Activation of the Hippo Pathway in Rana sylvatica: Yapping Stops in Response to Anoxia

Wood frogs (Rana sylvatica) display well-developed anoxia tolerance as one component of their capacity to endure prolonged whole-body freezing during the winter months. Under anoxic conditions, multiple cellular responses are triggered to efficiently cope with stress by suppressing gene transcription and promoting activation of mechanisms that support cell survival. Activation of the Hippo signaling pathway initiates a cascade of protein kinase reactions that end with phosphorylation of YAP protein. Multiple pathway components of the Hippo pathway were analyzed via immunoblotting, qPCR or DNA-binding ELISAs to assess the effects of 24 h anoxia and 4 h aerobic recovery, compared with controls, on liver and heart metabolism of wood frogs. Immunoblot results showed significant increases in the relative levels of multiple proteins of the Hippo pathway representing an overall activation of the pathway in both organs under anoxia stress. Upregulation of transcript levels further confirmed this. A decrease in YAP and TEAD protein levels in the nuclear fraction also indicated reduced translocation of these proteins. Decreased DNA-binding activity of TEAD at the promoter region also suggested repression of gene transcription of its downstream targets such as SOX2 and OCT4. Furthermore, changes in the protein levels of two downstream targets of TEAD, OCT4 and SOX2, established regulated transcriptional activity and could possibly be associated with the activation of the Hippo pathway. Increased levels of TAZ in anoxic hearts also suggested its involvement in the repair mechanism for damage caused to cardiac muscles during anoxia. In summary, this study provides the first insights into the role of the Hippo pathway in maintaining cellular homeostasis in response to anoxia in amphibians.