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. 2009 Nov 12;114(20):4546-51.
doi: 10.1182/blood-2009-05-224188. Epub 2009 Aug 11.

"V体育安卓版" Specific iron chelators determine the route of ferritin degradation

Ivana De Domenico  1 , Diane McVey Ward, Jerry Kaplan
Affiliations

Specific iron chelators determine the route of ferritin degradation

Ivana De Domenico (V体育官网入口) et al. Blood. .

Abstract

Deferoxamine (DFO) is a high-affinity Fe (III) chelator produced by Streptomyces pilosus. DFO is used clinically to remove iron from patients with iron overload disorders. Orally administered DFO cannot be absorbed, and therefore it must be injected. Here we show that DFO induces ferritin degradation in lysosomes through induction of autophagy. DFO-treated cells show cytosolic accumulation of LC3B, a critical protein involved in autophagosomal-lysosomal degradation. Treatment of cells with the oral iron chelators deferriprone and desferasirox did not show accumulation of LC3B, and degradation of ferritin occurred through the proteasome VSports手机版. Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, resulted in degradation of ferritin by the proteasome. These results indicate that ferritin degradation occurs by 2 routes: a DFO-induced entry of ferritin into lysosomes and a cytosolic route in which iron is extracted from ferritin before degradation by the proteasome. .

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Figures

Figure 1
Figure 1
DFO-induced ferritin loss is prevented by chloroquine treatment. HEK293T-Fpn cells were incubated with FAC (10μM Fe) for 24 hours followed by incubation in the absence or presence of 10μM ponasterone A, 100μM DFO, desferasirox, or deferriprone. Chloroquine was added at the final concentration of 100μM. After 10 hours, cells were harvested. The ferritin content was determined by ELISA. Error bars represent SD from 3 different experiments.
Figure 2
Figure 2
DFO induces autophagy. (A) HeLa cells transfected with or without pCMV-Fpn-FLAG were incubated with FAC (10μM Fe) for 24 hours followed by incubation for 10 hours in the absence or presence of 10μM ponasterone A, 100μM DFO, desferasirox, or deferriprone. Cells were incubated with or without chloroquine for 5 hours. Cells were fixed and processed for immunofluorescence using mouse anti-LC3 antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The number of LC3B-positive cells was quantified (right panel) and error bars represent the analysis of greater than 5 fields per sample (at least 100 cells). Error bars represent SD from 3 different experiments. (B) HeLa cells expressing Fpn-FLAG were incubated with or without FAC (10μM) for 18 hours in the presence or absence of DFO at different concentrations (0.05, 0.1, 1, 10, 100μM). Cells were fixed and processed for immunofluorescence as in panel A. (C) HeLa cells were incubated with or without FAC (10μM) for 18 hours in the presence or absence of DFO (100μM) or DFO previously saturated with iron. Cells were incubated with or without chloroquine for 5 hours. Cells were fixed, processed for immunofluorescence, and quantified as in panel A.
Figure 3
Figure 3
DFO in lysosomes induces autophagy. (A) HeLa cells were incubated with or without FAC (10μM) for 18 hours followed by incubation with or without DFO (100μM) or dextran-DFO for 16 hours. Cells were harvested for measurement of ferritin by ELISA or fixed and processed for immunofluorescence for LC3B as described in the Figure 2 legend. The amount of ferritin in FAC-loaded cells was taken as 100%. Error bars represent SD from 3 different experiments. (B) HeLa cells were transfected with or without dynamin K44A and incubated in the presence or absence of FAC (10μM) for 18 hours. DFO and chloroquine were added to a final concentration of 100μM for 6 hours. Cells were fixed and processed for immunofluorescence as in panel A. Percentage of LC3B accumulation was determined by analyzing 100 cells. Error bars represent SD from 3 different experiments.
Figure 4
Figure 4
DFO does not induce pH changes in the lysosome. HeLa cells were incubated with fluorescein dextran for 6 hours and then incubated in the absence of dextran for 2 hours. Cells were then incubated with or without chloroquine, DFO, DFO saturated with iron (DFO-Fe), or DFO-dextran. Images were acquired on an epifluorescence microscope. The data are representative images from each condition found in 3 separate experiments.
Figure 5
Figure 5
Inhibition of DFO-induced autophagy results in ferritin degradation in the proteasome. HEK293T-Fpn cells were incubated with FAC (10μM) for 24 hours followed by incubation in the presence or absence of 10μM ponasterone A (PoA) for 12 hours. Cells were incubated with or without 3-methyladenine for 8 hours in the presence or absence of 100μM chloroquine or 10μM MG132. FAC-loaded cells were also incubated with DFO, desferasirox, or deferriprone at the final concentration 100μM with or without 3-methyladenine for 8 hours in the presence or absence of 100μM chloroquine or 10μM MG132. Cells were harvested and ferritin content was determined by ELISA. Error bars represent SD from 4 different experiments.
Figure 6
Figure 6
Ferritin degradation is mTOR and LAMP 1-2 independent. (A) Wt and LAMP1−/−2−/−cells were incubated with or without FAC (10μM) for 24 hours. Cells were then incubated with 100μM DFO or desferasirox in the presence or absence of 100μM chloroquine for 8 hours. Cells were harvested and ferritin content was determined by ELISA. Error bars represent SD from 3 different experiments. (B) HeLa cells treated as in panel A were incubated in the presence or absence of 100nM rapamycin for 12 hours. Cells were harvested and ferritin content was determined by ELISA. (C) Cells as in panel B were stained for LC3B and the percentage of LC3B-positive cells was determined. Error bars represent SD from 3 different experiments.
Figure 7
Figure 7
Model of alternate routes for ferritin degradation. The model shows that ferritin degradation can occur by different routes. Ferroportin (Fpn) or oral iron chelator (desferasirox)–mediated ferritin degradation occurs by the proteasome. In contrast, ferritin degradation induced by DFO occurs in the lysosome.
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"VSports" References

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