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Jared Iacovelli, Majda Hadziahmetovic, Ying Song, Steven Grieco, Irene Zohn, Joshua L. Dunaief; Role Of Ferroportin In Rpe Iron Export. Invest. Ophthalmol. Vis. Sci. 2011;52(14):925.
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© ARVO (1962-2015); The Authors (2016-present)
Dysregulated iron homeostasis can lead to retinal degeneration, as demonstrated by mice lacking Hfe, Hepcidin (Hepc), or the ferroxidases Ceruloplasmin (Cp) and Hephaestin (Heph). The proteins absent in the later two mouse strains (Hepc, Cp/Heph) directly affect the function of the only known mammalian iron exporter, Ferroportin (Fpn). Hepcidin induces the internalization and degradation of Fpn, while Cp and Heph convert ferrous iron to ferric iron, stimulating export, and in some cells, stabilizing Fpn on the cell membrane. To further our understanding of retinal Fpn function, we studied Flatiron (ffe) mice harboring a Fpn mutation leading to mislocalization. We also used ARPE-19 cells to assess the role of Hepc on membrane Fpn levels and Fpn-mediated iron export from these cells.
Retinas from Flatiron mice were studied by morphometric analysis of plastic sections, and immunofluorescence stain for H- and L- ferritin on 10 µm cryosections performed as previously described (Hahn et al., 2004; Iacovelli et al., 2009). Analysis of transcriptional changes in neural retina and isolated RPE was performed using a custom TaqMan low density array. Western Analysis for Fpn was performed using rabbit-anti-Fpn (Alpha Diagnostics) on cell-surface and intracellular fractions from ARPE-19. Cell-surface and intracellular fractions were separated using the Pierce Cell Surface Biotinylation Kit. Iron transport was performed on APRE-19 grown in low sera media on Human ECM coated transwells for 6 months
In 6 month old neural retinas of ffe mice we found a decrease in Id1 (p=0.0066) mRNA and an increase in Tfrc (p=0.0366), indicating iron deficiency. In contrast, the RPE had a decrease in both Slc11a2 (p=0.0316) and Tfrc (p=0.287) indicating elevated iron levels. No difference in either H- or L- ferritin immunofluorescence was found between 10 month old wild-type or ffe retinas. In ARPE 19, Hepc treatment reduced cell surface Fpn levels and iron export.
Taken together, these data support an important role for Fpn in iron export from RPE. Iron export from ARPE-19 is reduced by Hepc due to decreased cell surface Fpn. RPE iron accumulation and retinal iron deficiency occurs in ffe mice which have mis-localized Fpn.
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