Regulation of NF-κB, FHC and SOD2 in response to oxidative stress in the freeze tolerant wood frog, Rana sylvatica
Introduction
The wood frog, Rana sylvatica, is a model freeze tolerant amphibian extensively studied for its adaptations to natural freezing survival. While frozen during the winter, essential functions like muscle movement, breathing, blood flow, neural conduction, and heartbeat stop. As much as 65–70% of total body water freezes as extracellular ice, the movement of water out of cells and into ice causing extensive cell dehydration with increased intracellular osmolality and ionic strength [9,51]. At subzero temperatures (typically −0.5 to −2.0 °C), wood frogs initiate slow and controlled ice formation that allows the animal maximum time to activate mechanisms for freezing survival. The most crucial of these is a rapid activation of liver glycogenolysis that increases the concentration of glucose in blood and most tissues to >150 μmol/g wet weight. Glucose is distributed to all organs where it acts as a cryoprotectant. Freezing prevents oxygen from reaching organs and waste from being removed. Hence, wood frogs must be able to survive without oxygen for long periods of time [46,52]. Therefore, it can be said that freezing is a complex of multiple stresses: low temperature, anoxia, dehydration, ischemia, osmotic and ionic stress, and hyperglycemia. Animals initiate many protective pathways at the cellular level to combat damage caused due to stress and this has been studied extensively at both molecular and enzymatic levels [9,14,21,[57], [58], [59]].
Periods of freeze/thaw, ischemia/reperfusion, and anoxia/reoxygenation enhance the production of reactive oxygen species (ROS) and lead to oxidative stress, particularly during the recovery period. In addition to many stress/recovery cycles, free glucose (under high glucose concentration) is prone to autooxidation and leads to the formation of advanced glycation end products (AGE) [1]. AGE adversely affect the cellular processes by interfering with receptors and producing ROS [44]. Wood frogs have two strategies of antioxidant defense: the first is to maintain high constitutive activities of antioxidant enzymes, and the second is to modify some enzyme activities in response to freezing [23]. Oxidative stress and antioxidant defense mechanisms have been studied in wood frogs during freezing and anoxia [11,18,21,23,57]. Various transcription factors are activated in response to elevated ROS and act to minimize oxidative damage by inducing synthesis of antioxidant enzymes. The nuclear factor erythroid 2–related factor 2 (Nrf2) and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) are two important transcription factors that are activated in response to ROS [4,17,28,40,55,56].
NF-κB is an oxygen sensitive transcription factor that is involved in inflammatory, cellular proliferation, apoptosis, and immune responses [27,35,55]. It is a complex of five proteins, NF-κB1 (p50 and p105 as a precursor), NF-κB2 (p52 and p100 as a precursor), RelA (p65), c-Rel and RelB [19]. Under normal conditions, NF-κB is sequestered in the cytoplasm in association with its inhibitory protein IκB. When levels of ROS increase, IκB is phosphorylated by IκB kinase complex at Ser32 and Ser36, and later ubiquitinated and degraded by the proteasomal pathway [19,27,35,55]. This frees NF-κB to translocate to the nucleus and activate stress responsive target genes. Amongst the various combinations, the p50-p65 combination is most common, and their homodimers and heterodimers lead to different outcomes [34]. NF-κB can be activated by either of two pathways. Under the classical pathway, p50/RelA (p65) and p50/c-Rel dimers predominantly translocate to the nucleus via degradation of IκB whereas in the alternative pathway NF-κB gets activated by precursor p100, and p52/RelB gets translocated to the nucleus [27,54]. NF-κB also turns on the expression of its repressor, IκBα which re-inhibits NF-κB via an auto feedback loop that involves IκBα moving into the nucleus and stopping NF-κB expression of target genes by moving it back into the cytoplasm [31].
Amongst multiple targets of NF-κB, manganese superoxide dismutase (MnSOD) and ferritin heavy chain (FHC) play essential roles in antioxidant activity [10,12,39]. MnSOD is a mitochondrial enzyme that is synthesised in the cytosol and then imported into mitochondria by the TIM/TOM complex [6]. It is a superoxide scavenger enzyme that neutralizes superoxide synthesised during oxidative phosphorylation due to electron leakage from complex I and III [22]. Superoxide radicals are toxic to the cell and react quickly with various targets to produce other toxic compounds such as hydroxyl radicals and peroxynitrite. SODs outcompete all harmful reactions of superoxide to detoxify superoxide and therefore play a critical role in antioxidant defense. MnSOD also protects against apoptosis, protects mtDNA from damage due to ROS, and helps to maintain cellular lipid integrity [6].
FHC is a ferroxidase, which converts free iron in the form of Fe(II) to the non-toxic form of Fe(III) that is stored in the mineral core of ferritin whereas the light chain subunit of ferritin is responsible for modifying the protein environment to sequester more iron by iron nucleation [25,37]. Iron is a critical trace element that all organisms utilize for many cellular functions in metabolism, oxygen transport, and energy production. Too much iron is toxic to cells because excess free iron in the form of Fe(II) catalyzes the formation of reactive oxygen species, namely the hydroxyl radical, via the Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH− + OH.) which damage DNA and proteins [25,37]. One mechanism by which organisms cope with excess iron is through the action of the ferritin protein. FHC is an iron storage protein, that sequesters iron to limit the Fenton reaction (generation of hydroxyl free radicals from hydrogen peroxide mediated by iron) and formation of AGEs [38].
The present study focuses on the NF-κB pathway in response to freezing stress. The study further investigates two downstream targets of NF-κβ (FHC and MnSOD) in the liver and skeletal muscles of the wood frog, Rana sylvatica, under 4 h and 24 h freezing conditions as compared with 5 °C acclimated controls.
Section snippets
Animal experiments
Male wood frogs, Rana sylvatica, were collected from breeding ponds in the Ottawa area during early spring. The frogs were washed in a tetracycline bath and were kept in plastic containers of damp sphagnum moss at 5 °C for 1–2 weeks before use. Control frogs were sampled from this group. For freezing exposure, other frogs were transferred to plastic containers lined with damp paper towels and placed inside a −2.5 °C incubator. Ice formation on the paper towel allowed for rapid and uniform
Effect of freezing on levels of NF-κB subunits p50, p65 and phosphorylated p65
Immunoblotting was used to analyze p50, p65, and phospho-p65 (Ser536) and single bands at around 50 kDa, 65 kDa, and 65 kDa, respectively, were observed in both liver and skeletal muscle extracts under different conditions. In the liver of 24 h frozen frogs a significant increase in p50 levels of 2.64 ± 0.25 fold relative to controls was seen but no change was observed after just 4 h (Fig. 1a). In skeletal muscle, the opposite effect was noticed as 4 h freezing produced a significant increase
Discussion
The wood frog's adaptation strategy for winter survival involves the tolerance of ice formation extracellularly while maintaining unfrozen cytoplasm. Harsh conditions posed by the environment triggers adaptations that help animals to deal with and survive through the winter. Major adaptations include ice nucleation below 0 °C by ice nucleating proteins to control ice formation, accumulation of glucose as a cryoprotectant to sustain a minimum intracellular volume, maintaining tissue viability by
Acknowledgments
We thank J.M. Storey for editorial review of this manuscript. Research was supported by a Discovery grant from the Natural Sciences and Engineering Council of Canada (#6793); KBS holds the Canada Research Chair in Molecular Physiology.
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