Inhibition of PI3K or AKT Differently Regulates Mesodermal Differentiation in P19CL6 Cells

The directional differentiation of stem cells into cardiomyocytes displays distinct stage-specific characteristics. Multiple signaling pathways exhibit differential effects at various developmental stages in the regulation of cardiac differentiation. This study investigated the role and underlying mechanisms of the PI3K-AKT signaling pathway during both early and late stages of myocardial differentiation. Our results demonstrated that PI3K inhibitors promote early myocardial differentiation and enhance the expression of the mesodermal differentiation marker gene Brachyury T in P19CL6 teratoma cells. In contrast, AKT inhibitors exert the opposite effects. AKT activity is required for Wnt activation-induced Brachyury T expression, and Wnt inhibition also decreased AKT phosphorylation. Notably, both PI3K and AKT inhibitors suppress the late-stage myocardial differentiation. Mechanistically, PI3K inhibition promotes mesodermal differentiation in association with enhanced phosphorylation of GSK3β and activation of β-catenin. AKT kinase activity is indispensable for GSK3β phosphorylation and β-catenin activation. Furthermore, the subcellular localization of the PI3K p85 regulatory subunit undergoes significant changes before and after differentiation. Transcriptome sequencing revealed that treatment with PI3K inhibitors regulates multiple signaling pathways, including Wnt, Notch, and Nodal. Wnt3a expression is significantly upregulated upon PI3K inhibition. PI3K inhibitors did not affect the expression of pluripotency-related genes such as Oct4 and Sox2 but significantly suppressed the expression of growth factor receptor adaptor proteins such as Grb7. These findings suggest that PI3K plays a pivotal role in maintaining stem cell self-renewal and that inhibition of the PI3K pathway can activate Wnt, Notch, and Nodal signaling to promote mesodermal differentiation, thereby favoring the initiation of cardiac differentiation. AKT activation during mesoderm differentiation may occur independently of the PI3K pathway.