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Department of Nutrition, Harvard School of Public Health, Boston, MA 02115 and * Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN 55812
2To whom correspondence should be addressed. E-mail: wessling{at}hsph.harvard.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • copper deficiency • ferroportin-1 • iron export
Copper deficiency impairs normal iron metabolism, resulting in microcytic, hypochromic anemia and associated iron-loading in key tissues such as liver (1). Copper is required as a cofactor for the plasma protein ceruloplasmin, which catalyzes the oxidation of Fe(II) such that Fe(III) is available to bind transferrin, thereby facilitating iron release to the circulation (2). However, the function of copper in iron metabolism is not fully explained by its role in the activity of ceruloplasmin alone because although ceruloplasmin knockout mice accumulate liver iron, they do not develop (3) or display only mild iron-deficiency anemia (4). Ceruloplasmin itself is not a metal transporter and is more likely to act in concert with a transmembrane transport protein. Recently, the first mammalian iron export protein, ferroportin-1 (FPN1),3 was identified (5). FPN1 [also called MTP1 (6) or Ireg1 (7)] is highly expressed in the spleen, liver, kidney, and duodenum. Mutations in the human FPN1 gene are associated with iron-loading disorders (8–11), reflecting the function of this transporter in iron release. Expression of FPN1 is regulated in response to iron status such that iron deficiency reduces FPN1 levels in liver (6), whereas it increases expression in the intestine (7,12,13). Conversely, iron overload in hemochromatosis patients (7,12,14) and mouse models (15) appears to increase hepatic and decrease duodenal FPN1 mRNA expression.
We (16) and others (17) recently established in vitro evidence for the upregulation of FPN1 expression in response to copper treatment 
10 µmol/L. Induction of FPN1 mRNA and protein by copper suggests that iron export in the copper-deficient state might be attenuated via downregulation of FPN1 expression by this metal. To test this prediction, we examined the effect of copper deficiency on FPN1 expression in vivo. Steady-state levels of FPN1 mRNA in copper-deficient (-Cu) and copper-adequate (+Cu) mice were compared in tissues that are critical in maintaining systemic iron homeostasis, i.e., duodenum, liver, kidney, and spleen.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Adult male and female ND4 Swiss Webster mice were purchased commercially (Harlan Sprague Dawley). Mice were administered one of two dietary treatments, copper-deficient (-Cu) or copper-adequate (+Cu), consisting of a copper-deficient purified diet (Teklad Laboratories) and either low-copper drinking water or copper-supplemented drinking water, respectively. The purified diet was similar to the AIN-76A diet (18,19). The purified diet contained 0.36 mg Cu/kg and 44 mg Fe/kg by chemical analysis. Mice fed the -Cu treatment drank deionized water, whereas +Cu treatment groups drank water that contained 20 mg Cu/L by adding CuSO4. Mice had free access to diet and drinking water. All mice were maintained at 24°C with 55% relative humidity on a 12-h light:dark cycle (lights on, 0700–1900 h). All protocols were approved formally by the University of Minnesota Animal Care Committee.
Breeding units were set up and dietary treatments were delayed until gestation d 17. Offspring were weaned when 3 wk old andadministered the same treatment as their respective dams for an additional week after the transfer to stainless steel cages. A total of 8 litters (4 +Cu and 4 -Cu) were sampled. Offspring (1 male and 1 female from each litter) were killed at postnatal d 28 (P28) to establish copper status. This experimental paradigm is similar to that described previously (20).
After decapitation and blood collection, the left kidney, whole spleen, a portion of the liver central lobe, and the first 1.5 cm of duodenum were rapidly removed, transferred to a tube containing RNA later (Ambion), and shipped to Harvard for further analyses. The right kidney, another portion of liver, and the next 3.5 cm of duodenum were processed for metal analyses. Duodenal samples for metal analyses were flushed of contents with deionized water, blotted, and weighed fresh.
A follow-up study was conducted in which 4 male P28 offspring of each treatment raised exactly as in the first experiment were killed and portions of the liver were quick frozen and stored at -75°C until shipped to Harvard for Western immunoblot analyses. Another portion was used for metal analysis.
Biochemical measurements.
Plasma from the hematocrit tubes was used to measure ceruloplasmin activity by following the oxidation of o-dianisidine; hemoglobin was determined as metcyanohemoglobin (21). Tissues and diet were wet-digested with HNO3 (TraceMetal grade, Fisher Scientific) and analyzed by flame atomic absorption spectroscopy (Model 2380, Perkin-Elmer). The method was checked with a certified standard, U.S. National Bureau of Standards 1577 bovine liver.
Northern blot analyses of FPN1.
Total RNA was isolated using RNA-Bee (Tel-Test) and Northern blot analysis was performed with 15–30 µg of total RNA as previously described using randomly primed FPN1 cDNA (22). The FPN1 cDNA probe was prepared using pCMV-SPORT6-mouse FPN1 (GenBank accession number BE554084, ATCC) as a template. After hybridization (2.2 x 109 dpm/L), blots were washed 4 times at room temperature for 15 min each in 0.1X SSC (0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate) and 0.15% SDS. Radioactivity was detected by phosphorimaging (Personal Molecular Imager FX, Bio-Rad) and quantified using Quantity One software (version 4.1.0, Bio-Rad). Blots were stripped and rehybridized with a 32P-labeled cDNA probe for the ribosomal phosphoprotein 36B4 (23) to verify equal loading and transfer of RNA. Preliminary experiments on mouse tissue indicated that copper status did not influence expression of 36B4.
Western blot analyses.
Liver tissues were homogenized in buffer containing 0.170 mol/L Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 10% ß-mercaptoethanol, and protease inhibitors (Complete Mini protease inhibitor cocktail, Roche) using Tissue-tearor (Biospec Products). The homogenates were centrifuged at 10,000 x g for 30 min at 4°C. DNA was sheared by sonication for 10 s. Protein content was determined using RC DC Protein Assay (Bio-Rad). For analyses of FPN1 and copper chaperone for superoxide dismutase (CCS), 50-µg samples were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred to an Optitran nitrocellulose membrane (Schleicher & Schuell). For the analyses of transferrin receptor (TfR) and ferritin, 50-µg samples were boiled for 10 min and loaded onto a separate 10% SDS-polyacrylamide gel. Equal loading and the integrity of transfer were confirmed by Ponceau-S staining. As a control, each gel was also immunoblotted for ß-actin. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline solution containing 0.01% Tween-20, pH 7.4 (TBS-T) for at least 2 h, and incubated with primary antibody overnight at 4°C. Blots were then washed for 30 min with TBS-T, incubated with horseradish peroxidase-linked secondary antibody, washed with TBS-T for 30 min, and visualized using enhanced chemiluminescence (SuperSignal WestPico, Pierce).
Statistics.
Values are means ± SEM. Comparisons of tissue metal levels, hematological values, and FPN1 mRNA expression between -Cu and +Cu mice and males and females between and within these groups were evaluated using Student’s unpaired t test (P < 0.05).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Total body weights did not differ between groups (Table 1). With one exception, male +Cu mice had higher kidney copper levels than female +Cu mice [63.7 ± 0.20 vs. 51.99 ± 2.36 nmol/g (n = 4/sex), P < 0.01]; otherwise, female and male mice did not differ in all of the variables examined. The data provided in Table 1 are therefore pooled from 4 male and 4 female mice in each group. Copper deficiency in -Cu mice was verified by significant reductions in tissue copper levels in duodenum (63%), liver (50%), and kidney (27%) as well as profoundly reduced serum ceruloplasmin activity (loss of >95%). Although duodenal iron levels were not altered by copper deficiency, -Cu mice had significantly reduced hematological iron values, with increased hepatic iron levels 
onefold higher than +Cu mice, consistent with previous reports (20). Thus, -Cu mice displayed copper-deficiency anemia and associated liver iron overload.
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Effects of copper deficiency on FPN1 mRNA levels.
Northern blot analyses were performed to compare the steady-state levels of FPN1 mRNA in duodenum, liver, kidney, and spleen between -Cu and +Cu mice. As anticipated (5,6), FPN1 mRNA was readily detected in all four tissues examined (Fig. 1). To correct for RNA loading and transfer efficiency, densities of FPN1 mRNA bands were normalized to mRNA levels for the ribosomal phosphoprotein 36B4 (23). From this quantitation, FPN1 mRNA levels in duodenum and spleen of -Cu mice were 90 and 106%, respectively, of tissues in +Cu mice, levels that did not differ. Although kidney and liver transcript levels were slightly higher in -Cu mice (125 and 122% of +Cu tissues, respectively), these differences were not significant (P = 0.076, kidney; P = 0.075, liver). FPN1 mRNA expression in liver tissues that were more severely depleted with copper (obtained from the second set of mice described above) also did not differ from +Cu mice (data not shown). Finally, a direct comparison of FPN1 mRNA levels and respective tissue copper and iron concentrations of individual mice did not reveal any correlation (data not shown), further confirming that copper deficiency had no effect on the relative expression of FPN1 mRNA.
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Because it was shown that iron-loading increases FPN1 protein in the liver of hemochromatosis patients (14), and iron status was suggested to control FPN1 synthesis post-transcriptionally (6,7,24), we further examined whether hepatic iron loading induced by copper deficiency alters FPN1 expression at the protein level. Western blot analyses were performed to compare FPN1 protein levels in liver tissues obtained from the second experiment in which -Cu mice had more than threefold higher liver iron than +Cu mice. Hepatic iron accumulation in the -Cu mice was con-firmed by significantly increased levels of the iron storage protein ferritin with concomitantly decreased TfR levels compared with +Cu mice (Fig. 2). Ferritin and TfR are reciprocally regulated in response to iron status (25). Depletion of hepatic copper in -Cu mice was also verified by induction of CCS (Fig. 2). Elevation of liver CCS accompanies dietary copper deficiency (26,27). In contrast, the level of FPN1 protein expression in copper-deficient, iron-loaded liver tissue was not affected (Fig. 2), suggesting that regulation of FPN1 expression by iron is impaired in -Cu mice.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A major finding from this study is that FPN1 mRNA expression does not vary in the intestine, kidney, spleen or liver of copper-deficient mice relative to copper-adequate controls. Intestine, kidney, and liver copper concentrations of the -Cu mice were significantly diminished (Table 1), and it is probable that spleen copper levels were also reduced based on previous observations (33). Thus, we conclude that depletion of tissue copper does not reduce FPN1 mRNA levels. These results contrast with previous reports showing that high-copper treatment significantly increased FPN1 mRNA and protein levels in J774 (16) and Caco-2 (17) cell lines. An obvious difference between our current investigation and these other studies is that a model of copper depletion rather than overload was examined. Although it is possible that FPN1 levels are modulated only in response to high copper, FPN1 expression might not be affected unless intracellular copper concentrations become more severely depleted (15.7–42.4 nmol/g of copper was still available in tissues of copper-deficient mice). Alternatively, whether in vitro studies of FPN1 expression faithfully reflect in vivo regulation in response to metal status is largely unknown. Thus, future studies should examine whether the accumulation of copper induces upregulation of FPN1 expression in vivo, for example, in patients with Wilson’s or Menkes’ disease (34), or in liver tissues of the Long-Evans-Cinnamon rat (35).
The apparent lack of change in FPN1 expression in copper-deficient mice does raise some puzzling questions. Intestinal FPN1 mRNA levels are reportedly increased in anemic mice (7) and anemic humans (12). Despite the fact that -Cu mice displayed anemia, intestinal FPN1 expression was unaffected (Fig. 1). Of particular note is that intestinal tissue iron levels in -Cu mice did not differ from +Cu mice, suggesting perhaps that tissue iron status might be the dominant factor upregulating intestinal FPN1 expression in vivo (7). However, studies by Chen et al. (36), comparing iron transport regulation in genetic and nutritional iron deficiencies, have rather convincingly demonstrated that FPN1 expression is regulated by systemic signals rather than enterocyte iron levels. If the latter model is correct, our observations suggest that copper adequacy is necessary for proper regulation of duodenal FPN1 expression in response to body iron requirements. Perhaps even more striking was our observation that levels of FPN1 mRNA and protein in livers of -Cu mice were not altered even though the copper-deficient mice had two- to threefold higher hepatic iron content. Upregulation of FPN1 transcripts was demonstrated in iron-loaded livers of HFE knockout mice (15) and in normal (6,13) and HFE-deficient (13) mice injected with iron-dextran. More recently, Adams et al. (14) showed that FPN1 protein is increased in iron-loaded livers of hemochromatosis patients. The idea that iron loading enhances FPN1 expression is further supported by studies demonstrating that iron treatment in vitro increases FPN1 levels in BEAS-2B bronchial epithelial cells (37), freshly isolated lung macrophages (37), and J774 macrophage cells (22). Although a trend for increased liver FPN1 mRNA levels was observed in -Cu mice, no differences were detected by assays used in the present study between hepatic levels of mRNA and protein for FPN1 in -Cu and +Cu mice. Thus, inappropriately low levels of FPN1 expression in the intestine and liver of -Cu mice may foster the microcytic anemia and hepatic iron-loading observed in the copper-deficient state.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CCS, copper chaperone for superoxide dismutase; -Cu, copper-deficient; +Cu, copper-adequate; FPN1, ferroportin 1; P28, postnatal day 28; TBS-T, Tris-buffered saline with Tween-20; TfR, transferrin receptor.
Manuscript received 28 October 2003. Initial review completed 26 November 2003. Revision accepted 18 December 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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