| Summary: | During the course of development, specification of the three embryonic germ layers, ectoderm, mesoderm and definitive endoderm (DE), is a critical process by which pluripotent cells acquire the temporal and spatial information needed to form specialised tissues. Of these initial germ layers, the DE arises during gastrulation, which latterly gives rise to the liver, pancreas, lung and epithelial lining of the digestive tract. Elucidation of the molecular mechanisms that govern DE specification not only facilitates our understanding of developmental biology but also aids in the differentiation of human pluripotent stem cells to specific cell types for disease modelling and regenerative therapies. DE formation is largely driven by the cooperation of Activin/Nodal and Wnt/β-catenin signalling, however recent evidence has additionally implicated PI3K/Akt signalling in modulating this process. Although it has been previously reported that PI3K activation acts to antagonise the in vitro differentiation of DE, the molecular mechanisms responsible for this effect remains unclear. To address this issue, this study utilises pluripotent human embryonic stem cells (hESCs) as an in vitro model to interrogate the molecular underpinnings of DE formation through a fully defined differentiation protocol. Modulation of PI3K activity was found to reciprocally downregulate the activation of Smad2/3, which was mitigated in the presence of the PI3K inhibitor LY294002 (LY). Suppression of PI3K/Akt signalling prolongs the activation of Smad2/3 in response to Activin, promoting their nuclear accumulation and the enhancement of transcriptional activity, resulting in the upregulation of mesendoderm and DE gene expression. Activation of PI3K negatively impacts the activity of Smad2/3 via phosphorylation of the Smad2/3 linker T220/T179 residue, which is fully independent of Erk and CDK activity. Phosphorylation of this residue induces the recruitment of the E3 ubiquitin ligase Nedd4L to activated Smad2/3, which in turn promotes their ubiquitin-mediated degradation and attrition of activity. Inhibition of mTORC2 activity by both inhibitor supplementation and genetic manipulation, rather than modulation of Akt or mTORC1 activity, recapitulates the LY-mediated reduction of T220/T179 phosphorylation and increases the duration of Smad2/3 transcriptional activity, promoting a more robust mesendoderm and endoderm differentiation. These findings reveal a new and novel connection between the PI3K/mTOR and TGFβ/Activin pathways, which will greatly impact our understanding of both cell fate determination and the preservation of normal cellular functions. Notably, identification of mTORC2 as a key player in the regulation of this differentiation provides new avenues through which hESCs differentiation protocols can be improved for both regenerative and biomedical applications.
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