Inhibitory action of an ERK1/2 inhibitor on primitive endoderm cell differentiation from mouse embryonic stem cells

Abstract

A combination of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3β (GSK3β) inhibitors, called 2i, is widely used for maintaining the pluripotency of mouse embryonic stem cells (ESCs) in vitro. Without 2i, a few mouse ESCs spontaneously gives rise to primitive endoderm (PrE) cells, whereas 2i completely blocks this PrE cell differentiation. However, the molecular mechanisms underlying the inhibitory action of 2i on PrE cell differentiation remain unclear. Robust PrE cell induction is achieved by enforced expression of the transcription factor Gata4. Here, we analyzed how 2i inhibits the PrE cell differentiation using mouse ESCs carrying an inducible Gata4 expression cassette. We found that 2i effectively inhibited the Gata4-induced PrE cell differentiation and the ERK1/2 inhibitor was responsible for this effect. We further revealed that the transcriptional activation ability of Gata4 was necessary for PrE cell induction and its disruption by the ERK1/2 inhibitor. The phosphorylation of Ser105, Ser266, and Ser411 of the Gata4 protein was not involved in the PrE cell induction. Overexpression of Klf4, an ERK1/2 substrate, inhibited the Gata4-mediated transcriptional activation. Our data indicated that ERK1/2 supported the PrE cell induction via the indirect transcriptional activation of Gata4.