N-cadherin mediates neuronal cell survival through Bim down-regulation.
ABSTRACT N-cadherin is a major adhesion molecule involved in the development and plasticity of the nervous system. N-cadherin-mediated cell adhesion regulates neuroepithelial cell polarity, neuronal precursor migration, growth cone migration and synaptic plasticity. In vitro, it has been involved in signaling events regulating processes such as cell mobility, proliferation and differentiation. N-cadherin has also been implicated in adhesion-dependent protection against apoptosis in non-neuronal cells. In this study, we investigated if the engagement of N-cadherin participates to the control of neuronal cells survival/death balance. We observed that plating either primary mouse spinal cord neurons or primary rat hippocampal neurons on N-cadherin recombinant substrate greatly enhances their survival compared to non-specific adhesion on poly-L-lysine. We show that N-cadherin engagement, in the absence of other survival factors (cell-matrix interactions and serum), protects GT1-7 neuronal cells against apoptosis. Using this cell line, we then searched for the signaling pathways involved in the survival effect of N-cadherin engagement. The PI3-kinase/Akt survival pathway and its downstream effector Bad are not involved, as no phosphorylation of Akt or Bad proteins in response to N-cadherin engagement was observed. In contrast, N-cadherin engagement activated the Erk1/2 MAP kinase pathway. Moreover, N-cadherin ligation mediated a 2-fold decrease in the level of the pro-apoptotic protein Bim-EL whereas the level of the anti-apoptotic protein Bcl-2 was unchanged. Inhibition of Mek1/2 kinases with U0126, and the resulting inhibition of Erk1/2 phosphorylation, induced the increase of both the level of Bim-EL and apoptosis of cells seeded on the N-cadherin substrate, suggesting that Erk phosphorylation is necessary for cell survival. Finally, the overexpression of a phosphorylation defective form of Bim-EL prevented N-cadherin-engagement induced cell survival. In conclusion, our results show that N-cadherin engagement mediates neuronal cell survival by enhancing the MAP kinase pathway and down-regulating the pro-apoptotic protein Bim-EL.
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ABSTRACT: In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex.Journal of Neuroscience 11/2013; 33(46):18149-60. · 6.91 Impact Factor
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ABSTRACT: N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.Oncogene advance online publication, 26 August 2013; doi:10.1038/onc.2013.310.Oncogene 08/2013; · 8.56 Impact Factor
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ABSTRACT: Fbxo45 is an atypical E3 ubiquitin ligase which specifically targets proteins for ubiquitin-mediated degradation. Fbxo45 ablation results in defective neuronal differentiation and abnormal formation of neural connections, however the mechanisms underlying these defects are poorly understood. Using an unbiased mass spectrometry-based proteomic screen, we show here that N-cadherin is a novel interactor of Fbxo45. N-cadherin specifically interacts with Fbxo45 through two consensus motifs overlapping the site of calcium-binding and dimerization of the cadherin molecule. N-cadherin interaction with Fbxo45 is significantly abrogated by calcium treatment. Surprisingly, Fbxo45 depletion by RNAi-mediated silencing results in enhanced proteolysis of N-cadherin. Conversely, ectopic expression of Fbxo45 results in decreased proteolysis of N-cadherin. Fbxo45 depletion results in dramatic reduction in N-cadherin expression, impaired neuronal differentiation and diminished formation of neuronal processes. Our studies reveal an unanticipated role for an F-box protein which inhibits proteolysis in the regulation of a critical biological process.The Journal of biological chemistry. 08/2014;
N-Cadherin Mediates Neuronal Cell Survival through Bim
Elise C. Lelie `vre1,2,3., Charlotte Plestant1,2,3., Ce ´cile Boscher1,2,3¤, Emeline Wolff1,2,3, Rene ´-Marc
Me `ge1,2,3*", He ´le `ne Birbes1,2,3*"
1INSERM, UMRS-839, Paris, France, 2Universite ´ Pierre et Marie Curie-Paris 6, Paris, France, 3Institut du Fer a ` Moulin, Paris, France
N-cadherin is a major adhesion molecule involved in the development and plasticity of the nervous system. N-cadherin-
mediated cell adhesion regulates neuroepithelial cell polarity, neuronal precursor migration, growth cone migration and
synaptic plasticity. In vitro, it has been involved in signaling events regulating processes such as cell mobility, proliferation
and differentiation. N-cadherin has also been implicated in adhesion-dependent protection against apoptosis in non-
neuronal cells. In this study, we investigated if the engagement of N-cadherin participates to the control of neuronal cells
survival/death balance. We observed that plating either primary mouse spinal cord neurons or primary rat hippocampal
neurons on N-cadherin recombinant substrate greatly enhances their survival compared to non-specific adhesion on poly-L-
lysine. We show that N-cadherin engagement, in the absence of other survival factors (cell-matrix interactions and serum),
protects GT1-7 neuronal cells against apoptosis. Using this cell line, we then searched for the signaling pathways involved in
the survival effect of N-cadherin engagement. The PI3-kinase/Akt survival pathway and its downstream effector Bad are not
involved, as no phosphorylation of Akt or Bad proteins in response to N-cadherin engagement was observed. In contrast, N-
cadherin engagement activated the Erk1/2 MAP kinase pathway. Moreover, N-cadherin ligation mediated a 2-fold decrease
in the level of the pro-apoptotic protein Bim-EL whereas the level of the anti-apoptotic protein Bcl-2 was unchanged.
Inhibition of Mek1/2 kinases with U0126, and the resulting inhibition of Erk1/2 phosphorylation, induced the increase of
both the level of Bim-EL and apoptosis of cells seeded on the N-cadherin substrate, suggesting that Erk phosphorylation is
necessary for cell survival. Finally, the overexpression of a phosphorylation defective form of Bim-EL prevented N-cadherin-
engagement induced cell survival. In conclusion, our results show that N-cadherin engagement mediates neuronal cell
survival by enhancing the MAP kinase pathway and down-regulating the pro-apoptotic protein Bim-EL.
Citation: Lelie `vre EC, Plestant C, Boscher C, Wolff E, Me `ge R-M, et al. (2012) N-Cadherin Mediates Neuronal Cell Survival through Bim Down-Regulation. PLoS
ONE 7(3): e33206. doi:10.1371/journal.pone.0033206
Editor: Masuko Ushio-Fukai, University of Illinois at Chicago, United States of America
Received December 9, 2010; Accepted February 11, 2012; Published March 12, 2012
Copyright: ? 2012 Lelie `vre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by institutional funding from INSERM (Institut National de la Sante ´ et Recherche Me ´dicale), Universite ´ Pierre et Marie Curie, as
well as grants from AFM (Association Franc ¸aise contre les Myopathies) and ARC (Association Franc ¸aise de Recherche contre le Cancer). Dr. Lelie `vre, Dr. Plestant
and Dr. Boscher have been supported by French Research Ministry graduate student fellowships. ARC provided also Dr. Boscher with a PhD fellowship. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (RMM); email@example.com (HB)
¤ Current address: Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
Cadherins are a family of transmembrane proteins that mediate
calcium-dependent homophilic cell-cell contacts. They provide
anchorage in between neighboring cells by interacting with the
actin cytoskeleton through catenins . Cadherins play crucial
roles in embryonic morphogenesis by affecting cell shape,
differentiation, migration and proliferation . In addition,
cadherin-mediated cell-cell interactions may regulate the balance
between cell survival and cell death during development and
maintenance of tissue homeostasis. Indeed several studies have
reported that disruption of cadherin adhesion initiates apoptosis in
various epithelial [3,4,5] and tumor cells [6,7,8,9]. However, the
mechanisms whereby cadherin adhesion contributes to cell fate by
regulating the survival/cell death balance is poorly understood by
comparison to the well-known molecular mechanism of anoı ¨kis
(apoptosis induced by the loss of cell contacts with the extracellular
matrix (ECM)) . Depending on cell types, the initiation and
execution of anoı ¨kis, which is refrained in physiological conditions
by integrin engagement, is mediated by different pathways that all
converge to the regulation of anti-apoptotic and pro-apoptotic Bcl-
2 family proteins, leading to activation of caspases and subsequent
activation of endonucleases, DNA fragmentation and eventually
cell death .
Proteins of the Bcl-2 family are major regulators of apoptosis
. Anti-apoptotic members, such as Bcl-2, Bcl-xL, and Mcl-1
contain four Bcl-2 homology (BH) domains while the pro-
apoptotic proteins fall into two categories: the Bax-like proteins,
such as Bax and Bak that contain multiple BH domains, and the
BH3-only members, Bad, Bid, Bmf, and Bim that contain only the
BH3 domain. These pro-apoptotic proteins promote the release of
apoptogenic factors from the mitochondria. Several studies
PLoS ONE | www.plosone.org1 March 2012 | Volume 7 | Issue 3 | e33206
indicate that the BH3-only members bind to the pro-survival Bcl-2
proteins and neutralize them, thereby allowing Bax-like proteins to
initiate apoptosis . The different BH3-only members respond
to different forms of cellular stress and are subject to regulation at
both transcriptional and posttranslation levels. During anoı ¨kis,
activation of MAP kinase, PI-3 kinase/Akt and JNK pathways
regulate Bcl-2 family members such as the anti-apoptotic Bcl-2
and Bcl-xL proteins or the pro-apoptotic proteins Bad or Bim .
Bim primary transcripts undergo alternative splicing to generate
three major isoforms: Bim-S, Bim-L and Bim-EL, the latter
representing the predominant isoform in most tissues . Bim-
EL is up-regulated during anoı ¨kis while its down-regulation by
RNA interference inhibits anoı ¨kis . It has been established that
[13,15,16,17]. Indeed, Putcha et al., have reported that Nerve
Growth Factor (NGF) deprivation in sympathetic neurons rapidly
induced expression of Bim-EL . Conversely, Bim deletion
conferred protection against cytochrome c release and neuronal
apoptosis upon NGF deprivation. Similar protective effect of Bim
deletion against apoptosis was observed in cerebellar granule
neurons subjected to K+withdrawal .
N-cadherin is the predominant form of cadherin expressed
ubiquitously in the early neural tube . N-cadherin participates
to the development and functional organization of the adult neural
tissue . This receptor is necessary for the polarisation of the
neuroepithelial sheets, the radial organization of developing
cortical plate and likely the migration of neuronal precursors
. It is also implicated in neurite outgrowth, dendritic
arborization, axon guidance, and in the early stages of
synaptogenesis . Later in development, N-cadherin localizes
at synapses , where it not only plays an adhesive role but also
participates to the regulation of synaptic function and plasticity
[23,24,25]. In this study, we assessed whether cadherin-mediated
cell-cell adhesion provides a survival signal for neuronal cells and
thereby focused our study on the contribution of N-cadherin. For
that purpose, we used an immobilized N-cadherin (N-cad)
recombinant protein coated on culture plates mimicking cell-cell
contact formation to tightly control cadherin engagement [26,27].
Controlled mobilization of N-cadherin was shown to induce cell
cycle exit and myogenic differentiation  and to promote
neurite outgrowth [29,30]. Our work demonstrates that N-
cadherin-mediated adhesion provides neuronal cells with pro-
survival signal, protecting them from apoptosis in the absence of
growth factors and extracellular matrix stimuli. In addition, we
describe a molecular pathway by which cadherin regulates cell
survival. Indeed, we report that the engagement of N-cadherin
results in the phosphorylation of Erk1/2 and the down-regulation
of the pro-apoptotic protein Bim, whereas the PI3-kinase/Akt
survival pathway and Bcl-2 protein are not affected.
Materials and Methods
Antibodies and reagents
The following antibodies were used for western blotting : rabbit
anti-Akt (1:1000; Cell Signaling Technology, Beverly, MA), rabbit
anti-phospho-Akt (1:1000; Cell Signaling Technology), rabbit anti-
Erk1/2 (1/10000; Upstate Biotechnology, Inc. Lake Placid, NY),
mouse anti-phospho-Erk1/2 (1/10000; Sigma), mouse anti-Bcl-2
(1/500; Transduction Laboratories, Becton Dickinson Europe, Le
Pont de Claix, France), rabbit anti-Bcl-xL (1/1000; Cell Signaling
Technology), rabbit anti-Bim (1/1000; Pharmingen, San Diego,
CA), rabbit anti-phospho-Bad (1/1000; Cell Signaling Technol-
ogy), rabbit anti-PARP (1/1000; Cell signalling Technology),
mouse anti-b-actin (1/20000; clone AC-15; Sigma), mouse anti-a-
tubulin (1:20000, clone E7, Developmental Studies Hybridoma
Bank). Mouse anti-cytochrome c (1/200, clone 6H2.B4; Pharmin-
gen) anti-bIII-tubulin (1/1000, clone Tuj1; Covance, CA) and
anti-paxillin antibodies (1/100, Upstate Biotechnologies) mono-
clonal antibodies were used for immunostaining. 49,6-diamino-2-
phenylindole, dihydrochloride (DAPI) was from Molecular Probes
(Eugene, OR). U0126 and LY294002 were from Cell Signaling
Technology, PMA was from Sigma, Triton X-100 was from
The expression vector encoding for the dominant negative
mutant form of N-cadherin deleted of its extracellular domain
(DN-Ncad), initially described in , has been modified by
replacing the Myc tag by the DsRed protein coding sequence .
The expression vectors coding for wild type Bim-EL and the ERK-
dependent phosphorylation site defective mutant Bim-EL (S69G)
have been described in .
Mouse GT1-7 cells  were grown at 37uC, 5%CO2, in
Dulbecco’s Modified Eagle Medium with 4.5 g/l glucose (DMEM,
Life Technologies) supplemented with 10% fetal bovine serum
(FBS, Life Technologies) and antibiotics (100 IU/ml penicillin and
100 mg/ml streptomycin, Life Technologies). GT1-7 cells were
transfected using Lipofectamine 2000 (Life Technologies). Cells
were seeded at 8.104cells/cm2and transfected 24 hours later
according to manufacturer’s instructions.
Primary mouse ventral spinal cord and rat hippocampal
neurons were prepared as previously described [35,36]. Briefly,
ventral spinal cord of E12.5 mouse embryos from the OF1
pregnant mice were isolated and digested with trypsin for 10 min
at 37uC in Ham F10 modified medium containing 0.025% trypsin.
Tissues were then mechanically dissociated in L15 medium
containing DNAse I and the supernatant was laid on a BSA 4%
solution and centrifuged. Spinal cells were seeded on coated
coverslips at 2.104cells/cm2in Neurobasal medium containing
0.5 mM L-glutamine and cultivated for 24 hours to 48 hours at
37uC, 5% CO2. Hippocampi from E18.5 rat embryos (Sprague
Dawley pregnant rat, R. Janvier laboratories) were isolated in
HBSS-20 mM Hepes and digested with trypsin 0,05% 10 min at
37uC, then mechanically dissociated. Hippocampal cells were
recovered in MEM medium supplemented with 2 mM glutamine,
1 mM sodium pyruvate and seeded on coated coverslips at 2.104
cells/cm2and cultivated for 24 hours at 37uC, 5% CO2. All
experiments with animals were in accordance with the guidelines
of the French Agriculture and Forestry Ministry for handling
animals (decree 87849, license 75–765-Renouvellement) and
approved by the ‘‘Charles Darwin ethical committee in animal
experimentation, Paris’’ under permit number Ce5/2010/064.
Adhesive substrates were prepared as described in . Briefly,
glass coverslips or thermosterilized bacterial 35-mm Petri dishes
(Falcon, Becton Dickinson Europe) were incubated with goat anti-
mouse Fcc fragment antibody (Jackson ImmunoResearch, West
Grove, PA) at 1 mg/cm2in 0.1 M borate buffer pH 8.0 overnight
at 4uC. Plates were washed and then incubated with purified
Ncad-Fc chimera (extracellular domain of the chicken N-cadherin
fused to the mouse IgG2b Fc fragment ) at a concentration of
1 mg/cm2for 2 hours at room temperature. For the control
samples, glass coverslips or dishes were incubated with poly-L-
lysine (PL, 0.01%, Sigma) or fibronectin (FN, 10 mg/cm2,
Invitrogen), in water overnight at 4uC. After washes, surfaces
were then saturated with 1.5% purified BSA (Sigma) in PBS for 5–
10 min at room temperature.
N-Cadherin Anti-Apoptotic Signaling
PLoS ONE | www.plosone.org2March 2012 | Volume 7 | Issue 3 | e33206
Controlled cell adhesion assays
Specific cell adhesion and spreading on coated surfaces were
performed according to . Briefly, to preserve cell-surface
cadherins, twenty-four hours serum-starved GT1-7 cells were
mechanical dissociated in trypsin-free conditions with PBS,
3.5 mM EDTA, 1.5% BSA on ice. Cells were then plated on
the different adhesion substrates in serum-free conditions to
prevent growth factors-mediated survival and at low cell density
(8.104cells/cm2) to prevent cell-cell contacts between neighboring
cells. For experiments using inhibitors, cells were incubated 1 hour
with the specific inhibitor prior to mechanically dissociation and
throughout culture on the different substrates with inhibitors at the
concentrations and times indicated. To induce Bad and Erk1/2
protein phosphorylation, confluent cells were treated with Phorbol
12-myristate 13-acetate (PMA) at 10 ng/ml for 1 hour.
Calcium switch assays
Confluent GT1-7 cell monolayers were deprived of serum for
24 hours, and the N-cadherin-mediated cell-cell contacts were
disrupted by treatment with 4 mM EGTA in DMEM for 40 min
at 37uC. The calcium-free medium was removed and N-cadherin
contacts were allowed to re-establish by addition of DMEM,
containing 1.8 mM Ca2+. The time of re-addition of Ca2+was
considered as 0 min. Following calcium restoration at 37uC, cells
were harvested, lysed, protein extracts separated by SDS-PAGE
and immunoblotted as described below.
Alternatively, serum deprived GT1-7 cells were maintained in
suspension in 1% agarose coated 60 mm culture dishes (10.104
cells/dish) to prevent cell attachment. Cells were either incubated
in the presence of 1.8 mM Ca2+(provided by the DMEM
medium) or in the presence of DMEM plus 2.5 mM EDTA
(0 mM free Ca2+) to prevent cadherin-dependent cell-cell
adhesion. At various time points, cells were washed once with
cold PBS, collected and processed for analysis of PARP cleavage
Cells were lysed in RIPA modified buffer (50 mM Tris-HCl,
pH 7.4, 1% NP-40, 0.25% DOC, 150 mM NaCl, 20 mM sodium
pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthova-
nadate and protease inhibitor cocktail (Roche)). Lysates were
vortexed 20 min at 4uC and then cleared by centrifugation at 10,
000 rpm for 15 min at 4uC. Equal amounts of proteins (20 mg
determined by micro-BCA kit, Pierce) were loaded and separated
by SDS-PAGE. After transfer at 4uC, nitrocellulose membranes
were blocked with 5% nonfat milk in Tris-buffered saline, pH 8.0,
containing 0.1% Tween 20 (TBST) prior to addition of the
corresponding primary antibody and followed with IRDye-
coupled secondary antibodies (Rockland) against rabbit or mouse
immunoglobulins. For detection of phosphorylated proteins,
antigens were detected with phospho-specific antibodies diluted
in 5% BSA in TBST at 4uC. Protein bands were identified with
Odyssey Imaging System (LI-COR Biosciences). Signals were
quantified with LI-COR software. The membranes were reprobed
with an anti-b-tubulin, anti-b-actin monoclonal or anti-pan Erk1/
2 polyclonal antibodies for normalization.
Immunocytochemistry and TUNEL assay
Cytochrome c release was followed by immunostaining as
described previously . Briefly, at the end of the treatment time,
cells were washed gently once with PBS followed by fixation in 4%
formaldehyde for 15 min. The fixed cells were washed three times
with PBS 5 min each, followed by permeabilization in 0.15%
Triton X-100 in PBS for 15 min. The cells were then blocked for
60 min in blocking buffer (2% BSA in PBS) followed by 4 hours
incubation with a mouse monoclonal antibody against cytochrome
c. The cells were washed three times for 10 min each in blocking
buffer followed by 1 hour incubation with a rhodamine-labeled
goat anti-mouse IgG (1/500, Jackson Immunology). The cells were
washed three times for 10 min in PBS, counterstained with 0.5–
1 mg/ml DAPI, to examine nuclear morphology and quantify
apoptosis. TUNEL assay was performed using the In Situ Cell
Death Detection Kit, TMR Red (Roche Diagnostics) following the
manufacturer’s protocol. Cells were viewed under a fluorescence
microscope (Leica DM6000) and images were captured with a
digital camera using Metamorph software. Different fields (at least
200 cells) were counted for each experiment.
Data were compared by ANOVA followed by protected t test
for multiple comparisons. Paired sample means were compared
using the t test. P values of 0.05 or less were considered statistically
N-cadherin engagement promotes survival of primary
In primary neuron culture studies, most culture protocols have
been optimized to grow neurons on poly-ornithine (PO) or poly-L-
lysine (PL) attachment factors. Associated culture media have been
adapted by adding sera and/or B27 supplement. We observed that
when these media additives were omitted from E18.5 rat
hippocampus neurons cultured on PL, although cells initially
bound to the plate, they rapidly died (Fig. S1). In contrast,
replacing PL by recombinant Ncad-Fc (N-cad) as a substrate
allowed maintaining healthy neurons in culture for at least 24 to
48 hours (Fig. S1). Although cells were seeded in similar conditions
for each substratum, the observed reduction in healthy neurons on
PL might be due to a deficit in the initial adhesion of these cells on
PL. To assess this point, we counted the actual number of cells
attached at 1 hour, 4 and 24 hours after seeding, and observed
that there was no deficit of cell binding on PL compared to N-cad
at early time points (Fig. S2A). This was confirmed by the fact that
we were able to observe by time-lapse microscopy that cells
initially attach then die (data not shown).
To further characterize the effect of N-cadherin on survival of
rat hippocampal neurons in the absence of B27 additive, cultures
were fixed after 24 hours and stained for bIII-tubulin to identify
neuronal cells. The total number of remaining bIII-tubulin
positive cells was dramatically lower on the PL substrate (below
20%) in comparison to the number of cells remaining on the N-
cad substrate (Fig. 1A, 2A), confirming that in these basal
conditions, N-cad had a survival effect on primary hippocampal
neurons. Similar observations were made for primary murine
spinal cord cultures (Fig. 1B, 2B). In this case, the difference in the
total number of bIII-tubulin positive cells between the PL and N-
cad substrate was not as dramatic after 24 hours. However, DAPI
staining revealed that the percentage of presumably apoptotic
condensed nuclei was higher on PL (5462% of bIII-tubulin
positive cells with condensed nuclei) than on N-cad substrates
(2563% of bIII-tubulin positive cells with condensed nuclei). In
addition, when we analyzed cell death by terminal deoxynucleo-
tidyl transferase dUTP nick end labeling (TUNEL) we observed
that the number of TUNEL positive cells in hippocampal cultures
increased with time on PL whereas it remained very low on N-cad
(Fig. S2, B). Altogether these observations show that primary
N-Cadherin Anti-Apoptotic Signaling
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N-Cadherin Anti-Apoptotic Signaling
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neurons die, likely by apoptosis, in these stringent conditions when
plated on PL, while they displayed a high survival rate when plated
on N-cad. They suggest thus that N-cadherin engagement
promotes their survival.
N-cadherin engagement protects GT1-7 cells from
However, molecular and biochemical studies on primary
culture models are difficult to perform due to the limited amount
of biological material. Therefore to study the molecular pathway
whereby N-cadherin mediates cell survival, we decided to use
GT1-7 immortalized hypothalamic neurons , which express
N-cadherin. These cells usually grow as confluent layers in serum-
supplemented media which provides them cell-cell adhesion as
well as diffusive (growth factors) and anchorage-dependent
(fibronectin/vitronectin) survival factors.
To investigate the contribution of cadherin-mediated cell-cell
adhesion to neuronal cell survival, we forced serum-starved GT1-7
cells to grow in suspension by plating them on agarose-coated
dishes, as previously reported [7,8]. We then manipulated
extracellular calcium concentrations to allow (1.8 mM Ca2+) or
inhibit (0 mM free Ca2+) calcium–dependent cell-cell adhesion.
The presence of a physiological concentration of Ca2+induced the
expected cell aggregation, while GT1-7 cells only loosely
aggregated in the absence of free Ca2+consistent with a massive
inhibition of cell-cell adhesion (Fig. 3A). To determine the
involvement of calcium-dependent cell adhesion on cell survival,
we then analyzed the cleavage of poly (ADP-ribose) polymerase
(PARP) induced in apoptotic cells by activated caspases. The
inhibition of calcium-dependent cell-cell adhesion specifically
induced the PARP cleavage whereas PARP cleavage remained
very low in cells cultured in the presence of calcium (Fig. 3B).
These results show that cells forced to grow under serum-free
anchorage-independent conditions survive as multicellular aggre-
gates and that Ca2+-dependent cell adhesion sustain cell survival in
these conditions, as previously reported in other cellular models
Next, we set up more controlled cell culture conditions to
investigate specifically the effect of N-cadherin adhesion on cell
survival. GT1-7 cells were seeded either on N-cad substrate to
specifically engage N-cadherin homophilic liganding or on PL to
deprive them of specific adhesion, in (i) serum-free conditions to
exclude cell-ECM and growth factors activated signaling, and (ii)
at low density to prevent uncontrolled cell-cell contact formation
(Fig. S3). In some control experiments, cells were seeded on
fibronectin substrate (FN) to allow for controlled integrin
mobilization, a situation known to mediate cell survival.
GT1-7 cell death was first evaluated by the nuclear condensa-
tion after DNA staining (Fig. 4). 72% of cells seeded on PL were
positive for nuclear condensation whereas only 18% of cells seeded
on N-cad exhibited condensed nuclei after 24 hours (Fig. 5A).
Interestingly, a similar low proportion of condensed nuclei (,20%)
was also obtained among cells seeded on fibronectin substrate
(FN), where cell-matrix-anchorage was expected to induce an
integrin-mediated cell survival pathway. To exclude the possibility
that cell survival observed on N-cad was the consequence of
integrin engagement induced by the deposition of ECM
components by GT1-7 cells themselves, we evaluated integrin
pathway activation by phosphorylated FAK and paxillin immu-
nostaining. We did not detect paxillin (Fig. S4) nor phosphorylated
forms of FAK (data not shown) accumulation at the ventral face of
the cells seeded on PL or N-cad while both stainings were strongly
Figure 1. N-cadherin engagement sustains spinal cord and hippocampal neuronal cell survival in minimal medium conditions.
Neurons were dissociated from E18.5 rat hippocampus (A) or E12.5 mouse ventral spinal cords (B) and cultured on N-cad or PL in MEM and
Neurobasal basal medium, for hippocampal and spinal neurons, respectively. After 24 hours, they were fixed, and stained with anti-bIII tubulin
antibody to identify neurons and with DAPI to assess the nuclear morphology. Arrows point toward cells dying with condensed nuclei while asterisks
indicate cells with healthy nuclei. Scale bar: 10 mm in A; 20 mm in B.
Figure 2. N-cadherin engagement sustains spinal cord and hippocampal neuronal cell survival. (A) In 24 hours hippocampal cultures,
the number of bIII tubulin-positive cells per mm2were counted on each substrate and expressed as a percentage of that on N-cad substrates,
arbitrarily fixed to 100%. (B) The numbers of bIII tubulin-positive cells with condensed (white bars) or non-condensed nuclei (dark bars) were
determined in ventral spinal cords cultures after 24 hours on each substrate. The number of bIII tubulin-positive cells per mm2was expressed as a
percentage of the number of bIII tubulin-positive cells per mm2on N-cad substrates, arbitrarily fixed to 100%. Results are expressed as the mean 6 SD
of two independent experiments.
N-Cadherin Anti-Apoptotic Signaling
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accumulated at focal adhesions in GT1-7 cells seeded on FN (Fig.
S4 and data not shown). To confirm that the survival effect was the
result of a specific engagement of N-cadherin, we tested the ability
of a dominant negative mutant form of N-cadherin  to perturb
cell survival triggered by N-cadherin. This DN-Ncad mutant was
expressed in GT1-7 cells that were then seeded on N-cad
substrate. While most of the control GT1-7 cells transfected with
a GFP-encoding plasmid displayed healthy nuclei morphology
after 24 hours on the N-cad substrate, more than 50% of the
DsRed positive cells displayed condensed nuclei (Fig. S5),
indicating that the expression of DN-Ncad counteracts the
protective effect of the N-cad substrate. These results suggest that
N-cadherin engagement protects cells against apoptosis in a
To gain insight on the specific cell death pathway activated,
cytochrome c release from mitochondria to the cytosol was
evaluated by immunostaining (Fig. 4). After 24 hours, a vast
majority of cells (80%) seeded on PL showed a cytochrome c
distribution characteristic of cytosolic localization and very often a
total loss of cytochrome c, as reported in , (Fig. 5B). In
contrast, most of the cells seeded on N-cad showed a punctuated
or tubular perinuclear pattern characteristic of the mitochondria
network and only 22% of the cells had released their cytochrome c
out of mitochondria. Similar results were obtained with cells
seeded on FN (positive control of cell survival), with less than 20%
of cells showing diffuse cytochrome c staining (Fig. 5B). Time
course studies showed that nuclear condensation and cytochrome
c release were already high 6 hours after plating cells on PL
whereas the percentage of apoptotic figures remained low and
constant for cells on N-cad (Fig. S6).
To confirm that these cytological changes were part of an
ongoing apoptotic process that was repressed by N-cadherin
Figure 3. Inhibition of calcium-dependent cell-cell adhesion induces apoptosis of GT1-7 cells in suspension. Serum-starved GT1-7 cells
were cultured in suspension in agarose-coated dishes in the presence of calcium (1.8 mM) or without calcium (2.5 mM EDTA). (A) Phase-contrast
photomicrographs were taken at time 0 and 24 hr of incubation. (B) Cells lysates were then analyzed by western blotting for PARP cleavage. Western
blot representative of two independent experiments.
N-Cadherin Anti-Apoptotic Signaling
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engagement, we analyzed cell death by two complementary
approaches: (i) TUNEL staining and (ii) PARP cleavage. After
4 hours of culture, the number of TUNEL positive GT1-7 cells
was significantly higher on PL (8.660.9%) than on N-cad
(2.160.6%) indicating that the deprivation of specific cell adhesion
(PL substrate) induced apoptotic cell death (Fig. 5C). Moreover,
when GT1-7 cells were seeded on PL, PARP cleavage was
detected by the appearance of the characteristic 85 kDa fragment.
By contrast, cells seeded on N-cad showed a reduced PARP
cleavage (Fig. 5D). Altogether, these results indicate that N-
cadherin engagement is sufficient to sustain GT1-7 cell survival by
protecting them from apoptotic cell death.
PI3-kinase/Akt pathway is not involved in N-cadherin
To elucidate the pathways involved in the protective effects of
N-cadherin mobilization, we studied the activation of the PI3-
kinase/Akt pathway, a key regulator of cell survival. First, we
investigated whether N-cadherin mobilization could activate Akt
kinase by phosphorylation on Ser-473. We previously showed that
cells require at least 2 hours in order to spread on N-cadherin
substrate; therefore, Akt phosphorylation was investigated by
western blotting analysis using a phospho-specific antibody starting
at 2 hours after plating. As cell death had reached a plateau at
6 hours, phosphorylation studies were conducted up to 6 hours.
Spreading of cells on N-cad did not affect Akt phosphorylation at
any time point studied (Fig. 6A). To confirm these results, we used
a calcium switch approach with GT1-7 grown at confluency.
Although less specific than Ncad-Fc mediated engagement,
calcium restoration rapidly initiates cadherin-dependent cell-cell
contact formation  allowing study of earlier time points.
However, Akt phosphorylation was not changed even at earlier
time points (Fig. 6B), confirming that Akt phosphorylation was not
activated by N-cadherin engagement.
Alternatively, Akt could be a direct substrate for caspases.
Indeed, it has been shown that Akt is cleaved by caspases leading
to a reduction in Akt protein level in MDCK cells detached from
the ECM. In turn this cleavage of Akt contributes to anoı ¨kis .
To test this hypothesis, we assessed the total amount of Akt at
various time points, but cellular levels of total Akt protein were not
decreased on PL substrate compared with those on N-cad
(Fig. 6C). Altogether, these observations suggest that the PI3-
kinase/Akt pathway is not involved in the anti-apoptotic effect of
N-cadherin in GT1-7 cells. In addition, the PI3-kinase inhibitor
LY294002 had no effect on cell survival, nuclear condensation and
TUNEL labeling of cell seeded on N-cad (data not shown). Thus,
we investigated the involvement of other pathways, such as MAP
kinase signaling, which has been reported to mediate cell survival
MAP kinase Erk signaling is required for cell survival
induced by N-cadherin engagement
To examine whether N-cadherin engagement could trigger a
signal transduction pathway involving the MAP kinases, we
evaluated the phosphorylation state of Erk1 and Erk2 using a
phospho-specific antibody. The phosphorylation of Erk1/2 was
increased in a time-dependent manner in cells seeded on N-cad
substrate as compared to cells seeded on PL. Phosphorylation of
Erk1 and Erk2 was maximal at 4 hours with a 2.5 fold increase
compared to cells seeded on PL (Fig. 7A). Furthermore, the
restoration of intercellular adhesion in the calcium switch assay
induced a strong elevation of Erk1 and Erk2 phosphorylation with
a 6-fold increase observed at 30 min (Fig. 7B). These results
support an activation of the Erk1/2 pathway in response to N-
cadherin engagement in accordance with our previous report on
the activation of Erk1/2 by N-cadherin engagement in primary
spinal cord neurons.
To confirm the involvement of the MAP kinase pathway in
relaying the anti-apoptotic signal triggered by N-cadherin
engagement, we examined the effect of U0126, a specific inhibitor
of the Mek1/2 kinase activity. We used this inhibitor at 20 mM.
Indeed this concentration was sufficient to abolish Erk1/2
phosphorylation during the calcium switch (Fig. S7). U0126-
treated cells were seeded either on PL or N-cad and apoptotic cells
were scored at 6 and 24 hours using nuclear condensation and
cytochrome c release markers (Fig. 8). U0126 treatment did not
affect cell death on PL. However, this treatment reversed the
protective effects of N-cadherin since a similar apoptosis level as
for cells seeded on PL was observed on the Ncad substrate (Fig. 8B,
C). These results indicate that N-cadherin protection is lost in the
presence of Mek1/2 inhibitor and support the hypothesis that the
Erk1/2 pathway is involved cell survival downstream of N-
N-cadherin engagement does not regulate Bcl-2 or Bad
We next aimed at determining the downstream targets of the
MAP kinases involved in cell survival pathway induced by the N-
Figure 4. N-cadherin engagement protects neuronal cells from
nuclear condensation and cytochrome c release. Serum-starved
GT1-7 cells were seeded at low density on FN, N-cad or PL substrates and
grown in serum-free medium. After 24 hours, cells were fixed and
immunostained for cytochrome c (right) while nuclei were stained with
DAPI (left). Arrows point toward cells showing condensed nuclei and
healthy nuclei and mitochondrial cytochrome staining. Scale bar: 20 mm.
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cadherin engagement. Several studies have reported that, during
anoı ¨kis, Bcl-2 family members such as the anti-apoptotic Bcl-2 and
Bcl-xL proteins or the pro-apoptotic proteins Bad or Bim are
regulated by activated MAP Kinases . Therefore, we
measured the Bcl-2 protein levels by immunoblotting at several
time points. Bcl-2 protein levels remained constant after N-
cadherin engagement and at levels equivalent to those of cells
seeded on PL (Fig. 9A). Consistent results were obtained with the
calcium switch assay (Fig. 9B), suggesting that cell survival induced
by N-cadherin engagement was not mediated by an increase of the
anti-apoptotic Bcl-2 protein levels.
It has also been demonstrated that the early phosphorylation of
the pro-apoptotic protein Bad on Ser-112 by MAP kinases leads to
its inactivation  resulting in cell survival. However, we did not
detect any increase of Bad phosphorylation in cells seeded on N-
cad at the earlier time point (4 hours) as compared to cells seeded
on PL (Fig. 9C). Moreover, calcium restoration in confluent GT1-
7 cells did not induce Bad phosphorylation at any time point tested
(Fig. 9D). These results indicate that Bcl-2 and Bad proteins take
not part in the survival pathway induced by N-cadherin
N-cadherin engagement down-regulates the pro-
apoptotic protein Bim-EL
The Bim protein is another critical regulator of apoptosis
downstream of MAP kinase signaling. We determined whether N-
cadherin engagement affects Bim protein levels. In confluent GT1-
7 cells, Bim was expressed at low levels and Bim-EL was the only
form of Bim detected. However in cells deprived of cell-matrix and
cell-cell contacts (PL substrate) Bim-EL was up-regulated, while it
remained low in cells seeded on N-cad. At 24 hours Bim-EL
protein level was 3.5 fold higher in cells seeded on PL than on cells
seeded on N-cad (Fig. 10A), suggesting that N-cadherin engage-
ment might favor cell survival by down-regulating Bim-EL. To
ascertain whether Erk1/2 pathway acts downstream of N-
cadherin to regulate the level of Bim-EL protein, N-cad seeded
cells were treated with 20 mM U0126. This treatment induced a 3
fold increase in Bim-EL levels thereby reaching similar levels to
those in cells seeded on PL (Fig. 10A). The increase of this pro-
apoptotic protein was consistent with the raise of the apoptotic
index in cells seeded on N-cad and treated with U0126 (Fig. 8).
Altogether these findings indicate that MAP kinase activation
following N-cadherin engagement could sustain GT1-7 cell
survival by maintaining Bim-EL protein levels low. Interestingly,
we observed that adhesion of GT1-7 cells to the pro-survival FN
substrate down-regulates Bim-EL in a similar Erk1/2-dependent
manner than N-cadherin (Fig. S8). Luciano et al. showed that
phosphorylation of Bim-EL by Erk1/2 on Ser-69 triggers its
degradation by the proteasome pathway and promotes cell
survival . We thus searched for changes in the status of Bim-
EL phosphorylation. As a positive control of Bim-EL phosphor-
ylation, cells were treated with PMA. Addition of PMA induced a
Figure 5. N-cadherin engagement protects neuronal cells from apoptosis. GT1-7 cells were cultured as indicated in Figure S2 for 24 (A, B) or
4 hours (C) and processed for nuclear condensation (A), cytochrome c release (B) as well TUNEL libeling (C). PARP cleavage was analyzed by western
blotting of equivalent protein content from cell lysates (D). Results in A, B and C are expressed as the mean 6 SD of four independent experiments.
Western blotting in D is representative of three independent experiments. Asterisks (*) or (***) indicate a significant difference (P,0.05) or (P,0.01)
respectively as compared with PL.
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mobility shift of Bim-EL and was correlated with the expected
phosphorylation of Erk1/2 (Fig. 10B). This mobility shift of Bim-
EL observed in GT1-7 cells was quite similar to the one observed
by Luciano et al. in different cell lines and tissues as a result of
Bim-EL phosphorylation . At early time points (4 hours), we
detected the same mobility shift of the Bim-EL protein in cells
spread on N-cad which was abrogated by U0126 treatment
(Fig. 10C). These results suggest that upon N-cadherin engage-
ment, Bim-EL is phosphorylated by Erk1/2 kinases.
Figure 6. Akt is not involved in N-cadherin-mediated GT1-7 cell
survival. Serum-starved cells were seeded at low density on PL or N-
cad and grown for the indicated time in serum-free medium (A, C). In
(B), confluent cell cultures were subjected to a calcium switch (see
‘‘Experimental Procedures’’). Equivalent amount of cell lysates was
separated on 4–12% SDS-PAGE, transferred onto nitrocellulose mem-
branes, and immunobloted with antibodies to phospho-specific Ser473
Akt, total Akt and b-actin. The relative densities of phospho-Akt were
normalized to total Akt, and the relatives densities of Akt were
normalized to b-actin. Results are expressed as the mean 6 SD of three
Figure 7. Erk1/2 phosphorylation is up-regulated by N-
cadherin engagement. Serum-starved GT1-7 cells were seeded at
low density on PL or N-cad and grown for the indicated time in serum-
free medium (A); alternatively confluent GT1-7 cell cultures were
subjected to a calcium switch (B). Equivalent amount of proteins were
blotted onto nitrocellulose membranes that were incubated either with
anti-phospho Erk1/2, anti-total Erk1/2, or anti-a-tubulin. The relative
densities of phospho-Erk1/2 were normalized to total Erk1/2 (A) or to a-
tubulin (B). Histograms present the data of three independent
experiments (mean 6 SD).
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Overexpression of a S69G phosphorylation defective
form of Bim-EL prevents N-cadherin cell survival
In order to test whether the phosphorylation and down-
regulation of Bim-EL was involved in mediating N-cadherin
engagement-induced cell survival, we looked at the effect of
overexpression of an Erk-dependent phosphorylation site defective
mutant of Bim-EL (Bim-EL S69G)  on the survival of GT1 cells
seededonN-cadherin(Fig.11). Toprevent massive accumulationof
the pro-apoptotic protein in cells before their settlement on N-
cadherin, Bim-EL expression vector were lipofected 2 hours before
plating on the N-cadherin substrate. To identify transfected cells,
the Bim-EL vector was lipofected together with a GFP expressing
vector. We first assessed the expression of Bim-EL S69G protein in
confluent GT1-7 cells 8 hours and 24 hours post-transfection
(Fig. 11A). While low levels of endogenous Bim-EL were detected
in GFP alone transfected cells, a 2.5 to 3 fold increase in Bim
expression was observed in Bim-EL S69G transfected cell cultures,
for a percentage of transfected cells around 10% of the cell culture
population, indicating that the Bim-EL mutated protein indeed
highly accumulates in individual transfected cells.
Figure 8. Mek 1/2 inhibitor induces apoptosis on cells seeded on N-cadherin. Serum-starved GT1-7 cells were pretreated with 20 mM U0126
or vehicle for 1 hour, then seeded at medium density on N-cad with or without 20 mM U0126 for 6 hours (A). Apoptosis was evaluated by
determination of nuclei condensation following DAPI staining and cytochrome c release following immunostaining. Arrows point toward cells
showing condensed nuclei and absence of cytochrome c staining while asterisks indicate a cell with healthy nuclei and cytochrome c staining. Scale
bar: 20 mm. (B, C). The percentage of cells with condensed nuclei and diffuse/absent cytochrome c labeling was quantified at 6 and 24 hours in
treated and non-treated cells seeded on PL or N-cad. Results are expressed as the mean 6 SD of four independent experiments. Asterisks indicate a
significant difference (P,0.05) as compared with PL.
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We then analyzed the effect of Bim-EL S69G overexpression on
the survival of cells seeded on N-cadherin by determining the
percentage of transfected cells with a condensed nucleus 6 hours
and 24 hours after plating (Fig. 11B). The expression of GFP alone
did not lead to an increase in the occurrence of dying cells
compared to un-transfected cells plated on N-cadherin (compare
with Figure 5). In contrast, the overexpression of Bim-EL S69G
induced a significant increase in the number of apoptotic cells both
at 6 and 24 hours. The overexpression of the wild type form of
Bim-EL expressed at similar levels (Fig. 11A) had no significant
effect on GT1-7 cells plated on N-cadherin at 6 hours, although it
impaired the survival at 24 hours (Fig. 11B), suggesting that the
accumulation with time of wild time Bim-EL may also lead to
apoptosis due to the overwhelming of the Bim-EL phosphorylation
and degradation pathway. Altogether, these data indicate that the
accumulation of Bim-EL and in particular of its Erk- phosphor-
ylation defective mutant (S69G) prevents N-cadherin-induced cell
survival, supporting the hypothesis that N-cadherin triggers
neuronal cell survival through a Bim-EL down-regulation.
In this study, we accumulate convergent arguments to support
the hypothesis that N-cadherin engagement provides neuronal
cells with a pro-survival signal. We cannot formally exclude the
contribution of other factors or adhesion molecules to this process.
However, the anti-apoptotic effect of N-cadherin engagement was
observed in tightly controlled conditions, that is in isolated cells, in
the absence of growth factors, and in the absence of detectable
integrin activation in focal adhesion complexes. Furthermore the
survival effect of the N-cad substrate was alleviated by the
expression of a dominant negative form of N-cadherin. These
results strongly support the hypothesis that N-cadherin initiates an
anti-apoptotic pathway in neurons and neuronal cells that is
Figure 9. N-cadherin engagement does not affect Bcl-2 protein level and Bad phosphorylation. Serum-starved GT1-7 cells were seeded
at low density on PL or N-cad and grown for the indicated time (A) or for 4 hours (C) in serum-free medium. In B-D, cells were subjected to a calcium
switch protocol. As a positive control, cells were treated with 10 ng/ml of PMA or DMSO for 1 hour. Equivalent amount of proteins were
immunobloted with antibodies to Bcl-2 and phospho-Bad. Relative densities of Bcl-2 and phospho-Bad were normalized with either a-tubulin, or b-
actin. Results are the mean 6 SD of three independent experiments.
N-Cadherin Anti-Apoptotic Signaling
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sufficient to sustain cell survival when they are deprived of other
survival factors such as integrin interactions with the ECM. We
further report that N-cadherin engagement mediates cell survival
by enhancing the Erk1/2 kinase pathway and down-regulating the
pro-apoptotic protein Bim-EL (Fig. 12). Bim-EL has been
extensively involved in apoptotic pathways in neurons upon
NGF deprivation or after K+withdrawal . Thus, we propose
that N-cadherin engagement may antagonize a common apoptotic
pathway activated in neurons upon various stress conditions.
The involvement of cadherins in cell survival has been described
previously in different cell types either normal or tumoral
[3,7,42,43,44]. However, the molecular mechanism whereby
cadherin adhesion contributes to cell survival is still poorly
understood and controversial. N-cadherin engagement has been
shown to promote survival through the activation of the PI3-
kinase/Akt and subsequent phosphorylation of the pro-apoptotic
protein Bad in metastatic cancer cells  and in vein smooth
muscle cells . The PI3-kinase pathway has also been
implicated in E-cadherin-mediated cell survival in Ewing Tumor
cells  and in epithelial cells [4,5]. Therefore, we examined the
role of PI3-kinase/Akt pathway. N-cadherin ligation did not affect
Akt and Bad phosphorylation. This was observed at different time
points from 30 min to 24 hours and using two different
approaches, calcium switch assay and biomimetic substrates. In
addition, the inhibitor of PI3-kinase, LY294002, did not block the
GT1-7 cell survival after N-cadherin engagement. In agreement
with our results, Skaper et al. showed that the inhibition of PI3-
kinase/Akt pathway failed to block the survival of hippocampal
and cerebellar granule neurons induced by N-cadherin agonist
. N- and E-cadherin engagements have been shown to up-
regulate Bcl-2 protein [7,8,42]. However, in our model, the levels
of Bcl-2 protein were not affected by N-cadherin engagement.
These results suggest that signaling pathways that affect cell
survival in response to N-cadherin engagement in neuronal cells
are independent of the PI3-kinase and Bcl-2 proteins and may
involve alternative pathways such as MAP kinases.
Among the MAP kinases, Erk1 and 2 have been widely
associated with neuronal cell survival, while JNK and p38 are
often implicated in cell death . Several studies suggest that
Erk1/2 mediates neuroprotective activity of extracellular factors
such as integrins and neurotrophins, as well as during survival of
damaged neurons [5,48]. We show that N-cadherin adhesion
induces an increase of Erk1/2 phosphorylation and a subsequent
inhibition of GT1-7 cell death. Pharmacological inhibition of the
MAP kinase pathway using U0126 resulted in the inhibition of
Erk1/2 phosphorylation and an increased number of apoptotic
cells on N-cadherin. Therefore our results suggest that N-cadherin
protects neurons against cell death by activating specifically Erk1/
2. Similar observations were reported in ovarian carcinoma cells
 and in epithelial cells  where E-cadherin engagement
activates the Ras/Erk1/2 cascade but not the PI3-kinase/Akt
One critical regulator of apoptosis and target of the MAP kinase
pathway is the pro-apoptotic protein Bim. The regulation of Bim
activity is complex, involving both transcriptional and posttran-
scriptional mechanisms. Bim is normally expressed at low levels,
and its pro-apoptotic activity kept in check by rapid phosphory-
lation by Erk1/2 resulting in its ubiquitination and proteosomal
degradation. Reginato et al. have shown that attached MCF-10A
cells contain low levels of Bim-EL and undetectable levels of Bim-
L and Bim-S. All three isoforms were highly up-regulated after cell
detachment from the ECM . Fukazawa et al. reported that
Bim-EL is the only form expressed in human HBC4 and MDA-
MB231 cancer cell lines . They showed that treatment with
MAP Kinase inhibitors, which prone cells for apoptosis in the
absence of cell anchorage, induced a reduction of Bim-EL
phosphorylation together with an increase of Bim-EL protein
levels. Similarly, we found that Bim-EL was the only form of Bim
expressed, albeit at low level, in GT1-7 cells. Upon cell deprivation
in cell-matrix and cell-cell contacts (PL substrate) Bim-EL was up-
regulated. Conversely, N-cadherin engagement down-regulated
Bim-EL protein levels in association with its phosphorylation as
revealed by mobility shift . Moreover, the Mek1/2 inhibitor
U0126 prevented this phosphorylation, indicating that Bim-EL
undergoes Erk1/2 dependent phosphorylation upon N-cadherin
engagement, which may be responsible for its down-regulation.
Interestingly, plating GT1-7 cells on fibronectin also elicited the
Figure 10. N-cadherin engagement down-regulates Bim-EL
protein levels in a Mek1/2 dependent manner. Serum-starved
cells were seeded at medium density on PL or N-cad and incubated
with or without 20 mM U0126 for 24 hours (A) or 4 hours (C), then
protein were extracted and analyzed by western blotting with either
anti-Bim, anti-phospho-Erk1/2, anti-Erk1/2, or anti a-tubulin antibodies.
Relative densities of Bim-EL were normalized to a-tubulin. As a positive
control of Bim-EL mobility shift, adherent cells were treated with 10 ng/
ml of PMA for 1 hour (B). Represented data are the mean of three
independent experiments. Asterisks indicate a significant difference
(P,0.05) as compared with no treatment. Arrows show Bim-EL mobility
N-Cadherin Anti-Apoptotic Signaling
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down-regulation of Bim-EL, suggesting that integrin-mediated
inhibition of anoikis may similarly rely, to some extent, on a Bim-
EL down-regulation in neuronal cells as previously described in
MCF-10A cells .
The MAP kinase pathway has been implicated in the regulation
of Bim-EL expression in various other cell types like hematopoietic
cells , fibroblasts , epithelial cells  and neurons .
Studies in PC12 neural cells have shown that NGF protects
neurons against apoptosis by acute Mek1/2-dependent phosphor-
ylation of Bim-EL and subsequent down-regulation of this specific
isoform . In epithelial  and cancer cells lines ,
inhibition of the MAP Kinase pathway was also reported to
abolish Bim-EL phosphorylation and upregulate Bim protein
levels in association with anoı ¨kis. Phosphorylation of Bim-EL by
Erk1/2 on Ser-69 has been reported to promote its degradation
via the proteasome pathway [33,55,56,57]. However, in our
model, we were not able to detect an accumulation of Bim-EL
upon treatment with proteasome inhibitors, neither we were able
to detect Bim-EL ubiquitination (data not shown). Recently, an
alternative mechanism was proposed by Ewings et al . Erk1/2-
dependent phosphorylation of Bim-EL promotes its rapid
dissociation from Bcl-xL and Mcl-1 proteins and this dissociation
may contribute to Bim-EL degradation . Therefore, it remains
to elucidate whether, in neuronal cells, Bim-EL phosphorylation
drives its subsequent degradation and by which mechanisms.
Nevertheless, in support to en essential role of Erk-dependent Bim-
EL phosphorylation in triggering N-cadherin engagement-medi-
ated neuronal cell survival, we observed an increased apoptosis on
N-cadherin of cells overexpressing Bim-EL S69G. Interestingly,
the overexpression of the wild type form of Bim-EL also impaired
with a delay the survival of GT1-7 cells plated on N-cadherin.
Altogether, these data indicate that the accumulation of Bim-EL
prevents N-cadherin-induced cell survival, supporting the hypoth-
esis that N-cadherin triggers neuronal cell survival through Bim-
It has long been recognized that cadherin function in close
cooperation with, and organize, the cytoskeleton (for review: ).
In turn, cytoskeleton and in particular microtubules may serve as
Figure 11. Overexpression of Bim-EL S69G prevents N-cadherin induced cell survival. Expression vectors coding for wild type Bim-EL and
Bim-EL S69G were lipofected in 24 hours starved GT1-7 cells together with pEGFP at a 2/1 ratio. Cell lysates of equivalent protein content were
analyzed by western blotting at 8 hours and 26 hours post-transfection for Bim and GFP expression; actin was analyzed as loading control (A). Two
hours after lipofection, cells were harvested from transfected cultures and seeded on N-cadherin. Preparations were fixed 6 hours and 24 hours later
(corresponding to 8 hours and 26 hours post-transfection) and nuclei stained. The percentage of GFP positive cells with condensed nuclei was then
determined for each condition in duplicates ($100 GFP+cells counted each time). Represented data are the mean of two independent experiments.
Asterisks indicate a significant difference (P,0.05) as compared with GFP expression alone.
Figure 12. Hypothetical model for N-cadherin mediated cell
survival signaling in neurons.
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scaffolds for signaling molecules that regulate apoptosis, such as
Bim. It has been reported that Bim binds to the dynein light chain
LC8 and is sequestered to the microtubule-associated dynein
motor complex. Under apoptotic stimuli, Bim may be released and
neutralizes Bcl-2 proteins . A potential link between cadherins
and microtubules has been postulated (for review: ).
Therefore, it will be interesting to study if N-cadherin engagement
affects Bim association with microtubules and whether this process
is affected by Mek1/2-dependent Bim phosphorylation, although
the quality of the available immunological reagents did not
allowed us to test this hypothesis.
In conclusion, this study proposes a novel role of cell-cell
contacts mediated by N-cadherin in signaling neuronal survival
and provides insight into the underlying molecular mechanisms. It
identifies MAP kinase activation and subsequent down-regulation
of the pro-apoptotic protein Bim as a major signaling pathway in
this process. To our knowledge, this is the first report that N-
cadherin engagement mediates neuronal cell survival. The fact the
GT1-7 cells response to cadherin engagement is shared with
embryonic spinal cord and hippocampal neurons strongly support
the hypothesis that promotion of neuronal survival by N-cadherin
engagement is a largely spread regulation process. We cannot
presently assign the relative contribution of this cell-cell contact
signaling and the well-known contribution of cell-matrix adhesion
and growth factors signaling in neuronal cell survival. However N-
cadherin is a major mediator of cell-cell interactions during the
development of the nervous system. Thus, our observations should
have potential relevance for the control of the survival/death
balance of neurons throughout development and plasticity of the
mammalian nervous system.
non supplemented medium affects cell survival. E18.5 rat
hippocampus neurons were cultured in MEM medium in the
presence of B27 additive and serum (+B27) either on PL (poly-L-
lysine) or N-cad (immobilized Ncad-Fc). Alternatively they were
cultured on the same types of substrate but in the absence of serum
and B27 (2B27). After 24 hours, cells were fixed and stained with
anti-bIII tubulin antibody to identify neurons. Although neurons
grew well on PL as well as on N-cad in the presence of B27, they
died on PL in the absence of the additive and serum. In addition,
neurite extension was greatly increased on N-cad compared to PL
even in the presence of B27 as expected from previous reports.
Similar observations were made with cells cultured from 12.5
mouse ventral spinal cords (not shown). Scale bar: 20 mm.
Culture of primary neurons in serum free
hippocampal neurons on PL versus Ncad-Fc substrates.
E18.5 rat hippocampus neurons were cultured in MEM medium
either on PL or N-cad in the absence of serum and B27 additive as
reported in Figure 1A. Cultures were fixed at 1, 4 and 24 hours
post-seeding and adherent cells were counted (A). Alternatively,
preparations were processed for TUNEL labeling and the
percentage of TUNEL positive cells determined (B).
Compared adhesion/survival of primary
herin engagement. In regular cell culture conditions (upper
left), cells adhere to the substratum via adsorbed fibronectin (FN)
and vitronectin (VN) provided by the serum (cell matrix adhesion)
and adhere to each other via cadherins and other cell adhesion
molecules (CAMs, cell-cell adhesion). To specifically activate
Experimental set up for controlled N-cad-
either N-cadherin or fibronectin, GT1-7 cells were plated at low
density on N-cad or FN, respectively. To deprive cells of specific
cell adhesion, they were seeded on PL which mediates electrostatic
the integrin pathway. GT1-7 cells were cultured on N-cad for
4 hours, then fixed and fluorescently stained for F-actin with
phalloı ¨din (red) and with anti-paxillin antibodies (green). Paxillin
staining remained diffuse in the cytoplasm of these cells indicating
that integrins were not mobilized on this substrate. In contrast,
when GT1-7 cells were cultured on fibronectin (FN) for 4 hours,
they displayed expected stress fibers and focal adhesions in which
both stainings were strongly accumulated, indicated that indeed
integrins have been mobilized and activated in focal adhesions in
these culture conditions. Scale bars: 20 mm.
Spreading of cells on N-cad does not mobilize
of N-cadherin drastically impairs the protective effect of
N-cad on GT1-7 cells. GT1-7 cells were lipofected with either a
dominant negative form of N-cadherin fused to DsRed (DN-
Ncad), or GFP alone, starved for 24 hours, then spread on the N-
cad substrate. After 24 hours, cells were fixed and processed for
nuclei staining. The percentage of transfected cells with normal
and condensed nuclei was determined for each condition in three
independent experiments and given as mean 6 SD.
Overexpression of a dominant negative form
cells were seeded at low density on PL or N-cad and grown for the
indicated time in serum-free medium. Cell death was measured by
nuclear condensation (A) and cytochrome c release (B). Asterisks
indicate a significant difference (P,0.05) as compared with PL.
Time course of GT1-7 cell death. Serum-starved
Serum starved GT1-7 cells were treated (I) or not (C) with the
U0126 inhibitor. Cultures were then subject to the calcium switch
protocol and harvested just after (09) or 30 minutes (309) after
calcium restoration. Proteins were extracted and analyzed for P-
Erk1/2 content by western blotting. The results of two
independent experiments are shown. The tubulin content in the
extracts was used as a gel loading control.
Dose dependent action of U0126 inhibitor.
down-regulates Bim-EL protein levels. Serum-starved cells
were seeded at medium density on PL or FN and incubated with or
without 20 mMU0126 for24 hours.Equalamountofproteinswere
immunoblotted with either anti-Bim or anti-a-tubulin antibodies.
Relative densities of Bim-EL were normalized to a-tubulin.
Spreading of GT1-7 cells on fibronectin also
We thank Dr S. El Bawab, Dr H. Enslen and Dr A. Sobel for their critical
reading of the manuscript, and Thibault Deville for technical assistance.
We thank O. Thoumine and F. Luciano for providing the DN N-cadherin
and mutant Bim-EL expression vectors, respectively. We would like to
thank Mireille Lambert and other members of the team for fruitful
discussions during the initial phase of this work.
Conceived and designed the experiments: RMM HB. Performed the
experiments: EL CP CB EW RMM HB. Analyzed the data: EL CP CB
RMM HB. Wrote the paper: EL RMM HB.
N-Cadherin Anti-Apoptotic Signaling
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N-Cadherin Anti-Apoptotic Signaling
PLoS ONE | www.plosone.org15 March 2012 | Volume 7 | Issue 3 | e33206