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Department of Chemistry and Biochemistry and Institute for Molecular Biology and Nutrition, California State University, Fullerton, CA 92834-6866
3To whom correspondence should be addressed. E-mail: mlinder{at}fullerton.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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30%. In repeated studies, no differences in the expression of hepatic ceruloplasmin mRNA were detected. Treatment of rats of both sexes with additional iron (25 mg as iron dextran) 5–14 d before killing increased liver ferritin but did not alter liver ceruloplasmin mRNA expression or levels of circulating ceruloplasmin. We conclude that iron status is not an important factor in the expression of plasma ceruloplasmin made by the liver. However, it does have modest effects on steady-state levels of liver ferritin mRNA.
KEY WORDS: • iron deficiency • ceruloplasmin expression • ferritin expression • iron treatment • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). A major historic explanation, first proposed by Frieden (4
,5
), has been that iron accumulates in response to a lack of active ceruloplasmin in the circulation. This is based upon two kinds of experimental observations. First, ceruloplasmin, an 
2-glycoprotein that accounts for about two thirds of the copper in human blood plasma (6
), has the ability to oxidize Fe(II) (7
,8
). It is thus also known as a ferro-O2-oxidoreductase (EC 1.16.31), or ferroxidase I. This enables it to convert reduced iron released from storage sites (such as ferritin) to the oxidized form. This, in turn allows it to bind to its plasma transport protein, transferrin. Second, copper deficiency leads to a severe reduction in plasma ceruloplasmin concentrations, and in this condition, the intravenous administration of copper-containing (and thus ferroxidase-active) ceruloplasmin immediately results in release of iron into the circulation from the liver (9
–11
). The latter was demonstrated convincingly many years ago by two different laboratories, using perfused livers of pigs (9
) and dogs (10
). Other forms of copper or related factors did not have the same rapid effect (11
,12
), confirming the specificity of ceruloplasmin. Nevertheless, promotion of iron efflux from cells is not the only function of ceruloplasmin (13
,14
), and normally, the concentration of this protein in the plasma is orders of magnitude higher than what is needed to maintain iron release (12
). Moreover, in inflammation, ceruloplasmin synthesis and blood levels increase at the same time that iron transport between tissues is markedly reduced (15
,16
). Thus, except in severe copper deficiency, there appears to be no relationship between plasma ceruloplasmin concentrations and plasma iron transport.
The recent discovery of some families with aceruloplasminemia has led to findings about iron supporting a role of ceruloplasmin in cellular iron efflux. Individuals that genetically cannot express ceruloplasmin (for which there is a single gene) gradually acquire an excess of iron in many of their tissues (17
–19
).
The story of the involvement of ceruloplasmin in iron efflux and oxidation seemed to be straightforward until a few years ago, when the components of the iron uptake system of yeast began to be identified. In the process, it was found that a membrane bound protein (FET3), in the family of the blue copper proteins to which ceruloplasmin belongs, was necessary for iron uptake (20
–23
). Additional findings reported in 1999, using human hepatoma cells (HepG2) (24
) and then also K 562 cells (25
), indicated that ceruloplasmin could indeed enhance iron uptake. However, this occurred only when the cells were made iron deficient. Also, only the uptake of nontransferrin bound iron was enhanced, and uptake appeared to involve a trivalent cation transporter (25
). Most strikingly, iron deficiency doubled the levels of ceruloplasmin mRNA in the hepatoma cells, and there was evidence that this was accompanied by increased iron uptake by the cells. Thus, there would appear to be solid evidence on both sides, that 1) ceruloplasmin promotes efflux of iron from the liver in vivo in animals, and 2) it promotes influx of iron into iron-deficient cells.
These apparent contradictions prompted us to examine whether observations made with cultured malignant cells would apply in vivo, in whole animals. The results reported here for studies in rats indicate that neither iron deficiency nor excess appears to affect the expression of ceruloplasmin mRNA in the liver or the level of circulating ceruloplasmin in the blood plasma, although there may be small effects on its oxidase activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Weanling male and female Sprague-Dawley rats from Simonson Laboratories (Gilroy, CA) were made iron deficient by feeding them (usually in groups of 10 at a time) a pelleted, starch-based low iron diet for 6–8 wk along with distilled water. The diet was designed by E.A. Ulman in accordance with AIN guidelines (26
) and produced by Research Diets (New Brunswick, NJ). It contained 5 mg/kg Fe, in a mixture of 200 g/kg protein (casein, +3g/kg DL-methionine), 660 g/kg carbohydrate (45% cornstarch, 10% maltodextrin, 10% sucrose, 5.0% cellulose), 50 g/kg fat (corn oil), 35 g/kg salt mix S18101 without added iron, 10 g/kg vitamin mix V10001 (+2 g/kg choline bitartrate). Iron deficiency was reversed in half of each group of rats by intraperitoneal injection of 5 mg of iron as iron dextran (Iron Dextran injection, Phoenix, St. Joseph, MO) after 2–3 and sometimes also 4–5 wk of treatment. In some cases, rats reared on normal diets were, or were not, injected with excess iron (25 mg in the form of iron dextran) 5 d before killing. Some other normal rats were acutely depleted of iron by bleeding from the tail vein and used 24–48 h later. Iron-deficient rats ate less and had lower body weights than the iron-normal and excess iron–treated rats. Actual food intakes were not recorded. Body weights (mean ± SD) for the three types of rats were 238 ± 35 g (n = 22) for iron-deficient rats; 257 ± 22 g (n = 42) for iron-normal rats; and 285 ± 35 g (n = 19) for rats treated with excess iron. Rats were killed by exsanguination under pentobarbital anesthesia, as previously described (14
). The university’s institutional review board for care and use of animals in research approved all protocols. Hematocrits were measured in capillary tubes after centrifugation. Livers were immediately frozen in liquid N2 before storage at -85°C. Blood plasma was collected and stored at -20°C.
Measurements of ferritin-iron.
This was determined as previously described (27
). In brief, liver homogenates (100 g/L) were made so that the final concentrations of added components were 10 mmol/L K phosphate, pH 7.0, and 0.02% NaN3. Heat supernatants were prepared after heating to 70°C for 10 min. Portions of the heat supernatant were titrated with specific rabbit antibody against horse spleen ferritin (Sigma Chemical, St. Louis, MO), which cross-reacts with, and precipitates, rat liver ferritin (27
). Precipitates were washed twice with cold 10 mmol/L PBS, pH 7, and precipitates were assayed for iron with ,'-bipyridyl, upon reduction with bisulfite in a boiling water bath (27
).
Copper and ceruloplasmin determinations.
Copper was determined by furnace atomic absorption spectrometry, using a Varian Zeeman 800 instrument (Sugarland, TX), as described previously (28
). The copper solution used as the standard was obtained from Fisher Scientific (Fair Lawn, NJ). Plasma/serum samples were assayed directly; liver samples were wet-ashed in trace-element grade nitric acid (Fisher Scientific) plus H2O2 before analysis. Ceruloplasmin oxidase activity was determined with p-phenylenediamine, as previously described (28
).
RNA extraction and Northern analysis.
Total RNA was extracted from liver tissue with RNAzol B (Tel-Test, Friendswood, TX). The purity of RNA was assessed by ratios of absorbance at 260:280 nm 
1.8. Portions of total RNA (20–40 µg) were separated in formaldehyde gels containing 1.0 or 1.2% agarose (29
) and transferred to nylon membranes (Zeta-Probe; BioRad, Hercules, CA) by capillary action. Membranes were hybridized with random-primer labeled cDNA plasmid inserts, using 32P-dCTP and the RapidHyb reagents and protocol of Amersham Pharmacia Biotech (Piscataway, NJ). The rat ceruloplasmin plasmid containing a 2 kb insert was provided by Dr. Julian Mercer (Deakin University, Burwood, Victoria, Australia). The rat ferritin L and H plasmids were obtained from Dr. Elizabeth Leibold (University of Utah, Salt Lake City, UT). After high stringency washing (once in 2X SSC, 0.1% SDS, 20 min at room temperature, then twice in 0.1 SSC, 0.1% SDS, at 42° for 15 min), membranes were exposed to X-ray film (Kodak X-Ormat, Rochester, NY) for autoradiography. Loading was assessed by comparing ethidium bromide staining of 28 S and 18 S rRNA bands on the gels and membranes (evaluated by densitometry) and directly by hybridizing the same membranes and/or parallel membranes with cDNA for rat GPDH or 18 S rRNA, as previously described (30
). Densitometry was performed on data obtained from the autoradiographs with the SpeedLight Photoimaging System (San Diego, CA), using Adobe Photoshop software.
Statistical methods.
The significance of differences between means for groups of rats treated in parallel was assessed by ANOVA and Duncan’s New Multiple Range Test. Data are presented as means ± SD. Differences with P < 0.05 were considered significant. Only differences that were significant are mentioned in the text.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The iron status of sets of male rats from five different experiments are summarized in Table 1
. The "iron-normal" rats (see Methods) had normal hematocrits and normal amounts of ferritin iron and ferritin protein, based on values reported previously (31
). In contrast, rats fed the low iron diet without iron supplementation were severely anemic, with hematocrits less than half normal and no detectible ferritin in the liver. Treatment of the iron-normal rats with excess iron did not alter hematocrits of male or the female rats so treated (Table 1)
, but it increased the concentrations of liver ferritin iron and ferritin protein about threefold in the males.
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Iron status had little effect on plasma copper and ceruloplasmin (Table 2
). The deficient rats had 30% lower levels of ceruloplasmin oxidase activity, but there was no difference at the level of ceruloplasmin antigen, determined by rocket immunoelectrophoresis. Plasma copper concentrations were also 30% lower (which would account for the lower oxidase activity). However, treatment with extra iron also slightly (18%) but significantly decreased ceruloplasmin oxidase activity in the male rats, with no alterations in ceruloplasmin antigen concentration. In female rats, iron treatment did not affect either variable, and in rats of both sexes, iron treatment did not change plasma copper concentrations. Within the iron-deficient groups, a direct comparison of ceruloplasmin levels and hematocrits of individual rats did not reveal any correlation. Thus, iron status made very little difference to the levels or activity of plasma copper and ceruloplasmin.
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–35
), we found that iron deficiency increased concentrations of liver copper in male rats (Table 2)
. Indeed, copper concentrations were about twice normal, which represents a major change. Treatment of female rats with extra iron, however, had no effect. Expression of ferritin L and H mRNA in livers of rats with different iron status.
Expression of ferritin mRNA was also affected by iron status. This was not expected because ferritin protein expression is regulated by iron primarily at the translational level and ferritin mRNA is fairly stable (36
–38
). The example in Figure 1A
shows that severe deficiency (D) lowered the level of ferritin L mRNA compared with normal rats (N). Similar data were obtained for ferritin H mRNA (data not shown). Based upon densitometry, the difference was about twofold (P < 0.01), and the degree of difference was essentially the same whether densities for the ferritin mRNA bands were compared with those for GPDH or 18 S rRNA, or with the density of the ethidium bromide–stained 18 S and 28 S rRNA detected on the membranes after transfer. Rats with and without extra iron were also examined. As shown by the data in Figure 1
B, there appeared to be a positive relationship between ferritin mRNA levels and iron availability, in this case showing an example for liver ferritin H mRNA. The same kind of relationship was seen with ferritin L (data not shown). To our knowledge, these are the first observations that concentrations of liver ferritin mRNA vary in relation to chronic differences in nutritional iron status.
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Total liver RNA was also hybridized with cDNA for rat ceruloplasmin. Although our initial experiments suggested that there was an enhancing effect of iron deficiency, repeated further studies (presented here) failed to confirm this finding. Instead, no consistent, significant effect on ceruloplasmin mRNA levels could be documented. Some examples of the Northern blots obtained are given in Figures 2
and 3, in which data for normal and deficient and normal and excess iron–treated rats are compared, respectively. Clearly, there was little or no difference in expression when the severely iron-deficient and normal rats were compared (Fig. 2
A and B), taking into consideration some differences in RNA loading (in this case based on GPDH). Densitometric scans on numerous Northern blots (corrected for loading with either GPDH or 18 S rRNA) indicated that expression was essentially identical. Measurement of RNA from deficient and normal livers (as a percentage of values for normal liver in the same blot) gave means (± SD) of 97 ± 13% (n = 18) and 100 ± 6% (n = 19), respectively.
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. Livers of rats given extra iron (X) had about the same proportion of total RNA (here indicated by the ethidium bromide fluorescence of the membranes after transfer) hybridizing with ceruloplasmin cDNA as the normal rats (N; Fig. 3
A and B). Densitometric results for 14 Northern blots (corrected for loading by comparison with rRNA or GPDH) indicated no significant difference, with values (means ± SD) of 96 ± 13 and 100 ± 12% of normal values in the same blot for the iron-treated and iron-normal rats, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
,17
19
). The ferroxidase activity of ceruloplasmin (8
,10
), coupled with reports from two different laboratories that intravenous infusion of ceruloplasmin leads to release of iron into the circulation (9
–12
), established the concept that one of the functions of this copper-containing plasma protein is to aid in the release of iron from liver cells and perhaps also from cells of other tissues (1
,4
,5
). Evidence that many cells have specific receptors for ceruloplasmin in their plasma membranes (1
,39
–41
), plus the recent findings of some glycosylphosphatidylinositol-anchored ceruloplasmin (at least in the brain) (42
,43
), support the idea that ceruloplasmin aids the release of iron from cells at the cell membrane by oxidizing Fe(II), i.e., iron fluxing in and out of cells and its storage molecule, ferritin, is likely to be in the ferrous form because, at least in the test tube, ferritin iron is released only upon reduction and chelation (27
,3
7
), but the bulk of the iron carried in the blood is in the ferric form, bound to transferrin. How the iron might be traversing the cell plasma membrane on its way out, however, is still far from clear, although evidence for the existence of an ATPase that could be involved has just been reported (44
).
In yeast, a copper binding protein with similarities to ceruloplasmin is also involved in transmembrane iron transport (20
–23
). However, in this case, it plays a role in iron uptake rather than efflux. Iron transport and metabolism in yeast differ from those in mammalian cells in other ways as well. Yeast takes up inorganic iron and transfers it across the cell membrane through a membrane channel (20
–23
), whereas mammalian cells take most (or all) of their iron from transferrin and through receptor-mediated endocytosis (45
). Mammalian cells store iron in ferritin; yeasts are perhaps unique among organisms in not making ferritin. Reports that, at least in cell culture, the iron status of mammalian cells would influence expression of ceruloplasmin (24
,25
), and that ceruloplasmin stimulated uptake of iron by these cells, were therefore intriguing, suggesting that under certain conditions at least, mammalian cells might act like yeast, and that ceruloplasmin might play a role in both uptake and release of iron by cells in mammals.
The results reported here indicate that in a whole-animal model, iron status has little or no influence on the levels and activity of plasma ceruloplasmin, nor on expression of its mRNA by the liver, which makes this plasma protein. Looking at the full spectrum, from severe iron deficiency to levels of excess iron several times those of the normal control, there were no apparent differences in liver ceruloplasmin mRNA concentrations, nor were there differences in the levels of circulating ceruloplasmin protein, determined by immunoassay. Thus, in contrast to what was reported for HepG2 ad K562 cells in culture (24
,25
), iron deficiency in rats did not increase the expression of ceruloplasmin mRNA, nor did it increase levels of ceruloplasmin in the blood plasma. The reverse, namely, a potentially depressing effect on ceruloplasmin expression by excess iron, was not observed either. Although the iron-deficient rats did not eat as much and were smaller, it seems unlikely that the calculated lack of an increase in ceruloplasmin mRNA levels in the liver was due to a coincident increase in total RNA that exactly balanced a change in ceruloplasmin. Malnutrition usually results in decreases in liver RNA (per g) (46
), and this would have amplified, rather than neutralized, any increase.
It is worth noting, however, that there appeared to be some differences in ceruloplasmin oxidase activity relating to iron status. Oxidase activities (but not levels of ceruloplasmin protein) were slightly lower (18%) in the male rats treated with excess iron. They were also somewhat lower, however, in the rats with iron deficiency (30%). The reasons for these discrepancies between ceruloplasmin oxidase activity and levels of ceruloplasmin protein (measured by rocket immunoelectrophoresis) are unclear and remain to be explored. However, somewhat lower than normal ceruloplasmin oxidase activities in the plasma of rats with excess iron have previously been reported (47
–49
). A similar, small decrease in plasma ceruloplasmin levels was reported by Cairo et al. (50
) for patients with hemochromatosis, this time also using immunoassays. However, in the same study, a group of subjects with nongenetic iron overload had normal ceruloplasmin levels. In the absence of changes in ceruloplasmin protein concentrations, changes in ceruloplasmin oxidase activities can occur indirectly, particularly when the redox state is perturbed. For example, high intakes of ascorbate by humans will reduce the ceruloplasmin oxidase activities measured in the plasma (51
) without changing levels of ceruloplasmin protein. The upregulation of ceruloplasmin expression in cultured cells by iron deficiency (24
,25
) could be the result of the activation of hypoxia-inducible factor (HIF). Hypoxia (and the resulting activation of HIF-regulated enzymes and other proteins) is another important regulator of iron transport and metabolism (52
–54
), including red blood cell production and iron absorption (which are increased by HIF activation). It is now clear that the ceruloplasmin promoter contains a HIF-responsive element, indicative of HIF-regulation (55
). Alternatively, or in connection with HIF, the upregulation of ceruloplasmin observed in tissue culture may have resulted from very low levels of iron achievable only in culture and not in vivo. (Levels of iron in the cultured cells were not reported.)
Our findings of lower levels of ferritin L and H mRNAs in livers of the iron-deficient rats are consistent with the need for less ferritin production in conditions in which there is little or no excess iron to be stored. Our current studies indicate that mRNA levels were about double those present in rats with normal iron nutriture. To our knowledge, an examination of potential effects of chronic iron deficiency on ferritin mRNA expression has not been previously examined. Effects of chronic excess iron have also not been investigated before. Thus, although iron regulates levels of ferritin protein synthesis primarily by acutely altering rates of translation of its mRNA (36
–38
), and inhibitors of transcription do not block increased ferritin synthesis and accumulation (55
–57
), our data indicate that there are modest differences in the "stable" levels of ferritin mRNA related to the extremes of nutritional iron status. These findings are also consistent with earlier reports that, apart from enhancing translation of existing ferritin mRNAs, iron increases their rate of transcription (56
–59
). This might result in higher steady-state levels of ferritin mRNAs in tissues of rats with excess iron, and the lower levels in iron deficiency.
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FOOTNOTES |
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2 Supported in part by NSF RUI grant MCB 973745 and National Institutes of Health grant DK53080.
Manuscript received 30 April 2001. Initial review completed 19 July 2001. Revision accepted 26 November 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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