Regulation of the MEF-2 and the SMAD family of transcription factors in the freeze tolerant wood frog, Rana sylvatica
The wood frog, Rana sylvatica is a native North America capable of withstanding full body freezing when ambient temperatures drop below 0°C. During the freeze exposure, approximately 65-70% of the extracellular fluid gets converted into ice. Anoxia, ischaemia, osmotic and oxidative stress are some of the consequences which result from a freezing cycle. The dynamic nature of cells allows them to adapt to a wide array of stress conditions at different organizational levels. Transcription factors are key regulators of gene expression responsible for adaptation. In the present study, the MEF2 and SMAD family of transcription factors are demonstrated to have importance in the wood frog during freezing. The proteins were initially associated with developmental controls, however recent studies have found them to be involved in stress responses. Western blots were used to assess the expression and phosphorylation levels of MEF2A, MEF2C, SMAD1, SMAD2, SMAD3, and SMAD4 during torpor. It was generally found that MEF2A, MEF2C, and SMAD3 were post-translationally (phosphorylated) at Thr312, thr300, ser425 sites, respectively during 24h and 8h thawing. RT-PCR analysis of MEF2 and SMAD target genes (calreticulin, glucose transporter-4, creatine kinase (brain and muscle) and serpine1, myostatin, tsc22d3, respectively) revealed a modest up-regulation during 24h freezing in wood frog brain, heart, skeletal muscle, liver and kidney in selected transcripts. These results show that the two families of transcription factors are transcriptionally active during freezing, which comes as no surprise given the signals which regulate these proteins as well as the functions of the genes they activate.