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p38 Mitogen-Activated Protein Kinase Activity Commits Embryonic Stem Cells to Ei

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发表于 2009-3-5 00:06 |显示全部帖子
作者:Myriam Aouadi, Frdric Bost, Leslie Caron, Kathiane Laurent, Yannick Le Marchand Brustel, Bernard Bintruy作者单位:Institut National de la Sant et de la Recherche Mdicale U, Institut Fdratif de Recherches , Facult de Mdecine, Universit de Nice Sophia Antipolis, Nice, France * v$ m! ~+ l# B/ b4 d' x
                  
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          【摘要】
- g$ w( o9 P' B: ^3 m- [      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 10¨C7 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 required RA treatment, p38 ¨C1¨C 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).
4 o/ w: S; k+ w* o  l- I% a          【关键词】 Embryonic stem cells p mitogen-activated protein kinase Neurogenesis Cardiomyogenesis, X  |/ ^, G2 |6 z) P0 ^! |
                  INTRODUCTION/ K( s% F- n; Z, f4 Q- Q' b
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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, skeletal-muscle cells, neurons or adipocytes .
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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 . 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) W0 J; B1 v1 W3 E: \6 \' b
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Differentiation of ES Cells
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* i) N* @. p/ V- d0 UMouse embryonic stem cells CGR8 . 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 µM all-trans retinoic acid (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and then cells were seeded onto Petri dishes. For p38MAPK inhibition, 10 µM 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 cardio-myocytes, which were visible at day 11. Numbers of EBs with neurons and cardiomyocytes were determined at day 11.
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# h# ^" w+ d  v" o8 aThe DBAp38C69 ES cell line was obtained from Dr. C. Gabel (Pfizer Laboratory, Pfizer Inc., Groton, CT, http://www.pfizer.com)  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.) ?1 `/ H) s& `7 t! T* D; a
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Western Blot Analysis
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For p38MAPK activity analysis, EBs were lysed as described . 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).
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4 n# I4 c2 o; }+ b- P" hMicroscopic Analysis of Cardiomyocytes and Neurons7 [1 h. t% ~0 A, B- r' R/ u$ I2 O
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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.1 i+ p/ s5 w; w9 e; O6 Y7 K8 j

5 ^; d3 o* T" b0 D! y7 CImmunofluorescence Staining
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! J9 @% E& T  R7 nDifferentiated 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¡ãC. Cell membranes were permeabilized in phosphate-buffered saline (PBS)-0.1% Triton X-100 for 15 min at 4¡ãC 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¡ãC. 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).
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' A& Q9 t- e( z" [RNA Analysis
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Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). For Northern Blot analysis, we used 10¨C15 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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3 \! I, A9 J/ R7 P  ]% eRA Inhibits the Early Spontaneous Activation of p38MAPK During CGR8 ES Cell Differentiation+ e) N9 \6 ]- {  e

# s9 P1 T9 T" G. p& r( \7 c& W2 dTo investigate the role of p38MAPK in ES cell commitment, we measured p38MAPK activity during early stages of differentiation. Since the 10¨C 7 M RA treatment of EBs induces neuro-genesis 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, ¨C 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.. r) U7 D" E" t% F2 [
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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¨C7 M RA ( ) or not treated (¨C). Cells were lysed, and the whole-cell extract was used for Western blot using anti-phos-pho-p38MAPK and anti-p38MAPK antibodies. (B): Quantification of p38MAPK activity from four independent experiments ¡À SEM. Data are expressed in arbitrary units. *, p  r! G: H/ E/ B. k
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p38¨C/¨C DBA-252 ES Cells Differentiated Spontaneously into Neurons but Not into Cardiomyocytes
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& ~; r  n: d6 |& @8 [To investigate the role of p38  in ES cells, we investigated the differentiation capacities of p38 ¨C/¨C DBA-252 ES cells , we found no p38MAPK activity in p38 ¨C/¨C cells, whereas p38MAPK is activated by anisomycin in p38 /  DBA-252 ES cells (Fig. 2A). We then analyzed the differentiation capacities of p38 ¨C/¨C DBA-252 ES cells at day 11, as compared with wild-type p38  cells (Fig. 2B¨C2D). In p38 /  control cells, in absence of RA, 65% of EBs formed cardiomyocytes. In contrast, cardiomyogenesis was strongly impaired in p38 ¨C/¨C 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./ N& z3 X! s9 q; z4 d

2 S  B& `# q) ^1 S4 E- E7 y  F7 mFigure 2. p38 ¨C/¨C DBA-252 embryonic stem (ES) cells differentiated spontaneously in neurons but not in cardiomyocytes. (A): Western blot analysis of EBs from p38  /  and p38 ¨C/¨C 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 ¨C7 M RA between day 2 and day 5. Percentage of EBs with cardiomyocytes at day 11 (mean of three independent experiments ¡À SEM ) and real-time RT-PCR analysis of MAP2 expression after indicated treatments. mRNA expression is normalized with 36B4 (bottom panel). *, p' W$ ?# w& [) [$ M% }

% Q: u6 ?: S9 C3 oThe formation of neurons and the expression of neuron markers were analyzed in p38 ¨C/¨Cand p38  /  DBA-252 ES cells (Fig. 2C, 2D). Whereas control cells had to be treated by RA to form neurons, p38 ¨C/¨Ccells 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 ¨C/¨C cells. In p38 ¨C/¨C 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 ¨C/¨C ES cells. MAP2 expression paralleled the phenotype of the cultures, with a characteristic strong expression in untreated p38 ¨C/¨C cells (Fig. 2D). Thus, analysis of p38 ¨C/¨C DBA-252 ES cells demonstrated that p38MAPK is necessary for cardiomyogenesis and, conversely, inhibits RA-induced neurogenesis.
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! X5 c( t! c9 H- ]6 }An Early Regulation of p38MAPK Activity Determines ES Cell Commitment1 f6 @/ x% W6 Z& w; ?

2 B! {: N6 M$ q" f4 S' z  UTo 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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, L$ |: h( X( fFigure 3. p38MAPK inhibitor prevents cardiomyocyte formation. EBs were treated with or without 10 µM PD169316 between day 2 and day 5 in the presence ( ) or absence (¨C) of 10¨C7 M RA. (A): Percentage of EBs with cardiomyocytes at day 11 (mean of three independent experiments ¡À 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  /¨C 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. N8 S  `. j+ o& j# W; i& B
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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¨C4C). 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.+ r' K' i+ ?- T$ L+ R2 b* L

% l/ k, k+ f; i# oFigure 4. Inhibition of p38MAPK promotes neurogenesis. CGR8-derived EBs were treated with ( ) or without (¨C) 10µM PD169316 between day 2 and day 5 in the presence ( ) or absence (¨C) of 10¨C7 M of RA. (A): Percentage of EBs with neurons at day 11. Results are the mean of four independent experiments ¡À SEM. (B): Analysis by indirect immuno-fluorescence, cultures were stained with -MAP2 antibody, specific for neurons, 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  /¨CRA 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 ¡À SEM.*, p: K- L, h8 F' m. H1 S0 C. O

0 M* e1 b& |) @+ e# }2 h) STherefore, 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 suggest that RA induces neurogenesis via the inhibition of p38MAPK in ES cells.
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/ T( _$ n) d  O0 i7 U5 |7 FTo 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 ) 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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6 H8 `5 |. z" VFigure 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 ¡À 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 ¡À SEM are given. p
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6 i' Y8 f) E, EDISCUSSION
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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 . In this study, we provide evidence for a role of p38MAPK as an early switch of ES cell commitment into cardiomyocytes or neurons.
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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 . Further studies are required to understand the molecular mechanisms underlying these various effects.
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- ^0 s/ y' u" u5 R7 b4 J0 _; UWe found that either deletion or specific inhibition of the peak of p38MAPK activity partially mimicked the cardiomyo-genesis inhibition by 10¨C7 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 .; r; N' P4 _: [

  r$ y; G  Q$ C; ^; }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 . 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¨C/¨C 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¨C/¨C, suggesting that in ES cells, RA induced neurogenesis mainly via p38MAPK inhibition.
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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 . 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.
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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¨C8 or 10¨C9 M RA-induced cardiac differentiation." Q  L) g; T" M+ v, C! y: w( z7 R
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ACKNOWLEDGMENTS' J) W+ k% C) m
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We thank J.F. Tanti and D. Aberdam for reading the manuscript and M. Prot for technical assistance and L. Hue for help in p38MAPK biochemical analysis. M.A. is supported by a fellowship from Institut National de la Sant¨¦ et de la Recherche M¨¦dicale-Provence Alpes Cote d¡¯Azur./ Z7 {& l' h; u! v, N; j* x

8 L& E5 s  v; ?6 ^/ V) sDISCLOSURES
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! j) r) s* g5 B* h* vThe authors indicate no potential conflicts of interest.' Y- v9 f& a8 W# c6 h9 M) H8 j3 e6 h
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Palm-Leis A, Singh US, Herbelin BS et al. Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes. J Biol Chem 2004;18:18.
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发表于 2015-6-14 01:18 |显示全部帖子
哎 怎么说那~~  

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发表于 2015-6-27 21:13 |显示全部帖子
希瑞干细胞
哈哈 我支持你

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发表于 2015-7-9 19:28 |显示全部帖子
干细胞之家微信公众号
在线等在线等  

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加油啊!!!!顶哦!!!!!支持楼主,支持你~  

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发表于 2015-8-13 15:54 |显示全部帖子
站个位在说  

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发表于 2015-8-21 11:43 |显示全部帖子
不对,就是碗是铁的,里边没饭你吃啥去?  

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发表于 2015-8-22 20:00 |显示全部帖子
干细胞研究还要面向临床

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不错不错.,..我喜欢  

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