Journal of Undergraduate Research
Volume 6, Issue 6 - March 2005

Hepcidin Decreases Ferroportin 1 Expression in a Mouse Macrophage Cell Line

Lindsey Koss

ABSTRACT

Macrophage cells play a major role in iron metabolism by recycling iron from senescent red blood cells. The objective of the present study was to assess the effect of hepcidin on proteins involved in macrophage iron metabolism. Hepcidin is a recently identified peptide hormone that is secreted by the liver in response to inflammation and iron loading. We tested the hypothesis that hepcidin would decrease levels of the iron-export protein, ferroportin (FPN1), and as a consequence, increase the levels of the iron-storage protein, ferritin. Mouse macrophage J774 cells were treated with various doses of hepcidin (0.04 - 1.8 μM) for various times (0.5 – 22 h), and levels of FPN1 and ferritin protein were determined by Western analysis. Because macrophages in vivo obtain a constant supply of iron through phagocytosis of senescent red blood cells, the effect of hepcidin was also assessed after macrophages ingested red cells. We found that hepcidin (0.04 μM) dramatically reduced FPN1 protein levels, with higher doses causing no further decreases. Treatment of cells with 0.70 μM hepcidin after erythrophagocytosis markedly decreased FPN1 protein levels after 3, 6, 12 and 22 h. Despite the large changes in FPN1 protein levels after hepcidin treatment, no changes in ferritin protein levels were observed at any dose or time, with or without erythrophagocytosis. The lack of change in ferritin protein levels suggests that cytosolic iron concentrations did not change or that the amount of ferritin was sufficient to accommodate any increases in cellular iron content subsequent to down-regulation of FPN1. We conclude that macrophage FPN1 is a target for hepcidin. The rapid and pronounced decrease in this iron-export protein in the presence of hepcidin is likely to contribute to the perturbations in macrophage iron metabolism observed in inflammation.

INTRODUCTION

Disturbances of iron metabolism are among the most prevalent disorders affecting humans. In the United States, approximately 1 in 200 people are genetically at risk for developing hemochromatosis (1), an iron-overload disease, whereas the anemia of inflammation is the most common type of anemia in hospitalized patients (2). Both of these disorders are characterized by altered macrophage iron metabolism. Macrophages of the liver, spleen, and bone marrow are specialized cells of the immune system that play a central role in iron metabolism by capturing and ingesting senescent or damaged erythrocytes from the circulation. After erythrophagocytosis, the red cell is degraded, heme is released from hemoglobin, and iron is released from heme. The iron thus liberated is either stored in the cell or released into the circulation, where it is taken up mainly by the bone marrow and re-incorporated into heme during red cell synthesis. Each day, approximately 20 mg of iron are recycled by macrophages (3).

In the anemia of inflammation, serum iron levels decrease and iron accumulates in macrophages (2). The accumulation of macrophage iron most likely results from a decrease in macrophage iron export, which appears to be mediated by the recently identified protein, ferroportin 1 (FPN1) (4-6). In the duodenum, FPN1 is localized to the basolateral membrane, where it exports cellular iron into the portal blood circulation. The abundant expression of FPN1 in macrophages (7) suggests that it may also export iron from these cells. Consistent with the idea that FPN1 exports iron from the macrophage are studies of patients with mutations in FPN1 (reviewed by Pietrangelo (8)). These patients accumulate iron in macrophages and/or tissues and develop iron overload.

A great advance in our understanding of the regulation of whole-body iron metabolism has come from the recent discovery of hepcidin, a small antimicrobial peptide produced by the liver (9). Hepcidin levels increase in response to iron loading (10) and inflammation (11). It has been proposed that increased levels of circulating hepcidin in response to inflammation signal the macrophage to down-regulate the release of iron into the plasma and the small intestine to decrease the absorption of dietary iron (12). Both of these effects would decrease levels of iron in the plasma, and if continued over time, may lead to the anemia of inflammation. Recent studies using a human embryonic kidney cell line (HEK293) suggest that hepcidin mediates these effects by interacting with FPN1 (13). Specifically, hepcidin was shown to bind to FPN1, cause its degradation, and lead to a decrease in the export of iron from cells loaded with 59Fe-labeled transferrin.

The aim of the present study was to examine the effect of hepcidin on iron metabolism in J774 cells, a mouse macrophage cell line. We tested the hypothesis that hepcidin treatment would decrease FPN1 expression and increase iron storage within the cell. Changes in cellular iron concentrations were assessed by measuring levels of the iron-storage protein, ferritin. Because macrophages continually ingest senescent red blood cells in vivo, the effects of hepcidin were also assessed after erythrophagocytosis.

METHODS

Cell Culture and Hepcidin Treatment

Mouse macrophage J774 cells were grown in alpha-minimum earl’s medium (Mediatech) fortified with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 _g/mL streptomycin at 37óC in 5% CO2. To investigate the effect of hepcidin on macrophage iron metabolism, J774 cells were treated with human hepcidin (Peptides International). Hepcidin was added to the cell culture media at the indicated doses and incubated for the indicated amounts of time.

Erythrophagocytosis by J774 cells

Rat erythrocytes were opsonized by incubating with goat anti-rat red blood cell immunoglobulin G (IgG) for 20 min at 37óC. Opsonized erythrocytes (1 x 107) were added to J774 cell monolayers and incubated at 37óC for 1.5 h. The noningested red blood cells were hypotonically lysed and removed by adding sterile water for 10 s (double processed, tissue-culture water, Sigma-Aldrich) and by washing twice with Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich). Cells were incubated at 37óC until harvesting for Western analysis as described below.

Western Blot Analysis

Cell monolayers were washed once in ice-cold PBS and lysed in ice-cold SDS lysis buffer [60 mM Tris-HCl, pH 6.8/2% SDS/5% glycerol/5% β-mercaptoethanol, and complete mini protease inhibitor mixture (Roche)]. Cell lysates were sonicated for 5 s and stored at -80°C until analysis. Protein content was determined by using the RC DC protein assay (Bio-Rad). Samples (60–120 mg protein) were mixed with SDS lysis buffer containing bromophenol blue and, without prior heating, were electrophoretically separated on a 10% SDS polyacrylamide gel. Molecular weight standards (Precision Plus, dual color, Bio-Rad) were run in parallel. The protein was transferred to Optitran nitrocellulose membrane (Schleicher & Schuell). After incubating blots for 1 h in blocking solution [5% nonfat dry milk in Tris-buffered saline, pH 7.4, containing 0.01%Tween 20 (TBST)], blots were incubated overnight at 4°C with affinity-purified rabbit anti-FPN1 peptide antibodies (2.5 μg/ml blocking solution). Blots were washed in TBST and then incubated for 40 min with a 1:2,000 dilution of horseradish peroxidase-linked donkey anti-rabbit IgG (Amersham Pharmacia). Immunoreactivity was visualized by using enhanced chemiluminescence (SuperSignal WestPico, Pierce) and autoradiographic film. To control for loading, blots were stripped for 5 min in 0.5 M glycine (pH 2.8), 0.5 M NaCl, washed in TBS, blocked for 1 h in blocking buffer, then reprobed by using a 1:10,000 dilution of mouse monoclonal anti-α-tubulin clone B-5-1-2 (Sigma), and a 1:10,000 dilution of ZyMax goat anti-mouse IgG horseradish peroxidase conjugate (Zymed). Ferritin protein levels were determined as above, except that protein samples (30 μg) were boiled for 5 min before electrophoretic separation on a 12%
SDS-polyacrylamide gel, and rabbit anti-horse spleen ferritin antiserum (Sigma), 1:1,000 dilution, was used at room temperature for 1 h. Autoradiograms were digitized by using GENEFLASH gel documentation system (SynGene), and signal intensities were quantified by densitometry by using GENETOOLS software (SynGene).

RESULTS

Western analysis using an affinity-purified anti-FPN1 antibody detected an immunoreactive band at ~65 kDa (Fig. 1), similar to previous studies (14). The band at 65 kDa is consistent with the predicted molecular mass of FPN1 (62 kDa). Treatment of J774 macrophages with 1.4 μM hepcidin for 3-12 h markedly reduced FPN1 protein levels (Fig. 1A). The down-regulation of FPN1 expression was found to occur within 30 min after hepcidin treatment (Fig. 1B). To assess the dose-response effect of hepcidin on FPN1 protein levels, J774 cells were treated with 0.04 to 1.4 _M hepcidin for 8 h (Fig. 1C). Treatment of cells with 0.04 μM hepcidin markedly decreased FPN1 protein levels, with hepcidin doses up to 1.4 μM causing no further decreases.

Figure 1. Hepcidin treatment markedly decreases FPN1 protein levels in J774 macrophages.

Figure 1. Hepcidin treatment markedly decreases FPN1 protein levels in J774 macrophages. J774 cells were treated with 1.4 mM hepcidin (HEPC) for 3-12 h (A) and 0.5-3 h (B). Proteins in cell lysates (50 mg protein per lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was probed with anti-FPN1 antibody as described in “Materials and Methods.” As a control for lane loading, the blot was stripped and reprobed for tubulin. The position and masses (in kDa) of molecular weight markers are indicated on the right in Panel A. In Panel C, J774 cells were treated with 0.04 - 1.4 μM hepcidin for 8 h.

Ferritin is a large iron-storage protein, consisting of 24 subunits with two types, light-chain ferritin and heavy-chain ferritin (15). Light chain ferritin (LFt) is the main subunit involved in iron storage and increases upon iron loading (16). To determine if hepcidin affects LFt protein levels, J774 cells were treated with 0.90-1.8 _M hepcidin for 6 h. As shown in Fig. 2, Western analysis indicates that hepcidin treatment did not alter levels of LFt protein in J774 cells. Treatment of cells with 800 _M Fe-NTA, as a positive control for LFt induction, increased LFt levels (Fig. 2).

Figure 2. Hepcidin treatment does not affect light-chain ferritin protein levels in J774 macrophages.

Figure 2. Hepcidin treatment does not affect light-chain ferritin protein levels in J774 macrophages. J774 cells were treated with 0-1.8 μM hepcidin for 6 h and probed for light-chain ferritin (LFt). Cell lysates (50 μg protein per lane) were probed with anti-ferritin antiserum. As a positive control for induction of LFt, cells were treated with 800 μM ferric nitrilotriacetic acid (Fe-NTA) for 6 h (far right lane, 15 μg protein). The position and masses (in kDa) of molecular weight markers are indicated on the right.

Previous studies have shown that FPN1 expression increases after erythrophagocytosis by J774 macrophages (14). To test if hepcidin would decrease FPN1 protein levels after induction by erythrophagocytosis, 0.70 _M hepcidin was added to J774 cells immediately after red cell ingestion. Erythrocytes were added to J774 cells and incubated for 1.5 h before removal of non-ingested red cells. Consistent with previous studies, FPN1 protein levels increased after erythrophagocytosis (Fig. 3; lanes 1 vs. lanes 3, 5). The addition of hepcidin, however, prevented the induction of FPN1 after erythrophagocytosis. This effect was observed at all time points after red cell ingestion.

Figure 3. Hepcidin treatment markedly decreases FPN1 protein levels but does not affect light-chain ferritin levels after erythrophagocytosis (EP).

Figure 3. Hepcidin treatment markedly decreases FPN1 protein levels but does not affect light-chain ferritin levels after erythrophagocytosis (EP). J774 cells were incubated with or without opsonized erythrocytes (EIgG) for 1.5 h. After removal of noningested EIgG, macrophages were treated with or without 0.70 μM hepcidin and incubated for 3–6 h (Left) or 6–22 h (Right). Cell lysates (60 μg and 30 μg per lane) were blotted with anti-FPN1 antibody and anti-ferritin antiserum, respectively.

In light of the marked reductions in the levels of the iron-export protein, FPN1, in response to hepcidin, it seemed probable that intracellular iron concentrations would increase, resulting in an increase in the cytosolic iron-storage protein LFt. However, we observed no increase in LFt in response to various doses of hepcidin (Fig. 2). The lack of an effect of hepcidin may have been due to the low basal cytosolic concentrations of iron in J774 cells. Thus, to test the effect of hepcidin when cells are loaded with iron, hepcidin was added after the J774 macrophages had ingested red blood cells. Although LFt levels markedly increased upon iron loading via erythrophagocytosis (Fig. 4), no further increases in LFt levels were observed in cells treated 0.9 or 1.8 μM hepcidin.

Figure 4. Treatment of J774 macrophages with increasing doses of hepcidin after erythrophagocytosis (EP) does not affect light-chain ferritin protein levels.

Figure 4. Treatment of J774 macrophages with increasing doses of hepcidin after erythrophagocytosis (EP) does not affect light-chain ferritin protein levels. J774 cells were incubated with or without opsonized erythrocytes (EIgG) for 1.5 h. After removal of noningested EIgG, macrophages were treated with or without increasing doses of hepcidin for 6 h.

DISCUSSION

A main finding of the present study was that hepcidin caused a rapid and dramatic decrease in FPN1 protein levels in J774 macrophage cells. The hepcidin-mediated reduction in FPN1 levels occurs also after erythrophagocytosis, which induces FPN1 expression. This decrease in FPN1 is consistent with a recent report by Nemeth et al. (13) in which hepcidin was found to promote the degradation of FPN1 in HEK293 cells.

The present study is the first to assess the effect of hepcidin on macrophage iron metabolism. Hepcidin, a peptide hormone secreted by the liver into the circulation, has been recently shown to regulate systemic iron metabolism. Mice lacking hepcidin gene expression develop massive iron overload (17), and transgenic mice overexpressing hepcidin are severely anemic. These observations suggest that hepcidin is a negative regulator of iron metabolism. Accordingly, hepcidin levels increase in response to iron overload (10) and inflammation (17,18). Using mouse models of acute and chronic inflammation, Yang et al. (7) found that the reduced serum iron levels were associated with decreased FPN1 expression in reticuloendothelial macrophages of the spleen, liver and bone marrow, suggesting a causative role for FPN1. The hypoferremia of inflammation does appear to require the participation of hepcidin because the drop in serum iron levels secondary to inflammation induced by turpentine injection was shown to be completely blunted in hepcidin-deficient mice (11). In the present study, we establish a direct relationship between hepcidin and macrophage FPN1 expression.

Changes in cytosolic iron were assessed by measuring levels of the iron-storage protein, ferritin. We found that ferritin levels did not change after hepcidin treatment, which indicates that either cytosolic iron levels did not change, or that ferritin levels were adequate to handle any increase in cytosolic iron resulting from changes in FPN1 expression. Hepcidin was added after erythrophagocytosis to model physiologic iron recycling, and again, FPN1 protein levels were considerably decreased and ferritin protein levels remained unchanged. Although these observations are opposite to what we had originally hypothesized, they are consistent with a very recent report that used small interfering RNAs to silence FPN1 in human macrophages (19). The decrease of FPN1 mRNA did not affect light-chain ferritin protein levels when cells were loaded with iron using ferric gluconate. To explain the lack of light-chain ferritin induction, the authors noted that light-chain ferritin levels were probably saturated by the iron treatment. This possibility does not seem to explain our data, however, because light-chain ferritin levels 3 hours after erythrophagocytosis were clearly not saturated (i.e., their levels continued to increase by 6 hours after erythrophagocytosis).

Overall, we conclude that the macrophage iron-export protein, FPN1, is a target for hepcidin. The decrease in FPN1 protein levels in response to hepcidin is likely to contribute to the perturbations in macrophage iron metabolism observed in inflammation.

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