Identifying the Biphasic Role of Calcineurin/NFAT Signaling Enables Replacement of Sox2 in Somatic Cell Reprogramming

Abstract

Induction of pluripotency with defined factors (octamer-binding transcription factor 4 [Oct4], SRY (sex determining region Y)-box 2 [Sox2], Kruppel-like factor 4 [Klf4], c-Myc) raises hopes for successful clinical trials. Despite considerable efforts, the molecular mechanism of reprogramming remains poorly understood. The aim of the present study was to identify the role of calcineurin/nuclear factor of activated T cells (NFAT) in reprogramming. Our results demonstrated a biphasic role for calcineurin/NFAT signaling during reprogramming. In the early phase of reprogramming, calcineurin activity is required to maintain proper cell cycle division and for mesenchymal-epithelial transition. In the late phase, calcineurin exerts a negative effect that is mediated by NFATc2. NFATc2 interacts with Hdac3, Ezh2, and Suv39h1 to increase H3K9me3 and H3K27me3 over the Sox2 enhancer and Klf2 promoter, respectively, resulting in the downregulation of their expression. Moreover, Gαq was identified as a positive upstream regulator for calcineurin. The Gαq/calcineurin/NFATc2 axis negatively regulates the late step of reprogramming. By inhibiting NFATc2 or calcineurin, induced pluripotent stem cells could be established without exogenous Sox2. Thus, the present study revealed another regulatory level of reprogramming, and proposes a biological axis that could be useful for cancer therapy. Stem Cells 2017;35:1162-1175.

miR-203 imposes an intrinsic barrier during cellular reprogramming by targeting NFATC2

“…In line with the reported biphasic role of NFATC2 on cellular preprogramming (Khodeer & Era, 2017, Sun, Liao et al, 2016, Nfatc2 transcripts were slightly induced during reprogramming in wild-type cells ( Figure 4A). These transcripts were repressed in Cola1(miR-203) cells and dramatically upregulated in miR-203(/) cells at intermediate stages during reprogramming to be finally silenced in stable iPS clones ( Figure 4A).…”

“…These transcripts were repressed in Cola1(miR-203) cells and dramatically upregulated in miR-203(/) cells at intermediate stages during reprogramming to be finally silenced in stable iPS clones ( Figure 4A). At the protein level, NFATC2 was induced by day 5 in wild-type cells as reported previously (Khodeer & Era, 2017, Sun et al, 2016, and this induction was enhanced in miR-203 knock-out cells ( Figure 4B). Members of the NFAT transcription factor family, including NFATC2, are normally present in the cytoplasm in a hyperphosphorylated and inactive form.…”

“…NFAT family proteins are known to regulate lineage-specific transcription factors during differentiation of T helper 1 (Th1), Th2, Th17, regulatory T (Treg), and follicular helper T cells (Tfh) (Lee, Kim et al, 2018). Interestingly a biphasic role for calcineurin/NFAT signaling during reprogramming has been reported (Khodeer & Era, 2017, Sun et al, 2016. At early and decisive phases of reprogramming, calcineurin-NFAT signaling is transiently activated and such activation is required for effective reprogramming.…”

“…NFATC2 interacts with HDAC3, EZH2 and SUV39H1 to increase H3K9me3, and H3K27me3 over the SOX2 enhancer and KLF2 promoter, respectively, resulting in down-regulation of their expression. At this latest point, calcineurin/NFATC2 signaling becomes a barrier for pluripotency and NFATC2 is typically downregulated at later stages during reprogramming (Khodeer & Era, 2017, Sun et al, 2016.…”

Calcineurin A gamma and NFATc3/SRPX2 axis contribute to human embryonic stem cell differentiation

“…A previous study of our laboratory has shown that calcineurin‐NFAT signaling plays a critical role in regulating early lineage specification of mouse ESCs, through controlling Src expression and the epithelial–mesenchymal transition (EMT) process (X. Li, Zhu, et al, 2011). Moreover, it was later reported that calcineurin‐NFAT signaling also functioned in somatic cell reprogramming (Khodeer & Era, 2017; Sun et al, 2016). However, the function of calcineurin‐NFAT signaling in hESCs remains unknown.…”