Fish Physiology and Biochemistry

, Volume 41, Issue 3, pp 745–759 | Cite as

Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure

  • Ignacio Fernández
  • Parameswaran Vijayakumar
  • Carlos Marques
  • M. Leonor Cancela
  • Paulo J. Gavaia
  • Vincent Laizé
Article

Abstract

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.

Keywords

Vitamin K epoxide reductase In vitro cell systems Gene expression Warfarin Vitamin K Danio rerio 

References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI–BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedCentralPubMedGoogle Scholar
  2. Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250Google Scholar
  3. Atkins GJ, Welldon KJ, Wijenayaka AR, Bonewald LF, Findlay DM (2009) Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms. Am J Physiol Cell Physiol 297:C1358–C1367CrossRefPubMedGoogle Scholar
  4. Azuma K, Casey SC, Ito M, Urano T, Horie K, Ouchi Y, Kirchner S, Blumberg B, Inoue S (2010) Pregnane X receptor knockout mice display osteopenia with reduced bone formation and enhanced bone resorption. J Endocrinol 207:257–263CrossRefPubMedGoogle Scholar
  5. Bainy ACD, Kubota A, Goldstone JV, Lille-Langøy R, Karchner SI, Celander MC, Hahn ME, Goksøyr A, Stegeman JJ (2013) Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in zebrafish (Danio rerio). Aquat Toxicol 142–143:447–457CrossRefPubMedGoogle Scholar
  6. Bertrand S, Thisse B, Tavares R, Sachs L, Chaumot A, Bardet PL, Escrivà H, Duffraisse M, Marchand O, Safi R, Thisse C, Laudet V (2007) Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression. PLoS Genet 3:e188CrossRefPubMedCentralPubMedGoogle Scholar
  7. Boglione C, Gavaia P, Koumoundouros G, Gisbert E, Moren M, Fontagné S, Witten PE (2013a) A review on skeletal anomalies in reared European larvae and juveniles. Part 1: normal and anomalous skeletogenic processes. Rev Aquac 5:S99–S120CrossRefGoogle Scholar
  8. Boglione C, Gisbert E, Gavaia P, Witten PE, Moren M, Fontagné S, Koumoundouros G (2013b) A review on skeletal anomalies in reared European larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev Aquac 5:S121–S167CrossRefGoogle Scholar
  9. Bresolin T, de Freitas Rebelo M, Bainy ACD (2005) Expression of PXR, CYP3A and MDR1 genes in liver of zebrafish. Comp Biochem Phys Part C 140:403–407CrossRefGoogle Scholar
  10. Chen Y, Ferguson SS, Negishi M, Goldstein JA (2004) Induction of human CYP2C9 by rifampicin, hyperforin, and phenobarbital is mediated by the pregnane X receptor. J Pharmacol Exp Ther 308:495–501CrossRefPubMedGoogle Scholar
  11. Chen Y, Tang Y, Guo C, Wang J, Boral D, Nie D (2012) Nuclear receptors in the multidrug resistance through the regulation of drug-metabolizing enzymes and drug transporters. Biochem Pharmacol 83:1112–1126CrossRefPubMedCentralPubMedGoogle Scholar
  12. Corbett EF, Oikawa K, Francois P, Tessier DC, Kay C, Bergeron JJM, Thomas DY, Krause KH, Michalak M (1999) Ca2+ regulation of interactions between endoplasmic reticulum chaperones. J Biol Chem 274:6203–6211CrossRefPubMedGoogle Scholar
  13. Czogalla KJ, Biswas A, Wendeln A-C, Westhofen P, Müller CR, Watzka M, Oldenburg J (2015) Human VKORC1 mutations cause variable degrees of 4-hydroxycoumarin resistance and affect putative warfarin binding interfaces. Blood 122:2743–2750CrossRefGoogle Scholar
  14. Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang JM, Taly JF, Notredame C (2011) T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res 39:W13–W17CrossRefPubMedCentralPubMedGoogle Scholar
  15. Ekins S, Reschly EJ, Hagey LR, Krasowski MD (2008) Evolution of pharmacologic specificity in the pregnane X receptor. BMC Evol Biol 8:103Google Scholar
  16. Ferland G (2012) Vitamin K and the nervous system: an overview of its actions. Adv Nutr 3:204–212CrossRefPubMedCentralPubMedGoogle Scholar
  17. Fernández I, Santos A, Cancela ML, Laizé V, Gavaia PJ (2014a) PXR gene expression patterns and warfarin side effects in zebrafish: long-term warfarin exposure affects larval development and expression of PXR, vitamin K cycle- and vitamin K dependent protein genes. Environ Pollut 194:86–95CrossRefPubMedGoogle Scholar
  18. Fernández I, Tiago DM, Laizé V, Cancela LM, Gisbert E (2014b) Retinoic acid differentially affects in vitro proliferation, differentiation and mineralization of two fish bone-derived cell lines: different gene expression of nuclear receptors and ECM proteins. J Steroid Biochem Mol Biol 140:34–43CrossRefPubMedGoogle Scholar
  19. Force A, Lynch M, Pickett FB, Amores A, Yan YL, Postlethwait J (1999) Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151:1531–1545PubMedCentralPubMedGoogle Scholar
  20. Goldstein JA, de Morais SM (1994) Biochemistry and molecular biology of the human CYP2C subfamily. Pharmacogenetics 4:285–299CrossRefPubMedGoogle Scholar
  21. Goodstadt L, Posting CP (2004) Vitamin K epoxide reductase: homology, active site and catalytic mechanism. Trends Biochem Sci 29:289–292CrossRefPubMedGoogle Scholar
  22. Hammed A, Matagrin B, Spohn G, Prouillac C, Benoit E, Lattard V (2013) VKORC1L1, an enzyme rescuing the VKOR activity in some extrahepatic tissues during anticoagulation therapy. J Biol Chem 288:28733–28742CrossRefPubMedCentralPubMedGoogle Scholar
  23. Hanumanthaiah R, Thankavel B, Day K, Gregory M, Jagadeeswaran P (2001) Developmental expression of vitamin K-dependent γ-carboxylase activity in zebrafish embryos: effect of warfarin. Blood Cell Mol Dis 27:992–999CrossRefGoogle Scholar
  24. Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S (2006) Steroid and xenobiotic receptor SXR mediates vitamin K2-activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells. J Biol Chem 281:16927–16934CrossRefPubMedGoogle Scholar
  25. Jeong HM, Cho DH, Jin YH, Chung JO, Chung MN, Chung DJ, Lee KY (2011) Inhibition of osteoblastic differentiation by warfarin and 18-α-glycyrrhetinic acid. Arch Pharm Res 34:1381–1387CrossRefPubMedGoogle Scholar
  26. Krossøy C, Waagbø R, Fjelldal PG, Wargelius A, Lock EJ, Graff IE, Ørnsrund R (2009) Dietary menadione nicotinamide bisulphite (vitamin K3) does not affect growth or bone health in first-feeding fry of Atlantic salmon (Salmo salar L.). Aquac Nutr 15:638–649CrossRefGoogle Scholar
  27. Krossøy C, Lock EJ, Ørnsrud R (2010) Vitamin K-dependent γ-glutamylcarboxylase in Atlantic salmon (Salmon salar L.). Fish Physiol Biochem 36:627–635CrossRefPubMedGoogle Scholar
  28. Krossøy C, Waagbø R, Ørnsrund R (2011) Vitamin K in fish nutrition. Aquac Nutr 17:585–594CrossRefGoogle Scholar
  29. Kulman JD, Harris JF, Nakazawa N, Ogasawara M, Satake M, Davie EW (2006) Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade. Proc Natl Acad Sci USA 103:15794–15799CrossRefPubMedCentralPubMedGoogle Scholar
  30. Laizé V, Gavaia PJ, Cancela ML (2015) Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities. Drug Discov Today Dis Models (in press). doi:10.1016/j.ddmod.2014.08.001
  31. Lee JH, Kwon EJ, Kim DH (2013) Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes. Biochem Biophys Res Commun 439:327–332CrossRefPubMedGoogle Scholar
  32. Menger H, Lin AE, Toriello HV, Bernert G, Spranger JW (1997) Vitamin K deficiency embryopathy: A phenocopy of the warfarin embryopathy due to a disorder of embryonic vitamin K metabolism. Am J Med Genet 72:129–134Google Scholar
  33. Müller E, Keller A, Fregin A, Müller CR, Rost S (2014) Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling. BMC Genet 15:17CrossRefPubMedCentralPubMedGoogle Scholar
  34. Oldenburg J, Marivona M, Müller-Reible C, Waltzka M (2008) The vitamin K cycle. Vitam Horm 78:35–62CrossRefPubMedGoogle Scholar
  35. Pelz H-J, Rost S, Hünerberg M, Fregin A, Heiberg A-C, Baert K, MacNicoll AD, Prescott CV, Walker A-S, Oldenburg J, Müller CR (2005) The genetic basis of resistance to anticoagulants in rodents. Genetics 170:1839–1847CrossRefPubMedCentralPubMedGoogle Scholar
  36. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pairwise correlations. Biotechnol Lett 26:509–515CrossRefPubMedGoogle Scholar
  37. Potischman N, Freudenheim JL (2003) Biomarkers of nutritional exposure and nutritional status: an overview. J Nutr 133:873S–874SGoogle Scholar
  38. Rafael MS, Marques CL, Parameswaran V, Cancela ML, Laizé V (2010) Fish bone-derived cell lines: and alternative in vitro cell system to study bone biology. J Appl Ichthyol 26:230–234CrossRefGoogle Scholar
  39. Richard N, Fernández I, Wulff T, Hamre K, Cancela LM, Conceição LEC, Gavaia PJ (2014) Dietary supplementation with vitamin K affects transcriptome and proteome of Senegalese sole, improving larval performance and quality. Mar Biotechnol 16:522–537CrossRefPubMedGoogle Scholar
  40. Rishavy MA, Usubalieva A, Hallgren KW, Berkner KL (2011) Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR). J Biol Chem 286:7267–7278CrossRefPubMedCentralPubMedGoogle Scholar
  41. Rishavy MA, Hallgren KW, Wilson LA, Usubalieva A, Runge KW, Berkner KL (2013) The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation. J Biol Chem 288:31556–31566CrossRefPubMedCentralPubMedGoogle Scholar
  42. Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hortnagel K, Pelz HJ, Lappegard K, Seifried E, Scharrer I, Tuddenham EG, Muller CR, Strom TM, Oldenburg J (2004) Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 427:537–541CrossRefPubMedGoogle Scholar
  43. Rost S, Fregin A, Hünerberg M, Bevans CG, Müller CR, Oldenburg J (2005) Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORC1: evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin. J Throm Haemost 94:780–786Google Scholar
  44. Rost S, Pelz H-J, Menzel S, MacNicoll AD, León V, Song K-J, Jäkel T, Oldenburg J, Müller CR (2009) Novel mutations in the VKORC1 gene of wild rats and mice—a response to 50 years of selection pressure by warfarin? BMC Genet 10:4CrossRefPubMedCentralPubMedGoogle Scholar
  45. Sahoo SK, Kim DH (2010) Characterization of calumenin in mouse heart. BMB Rep 43:158–163CrossRefPubMedGoogle Scholar
  46. Sahoo SK, Kim T, Kang GB, Lee JG, Eom SH, Kim DH (2009) Characterization of calumenin-SERCA2 interaction in mouse cardiac sarcoplasmic reticulum. J Biol Chem 284:31109–31121CrossRefPubMedCentralPubMedGoogle Scholar
  47. Schulman S, Wang B, Li W, Rapoport TA (2010) Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners. Proc Natl Acad Sci USA 107:15027–15032CrossRefPubMedCentralPubMedGoogle Scholar
  48. Shearer MJ (2009) Vitamin K in parental nutrition. Gastroenterology 137:S105–S118Google Scholar
  49. Spohn G, Kleinridders A, Wunderlich FT, Watzka M, Zaucke F, Blumbach K, Geisen C, Seifried E, Müller C, Paulsson M, Brüning JC, Oldenburg J (2009) VKORC1 deficiency in mice causes early postnatal lethality due to severe bleeding. Thromb Haemost 101:1044–1050PubMedGoogle Scholar
  50. Stafford DW (2005) The vitamin K cycle. J Thromb Haemost 3:1873–1878CrossRefPubMedGoogle Scholar
  51. Tabb MM, Sun A, Zhou C, Grün F, Errandi J, Romero K, Pham H, Inoue S, Mallick S, Lin M, Forman BM, Blumberg B (2003) Vitamin K2 regulation of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR. J Biol Chem 278:43919–43927CrossRefPubMedGoogle Scholar
  52. Tie J-K, Jin D-Y, Stafford DW (2012) Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms. J Biol Chem 287:33945–33955CrossRefPubMedCentralPubMedGoogle Scholar
  53. Tie J-K, Jin D-Y, Stafford DW (2014) Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration. J Biol Chem 289:9396–9407CrossRefPubMedCentralPubMedGoogle Scholar
  54. Udagawa M (2004) The effect of parental vitamin K deficiency on bone structure in mummichog Fundulus heteroclitus. J World Aquac Soc 35:366–371CrossRefGoogle Scholar
  55. Vijayakumar P, Laizé V, Cardeira J, Trindade M, Cancela ML (2013) Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralization. Zebrafish 10:500–509CrossRefPubMedCentralPubMedGoogle Scholar
  56. Wajih N, Sane DC, Hutson SM, Wallin R (2004) The inhibitory effect of calumenin on the vitamin K-dependent γ-carboxylation system. J Biol Chem 279:25276–25283CrossRefPubMedGoogle Scholar
  57. Wajih N, Hutson SM, Wallin R (2007) Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. J Biol Chem 282:2626–2635CrossRefPubMedGoogle Scholar
  58. Wallin R, Sane DC, Hutson SM (2002) Vitamin K 2,3-epoxide reductase and the vitamin K-dependent g-carboxylation system. Thromb Res 108:221–226CrossRefPubMedGoogle Scholar
  59. Watzka M, Geisen C, Bevans C, Sittinger GK, Spohn G, Rost S, Seifried EC, Müller R, Oldenburg J (2010) Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment. J Thromb Haemos 9:109–118CrossRefGoogle Scholar
  60. Weigt S, Huebler N, Strecker R, Braunbeck T, Broschard TH (2012) Developmental effects of coumarin and the anticoagulant coumarin derivative warfarin on zebrafish (Danio rerio) embryos. Reprod Toxicol 33:133–141CrossRefPubMedGoogle Scholar
  61. Westhofen P, Watzka M, Marinova M (2011) Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function. J Biol Chem 286:15085–15094CrossRefPubMedCentralPubMedGoogle Scholar
  62. Zhu A, Sun H, Raymond RM, Furie BC, Furie B, Bronstein M, Kaufman RJ, Westrick R, Ginsburg D (2007) Fatal hemorrhage in mice lacking γ-glutamyl carboxylase. Blood 109:5270–5275CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Ignacio Fernández
    • 1
  • Parameswaran Vijayakumar
    • 1
    • 2
  • Carlos Marques
    • 1
  • M. Leonor Cancela
    • 1
    • 3
  • Paulo J. Gavaia
    • 1
  • Vincent Laizé
    • 1
  1. 1.Centre of Marine Sciences (CCMAR)University of AlgarveFaroPortugal
  2. 2.Centre for Ocean ResearchSathyabama UniversityChennaiIndia
  3. 3.Department of Biomedical Sciences and Medicine (DCBM)University of AlgarveFaroPortugal

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