Regulation of NF-κB, FHC and SOD2 in response to oxidative stress in the freeze tolerant wood frog, Rana sylvatica

The wood frog, Rana sylvatica, is the primary model animal used for studying vertebrate freeze tolerance. Freeze tolerance adaptations by wood frogs are mediated by a set of well-tuned regulatory controls at the molecular level, starting from cell signal transduction and gene expression events that are ultimately reflected in protective responses by multiple cell systems. Previous studies provided excellent presumptive evidence for the involvement of the NF-κB transcription factor in freeze tolerance. The present study of the NF-κB pathway focussed on freezing time points, 4 h frozen and 24 h frozen for liver and skeletal muscle in wood frog. The total protein levels of the major NF-κB subunits p50 and p65, its inhibitor, p-IκB, and downstream targets, ferritin heavy chain (FHC) and manganese superoxide dismutase (MnSOD) were quantified using western blots. Results showed a significant increase in the levels of NF-κB subunits and its downstream targets during freezing. Nuclear distributions of NF-κB subunits and transcript levels of FHC were analysed to delve deeper into the pathway. Results obtained from nuclear distribution analysis were consistent with the total protein levels showing increased levels of p50 and p65 during 24 h freezing conditions compared to controls but no change in phospho-p65 levels. Further, FHC transcript levels increased in 24 h frozen liver but did not change in frozen muscles. These findings suggest the activation of NF-κB antioxidant defenses in wood frogs during freezing in potential anticipation of high oxidative stress during reperfusion during thawing.