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Ferroportin Is a Manganese-Responsive Protein That Decreases Manganese Cytotoxicity and Accumulation

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Ferroportin Is a Manganese-Responsive Protein That Decreases Manganese Cytotoxicity and Accumulation

Zhaobao Yin et al. J Neurochem.

Abstract

Although manganese (Mn) is an essential trace element for human development and growth, chronic exposure to excessive Mn levels can result in psychiatric and motor disturbances, referred to as manganism. However, there are no known mechanism(s) for efflux of excess Mn from mammalian cells. Here, we test the hypothesis that the cytoplasmic iron (Fe) exporter ferroportin (Fpn) may also function as a Mn exporter to attenuate Mn toxicity. Using an inducible human embryonic kidney (HEK293T) cell model, we examined the influence of Fpn expression on Mn-induced cytotoxicity and intracellular Mn concentrations. We found that induction of an Fpn-green fluorescent protein fusion protein in HEK293T cells was cytoprotective against several measures of Mn toxicity, including Mn-induced cell membrane leakage and Mn-induced reductions in glutamate uptake. Fpn-green fluorescent protein mediated cytoprotection correlated with decreased Mn accumulation following Mn exposure. Thus, Fpn expression reduces Mn toxicity concomitant with reduced Mn accumulation. To determine if mammalian cells may utilize Fpn in response to increased intracellular Mn concentrations and toxicity, we assessed endogenous Fpn levels in Mn-exposed HEK293T cells and in mouse brain in vivo. We find that 6 h of Mn exposure in HEK293T cells is associated with a significant increase in Fpn levels. Furthermore, mice exposed to Mn showed an increase in Fpn levels in both the cerebellum and cortex. Collectively, these results indicate that (i) Mn exposure promotes Fpn protein expression, (ii) Fpn expression reduces net Mn accumulation, and (iii) reduces cytotoxicity associated with exposure to this metal.

Figures

Figure 1
Figure 1. Fpn protein overexpressed in ponasterone A induced HEK293T cells
HEK293T cells were maintained in DMEM with the supplement of 10% FBS. FPN expression was undetectable in WT HEK293T cells and ponasterone A uninduced HEK293T-Fpn-GFP cells. With ponasterone A, HEK293T-Fpn-GFP cells expressed high level of Fpn protein, detected by anti-FPN (A) and anti-GFP (B), as well as visible under fluorescent microscope (C).
Figure 1
Figure 1. Fpn protein overexpressed in ponasterone A induced HEK293T cells
HEK293T cells were maintained in DMEM with the supplement of 10% FBS. FPN expression was undetectable in WT HEK293T cells and ponasterone A uninduced HEK293T-Fpn-GFP cells. With ponasterone A, HEK293T-Fpn-GFP cells expressed high level of Fpn protein, detected by anti-FPN (A) and anti-GFP (B), as well as visible under fluorescent microscope (C).
Figure 1
Figure 1. Fpn protein overexpressed in ponasterone A induced HEK293T cells
HEK293T cells were maintained in DMEM with the supplement of 10% FBS. FPN expression was undetectable in WT HEK293T cells and ponasterone A uninduced HEK293T-Fpn-GFP cells. With ponasterone A, HEK293T-Fpn-GFP cells expressed high level of Fpn protein, detected by anti-FPN (A) and anti-GFP (B), as well as visible under fluorescent microscope (C).
Figure 2
Figure 2. Fpn protein expression inversely associated with Mn toxicity to HEK cells
Six hour exposure of Mn (100, 200, or 500 µM) induced LDH leaking from cell membrane to media in a concentration-dependent manner in WT HEK293T control cells and ponasterone A uninduced HEK293T-Fpn-GFP cells; a significant increase in LDH release occurred only at 500 µM Mn treatment in ponasterone A induced HEK293T-Fpn-GFP cells. The extent of LDH leakage was significantly lower (p<0.05 or 0.001) in ponasterone A induced HEK293T-Fpn-GFP cells than in WT HEK293T and in uninduced HEK293T-Fpn-GFP cells. Data were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test with statistical significance set at p < 0.05. Values are mean ± SEM derived from three independent experiments conducted in triplicates. * p<0.05, ** p<0.01, *** p<0.001 compared to control; Δ p<0.05, ΔΔΔ p<0.001 compared between treatments at the same concentration in the different groups.
Figure 3
Figure 3. Fpn protein expression enhanced glutamate uptake in Mn treated HEK cells
Six hour of Mn exposure (100, 200, or 500 µM) induced decrease of glutamate uptake in a concentration-dependent manner in WT HEK293T and ponasterone A uninduced HEK293T cells; increased Fpn protein expression in ponasterone A induced HEK293T-Fpn-GFP cells was associated with a reversal of the Mn-induced (100 and 250 µΜ) decrease in glutamate uptake. High level (500 µM) of Mn on glutamate uptake remained significantly decreased in induced HEK293T-Fpn-GFP cells compared with WT cells. Glutamate uptake in ponasterone A induced HEK293T-Fpn-GFP cells was significantly higher vs. WT HEK293T and ponasterone A uninduced HEK293T-Fpn-GFP cells at the same Mn treatments. Values are mean ± SEM derived from three independent experiments with three or more samples per experiment. * p<0.05, *** p<0.001 compared to control; Δ p<0.05, ΔΔΔ p<0.001 compared between treatments at the same concentration in the different groups.
Figure 4
Figure 4. Fpn protein expression reduced intracellular Mn concentration
Mn exposure resulted in increase of intracellular Mn level in a concentration-dependent manner in all three WT HEK293T, ponasterone A uninduced and induced HEK293T-Fpn-GFP cells. However, intracellular Mn concentrations were significantly lower in ponasterone A induced HEK293T-Fpn-GFP cells vs. ponasterone A uninduced HEK293T-Fpn-GFP or WT HEK293T cells at the same concentration of Mn treatments. Values are mean ± SEM derived from three independent experiments with three or more samples in each experiment. ** p<0.01, *** p<0.001 compared to control; Δ p<0.05, ΔΔΔ p<0.001 compared between the same concentration treatment in the different groups.
Figure 5
Figure 5. Mn increased Fpn protein expression in HEK293T cells
Mn treatment (500 µM) for 6 hour resulted in significant increase of Fpn protein expression in WT HEK293T cells. Values are mean ± SEM derived from three independent experiments each carried out in triplicates. * p<0.05 compared to non-Mn treated WT HEK293T cells
Figure 6
Figure 6. Mn injection increased Fpn expression in mice brains
Mn injection (100 mg/kg body weight) 1 dose or three doses increased Fpn expression significantly in mice brain cortices (A) or cerebella (B). Values are mean ± SEM of 4–6 animals in each group. * p<0.05, ** p<0.01 compared to control.
Figure 6
Figure 6. Mn injection increased Fpn expression in mice brains
Mn injection (100 mg/kg body weight) 1 dose or three doses increased Fpn expression significantly in mice brain cortices (A) or cerebella (B). Values are mean ± SEM of 4–6 animals in each group. * p<0.05, ** p<0.01 compared to control.

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