Division of Life Sciences and Medicine, The First Affiliated Hospital of University of Science & Technology of China, and CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network. Hefei, Anhui, People's Republic of China.
Translational Research Institute, Henan Provincial People's Hospital, Academy of Medical Science, Zhengzhou University, Zhengzhou, Henan, People's Republic of China.
Stem Cells. 2022 Oct 21;40(10):892-905. doi: 10.1093/stmcls/sxac050.
Exploiting the pluripotent properties of embryonic stem cells (ESCs) holds great promise for regenerative medicine. Nevertheless, directing ESC differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks. Here, we reveal the involvement of the p21-activated kinase 4 (Pak4), a serine/threonine kinase, in sustaining murine ESC (mESC) pluripotency. Pak4 is highly expressed in R1 ESC cells compared with embryonic fibroblast cells and its expression is progressively decreased during differentiation. Manipulations using knockdown and overexpression demonstrated a positive relationship between Pak4 expression and the clonogenic potential of mESCs. Moreover, ectopic Pak4 expression increases reprogramming efficiency of Oct4-Klf4-Sox2-Myc-induced pluripotent stem cells (iPSCs) whereas Pak4-knockdown iPSCs were largely incapable of generating teratomas containing mesodermal, ectodermal and endodermal tissues, indicative of a failure in differentiation. We further establish that Pak4 expression in mESCs is transcriptionally driven by the core pluripotency factor Nanog which recognizes specific binding motifs in the Pak4 proximal promoter region. In turn, the increased levels of Pak4 in mESCs fundamentally act as an upstream activator of the Akt pathway. Pak4 directly binds to and phosphorylates Akt at Ser473 with the resulting Akt activation shown to attenuate downstream GSK3β signaling. Thus, our findings indicate that the Nanog-Pak4-Akt signaling axis is essential for maintaining mESC self-renewal potential with further importance shown during somatic cell reprogramming where Pak4 appears indispensable for multi-lineage specification.
胚胎干细胞 (ESCs) 的多能性特性为再生医学带来了巨大的希望。然而，将 ESC 分化为特定的细胞谱系需要复杂的控制，这既受到内在和外在因素的影响，也受到特定信号网络的作用。在这里，我们揭示了 p21 激活激酶 4（Pak4）在维持小鼠胚胎干细胞（mESC）多能性中的作用。Pak4 在 R1 ESC 细胞中的表达水平明显高于胚胎成纤维细胞，并且在分化过程中其表达水平逐渐降低。使用敲低和过表达的操作表明，Pak4 的表达与 mESC 的克隆形成潜力之间存在正相关关系。此外，异位表达 Pak4 可提高 Oct4-Klf4-Sox2-Myc 诱导的多能干细胞（iPSC）的重编程效率，而 Pak4 敲低的 iPSC 则基本上无法生成含有中胚层、外胚层和内胚层组织的畸胎瘤，表明分化失败。我们进一步证实，mESC 中的 Pak4 表达是由核心多能性因子 Nanog 转录驱动的，Nanog 可以识别 Pak4 近端启动子区域的特定结合基序。反过来，mESC 中 Pak4 水平的升高从根本上充当 Akt 途径的上游激活剂。Pak4 直接与 Akt 在 Ser473 结合并使其磷酸化，导致 Akt 激活，从而减弱下游 GSK3β 信号。因此，我们的研究结果表明，Nanog-Pak4-Akt 信号轴对于维持 mESC 的自我更新潜力至关重要，并且在体细胞重编程过程中显示出进一步的重要性，其中 Pak4 似乎对于多谱系特化是不可或缺的。
