OPEN
A p38mapk-p53 cascade regulates mesodermal
differentiation and neurogenesis of embryonic
stem cells
Y Hadjal1,2
, O Hadadeh1,2
, CEl Yazidi1,2
, E Barruet1,2
and B Bine´truy*,1,2
Embryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular
therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward speciﬁc lineages need to
be speciﬁed. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and
is necessary to mesodermal formation during the critical ﬁrst 5 days of mouse ESC commitment. This period corresponds to the
expression of speciﬁc master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic
approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering
directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections
stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections.
Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting
cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53À / À
ESCs compared with their
wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of
anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene
expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression,
p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part,
the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages.
Cell Death and Disease (2013) 4, e737; doi:10.1038/cddis.2013.246; published online 25 July 2013
Subject Category: Experimental Medicine
Because of their pluripotency and unlimited proliferation
properties, embryonic stem cells (ESCs) have been proposed
as cellular therapy for human diseases. However, this
potential is impaired by the poor efﬁciency of ESC differentiation
into mature differentiated cells and by the persistence of
undifferentiated cells with a tumorigenic capacity within
differentiated cultures. Therefore, there is a crucial need for
a better understanding of molecular mechanisms governing
ESC commitment into speciﬁc lineages. Self-renewal of
mouse ESCs is dependent on the intracellular pathways
involving a complex interplay between speciﬁc epigenetic
processes, miRNAs and transcription factors, such as Oct4,
Nanog or Sox2 (see for reviews1,2
). In vitro ESC differentiation
can be induced by various experimental protocols resulting in
the commitment into a variety of mature differentiated cell
types (see for reviews3–5
). This process can be modulated
between the 2nd and 5th day of differentiation by the potent
morphogen retinoic acid (RA) that induces differentiation
into neurons and adipocytes6–8
and, conversely, inhibits
cardiomyocyte and skeletal myotube formation.6,9,10
Although
ESC differentiation experimental models recapitulate the
successive steps from pluripotency to well-differentiated cells,
the molecular mechanisms underlying these processes are
poorly understood.
p38 Mitogen-activated protein kinase (p38MAPK) is
activated by phosphorylation in response to osmotic stress,
UV and various cytokines involved in inﬂammatory responses
and has been proposed to regulate several cellular processes,
such as proliferation, cell survival and differentiation.11,12
Deletion of the p38a gene leads to early embryonic lethality
between 11.5 and 12.5 days,13,14
demonstrating an essential
role of p38MAPK in development. We previously found that
the p38MAPK pathway is necessary at two successive early
steps during the differentiation process of ESC commitment
into cardiomyocyte, endothelial cells, smooth muscle and
skeletal muscle cell lineages, whereas it inhibits neuro-
genesis.10,15
Particularly, this p38MAPK control takes
place during the critical ﬁrst 5 days of ESC commitment,
concomitantly with the expression of speciﬁc master genes
that direct ESC into each of the three embryonic layers.
To better understand the molecular mechanisms underlying
these processes, we treated ESCs with a p38MAPK-speciﬁc
1
INSERM U910, Faculte´ de Me´decine, 27 Boulevard Jean Moulin, Marseille Cedex 5, France and 2
Faculte´ de Me´decine, Aix-Marseille Universite´, 27 Boulevard Jean
Moulin, Marseille Cedex 5, France
*Corresponding author: B Bine´truy, INSERM U910, Faculte´ de Me´decine La Timone, 27 Boulevard Jean Moulin, Marseille Cedex 5 13385, France.
Tel: 33 04 9132 4886; Fax: 33 04 9182 6958; E-mail: Bernard.Binetruy@univ-amu.fr
Received 31.5.13; revised 05.6.13; accepted 06.6.13; Edited by G Raschella`
Keywords: mouse embryonic stem cells; p53; p38MAPK; neurogenesis; cardiomyogenesis; skeletal myogenesis
Abbreviations: ERK, extracellular signal-regulated kinase; EB, embryoid body; ESC, embryonic stem cell; MAPK, mitogen-activated protein kinase; MyH1, myosin
heavy chain-1; RA, retinoic acid; RT-qPCR, reverse-transcriptase quantitative PCR; PBS, phosphate-buffered saline
Citation: Cell Death and Disease (2013) 4, e737; doi:10.1038/cddis.2013.246
& 2013 Macmillan Publishers Limited All rights reserved 2041-4889/13
www.nature.com/cddis
inhibitor and performed microarray experiments on mRNAs
extracted from treated versus untreated cells. Interestingly,
one of the regulated targets is Bcl2, a well-known
p53-inhibited gene.
The p53 protein is a short-lived, latent transcription factor,
which is activated and stabilized in response to a wide range
of cellular stresses; exacerbated p53 activation leads
to dramatic cellular responses such as cell cycle arrest,
apoptosis or senescence. Furthermore, p53 is a crucial tumor
suppressor that is somatically mutated in about half of all the
human cancers and has multiple roles in maintaining genomic
stability in somatic cells (see for review16
). Its role in ESC
biology seems paradoxical, indeed undifferentiated ESCs fail
to undergo p53-dependent cell cycle arrest, apoptosis and
senescence after DNA damage. By contrast, p53 can induce
ESC differentiation by directly suppressing the expression of
Nanog, one of the master transcriptional factor maintaining
ESC self-renewal (Lin et al.17
and see for review18
). However,
its precise role in ESC commitment toward speciﬁc lineages is
unknown.
We analyzed the role of p53 in ESC differentiation and
found that p53 is an important mediator of p38MAPK-induced
control of ESC commitment.
Results
Differential display experiments identify p53 as a
potential mediator of the p38MAPK pathway. CGR8
mouse ESCs were induced to differentiate by forming
embryoid bodies (EBs) and, at day 3, treated or not by the
speciﬁc p38MAPK inhibitor PD169316, and mRNAs were
extracted and analyzed by microarray experiments 48 h later.
For each condition, three mRNA batches were hybridized
on DNA chips spotted with 44 K mouse gene-speciﬁc
sequences, and regulations of genes of interest were
conﬁrmed by reverse-transcriptase quantitative PCR
(RT-qPCR) technique (data not shown). Among the identiﬁed
targets, we focused our attention on one gene upregulated in
our experiments, Bcl2, a well-known p53-inhibited gene.19,20
Interestingly, Bcl2 has already been described as a
p38MAPK-inhibited gene and involved in ESC neuro-
genesis.21
Altogether, these data suggested that p53 could
mediate the p38MAPK control on ESC differentiation
and prompted us to verify this hypothesis.
Chemical inhibition of p53 mimics the effects
of p38MAPK inhibition on target genes and ESC
commitment. CGR8 ESCs induced to differentiate were
treated by either the p38MAPK inhibitor or piﬁthrin-a, a
speciﬁc p53 chemical inhibitor,22
and compared with
untreated controls. As expected, p38MAPK inhibitor treatment
induces Bcl2 gene expression and enhances neurogenesis,
characterized by MAP2 gene expression;
interestingly, p53 inhibition induces similar effects (Figures
1a and b). Conversely, cardiomyocyte formation
was repressed by these treatments; as judged by Mesp1
expression (Figure 1c), a cardiac mesodermal master
gene involved in ESC cardiomyogenesis23
and TroponinT2
Figure 1 Chemical inhibition of p53 mimics the effects of p38MAPK inhibition. EBs from wild-type CGR8 ESCs at day 0 were induced to differentiate and treated or not,
between day 3 and 10, by either the PD169316 p38MAPK inhibitor or the piﬁthrin-a p53 inhibitor. mRNAs from EBs were extracted at day 10 (a and b) or at various time as
indicated (c and d) and analyzed by real-time RT-PCR for the expression of Bcl2 (a), MAP2 (b), Mesp1 (c) and TroponinT2 (d) gene expression. Results are expressed in
arbitrary units, with the values of untreated CGR8 control cells (Ct) taken as 1, and are the mean±S.E.M. of at least three independent experiments. Signiﬁcance to Ct values
is given as: *Po0.05 and **Po0.01
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
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expression (Figure 1d), a cardiomyocyte-speciﬁc
marker. These data suggest that the p38MAPK role in ESC
differentiation could indeed be mediated by a p38MAPK-p53
cascade.
The p38 MAPK pathway regulates the stability of
endogenous p53 proteins at day 3 of differentiation.
In response to osmotic stress, p53 proteins have been shown
to be directly phosphorylated by p38MAPK on several
serine residues,24,25
and, in addition, p53 acetylation has
been involved in human ESC differentiation.26
Therefore,
we analyzed the consequences of p38MAPK inhibition on p53
protein expression and post-translational regulation. We could
not ﬁnd any signiﬁcant effect of p38MAPK inhibition neither on
phosphorylation of p53 serine residues 15 and 392 or on p53
acetylation (data not shown). As already shown,17
we found
that the p53 protein level decreases rapidly as ESCs
differentiated (Figure 2a); however, at day 3 of differentiation,
a stronger decrease is systematically observed 24h after
p38MAPK inhibition (Figure 2b). Noteworthy, this effect is not
detectable after 4h of inhibition. This regulation is not at the
p53 transcriptional level, neither at day 3 (Figure 2b, lower
panel) or during the ﬁrst 5 days of differentiation (Figure 2c),
and is not associated to a control of the cytoplasmic/nucleic
localization of p53 proteins (Figure 2d). Finally, we found that
this effect is not mediated by a p38MAPK-dependent regulation
of the proteasome activity. Indeed, although inhibition of
the proteasome efﬁciently stabilizes p53 proteins, the destabilization
of p53 proteins induced by p38MAPK inhibition is still
present after treatment with the proteasome inhibitor MG132
(Figure 2e). Although additional works are necessary to
understand the molecular mechanism by which the p38MAPK
pathway augments p53 protein stability, the experiments
shown suggest that p38MAPK inhibition leads to a shorter
half-life of p53 protein. Furthermore, the fact that this regulation
necessitates 24h (Figure 2b) strongly suggests that it
corresponds to an indirect mechanism involving intermediate
partner(s).
To precise the role of p53 in ESC differentiation and to avoid
potential unspeciﬁc effects of chemical compounds, we then
Figure 2 The p38MAPK regulates the stability of p53 proteins in CGR8 ESCs. (a) ESCs were induced to differentiate, and total cellular proteins were extracted at various
time from day 0 to 5, as indicated, and analyzed by western blotting with either a-p53 antibodies or a-extracellular signal-regulated kinase (a-ERK) antibodies as loading
control. (b) ESCs were induced to differentiate and, at day 3, treated or not, as indicated, by the p38MAPK inhibitor. Total cellular proteins or mRNAs were extracted 0, 4 or
24 h later and analyzed either by western blotting with a-p53 antibodies or a-ERK antibodies as loading control (upper panels) or by real-time RT-PCR for the expression of p53
gene (lower panel). (c) ESCs were induced to differentiate and treated or not, as indicated, by the p38MAPK inhibitor either from day 0 to 5 or from day 3 to 5. mRNAs were
extracted at various time, as indicated, and analyzed by real-time RT-PCR for the expression of p53 gene. Results are expressed in arbitrary units, with the values of untreated
CGR8 control cells at day 0 taken as 1, and are the means±S.E.M. of at least three independent experiments. (d) ESCs were induced to differentiate and, at day 3, treated or
not, as indicated, by the p38MAPK inhibitor. Cytoplasmic and nuclear cellular proteins were extracted 0, 4 or 24 h later and analyzed by western blotting with either a-p53
antibodies or a-ERK antibodies as loading control for cytoplasmic extracts, or a-LaminB1 as loading control for nuclear extracts. (e) ESCs were induced to differentiate until
day 3 and treated or not either by the p38MAPK inhibitor for 24 h or by 20mM of proteasome inhibitor MG132 for 5 h (left panel); in parallel experiments, EBs were treated either
by the proteasome inhibitor alone for 5 h or by the p38MAPK inhibitor for 24 h and then by MG132 for 5 h (right panel). Total cellular proteins were extracted and analyzed
by western blotting with either a-p53 antibodies or a-ERK antibodies as loading control
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
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Cell Death and Disease
used two genetic approaches: transient transfections of
anti-p53 siRNAs and analysis of differentiation capacities
of ESC knockout for the p53 gene.27
siRNAs against p53 efﬁciently inhibit p53 expression
and activity in ESCs. Anti-mouse-p53 siRNAs (siRNA
SMART pool; Dharmacon, Waltham, MA, USA) were
transiently transfected into undifferentiated CGR8 ESCs
and p53 mRNA expression analyzed by RT-qPCR 24 and
48 h later. A strong decrease in expression is consistently
observed compared with cells transfected with control
siRNAs, with only 8.5–10% of p53 mRNA left (Figure 3a).
This p53 inhibition is also detected at the protein level,
as shown by western blot experiment (Figure 3b).
We then analyzed the p53 repression during differentiation;
undifferentiated ESCs were transfected with control or
anti-p53 siRNAs, the day after (day 0) ESCs were induced
to differentiate, and p53 mRNAs were analyzed at days 0, 3,
4, 5, 7 and 12 of differentiation. A signiﬁcant p53 repression
persists until day 5 of the protocol, and is only lost at day 7
(Figure 3c). Importantly, this inhibitory effect last long enough
to cover the ﬁrst 5 days of critical period for ESC
commitment. We then investigated the effects of anti-p53
siRNA transfections on p53 activity. As expected, compared
with control cells, transfections of anti-p53 siRNAs in ESCs
lead to a 3–4-fold increase in Bcl2 expression (Figure 3d),
whereas the expression of p21 gene, a positively regulated
p53 target, is decreased by 70% at 24 h and 60% at 48 h
(Figure 3e).
siRNA-mediated inhibition or homozygote gene
knockout of p53 interfere with ESC differentiation.
Differentiation capacities of CGR8 ESCs transfected by
anti-p53 siRNAs compared with control siRNAs transfection
and those of p53À / À
ESCs compared with their wild-type
counterparts were then analyzed. Anti-p53 siRNAtransfected
CGR8 ESCs and p53À / À
ESCs display, on the
one hand, augmented neurogenesis (Figure 4a) and, on
the other hand, a strong inhibition of cardiomyogenesis,
illustrated by the decrease in gene expression of both the
cardiac transcription factor Nkx2.5 (Supplementary Figure
S1A) and the cardiac marker TroponinT2 (Figure 4b)
compared with controls. This cardiac inhibition is also clearly
detected by immunoﬂuorescence experiments using
a-troponinT2 antibodies (Figures 4c and d). In addition to
ESC neurogenesis and cardiomyogenesis, the p38MAPK
pathway controls also endothelial cell, smooth and skeletal
muscle cell10
and adipocyte28
lineage formation. Similarly to
p38MAPK inhibition, we found that the two p53-inhibition
approaches induce a strong inhibition of skeletal myogenesis
compared with controls; as judged by the decrease in
myosin heavy chain-1 (MyH1) mRNA (Figure 5a) and protein
(Figure 5b) expression and by immunoﬂuorescence
experiments using a-MyH1 antibodies (Figures 5c and d).
By contrast, although p38MAPK inhibition leads to a
decrease in endothelial and smooth muscle cell formation
and to an increase in adipogenesis, these three cell lineages
are not signiﬁcantly affected by anti-p53 siRNA transfections.
These lineages were characterized by gene expression of
the following speciﬁc markers: Tie2 and Flk1 for endothelial
differentiation (Supplementary Figures S1B and C, left parts);
a-smooth muscle actin for smooth muscle cells (Figure 6a,
left part); and peroxisome-proliferative-activated receptor
gamma and adipocyte-speciﬁc lipid-binding protein
for adipocytes (Figures 6b and c, left parts). Similarly,
endothelial differentiation is not modiﬁed in p53À / À
ESCs
compared with their wild-type counterparts (Supplementary
Figures S1B and C, right parts). Surprisingly, smooth muscle
Figure 3 siRNAs against p53 inhibit p53 expression and activity in CGR8 ESCs. (a and b) Undifferentiated ESCs were transfected overnight with control siRNAs (si Ct) or
anti-p53 siRNAs (si p53), kept in undifferentiated conditions. mRNAs (a) or proteins (b) were extracted after either 24 or 48 h, as indicated, and analyzed by real-time RT-PCR
or western blotting for the expression of p53 gene. (a) Results are expressed in arbitrary units, with the values of untreated CGR8 control cells taken as 1, and are the
means±S.E.M. of at least three independent experiments; residual p53 mRNA expression is indicated in %. (b) Total proteins were analyzed with either a-p53 antibodies or
a-actin antibodies as loading control. (c) Undifferentiated ESCs were transfected overnight with si Ct or si p53 and induced to differentiate. mRNAs were extracted at various
time from day 0 to 12, as indicated, and analyzed by real-time RT-PCR for the expression of p53 gene. Results are expressed in arbitrary units, with the values of control
siRNA-transfected control cells at day 0 taken as 1, and are the means±S.E.M. of at least three independent experiments. (d and e) Undifferentiated ESCs were transfected
overnight with si Ct or si p53 and kept in undifferentiated conditions. mRNAs were extracted 24 or 48 h later and analyzed by real-time RT-PCR for the expression of Bcl2 (d) or
p21 (e) genes. Results are expressed in arbitrary units, with the values of control siRNA-transfected control cells taken as 1, and are the means±S.E.M. of at least three
independent experiments
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
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cell formation and adipogenesis are strongly inhibited in
p53À / À
ESCs (Figures 6a–c, right parts). These data reveal
different biological effects of transient p53 inhibition by
siRNAs versus p53 homozygote gene knockout, suggesting
variable successive roles for this gene during ESC differentiation.
Altogether, our results also suggest the existence
of other mediators than p53 for the p38MAPK pathway to
account for all its effects on ESC differentiation.
To precise the p53 role in ESC differentiation, we analyzed
the effects of p53 inhibition on expression of master genes
important during embryonic development and ESC commitment.
We focused our analysis on master genes whose gene
expression peaked during the day 0–5 period of ESC
differentiation, and therefore susceptible to be regulated by
siRNA transient transfection experiments. Consistent
with neurogenesis regulation, the peak expression of the
Pax6 ectodermal master gene is augmented by anti-p53
siRNA transfections (Figure 7a). We also investigated the
expression of three master genes relevant to mesodermal
lineages: Brachyury, Mesp1 and TBX1. Gene expressions of
Brachyury and Mesp1, which peak at day 5, are almost
completely abolished by p53 inhibition, either after anti-p53
siRNA transfections or in p53À / À
cells (Figures 7b–e).
These inhibitions could account for the p53 effects on
mesodermal-derived differentiations. By contrast, although
TBX1 is not signiﬁcantly affected by anti-p53 siRNAs
(Figure 7f), expression of this gene is strongly downregulated
in knockout cells (Supplementary Figure S2A), suggesting for
this gene a complex regulation by p53. Noteworthy, the peak
of expression of TBX1 appears at day 7, later than Brachyury
and Mesp1, which could explain the lack of regulation by
anti-p53 siRNAs.
p53-dependent Nanog repression contributes partially
to the p53 effects on ESC differentiation. ESC differentiation
is accompanied by the rapid downregulation of
Nanog gene expression, a crucial gene involved in ESC
self-renewal. It has been shown that this regulation is
mediated by p53, which, upon differentiation, gets activated
and represses Nanog expression by direct binding to its
promoter.17
We tested the hypothesis that this p53-dependent
Nanog repression would contribute to p53 regulation of
ESC differentiation. Consequently, Nanog expression should
be augmented in p53À / À
ESCs, and interfering with this
expression should affect their differentiation capacities.
Indeed, we found that a signiﬁcant percentage of the level
of Nanog expression in undifferentiated cells persists until
day 12 of the differentiation of p53À / À
ESCs, whereas this
Figure 4 Effects of anti-p53 siRNAs or homozygote gene knockout of p53 on ESC neurogenesis and cardiomyogenesis. Undifferentiated CGR8 ESCs were transfected
overnight with control siRNAs (si Ct) or anti-p53 siRNAs (si p53). The day after, day 0, these cells and p53À / À
ESCs (p53À / À
) and their wild-type counterparts (p53þ / þ
)
were induced to differentiate, cultivated until day 7 and replated on petri dishes until day 12 for analysis of neurogenesis (a) or cardiomyogenesis (b–d). mRNAs were extracted
and analyzed by real-time RT-PCR for MAP2 (a) or TroponinT2 (b) gene expression. Results are expressed in arbitrary units, with the values of untransfected CGR8 control
cells or wild-type control ESCs taken as 1, and are the means±S.E.M. of at least three independent experiments. Signiﬁcance to Ct values is given as: *Po0.05, **Po0.01
and ***Po0.001. For immunoﬂuorescence analysis, differentiated cell cultures were ﬁxed and incubated with a-TroponinT2 (c and d) antibodies (upper panels), and nuclei
were counterstained with DAPI (lower panels); representative ﬁelds are shown
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
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Cell Death and Disease
expression is barely detected after day 5 into their wild-type
counterparts (Figure 8a). This persistence of Nanog expression
seems to profoundly perturb the commitment of p53À / À
ESCs. In addition to Nanog, the expression of Oct4, another
key self-renewal transcription factor, is also persisting during
differentiation instead of decreasing rapidly (Supplementary
Figure S2B). This result is most likely due to the known
capacity of these transcription factors to activate each other.1
To test the role of Nanog during p53À / À
ESC differentiation,
we performed transient transfections of anti-Nanog siRNAs
at day 0 and found that Nanog mRNA expression is inhibited
by 70% (Figure 8b). This Nanog inhibition is sufﬁcient to
restore signiﬁcant Brachyury and Mesp1 mRNA expressions,
at days 5 (Figure 8c) and 7 (Figure 8d), respectively, of the
differentiation process; by contrast, it has no effect on Pax6,
Bcl2 (Supplementary Figures S3A and B) or TBX1 (data not
shown) gene expressions. Subsequently, we observed the
restoration of p53À / À
ESC differentiation capacities toward
cardiomyocyte and smooth muscle cell formation (Figures 8e
and f and Supplementary Figure S3C). However, it is worth to
note that this restoration is partial, reaching only 30–50% of
the level of their wild-type counterparts. Importantly, in these
experiments, anti-Nanog siRNAs display no signiﬁcant effect
on neurogenesis (Figure 8g), which correlates with the lack
of effect on Bcl2 and Pax6 gene expression. Likewise,
although activating Brachyury expression, anti-Nanog
siRNAs do not restore skeletal myogenesis (Figure 8h),
showing that Brachyury regulation is not sufﬁcient for this
lineage. Altogether, our results suggest that p53-dependent
Nanog repression is involved in p53 activation of cardiomyogenesis
and smooth muscle formation, whereas neurogenesis
inhibition and skeletal myogenesis activation are Nanog
independent.
Discussion
We previously described a role for p38MAPK as an early
switch of ESC commitment into mesodermal lineages
(p38MAPK on) or neurons (p38MAPK off).10,15
This control
takes place during the critical ﬁrst 5 days of ESC commitment,
concomitantly with the expression of speciﬁc master genes
that direct ESC into each of the three embryonic layers. We
found now that this effect is partially mediated by the
transcription factor p53, unveiling an important p38MAPKp53
cascade regulating the early commitment of ESCs.
During the ESC differentiation process, p38MAPK induces a
stabilization of p53 protein and increases its transcriptional
activity. This cascade, regulating the expression of speciﬁc
Figure 5 Effects of anti-p53 siRNAs or homozygote gene knockout of p53 on ESC skeletal myogenesis. Undifferentiated CGR8 ESCs were transfected overnight with
control siRNAs (si Ct) or anti-p53 siRNAs (si p53). The day after, day 0, these cells and p53À / À
ESCs (p53À / À
) and their wild-type counterparts (p53þ / þ
) were induced to
differentiate, cultivated until day 7 and replated on petri dishes until day 26 for skeletal myotube formation. (a) mRNAs were extracted and analyzed by real-time RT-PCR
for MyH1 gene expression. Results are expressed in arbitrary units, with the values of untransfected CGR8 control cells or wild-type control ESCs taken as 1, and are the
means±S.E.M. of at least three independent experiments. Signiﬁcance to Ct values is given as: *Po0.05, **Po0.01 and ***Po0.001. (b) Total cellular proteins were
extracted and analyzed by western blotting with either a-MyH1 antibodies or a-extracellular signal-regulated kinase (a-ERK) antibodies as loading control. (c and d) For
immunoﬂuorescence analysis, differentiated cell cultures were ﬁxed and incubated with a-MyH1 antibodies (upper panels), and nuclei were counterstained with DAPI
(lower panels); representative ﬁelds are shown
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
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master genes of ESC commitment, inhibits ectodermal and,
conversely, activates several mesodermal lineages.
Homozygous p53-knockout mice were found to survive to
birth, but then to succumb rapidly to tumors,29
suggesting, at
ﬁrst, that the p53 gene is dispensable during the embryonic
development but crucial as tumor-suppressor gene during the
adult life. However, a high rate of developmental abnormalities
is associated with p53 deﬁciency, which result to defects
Figure 6 Effects of anti-p53 siRNAs or homozygote gene knockout of p53 on ESC smooth muscle cell and adipocyte formation. Undifferentiated CGR8 ESCs were
transfected overnight with control siRNAs (si Ct) or anti-p53 siRNAs (si p53). The day after, day 0, these cells and p53À / À
ESCs (p53À / À
) and their wild-type counterparts
(p53þ / þ
) were induced to differentiate, cultivated until day 7 and replated on petri dishes until either day 12 for smooth muscle analysis (a) or day 26 for adipocyte analysis
(b and c). mRNAs were extracted and analyzed by real-time RT-PCR for a-smooth muscle actin (a-SMA) (a), adipocyte-speciﬁc lipid-binding protein (aP2) (b) and
peroxisome-proliferative-activated receptor gamma (PPARg) (c) gene expression. Results are expressed in arbitrary units, with the values of untransfected CGR8 control cells
or wild-type control ESCs taken as 1, and are the means±S.E.M. of at least three independent experiments. Signiﬁcance to Ct values is given as: *Po0.05, **Po0.01 and
***Po0.001
Figure 7 Effects of anti-p53 siRNAs or homozygote gene knockout of p53 on the expression of master genes of ESC commitment. Undifferentiated CGR8 ESCs were
transfected overnight with control siRNAs (si Ct) or anti-p53 siRNAs (si p53). The day after, day 0, these cells and p53À / À
ESCs (p53À / À
) and their wild-type counterparts
(p53þ / þ
) were induced to differentiate. mRNAs were extracted at various time from day 0 to 7, as indicated, and analyzed by real-time RT-PCR for the expression of Pax6 (a),
Brachyury (b and c), Mesp1 (d and e) and TBX1 (f) genes. Results are expressed in arbitrary units, with the values of untransfected CGR8 control cells (a, b, d and f) or
wild-type control ESCs (c–e) taken as 1, and are the means±S.E.M. of at least three independent experiments. Signiﬁcance to Ct values is given as: *Po0.05, **Po0.01
and ***Po0.001
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
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in neural tube closure, overgrowth of neural tissues and
exencephaly, therefore suggesting a role for p53 in develop-
ment.30,31
This apparent discrepancy could be due to
compensation by the p53 family members p63 and p73,
which are expressed in early mouse embryos, resulting in the
incomplete penetrance of the developmental phenotype
observed. Furthermore, in vitro, depending on the cellular
model analyzed, p53 can either facilitate or suppress cell
differentiation.32
In agreement with the in vivo negative role of
p53 on neurogenesis, we found that this transcription factor is
a potent early inhibitor of in vitro ESC neurogenesis and
that this effect can be explained, at least partially, by the
transcriptional repression of two genes Bcl2 and Pax6.
Indeed, Bcl2 is known to be targeted by p38MAPK and is
critical for neuronal commitment of ESCs,21
and Pax6 is a
transcription factor long known to be essential for cortical
development. Interestingly, it has been shown recently
that Pax6À / À
ESCs displayed impaired neurogenesis.33
Therefore, we hypothesize that, in our experiments, p53
inhibition leads to Bcl2 and Pax6 overexpressions, which in
turn augment ESC neurogenesis. These regulations seem
independent of the p53-induced repression of Nanog gene
expression observed during differentiation, suggesting
that the p53 effect is mediated by another mechanism.
p53 is necessary for myotube formation in C2C12
myoblast34
and mesenchymal stem cell35
cellular models.
Here we found a p53 requirement in skeletal myogenesis
early on during ESC commitment, demonstrating a p53
control all along this process. This effect is associated to
the control of the mesodermal master gene Brachyury, and we
found that the p53-dependent repression of Nanog participates
in this regulation. However, even if anti-Nanog siRNA
transfections are capable to restore Brachyury expression,
they do not restore skeletal myogenesis of p53À / À
ESCs.
This result could be due to a partial Nanog downregulation or,
alternatively, the involvement of additional molecular mechanisms
corresponding to later roles of p53.
Not much was known about p53 and in vitro cardiomyocyte
formation; however, it has been shown recently that
the pharmacological compound icariin augments ESC cardiomyogenesis
via activation of both p5336
and p38MAPK.37
Our results show that the p38MAPK-p53 cascade is required
for cardiomyocte formation from mouse ESCs. Furthermore,
inhibition of Nanog gene expression in differentiated p53À / À
ESCs restore both a cardiac phenotype and the expression of
two master genes important for cardiac development,
Brachyury and Mesp1.
In conclusion, our results identify a p38MAPK-p53-Nanog
cascade important for the ESC early commitment toward
several lineages. However, Nanog repression does not
account for all the effects of p53, and, likewise, the p53
activation is not sufﬁcient to account for all the effects of the
Figure 8 Role of p53-dependent Nanog repression on ESC differentiation. (a) p53À / À
ESCs (p53À / À
) and their wild-type counterparts (p53þ / þ
) were induced to
differentiate until day 12 and analyzed for endogenous Nanog expression at various time from day 0 to 12, as indicated. mRNAs were extracted and analyzed by real-time
RT-PCR. Results are expressed in arbitrary units, with the values of wild-type control ESCs at day 0 taken as 1, and are the means±S.E.M. of at least three independent
experiments. (b–h) Undifferentiated p53À / À
ESCs were transfected overnight with control siRNAs (si Ct) or anti-Nanog siRNAs (si Nanog) and were kept in undifferentiated
conditions (b), or induced to differentiate until day 5 (c), day 7 (d), day 12 (e–g) or day 26 (h) and compared with differentiated p53þ / þ
ESC counterparts. mRNAs were
extracted 24 h later (b), at days 0, 3, 5 and 7 (c and d), or after differentiation (e–h), and analyzed by real-time RT-PCR for Nanog (b), Brachyury (c), Mesp1 (d), TroponinT2
(e), Alpha-SMA (f), MAP2 (g) and MyH1 (h) gene expression. Results are expressed in arbitrary units, with the values of si Ct control ESCs taken as 1 (b–d), or with the values
of wild-type control ESCs taken as 1 (e–h), and are the means±S.E.M. of at least three independent experiments. Signiﬁcance to values obtained with p53þ / þ
cells or siCt
conditions is given as: *Po0.05 and **Po0.01
ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
8
Cell Death and Disease
p38MAPK signal transduction pathway on ESC differentiation,
suggesting the existence of additional molecular
mechanisms for these processes.
Materials and Methods
Differentiation of ESCs. Mouse ESCs CGR838
and p53À / À
and their
wild-type counterpart ESCs27
were grown on gelatin-coated plates and were
induced to differentiate as described previously.38
Brieﬂy, 1 Â 103
ESCs were
aggregated without leukemia-inhibitory factor to form EBs at day 0, cultivated in
suspension until day 7, treated (for neurogenesis and adipogenesis) or not (for
cardiomyogenesis, endothelial differentiation, smooth and skeletal myogenesis)
with 10À 7
M all-trans RA (Sigma, Saint-Quentin-Fallavier, France) daily between
day 3 and 5, plated at day 7 on gelatin-coated six-well plate and treated with
85 nM insulin (Sigma) and 2 nM 3,30
,5-tri-iodothyronine (Sigma) to induce terminal
differentiation. Fresh medium containing the inducers was applied every 2 days.
Neurons, cardiomyocytes, endothelial and smooth muscle cells were analyzed at
day 12, and skeletal myotubes and adipocytes were analyzed at day 26.10
During EB differentiation, 10 mmol/l of the p38 inhibitor PD169316
(Sigma) or 10 mmol/l of piﬁthrin-a (Sigma) were added to the culture media as
indicated.
RNA analysis. Total RNA was prepared using trizol reagent (Invitrogen, Life
Technologies, Saint-Aubier, France). Real-time RT-PCR was performed with the
ABI Prism7300 (Applied Biosystems, Life Technologies) and Taqman or Mesa
Green MasterMix (Eurogentec, Angers, France). The relative amounts of the
different mRNAs were quantiﬁed by using the comparative CT method (2À (DDCT)
).
36B4 was used as housekeeper transcripts, and gene expressions were
normalized using 36B4 RNA levels. Primer sequences (Eurogentec) are given in
Supplementary Table 1.
siRNAs experiments. ESCs grown without antibiotics were incubated at day
0 with siRNAs against either total p53 SMART pool (Dharmacon), Nanog SMART
pool (Dharmacon) or control siRNAs (Dharmacon). Transfection of siRNAs was
performed using Lipofectamine2000 (Invitrogen) according to the manufacturer’s
instructions. Brieﬂy, 30 ml of lipofectamine was incubated for 5 min in 1.5 ml
of antibiotic-free cell culture medium plus serum, and 1 nmol of siRNA was added
to the mix and incubated for 15 min; the mix was then added to 1 Â 106
cells.
p53 knockdown was determined either by immunoblotting with anti-p53 antibody
(Cell Signaling, Ozyme, Saint-Quentin en Yvelines, France) or by quantitative realtime
RT-PCR.
Western blot analysis. For p53 protein analysis, EBs were lysed as
described.38
Samples (40 mg) were separated by SDS-polyacrylamide gel
electrophoresis on a 10% gel (Invitrogen) and transferred onto polyvinylidene
diﬂuoride membranes (Millipore, Molsheim, France). Membranes were incubated
with antibodies against either mouse p53 (Cell Signaling), Lamin B1 (BioLegend,
Ozyme) or ERK2 (Santa Cruz Biotechnology, Clinisciences, Nanterre, France).
Immunoﬂuorescence staining. Differentiated EBs were washed and
ﬁxed at day 7 in 3% paraformaldehyde for 15 min at room temperature (RT).
Cell membranes were permeabilized in phosphate-buffered saline (PBS)-0.1%
Triton X-100 for 15min at RT and incubated with an anti-Troponin T (CT3) or an
anti-MHC (MF2Ds) (DSHB, Iowa City, IA, USA) in PBS-10% fetal bovine serum for
1 h. After three washes in PBS, cells were incubated with anti-mouse ALEXA488
or ALEXA546 (Invitrogen) for 1 h at RT. Cell imaging was performed using a Nikon
microscope Eclipse 90I. Z-stack pictures were obtained by using a Nikon Digital
Sight DS-1QM camera controlled by NIS element AR software (Nikon, Champigny
sur Marne, France).
Statistical analyses. All experiments were performed at least three times.
All data are expressed as the mean±S.E.M. Treatments were compared with
their respective controls, and signiﬁcant differences among the groups were
determined using unpaired Student’s t-test. A value of Po0.05 was taken as an
indication of statistical signiﬁcance.
Conﬂict of Interest
The authors declare no conﬂict of interest.
Acknowledgements. We thank Dr. Blattner (Karlsruhe, Germany) for the gift
of p53 wild-type and knockout ESC lines.
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ES differentiation control by a p38MAPK-p53 cascade
Y Hadjal et al
10
Cell Death and Disease