p38 Mitogen-Activated Protein Kinase Activity ... - Wiley Online Library

EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES p38 Mitogen-Activated Protein Kinase Activity Commits Embryonic Stem Cells to Either Neurogenesis or Cardiomyogenesis MYRIAM AOUADI, FRE´ DE´ RIC BOST, LESLIE CARON, KATHIANE LAURENT, YANNICK LE MARCHAND BRUSTEL, BERNARD BINE´ TRUY Institut National de la Sante´ et de la Recherche Me´dicale U568, Institut Fe´de´ratif de Recherches 50, Faculte´ de Me´decine, Universite´ de Nice Sophia Antipolis, Nice, France Key Words. Embryonic stem cells • p38 mitogen-activated protein kinase • Neurogenesis • Cardiomyogenesis

ABSTRACT Mouse embryonic stem (ES) cells can be differentiated, in vitro into a variety of cell types including cardiac cells and neurons. This process is strictly controlled by the potent morphogen retinoic acid (RA). At a concentration of 10ⴚ7 M, RA induces ES cell differentiation into neurons and, conversely, inhibits cardiomyogenesis. We found that p38 mitogen-activated protein kinase (p38MAPK) activity peaked spontaneously, between day 3 and day 5, during ES cell differentiation and that RA completely inhibited this peak of activity. In contrast to wild-type cells, which re-

quired RA treatment, p38␣ⴚ/ⴚ ES cells differentiated spontaneously into neurons and did not form cardiomyocytes. Moreover, inhibition of the peak of p38MAPK activity by a specific inhibitor, PD169316, committed ES cells into the neuronal lineage and blocked cardiomyogenesis. By genetic and biochemical approaches, we demonstrate that, in two different ES cell lines, the control of p38MAPK activity constitutes an early switch, committing ES cells into either neurogenesis (p38 off) or cardiomyogenesis (p38 on). STEM CELLS 2006;24:1399 –1406

INTRODUCTION

5th and 7th day resulted in a slight induction of cardiomyogenesis. Moreover, without RA treatment, ES cells differentiate spontaneously in cardiomyocytes and myocytes [3, 4]. Our laboratory previously demonstrated that 10⫺7 M RA-induced extracellular signal-regulated kinase (ERK) activation is specifically required for ES cell commitment into adipocyte lineage [5]. ERK, stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK) are conserved members of signal transduction pathways activated in response to growth factors or environmental stresses [6]. ERK appears to play a major role in cell proliferation and differentiation (reviewed in ref. [7]), whereas JNK is involved in apoptosis [8]. The p38MAPK family includes four genes (p38␣, p38␤, p38␥, and p38␦) encoding four proteins. These proteins are activated by phosphorylation in response to osmotic stress, UV, and various cytokines involved in inflammatory responses. p38MAPKs, mainly p38␣, have been proposed to regulate several cellular processes, such as proliferation, cell survival, and differentiation [9]. For example, p38MAPK is involved in differentiation and/or survival of several cell types, including neurons and myoblasts [10, 11].

Embryonic stem (ES) cells are pluripotent and retain the potential for unlimited proliferation. Transplantation of ES cells or their derivatives has been proposed as a future therapy for various human diseases. However, molecular mechanisms governing the commitment of ES cells into a specific lineage are poorly understood, and their comprehension would improve efficiency of differentiation into specific lineages. ES cells are maintained in an undifferentiated state in presence of leukemia inhibitory factor (LIF). Removing LIF and adding appropriate differentiation agents results in the commitment of ES cells into a variety of cell lineages, including cardiac cells, skeletalmuscle cells, neurons or adipocytes [1]. Retinoic acid (RA) is a potent morphogen involved in vivo in the regulation of many developmental processes and can modulate cellular differentiation in various experimental models. Retinoic acid influences, in a time- and concentration-dependent manner, the pattern of differentiation of ES cells [1]. For example, the 10⫺7 M RA treatment between the 2nd and 5th day is necessary for ES cell differentiation into neurons and adipocytes [2]. However, at a low concentration (10⫺8 or 10⫺9 M), RA treatment between the

Correspondence: Bernard Bine´truy, Ph.D., INSERM U626, Faculte´ de Me´decine, Universite´ de Marseille II, 27 Bld J Moulin, 13385 Marseille, France. Telephone: (33) 4-91-32-44-06; Fax: (33) 4-91-25-43-36; e-mail: [email protected] Received August 17, 2005; accepted for publication January 7, 2006; first published online in STEM CELLS EXPRESS January 19, 2006. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0398

STEM CELLS 2006;24:1399 –1406 www.StemCells.com

p38MAPK Activity Is a Switch in ES Cell Commitment

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Deletion of the p38␣ gene leads to early embryonic lethality at between 11.5 and 12.5 days due to erythropoiesis deficiency [12] and/or to abnormal placental development [13]. These results show an essential role of p38MAPK in development. In this study, we investigated the role of p38MAPK pathway in ES cell commitment into cardiomyocytes and neurons using p38␣deleted cells and a highly specific p38MAPK chemical inhibitor, PD169316. In ES cells, p38␣ accounts for most of p38MAPK activity since p38a⫺/⫺ ES cells present no remaining p38MAPK activity [14]. We show that p38MAPK activity constitutes an early switch in ES cell commitment into neurogenesis (p38 off) versus cardiomyogenesis (p38 on).

MATERIALS

AND

METHODS

branes were permeabilized in phosphate-buffered saline (PBS)0.1% Triton X-100 for 15 min at 4°C and incubated with either polyclonal anti-MAP2 antibody (Sigma-Aldrich) or monoclonal ␣-troponin T antibody (CT3; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/⬃dshbwww) in PBS-1% bovine serum albumin overnight at 4°C. After three washes in PBS, cells were incubated with anti-mouse Texas Red-conjugated antibody (to label ␣-troponin and ␣-MAP2 antibodies) for 1 hour at room temperature. Cell imaging was performed using an Axiovert 200 microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) equipped with a ⫻20 Apoplan objective (Carl Zeiss) and a cooled digital CCD CoolSNAP HQ camera (Roper Scientific), using the Metamorph image analysis software (Universal Imaging Corporation).

Differentiation of ES Cells

RNA Analysis

Mouse embryonic stem cells CGR8 [15] were grown on gelatincoated plates, and p38⫺/⫺ and p38⫹/⫹ DBA-252 cells were grown on feeder cells treated with mitomycin C. For differentiation, cells were maintained in culture media without LIF as described previously [5]. Briefly, between day 0 and day 7, embryoid bodies (EBs) were formed in suspension and treated daily between day 2 and day 5 with 0.1 ␮M all-trans retinoic acid (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and then cells were seeded onto Petri dishes. For p38MAPK inhibition, 10 ␮M PD169316 (Calbiochem, San Diego, http:// www.emdbiosciences.com) was added to the culture media, which was renewed every day between day 2 and day 5 of the differentiation protocol. After RA treatment, neurons were detectable at day 11. Without RA treatment, EBs formed cardiomyocytes, which were visible at day 11. Numbers of EBs with neurons and cardiomyocytes were determined at day 11. The DBAp38C69 ES cell line was obtained from Dr. C. Gabel (Pfizer Laboratory, Pfizer Inc., Groton, CT, http://www. pfizer.com) [14] deleted for the p38␣ isoform gene and the DBA-252 wild-type cells. Prior differentiation, these cells were separated from feeders cells by a 30-min preplating and induced to differentiate according to the same protocol.

Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). For Northern Blot analysis, we used 10 –15 ␮g of total RNA. Hybridization signals were analyzed with a Molecular Dynamics radioimager, quantified and normalized to S26 signal using the ImageQuant 5.0 software. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with the ABI Prism7000 (Applied BioSystems, Foster City, CA, http://www. appliedbiosystems.com) and SYBR Green or Taqman reagents (Eurogentec, Seraing, Belgium, http://www.eurogentec.be) according to the manufacturers’ recommendations. We used 36B4 as an internal control.

Western Blot Analysis For p38MAPK activity analysis, EBs were lysed as described [5]. Samples (150 ␮g) were separated by SDS-polyacrylamide gel electrophoresis on a 12% gel and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, http:// www.millipore.com). Membranes were incubated with primary antibody against either p38MAPK phosphorylated on Thr180/ Tyr182 or all p38 isoforms (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com).

Microscopic Analysis of Cardiomyocytes and Neurons EBs were examined microscopically for the presence of cardiomyocytes (beating heart) and neurons (cells with neurites). Quantification was given as the percentage of EBs with cardiomyocytes and neurons.

Immunofluorescence Staining Differentiated EBs were trypsinized and seeded at day 7 on Petri dishes coated with laminin (Sigma-Aldrich). Cells were then fixed in 4% paraformaldehyde for 20 min at 4°C. Cell mem-

RESULTS RA Inhibits the Early Spontaneous Activation of p38MAPK During CGR8 ES Cell Differentiation To investigate the role of p38MAPK in ES cell commitment, we measured p38MAPK activity during early stages of differentiation. Since the 10⫺7 M RA treatment of EBs induces neurogenesis and blocks cardiomyogenesis, we analyzed the effect of RA on p38MAPK activity between day 2 and day 7. In the absence of RA treatment, p38MAPK activity peaked spontaneously by sixfold, 5 days after the formation of CGR8-derived EB (Fig. 1A, 1B, ⫺ lanes). Interestingly, RA completely inhibited the peak of p38MAPK activation without affecting p38MAPK protein expression level. Thus, permissive cardiomyogenic culture conditions (Fig. 1, NoRA) are associated with a high p38MAPK activity, whereas, conversely, neurogenic permissive conditions (Fig. 1, ⫹RA) are associated with a repressed activity. These results suggest a functional role for p38MAPK at this stage of ES cell differentiation.

p38␣ⴚ/ⴚ DBA-252 ES Cells Differentiated Spontaneously into Neurons but Not into Cardiomyocytes

To investigate the role of p38␣ in ES cells, we investigated the differentiation capacities of p38␣⫺/⫺ DBA-252 ES cells [14] into cardiomyocytes and neurons. First, to verify that there is no residual p38MAPK activity due to other p38 isoforms, p38␣⫺/⫺ DBA-252 ES cells were treated by anisomycin, a potent activator of this MAPK pathway. In agreement with Allen et al. [14], we found no p38MAPK activity in p38␣⫺/⫺ cells, whereas p38MAPK is activated by anisomycin in p38⫹/⫹ DBA-252 ES

Aouadi, Bost, Caron et al.

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An Early Regulation of p38MAPK Activity Determines ES Cell Commitment

Figure 1. RA treatment inhibits early p38MAPK activation. (A): CGR8-derived embryoid bodies from day 2 to day 7 after formation were treated with 10⫺7 M RA (⫹) or not treated (⫺). Cells were lysed, and the whole-cell extract was used for Western blot using anti-phospho-p38MAPK and anti-p38MAPK antibodies. (B): Quantification of p38MAPK activity from four independent experiments ⫾ SEM. Data are expressed in arbitrary units. ⴱ, p ⬍ .05; ⴱⴱ, p ⬍ .005 (Student’s t test). Abbreviations: p38MAPK, p38 mitogen-activated protein kinase; RA, retinoic acid.

cells (Fig. 2A). We then analyzed the differentiation capacities of p38␣⫺/⫺ DBA-252 ES cells at day 11, as compared with wild-type p38␣ cells (Fig. 2B–2D). In p38␣⫹/⫹ control cells, in absence of RA, 65% of EBs formed cardiomyocytes. In contrast, cardiomyogenesis was strongly impaired in p38␣⫺/⫺ cells, with only 5% of EBs forming cardiomyocytes (Fig. 2B). Noteworthy, this very low cardiomyogenesis was observed throughout the differentiation process (data not shown), indicating that this defect does not correspond to a simple shift in the apparition of cardiomyocytes. In agreement with the phenotypic analysis, real-time RT-PCR analysis showed that deletion of p38␣ strongly decreased the expression of the cardiomyocyte-specific genes MLC2a, MHC␣, and MEF2C (Fig. 2B). These results clearly demonstrated that the p38MAPK pathway is required for cardiomyogenesis of ES cells. The formation of neurons and the expression of neuron markers were analyzed in p38␣⫺/⫺ and p38␣⫹/⫹ DBA-252 ES cells (Fig. 2C, 2D). Whereas control cells had to be treated by RA to form neurons, p38␣⫺/⫺ cells differentiated spontaneously in neurons, without the need of RA treatment (Fig. 2C). No phenotypical differences were noticed between the neurons observed in RA-treated p38␣⫹/⫹ ES cells and untreated p38␣⫺/⫺ cells. In p38␣⫺/⫺ EBs, the percentage of neurons was similar with or without RA treatment (Fig. 2D), suggesting that the RA treatment is not required for neurogenesis in p38␣⫺/⫺ ES cells. MAP2 expression paralleled the phenotype of the cultures, with a characteristic strong expression in untreated p38␣⫺/⫺ cells (Fig. 2D). Thus, analysis of p38␣⫺/⫺ DBA-252 ES cells demonstrated that p38MAPK is necessary for cardiomyogenesis and, conversely, inhibits RA-induced neurogenesis. www.StemCells.com

To define whether the role of p38␣ in ES cell differentiation is due to its peak of activity at days 4 and 5, we analyzed the effect of the p38MAPK-specific inhibitor PD169316. To determine whether a causal relationship exists between the peak of p38MAPK activity between days 2 and 5 and cardiomyocyte formation, CGR8-derived EBs were treated during this period with or without PD169316 and RA. Cardiomyogenesis was analyzed at day 11. Eighty-five percent of the EBs formed cardiomyocytes in absence of RA (spontaneous cardiomyogenesis), and RA completely abolished cardiomyocyte differentiation (Fig. 3A). Inhibition of the peak of p38MAPK activity by PD169316 resulted in a strong reduction of cardiomyogenesis, similar to the RA treatment (Fig. 3A). Cardiomyogenesis was further analyzed by immunofluorescence with a specific marker of cardiomyocytes, troponin T. As shown in Figure 3B, RA and/or PD169316 treatment inhibited the formation of troponin T expressing cells. Therefore, these experiments demonstrate that the addition of RA or PD169316 not only inhibits the phenotypic appearance of beating hearts but block the formation of cardiac cells at an early stage. Real-time RT-PCR analysis of the cardiomyocyte markers MLC2a and MHC␣ showed high expressions in untreated EBs, whereas the p38MAPK inhibitor alone led to a strong reduction of expressions (Fig. 3C). Noteworthy, in CGR8 cells, the inhibitory effects of RA or PD169316 on MLC2a are partial and less pronounced than in DBA-252 cells (compare Fig. 2B to Fig. 3C). This observation could correspond to nonfunctional cardiomyocytes that express low levels of MLC2a. Our results indicate that the peak of p38MAPK activity is required for cardiomyogenesis in CGR8 ES cells and suggest that RA acts, at least partially, through the inhibition of p38MAPK to inhibit cardiomyogenesis. Since retinoic acid is required for the formation of neurons and inhibits the peak of p38MAPK activity, we analyzed the commitment of CGR8 ES cells into neurons in the absence or presence of PD169316 between day 2 and day 5 (Fig. 4A– 4C). In control RA-treated cells, neuron formation was observed on day 11. Importantly, the addition of PD169316 alone induced as much as neurite outgrowth (80% of EBs contained neurons) as the RA treatment (70% of EBs formed neurons) (Fig. 4A, 4B). Co-treatment of EBs with RA and PD169316 had no additional effect on the RA-induced or PD169316-alone-induced neurogenesis. These phenotypical observations were confirmed with anti-MAP2 antibody specific for neurons. ES cells were differentiated and neuronal networks were visualized by staining of neuritis and cellular bodies. Only ES cell cultures treated with RA and/or PD169316 presented neuronal networks (Fig. 4B). Analysis of the expression of two specific neuronal markers, MAP2 and NF-M, showed that PD169316 led to a strong induction of MAP2 and NF-M expression similar to RA treatment (Fig. 4C). Co-treatment of EBs with RA and PD169316 had no significant additive effect. These results indicate that inhibition of the peak of p38MAPK activity is necessary and sufficient to commit ES cells into neurogenesis and to induce the expression of specific neuron genes. Therefore, regarding ES cell differentiation, inhibition of p38MAPK by PD169316 between day 2 and day 5 mimics the deletion of the p38␣ gene. Furthermore, our results strongly

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p38MAPK Activity Is a Switch in ES Cell Commitment Figure 2. p38␣⫺/⫺ DBA252 embryonic stem (ES) cells differentiated spontaneously in neurons but not in cardiomyocytes. (A): Western blot analysis of EBs from p38⫹/⫹ and p38⫺/⫺ ES cells treated with anisomycin or untreated. Cells were lysed, and the whole-cell extracts were used for p38MAPK Western blot using anti-phospho-p38MAPK and anti-p38MAPK antibodies. Tubulin was used as a control of protein levels. (B): EBs were treated with or without 10⫺7 M RA between day 2 and day 5. Percentage of EBs with cardiomyocytes at day 11 (mean of three independent experiments ⫾ SEM [left panel]). Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of MLC2a (white bars), MHC␣ (black bars), and MEF2C (grey bars) expression after indicated treatments; mRNA expression is normalized with 36B4 (right panel). (C): EBs were treated with or without 10⫺7 M RA between day 2 and day 5. Photomicrographs of EBs with neurons at day 11, after the indicated treatments, are shown (arrows point to the neurites). Magnification ⫻10. (D): Percentage of EBs with neurons at day 11 (mean of three independent experiments ⫾ SEM [top panel]) and real-time RT-PCR analysis of MAP2 expression after indicated treatments. mRNA expression is normalized with 36B4 (bottom panel). *, p ⬍ .05; **, p ⬍ .005. Abbreviations: EB, embryoid body; MAP, mitogen-activated protein; MEF2c, myocyte enhancer factor 2c; MHC, myosin heavy chain; MLC, myosin light chain; NoRA, no retinoic acid treatment; RA, retinoic acid.

suggest that RA induces neurogenesis via the inhibition of p38MAPK in ES cells. To rule out a nonspecific effect of PD169316, CGR8-derived EBs were treated with PD169316 for different periods between day 0 and day 7, without any RA treatment (Fig. 5). The presence of neurons was analyzed at day 11. The addition of PD169316 between day 2 and day 5 promoted neuron differentiation, as did a treatment from day 0 to day 7. Interestingly, the addition of PD169316 when p38MAPK is not activated (i.e., before [day 0 –2] or after [day 5–7]) did not induce neurogenesis (Fig. 5A). Northern blot analysis confirmed that MAP2 expression was induced by a treatment with PD169316

only when the inhibitor is applied between day 2 and day 5 or between day 0 and day 7, leading to similar conclusion (Fig. 5B, 5C). Therefore, our results demonstrate that the effect of the inhibitor strictly correlates with the inhibition of the peak of p38MAPK spontaneous activity in EBs (Fig. 1).

DISCUSSION It is well documented that the duration and strength of signal transduction activation has profound influences on cellular responses. For example, sustained activation of ERK is associated with differentiation, whereas transient activation of this pathway is required for proliferation [16]. In this study, we provide


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Figure 3. p38MAPK inhibitor prevents cardiomyocyte formation. EBs were treated with or without 10 ␮M PD169316 between day 2 and day 5 in the presence (⫹) or absence (⫺) of 10⫺7 M RA. (A): Percentage of EBs with cardiomyocytes at day 11 (mean of three independent experiments ⫾ SEM). (B): At day 7, embryoid bodies were trypsinized and seeded in Petri dishes. Cultures were fixed with paraformaldehyde and analyzed by indirect immunofluorescence. Cultures were stained with ␣-cardiac troponin T antibody specific for cardiomyocytes and visualized in red (right panels). Phase-contrast images of the same fields (left panels) are given. Acquisition parameters (exposition time) of the images were kept constant in ⫹/⫺RA and PD169316 conditions. (C): Real-time reverse transcription-polymerase chain reaction analysis of MLC2a (white bars) and MHC␣ (black bars) expression after indicated treatments; mRNA expression is normalized with 36B4. *, p ⬍ .05; **, p ⬍ .005. Abbreviations: EB, embryoid body; MHC, myosin heavy chain; MLC, myosin light chain; NoRA, no retinoic acid treatment; RA, retinoic acid.

evidence for a role of p38MAPK as an early switch of ES cell commitment into cardiomyocytes or neurons. We found that p38MAPK is spontaneously activated between day 3 and day 5 after LIF withdrawal without any change in p38MAPK expression. Previous reports have shown that in ES cells cultivated in monolayer, p38MAPK is activated upon LIF withdrawal [17]. Interestingly, we found that RA treatment inhibited this peak of activation. Few studies have shown that retinoic acid modulates MAPK activity; however, a recent report demonstrated that RA inhibits cyclic stretch induced activity in neonatal cardiomyocytes [18]. Furthermore, RA is known to activate JNK in P19 mouse embryonal carcinoma cells during differentiation [19] and ERK in ES cells [5]. Further studies are required to www.StemCells.com

understand the molecular mechanisms underlying these various effects. We found that either deletion or specific inhibition of the peak of p38MAPK activity partially mimicked the cardiomyogenesis inhibition by 10⫺7 M RA treatment and strongly reduced the MLC2a and MHC␣ expression. Furthermore, deletion of p38␣ also reduced MEF2C expression, which is an important transcription factor acting on many genes encoding cardiac structural proteins. Interestingly, p38MAPK is a well known regulator of this transcription factor [20 –23], suggesting that the p38MAPK effect could be directly due to MEF2C regulation. Our results demonstrate that p38MAPK activation is required for cardiomyogenesis in ES cells and suggest that 10⫺7 M RA treatment blocks cardiomyogenesis via p38MAPK inhibition. In

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Figure 4. Inhibition of p38MAPK promotes neurogenesis. CGR8-derived EBs were treated with (⫹) or without (⫺) 10␮M PD169316 between day 2 and day 5 in the presence (⫹) or absence (⫺) of 10⫺7 M of RA. (A): Percentage of EBs with neurons at day 11. Results are the mean of four independent experiments ⫾ SEM. (B): Analysis by indirect immunofluorescence, cultures were stained with ␣-MAP2 antibody, specific for neurons, visualized in red (right panels). Phasecontrast images of the same fields (left panels) are given. Acquisition parameters (exposition time) of the images were kept constant in ⫹/⫺RA and PD169316 conditions. (C): Expression of MAP2 and NFM genes was analyzed by Northern blots, and signals were quantified and normalized to S26 control signal. The histogram represents the mean of four independent experiments ⫾ SEM. ⴱ, p ⬍ .05; ⴱⴱ, p ⬍ .005 (Student’s t test). Abbreviations: EB, embryoid body; MAP, mitogen-activated protein; NoRA, no retinoic acid treatment; RA, retinoic acid.

contrast, using specific inhibitors, we found that inhibition of ERK or JNK did not affect cardiomyogenesis in ES cells model ([5]; unpublished data). Consistent with our results, a role for p38␣ in various aspects of cardiomyogenesis, including the regulation of cardiomyocyte differentiation, apoptosis, and hypertrophy, has been described [24]. For example, Davidson et al. showed that p38MAPK is necessary during the early stages of cardiomyogenesis of P19 embryonal carcinoma cell line [25]. A recent study showed that ERK, JNK, and p38MAPK are activated in a coordinated and sustained manner and contribute to proliferation and cardiomyocyte differentiation of P19 cells

[26]. Interestingly, according to our results, p38␣⫺/⫺ embryos present a massive reduction of the myocardiac muscle attributed to a defect in placental development [13]. Former investigations concerning the role of p38MAPK in neurogenesis were carried out in PC12 and P19 cells. In these cell lines, p38MAPK activation is required for neurite formation and neuron survival during late stages of differentiation [27]. For example, it was shown that nerve growth factor and bone morphogenetic protein-2 act through p38MAPK activation to induce neuron differentiation of PC12 cells [10, 28]. In P19, p38MAPK activity has been shown to prevent cell death during


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stages of differentiation. Indeed, PC12 cells are already committed into the neuronal lineage, and P19 is a multipotent embryonic cell line that terminally differentiates into neurons after RA treatment. In contrast, in our study, we analyzed the role of p38MAPK in the early stages of neuron differentiation, during ES cell commitment. We found that inhibition of p38MAPK using PD169316 or p38␣⫺/⫺ cells is sufficient to induce, spontaneously, a high level of neurogenesis, similar to the one induced by RA. Interestingly, RA did not affect significantly neurogenesis in nontreated p38␣⫺/⫺, suggesting that in ES cells, RA induced neurogenesis mainly via p38MAPK inhibition. Altogether, these results suggest that p38MAPK may exert different roles depending on the stage of neuronal differentiation: inhibitory during cell commitment and anti-apoptotic during the late stages of differentiation. Such opposite roles during the differentiation process have also been found for the ERK pathway [31]. It is very likely that the molecular mechanisms underlying these distinct functions are different, and their identification should be of great interest for the development of ES cell use. To our knowledge, this is the first report describing a role for p38MAPK in ES cell commitment. In conclusion, we produce genetic and biochemical evidence that in two different ES cell lines (CGR8 and DBA-252), the control of p38MAPK activity constitutes an early switch in ES cell commitment into cardiomyocytes (p38 on) and neurons (p38 off). In future studies, it would be of interest to analyze p38MAPK activity following 10⫺8 or 10⫺9 M RA-induced cardiac differentiation. Figure 5. PD169316 treatment between day 2 and day 5 specifically induces neurogenesis. EBs were treated between day 0 and day 7 with or without PD169316 and/or RA as indicated. (A): Results are expressed as the percentage of EBs with neurons; the means of four independent experiments ⫾ SEM are given. (B): Representative Northern blot analysis of MAP2 and S26 expressions. (C): Quantification of from MAP2 signals normalized to S26 signals; the means of four independent experiments ⫾ SEM are given. p ⬍ .05. Abbreviations: EB, embryoid body; MAP, mitogen-activated protein; RA, retinoic acid.

neuronal differentiation [29]. It is noteworthy that these studies were performed with the chemical inhibitor SB203580, which is less potent and less specific than PD169316 [30]. More importantly, the role of p38MAPK in these cells is restricted to the late

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DISCLOSURES The authors indicate no potential conflicts of interest.

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