THE JOURNAL OF BIOLOGICAL CHEMISTRY
5460 –5467, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CCAAT/Enhancer-binding Protein ␤ Plays a Regulatory Role in
Differentiation and Apoptosis of Neuroblastoma Cells*
Received for publication, September 11, 2001, and in revised form, November 21, 2001
Published, JBC Papers in Press, December 3, 2001, DOI 10.1074/jbc.M108761200
Marta Cortés-Canteli‡§, Miguel Pignatelli‡¶, Angel Santos储**, and Ana Perez-Castillo‡ ‡‡
From the ‡Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientı́ficas-Universidad Autónoma
de Madrid, 28029-Madrid, Spain and the 储Departamento de Bioquı́mica y Biologı́a Molecular, Facultad de Medicina,
Universidad Complutense de Madrid, 28040-Madrid, Spain
The C/EBP␤ (CCAAT/enhancer-binding protein ␤) is a
transcription factor that belongs to basic region-leucine
zipper class DNA-binding proteins. There is a significant
body of evidence that suggests that this protein plays a
central role in adipocytic and eosinophilic differentia-
tion. However, there is no information available regard-
ing the role of this transcription factor in the develop-
ment of mammalian neuronal tissues. In this study, we
have examined the effect of C/EBP␤ overexpression on
the differentiation and survival of mouse Neuro2A cells.
We found that C/EBP␤ induces neuronal differentiation
and that this process is inhibited by transfection with
the C/EBP homologous protein 10 (CHOP), strongly sug-
gesting that the extension of neurites is indeed due to
the C/EBP␤ transcriptional activity. As it has been sug-
gested in adipocyte differentiation, here we show that
C/EBP␤ induces the expression of the endogenous
C/EBP␣ gene and that this protein by itself is also able to
induce a differentiated phenotype in Neuro2A cells.
Neuronal differentiation induced by C/EBP␤ requires
activation of the phosphatidylinositol 3-kinase signaling
pathway, whereas inhibition of the mitogen-activated
protein kinase signaling does not have any effect. In
addition, we show that C/EBP␤ is expressed in the brain
of neonatal rats, suggesting that this protein could play
an important role in neuronal maturation. Finally, cell
death was also induced by C/EBP␤ through activation of
the p53 protein and the cdk inhibitor p21.
Neuritogenesis is a central event in brain development, and
its regulation during neuronal differentiation is complex and
likely to involve multiple signaling systems and the regulation
of a wide set of genes. Research over the past few years has
given us a glimpse of the molecules involved in dendritic mor-
phogenesis. Proper dendrite development relies on extracellu-
* This work was supported by Grants PM97-0063 (to A. P. C.) and
PM99-0057 (A. S.) from the Dirección General de Enseñanza Superior e
Investigación Cientı́fica and by Grants 08.5/0003/1997 and 08.5/0078/
2000 (to A. P. C. and A. S.) from the Comunidad de Madrid. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A fellow of the Comunidad de Madrid.
¶ A fellow of the Fondo de Investigaciones Sanitarias de la Seguridad
Social.
** To whom correspondence may be addressed: Departamento de
Bioquı́mica y Biologı́a Molecular, Facultad de Medicina, Universidad
Complutense, 28040-Madrid, Spain. Tel.: 34-91-3941446; Fax: 34-91-
3941691; E-mail: piedras3@eucmax.sim.ucm.es.
‡‡ To whom correspondence may be addressed: Instituto de Investi-
gaciones Biomédicas, Consejo Superior de Investigaciones Cientı́ficas-
Universidad Autónoma, Arturo Duperier, 4, 28029-Madrid, Spain. Tel.:
34-91-5854625; Fax: 34-91-5854587; E-mail: aperez@iib.uam.es.
5460
Vol. 277, No. 7, Issue of February 15, pp.
Printed in U.S.A.
lar signals and the signal transduction that lies downstream of
them (1). This signal transduction includes local regulation of
dendritic structures via the cytoskeleton and regulation medi-
ated through changes in the transcription of target genes.
Therefore, the identification of molecules involved in dendritic
morphogenesis is an essential step toward understanding how
dendrites develop. Due to the complexity of the developing
nervous system, many studies, including the one described
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here, have used in vitro model systems to gain a better under-
standing of the mechanisms that regulate neuritogenesis (2–5).
These in vitro systems have also been widely used to study
neuronal programmed cell death and the characterization of
the genes implicated in its regulation. The present study in-
vestigates the role of the CCAAT/enhancer-binding protein ␤
(C/EBP␤)1 gene on neurite outgrowth and apoptosis of
Neuro2A (N2A) cells. The C/EBP family of transcription factors
is composed of at least five distinct members (C/EBP␣,
C/EBP␤, C/EBP␦, C/EBP⑀, and C/EBP␥) (6). These proteins are
known to be involved in the regulation of cell growth and
differentiation of several cell types. Common structural fea-
tures of these proteins are highly conserved DNA binding and
leucine zipper dimerization domains at the carboxyl terminus
and divergent amino termini containing regulatory and trans-
activation domains (7, 8). Initially, C/EBP␤ has been linked to
hepatocyte-specific gene regulation because it shows high ex-
pression in liver cells (9) and binds to regulatory elements in
the promoter of liver-specific genes (10). In addition, it has been
suggested that C/EBP␤ contributes to regulate the acute phase
response of the liver (11). Lately, it has become clear that
C/EBP␤ also plays a role in other tissues. Results derived from
experiments in cell culture and with knockout mice have dem-
onstrated that this protein is critical for lymphocyte (12, 13)
and adipocyte differentiation (14, 15). The role of different
C/EBP proteins during adipocyte differentiation is thought to
follow a sequential model during which C/EBP␦ and C/EBP␤
expression precede expression of C/EBP␣ and correlates with
induction of differentiative events (15). In fact, expression of
C/EBP␣ is primarily restricted to highly differentiated cells
such as hepatocytes, adipocytes, and type II cells of the lung
(16). This sequential expression of C/EBP isoforms, together
with the fact the C/EBP␣ promoter contains a C/EBP response
element, has led to the idea that C/EBP␤ can activate the
expression of the C/EBP␣ promoter during differentiation pro-
cesses such as adipose conversion.
1
The abbreviations used are: C/EBP␤, CCAAT/enhancer-binding
protein ␤; ApC/EPB, Aplysia C/EPB; N2A, Neuro2A; CHOP, C/EBP
homologous protein 10; PI3K, phosphatidylinositol 3-kinase; GFP,
green fluorescent protein; ERK, extracellular signal-regulated kinase;
MAP, microtubule-associated protein; MAPK, mitogen-activated pro-
tein kinase; MEK, MAPK/ERK kinase.
This paper is available on line at http://www.jbc.org
 Neuronal Differentiation Induced by C/EBP␤
The activity of the transcription factor C/EBP␤ can be con-
trolled by phosphorylation. Previous studies have shown that
extracellular stimuli can alter the phosphorylation of several
sites in C/EBP␤ and can modify their effects on promoter ac-
tivity. Increased transcriptional activities through phosphoryl-
ation of distinct sites in rat, chicken, or human C/EBP␤ are
regulated by many different stimuli of cell proliferation and
differentiation, such as phorbol esters/protein kinase C-␣,
v-erbB, and ras (17, 18), suggesting a link among these post-
translational modifications, transcriptional activation, and cell
function. It has also been reported that phosphorylation of the
C/EBP␤ protein after insulin stimulation reduces transactiva-
tion by interfering with DNA binding or by disrupting interac-
tions with p300/CBP proteins (19). Regarding a possible role of
C/EBP␤ in the brain, it has been shown that C/EBP␤ mRNA is
widely expressed in the adult mouse brain (20). Expression of
this protein has also been reported in the PC12 cell line, which
undergoes differentiation to neuron-like cells in response to
nerve growth factor (20). In these cells, nerve growth factor
receptor signaling stimulates activity of C/EBP␤. However, the
function of C/EBP␤ within the mammalian nervous system is
unknown. In the invertebrate Aplysia, it has been shown that
the transcription factor Aplysia CCAAT enhancer-binding pro-
tein (ApC/EBP) plays an essential role in the consolidation of
stable long term synaptic plasticity (21). More recent studies in
mammals have suggested an implication of C/EBP␤ and
C/EBP␦ in long term synaptic plasticity and memory consoli-
dation in rat hippocampus (22). Here we show that stable-
transfected N2A cells overexpressing the C/EBP␤ protein pres-
ent a differentiated phenotype and extend very long neurites
even when growing in the presence of serum. This induction of
differentiation is accompanied by an increase in the expression
of genes associated with the differentiated state and requires
activation of phosphatidylinositol 3-kinase (PI3K). The neurite
outgrowth activity was specific to C/EBP␤ transactivation ca-
pacity because CHOP (C/EBP homologous protein 10), a dom-
inant negative of this protein, inhibited this outgrowth. In
addition, C/EBP␤ promotes N2A apoptosis, which is dependent
on p53. Finally, we also found that the C/EBP␤ protein is
broadly distributed in the brain of neonatal rats during a
period of intense neuronal maturation. Together, these results
suggest that the C/EBP␤ protein may play an important role in
neuronal maturation and cell death during mammalian brain
development.
EXPERIMENTAL PROCEDURES
Cell Culture and Transfection—Mouse N2A cells were grown in
Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum (In-
vitrogen) and 2 mM glutamine at 37 °C. For the selection of stably
transfected cells, 0.5 ⫻ 106 cells were seeded into a 6-cm diameter tissue
culture plate and incubated overnight. The following day, the medium
was aspirated, and the cells were washed twice with TD buffer (20 mM
Tris-HCl, pH 7.4, 1 mM Na2HPO4, 140 mM NaCl, 5 mM KCl) and
transfected with 2 ␮g of pMSV-C/EBP␤ (kindly provided by Dr.
McKnight, University of Texas, Dallas, TX) by the calcium phosphate
precipitation technique as described previously (23). Twelve h after
transfection, the medium was replaced with regular growth medium,
and the cells were incubated for 24 h, after which they were subcultured
at 1:10 dilution with the addition of geneticin (1 mg/ml). The growth
medium was renewed every 3 days, and fresh geneticin was added.
Cultures were expanded and then screened for C/EBP␤ expression. For
transient transfection experiments (C/EBP␣ promoter, pG13-luc, and
MG15-luc), semiconfluent cells were transfected by the calcium phos-
phate precipitation technique in 24-well plates. After 8 h of exposure to
the Ca2⫹/DNA mixture, cells were washed with TD buffer, incubated for
another 24 h in regular medium, and harvested for determination of
luciferase activity as described previously (24). Each transient trans-
fection experiment was repeated at least three times in duplicate. For
the experiment shown in Fig. 3B, the p4 pool was cotransfected with a
CHOP expression vector together with a GFP (green fluorescent pro-
5461
tein) construct (CLONTECH, Palo Alto, CA) at a ratio of 5:1 by the
calcium phosphate method. Twenty-four h after transfection, cell mor-
phology was examined with a Zeiss Axiovert microscope. For transient
transfection experiments with the constitutively active p110 subunit
(K227E) of PI3K, N2A and p4 were transfected using Transfast (Pro-
mega, Madison, WI), following the manufacturer’s instructions to
achieve a high efficiency of transfection (⬎80%).
For overexpression of C/EBP␣ protein, full-length C/EBP␣ was
cloned into the pLXSN retroviral vector (CLONTECH). BOSC23 pack-
aging cells were grown in 60-mm dishes and transfected by the calcium
phosphate method. Recombinant retroviral particles were collected
from the supernatant 2 days later and used to infect N2A and p4 cells.
Two days later, cell morphology was examined with a Zeiss Axiovert
microscope.
The rat C/EBP␣ promoter construct pC/EBP⌬380 has been described
previously (24). pG13 and MG15 reporter plasmids were provided by B.
Vogelstein (The Johns Hopkins Oncology Center, Baltimore, MD) (25),
and CHOP, C/EBP␣, and K227E p110 expression constructs were pro-
vided by N. Holbrook (National Institutes of Health, Baltimore, MD),
Dr. McKnight, and J. Downward (Imperial Cancer Research Fund,
London, UK), respectively (14, 26, 27). For morphological analysis, cells
were seeded at a density of 20,000/cm2 and grown for 24 h in complete
medium, and then some of the plates were placed in serum-free medium
and observed 6 h after serum withdrawal. Some cultures were treated
with LY294002 (Calbiochem, Nottingham, UK), a reversible inhibitor of
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PI3K used at 10 ␮M, or PD98059 (Calbiochem), an inhibitor of MEK1
and MEK2 used at 80 ␮M.
Antibodies—The DF77 polyclonal antibody specific for the C/EBP␤
was obtained by injecting rabbits with a peptide corresponding to the
amino-terminal portion of this protein (nucleotides 108 – 419) fused to
the glutathione S-transferase protein. The specificity of the antibody
was confirmed by incubating the antibody with immunizing peptide (1
␮M) for 4 h at 4 °C before Western blotting and also by comparing the
signal obtained with this antibody with an anti-C/EBP␤ commercially
available antibody and an anti-C/EBP␤ kindly provided by Dr.
McKnight. Monoclonal anti-MAP2 and anti-NeuN antibodies were from
PharMingen (San Diego, CA) and Chemicon (Temecula, CA), respec-
tively. The polyclonal anti-C/EBP␣, commercial anti-C/EBP␤, anti-p21,
and monoclonal anti-p53 antibodies were from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA).
Immunoblotting—N2A cells were washed twice with TD followed by
centrifugation at 200 ⫻ g for 5 min. The cell pellet was resuspended in
500 ␮l of extraction buffer (phosphate-buffered saline containing 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease in-
hibitors). Cells were disrupted by repeated aspiration through a 21-
gauge needle, incubated on ice for 30 min, and spun at 12,000 ⫻ g for 20
min. Supernatants were removed and stored at ⫺70 °C until analysis
by gel electrophoresis. Brain protein extracts were obtained as de-
scribed previously (28). Twenty ␮g of protein extracts were loaded onto
each lane of a 10% SDS-polyacrylamide gel, separated, and then blotted
to nitrocellulose membranes at 90 mA overnight in transfer buffer (25
mM Tris, pH 8.3, 190 mM glycine, 20% methanol, 0.5% SDS). Protein
loading and transference were checked by Red Ponceau staining of the
membranes. For immunoblotting, membranes were blocked with 5%
non-fat milk in TBST buffer (20 mM Tris-HCl, pH 7.6, 130 mM NaCl,
0.1% Tween 20) for 1 h at room temperature, incubated with the
corresponding primary antibody in TBST buffer with 5% non-fat milk
for 2 h, and after several washes, subsequently incubated with a horse-
radish peroxidase-coupled secondary antibody (1:5000) in the same
buffer. After washing, immunoreactive bands were visualized using the
ECL detection kit (Amersham Biosciences, Inc.) according to the man-
ufacturer’s instructions.
Immunofluorescence Studies—Cells were grown on glass coverslips
for 24 h, fixed for 10 min with methanol at ⫺20 °C, and permeabilized
with 0.1% Triton X-100 for 30 min at 37 °C. After a 1-h incubation with
the primary antibody, cells were washed with phosphate-buffered sa-
line and incubated with the corresponding fluorescein-labeled second
antibody for 45 min at 37 °C. The cells were then examined with a Zeiss
Axiophot microscope.
Apoptosis Assay—To calculate the percentage of cell death, 0.5 ⫻ 106
cells were seeded in 6-cm diameter tissue culture plates and grown in
Dulbecco’s modified Eagle’s medium for 24 h before switching to Dul-
becco’s modified Eagle’s medium without fetal bovine serum for a fur-
ther 24 h. For analysis, both the floating cells in the supernatant and
the phosphate-buffered saline wash containing the cells were collected
from each plate. Apoptosis was assayed using PI staining and fluores-
cence-activated cell sorter analysis. Cells were analyzed in a FACSscan
flow cytometer (Becton Dickinson and Co., Bedford, MA), and the for-
5462 Neuronal Differentiation Induced by C/EBP␤
FIG. 1. Expression of C/EBP␤ protein in brain. Western blot
analyses of C/EBP␤ were carried out with 20 ␮g of total cellular pro-
teins extracted from different brain regions of 15-day-old rats. C/EBP␤
protein was detected by incubation with the DF77 polyclonal antibody
as described under “Experimental Procedures.” A representative West-
ern blot is shown. C, cerebellum; CC, cerebral cortex; S, striatum; H,
hippocampus; M, midbrain; B, brainstem; T, thalamus.
ward scatter was plotted against the propidium iodide fluorescence. The
data were analyzed using the Cell Quest software package (Becton,
Dickinson and Co.).
Statistical Analysis—The data were analyzed using the Excell soft-
ware package. Significant effects were determined using Student’s t
test. A statistically significant difference was considered to be present
at p ⬍ 0.05.
RESULTS
Expression of the C/EBP␤ Protein in Different Brain Re-
gions—The results of in situ hybridization showing a high level
of expression of the C/EBP␤ mRNA in neural tissue (20)
prompted us to investigate the presence of the C/EBP␤ protein
in different brain regions. For this purpose, we raised a poly-
clonal antibody against a peptide corresponding to the amino-
terminal portion of the C/EBP␤ protein, which corresponds to
the most divergent peptide sequence from the other homolo-
gous members of the C/EBP family of transcription factors.
Western blot analysis of total protein extracts prepared from
different brain regions isolated from 15-day-old rats showed a
prominent expression of the C/EBP␤ protein in all the regions
studied. However, this expression was slightly higher in the
cerebellum (Fig. 1). C/EBP␤ protein was also present in the
brain of 5-day-old neonates (data not shown). The same pattern
of expression was obtained with a commercial anti-C/EBP␤
antibody (Santa Cruz Biotechnology) and with the c76 poly-
clonal anti-C/EBP␤ antibody provided by Dr. McKnight (data
not shown). These data, together with the results presented in
Fig. 2A showing a high level of expression of the C/EBP␤
protein in the stable transfected N2A cells, demonstrate the
specificity of the DF77 antibody.
Forced Expression of C/EBP␤ Leads to Premature Neuronal
Differentiation—To directly assess the participation of the
C/EBP␤ protein in neuronal function, we generated stably
transfected N2A cell lines. The complete C/EBP␤ cDNA, cloned
into an murine sarcoma virus (MSV)-driven expression vector,
was transfected into N2A cells, and different pools of G418-
resistant clones were tested for C/EBP␤ expression using the
DF77 polyclonal antibody. For the rest of the experiments, we
chose to use pool p4, which showed very high levels of C/EBP␤
protein (Fig. 2A) as compared with parental N2A cells. The
differences in the amount of C/EBP␤ protein between parental
N2A and p4 cells can also be observed by immunofluorescence
analysis (Fig. 2B). In parental N2A cells, a weak and diffuse
signal can be observed in contrast with a much stronger signal
detected in pool p4, which is clearly localized in the nucleus.
Because the C/EBP␤ protein is a member of a diverse group of
transcription factors, its nuclear localization is obviously essen-
tial for its subsequent transactivation of target genes. The
morphologies of N2A wild type and p4 cells were observed 24 h
after they were plated in the presence of 10% fetal bovine
serum. As is apparent from the micrographs in Fig. 3A, over-
expression of C/EBP␤ induced a dramatic morphological
change. We observed a pronounced difference in the phenotype
of the parental N2A cells and pool p4 overexpressing the
C/EBP␤ protein. More than 90% of the control cells showed a
round morphology, typical of an undifferentiated state,
whereas around 75% of neuroblastoma cells expressing the
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FIG. 2. Expression of C/EBP␤ protein in parental N2A cells and
pool p4 (p4). A, Western blot analysis of C/EBP␤ carried out with 20 ␮g
of total cellular proteins extracted from N2A cells and pool p4. C/EBP␤
protein was detected by incubation with the DF77 polyclonal antibody
as described under “Experimental Procedures.” A representative West-
ern blot is shown. B, immunofluorescence analysis of C/EBP␤ subcel-
lular localization. Pictures were taken at a 400-fold magnification. Bar
scale, 10 ␮m.
C/EBP␤ protein displayed a more differentiated phenotype.
Moreover, when the cells were grown for a further 6 h in
serum-free medium (serum starvation induces spontaneous
process outgrowth in these cells), almost 100% of p4 cells ex-
pressing C/EBP␤ had very long and branched neurites, at least
two cell bodies in diameter, whereas parental N2A cells only
started to extend short projections. The transcription factor
CHOP (C/EBP homologous protein 10, also known as
GADD153) acts as a dominant negative regulator of C/EBPs by
forming stable heterodimers with these proteins and prevent-
ing them from binding to DNA (29, 30). Therefore, to ensure
that the neurite outgrowth activity in the p4 pool was in fact
mediated by the expression of the C/EBP␤ protein, pool p4 was
transiently cotransfected with a plasmid expression vector for
CHOP and GFP. After transfection, cells were maintained in
culture under growth conditions for 24 h. Transfected cells
could be identified by GFP fluorescence. Twenty four h after
transfection, most GFP-expressing cells (80%) adopted a round
morphology. In contrast, we rarely found GFP-expressing cells
with a differentiated phenotype, suggesting that the phenotype
observed was indeed due to C/EBP␤ overexpression (Fig. 3B).
To examine whether phosphorylation of C/EBP␤ is required for
the morphological changes observed in p4, we used inhibitors of
MAPK (Erks) and PI3K signaling pathways. As shown in Fig.
4, the change in cell morphology induced by C/EBP␤ in the p4
cells was completely blocked by pretreatment of cells with the
PI3K inhibitor LY294002 but not with the MEK inhibitor
PD98059, suggesting that the PI3K pathway, but not the MAP
kinase pathway, is required for C/EBP␤-induced neurite out-
growth. These results were further confirmed by transient trans-
fection of p4 cells with a constitutively active p110 subunit
(K227E) of PI3K. As shown in Fig. 4, transfection with the Myc-
tagged K227E p100 plasmid significantly increased the number
of cells with a differentiated phenotype, as well as the number of
neurites/cell and the length of these prolongations. In contrast,
 Neuronal Differentiation Induced by C/EBP␤
FIG. 3. Effect of C/EBP␤ overexpression on the phenotype of
Neuro2A cells. Pool 4 and parental N2A cells were seeded and grown
in medium containing 10% fetal bovine serum (FBS) during 24 h (A).
Then, some of the cultures were induced to differentiate by serum
withdrawal. Phase-contrast photomicrographs, taken in basal condi-
tions and 6 h after growing in serum-free medium, are shown at a
300-fold magnification. Bar scale, 20 ␮m. Some cultures of pool 4 were
cotransfected with a CHOP expression vector and GFP, as indicated (B).
They are shown by phase contrast and fluorescence at a 300-fold mag-
nification. Bar scale, 20 ␮m.
parental N2A cells transfected with the same construct did not
show any significant change in cell morphology, suggesting that
in fact, C/EBP␤ is required for the effects triggered by PI3K
activation. We next examined the levels of two neuronal-specific
markers: microtubule-associated protein 2 (MAP2) and NeuN in
parental N2A and p4 cells. A striking difference was observed
between parental N2A cells and p4. Under basal conditions (cells
growing in the presence of fetal bovine serum for 24 h), only a low
amount of MAP2 protein could be found in N2A non-transfected
cells, and very few immunoreactive MAP2-expressing cells were
observed (Fig. 5, A and B), which is in agreement with the
undifferentiated phenotype presented by these cells. On the con-
trary, Western blot analysis showed a significant increase in the
amount of MAP2 protein in cells overexpressing C/EBP␤. In-
creased expression of MAP2 protein was also confirmed by im-
munofluorescence wherein pool p4 showed a very strong signal,
in contrast to the very weak signal observed in N2A progenitor
cells. Similar results were observed with NeuN, a neuronal cell
type-specific nuclear protein expressed in postmitotic neurons
(31).
As can be shown in Fig. 5A, basal levels of NeuN immuno-
reactivity can be found in parental N2A cells, and these levels
are significantly enhanced in p4 cells. Immunofluorescence
studies revealed that this protein is localized in the nucleus, as
expected (Fig. 5C). We next determined whether, as happens
during adipocyte differentiation, the expression of C/EBP␣
could be increased in N2A cells overexpressing C/EBP␤ protein
and therefore whether C/EBP␣ could play a role in maintaining
the neuronal differentiated phenotype. Our results show a
striking increase in the amount of endogenous C/EBP␣ protein
in p4 cells, as determined by immunofluorescence (Fig. 6A) and
5463
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FIG. 4. Effect of protein kinase inhibitors on the phenotype of
N2A and pool 4. Pool p4 and parental N2A cells were seeded and
grown for 24 h in medium containing 10% fetal bovine serum (B). Some
cultures were incubated with the kinase inhibitors PD98059 (MEK1/2)
and LY294002 (PI3K) or transfected with the constitutively active p110
subunit (K227E) of PI3K as described under “Experimental Proce-
dures.” Phase-contrast photomicrographs are shown at a 300-fold mag-
nification. Bar scale, 20 ␮m.
Western blot (Fig. 6B) analysis. We also tested whether the
increase in the levels of C/EBP␣ protein could be due to a direct
effect of C/EBP␤ on C/EBP␣ promoter activity. To this end, we
performed transient transfections in parental N2A and in p4
cells with a reporter construct containing 380 bp of the pro-
moter region of the C/EBP␣ linked to a luciferase reporter
gene. As shown in Fig. 6C, transactivation of the reporter
construct was significantly increased (5.5-fold) in cells stably
expressing the C/EBP␤ protein, suggesting that the observed
increase in the amount of C/EBP␣ protein is due, at least in
part, to an induction in the transcription of this gene by
C/EBP␤. Finally, we tested whether C/EBP␣ itself could induce
parental N2A differentiation and also produce a more differen-
tiated phenotype in cells overexpressing C/EBP␤. Conse-
quently, we infected parental N2A and p4 cells with a retrovi-
ral expression vector for C/EBP␣. As shown in Fig. 7, 2 days
after viral infection, more than 60% of parental N2A cells had
a differentiated phenotype, as compared with 15% in N2A cells
transfected with the empty vector. P4 cells overexpressing
C/EBP␣ presented very long neurites, a feature of mature
neurons. Altogether, these results suggest a potential role of
the C/EBP␣ gene in the induction and maintenance of the
neuronal-differentiated state.
Effect of C/EBP␤ Overexpression on Cell Death—Apoptotic
cell death of differentiating cells is found widely in the devel-
oping brain. In vitro, apoptosis is also observed during the
course of neuronal differentiation of N2A cells. We have there-
fore analyzed whether C/EBP␤ could induce this process in
N2A cells. To test this premise, we examined apoptotic cell
death with PI staining and fluorescence-activated cell sorter
5464 Neuronal Differentiation Induced by C/EBP␤
FIG. 5. Effect of C/EBP␤ on neuronal marker expression in
N2A cells and pool 4. A, Western blot analysis of protein extracts
isolated from N2A cells and pool 4 cultured in regular growth medium
for 24 h. Immunostaining was performed with the indicated antibodies
as described under “Experimental Procedures.” B and C, fluorescence
microphotographs of N2A and pool 4 cultured in basal conditions for
24 h and immunostained with MAP2 (B) and NeuN (C). Pictures were
taken at a 600-fold magnification. Bar scale, 10 ␮m.
analysis to score the number of apoptotic cells in N2A progen-
itor cells and p4 cells 24 h after serum withdrawal. In cultures
growing in regular medium, fewer than 8% of the N2A cells
manifested apoptotic features (data not shown). After 24 h of
serum withdrawal, up to 11.8% of the parental N2A cells were
PI⫹, and a significant enhancement of the apoptotic population
can be observed in p4 cells (19.6%) (Fig. 8). These data indicate
that, in addition to inducing neuronal differentiation, C/EBP␤
is a potent inducer of programmed cell death in N2A cells.
Tumor suppressor p53 plays a key role in the control of
apoptosis, and its inactivation is a common event in human
tumors (32). Expression of p53 protein results in apoptosis in
numerous cell lines. To investigate whether the enhanced apo-
ptosis observed in p4 cells could be mediated by the p53 pro-
tein, we first performed Western blot analysis with a specific
anti-p53 antibody. As shown in Fig. 9A, although the level of
p53 protein is not altered in p4 cells as compared with parental
N2A cells, we could observe a shift in the mobility of the protein
in the cells overexpressing C/EBP␤, suggesting a possible phos-
phorylation/activation of this factor. To test this hypothesis, we
next performed transient transfection experiments using the
reporter construct pG13-luc, which contains multiple p53 bind-
ing sites linked to a basal promoter (25). As can be seen in Fig.
9B, when the p53 reporter construct pG13-luc was transfected
into N2A cells and the p4 pool, a clear induction of luciferase
activity was observed in p4 cells, suggesting that, although we
did not observe any increase in the amount of p53, there is an
activation of this protein in the stably transfected cells. As a
control, when these cells were transfected with the reporter
construct MG15-luc (containing mutations in the p53 binding
sites), no significant increase in luciferase activity was ob-
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FIG. 6. Effect of C/EBP␤ overexpression on C/EBP␣ expres-
sion. A, fluorescence micrographs of N2A and pool 4 cultured in basal
conditions and immunostained with a C/EBP␣ antibody. Pictures were
taken at a 600-fold magnification. Bar scale, 10 ␮m. B, Western blot
analysis of protein extracts isolated from N2A cells and pool 4 cultured
in basal conditions. Immunostaining was performed with the same
antibody as in panel A. C, C/EBP␤-induced transactivation of a C/EBP␣
reporter gene. Reporter plasmid pC/EBP⌬380 (24) was transfected into
parental N2A cells or pool 4, and the resulting luciferase activity was
determined. The bar graph results display the average of at least three
separate experiments, and the S.D. is shown. ***, p ⬍ 0.001.
FIG. 7. Effect of C/EBP␣ overexpression on the phenotype of
N2A and p4 cells. Parental N2A and pool p4 were infected with
C/EBP␣ or empty vector retrovirus, as indicated under “Experimental
Procedures,” and 2 days after infection, pictures were taken at a 300-
fold magnification. Bar scale, 20 ␮m.
served in the N2A overexpressing the C/EBP␤ protein. To
confirm the activation of p53 in p4, we analyzed the levels of
p21, a well known downstream gene of p53. Using the same
protein extracts, we could observe a significant increase in the
levels of p21 in p4 cells as compared with parental N2A cells
(Fig. 9A). These experiments clearly demonstrate the ability of
C/EBP␤ to induce the activity and/or expression of two cell
cycle-related proteins shown to play a role in the apoptotic
pathway.
DISCUSSION
The molecular mechanisms regulating the differentiation
and death of neuronal cells during the development of the
 Neuronal Differentiation Induced by C/EBP␤
FIG. 8. Effect of C/EBP␤ overexpression on apoptosis of
Neuro2A cells. Cells were cultured for 24 h in serum-free medium and
stained with PI, and the frequency of apoptotic and necrotic cells was
determined by flow cytometry. Values (⫾S.D.) represent the mean of
triplicate experiments. **, p ⬍ 0.01.
FIG. 9. A, expression of p53 and p21 in parental N2A cells and p4.
Total protein extracts were prepared from cells incubated for 24 h in
complete medium and analyzed by immunoblot using the anti-p53 or
anti-p21 antibodies as described under “Experimental Procedures.” B,
N2A cells and p4 cells were transfected with 0.5 ␮g of pGL3-luc or
pMG15-luc vectors, and 24 h after transfection, luciferase activity of cell
lysates was determined. The bar graph results display the average of at
least three separate experiments in triplicate, and the S.D. is shown. *,
p ⬍ 0.05.
nervous system are very complex, and their regulation impli-
cates the action of numerous genes (1). In this study, we have
used Neuro2A cells, a well studied model of differentiation and
cell death (3), to show that overexpression of the transcription
factor C/EBP␤ clearly promotes neuronal differentiation. This
promotion of neurite outgrowth is accompanied by an increase
in the expression of neuronal differentiation markers such as
MAP2 and NeuN. We also show that the C/EBP␤ protein is
widely distributed in the brain of postnatal rats, a period of
time during which the dendritic arbor of neurons develops (33).
In addition, C/EBP␤ overexpression significantly increases pro-
grammed cell death, probably through a mechanism involving
5465
the cell cycle-dependent proteins p53 and p21. These results
suggest that C/EBP␤ may play an important role in the regu-
lation of neuronal differentiation and cell death.
In the brain, recent in situ hybridization studies have shown
that C/EBP␤ mRNA is widely expressed in the adult mice and
is mainly located in neurons (20). In agreement with these
results, we show here that the C/EBP␤ protein is also widely
expressed in the neonatal rat brain (Fig. 1), a period of intense
neuronal maturation. Regarding a possible role of C/EBP␤ in
the brain, studies of long term facilitation using cultured neu-
rons of the marine mollusk Aplysia have provided evidence for
a role of an Aplysia C/EBP homologue (ApC/EBP) in synaptic
plasticity. Stimulation of cyclic AMP signaling pathways in-
duced the expression of this protein, and injection of antiserum
against ApC/EBP demonstrated its critical role in the estab-
lishment of long term facilitation (21). In mammals, it has been
shown that the expression and DNA binding activities of
C/EBP␤ and C/EBP␦ are enhanced by the stimulation of cAMP
or Ca2⫹ signals in cultured hippocampal neurons (22), suggest-
ing a possible involvement of these genes in long term plasticity
in mammalian brain. It has been also shown that rat C/EBP␤
and C/EBP␦ proteins are induced after inhibitory avoidance
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learning and that they colocalize with phosphorylated cAMP-
response element-binding protein in the hippocampus (34).
This induction is blocked by fornix lesions, a manipulation that
is known to impair memory consolidation. Very recently,
Taubenfeld et al. (35) have directly demonstrated that in mam-
mals, the consolidation of new but not reactivated memory
requires C/EBP␤.
Here we show that overexpression of C/EBP␤ in N2A cells
promotes neuronal differentiation, as measured by neurite out-
growth and expression of the neuronal markers MAP2 and
NeuN. These observations, along with the presence of C/EBP
protein in the brain of developing rats and its neuronal local-
ization (according to the in situ studies (20)) suggest that this
protein could participate in the regulation of growth of neuro-
nal processes in the developing brain. The effect of C/EBP␤ on
neurite outgrowth observed here is specific to this protein and
mediated by its transactivation capacity because the abroga-
tion of this transactivation potential (by the dominant negative
CHOP) completely blocks neurite extension. These results are
in line with previous studies showing an implication of the
C/EBP␤ protein in the differentiation of non-neuronal tissues
(12, 36).
Neurite outgrowth depends on the increased assembly of
microtubules, which constitute the cytoskeletal scaffolding of
developing neurites (2, 37, 38). During this event, both an
increase in the expression of microtubule proteins and post-
transcriptional modifications take place (2, 39, 40). The micro-
tubule-associated protein MAP2 plays an important role in
tubuline polymerization and is necessary for the initial forma-
tion of neurites (39, 41). Expression of MAP2 antisense tran-
scripts in P19 embryonic carcinoma cells undergoing retinoic
acid-induced neuronal differentiation results in a significant
reduction in the number of neurites observed (42). Also, it has
been reported that blocking the expression of MAP2 with an-
tisense oligonucleotides results in a suppression of the out-
growth of neurites in cultured cerebellar macroneurons (43).
The results presented here show that C/EBP␤ regulates the
expression of MAP2 because a robust increase in the levels of
MAP2 protein is observed in cells overexpressing C/EBP␤, sug-
gesting that MAP2 is involved in C/EBP␤ action on neurite
extension.
In non-neuronal cells, such as 3T3-L1, it has been shown
that C/EBP␤ is expressed early in the differentiation program
(36), and this induction is thought to be responsible for the
5466 Neuronal Differentiation Induced by C/EBP␤
later transcriptional activation of C/EBP␣, another member of
this family. Among other effects, it has been described that the
expression of C/EBP␣ is necessary for the maintenance of the
differentiated state of adipose and hepatic tissues (44), and
ectopic expression of this protein in several fibroblastic cell
lines is sufficient to induce adipocyte differentiation (45). Here
we show that C/EBP␤ induces the expression of the endogenous
C/EBP␣ gene in N2A cells because a significant increase in the
endogenous levels of this protein is observed in the C/EBP␤-
overexpressing cells. This is associated with an enhancement of
the promoter activity of the C/EBP␣ gene, which contains a
C/EBP␤ consensus binding site. In addition, when p4 cells are
infected with a retroviral expression vector for C/EBP␣, a more
differentiated phenotype can be observed. Interestingly, over-
expression of C/EBP␣ in parental N2A also results in an induc-
tion of differentiation, suggesting that this protein by itself is
able to trigger neuronal differentiation. Altogether, these re-
sults suggest that, similarly to the proposed model for adipo-
cyte differentiation, C/EBP␤ seems to regulate C/EBP␣ gene
expression in neuroblastoma cells, and this in turn would also
play an important role in the neuronal differentiation of these
cells.
Transcription factor phosphorylation is a common way of
control of transcriptional activity (46, 47). It is well established
that C/EBP␤ is a nuclear end point for several protein kinase-
mediated pathways (19, 47, 48). Activation of cAMP-dependent
protein kinase in rat pheochromocytoma PC12 cells increases
phosphorylation and nuclear translocation of C/EBP␤ and a
concomitant induction of c-fos transcription (48). Site-selective
phosphorylation of C/EBP␤ at Ser105 by protein kinase C en-
hances C/EBP␤ transactivation activity in HepG2 cells (49).
More recently, Piwien-Pilipuk et al. (50) have also shown that
growth hormone regulates the phosphorylation and function of
C/EBP␤ by modulating the PI3K pathway. The results pre-
sented here clearly show that activation of the PI3K signaling
pathway by expression of the constitutively activated K227E
mutant increased neuronal differentiation in cells overexpress-
ing C/EBP␤, whereas activation of this pathway had no effect
on parental N2A cells. Consistently, a PI3K inhibitor,
LY294002, inhibited neurite outgrowth. This process was un-
affected by the MEK inhibitor PD98059. Taken together, these
data suggest that C/EBP␤-mediated neuronal differentiation
requires activation of the PI3K pathway.
At a certain stage during the development and differentia-
tion of the vertebrate nervous system, the massive scale death
of neurons and oligodendrocytes occurs as part of the normal
physiological process (51, 52). This neuronal cell death has
been shown to occur via apoptosis (53). The results presented
here demonstrate that overexpression of C/EBP␤ causes a sig-
nificant increase in the apoptotic population of N2A cells,
which is in agreement with previous reports showing a role of
the C/EBP␤ protein in the induction of apoptosis in hematopoi-
etic and myeloma cells (12, 54). This apoptotic process appears
to implicate at least two cell cycle-dependent proteins in the
mediation of this effect, p53 and p21. C/EBP␤ does not enhance
the levels of p53 protein, but it is able to trigger its activation,
as shown by the observed increase in the transactivation of a
p53-responsive promoter in C/EBP␤-overexpressing cells (Fig.
9). It has been shown that the DNA binding and transcriptional
activity of p53 can be modulated by phosphorylation (55–57)
and that this phosphorylation can induce a shift in the migra-
tion of this protein in an SDS gel (58), similar to the one
described here. These data suggest that the apoptosis induced
by C/EBP␤ in N2A cells could be mediated by an increase in
p53 phosphorylation and the subsequent enhancement in its
transactivation activity. The increase in p21 protein levels
detected in C/EBP␤ overexpressing cells could be due to an
increase in p53 activity or to a direct regulation of p21 gene
expression by the C/EBP␤ itself because, although it is well
established that p53 regulates the expression of the p21 gene
(59, 60), it has also been shown that p21 can be directly regu-
lated by members of the C/EBP family in other cell systems
(61, 62).
In summary, our results identify C/EBP␤ as a potent inducer
of neurite outgrowth and apoptosis of neuroblastoma cells and
suggest a potential role of this transcription factor in the de-
veloping brain because the C/EBP␤ protein is present in this
organ during the developmental period.
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CCAAT/Enhancer-binding Protein β Plays a Regulatory Role in Differentiation and
Apoptosis of Neuroblastoma Cells
Marta Cortés-Canteli, Miguel Pignatelli, Angel Santos and Ana Perez-Castillo
J. Biol. Chem. 2002, 277:5460-5467.
doi: 10.1074/jbc.M108761200 originally published online December 3, 2001

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