Coping with the stress: Expression of ATF4, ATF6, and downstream targets in organs of hibernating ground squirrels

Perturbation of the endoplasmic reticulum (ER) protein folding apparatus via any one of several environmental or metabolic stresses rapidly triggers a complex program of cellular responses that is termed the unfolded protein response (UPR). Stresses that trigger this response in mammals can include low temperature, hypoxia, ischemia, and oxidative stress. All of these can be natural features of mammalian hibernation, and hence the UPR might be integral to long term survival in a state of cold torpor. The present study analyzes changes in gene and/or protein expression of multiple markers of the UPR in tissues of euthermic (control) versus hibernating ground squirrels, Spermophilus tridecemlineatus. Immunoblot analysis of ATF4 protein expression revealed strong increases of 1.9- to 2.5-fold in brown adipose tissue, skeletal muscle, and brain during hibernation. However, transcript levels of atf4 were unchanged or lowered which suggests that ATF4 protein levels were regulated at the translational level. Subcellular localization studies showed that ATF4 translocated into the nucleus during hibernation, as did its cofactor, the phosphorylated form of CREB-1, which rose by 25- to 39-fold in nuclear extracts of brain and skeletal muscle of torpid animals. The responses of other proteins involved in the UPR including p-PERK, ATF6, GADD153, and GADD34 were also evaluated. The data suggest that ATF4 up-regulation may play an important role in coordinating gene expression responses that support the hibernating phenotype.