Redox Regulation of a Canonical Wnt Signaling Pathway
Professor Greg Kelly
Professor of Biology
Tuesday, November 19 , 20133:30 p.m.
C2-361 (Reading Room)
Abstract: The first epithelial-to-mesenchymal transition (EMT) in the mouse embryo occurs when cells of the inner cell mass are induced to form primitive endoderm. F9 teratocarcinoma stem cells treated with retinoic acid mimic this process in vitro. Retinoic acid regulates the expression of a number of genes including Wnt6, which encodes a protein that will activate the Wnt/β-catenin pathway leading to primitive endoderm formation. Differentiation is preceded by an increase in the levels of reactive oxygen species (ROS) and evidence exists that these species are sufficient and necessary for the EMT to proceed. In Xenopus embryos ROS are known to modulate the Wnt/β-catenin pathway, acting at the level of nucleoredoxin (NRX) and Dishevelled (Dvl) (Funato et al. 2006). We tested whether this modulation of the Wnt pathway exists during the early events in mouse embryogenesis, and found using our F9 stem cell model that primitive endoderm formation is dependent on the redox state of NRX. In undifferentiated F9 cells NRX interacted with Dvl2 and while this association was enhanced under reduced conditions, it decreased following H2O2treatment, leading to differentiation. Depleting NRX levels using an shRNA approach resulted in morphological changes like those induced by retinoic acid, while increasing PKA activity caused these primitive endoderm cells to complete the EMT and form parietal endoderm. Depleting cells of NRX also correlated to an increase in Tcf-Lef transcription, indicative of canonical Wnt signaling. Together these results indicate that a mechanism exists whereby NRX maintains canonical Wnt signaling in the “off” state in these F9 stem cells. Increasing the levels of ROS lift these constraints allowing Dvl, no longer bound to NRX, to prime the Wnt pathway(s) required for primitive endoderm formation.