Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells

Abstract

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ESderived differentiated cells, together with p38a mitogenactivated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

Bcl2, a transcriptional target of p38α, is critical for neuronal commitment of mouse embryonic stem cells

“…We have previously shown that overexpression of functional antiapoptotic enhanced green fluorescent protein (EGFP)-BCL2 protein does not affect expression of early mesodermal (Brachyury) or pro-neuronal (Nestin) markers during heterogeneous differentiation triggered upon LIF withdrawal for a week. 9 However, a slight increase in TUJ1-positive cells was observed by immunolabelling staining (data not shown). To further explore whether this pro-neuronal differentiation effect of BCL2 was additive with repression of p38 activity, we investigated their combined effects on neuronal differentiation.…”

“…In addition, to identify genes potentially involved in differential apoptosis regulation, we compared SB-regulated genes to the previously reported PD-target genes. 9 Supplementary Tables 1A and B depicted pair-wise comparisons of genes between d3 (differentiation and apoptosis) and d3SB (differentiation with enhanced apoptosis) conditions. Genes, whose expression was similarly modulated by the PD169316 compound, are bolded with an asterisk.…”

“…To identify the common and specific target genes of each inhibitor and to characterize those that are potentially involved in differential apoptosis regulation, we undertook a microarray analysis with the SB compound and compared the results with previous work performed with the PD compound. 9 Gene profiling experiments were carried out with mRNAs prepared from two different ES cell lines (S1 and D4) grown for 3 days in cell medium without LIF (d3) in the absence or in the presence of 10 mM SB203580 (d3SB). Total RNAs from plated ES cell lines grown under these conditions (d3 or d3SB) were processed and hybridized on the mouse MG_U74A and MG_U74AV2 chips (Affymetrix), which include 10 043 transcripts (Materials and Methods).…”

“…37 In addition, it is noteworthy that none of the genes encoding Ca 2 þ binding and heavy metal-detoxifying proteins were induced by SB, in contrast to the PD compound, which increases expression of many of such genes. For example, the antiapoptotic Met1 gene, specifically induced by the PD169316 compound and shown to decrease apoptosis of ES-derived differentiated cells, 9 was not upregulated by SB203580. This different subset of PD-and SB-modulated genes is most likely related to the differential apoptotic response triggered by these inhibitors.…”

“…7,8 When this first wave of apoptosis is blocked by the PD169316 compound, a p38 inhibitor, the expression of differentiation markers is altered, emphasizing the importance of p38 activation and apoptosis to control early steps of cell differentiation. 9 Neuronal differentiation is efficiently triggered from EBs or plated ES cells treated with retinoic acid (RA), in the absence of serum. 10,11 Growth factors and/or defined medium-based procedures, allowing a gradual differentiation of EBs to neuronal precursor cells, have also been described (Bouhon et al, 12 Gossrau et al 13 and reference therein).…”