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* Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720-3104; Biochemistry Department, School of Medicine, American University, Beirut, Lebanon; ** Department of Molecular Medicine, King's College, London SE59NU, UK; and The Queensland Institute of Medical Research and the University of Queensland, Post Office Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia
2 To whom correspondence should be addressed. E-mail: vulpe{at}berkeley.edu.
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ABSTRACT |
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50% of controls) ferroxidase activity. Liver hepcidin expression was significantly downregulated by copper deficiency (60% of controls), and enterocyte mRNA and protein levels of ferroportin1 were increased to 2.5 and 10 times, respectively, relative to controls, by copper deficiency, indicating a systemic iron deficiency in the copper-deficient mice. Interestingly, hephaestin protein levels were significantly decreased to 40% of control, suggesting that decreased enterocyte copper content leads to decreased hephaestin synthesis and/or stability. We also examined the effect of copper deficiency on hephaestin in vitro in the HT29 cell line and found dramatically decreased hephaestin synthesis and activity. Both in vivo and in vitro studies indicate that copper is required for the proper processing and/or stability of hephaestin.
KEY WORDS: • copper • iron • hephaestin • ceruloplasmin • ferroportin1
Disturbances of copper metabolism lead to altered iron homeostasis (1). Rodents, pigs, or people consuming copper-deficient diets develop iron deficiency anemia in addition to accumulating iron in the gut, liver, and spleen (2–4). The multicopper ferroxidases, ceruloplasmin (Cp)3 and hephaestin (Hp) represent pivotal links between iron and copper homeostasis (1,5,6). Both have been implicated in the efflux of iron from mammalian cells (7,8). Cp is synthesized in the liver for secretion into the plasma and accounts for as much as 95% of plasma copper. Individuals with little or no Cp (aceruloplasminemia) develop iron overload in the brain, liver, pancreas, and other tissues (9). Similarly, aceruloplasminemic mice progressively accumulate iron in serum ferritin, reticuloendothelial cells, and hepatocytes secondary to impaired iron efflux (10). In contrast, defects in Hp, which is expressed predominantly in the gut, as seen in the sex-linked anemia (sla) mouse (11), lead to intestinal iron accumulation and systemic iron deficiency. Sla mice normally take up iron from the intestinal lumen into mature epithelial cells, but are unable to export it adequately into the circulation.
Both Cp and Hp are multicopper ferroxidases involved in iron efflux from cells; they require copper for structural and enzymatic activity (10,12,13). However, the in vivo functions of Hp and Cp are quite distinct. Cp is primarily a soluble serum ferroxidase that plays a role in iron homeostasis in the liver and several other tissues, whereas Hp is membrane bound, highly expressed in the small intestine, and required for efficient dietary iron absorption. Hp therefore plays a role in the uptake of iron from the diet, whereas Cp helps in the redistribution of iron from the liver and other internal organs.
Iron exits from the enterocytes of the small intestine into the circulation through an export pathway that involves both ferroportin1 (FPN1), the basolateral iron exporter (14–16), and Hp. This process is in turn regulated systemically by the liver-derived peptide hepcidin. Liver hepcidin mRNA expression is increased in mice with dietary iron overload (17), and disrupted hepcidin expression in mice leads to iron overload (18). A temporal association exists between a decrease in liver hepcidin mRNA levels and an increase in intestinal iron transporter gene expression (19,20). An in vitro study suggested that FPN1 is internalized in response to increased hepcidin levels (21), whereas another study showed that divalent metal transporter1 expression and apical transport were decreased by increased hepcidin levels but not FPN1 (22). Previous in vivo studies of copper deficiency are consistent with a functional defect in Hp (2) because iron absorption is impaired in copper-deficient animals, but there are no direct studies of the effect of copper deficiency on the Hp function. In this report, we investigate the effect of in vivo and in vitro copper deficiency on Hp and FPN1. While this manuscript was in preparation, a study by Gitlin et al. was published (23) that also examined the effect of copper deficiency on Hp protein levels in a cell culture model. We compare the results of these 2 complementary studies in the discussion.
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MATERIALS AND METHODS |
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Mouse tissue collection. At the end of the dietary treatment, mice were anesthetized with Aerrane after overnight food deprivation. Blood was collected by cardiac puncture and brain, heart, liver, spleen, kidney, upper small intestine, colon, and right tibia were dissected. Serum was collected by Serum-gel tubes (SARSTEDT catalog 41.1500.005). The copper and iron content of tissues was measured using a Vista AX Simultaneous ICP-AES (Varian) according to the nitric acid digestion method (25). The enterocyte isolation procedure was as described (25). Serum, liver, and enterocytes were snap-frozen in liquid nitrogen for subsequent RNA and protein analysis.
HT29 cell culture. The human colonic carcinoma cell line HT29 was cultured in McCoy's 5A Medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C with 5% CO2. For differentiation studies, HT29 cells grown to 70% confluence were treated with 5 mmol/L sodium butyrate for 72 h. Chelation of trace copper in HT29 cells with bathocuproine sulfonate (BCS) over a time course (2, 4, 8, and 24 h for nondifferentiated cells or 1, 3, 5, and 7 d for differentiated cells) was used to reduce intracellular copper levels. When required, additional copper was added as a 50 µmol/L copper-histidine complex (Cu-His) for 24 h to induce copper overload. Cell viability was quantified using trypan blue exclusion and was expressed as the percentage of viable cells relative to the control group. Before the analytical procedures described below, cultured cells were washed twice with ice-cold PBS and used immediately.
Superoxide dismutase (SOD1) activity assay. SOD1 activity was assayed using a SOD assay kit (Cayman Chemicals). Cells were collected by centrifugation at 1000 x g for 10 min at 4°C. Cell pellets were lysed in cold 20 mmol/L HEPES buffer, pH 7.2, containing 1 mmol/L EGTA, 210 mmol/L mannitol, and 70 mmol/L sucrose for 20 min. Cells were centrifuged at 1500 x g for 5 min at 4°C. Cell extracts were incubated with xanthine oxidase for 20 min, and the absorbance of the reaction mixtures was measured at 450 nm following the manufacturer's protocol. The protein concentration of the extracts was determined by the Bradford method (Biorad). For all of the SOD1 experiments, duplicate independent samples were tested.
Northern blot analysis. Northern blot analysis of mouse liver and enterocyte mRNA was conducted as described (25) using a mouse Heph probe corresponding to positions 2068–2861 (Genbank AF082567), a Cp probe corresponding to positions 268–788 (Genbank NM_007752), a Fpn1 (Ferroportin1 cDNA) probe corresponding to nucleotides 929–1605 (Genbank AF231120), a Hamp1 probe corresponding to positions 51–361 (Genbank BC021587), and a mouse ß-actin cDNA probe purchased from Clontech (catalog 5408–1). For the HT29 cell line, a Heph probe corresponding to positions 1981–2488 of the human gene (Genbank AF148860) was used. The ß-actin signal was used as a loading control.
Hp, FPN1 and ferritin antibodies. Polyclonal rabbit anti-mouse Hp IgG was raised to the C-terminal amino acids (QHRQRKLRRNRRSIL) of Hp as described in Chen et al. (25). Rabbit anti-FPN1 (CGKQLTSPKDTEPKPLEGTH) was made using the same protocol, and rabbit anti-mouse ferritin (recognizes both L and H subunits) purchased from Roche (catalog 605 022) was utilized. Peroxidase-labeled anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology.
Immunoblot analysis. Mouse enterocytes and treated HT29 cells were lysed as described (25). For all studies, except when FPN1 analysis was carried out, samples containing 100–200 µg protein were denatured by boiling for 5 min in 2X SDS sample buffer; however, samples for FPN1 Western blot analysis were not boiled (25) before electrophoresis. The proteins were separated by SDS-PAGE (7.5% acrylamide running gel) and transferred to nitrocellulose membranes. Blots were first incubated for 1 h with blocking buffer (containing PBS, 0.1% Tween-20, and 10% nonfat dry milk), and then incubated with primary antibodies for 1 h at room temperature. Primary antibodies were used at the following concentrations: 1:3000 for rabbit anti-Hp, 1:3000 for rabbit anti-FPN1, and 1:500 for rabbit anti-ferritin. Blots were then washed 3 times in 0.1% PBS-T, incubated for 1 h at room temperature with 1:40,000 diluted peroxidase-labeled anti-rabbit secondary antibodies, and signals were visualized by enhanced chemiluminescence.
p-Phenylene diamine oxidase activity assay. The oxidase activity of Hp was determined in mouse enterocytes and HT29 cells. Cells were washed and lysed as described above. Cell homogenates were centrifuged at 10,000 x g for 20 min to remove unlysed cells and nuclei. The clear lysate (50 µg of protein) was applied to a native nonreducing, nondenaturing 4–20% Tris-glycine PAGE gel (Invitrogen) and separated electrophoretically in native Tris-glycine electrophoresis buffer (25 mmol/L Tris, 250 mmol/L glycine). The gels were then incubated with 0.1% pPD in 0.1mol/L acetate buffer, pH 5.45, for 2 h and air-dried in the dark. Purified human Cp (Vital Products) was used as a positive control. For the in-tube assay, cell extracts (100 µg of protein) were incubated with 0.01% p-phenylenediamine in 0.1 mol/L acetate buffer (pPD) substrate in acetate buffer, pH 5.45, for 2 h at 37°C in the dark. Color development was monitored as absorbance at 530 nm. For all of the cell culture experiments, the studies were carried out in at least triplicate independent samples.
Ferroxidase activity assay. The ferroxidase-specific assay differs from the pPD gel assay only in the final assay step. The gels are placed for 2 h at 37°C in a fresh solution of 0.00784% Fe(NH4)2(SO4)2·6H2O in 100 mmol/L sodium acetate, pH 5.0. Gels were then rehydrated with 15 mmol/L ferrozine solution in the dark. Color development was then monitored continuously and quantified by scanning densitometry. Cp activity was detected with this in-gel assay and served as a positive control.
Statistical analysis. In all mouse experiments, 7–10 mice were tested individually. Significant treatment differences were determined by 1-way ANOVA and differences among groups by the Bonferroni post-hoc test. Data are presented as means ± SD. Differences were considered significant at P < 0.05.
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RESULTS |
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2-fold (P < 0.05) in mice fed the copper-deficient diet (Fig. 2C).
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0 after 24 h of 40 µmol/L BCS treatment. In differentiated confluent HT29 cells, Hp protein levels and pPD activity were decreased by copper chelator treatment (Fig. 4C).
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DISCUSSION |
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Previous investigations demonstrated that nutritional copper deficiency leads to defects in iron metabolism including anemia and increased intestinal and liver iron (3,37,38). The increased liver iron can be explained by an effect of copper deficiency on Cp function (9,10,39–41). As in other studies, we found that copper deficiency does not affect Cp mRNA expression (42,43) but dramatically decreases enzymatic activity. In this study, we showed that in vivo copper deficiency did not significantly alter Heph mRNA expression, yet Hp protein and ferroxidase activity were dramatically reduced. In contrast, FPN1 mRNA expression and protein levels were increased. Because we showed previously that FPN1 expression (25) increases in response to iron deficiency, we suggest that the FPN1 expression changes in copper deficiency represent a secondary response to the iron deficiency. The increased FPN1 mRNA and protein levels in the enterocytes of copper-deficient mice are consistent with the hypothesis that the enterocyte responds to a systemic signal of iron deficiency (with reduced hepcidin levels as the strongest candidate) rather than local cues (25,44). Indeed, we noted decreased expression of Hamp1 in the liver of the copper-deficient mice. Our results are in agreement with recent studies that have also noted decreased Hp protein levels in rats (45,46) and cell culture (23) in the presence of copper deficiency. Reeves et al. (45,46) also found increased iron in duodenal enterocytes. In 2 additional studies in rats (47) and mice (48), Cp activity and hematocrit were reduced and liver iron increased in copper deficiency as seen in our study and by Reeves et al. (45,46); in contrast, duodenal mucosal iron and ferritin were reduced. Similarly, in those 2 studies, there was no change in duodenal mucosal FPN1 expression, whereas we found a dramatic increase in FPN1 protein levels. We suggest that the differences could arise from the use of whole gut in those studies because it contains a variety of cell types. In our work and that of Reeves et al. (45,46), isolated duodenal enterocytes were used.
We hypothesize that copper deficiency chronically impairs Hp activity, which leads to inefficient basolateral intestinal iron export as evidenced by increased enterocyte ferritin levels and systemic iron deficiency and anemia. The systemic iron deficiency and anemia lead to decreased hepatic Hamp1 expression despite the increased hepatic iron levels (49,50). A reduction in hepcidin expression would manifest itself in the gut as increased FPN1 expression (and presumably persistent basolateral localization) in an attempt to increase iron absorption (21). Our in vivo findings provide an explanation for the longstanding observation that copper deficiency results in defects in intestinal iron export (2).
We also found that copper deficiency does not affect Heph transcript levels in HT29 cells but results in dramatic decreases in Hp protein and pPD oxidase activity. An elegant study by Nittis and Gitlin also found decreased protein levels in copper deficiency in another cell culture cell line, T84, and in MDCK cells transfected with the human cDNA (23). In addition, they demonstrated that this decrease in Hp protein level results from increased proteosome-mediated degradation of Hp protein in copper deficiency, likely related to decreased stability of apoHp in copper deficiency. Together these studies provide compelling evidence that copper deficiency does not change mRNA levels, but decreases Hp protein levels and subsequently oxidase activity.
The mechanisms of copper loading into copper proteins in the secretory pathway are not known. Proper assembly of copper into the primarily cytosolic SOD1 requires the copper chaperone for SOD1 (CCS) (48), but evidence for similar mechanisms for secreted or membrane-bound copper proteins is lacking. In fact, copper is likely not necessary for the proper folding or assembly of most copper proteins, including the multicopper oxidases, nor does it dramatically alter their subsequent stability (49,50). Genetic or nutritional copper deficiency does not change the levels of inactive apoCp (42,51), although the protein has a decreased plasma half-life (52,53). In contrast, the finding that copper deficiency dramatically decreased Hp protein levels suggests an unexpected and unusual link between copper availability and protein stability.
The study by Nittis and Gitlin (23) and our study suggest that copper may be required for the stability of apoHp synthesized under low-copper conditions. In the other multicopper ferroxidases, site-directed mutagenesis studies of Cp and Fet3p support an "all or none" mechanism of copper incorporation into these proteins (54). Mutation of a single copper binding site prevents loading of copper into any site, suggesting a cooperative mechanism of copper loading. Our previous finding of a partially active Hp protein in sla mice, which is missing an entire T1 Cu domain, suggests more independence of the copper sites (12) in Hp. Alternatively, the removal of an entire copper site may allow the remaining sites to cooperatively bind copper. In contrast to Cp and Fet3p, mutations of the copper-binding residues in tyrosinase lead to dramatic decreases in protein levels (55) even though copper deficiency does not (49). Given the close structural similarity of Hp and Cp (56), it is not clear why one apoprotein would be more "unfolded" in the absence of copper and recognized by this pathway, but not the other. Because copper is presumably delivered to the apoHp in the Golgi compartment, such a mechanism requires retrograde Golgi-ER communication/transport as was demonstrated previously (57). The inhibition of degradation of Hp by brefeldin A or bafilomycin A1 in the study by Nittis and Gitlin (23) supports such a hypothesis. Regardless of the mechanism, these results indicate a clear difference in the synthetic and post-translational modification pathways of Hp and those of Cp and Fet3p.
Another possibility is that the decreased protein levels reflect the copper-regulated degradation of Hp. Excess copper stimulates proteasome-dependent degradation of mouse CCS, whereas copper deficiency increases CCS protein but not mRNA levels (58). Similarly, copper stimulates the degradation of Ctr1p in yeast (59). Because Hp has no known role in copper metabolism, the rationale for copper regulation is unclear. Clearly, further study is warranted to determine whether Hp has a role in copper metabolism and whether the observed decreased protein levels are reflective of a copper-related regulatory process.
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FOOTNOTES |
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3 Abbreviations used: BCS, bathocuproine sulfonate; CCD, copper chaperone for SOD1; Cp, ceruloplasmin; FPN1, ferroportin1; Hp, hephaestin; pPD, 0.01% p-phenylenediamine in 0.1 mol/L acetate buffer; SOD, superoxide dismutase.
Manuscript received 11 October 2005. Initial review completed 31 October 2005. Revision accepted 6 February 2006.
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