Korean J Nutr.  2009 Jul;42(5):434-441. 10.4163/kjn.2009.42.5.434.

Effect of Copper on the Regulation of Ferroportin-1 Gene Expression

• Park BY
• Chung J

• KMID: 2267325
• DOI: http://doi.org/10.4163/kjn.2009.42.5.434

Abstract

Ferroportin-1 (FPN) is a transporter protein that is known to mediate iron export from macrophages. The purpose of this study was to investigate the effect of copper on the regulation of FPN gene expression in J774 mouse macrophage cells. J774 cells were treated with various concentrations of CuSO4 and RT-PCR analyses were performed to measure the steady-state levels of mRNAs for FPN and divalent metal transporter 1 (DMT1, an iron importer). Copper treatment significantly increased FPN mRNAs in a dose-dependent manner, but didn't change the levels of DMT1 mRNA. Experiments with transcriptional inhibitor actinomycin D (0.5 microgram/mL) revealed that copper treatment did not affect the half-life of FPN mRNAs in J774 cells. On the other hand, results from luciferase reporter assays showed that copper directly stimulated the promoter activity of FPN. In summary, our data showed copper induced FPN mRNA of macrophages via a transcriptional rather than post-transcriptional mechanisms.

Keyword

ferroportin-1; copper; macrophages

MeSH Terms

Animals
Copper
Dactinomycin
Gene Expression
Half-Life
Hand
Iron
Luciferases
Macrophages
Mice
RNA, Messenger
Copper
Dactinomycin
Iron
Luciferases
RNA, Messenger

Figure

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  Fig. 1 Diagrams of two reporter DNA constructs used in the present study.

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  Fig. 2 Effect of copper on the levels of mRNA for ferroportin (FPN) and for divalent metal transporter 1 (DMT1) in J774 macrophage cells. RT-PCR analyses were performed to determine mRNA for FPN, DMT1, and β-actin (upper panel). Levels of FPN and DMT1 mRNA were normalized to β-actin mRNA levels (lower panel).

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  Fig. 3 Effects of varying doses of copper on the viability of J774 macrophage cells. Cell viability were measured by MTT assays. Results are the mean ± s.d. of three independent experiments.

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  Fig. 4 Effects of copper on FPN mRNA stability in J774 macrophage cells. J774 cells were pretreated with 200 µM CuSO4 for 20 hr and throughly washed. Then actinomycin D (0.5 µg/mL) was added with CuSO4 (Cu+) or with medium alone (Cu-). Total RNA was isolated at 0, 2, 6, 9 hours after actinomycin D treatment, and RT-PCR analyses were performed (upper panel). The rate of FPN mRNA decay of untreated and copper-treated cells was determined after normalization with β-actin mRNA. Results are the mean ± s.d. of three independent experiments.

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  Fig. 5 Effects of copper on FPN promoter/5'-UTR driven luciferase reporter activity. HeLa cells were transiently transfected with A: FPN-Luc, B: FPNΔIRE-Luc, or C: pGL-control (empty) vector. Twelve hours later, cells were exposed to various agents for 20 h, and cell lysates were assayed for luciferase activity. Luciferase activities were normalized to protein concentraion of cell lysates and expressed as fold changes compared to untreated controls. 1, untreated; 2, copper; 3, iron; 4. iron chelator. Results are the mean ± s.d. of three independent experiments. *: p < 0.05 vs. untreated controls.

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