Effect of hepcidin on intracellular calcium in human osteoblasts.

Hepcidin is known to increase intracellular iron through binding to and degrading ferroportin, which is a transmembrane protein that transports iron from the intracellular to the outside. However, it is not clear whether hepcidin has a similar effect on intracellular calcium. Here, we investigated the influence of hepcidin on intracellular calcium in human osteoblasts, with or without high environmental iron concentrations. Our data showed that hepcidin (<100 nmol/L) could increase intracellular calcium, and this effect was more significant when cells were exposed to high environmental iron concentrations. To further explore its underlying mechanisms, we pretreated human osteoblasts with Nimodipine, a L-type calcium channel blocker, and Dantrolene, a ryanodine receptor antagonist to inhibit abnormal calcium release from the sarco-endoplasmic reticulum. These treatments had not resulted in any alteration of intracellular calcium in human osteoblasts. Thus, these findings indicate that the increase of intracellular calcium induced by hepcidin is probably due to calcium release from endoplasmic reticulum, which is triggered by calcium influx.

[The effect of ferric ion on the differentiation of osteoclast and bone resorption].

OBJECTIVE: To explore the effects of ferric ion on the differentiation from both RAW264.7 and bone marrow macrophages to osteoclast in vitro and bone resorption in vivo. METHODS: In the presence of 50 ng/ml receptor activator of nuclear factor kappa-B ligand (RANKL), RAW264.7 was treated with ferric ammonium citrate (FAC). The formation of osteoclast was observed by staining of tartrate-resistant acid phosphatase (TRAP) and the TRAP positive cell counted. The expression levels of TRAP, cathepsin-K, nuclear factor of activated T cells c1 (NFATc1) and (receptor activator of NF-κB) RANK were measured by reverse transcription-polymerase chain reaction (RT-PCR).The control and iron overload groups were established by the intraperitoneal injection of normal saline and FAC. In vivo imaging system was employed to determine the bone density of femoral midportion and the fourth lumbar vertebra. After that, the bone marrow cells of femurs were used for osteoclast culture. The serum levels of ferritin, TRAP-5b, RANKL, osteoprotegerin (OPG) and C-terminal cross-linking telopeptide (CTX) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Ferric ion could stimulate the formation of TRAP positive cells in a dose-dependent manner. The expression levels of TRAP, cathepsin-K and NFATc1 in the FAC treated group were significantly higher those of the control group (P < 0.05) while the expression of RANK showed no statistical difference among these groups (P = 0.967). The bone marrow density of femoral midportion and the fourth lumbar vertebra of the iron-overload group decreased significantly versus the control group. The concentrations of ferritin, TRAP-5b, RANKL and CTX of the iron overload group were markedly higher than those of the control group (P < 0.05). Moreover, the concentration of ferritin showed a positive correlation with TRAP-5b and CTX respectively in the iron-overload group (r = 0.65, r = 0.76, P < 0.05). But no significant differences existed in the concentration of OPG for two groups (P > 0.05). CONCLUSION: Ferric ion may enhance the differentiation of osteoclast in vitro as well as bone resorption in vivo.

Iron overload inhibits osteoblast biological activity through oxidative stress.

Iron overload has recently been connected with bone mineral density in osteoporosis. However, to date, the effect of iron overload on osteoblasts remains poorly understood. The purpose of this study is to examine osteoblast biological activity under iron overload. The osteoblast cells (hFOB1.19) were cultured in a medium supplemented with different concentrations (50, 100, and 200 µM) of ferric ammonium citrate as a donor of ferric ion. Intracellular iron was measured with a confocal laser scanning microscope. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescin diacetate fluorophotometry. Osteoblast biological activities were evaluated by measuring the activity of alkaline phosphatase (ALP) and mineralization function. Results indicated that iron overload could consequently increase intracellular iron concentration and intracellular ROS levels in a concentration-dependent manner. Additionally, ALP activity was suppressed, and a decline in the number of mineralized nodules was observed in in vitro cultured osteoblast cells. According to these results, it seems that iron overload probably inhibits osteoblast function through higher oxidative stress following increased intracellular iron concentrations.

A comparison of the biological activities of human osteoblast hFOB1.19 between iron excess and iron deficiency.

Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0-200 µmol/L ferric ammonium citrate and 0-20 µmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.