Ccar1 prevents β-catenin nuclear translocation to sustain ground-state pluripotency in mouse ESCs under R2i

Abstract

Dual inhibition of extracellular signal-regulated kinase (MEK) and transforming growth factor beta (TGFβ), known as R2i, sustains ground-state pluripotency in mouse embryonic stem cells (mESCs). To understand the molecular mechanisms of R2i, we analyzed the proteomic profile of mESCs cultured under R2i conditions in our previous study. Our data showed overexpression of the cell cycle and apoptosis regulator 1 (Ccar1) under R2i conditions. In this study, we investigated the role of Ccar1 in the pluripotency of mESCs through the loss-of-function approach. We hypothesize that Ccar1 contributes to the maintenance of pluripotency by interacting with β-catenin and preventing its translocation to the nucleus. Therefore, we used siRNA against Ccar1 and then analyzed the localization of β-catenin and the expression of its target genes by immunofluorescence assay and qRT-PCR. Immunofluorescence analysis demonstrated that siRNA-mediated downregulation of Ccar1 promoted the nuclear translocation of β-catenin. qRT-PCR analysis showed a significant reduction of pluripotency marker genes as well as some cell cycle markers such as Ccar1, c-myc, and Tbx3 in siRNA-treated cells. In addition, the expression level of the Wnt target genes (Cdx1, Wnt3a, Tbx1, Fgf4, Apc, Cdh1, Wnt3a) was significantly increased when Ccar1 was knocked down. We observed that upregulation of Ccar1 under R2i culture conditions could prevent nuclear translocation of β-catenin and maintain pluripotency and self-renewal of mESCs. These findings suggest that Ccar1 prevents nuclear β-catenin translocation to maintain pluripotency and self-renewal of mESCs under R2i conditions, although further direct interaction assays are required to confirm this mechanism.