Elsevier

Toxicology Letters

Volume 191, Issues 2–3, 15 December 2009, Pages 211-215
Toxicology Letters

Excess iron inhibits osteoblast metabolism

https://doi.org/10.1016/j.toxlet.2009.08.023Get rights and content

Abstract

Hemochromatosis is an iron overload disorder associated with osteopenia and osteoporosis. To learn more about the effects of iron on bone cells, we examined the effects of ferric ion on the proliferation, differentiation, and mineralization of two types of cultured osteoblasts, the cell line MC3T3-E1 and rat calvarial osteoblast-like (ROB) cells. We used ferric ammonium citrate (FAC) as a donor of ferric ion, and FAC inhibited the proliferation of MC3T3-E1 cells in a dose-dependent manner. FAC (0.1–1 μg/ml) inhibited indices of osteoblast differentiation, such as the expression of type I collagen (mRNA and protein), the activity of alkaline phosphatase, and the deposition of calcium by osteoblasts. These results suggest that iron overload might give rise to osteoporosis by inhibiting osteoblast proliferation and differentiation.

Introduction

Osteoblasts are bone-forming cells derived from undifferentiated, pluripotent mesenchymal cells. Bone formation involves a complex series of events, including the proliferation and differentiation of osteoprogenitor cells, and eventually results in the formation of a mineralized extracellular matrix. The sequential expression of type I collagen, alkaline phosphatase (ALPase), and osteocalcin and the deposition of calcium are markers of osteoblastic differentiation. Numerous cytokines, hormones, and growth factors control the formation of bone by regulating both the proliferation and the differentiation of osteoblasts. Bone metabolic diseases develop when there is an imbalance between the formation and resorption of bone, which depend on the interactions between osteoblasts and osteoclasts (Riggs, 1987). Several model systems have been developed for in vitro studies of the molecular biology of the proliferation and differentiation of osteoclasts (Hagiwara et al., 2008) and osteoblasts (Stein et al., 1990, Bredford et al., 1993, Liu et al., 1994). Here, we used a mouse clonal cell line MC3T3-E1 and rat calvarial osteoblast-like cells (ROB cells) that undergoes efficient osteoblastic differentiation through a cellular condensation and calcification stages (Naruse et al., 2002, Inoue et al., 2002, Hagiwara et al., 1996).

The total iron pool of the body is composed of the iron in the red cell mass (2.5 g), tissue iron (1 g), and a small amount of iron circulating in the plasma (4 mg). The iron stores in tissues (predominantly the liver, spleen, and bone marrow) are largely in the form of ferritin, a complex of ferric ion and the apoferritin protein; ferritin is a large multisubunit protein with eight Fe transport pores (Munro, 1990). Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide (Killip et al., 2007, Arcasoy et al., 1978). In contrast, when the body absorbs too much iron, hemochromatosis occurs (Edison et al., 2008); this disease is caused by the gradual build up of extra iron in tissues and organs. The most susceptible organs include the liver, adrenal glands, heart, and pancreas; patients can present with cirrhosis, adrenal insufficiency, heart failure, or diabetes. Osteoporosis has also been reported in patients with hemochromatosis (Weinberg, 2008, Kudo et al., 2008). However, the effects of excess iron on bone cell metabolism, especially osteoblastic metabolism, are not fully understood. Therefore, the objective of the present study is to elucidate the effects of the iron ion on bone metabolism, especially osteoblastic proliferation, differentiation, and calcification.

Section snippets

Materials

Ferric ammonium citrate (FAC) and desferrioxamine (DFO) were purchased from Nacalai tesque Inc. (Kyoto, Japan) and Sigma Chemical Co. (St Louis, MO), respectively. α-Minimum essential medium (α-MEM) and penicillin/streptomycin antibiotic mixture were obtained from Life Technologies, Inc. (Grand Island, NY, USA). Fetal bovine serum was from Moregate BioTech (Bulimba, Australia).

Culture of osteoblastic cells

Preosteoblastic MC3T3-E1 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). Cell were maintained in 55-cm2

Results

We evaluated the viability of MC3T3-E1 cell treated with FAC using the MTT assay. The exposure of MC3T3-E1 cells to 1 μg/ml FAC decreased the viability of cells by approximately 40% in 74 h, relative to control cultures that were treated with vehicle alone. The effects of FAC were dose-dependent (Fig. 1). Exposure of MC3T3-E1 cells to 1 μg/ml FAC did not affect cell morphology. To assess the effect of iron ion on differentiation and mineralization of MC3T3-E1 cells, we added FAC to the culture

Discussion

Hemochromatosis is an iron overload disorder and a known cause of osteoporosis (Weinberg, 2008). The aim of this study was to identify the effects of ferric ion on the metabolism of osteoblastic cells. We found that ferric ion inhibited not only cell proliferation but also the differentiation and mineralization of osteoblastic cells. The concentrations (0.1–1 μg/ml) of FAC that we used in the present study were lower than in the effective range of those (1.4 μg/ml) used in studies on the survival

Conflict of interest

None declared.

Acknowledgments

We would like to thank Mrs. K. Nakata for assistance with cell culture. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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