Cardiac differentiation in vitro is a complex, stepwise process that is rigidly governed by a subset of transcription factors and signaling cascades. In this study, we investigated the cooperation of cardiac-specific transcription factors Gata4 and Nkx2.5, as well as mitogen-activated protein kinase (MAPK) cascades. P19 embryonic carcinoma cells were induced into spontaneously beating cardiomyocytes utilizing a two-step protocol that comprised an early stage and a late stage of differentiation. During early-stage differentiation in suspension culture, P19 cells aggregated to form embryoid bodies (EBs), and the Gata4 and Nkx2.5 genes were induced. However, Gata4 expressed at the early stage of differentiation was incapable of activating downstream gene expression, as it was localized in the cytoplasm and prone to degradation. After EBs were plated for late-stage differentiation in adherent culture, the MAPK cascades were highly activated and contributed to the activation of Gata4 and Nkx2.5. Specifically, we revealed that p38 signaling participated in regulating the localization and stabilization of Gata4 and Nkx2.5. Additionally, the JNK cascade regulated late-stage cardiac differentiation; JNK kinase reduced Gata4 stabilization and conversely alleviated Nkx2.5 degradation by direct interaction and phosphorylation of Nkx2.5. Finally, we found that the C-terminal domain of Nkx2.5 was required for its stabilization under conditions of oxidative stress and JNK activation. Overall, our results indicated that the induction and activation of Gata4 and Nkx2.5 during early- and late-stage cardiac differentiation was closely associated with the function of the MAPK signaling cascades.

体外心脏分化是一个复杂的，逐步的过程，受转录因子和信号转导级联的严格控制。在这项研究中，我们调查了心脏特异性转录因子Gata4和Nkx2.5以及有丝分裂原激活的蛋白激酶（MAPK）级联的合作。利用包括分化的早期和晚期的两步方案，将P19胚胎癌细胞诱导为自发搏动的心肌细胞。在悬浮培养的早期分化过程中，P19细胞聚集形成胚状体（EB），以及Gata4和Nkx2.5基因被诱导。然而，在分化早期表达的Gata4不能激活下游基因表达，因为它位于细胞质中并易于降解。EBs在贴壁培养中用于后期分化的平板接种后，MAPK级联被高度激活并有助于Gata4和Nkx2.5的激活。具体来说，我们揭示了p38信号传导参与调节Gata4和Nkx2.5的定位和稳定。此外，JNK级联调节晚期心脏分化。JNK激酶通过直接相互作用和Nkx2.5的磷酸化降低了Gata4的稳定性并反过来减轻了Nkx2.5的降解。最后，我们发现Nkx2.5的C末端结构域是其在氧化应激和JNK活化条件下稳定所必需的。