Nutr Res Pract.  2009 Sep;3(3):192-199.

Cadmium increases ferroportin-1 gene expression in J774 macrophage cells via the production of reactive oxygen species

• Park BY
• Chung J

Abstract

Cadmium intoxication has been associated with the dysregulation of iron homeostasis. In the present study, we investigated the effect of cadmium on the expression of ferroportin 1 (FPN1), an important iron transporter protein that is involved in iron release from macrophages. When we incubated cadmium with J774 mouse macrophage cells, FPN1 mRNA levels were significantly increased in a dose- and time-dependent manner. Furthermore, the cadmium-induced FPN1 mRNA expression was associated with increased levels of FPN1 protein. On the other hand, cadmium-mediated FPN1 mRNA induction in J774 cells was completely blocked when cells were co-treated with a transcription inhibitor, acitomycin D. Also, cadmium directly stimulated the activity of the FPN1-promoter driven luciferase reporter, suggesting that the cadmium up-regulates FPN1 gene expression in a transcription-dependent manner. Finally, cadmium exposure to J774 macrophages increased intracellular reactive oxygen species (ROS) levels by ~ 2-fold, compared to untreated controls. When J774 cells were co-treated with antioxidant N-acetylcystein, the cadmium-induced FPN1 mRNA induction was significantly attenuated. In summary, the results of this study clearly demonstrated that cadmium increased FPN1 expression in macrophages through a mechanism that involves ROS production, and suggests another important interaction between iron and cadmium metabolism.

Keyword

Iron; cadmium; ferroportin 1; macrophages

MeSH Terms

Animals
Cadmium
Cation Transport Proteins
Gene Expression
Hand
Homeostasis
Iron
Luciferases
Macrophages
Mice
Reactive Oxygen Species
RNA, Messenger
Cadmium
Cation Transport Proteins
Iron
Luciferases
RNA, Messenger
Reactive Oxygen Species

Figure

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  Fig. 1 Effects of cadmium treatment on the viability of J774 macrophage cells. J774 cells were treated with CdCl2 with indicated concentrations and time. The cell viability was measured by MTT assay as described in Methods and Materials. Data shown are mean ± SD (n=6). *P < 0.05 vs. untreated controls

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  Fig. 2 Effects of cadmium treatment with various concentrations on FPN1 mRNA levels in J774 cells. J774 cells were incubated with the indicated concentrations of CdCl2 for 8 h. Total RNA were prepared and analyzed for FPN1 or β-actin mRNA by RT-PCR analyses. Data shown are representative agarose gels (upper panel) or means ± SD of three independent experiments (lower panel). *P < 0.05 vs. untreated controls

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  Fig. 3 Effects of cadmium treatment with various incubation time on FPN1 mRNA levels in J774 cells. J774 cells were incubated with 25 µM CdCl2 for the indicated time. Total RNA were prepared and analyzed for FPN1 or β-actin mRNA by RT-PCR analyses. Data shown are representative agarose gels (upper panel) or means ± SD of three independent experiments (lower panel). *P < 0.05 vs. untreated controls

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  Fig. 4 Effects of cadmium treatment on FPN1 protein levels in J774 cells. J774 cells were incubated with 0, 5, 10, or 25 µM CdSO4 for 8 h. Whole cell lysates were prepared and analyzed for FPN1 protein by Western blot. Upper panel: representative blot, Lower panel: band density detected by chemiluminescence was quantified (QuantityOne imaging software, Bio-Rad). Values are mean ± SD of three independent experiments. *P < 0.05 vs. untreated controls

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  Fig. 5 Effects of cadmium and actinomycin D on FPN1 mRNA levels in J774 cells. J774 cells were treated with 25 µM CdCl2 for 4 h in the presence or absence of 0.5 µg/ml actinomycin D. Total RNAs were prepared and analyzed for FPN1 or β-actin mRNA by RT-PCR. Upper panel: representative agarose gels, Lower panel: band intensity detected by ethidium bromide-staining was quantified (QuantityOne imaging software, Bio-Rad). Values are mean ± SD of three independent experiments. *P < 0.05 vs. untreated controls

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  Fig. 6 Effects of cadmium treatment on the luciferase activity driven by FPN1-promoter. HeLa cells were transiently transfected with FPN1 promoter/luciferase reporter constructs for 12 h. Cells were treated with 0, 5, 10, or 25 µM CdSO4 for another 8 h. Luciferase activity in cell lysates was then determined. Values are presented as mean ± SD of three independent experiments. *P < 0.05 vs. untreated controls

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  Fig. 7 Effects of cadmium treatment in J774 cells on the reactive oxygen species (ROS) generation. J774 cells were treated with 25 µM cadmium and intracellular ROS generation was detected by using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Values are presented as mean ± SD of three independent experiments. *P < 0.05 vs. untreated controls

• 

  Fig. 8 Effects of cadmium and N-acetylcystein (NAC) treatment on FPN1 mRNA levels in J774 cells. J774 cells were pretreated in the presence or absence of 1 mM NAC for 30 min, and then further treated with or without 25 µM CdCl2. Total RNA were prepared and analyzed for FPN1 or β-actin mRNA by RT-PCR analyses. Data shown are representative agarose gels (A) or means ± SD of three independent experiments (B). Intracellular GSH/GSSG ratio was measured as described in Methods and Materials (C). *P < 0.05 vs. untreated controls

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