Regulation of p53 by reversible post-transcriptional and post-translational mechanisms in liver and skeletal muscle of an anoxia tolerant turtle, Trachemys scripta elegans Jing Zhang, Kyle K. Biggar, Kenneth B. Storey ⁎ Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6 abstract article info Article history: Accepted 10 October 2012 Available online 1 November 2012 Keywords: Anoxia tolerance p53 Ischemia Reperfusion Oxidative stress MicroRNA The red-eared slider turtle (Trachemys scripta elegans) exhibits well-developed natural anoxia tolerance that depends on multiple biochemical adaptations, including anoxia-induced hypometabolism. We hypothesized that signaling by the p53 protein could aid in establishing the hypometabolic state by arresting the cell cycle, protecting against DNA damage as well as altering pathways of energy metabolism. Immunoblotting was used to evaluate the regulation and post-transcriptional modifications of p53 in liver and skeletal muscle of red-eared slider turtles subjected to 5 h or 20 h of anoxic submergence. Tissue specific regulation of p53 was observed with the liver showing a more rapid activation of p53 in response to anoxia as well as differ- ential expression of seven serine phosphorylation and two lysine acetylation sites when compared with skel- etal muscle. Protein expression of MDM2, a major p53 inhibitor, was also examined but did not change during anoxia. Reverse-transcriptase PCR was used to assess transcript levels of selected p53 target genes (14-3-3σ, Gadd45α and Pgm) and one microRNA (miR-34a); results showed down-regulation of Pgm and up-regulation of the other three. These findings show an activation of p53 in response to anoxia exposure and suggest an important role for the p53 stress response pathway in regulating natural anoxia tolerance and hypometabolism in a vertebrate facultative anaerobe. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Because of its critical role as the terminal electron acceptor in oxidative phosphorylation, oxygen is central to ATP energy produc- tion for most organisms on earth. When deprived, many animals will quickly suffer injury or death, however, some animals have well-developed capacities for anaerobiosis. Among vertebrates, several species of freshwater turtles exhibit high anoxia tolerance that aids a diving lifestyle and extended underwater submergence over the winter months. One such species is the red-eared slider turtle (Trachemys scripta elegans) that can endure many weeks of oxygen restriction (hypoxia) or absence (anoxia) (Clark and Miller, 1973; Jackson, 1968). Among the adaptations that support this toler- ance are large reserves of fermentative fuels (mainly glycogen) stored in the liver, buffering acid build-up by releasing calcium and magne- sium ions from the shell, storing the end product (lactate) in the shell, and strong metabolic rate depression (Jackson, 2002; Storey, 2007). A highly critical component of anoxia survival is the reduction in ATP demand brought about by metabolic suppression. Indeed, turtles submerged in cold water may have a metabolic rate that is only ~ 10% of the comparable normoxic value at the same temperature (Herbert and Jackson, 1985; Jackson, 1968). Various molecular mechanisms utilized by freshwater turtles to achieve such impressive metabolic depression have been elucidated including post-translational modifi- cations of transcription factors and enzymes that are involved in key survival pathways (Krivoruchko and Storey, 2010a,b; Storey, 2007). Assessment of specific signaling pathways, and their contribution towards hypometabolism, has proven to be critical in understanding anoxia tolerance, as demonstrated previously by examining the roles of MAPK signal transduction in anoxia survival by turtles (Greenway and Storey, 2000). The p53 transcription factor has major roles in regulating apoptosis, the cell cycle, DNA damage repair, and energy metabolism and, as such, is now known to be central to signaling networks during periods of cellular stress (Jones et al., 2005; Levine, 1997; Maeda et al., 2002; Okoshi et al., 2008; Vousden and Ryan, 2009). Previous research has suggested that p53 is involved in stress responses to hypoxia (e.g. protecting against DNA damage, inducing cell cycle arrest, and changes in oxygen-based mitochondrial metabolism) (Graeber et al., 1994). Therefore, similar to mammals, the activation of p53 could provide the protective adjustments required to prevent cellular damage to the anoxic turtle (Zhao et al., 2009). Three major post-translational modifications (ubiquitination, phos- phorylation and acetylation) are responsible for regulating the activity and stability of p53 (Barlev et al., 2001; Ito et al., 2001; Sakaguchi et al., 1998). Additionally, negative influences on p53 activity are imparted by a key regulatory protein, murine double minute 2 (MDM2). MDM2 Gene 513 (2013) 147–155 Abbreviations: Pgm, phosphoglycerate mutase; Gadd45α, growth arrest and DNA damage inducible α; MDM2, murine double minute 2. ⁎ Corresponding author. Tel.: +1 613 520 3678; fax: +1 613 520 3749. E-mail address: kenneth_storey@carleton.ca (K.B. Storey). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.10.049 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene