Abstract
Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF) cytokine. LIF starvation leads to cell commitment, and part of the ES-derived differentiated cells die by apoptosis together with caspase3-cleavage and p38α activation. Inhibition of p38 activity by chemical compounds (PD169316 and SB203580), along with LIF withdrawal, leads to different outcomes on cell apoptosis, giving the opportunity to study the influence of apoptosis on cell differentiation. By gene profiling studies on ES-derived differentiated cells treated or not with these inhibitors, we have characterized the common and specific set of genes modulated by each inhibitor. We have also identified key genes that might account for their different survival effects. In addition, we have demonstrated that some genes, similarly regulated by both inhibitors (upregulated as Bcl2, Id2, Cd24a or downregulated as Nodal), are bona fide p38α targets involved in neurogenesis and found a correlation with their expression profiles and the onset of neuronal differentiation triggered upon retinoic acid treatment. We also showed, in an embryoid body differentiation protocol, that overexpression of EGFP (enhanced green fluorescent protein)–BCL2 fusion protein and repression of p38α are essential to increase formation of TUJ1-positive neuronal cell networks along with an increase in Map2-expressing cells.
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Abbreviations
- EBs:
-
embryoid bodies
- ES:
-
embryonic stem
- ERK2:
-
extracellular-signal regulated kinase 2
- EGFP:
-
enhanced green fluorescent protein
- LIF:
-
leukemia inhibitory factor
- MAP2:
-
microtubule-associated protein 2
- MAPK:
-
mitogen-activated protein kinase
- RA:
-
retinoic acid
- RTQ-PCR:
-
real-time quantitative-polymerase chain reaction
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Acknowledgements
We thank all members of the UMR-CNRS-5164-CIRID and, in particular, Jean François Moreau for helpful insights and a careful reading of the paper. We thank C Gabel for the gift of the ES WT and ES p38α−/− cell lines, A Smith for the gift of the ES CGR8 WT and E14TG2aSox1-EGFP ES cell lines, A Dierich for the gift of the S1 ES cell line, R Kemler for the gift of the D3 ES cell line and Dr Miyazaki for the pCXN2 expression vector. We thank V Pitard, M Landry and B Guillotin, respectively, for very good advices concerning the flow cytometry experiments, the characterization of neuronal cells and RTQ-PCR experiments. Thanks also to E Préchais for preliminary data concerning the validation of the p38α target genes. HB thanks P Dubus for constant support and reading of the paper. This work was founded by the European consortium FungenES (6th Framework, project no LSHG-CT-2003-503494, Coordinator: Professor J Hescheler), CNRS, the Ministere of Research (Affymetrix microarray grant, IGBMC genopole platform, Strasbourg), University of Bordeaux 2, the Ligue National contre le Cancer, Comités Aquitaine-Charentes, the Region Aquitaine and the IFR66. MT, OF and MG were financed by a FungenES fellowship and CS by a Region Aquitaine fellowship.
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Trouillas, M., Saucourt, C., Duval, D. et al. Bcl2, a transcriptional target of p38α, is critical for neuronal commitment of mouse embryonic stem cells. Cell Death Differ 15, 1450–1459 (2008). https://doi.org/10.1038/cdd.2008.63
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DOI: https://doi.org/10.1038/cdd.2008.63
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