The MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification.. The MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification.

Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop. Overall design: mRNA profiles of wild type, Nox1-/-, Mapk7f/f, Mapk7-/-, Bcl6b-/-, Mapk14f/f and Mapk14-/- were generated by deep sequencing, in duplicate, using Illumina NextSeq 500.

活性氧簇（Reactive oxygen species, ROS）在多种干细胞的自我更新分裂过程中发挥关键调控作用。然而，目前学界尚未明确ROS信号如何与干细胞自我更新机制实现整合。本研究发现，MAPK14/MAPK7/BCL6B信号通路可通过ROS扩增构建正反馈环路，驱动精原干细胞（spermatogonial stem cell, SSC）的自我更新。在体外培养的精原干细胞中，MAPK14的激活能够诱导MAPK7发生磷酸化；而对Mapk14或Mapk7进行特异性敲除，会在精原干细胞移植后引发显著的精原干细胞缺陷。该信号通路的激活不仅可上调Nox1的表达，还能提升细胞内ROS水平。针对MAPK7下游靶点的化学筛选实验，发现了诸多依赖ROS的精原干细胞转录因子；其中BCL6B被证实可通过ETV5介导的核转位，上调Nox1的表达以启动ROS生成。鉴于过氧化氢处理或Nox1基因转染同样能够诱导BCL6B的核转位，本研究结果表明：BCL6B可通过形成正反馈环路，启动并放大ROS信号，进而激活依赖ROS的精原干细胞转录因子。整体实验设计：采用Illumina NextSeq 500测序平台，对野生型、Nox1-/-、Mapk7f/f、Mapk7-/-、Bcl6b-/-、Mapk14f/f及Mapk14-/-样本的mRNA表达谱进行了两次生物学重复的深度测序。