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Calcified Tissue International

, Volume 90, Issue 3, pp 219–229 | Cite as

Microarray Profiling of Diaphyseal Bone of Rats Suffering from Hypervitaminosis A

  • Thomas LindEmail author
  • Lijuan Hu
  • P. Monica Lind
  • Rachael Sugars
  • Göran Andersson
  • Annica Jacobson
  • Håkan Melhus
Original Research

Abstract

Vitamin A is the only known compound that produces spontaneous fractures in rats. In an effort to resolve the molecular mechanism behind this effect, we fed young male rats high doses of vitamin A and performed microarray analysis of diaphyseal bone with and without marrow after 1 week, i.e., just before the first fractures appeared. Of the differentially expressed genes in cortical bone, including marrow, 98% were upregulated. In contrast, hypervitaminotic cortical bone without marrow showed reduced expression of 37% of differentially expressed genes. Gene ontology (GO) analysis revealed that only samples containing bone marrow were associated with a GO term, which principally represented extracellular matrix. This is consistent with the histological findings of increased endosteal/marrow osteoblast number. Fourteen genes, including Cyp26b1, which is known to be upregulated by vitamin A, were selected and verified by real-time PCR. In addition, immunohistochemical staining of bone sections confirmed that the bone-specific molecule osteoadherin was upregulated. Further analysis of the major gene-expression changes revealed apparent augmented Wnt signaling in the sample containing bone marrow but reduced Wnt signaling in cortical bone. Moreover, induced expression of hypoxia-associated genes was found only in samples containing bone marrow. Together, these results highlight the importance of compartment-specific analysis of bone and corroborate previous observations of compartment-specific effects of vitamin A, with reduced activity in cortical bone but increased activity in the endosteal/marrow compartment. We specifically identify potential key osteoblast-, Wnt signaling-, and hypoxia-associated genes in the processes leading to spontaneous fractures.

Keywords

Retinol Microarray Diaphyseal bone Marrow Rat 

Notes

Acknowledgment

We thank Uppsala Array Platform for their technical assistance and Valeria Giandomenico for critical reading of the manuscript. This work was supported by the Swedish Society of Medicine (T. L.) and the Swedish Medical Research Council (H. M., G. A.).

Supplementary material

223_2011_9561_MOESM1_ESM.xls (34 kb)
Supplementary material 1 (XLS 34 kb)
223_2011_9561_MOESM2_ESM.xls (51 kb)
Supplementary material 2 (XLS 51 kb)

References

  1. 1.
    Collazo J, Rodriguez J (1933) Hypervitaminose II, exophtalmus und spontanfrakturen. Klin Wochschr 12:1768–1771CrossRefGoogle Scholar
  2. 2.
    Bomskov C, Seeman G (1933) Uber eine wirkung des vitamin A auf den mineralhaushalt. Z Ges Exp Med 89:771–779CrossRefGoogle Scholar
  3. 3.
    Strauss K (1934) Beobachtungen bei hypervitaminose A. Anat U Allgem Pathol 94:345–352Google Scholar
  4. 4.
    Moore T, Wang YL (1945) Hypervitaminosis A. Biochem J 39:222–228Google Scholar
  5. 5.
    Nieman C, Obbink H (1954) The biochemistry and pathology of hypervitaminosis A. Vitam Horm 12:69–99PubMedCrossRefGoogle Scholar
  6. 6.
    Rodahl K (1950) Hypervitaminosis in the rat. J Nutr 41:399–421PubMedGoogle Scholar
  7. 7.
    Johansson S, Lind PM, Hakansson H, Oxlund H, Orberg J et al (2002) Subclinical hypervitaminosis A causes fragile bones in rats. Bone 31:685–689PubMedCrossRefGoogle Scholar
  8. 8.
    Hixson EJ, Burdeshaw JA, Denine EP, Harrison SD Jr (1979) Comparative subchronic toxicity of all-trans- and 13-cis-retinoic acid in Sprague-Dawley rats. Toxicol Appl Pharmacol 47:359–365PubMedCrossRefGoogle Scholar
  9. 9.
    Kurtz PJ, Emmerling DC, Donofrio DJ (1984) Subchronic toxicity of all-trans-retinoic acid and retinylidene dimedone in Sprague-Dawley rats. Toxicology 30:115–124PubMedCrossRefGoogle Scholar
  10. 10.
    Forsyth KS, Watson RR, Gensler HL (1989) Osteotoxicity after chronic dietary administration of 13-cis-retinoic acid, retinyl palmitate or selenium in mice exposed to tumor initiation and promotion. Life Sci 45:2149–2156PubMedCrossRefGoogle Scholar
  11. 11.
    Teelmann K (1981) Experimental toxicology of the aromatic retinoid Ro 10-9359 (Etretinate). In: Orfanos C, Braun-Falco O, Farber E, Grupper C, Polano M, Schuppli R (eds) Retinoids: advances in basic research and therapy. Springer-Verlag, Berlin, pp 41–47Google Scholar
  12. 12.
    Kneissel M, Studer A, Cortesi R, Susa M (2005) Retinoid-induced bone thinning is caused by subperiosteal osteoclast activity in adult rodents. Bone 36:202–214PubMedCrossRefGoogle Scholar
  13. 13.
    Lind T, Lind PM, Jacobson A, Hu L, Sundqvist A et al (2011) High dietary intake of retinol leads to bone marrow hypoxia and diaphyseal endosteal mineralization in rats. Bone 48:496–506PubMedCrossRefGoogle Scholar
  14. 14.
    Li C, Wong WH (2001) Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 98:31–36PubMedCrossRefGoogle Scholar
  15. 15.
    Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ et al (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249–264PubMedCrossRefGoogle Scholar
  16. 16.
    Smyth GK (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3Google Scholar
  17. 17.
    Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W (eds) Bioinformatics and computational biology solutions using R and bioconductor. Springer, New York, pp 397–420CrossRefGoogle Scholar
  18. 18.
    Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57:289–300Google Scholar
  19. 19.
    Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nat Protoc 4:44–57CrossRefGoogle Scholar
  20. 20.
    Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W et al (2003) DAVID: database for annotation, visualization, and integrated discovery. Genome Biol 4:P3PubMedCrossRefGoogle Scholar
  21. 21.
    Hollberg K, Hultenby K, Hayman A, Cox T, Andersson G (2002) Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport. Exp Cell Res 279:227–238PubMedCrossRefGoogle Scholar
  22. 22.
    Wendel M, Sommarin Y, Heinegård D (1998) Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone. J Cell Biol 141:839–847PubMedCrossRefGoogle Scholar
  23. 23.
    Kamiya N, Kaartinen VM, Mishina Y (2011) Loss-of-function of ACVR1 in osteoblasts increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1. Biochem Biophys Res Commun 414:326–330PubMedCrossRefGoogle Scholar
  24. 24.
    Zhao X, Brade T, Cunningham TJ, Duester G (2010) Retinoic acid controls expression of tissue remodeling genes Hmgn1 and Fgf18 at the digit–interdigit junction. Dev Dyn 239:665–671PubMedCrossRefGoogle Scholar
  25. 25.
    Shimono K, Tung WE, Macolino C, Chi AH, Didizian JH et al (2011) Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists. Nat Med 17:454–460PubMedCrossRefGoogle Scholar
  26. 26.
    Bonewald LF (2011) The amazing osteocyte. J Bone Miner Res 26:229–238PubMedCrossRefGoogle Scholar
  27. 27.
    Easwaran V, Pishvaian M, Salimuddin, Byers S (1999) Cross-regulation of beta-catenin-LEF/TCF and retinoid signaling pathways. Curr Biol 9:1415–1418PubMedCrossRefGoogle Scholar
  28. 28.
    Engberg N, Kahn M, Petersen DR, Hansson M, Serup P (2010) Retinoic acid synthesis promotes development of neural progenitors from mouse embryonic stem cells by suppressing endogenous, Wnt-dependent nodal signaling. Stem Cells 28:1498–1509PubMedCrossRefGoogle Scholar
  29. 29.
    Kato M, Patel MS, Levasseur R, Lobov I, Chang BH et al (2002) Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 157:303–314PubMedCrossRefGoogle Scholar
  30. 30.
    Fujino T, Asaba H, Kang MJ, Ikeda Y, Sone H et al (2003) Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion. Proc Natl Acad Sci USA 100:229–234PubMedCrossRefGoogle Scholar
  31. 31.
    Kramer I, Halleux C, Keller H, Pegurri M, Gooi JH et al (2010) Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol Cell Biol 30:3071–3085PubMedCrossRefGoogle Scholar
  32. 32.
    Elizalde C, Campa VM, Caro M, Schlangen K, Aransay AM et al (2011) Distinct roles for Wnt-4 and Wnt-11 during retinoic acid-induced neuronal differentiation. Stem Cells 29:141–153PubMedCrossRefGoogle Scholar
  33. 33.
    Gong Y, Slee RB, Fukai N, Rawadi G, Roman–Roman S et al (2001) LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107:513–523PubMedCrossRefGoogle Scholar
  34. 34.
    Chang J, Sonoyama W, Wang Z, Jin Q, Zhang C et al (2007) Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK. J Biol Chem 282:30938–30948PubMedCrossRefGoogle Scholar
  35. 35.
    Li X, Liu H, Qin L, Tamasi J, Bergenstock M et al (2007) Determination of dual effects of parathyroid hormone on skeletal gene expression in vivo by microarray and network analysis. J Biol Chem 282:33086–33097PubMedCrossRefGoogle Scholar
  36. 36.
    Cho SW, Her SJ, Sun HJ, Choi OK, Yang JY et al (2008) Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts. Biochem Biophys Res Commun 367:399–405PubMedCrossRefGoogle Scholar
  37. 37.
    Sathi GA, Inoue M, Harada H, Rodriguez AP, Tamamura R et al (2009) Secreted frizzled related protein (sFRP)-2 inhibits bone formation and promotes cell proliferation in ameloblastoma. Oral Oncol 45:856–860PubMedCrossRefGoogle Scholar
  38. 38.
    von Marschall Z, Fisher LW (2010) Secreted frizzled-related protein-2 (sFRP2) augments canonical Wnt3a-induced signaling. Biochem Biophys Res Commun 400:299–304CrossRefGoogle Scholar
  39. 39.
    Kobayashi K, Luo M, Zhang Y, Wilkes DC, Ge G et al (2009) Secreted frizzled-related protein 2 is a procollagen C proteinase enhancer with a role in fibrosis associated with myocardial infarction. Nat Cell Biol 11:46–55PubMedCrossRefGoogle Scholar
  40. 40.
    Railo A, Pajunen A, Itäranta P, Naillat F, Vuoristo J et al (2009) Genomic response to Wnt signalling is highly context-dependent—evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets. Exp Cell Res 315:2690–2704PubMedCrossRefGoogle Scholar
  41. 41.
    Kalantari F, Miao D, Emadali A, Tzimas GN, Goltzman D et al (2007) Cellular and molecular mechanisms of abnormal calcification following ischemia-reperfusion injury in human liver transplantation. Mod Pathol 20:357–366PubMedCrossRefGoogle Scholar
  42. 42.
    Ingber D, Folkman J (1988) Inhibition of angiogenesis through modulation of collagen metabolism. Lab Investig 59:44–51PubMedGoogle Scholar
  43. 43.
    Tallman MS, Andersen JW, Schiffer CA, Appelbaum FR, Feusner JH et al (2000) Clinical description of 44 patients with acute promyelocytic leukemia who developed the retinoic acid syndrome. Blood 95:90–95PubMedGoogle Scholar
  44. 44.
    Zhang E, Jiang B, Yokochi A, Maruyama J, Mitani Y et al (2010) Effect of all-trans-retinoic acid on the development of chronic hypoxia-induced pulmonary hypertension. Circ J 74:1696–1703PubMedCrossRefGoogle Scholar
  45. 45.
    Carmeliet P, Tessier-Lavigne M (2005) Common mechanisms of nerve and blood vessel wiring. Nature 436:193–200PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Thomas Lind
    • 1
    Email author
  • Lijuan Hu
    • 1
  • P. Monica Lind
    • 2
  • Rachael Sugars
    • 3
  • Göran Andersson
    • 4
  • Annica Jacobson
    • 1
  • Håkan Melhus
    • 1
  1. 1.Department of Medical Sciences, Section of Clinical PharmacologyUppsala UniversityUppsalaSweden
  2. 2.Department of Medical Sciences, Section of Occupational and Environmental MedicineUppsala UniversityUppsalaSweden
  3. 3.Oral Biology, Department of Dental MedicineKarolinska InstitutetHuddingeSweden
  4. 4.Division of Pathology, Department of Laboratory MedicineKarolinska Institutet, Karolinska University HospitalHuddingeSweden

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