title: Iron overload induced death of osteoblasts in vitro: involvement of the mitochondrial apoptotic pathway.
abstract: BACKGROUND
  Iron overload is recognized as a new pathogenfor osteoporosis. Various studies demonstrated that iron overload could induce apoptosis in osteoblasts and osteoporosis in vivo. However, the exact molecular mechanisms involved in the iron overload-mediated induction of apoptosis in osteoblasts has not been explored.
  
  
  OBJECTIVE
  In this study, we attempted to determine whether the mitochondrial apoptotic pathway is involved in iron-induced osteoblastic cell death and to investigate the beneficial effect of N-acetyl-cysteine (NAC) in iron-induced cytotoxicity.
  
  
  METHODS
  The MC3T3-E1 osteoblastic cell line was treated with various concentrations of ferric ion in the absence or presence of NAC, and intracellular iron, cell viability, reactive oxygen species, functionand morphology changes of mitochondria and mitochondrial apoptosis related key indicators were detected by commercial kits. In addition, to further explain potential mechanisms underlying iron overload-related osteoporosis, we also assessed cell viability, apoptosis, and osteogenic differentiation potential in bone marrow-derived mesenchymal stemcells(MSCs) by commercial kits.
  
  
  RESULTS
  Ferric ion demonstrated concentration-dependent cytotoxic effects on osteoblasts. After incubation with iron, an elevation of intracelluar labile iron levels and a concomitant over-generation of reactive oxygen species (ROS) were detected by flow cytometry in osteoblasts. Nox4 (NADPH oxidase 4), an important ROS producer, was also evaluated by western blot. Apoptosis, which was evaluated by Annexin V/propidium iodide staining, Hoechst 33258 staining, and the activation of caspase-3, was detected after exposure to iron. Iron contributed to the permeabilizatio of mitochondria, leading to the release of cytochrome C (cyto C), which, in turn, induced mitochondrial apoptosis in osteoblasts via activation of Caspase-3, up-regulation of Bax, and down-regulation of Bcl-2. NAC could reverse iron-mediated mitochondrial dysfunction and blocked the apoptotic events through inhibit the generation of ROS. In addition, iron could significantly promote apoptosis and suppress osteogenic differentiation and mineralization in bone marrow-derived MSCs.
  
  
  CONCLUSIONS
  These findings firstly demonstrate that the mitochondrial apoptotic pathway involved in iron-induced osteoblast apoptosis. NAC could relieved the oxidative stress and shielded osteoblasts from apoptosis casused by iron-overload. We also reveal that iron overload in bone marrow-derived MSCs results in increased apoptosis and the impairment of osteogenesis and mineralization.
authors.affiliation: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tian
authors.affiliation: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Shilei
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Orthopedics, Henan Provincial People's Hospital, Zhengzhou, China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Dai
authors.affiliation: Department of Child Health, Changzhou Maternal and Child Health Care Hospital, Changzhou, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Qixin
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
keywords: Apoptosis; Iron overload; Mitochondria; Osteoporosis; Oxidative stress