines indicate the initial wound edges. Bar, 100 µm. (B) Wound closures of siNcad-treated and -untreated HER2VEKA cells were measured and are given as the percentage of the initial distance. Values are the means ± s.d. from at least three independent experiments. (C) Western blot analysis of HER2VEKA cells treated with siNcad for 24, 48 and 72 hours, compared with untreated HER2VEKA cells. (D) Immunofluorescence staining of HER2VEKA cells treated with siNcad siRNA for 24 and 48 hours, or left untreated. Bar, 20 µm.

 

Figure 6
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  		Fig. 6. Treatment of HER2VE cells with EGFR- and HER2-inhibitor results in the upregulation of N-cadherin and its recruitment to the cell membrane in (A) Western blot analysis of HER2VE-expressing cells treated with tyrphostin AG1478 (10 µM), AG825 (350 nM) or AEE788 (5 µM) for 24 hours. Lysates were immunoblotted using anti-N-cadherin antibody. {alpha}-tubulin was used as a loading control. (B) Immunofluorescence staining for N-cadherin after inhibitor treatment for 24 hours. Bar, 20 µm. (C) Wound-healing assay of HER2VE cells treated with tyrphostin AG1478 (10 µM), AG825 (350 nM) or AEE788 (5 µM) for 48 hours. (Left panels) Initial wound edges; (right panels) phase-contrast pictures taken after 48 hours. Solid lines indicate initial scratches; dashed lines indicate healed area. Bar, 100 µm.

 

Figure 7
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  		Fig. 7. EGFR-HER2 receptor complexes interact directly with N-cadherin. (A) Western blot analysis of HER2VE and HER2VEKA cells after immunoprecipitation using antibodies directed against total HER2 or EGFR (upper panel) and N-cadherin (lower panel). Precipitates were immunoblotted for N-cadherin (upper panel) and EGFR or HER2 (lower panel). (B) Western blot analysis of LN18 cells and HER2 clones after immunoprecipitation using antibodies directed against β-catenin. Precipitates were immunoblotted for tyrosine phosphorylation of β-catenin using the anti-phospho-tyrosine antibody PY20 (upper panel). Western blot analysis of P-β-catenin (phosphorylated at Thr41 and Ser45) and P-GSK3β (phosphorylated at Ser9 and Ser21) (lower panel). WB, western blot analysis; IP, immunoprecipitation; ctrl, LN18 wild-type control cells; N, HER2N cells; VE, HER2VE cells; VEKA, HER2VEKA cells.

 

Figure 8
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  		Fig. 8. Model of EGFR-HER2-receptor-mediated reorganisation of N-cadherin, and of changes in glial cell migration. (Left) EGFR-HER2VEKA complexes do not directly interact with N-cadherin–β-catenin, leaving N-cadherin expressed at the plasma membrane. Owing to dominant-negative HER2VEKA the PI3K/Akt pathway is not activated. Therefore, GSK3β is not inhibited and phosphorylates β-catenin, leading to β-catenin degradation, which, consequently, might lead to inhibition of migration. N-cadherin is stabilised at the cell surface and β-catenin stays in the adherens-junction complex. (Right) Constitutively activated EGFR-HER2VE receptor complexes directly interact with N-cadherin–β-catenin. Tyrosine phosphorylation of β-catenin leads to a decrease of N-cadherin–β-catenin complexes in the plasma membrane (Lilien and Balsamo, 2005Go). Activation of the PI3K/Akt signalling pathway inhibits GSK3β. Thus, β-catenin is not tagged for ubiquitylation. Moreover, it has been shown recently, that Akt can phosphorylate β-catenin at Ser552, leading to its transcriptional activation (Fang et al., 2007Go). The strong EGFR-HER2VE signal, therefore, leads to disruption of adherens junctions, by disassociation of its components from the cell surface.

 

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