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

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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.

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Acknowledgements

The authors would like to thank Pr. Josiane Cillard for helpful discussion. This work was supported by grants from the Région Bretagne (MD) and the “Société Française de Rhumatologie” Paris, France.

Conflicts of interest

None.

Author information

Authors and Affiliations

  1. INSERM, UMRU991, CHU Pontchaillou, 35033, Rennes Cedex, France

    M. Doyard, N. Fatih, M. L. Island, P. Leroyer, G. Chalès, O. Loréal & P. Guggenbuhl

  2. Université de Rennes 1, IFR 140, 35043, Rennes, France

    M. Doyard, N. Fatih, M. L. Island, P. Leroyer, G. Chalès, O. Loréal & P. Guggenbuhl

  3. CNRS UMR6061, Institut de Génétique et Développement, Université de Rennes, UEB, IFR140, 35043, Rennes, France

    A. Monnier & J. Mosser

  4. Plateforme Génomique Santé Biogenouest®, 35043, Rennes, France

    M. Aubry, R. Bouvet & J. Mosser

  5. Service de Génétique Moléculaire et Génomique, CHU, 35033, Rennes, France

    R. Bouvet & J. Mosser

  6. Service de Rhumatologie, Hôpital Sud, CHU, 35033, Rennes, France

    G. Chalès & P. Guggenbuhl

  7. Service des Maladies du Foie, Hôpital Pontchaillou, CHU, 35033, Rennes, France

    O. Loréal

  8. Service de Rhumatologie, Hôpital Sud, CHU, 35000, Rennes, France

    P. Guggenbuhl

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  1. M. Doyard
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  2. N. Fatih
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  10. O. Loréal
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Correspondence to O. Loréal or P. Guggenbuhl.

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Fig. S1

Iron 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)

Fig. S2

Impact 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)

Fig. S3

Inhibition 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)

Fig. S4

The 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)

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Doyard, M., Fatih, N., Monnier, A. et al. Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells. Osteoporos Int 23, 2435–2445 (2012). https://doi.org/10.1007/s00198-011-1871-z

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  • DOI: https://doi.org/10.1007/s00198-011-1871-z

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