Advertisement

Calcified Tissue International

, Volume 104, Issue 1, pp 70–78 | Cite as

Irp2 Knockout Causes Osteoporosis by Inhibition of Bone Remodeling

  • Yaru ZhouEmail author
  • Yu Yang
  • Yan Liu
  • Hengrui Chang
  • Kuanzhi Liu
  • Xiaojuan Zhang
  • Yanzhong ChangEmail author
Original Research
  • 162 Downloads

Abstract

It has been found that iron disorder may lead to osteoporosis. However, the mechanism has been little explored. In the present study, we try to investigate the effects of iron disorder on bone metabolism using Irp2 knockout (Irp2−/−) mice. Female Irp2−/− mice were used in this study. Bone mineral density (BMD) was measured by Micro-CT. Serum markers for bone turnover were measured by enzyme-linked immunosorbent assay. Content of iron was measured in bone and liver tissue, and Vitamin D 25-hydroxylase (CYP2R1) content was measured in liver tissue. Relative gene expression involved in iron export and uptake, and some genes involved in activities of osteoblast and osteoclast were all measured by real-time PCR and western blot. Compared to wild-type mice, Irp2−/− mice exhibited reduced BMD, bone iron deficiency, and hepatic iron overload. Serum levels of 25(OH)D3 and markers for bone formation such as bone alkaline phosphatase (Balp), bone-gla-protein (BGP), and type I collagen alpha1 chain (Col I α1) were decreased, while markers for bone resorption including cathepsin K (Ctsk) and tartrate-resistant acid phosphatase (Trap) were all significantly increased. Hepatic CYP2R1 level was decreased in Irp2−/− mice compared with wild-type control mice. Compared to wild-type C57BL6 control mice, the expression of genes involved in osteoblast activity such as Balp, BGP, and Col I α1 were all significantly decreased in bone tissue, while genes for osteoclast activity such as Ctsk and Trap were all markedly increased in Irp2−/− mice at mRNA level. Genes involved in iron storage, uptake, and exporting were also measured in bone tissue. Posttranscriptionally decreased ferritin (FTL), ferroportin 1 (FPN1), and increased transferrin receptor 1 (TfR1) gene expressions have been unexpectedly found in bone tissue of Irp2−/− mice. Irp2−/− mice exhibit reduced bone iron content and osteoporosis. Decreased circulating 25(OH)D3 levels promoted activity of osteoclast, while impaired activity of osteoblast may contribute to pathogenesis of osteoporosis. And, reduced bone iron content may not be totally caused by TfR1-dependent pathways.

Keywords

Osteoporosis Iron disorder Hepatic Bone tissue 

Notes

Acknowledgements

We acknowledge the Beijing Synchrotron Radiation Facility for the beam time. This work was supported by the National Natural Science Foundation of China (Grant Number 31471035).

Author Contributions

YZ and YY performed most of the experiments; YL, HC, KL, and XZ performed experiments and data analysis; YY wrote the manuscript; YZ, YL, and YC interpreted data and critically revised the manuscript. All authors approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

Yaru Zhou, Yu Yang, Yan Liu, Hengrui Chang, Kuanzhi Liu, Xiaojuan Zhang, and Yanzhong Chang declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

The experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee of Hebei Science and Technical Bureau in China.

Supplementary material

223_2018_469_MOESM1_ESM.pdf (46 kb)
Supplementary material 1 (PDF 46 KB)

References

  1. 1.
    Rossi F, Perrotta S, Bellini G et al (2014) Iron overload causes osteoporosis in thalassemia major patients through interaction with transient receptor potential vanilloid type 1 (TRPV1) channels. Haematologica 99(12):1876–1884CrossRefGoogle Scholar
  2. 2.
    Guggenbuhl P, Deugnier Y, Boisdet JF et al (2005) Bone mineral density in men with genetic hemochromatosis and HFE gene mutation. Osteoporos Int 16:1809–1814CrossRefGoogle Scholar
  3. 3.
    Valenti L, Varenna M, Fracanzani AL (2009) et, al. Association between iron overload and osteoporosis in patients with hereditary hemochromatosis. Osteoporos Int 20:549–555CrossRefGoogle Scholar
  4. 4.
    Sinigaglia L, Fargion S, Fracanzani AL et al (1997) Bone and joint involvement in genetic hemochromatosis: role of cirrhosis and iron overload. J Rheumatol 24(9):1809–1813Google Scholar
  5. 5.
    Medeiros DM, Plattner A, Jennings D et al (2002) Bone morphology, strength and density are compromised in iron-deficient rats and exacerbated by calcium restriction. J Nutr 132(10):3135–3141CrossRefGoogle Scholar
  6. 6.
    Medeiros DM, Stoecker B, Plattener A et al (2004) Iron deficiency negatively affects vertebrae and femurs of rats independently of energy intake and body weight. J Nutr 134(11):3061–3067CrossRefGoogle Scholar
  7. 7.
    Li GF, Pan YZ, Sirois P et al (2012) Iron homeostasis in osteoporosis and its clinical implications. Osteoporos Int 23:2403–2408CrossRefGoogle Scholar
  8. 8.
    Yamasaki K, Hagiwara H (2009) Excess iron inhibits osteoblast metabolism. Toxicol Lett 191:211–215CrossRefGoogle Scholar
  9. 9.
    He YF, Ma Y, Gao C et al (2013) Iron overload inhibits osteoblast biological activity through oxidative stress. Biol Trace Elem Res 152(2):292–296CrossRefGoogle Scholar
  10. 10.
    Hentze MW, Muckenthaler MU, Galy B et al (2010) Two to tango:regulation of Mammalian iron metabolism. Cell 9(1):24–38 142(CrossRefGoogle Scholar
  11. 11.
    Anderson CP, Shen M, Eisenstein RS et al (2012) Mammalian iron metabolism and its control by iron regulatory proteins. Biochim Biophys Acta 1823(9):1468–1483CrossRefGoogle Scholar
  12. 12.
    Sanchez M, Galy B, Schwanhaeusser B et al (2011) Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins. Blood 118(22):e168–e179CrossRefGoogle Scholar
  13. 13.
    Vashisht AA, Zumbrennen KB, Huang X et al (2009) Control of iron homeostasis by an iron-regulated ubiquitin ligase. Science 326(5953):718–721CrossRefGoogle Scholar
  14. 14.
    Salahudeen AA, Thompson JW, Ruiz JC et al (2009) An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis. Science 326(5953):722–726CrossRefGoogle Scholar
  15. 15.
    Moroishi T, Nishiyama M, Takeda Y et al (2011) The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo. Cell Metab 14(3):339–351CrossRefGoogle Scholar
  16. 16.
    Ishii KA, Fumoto T, Iwai K et al (2009) Coordination of PGC-1beta and iron uptake in mitochondrial biogenesis and osteoclast activation. Nat Med 15(3):259–266CrossRefGoogle Scholar
  17. 17.
    Yang Q, Jian J, Abramson SB et al (2011) Inhibitory effects of iron on bone morphogenetic protein 2-induced osteoblastogenesis. J Bone Miner Res 26:1188–1196CrossRefGoogle Scholar
  18. 18.
    Chang YZ, Qian ZM, Wang K et al (2005) Effects of development and iron status on ceruloplasmin expression in rat brain. J Cell Physiol 204(2):623–631CrossRefGoogle Scholar
  19. 19.
    Machado I, Bergmann G, Pistón M (2016) A simple and fast ultrasound-assisted extraction procedure for Fe and Zn determination in milk-based infant formulas using flame atomic absorption spectrometry (FAAS). Food Chem 194:373–376CrossRefGoogle Scholar
  20. 20.
    Shi ZH, Nie G, Duan XL, Rouault T, Wu WS, Ning B, Zhang N, Chang YZ, Zhao BL (2010) Neuroprotective mechanism of mitochondrial ferritin on 6-hydroxydopamine-induceddopaminergic cell damage: implication for neuroprotection in Parkinson’s disease. Antioxid Redox Signal 2010;13(6):783–796CrossRefGoogle Scholar
  21. 21.
    Winter WE, Bazydlo LA, Harris NS (2014) The molecular biology of human iron metabolism. Lab Med 45(2):92–102CrossRefGoogle Scholar
  22. 22.
    Li J, Hou Y, Zhang S et al (2013) Excess iron undermined bone load-bearing capacity through tumor necrosis factor-alpha-dependent osteoclastic activation in mice. Biomed Rep 1:85–88CrossRefGoogle Scholar
  23. 23.
    D’Amelio P, Cristofaro MA, Tamone C et al (2008) Role of iron metabolism and oxidative damage in postmenopausal bone loss. Bone 43:1010–1015CrossRefGoogle Scholar
  24. 24.
    Meyron-Holtz EG, Ghosh MC, Rouault TA (2004) Mammalian tissue oxygen levels modulate iron-regulatory protein activities in vivo. Science 306:2087–2090CrossRefGoogle Scholar
  25. 25.
    Meyron-Holtz EG, Ghosh MC, Iwai K et al (2004) Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis. EMBO J 23:386–395CrossRefGoogle Scholar
  26. 26.
    Kim HY, Klausner RD, Rouault TA (1995) Translational repressor activity is equivalent and is quantitatively predicted by in vitro RNA binding for two iron-responsive element binding proteins, IRP1 and IRP2. J Biol Chem 270:4983–4986CrossRefGoogle Scholar
  27. 27.
    Ghosh MC, Zhang DL, Jeong SY et al (2013) Deletion of iron regulatory protein 1 causes polycythemia and pulmonary hypertension in mice through translational derepression of HIF2α. Cell Metab 17(2):271–281CrossRefGoogle Scholar
  28. 28.
    Cooperman SS, Meyron-Holtz EG, Olivierre-Wilson H et al (2005) Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood 106(3):1084–1091CrossRefGoogle Scholar
  29. 29.
    Zhou J, Ye S, Fujiwara T et al (2013) Steap4 plays a critical role in osteoclastogenesis in vitro by regulating cellular iron/reactive oxygen species (ROS) levels and cAMP response element-binding protein (CREB) activation. J Biol Chem 288(42):30064–30074CrossRefGoogle Scholar
  30. 30.
    Guggenbuhl P, Fergelot P, Doyard M et al (2011) Bone status in a mouse model of genetic hemochromatosis. Osteoporos Int 22(8):2313–2319CrossRefGoogle Scholar
  31. 31.
    Doyard M, Chappard D, Leroyer P et al (2016) Decreased bone formation explains osteoporosis in a genetic mouse model of hemochromatosiss. PLoS ONE 11(2):e0148292CrossRefGoogle Scholar
  32. 32.
    Mediero A, Cronstein BN (2013) Adenosine and bone metabolism. Trends Endocrinol Metab 24(6):290–300CrossRefGoogle Scholar
  33. 33.
    Uemura H, Yasui T, Kiyokawa M et al (2002) Serum osteoprotegerin/osteoclastogenesis-inhibitory factor during pregnancy and lactation and the relationship with calcium-regulating hormones and bone turnover markers. J Endocrinol 174(2):353–359CrossRefGoogle Scholar
  34. 34.
    Price PA, Parthemore JG, Deftos LJ (1980) New biochemical marker for bone metabolism. Measurement by radioimmunoassay of bone GLA protein in the plasma of normal subjects and patients with bonedisease. J Clin Invest 66(5):878–883CrossRefGoogle Scholar
  35. 35.
    Zoch ML, Clemens TL, Riddle RC (2016) New insights into the biology of osteocalcin. Bone 82:42–49CrossRefGoogle Scholar
  36. 36.
    Song YE, Tan H, Liu KJ et al (2011) Effect of fluoride exposure on bone metabolism indicators ALP, BALP, and BGP. Environ Health Prev Med 16(3):158–163CrossRefGoogle Scholar
  37. 37.
    Kemper O, Herten M, Fischer J et al (2014) Prostacyclin suppresses twist expression in the presence of indomethacin in bone marrow-derived mesenchymal stromal cells. Med Sci Monit 20:2219–2227CrossRefGoogle Scholar
  38. 38.
    Inui T, Ishibashi O, Inaoka T et al (1997) Cathepsin K antisense oligodeoxynucleotide inhibits osteoclastic bone resorption. J Biol Chem 272(13):8109–8112CrossRefGoogle Scholar
  39. 39.
    Galy B, Ferring D, Minana B et al (2005) Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2). Blood 106(7):2580–2589CrossRefGoogle Scholar
  40. 40.
    Arosio P, Elia L, Poli M (2017) Ferritin, cellular iron storage and regulation. IUBMB Life 69(6):414–422CrossRefGoogle Scholar
  41. 41.
    Ganz T (2005) Cellular iron: ferroportin is the only way out. Cell Metab 1(3):155–157CrossRefGoogle Scholar
  42. 42.
    Rochette L, Gudjoncik A, Guenancia C et al (2015) The iron-regulatory hormone hepcidin: a possible therapeutic target? Pharmacol Ther 146:35–52CrossRefGoogle Scholar
  43. 43.
    Wang G, Shao A, Hu W et al (2015) Changes of ferrous iron and its transporters after intracerebral hemorrhage in rats. Int J Clin Exp Pathol 8(9):10671–10679Google Scholar
  44. 44.
    Rice AE, Mendez MJ, Hokanson CA et al (2009) Investigation of the biophysical and cell biological properties of ferroportin, a multipass integral membrane protein iron exporter. J Mol Biol 386(3):717–732CrossRefGoogle Scholar
  45. 45.
    Zumbrennen-Bullough KB, Becker L, Garrett L et al (2014) Abnormal brain iron metabolism in Irp2 deficient mice is associated with mild neurological and behavioral impairments. PLoS ONE 9(6):e98072CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of EndocrinologyThird Hospital of Hebei Medical UniversityShijiazhuangChina
  2. 2.Department of Orthopaedic SurgeryThird Hospital of Hebei Medical UniversityShijiazhuangChina
  3. 3.College of Life ScienceHebei Normal UniversityShijiazhuangChina

Personalised recommendations