title: Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells.
abstract: 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.
  
  
  BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: INSERM, UMRU991, CHU Pontchaillou, 35033 Rennes Cedex, France.
authors.forename: M
authors.initials: M
authors.lastname: Doyard
authors.forename: N
authors.initials: N
authors.lastname: Fatih
authors.forename: A
authors.initials: A
authors.lastname: Monnier
authors.forename: M L
authors.initials: ML
authors.lastname: Island
authors.forename: M
authors.initials: M
authors.lastname: Aubry
authors.forename: P
authors.initials: P
authors.lastname: Leroyer
authors.forename: R
authors.initials: R
authors.lastname: Bouvet
authors.forename: G
authors.initials: G
authors.lastname: Chalès
authors.forename: J
authors.initials: J
authors.lastname: Mosser
authors.forename: O
authors.initials: O
authors.lastname: Loréal
authors.forename: P
authors.initials: P
authors.lastname: Guggenbuhl
mesh_terms: D027682:Cation Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D019343:Citric Acid; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019190:Iron Overload / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010641:Phenotype; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D000098742:Ferroportin
chemical_list: D027682:Cation Transport Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; D000098742:Ferroportin; D019343:Citric Acid; C016600:ferrous citrate; C013531:ferric ammonium citrate