Lipid raft-dependent endocytosis: a new route for hepcidin-mediated regulation of ferroportin in macrophages.
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 raft-mediated endocytosis plays a role in hepcidin activity.


METHODS
Raft/detergent-resistant membranes from murine bone marrow-derived macrophages and J774a1 cells were analyzed by Western blotting. The effect of lipid raft- or clathrin-dependent endocytosis inhibitors was studied on 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 raft markers (caveolin 1, flotillin 1). Interestingly, iron overload strongly increased the presence of ferroportin in the lightest raft fraction. Moreover, lipid raft 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.
Auriac
Anne
A
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. fcanonne@icsn.cnrs-gif.fr
Willemetz
Alexandra
A
Canonne-Hergaux
François
F
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism; D004705:Endocytosis; D005290:Ferric Compounds / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D064451:Hepcidins; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D008856:Microscopy, Fluorescence; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D014168:Transferrin / Q000378:metabolism; D000098742:Ferroportin
D023181:Antimicrobial Cationic Peptides; D027682:Cation Transport Proteins; D051242:Caveolin 1; D005290:Ferric Compounds; C579599:Hamp protein, mouse; D064451:Hepcidins; D008565:Membrane Proteins; D014168:Transferrin; C106354:flotillins; D000098742:Ferroportin; D005372:Filipin; D002772:Cholera Toxin; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate