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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES |
Institut National de la Santé et de la Recherche Médicale U568, Institut Fédératif de Recherches 50, Faculté de Médecine, Université de Nice Sophia Antipolis, Nice, France
Key Words. Embryonic stem cells • p38 mitogen-activated protein kinase • Neurogenesis • Cardiomyogenesis
Correspondence: Bernard Binétruy, Ph.D., INSERM U626, Faculté de Médecine, Université 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: bernard.binetruy{at}medecine.univ-mrs.fr
Received August 17, 2005;
accepted for publication January 7, 2006.
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ABSTRACT |
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–1– 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).
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, p38ß, 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]. 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).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
Western Blot Analysis
For p38MAPK activity analysis, EBs were lysed as described [5]. Samples (150 µg) 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 membranes 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 x 20 Apoplan objective (Carl Zeiss) and a cooled digital CCD CoolSNAP HQ camera (Roper Scientific), using the Metamorph image analysis software (Universal Imaging Corporation).
RNA Analysis
Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). For Northern Blot analysis, we used 10–15 µg 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.
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RESULTS |
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, – 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.
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–/– DBA-252 ES Cells Differentiated Spontaneously into Neurons but Not into Cardiomyocytes 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 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.
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–/–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.
An Early Regulation of p38MAPK Activity Determines ES Cell Commitment
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. Cardiomyo-genesis 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.
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). 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 neuro-genesis. 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.
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gene. Furthermore, our results strongly 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).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 cardiomyo-genesis 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 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 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 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.
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ACKNOWLEDGMENTS |
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DISCLOSURES
The authors indicate no potential conflicts of interest.
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REFERENCES |
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