NOX1 and NOX4 are required for the differentiation of mouse F9 cells into extraembryonic endoderm

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm.

当用视黄酸（retinoic acid, RA）处理小鼠F9细胞时，其可分化为原始内胚层（primitive endoderm, PrE）。该分化过程伴随活性氧（reactive oxygen species, ROS）水平升高；向F9细胞施加抗氧化剂可减弱其分化能力，而仅用过氧化氢（H₂O₂）处理即可诱导PrE分化。我们将NADPH氧化酶（NADPH oxidase, NOX）复合物确定为该内源性ROS的潜在来源；在该基因家族中，Nox1与Nox4在分化进程中受RA诱导的上调幅度最为显著。编码胚外内胚层核心调控因子的Gata6基因同样受RA诱导上调，且我们的实验证据表明，在过表达Gata6的F9细胞中，NOX1与NOX4的蛋白水平会升高。泛NOX抑制剂与NOX1特异性抑制剂均可显著削弱RA诱导PrE分化的能力，这一结果可通过敲低Nox1和/或Nox4转录本的遗传学手段得以复现。值得注意的是，在未经过处理的F9细胞中单独过表达这两种基因均无法诱导分化，即便二者均可提升ROS水平。综上，本研究数据表明，PrE分化过程中产生的ROS在一定程度上依赖于NOX1与NOX4水平的升高，而该调控过程受GATA6的掌控。此外，上述结果提示，多种NOX蛋白的协同活性是F9细胞向原始内胚层分化的必要条件。