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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 23, 2002 as doi:10.1096/fj.02-0017fje. |
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Institute of Biochemistry and Departments of Biology and Chemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6
2Correspondence: Institute of Biochemistry and Departments of Biology and Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6. E-mail: kenneth_storey{at}carleton.ca
SPECIFIC AIMS
Identification and analysis of the biochemical adaptations that support winter freezing survival by selected amphibians and reptiles not only provides a greater understanding of this unusual phenomenon, but suggests molecular strategies that could be applicable in medical cryopreservation. The present study documents the freeze up-regulation of a novel gene, li16, in liver of the wood frog (Rana sylvatica), monitors changes in gene/protein expression under freezing, anoxia and dehydration stresses, demonstrates probable li16 regulation by a cGMP-dependent mechanism, and suggests a role for the protein in ischemia resistance during freezing.
PRINCIPAL FINDINGS
1. Freezing induces increased transcription of li16 mRNA
Differential screening of a wood frog liver cDNA library coupled with 5'RACE was used to obtain 446 base pairs of a 0.5 kb freeze-induced transcript from liver of frozen frogs. The sequence, named li16, contained a full open reading frame (348 base pairs, 115 amino acids), a start codon with Kozak sequence, a polyadenylation sequence, and a polyA tail. Blast analysis showed no significant similarity with any known gene and protein. Northern blots confirmed enhanced expression of li16 transcripts during freezing. Transcript levels rose by 1.92 ± 0.21-, 2.67 ± 0.24-, or 3.67 ± 0.34-fold when frogs were frozen for 4, 8, or 24 h at -2.5°C (Fig. 1
). Transcript levels also rose 
fourfold in heart and gut during freezing. Nuclear run-off assays confirmed that freezing enhanced the rate of li16 mRNA synthesis. Incorporation of 32P-UTP into elongating transcripts in nuclei isolated from liver of frozen frogs was 2.41 ± 0.12-fold higher than in nuclei from unfrozen controls.
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2. Confirmation of the protein coding capacity of the li16 transcript and characterization of the freeze-thaw effects on Li16 protein levels
To confirm that the li16 transcripts were translated into protein, rabbit antiserum was produced against a synthetic peptide corresponding to the 10 carboxyl-terminal amino acids of the putative Li16 protein. The antiserum cross-reacted with one major protein band of 14 kDa, consistent with the predicted molecular mass of Li16 (12.8 kDa). Li16 protein rose in wood frog liver during freezing, reaching 2.40 ± 0.22-fold higher than in unfrozen frogs after 24 h frozen (Fig. 2
A).The protein continued to accumulate in liver over the early hours of thawing at 5°C to reach a peak after 2 h that was 3.5-fold higher than the value in 24 h frozen liver (Fig. 2B
). With longer thawing, protein levels declined.
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3. Gene and protein expression responds to alternative stresses: anoxia and dehydration
Freezing places two major stresses on cells: anoxia/ischemia due to plasma freezing and dehydration due to the exit of a high percentage of intracellular water into extracellular ice masses. Previous studies have shown that various freeze-induced adaptations can be linked with either anoxia or dehydration triggers. Expression of li16 responded strongly to anoxia with transcript levels in liver rising by 3.04 ± 0.54-fold after 4 h and 7.55 ± 1.59-fold after 24 h of exposure under a nitrogen gas atmosphere at 5°C. The loss of 20% of total body water during dehydration at 5°C had no effect on li16 transcript levels, but greater water loss (40%, still within the survivable limit) increased transcript levels by 6.15 ± 1.50-fold.
Comparable responses occurred when Li16 protein was assessed. Li16 protein content in liver rose by 1.81 ± 0.38-fold after 4 h of anoxia exposure and reached 4.37 ± 0.55-fold higher than aerobic, controls after 24 h. Within 1 h after reoxygenation, however, Li16 content returned to near control levels. Protein levels were 1.76 ± 0.35- and 2.16 ± 0.20-fold higher than controls in frogs that had lost 20 or 40% of total body water; with rehydration, Li16 returned to near control values.
4. Li16 up-regulation after freezing exposure is indirect and may involve cGMP
An in vitro tissue explant system recently developed in our laboratory was used to assess the effects of external stressors and intracellular second messenger pathways on li16 regulation. Initial experiments on liver slices tested the effect of in vitro freezing as well as influences of two elements of freezing: extreme hyperglycemia (glucose at 500 mM, mimicking natural cryoprotectant levels) and dehydration (incubation with 1% w/v polyethylene glycol 8000). None of these treatments directly affected li16 transcript levels. Subsequent studies assessed effects of protein kinase second messengers by incubating liver slices at 10°C with phorbol 12-myristate 13-acetate (PMA) (at 2, 20, and 200 µM), dibutyryl-cGMP or dibutyryl-cAMP (both at 0.02, 0.2, and 2 mM). Analysis by Northern blotting revealed that li16 transcript levels responded to cGMP; transcript levels increased significantly in a time- (1–10 h) and dose-dependent manner. For example, li16 transcripts rose by 1.63 ± 0.22- and 2.53 ± 0.43-fold after treatment with 0.2 and 2 mM dibutyryl-cGMP, respectively. Neither dibutyryl-cAMP nor PMA affected li16 transcript levels.
DISCUSSION, CONCLUSIONS, AND SIGNIFICANCE
The present study describes the freeze-induced up-regulation of a novel gene, li16, in tissues of the freeze-tolerant wood frog and analyzes influences on its expression. The patterns of change in li16 transcripts and Li16 protein levels observed in liver during freeze-thaw suggest a function for the protein that either becomes increasingly important with long-term freezing or is required for recovery during the first few hours of thawing. Organ- and stress-specific expression of li16 also provided hints about the function of this novel protein. Transcripts of li16 were freeze induced in three organs (liver, heart, gut), but elevated protein was found only in liver and heart during freezing. The very limited tissue distribution and differential translation response to freezing suggests a restricted, organ-specific function for the protein. The presence and accumulation of Li16 in liver and heart is interesting because these two organs are the last ones to freeze in vivo. They are also two organs with high metabolic work loads while the frog is freezing. The heart continues to beat against rising peripheral resistance and blood viscosity as the freezing front penetrates inward through the frog’s body, whereas the liver synthesizes and exports massive amounts of cryoprotectant (glucose) and multiple proteins that provide freeze protection to itself and other organs. Once freezing shuts down breathing, the work of both organs continues under increasingly hypoxic, and perhaps anoxic, conditions that limit ATP production. A possible role for Li16 in ischemia/anoxia resistance might therefore be proposed.
This idea is further supported by the analysis of li16 gene and Li16 protein responses to anoxia and dehydration stresses in frog liver. A pronounced increase in gene and protein expression occurred during anoxia exposure with a pattern similar to the responses to freezing. Responses to dehydration occurred only at high levels of water loss (40%) and not at intermediate dehydration (20%). High water loss is actually associated with a significant hypoxia stress in frogs because reduced blood volume and increased blood viscosity make tissue oxygenation difficult. As a result, cellular energetics are compromised and lactate accumulates. Hence, the response of li16 transcripts to higher levels of dehydration could have been triggered by a low oxygen signal.
Finally, in vitro incubations of liver slices were used to further explore li16 gene expression. Direct exposure of tissue to freezing, dehydration, or hyperglycemia did not affect li16 transcript levels. The cryoprotectant biosynthesis response to freezing by wood frog liver is similarly unaffected by direct freezing exposure of the liver. This implies that both li16 induction and the hyperglycemia response are mediated by nervous or hormonal triggers that transduce the stimulus of freezing that begins at peripheral body sites, usually on the skin. Indeed, incubation studies with second messenger analogs implicated a cGMP-mediated pathway in the control of li16 expression whereas the gene was unresponsive to second messengers of protein kinases A or C. The cryoprotectant biosynthesis response to freezing in wood frog liver is cAMP mediated, so together these results implicate the actions of at least two signal transduction pathways in mediating responses to natural freezing in liver.
In conclusion, li16 joins another gene, fr10 (which encodes a 10 kDa protein), as the second example of a novel, freeze-induced gene in freeze-tolerant wood frogs. The two differ substantially in their organ distribution of mRNA and protein expression, the pattern of response over freeze-thaw cycles, and effects of other stresses (fr10 is freeze and dehydration responsive but not affected by anoxia). However, both represent novel proteins with potentially important roles in the freezing survival of vertebrate organs. Identification of these roles will not only contribute to our understanding of natural freeze tolerance, but may have useful applications for the improvement of medical organ cryopreservation technology. The dual responsiveness of li16 to freezing and anoxia suggests a possible function for the Li16 protein in ischemia resistance during freezing, a role that future studies will address.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0017fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.02-0017fje. 
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