Freeze tolerant frogs: expression and regulation of transcription factors of the unfolded protein response and the ER-associated degradation.
Wood frogs (Rana sylvatica) are the primary model used in the study of freeze tolerance in vertebrates and much is known about the adaptations of physiology, biochemistry and gene expression that support winter freezing survival, particularly their natural cryoprotective processes. Freezing and/or its components (anoxia and dehydration) places multiple stresses on cells; one of these is endoplasmic reticulum (ER) stress, a condition caused by accumulation of unfolded or misfolded proteins in the ER. The regulated expression of selected transcription factors, such as ATF4, that trigger genes that protect against ER stress is important for cell survival of freezing. During ER stress, the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway are triggered, which can potentially lead to apoptosis. Western blots were used to evaluate the responses by key protein components of the UPR and the ERAD under freezing, anoxia and dehydration stresses in two major organs of wood frogs (skeletal muscle and liver). The proteins analyzed included the activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and the EDEM and XBP1 proteins. Stress-induced redistribution of transcription factors between cytoplasmic and nuclear fractions was also evaluated. All three stresses triggered the UPR in both tissues but only freezing of skeletal muscle seemed to trigger the ERAD. Only anoxic treated skeletal muscle showed metabolic signs of potential apoptosis. It was concluded that wood frog organs activate the UPR as a means of stabilizing cellular proteins and shutting down global protein synthesis in order to survive freezing exposures without irreparable injury.