BMSEHA15
Published online 10 March 2010
(Haematologica 2010, 10.3324/haematol.2009.019992)
Copyright © 2010 by Ferrata Storti Foundation
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Article

Lipid rafts-dependent endocytosis: a new route for hepcidin-mediated regulation of ferroportin in macrophages

Anne Auriac1, Alexandra Willemetz1, François Canonne-Hergaux1,2

1 Centre de Recherche de Gif-sur-Yvette, UPR 2301, CNRS and
2 Institut de Chimie des Substances Naturelles (ICSN), Gif-sur-Yvette, FRANCE

Correspondence: François Canonne-Hergaux, Centre de Recherche de Gif-sur-Yvette, UPR 2301, CNRS, Institut de Chimie des Substances Naturelles (ICSN), Equipe 34, Bâtiment 27, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex France. Phone: international +33.1.69823081. Fax: international +33.1.69077247. E-mail: fcanonne{at}icsn.cnrs-gif.fr

ABSTRACT

Background: Expression of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and is decreased by hepcidin. We previously showed that ferroportin is present in specific cell surface domains suggestive of lipid rafts. Herein, we have clarified the localization of ferroportin in macrophage membranes and tested whether rafts-mediated endocytosis plays a role in hepcidin activity.

Design and Methods: Rafts/detergent-resistant membranes from murine bone marrow–derived macrophages and J774a1 cells were analyzed by Western blotting. The effect of lipid rafts- or clathrin-dependent endocytosis inhibitors was studied hepcidin activity. For this purpose, after treatment, ferroportin expression was analyzed by fluorescence microscopy, Western blotting of total protein extracts or plasma membrane protein samples and by quantitative immunofluorescence assay (In-Cell-Western).

Results: Macrophage ferroportin was mostly detected in detergent-resistant membranes containing rafts markers (caveolin 1, flotillin 1). Interestingly, iron overload strongly increased the presence of ferroportin in the lightestrafts fraction. Moreover, lipid rafts breakdown by cholesterol sequestration (filipin) or depletion (methyl-beta-cyclodextrin) decreased hepcidin activity on macrophage ferroportin. Cell surface biotinylation and immunofluorescence studies indicated that the process of both hepcidin mediated endocytosis and degradation of ferroportin were affected. By contrast, the inhibition of clathrin dependent endocytosis did not interfere with hepcidin effect.

Conclusions: Macrophage ferroportin is present in lipid rafts which contribute to hepcidin activity. These observations reveal the existence of a new cellular pathway in hepcidin mediated degradation of ferroportin and open a new area of investigation in mammalian iron homeostasis