Advertisement

Molecular and Cellular Biochemistry

, Volume 312, Issue 1–2, pp 121–129 | Cite as

Expression of Nrf2 and its downstream gene targets in hibernating 13-lined ground squirrels, Spermophilus tridecemlineatus

  • Pier Jr Morin
  • Zhouli Ni
  • David C. McMullen
  • Kenneth B. Storey
Article

Abstract

Mammalian hibernation is associated with wide variation in heart rate, blood flow, and oxygen delivery to tissues and is used as a model of natural ischemia/reperfusion. In non-hibernators, ischemia/reperfusion is typically associated with oxidative stress but hibernators seem to deal with potential oxidative damage by enhancing antioxidant defenses in an anticipatory manner. The present study assesses the role of the Nrf2 transcription factor in the regulation of antioxidant defenses during hibernation. Nrf2 mRNA and protein expression were enhanced in selected organs of 13-lined ground squirrels, Spermophilus tridecemlineatus during hibernation. Furthermore, Nrf2 protein in heart was elevated by 1.4–1.5 fold at multiple stages over a torpor–arousal bout including during entry, long term torpor, and early arousal. Levels returned to euthermic values when squirrels were fully aroused in interbout. Protein levels of selected downstream target genes under Nrf2 control were also measured via immunoblotting over the torpor–arousal cycle in heart. Cu/Zn superoxide dismutase and aflatoxin aldehyde reductase levels increased significantly during entry into torpor and then gradually declined falling to control levels or below in fully aroused animals. Heme oxygenase-1 also showed the same trend. This suggests a role for Nrf2 in regulating the antioxidant defenses needed for hibernation success. Heart nrf2 was amplified by PCR and sequenced. The deduced amino acid sequence showed high identity with the sequence from other mammals but with selected unique substitutions (e.g., proline residues at positions 111 and 230) that might be important for conformational stability of the protein at near 0°C body temperatures in the torpid state.

Keywords

Oxidative stress Antioxidant defense Ischemia resistance Torpor–arousal cycle NF-E2-related factor-2 Superoxide dismutase Heme oxygenase Aflatoxin aldehyde reductase Heart 

Notes

Acknowledgements

We thank Dr. J.M. Hallenbeck, National Institute of Neurological Disorders and Stroke, for supplying us with samples of ground squirrel tissues and Dr. John D. Hayes, University of Dundee, for providing the AFAR1 antibody. Thanks also to J.M. Storey for editorial review of the manuscript. Supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada; KBS holds the Canada Research Chair in Molecular Physiology.

References

  1. 1.
    Wang LCH, Lee TF (1996) Torpor and hibernation in mammals: metabolic, physiological and biochemical adaptations. In: Fregley MJ, Blatteis CM (eds) Handbook of physiology: environmental physiology. Oxford University Press, Oxford, pp 507–532 Google Scholar
  2. 2.
    Storey KB (2003) Mammalian hibernation: transcriptional and translational controls. Adv Exp Med Biol 543:21–38PubMedGoogle Scholar
  3. 3.
    Storey KB, Storey JM (2007) Putting life on ‘pause’ – molecular regulation of hypometabolism. J Exp Biol 210:1700–1714PubMedCrossRefGoogle Scholar
  4. 4.
    Ma YL, Zhu X, Rivera PM, Tøien Ø, Barnes BM, LaManna JC, Smith MA, Drew KL (2005) Absence of cellular stress in brain after hypoxia induced by arousal from hibernation in Arctic ground squirrels. Am J Physiol 289:R1297–R1306Google Scholar
  5. 5.
    Lee YJ, Hallenbeck JM (2006) Insights into cytoprotection from ground squirrel hibernation, a natural model of tolerance to profound brain oligaemia. Biochem Soc Trans 34:1295–1298PubMedCrossRefGoogle Scholar
  6. 6.
    Frank CL (1992) The influence of dietary fatty acids on hibernation by golden-mantled ground squirrels (Spermophilus lateralis). Physiol Zool 65:906–920Google Scholar
  7. 7.
    Gunstone FD (1996) Fatty acid and lipid chemistry. Aspen Publishers, MarylandGoogle Scholar
  8. 8.
    Stewart D, Killeen E, Naquin R, Alam S, Alam J (2003) Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium. J Biol Chem 278:2396–2402PubMedCrossRefGoogle Scholar
  9. 9.
    Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 13:76–86PubMedCrossRefGoogle Scholar
  10. 10.
    Venugopal R, Jaiswal AK (1996) Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci USA 93:14960–14965PubMedCrossRefGoogle Scholar
  11. 11.
    Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima YI (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Comm 236:313–322PubMedCrossRefGoogle Scholar
  12. 12.
    He CH, Gong P, Hu B, Stewart D, Choi ME, Choi AM, Alam J (2001) Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. J Biol Chem 276:20858–20865PubMedCrossRefGoogle Scholar
  13. 13.
    Kim IC, Masutani H, Yamaguchi Y, Itoh K, Yamamoto M, Yodoi J (2001) Hemin-induced activation of the thioredoxin gene by Nrf2. A differential regulation of the antioxidant responsive element by a switch of its binding factors. J Biol Chem 276:18399–18406PubMedCrossRefGoogle Scholar
  14. 14.
    Chanas SA, Jiang Q, McMahon M, McWalter GK, McLellan LI, Elcombe CR, Henderson CJ, Wolf CR, Moffat GJ, Itoh K, Yamamoto M, Hayes JD (2002) Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem J 365:405–416PubMedCrossRefGoogle Scholar
  15. 15.
    Park EY, Rho HM (2002) The transcriptional activation of the human copper/zinc superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through two different regulator sites, the antioxidant responsive element and xenobiotic responsive element. Mol Cell Biochem 240:47–55PubMedCrossRefGoogle Scholar
  16. 16.
    Alam J, Stewart D, Touchard C, Boinapally S, Choi AMK, Cook JL (1999) Nrf2, a cap‘n’collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 274:26071–26078PubMedCrossRefGoogle Scholar
  17. 17.
    Ellis EM, Hayes JD (1995) Substrate specificity of an aflatoxin-metabolizing aldehyde reductase. Biochem J 312:535–541PubMedGoogle Scholar
  18. 18.
    Ellis EM, Slattery CM, Hayes JD (2003) Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter. Carcinogenesis 24:727–737PubMedCrossRefGoogle Scholar
  19. 19.
    Lee YJ, Miyake SI, Wakita H, McMullen DC, Azuma Y, Auh S, Hallenbeck JM (2006) Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells. J Cereb Blood Flow Metabol 27:950–962Google Scholar
  20. 20.
    Morin P Jr, Storey KB (2005) Cloning and expression of hypoxia-inducible factor 1α from the hibernating ground squirrel, Spermophilus tridecemlineatus. Biochim Biophys Acta 1729:32–40PubMedGoogle Scholar
  21. 21.
    Carey HV, Frank CL, Seifert JP (2000) Hibernation induces oxidative stress and activation of NK-kappaB in ground squirrel intestine. J Comp Physiol B 170:551–559PubMedCrossRefGoogle Scholar
  22. 22.
    Chauhan VP, Tsiouris JA, Chauhan A, Sheikh AM, Brown WT, Vaughan M (2002) Increased oxidative stress and decreased activities of Ca2+/Mg2-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear. Life Sci 71:153–161PubMedCrossRefGoogle Scholar
  23. 23.
    Buzadzic B, Spasic M, Saicic ZS, Radojicic R, Petrovic VM, Halliwell B (1990) Antioxidant defenses in the ground squirrel Citellus citellus. 2. The effect of hibernation. Free Radic Biol Med 9:407–413PubMedCrossRefGoogle Scholar
  24. 24.
    Eddy SF, McNally JD, Storey KB (2005) Up-regulation of a thioredoxin peroxidase-like protein, proliferation associated gene, in hibernating bats. Arch Biochem Biophys 435:101–111CrossRefGoogle Scholar
  25. 25.
    Morin P Jr, Storey KB (2007) Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus. Arch Biochem Biophys 461:59–65PubMedCrossRefGoogle Scholar
  26. 26.
    Ohta H, Okamoto I, Hanaya T, Arai S, Ohta T, Fukuda S (2006) Enhanced antioxidant defense due to extracellular catalase activity in Syrian hamster during arousal from hibernation. Comp Biochem Physiol C 143:484–491Google Scholar
  27. 27.
    Okamoto I, Kayano T, Hanaya T, Arai S, Ikeda M, Kurimoto M (2006) Up-regulation of an extracellular superoxide dismutase-like activity in hibernating hamsters subjected to oxidative stress in mid- to late arousal from torpor. Comp Biochem Physiol C 144:47–56CrossRefGoogle Scholar
  28. 28.
    Drew KL, Toien O, Rivera PM, Smith MA, Perry G, Rice ME (2002) Role of the antioxidant ascorbate in hibernation and warming from hibernation. Comp Biochem Physiol C 133:483–492Google Scholar
  29. 29.
    Osborne PG, Hashimoto M (2006) Brain antioxidant levels in hamsters during hibernation, arousal and cenothermia. Behav Brain Res 168:208–214PubMedCrossRefGoogle Scholar
  30. 30.
    Chan K, Kan YW (1999) Nrf2 is essential for protection against acute pulmonary injury in mice. Proc Natl Acad Sci USA 96:12731–12736PubMedCrossRefGoogle Scholar
  31. 31.
    Cho HY, Jedlicka AE, Reddy SP, Kensler TW, Yamamoto M, Zhang LY, Kleeberger SR (2002) Role of NRF2 in protection against hyperoxic lung injury in mice. Am J Respir Cell Mol Biol 26:175–182PubMedGoogle Scholar
  32. 32.
    Katoh Y, Itoh K, Yoshida E, Miyagishi M, Fukamizu A, Yamamoto M (2001) Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription. Genes Cells 6:857–868PubMedCrossRefGoogle Scholar
  33. 33.
    Shen G, Hebbar V, Nair S, Xu C, Li W, Lin W, Keum YS, Han J, Gallo MA, Kong AN (2004) Regulation of Nrf2 transactivation domain activity. The differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. J Biol Chem 279:23052–23060PubMedCrossRefGoogle Scholar
  34. 34.
    Papaiahgari S, Kleeberger SR, Cho HY, Kalvakolanu DV, Reddy SP (2004) NADPH oxidase and ERK signaling regulates hyperoxia-induced Nrf2-ARE transcriptional response in pulmonary epithelial cells. J Biol Chem 279:42302–42312PubMedCrossRefGoogle Scholar
  35. 35.
    Pi J, Qu W, Reece JM, Kumagai Y, Waalkes MP (2003) Transcription factor Nrf2 activation by inorganic arsenic in cultured keratinocytes: involvement of hydrogen peroxide. Exp Cell Res 290:234–245PubMedCrossRefGoogle Scholar
  36. 36.
    Qiang W, Cahill JM, Liu J, Kuang X, Liu N, Scofield VL, Voorhees JR, Reid AJ, Yan M, Lynn WS, Wong PK (2004) Activation of transcription factor Nrf-2 and its downstream targets in response to moloney murine leukemia virus ts1-induced thiol depletion and oxidative stress in astrocytes. J Virol 78:11926–11938PubMedCrossRefGoogle Scholar
  37. 37.
    Tenhunen R, Marver HS, Schmid R (1968) The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 61:748–755PubMedCrossRefGoogle Scholar
  38. 38.
    Lee PJ, Jiang BH, Chin BY, Iyer NV, Alam J, Semenza GL, Choi AMK (1997) Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J Biol Chem 272:5375–5381PubMedCrossRefGoogle Scholar
  39. 39.
    Wang P, Chen H, Qin H, Sankarapandi S, Becher MW, Wong PC, Zweier JL (1998) Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury. Proc Natl Acad Sci USA 95:4556–4560PubMedCrossRefGoogle Scholar
  40. 40.
    Hermes-Lima M, Storey JM, Storey KB (1998) Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails. Comp Biochem Physiol B Biochem Mol Biol 120:437–448PubMedCrossRefGoogle Scholar
  41. 41.
    Kim YJ, Ahn JY, Liang P, Ip C, Zhang Y, Park YM (2007) Human prx1 gene is a target of Nrf2 and is up-regulated by hypoxia/reoxygenation: implication to tumor biology. Cancer Res 67:546–554PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Pier Jr Morin
    • 1
  • Zhouli Ni
    • 1
  • David C. McMullen
    • 1
  • Kenneth B. Storey
    • 1
  1. 1.Institute of Biochemistry and Departments of Chemistry and BiologyCarleton UniversityOttawaCanada

Personalised recommendations