Osteoporosis International

, Volume 23, Issue 10, pp 2435–2445 | Cite as

Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells

  • M. Doyard
  • N. Fatih
  • A. Monnier
  • M. L. Island
  • M. Aubry
  • P. Leroyer
  • R. Bouvet
  • G. Chalès
  • J. Mosser
  • O. Loréal
  • P. Guggenbuhl
Original Article

Abstract

Summary

In order to understand mechanisms involved in osteoporosis observed during iron overload diseases, we analyzed the impact of iron on a human osteoblast-like cell line. Iron exposure decreases osteoblast phenotype. HHIPL-2 is an iron-modulated gene which could contribute to these alterations. Our results suggest osteoblast impairment in iron-related osteoporosis.

Introduction

Iron overload may cause osteoporosis. An iron-related decrease in osteoblast activity has been suggested.

Methods

We investigated the effect of iron exposure on human osteoblast cells (MG-63) by analyzing the impact of ferric ammonium citrate (FAC) and iron citrate (FeCi) on the expression of genes involved in iron metabolism or associated with osteoblast phenotype. A transcriptomic analysis was performed to identify iron-modulated genes.

Results

FAC and FeCi exposure modulated cellular iron status with a decrease in TFRC mRNA level and an increase in intracellular ferritin level. FAC increased ROS level and caspase 3 activity. Ferroportin, HFE and TFR2 mRNAs were expressed in MG-63 cells under basal conditions. The level of ferroportin mRNA was increased by iron, whereas HFE mRNA level was decreased. The level of mRNA alpha 1 collagen type I chain, osteocalcin and the transcriptional factor RUNX2 were decreased by iron. Transcriptomic analysis revealed that the mRNA level of HedgeHog Interacting Protein Like-2 (HHIPL-2) gene, encoding an inhibitor of the hedgehog signaling pathway, was decreased in the presence of FAC. Specific inhibition of HHIPL-2 expression decreased osteoblast marker mRNA levels. Purmorphamine, hedgehog pathway activator, increased the mRNA level of GLI1, a target gene for the hedgehog pathway, and decreased osteoblast marker levels. GLI1 mRNA level was increased under iron exposure.

Conclusion

We showed that in human MG-63 cells, iron exposure impacts iron metabolism and osteoblast gene expression. HHIPL-2 gene expression modulation may contribute to these alterations. Our results support a role of osteoblast impairment in iron-related osteoporosis.

Keywords

Gene expression Hemochromatosis HHIPL-2 Iron overload Osteoblast Osteoporosis 

Supplementary material

198_2011_1871_MOESM1_ESM.pdf (18 kb)
Fig. S1Iron metabolism gene expression under basal situation in MG-63 cells. SLC40A1 (a), HFE (b) and TFR2 (c) mRNA levels under basal culture conditions. The results are expressed as a percentage of expression in caco-2 cells (PDF 220 kb)
198_2011_1871_MOESM2_ESM.pdf (404 kb)
Fig. S2Impact of FeCi exposure on the expression of osteoblast genes in MG-63 cells. mRNA levels of COL1A1 (a), BGLAP (b) and RUNX2 (c) in cells treated with FeCi and/or DFO for 72 h. The results are expressed as a percentage of their respective control (100%). Asterisk indicates p < 0.05 compared with the corresponding concentration of citrate; plus sign indicates p < 0.05 compared with FeCi 20 μM (PDF 454 kb)
198_2011_1871_MOESM3_ESM.pdf (225 kb)
Fig. S3Inhibition of mRNA HHIPL-2 expression by specific siRNAs. HHIPL-2 mRNA level after transfection of MG-63 cells with two HHIPL-2-specific siRNAs (si1 and si2) or with a control siRNA (Control) for 72 h. The results are expressed as a percentage of the control (100%). Asterisk indicates p < 0.05 compared with the control (PDF 224 kb)
198_2011_1871_MOESM4_ESM.pdf (66 kb)
Fig. S4The impact of iron exposure on GLI1 mRNA expression level in MG-63 cells. Expression of GLI1 mRNA after treatment with FAC and/or DFO for 72 h. The results are expressed as a percentage of the control (100%). Asterisk indicates p < 0.05 compared with the control (PDF 66.0 kb)

References

  1. 1.
    Weinberg ED (2006) Iron loading: a risk factor for osteoporosis. Biometals 19:633–635PubMedCrossRefGoogle Scholar
  2. 2.
    Pietrangelo A, Trautwein C (2004) Mechanisms of disease: the role of hepcidin in iron homeostasis—implications for hemochromatosis and other disorders. Nat Clin Pract Gastroenterol Hepatol 1:39–45PubMedCrossRefGoogle Scholar
  3. 3.
    Brissot P, Le Lan C, Lorho R, Gaboriau F, Lescoat G, Loréal O (2005) Genetic hemochromatosis update. Acta Gastroenterol Belg 68:33–37PubMedGoogle Scholar
  4. 4.
    Niederau C, Fischer R, Purschel A, Stremmel W, Haussinger D, Strohmeyer G (1996) Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 110:1107–1119PubMedCrossRefGoogle Scholar
  5. 5.
    Brissot P, Troadec MB, Bardou-Jacquet E, Le Lan C, Jouanolle AM, Deugnier Y, Loréal O (2008) Current approach to hemochromatosis. Blood Rev 22:195–210PubMedCrossRefGoogle Scholar
  6. 6.
    Pawlotsky Y, Le Dantec P, Moirand R, Guggenbuhl P, Jouanolle AM, Catheline M, Meadeb J, Brissot P, Deugnier Y, Chales G (1999) Elevated parathyroid hormone 44–68 and osteoarticular changes in patients with genetic hemochromatosis. Arthritis Rheum 42:799–806PubMedCrossRefGoogle Scholar
  7. 7.
    Guggenbuhl P, Albert JD, Chales G (2007) Rheumatic manifestations of genetic hemochromatosis. Presse Med 36:1313–1318PubMedCrossRefGoogle Scholar
  8. 8.
    Sinigaglia L, Fargion S, Fracanzani AL, Binelli L, Battafarano N, Varenna M, Piperno A, Fiorelli G (1997) Bone and joint involvement in genetic hemochromatosis: role of cirrhosis and iron overload. J Rheumatol 24:1809–1813PubMedGoogle Scholar
  9. 9.
    Guggenbuhl P, Deugnier Y, Boisdet JF, Rolland Y, Perdriger A, Pawlotsky Y, Chales G (2005) Bone mineral density in men with genetic hemochromatosis and HFE gene mutation. Osteoporos Int 16:1809–1814PubMedCrossRefGoogle Scholar
  10. 10.
    Valenti L, Varenna M, Fracanzani AL, Rossi V, Fargion S, Sinigaglia L (2009) Association between iron overload and osteoporosis in patients with hereditary hemochromatosis. Osteoporos Int 20:549–555PubMedCrossRefGoogle Scholar
  11. 11.
    Jensen CE, Tuck SM, Agnew JE, Koneru S, Morris RW, Yardumian A, Prescott E, Hoffbrand AV, Wonke B (1998) High prevalence of low bone mass in thalassaemia major. Br J Haematol 103:911–915PubMedCrossRefGoogle Scholar
  12. 12.
    Jensen CE, Tuck SM, Agnew JE, Koneru S, Morris RW, Yardumian A, Prescott E, Hoffbrand AV, Wonke B (1998) High incidence of osteoporosis in thalassaemia major. J Pediatr Endocrinol Metab 11(Suppl 3):975–977PubMedGoogle Scholar
  13. 13.
    Jian J, Pelle E, Huang X (2009) Iron and menopause: does increased iron affect the health of postmenopausal women? Antioxid Redox Signal 11:2939–2943PubMedCrossRefGoogle Scholar
  14. 14.
    Pawlotsky Y, Lancien Y, Roudier G, Hany Y, Louboutin JY, Ferrand B, Bourel M (1979) Bone histomorphometry and osteo-articular manifestations of idiopathic hemochromatosis. Rev Rhum Mal Osteoartic 46:91–99PubMedGoogle Scholar
  15. 15.
    Conte D, Caraceni MP, Duriez J, Mandelli C, Corghi E, Cesana M, Ortolani S, Bianchi PA (1989) Bone involvement in primary hemochromatosis and alcoholic cirrhosis. Am J Gastroenterol 84:1231–1234PubMedGoogle Scholar
  16. 16.
    de Vernejoul MC, Pointillart A, Golenzer CC, Morieux C, Bielakoff J, Modrowski D, Miravet L (1984) Effects of iron overload on bone remodeling in pigs. Am J Pathol 116:377–384PubMedGoogle Scholar
  17. 17.
    Matsushima S, Hoshimoto M, Torii M, Ozaki K, Narama I (2001) Iron lactate-induced osteopenia in male Sprague–Dawley rats. Toxicol Pathol 29:623–629PubMedCrossRefGoogle Scholar
  18. 18.
    Guggenbuhl P, Fergelot P, Doyard M, Libouban H, Roth MP, Gallois Y, Chales G, Loréal O, Chappard D (2011) Bone status in a mouse model of genetic hemochromatosis. Osteoporos Int 22:2313–2319PubMedCrossRefGoogle Scholar
  19. 19.
    Messer JG, Kilbarger AK, Erikson KM, Kipp DE (2009) Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures. Bone 45:972–979PubMedCrossRefGoogle Scholar
  20. 20.
    Yamasaki K, Hagiwara H (2009) Excess iron inhibits osteoblast metabolism. Toxicol Lett 191:211–215PubMedCrossRefGoogle Scholar
  21. 21.
    Yang Q, Jian J, Abramson SB, Huang X (2011) Inhibitory effects of iron on bone morphogenetic protein-2-induced osteoblastogenesis. J Bone Miner Res 26:1188–1196PubMedCrossRefGoogle Scholar
  22. 22.
    Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O (2001) A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 276:7811–7819PubMedCrossRefGoogle Scholar
  23. 23.
    Nicolas G, Bennoun M, Devaux I, Beaumont C, Grandchamp B, Kahn A, Vaulont S (2001) Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 98:8780–8785PubMedCrossRefGoogle Scholar
  24. 24.
    Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J (2004) Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 306:2090–2093PubMedCrossRefGoogle Scholar
  25. 25.
    Kasai K, Hori MT, Goodman WG (1990) Characterization of the transferrin receptor in UMR-106-01 osteoblast-like cells. Endocrinology 126:1742–1749PubMedCrossRefGoogle Scholar
  26. 26.
    Spanner M, Weber K, Lanske B, Ihbe A, Siggelkow H, Schutze H, Atkinson MJ (1995) The iron-binding protein ferritin is expressed in cells of the osteoblastic lineage in vitro and in vivo. Bone 17:161–165PubMedCrossRefGoogle Scholar
  27. 27.
    Choi HD, Noh WC, Park JW, Lee JM, Suh JY (2011) Analysis of gene expression during mineralization of cultured human periodontal ligament cells. J Periodontal Implant Sci 41:30–43PubMedCrossRefGoogle Scholar
  28. 28.
    Ruiz-Gaspa S, Nogues X, Enjuanes A, Monllau JC, Blanch J, Carreras R, Mellibovsky L, Grinberg D, Balcells S, Diez-Perez A, Pedro-Botet J (2007) Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures. J Cell Biochem 101:1430–1438PubMedCrossRefGoogle Scholar
  29. 29.
    Wang Y, Li LZ, Zhang YL, Zhu YQ, Wu J, Sun WJ (2011) LC, a novel estrone-rhein hybrid compound, concurrently stimulates osteoprotegerin and inhibits receptor activator of NF-kappaB ligand (RANKL) and interleukin-6 production by human osteoblastic cells. Mol Cell Endocrinol 337:43–51PubMedCrossRefGoogle Scholar
  30. 30.
    Grausova L, Kromka A, Burdikova Z, Eckhardt A, Rezek B, Vacik J, Haenen K, Lisa V, Bacakova L (2011) Enhanced growth and osteogenic differentiation of human osteoblast-like cells on boron-doped nanocrystalline diamond thin films. PLoS One 6:e20943PubMedCrossRefGoogle Scholar
  31. 31.
    Hershko C, Graham G, Bates GW, Rachmilewitz EA (1978) Non-specific serum iron in thalassaemia: an abnormal serum iron fraction of potential toxicity. Br J Haematol 40:255–263PubMedCrossRefGoogle Scholar
  32. 32.
    Breuer W, Ronson A, Slotki IN, Abramov A, Hershko C, Cabantchik ZI (2000) The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation. Blood 95:2975–2982PubMedGoogle Scholar
  33. 33.
    Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford A, Sadler PJ (1989) Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Biol Chem 264:4417–4422PubMedGoogle Scholar
  34. 34.
    Hentze MW, Muckenthaler MU, Andrews NC (2004) Balancing acts: molecular control of mammalian iron metabolism. Cell 117:285–297PubMedCrossRefGoogle Scholar
  35. 35.
    Galaris D, Pantopoulos K (2008) Oxidative stress and iron homeostasis: mechanistic and health aspects. Crit Rev Clin Lab Sci 45:1–23PubMedCrossRefGoogle Scholar
  36. 36.
    Avery SV Molecular targets of oxidative stress. Biochem J 434:201–10Google Scholar
  37. 37.
    Katoh Y, Katoh M (2006) Comparative genomics on HHIP family orthologs. Int J Mol Med 17:391–395PubMedGoogle Scholar
  38. 38.
    Chuang PT, McMahon AP (1999) Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397:617–621PubMedCrossRefGoogle Scholar
  39. 39.
    Bishop B, Aricescu AR, Harlos K, O’Callaghan CA, Jones EY, Siebold C (2009) Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat Struct Mol Biol 16:698–703PubMedCrossRefGoogle Scholar
  40. 40.
    Spinella-Jaegle S, Rawadi G, Kawai S, Gallea S, Faucheu C, Mollat P, Courtois B, Bergaud B, Ramez V, Blanchet AM, Adelmant G, Baron R, Roman-Roman S (2001) Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation. J Cell Sci 114:2085–2094PubMedGoogle Scholar
  41. 41.
    van der Horst G, Farih-Sips H, Lowik CW, Karperien M (2003) Hedgehog stimulates only osteoblastic differentiation of undifferentiated KS483 cells. Bone 33:899–910PubMedCrossRefGoogle Scholar
  42. 42.
    Shimoyama A, Wada M, Ikeda F, Hata K, Matsubara T, Nifuji A, Noda M, Amano K, Yamaguchi A, Nishimura R, Yoneda T (2007) Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function. Mol Biol Cell 18:2411–2418PubMedCrossRefGoogle Scholar
  43. 43.
    Mak KK, Bi Y, Wan C, Chuang PT, Clemens T, Young M, Yang Y (2008) Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression. Dev Cell 14:674–688PubMedCrossRefGoogle Scholar
  44. 44.
    Plaisant M, Fontaine C, Cousin W, Rochet N, Dani C, Peraldi P (2009) Activation of hedgehog signaling inhibits osteoblast differentiation of human mesenchymal stem cells. Stem Cells 27:703–713PubMedCrossRefGoogle Scholar
  45. 45.
    Oliveira F, Bellesini L, Defino H, da Silva HC, Beloti M, Rosa A (2011) Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells. J Cell Biochem. doi:10.1002/jcb.23345

Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2012

Authors and Affiliations

  • M. Doyard
    • 1
    • 2
  • N. Fatih
    • 1
    • 2
  • A. Monnier
    • 3
  • M. L. Island
    • 1
    • 2
  • M. Aubry
    • 4
  • P. Leroyer
    • 1
    • 2
  • R. Bouvet
    • 4
    • 5
  • G. Chalès
    • 1
    • 2
    • 6
  • J. Mosser
    • 3
    • 4
    • 5
  • O. Loréal
    • 1
    • 2
    • 7
  • P. Guggenbuhl
    • 1
    • 2
    • 6
    • 8
  1. 1.INSERM, UMRU991Rennes CedexFrance
  2. 2.Université de Rennes 1RennesFrance
  3. 3.CNRS UMR6061, Institut de Génétique et Développement, Université de RennesRennesFrance
  4. 4.Plateforme Génomique Santé Biogenouest®RennesFrance
  5. 5.Service de Génétique Moléculaire et GénomiqueRennesFrance
  6. 6.Service de Rhumatologie, Hôpital SudRennesFrance
  7. 7.Service des Maladies du Foie, Hôpital PontchaillouRennesFrance
  8. 8.Service de Rhumatologie, Hôpital SudRennesFrance

Personalised recommendations