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J. Iacovelli, I. De Domenico, S. S. Miller, J. L. Dunaief; Hepcidin Dependent Ferroportin Degradation in Cultured Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1862.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated that mice lacking the ferroxidases Ceruloplasmin (Cp) and Hephaestin (Heph) accumulate iron within the retina and develop retinal degeneration with features of AMD. Ferroportin (Fpn), an iron transport protein, functions with Cp/Heph to transport iron out of cells. Binding of hepcidin to Fpn induces its internalization and degradation, thus regulating iron efflux from cells (Nemeth et al., Science, 2004). Herein, we study the interaction of Hepcidin with Fpn in ARPE 19 and human fetal RPE cells.
ARPE19 and human fetal RPE (hfRPE) were cultured according to established methods. ARPE19 were transfected with wildtype or mutant FPN-GFP plasmids and treated with 700 nM hepcidin 24 hours post-transfection. 20 µg of protein lysate was analyzed by Western Analysis using an anti-GFP antibody. Localization of FPN-GFP following hepcidin treatment was analyzed by fluorescence microscopy. The response of endogenous Fpn to hepcidin was analyzed by Western Analysis on 30 -60 µg of protein lysate from ARPE19 and hfRPE using an anti-Fpn antibody. Localization of endogenous Fpn in hfRPE was analyzed by confocal microscopy.
Hepcidin treatment of transfected ARPE 19 induced a 50% reduction in transfected wildtype Fpn but did not reduce the protein levels of the hepcidin resistant Fpn mutant. Fpn-GFP was localized on the membrane of transfected ARPE19 and became cytosolic following hepcidin treatment. Hepcidin reduced the levels of endogenous Fpn in both ARPE19 and hfRPE. In hfRPE, in the absence of hepcidin Fpn was localized on the plasma membrane and became cytosolic following hepcidin treatment.
This in vitro study demonstrates that in response to hepcidin, both exogenous and endogenous Fpn in human RPE is internalized and degraded. Therefore, it is likely in vivo that RPE Fpn may respond to hepcidin either produced locally by the retina or produced by liver and transported to the retina by through choroidal blood vessels.
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