The role of Mek/Erk signalling inhibition and Krüppel-like factor 2 in mouse ground state pluripotency

Abstract

The maintenance of undifferentiated mouse embryonic stem cells (mESCs) requires the presence of LIF and serum. Interestingly, by using two chemical molecules to inhibit both the pro-differentiative Fgf/Mek/Erk and Gsk3/Tcf3 pathways in mESCs (dual inhibition or “2i”), a pluripotent “ground state”, resembling the mouse pre-implantation epiblast can be established without the need for LIF and serum. While Gsk3-inhibition is known to alleviate Tcf3-mediated repression of Esrrb, the molecular mechanism downstream of Mek/Erk inhibition remains to be identified. Here, it was uncovered that Erk2 phosphorylates the Krüppel-like factor 2 (Klf2), leading to Klf2 proteasomal degradation. Mek/Erk inhibition during 2i conditions thus serves to halt Klf2 phospho-degradation, leading to Klf2 protein stabilisation and maintenance of ground state pluripotency. Indeed, while Klf2-null mESCs are viable under LIF/Serum, they undergo apoptosis during 2i culture. Additionally, it was found that Klf2 overexpression is sufficient to replace Mek-inhibition, allowing for mESC self-renewal under Gsk3-inhibition alone. Taken together, this study highlights the importance of Klf2 during 2i conditions, and defines the Mek/Erk/Klf2 pathway with the Gsk3/Tcf3/Esrrb axis to establish ground state pluripotency.