Regulation of the Tb-E2F pathway in the freeze tolerant wood frog, Rana sylvatica
The North American wood frog, Rana sylvatica, is a primary model animal used in the study of freeze tolerance in vertebrates. During Canadian winters, wood frogs endure the freezing of about 65-70% of their total body water. Anoxia, ischemia, oxidative stress and many other consequences are a result of freeze/thaw cycles. A variety of adaptations are known that protect the frogs against potential freezing injuries as well as regulate their intermediary metabolism and gene expression to support survival. Selected transcription factors have critical roles to play in freezing survival by regulating the expression of genes that control the adaptations needed to handle freezing stress. In my present study, the retinoblastoma (Rb) protein coupled with the E2F transcription factor family were demonstrated to have roles in controlling the cell cycle in wood frog liver and skeletal muscle during freezing and associated stresses (anoxia, dehydration). Western blotting was used to quantify total Rb, phosphorylation or acetylation at different sites on Rb, E2F members and selected downstream targets under E2F control. Other central regulators of the cell cycle were also quantified including Cyclins, Cyclin dependent kinases (Cdks), and checkpoint proteins. Nuclear distributions of Rb-E2F and Cdk:Cyclins were also assessed. RT-PCR was used to quantify mRNA transcript levels of Cyclin D1 which decreased significant during freezing as well as c-Myc, a downstream target of E2F. The data indicate that the cell cycle is under regulation during freezing through E2F upregulation and Rb phosphorylation via Cdk:Cyclin activity.