Presence of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin

Abstract

Ferroportin, the only mammalian iron exporter identified to date, is highly expressed in duodenal enterocytes and in macrophages. Several lines of evidence indicate that in enterocytes the iron export mediated by ferroportin occurs and is regulated at the basolateral cell surface, where the transporter is strongly expressed. By contrast, in macrophages, ferroportin has been shown in intracellular vesicles. We used a high-affinity antibody to specify the localization of endogenous ferroportin expressed in primary culture of bone marrow-derived macrophages, in both basal and induced conditions. Our observations indicate that ferroportin is expressed in vesicular compartments that can reach the plasma membrane of macrophages. Of importance, when ferroportin expression was up-regulated through iron treatment or erythrophagocytosis, ferroportin expression was strongly enhanced at the plasma membrane of macrophages. Moreover, hepcidin dramatically reduced macrophage ferroportin protein levels. At the subcellular level, hepcidin was shown to induce rapid internalization and degradation of the macrophage iron exporter. These data are consistent with a direct iron export by ferroportin through the plasma membrane of macrophages and strongly support an efficient posttranscriptional down-regulation of ferroportin by hepcidin in these cells. [metal transporter protein-1], Slc40a1) is the sole iron exporter identified in mammals 1-3 that participates in iron release from both enterocytes of the duodenum and tissue macrophages. Ferroportin is highly expressed in absorptive duodenal enterocytes where it presents a strong basolateral subcellular localization 1-3 and in tissue macrophages in liver (Kupffer cells), spleen, and bone marrow. 1,4,5 Several lines of evidence highlight the importance of this protein in iron homeostasis. Inactivation of the ferroportin gene at the adult stage in mice leads to iron accumulation in enterocytes, Kupffer cells, and splenic macrophages. 6 Ferroportin mutations in human patients with type 4 hemochromatosis induce predominant macrophage iron overload. 7 Finally, ferroportin overexpressed in the macrophage cell line J774 stimulates iron release after erythrophagocytosis. 8 Recently, ferroportin has been shown to be the molecular target of hepcidin. 9 Hepcidin is a major systemic regulator of intestinal iron absorption and iron recycling from macrophages. 10 In epithelial cells, hepcidin was shown to act on the efflux of iron through a direct interaction with ferroportin at the cell surface, leading to internalization and degradation of the iron exporter. 9 Moreover, recent studies have shown that some hemochromatosis-associated ferroportin mutations are unresponsive to hepcidin-mediated internalization. [11][12][13] Of interest, in hepcidin-deficient mice, 14 ferroportin is strongly up-regulated in both enterocytes and macrophages. 15 Hepcidin is also assumed to regulate iron efflux from macrophages 10 because it has been shown that treatment of J774 macrophages with hepcidin decreases ferropo...

Differential regulation of iron homeostasis during human macrophage polarized activation

“…This is apparently not in line with the fact that M2 macrophages express 30-40% more TfR1 than M1 cells and hence should internalize more iron. However, greater iron uptake would presumably lead in turn to a higher iron export activity, because iron induces Fpn in macrophages [13,29,44].Overall, our results suggest that M2 macrophages lack the typical iron-withholding mechanisms present in classically activated M1 macrophages. Moreover, increased iron availability in the extracellular milieu of M2 macrophages may have relevant physiopathological consequences (see below).…”

“…As Fpn functions on the plasma membrane where it translocates after iron loading [29], we examined expression of Fpn in the membrane fraction. Figure 3E shows that Fpn protein levels were 30% lower in M1 cells and 2.5-fold higher in M2 macrophages, as compared with unpolarized macrophages.…”

“…This is apparently not in line with the fact that M2 macrophages express 30-40% more TfR1 than M1 cells and hence should internalize more iron. However, greater iron uptake would presumably lead in turn to a higher iron export activity, because iron induces Fpn in macrophages [13,29,44].…”

“…For the preparation of membrane extracts, homogenates were centrifuged at 2000 g for 10 min and the postnuclear supernatants were ultracentrifuged at 150 000 g to pellet crude membrane fractions [29].…”

“…The stronger difference in Fpn at the mRNA level (analysed at 18 h) than at the protein level (analysed at 72 h), prompted us to evaluate Fpn expression at an intermediate time point. Analysis of both Fpn mRNA and protein at 48 h (Fig 3C and D) showed a difference between the various macrophage populations similar to that observed at the other time points, thus indicating that the different extent of Fpn modulation at the mRNA and protein levels is maintained at times ranging from 18 to 72 h after polarization.As Fpn functions on the plasma membrane where it translocates after iron loading [29], we examined expression of Fpn in the membrane fraction. Figure 3E shows that Fpn protein levels were 30% lower in M1 cells and 2.5-fold higher in M2 macrophages, as compared with unpolarized macrophages.…”