Iron is essential to all cells due in part to its role in numerous redox reactions. The element is potentially toxic, however, because it can mediate uncontrolled oxidative damage(1) . Cells temper the activity of intracellular iron by sequestering it within the shell of the iron storage protein, ferritin. Ferritin can protect cells by sequestering up to 4500 iron atoms within the complex ferritin shell (2) . This ubiquitous protein, with an approximate molecular mass of 450 kDa, is composed of a mixture of 24 light (L) and heavy (H) subunits 19 and 21 kDa, respectively. Ferritin is structurally conserved, with high sequence homology in both animals and plants indicating evolution from a common ancestral gene(3) . The role of iron in the regulation of ferritin synthesis has been intensively investigated in cultured cells (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . The short term regulation of ferritin synthesis is largely translational, involving a specific protein-mRNA interaction. The regulatory components are (i) the iron-responsive element (IRE), ()a stem-loop in the mRNAs of ferritin, the transferrin receptor and erythroid -aminolevulinate synthase, and (ii) the IRE-BP, a 90-kDa binding protein, abundant in cytoplasm(14, 15, 16) . The interaction of the IRE-BP and IRE is modulated by an iron sulfur cluster located near the center of the protein(17) . The structural integrity of the iron-sulfur cluster depends upon the iron content of the cell. Iron depletion promotes binding of the IRE-BP to the IRE. When the IRE stem-loop is located in the 5`-UTR, as with the ferritin and erythroid -aminolevulinate synthase messages, translation of the mRNA is repressed. In contrast, IRE-BP binding to the IRE in the 3`-UTR of the transferrin receptor message protects the mRNA from degradation(20, 21, 22) . In iron-replete cells, the IRE-BP contains a cubane 4Fe-4S cluster that prevents binding to the IRE (18, 19, 20) . The iron-replete IRE-BP has aconitase enzymatic activity and a very low affinity for the IRE. In contrast, IRE-BP bound to the IRE lacks aconitase activity.

Ascorbate stimulates iron uptake from food, induces iron release from the ferritin shell, and prevents ferritin autophagy by lysosomes, thereby retarding the degradation of this iron storage protein (10, 11, 12) . However, ascorbate has a plethora of effects on other aspects of cell metabolism. First purified by Szent-Györgyi in 1928(23) , ascorbate (vitamin C) serves as a cofactor in numerous enzymatic reactions and affects the turnover of an array of proteins(13, 24) . Ascorbate is required for optimal activity of various enzymes involved in hydroxylation reactions associated with collagen formation, carnitine and norepinephrine synthesis, tryptophan, tyrosine, histamine, and cholesterol metabolism(24) . Ascorbate, like vitamin E, uric acid, and glutathione, directly protects cells from oxidative damage. The vitamin is a potent competitive inhibitor of carcinogenic nitrosoamine formation. Primates (including humans), guinea pigs, the Indian fruit bat, and some fish fail to produce ascorbate due to L-gulono--lactone oxidase conversion into a pseudogene by repeated mutations(25, 26) .

Cells in culture contain very little ascorbate since the vitamin is extremely labile in solution and is not added to most culture media (12) . We have now found that iron-mediated activation of ferritin message translation is markedly enhanced in cells grown in medium containing physiological concentrations of ascorbate relative to those grown under standard culture conditions. Base-line ferritin synthesis in cells that are replete or deficient in ascorbate is identical. Most earlier work on ferritin synthesis in tissue culture was done with ascorbate-depleted cells. The fact that iron metabolism in general, and ferritin synthesis in particular, is profoundly affected by ascorbate means that work must now be interpreted cautiously. Iron metabolism in intact animals is profoundly modified by ascorbate. We now demonstrate that the same is true for cells in culture.

MATERIALS AND METHODS

Cell Culture

Quantitation of the Steady State Level of Ferritin

Metabolic Labeling and Immunoprecipitation of Newly Synthesized Ferritin Molecules

Transient Transfection of K562 Cells

Metabolic Labeling and Immunoprecipitation of Newly Synthesized CAT Molecules

CAT Activity

Luciferase Activity

Northern Blot Analysis

RESULTS AND DISCUSSION

The human cell lines Hep3B (hepatoma) and HepG2 (hepatoblastoma) are relatively well differentiated, with regulated expression of many proteins, including ferritin(31, 32, 33) . They are, therefore, ideal reporter systems in which to assess iron-related cellular responses. As expected, steady state ferritin levels increased substantially in Hep3B or HepG2 cells loaded with iron and decreased when intracellular iron was chelated with desferrioxamine (Table 1, rows 1, 3, and 4). K562 cells responded similarly.

In contrast to the 16 h loading with iron (Table 1, row 3), a 2-h iron-treatment was insufficient to increase the levels of cellular ferritin (Table 1, row 2). However, if the cellular ascorbate deficiency was first corrected by supplementing the growth medium with physiological levels of the vitamin for 16 h, the ferritin content of the cells rose substantially with a 2-h iron pulse (Table 1, row 7). Correcting the cellular ascorbate content alone did not alter ferritin levels (Table 1, row 5). The increase in cellular ferritin content that occurred in ascorbate-deficient cells in response to 16 h of iron loading (Table 1, row 3) was augmented by ascorbate repletion for 2 h (Table 1, row 6). Ferritin content was unchanged in cells supplemented for 2 h with ascorbate that were not preloaded with iron (data not shown).

We concluded from these data that while ascorbate repletion alone did not affect cellular ferritin content, it potentiated the cellular response to iron-loading. Iron stimulation that was below the effective concentration in ascorbate-deficient cells significantly increased the ferritin content of ascorbate-replete cells (Table 1, rows 2 and 7). Ascorbate-deficient cells that had achieved a peak increase in ferritin content with prolonged iron loading increased ferritin levels further after a mere 2 h of ascorbate supplementation (Table 1, rows 2 and 6).

Earlier work in our laboratory (10, 11, 12) showed that ascorbate stabilizes cellular ferritin by retarding lysosomal autophagy of the protein. To determine whether the large increase in ferritin content in ascorbate-replete cells treated with iron was due only to stabilization of ferritin protein, we pulse labeled the cells with [S]methionine. The rate of ferritin synthesis increased by greater than 10-fold in the leukemia cells (Fig. 1A) and by more than 50-fold in hepatoma cells (Fig. 2A) cells treated for 16 h with ascorbate and subsequently pulsed for 2 h with iron. Loading cells with iron for 16 h increases ferritin synthesis (Fig. 1B and 2B). Iron loading followed by a 4-h incubation with ascorbate greatly enhanced the effect. Ascorbate alone does not alter ferritin synthesis after either 2 or 4 h (Fig. 2B). Time course experiments indicated that enhancement of iron-induced ferritin synthesis was negligible with a 30-min ascorbate pulse and increased to maximal values by 3-4 h (data not shown). In aggregate, these data indicated that enhanced ferritin synthesis was a major contributor to the higher ferritin content of ascorbate-replete cells treated with iron. The time delay to reach a maximum effect on ferritin synthesis after ascorbate treatment of iron-loaded cells suggested that cellular repletion with ascorbate was required. If, for instance, ascorbate worked merely to mobilize iron adsorbed nonspecifically to the plasma membrane, no time delay in the upturn in ferritin synthesis should occur.

Figure 1: The effect of iron and ascorbate on de novo ferritin biosynthesis in K562 human erythroleukemia cells. The cells were maintained in RPMI 1640 medium supplemented with 10% newborn calf serum, penicillin, and streptomycin in a density of 5 10 cells/ml. A, extended ascorbate pretreatment and iron-induced ferritin synthesis; 10 cells were incubated with 150 µM ascorbate (AA) for 16 h. The ascorbate was washed out and followed by a 10 µg/ml ferric ammonium citrate (Fe) treatment for 2 h. B, ascorbate effect on iron-loaded cells. Cells were incubated with 10 µg/ml ferric ammonium citrate for 16 h. The cells were washed free of iron and subsequently incubated with 150 µM ascorbate for 4 h. C, the effect of 150 µM dehydroascorbate (DHA) on iron-induced ferritin synthesis. After the treatments, the cells were metabolically labeled with [S]methionine, immunoprecipitated, and separated on a 15% SDS-phosphate-urea gel. All treatments were repeated several times, and a representative result is shown.

Figure 2: The effect of iron and ascorbate on de novo ferritin biosynthesis in Hep3B human hepatoma cells. Hep3B cells were maintained in -Dulbecco's modified Eagle's medium supplemented with 10% bovine serum, penicillin, streptomycin, and L-glutamine. 10 cells were treated as follows. A, the effect on ferritin synthesis induced by 5 µM ferrotransferrin (FeII)Tf or 10 µg/ml ferric ammonium citrate (FeIII) on cells pretreated with 150 µM ascorbate (AA). B, time-dependent effect of ascorbate on ferritin synthesis by iron-loaded cells. C, time-dependent effect of 150 µM dehydroascorbate (DHA) on ferritin synthesis by iron-loaded cells. After the treatments and metabolic labeling with [S]methionine, the cells were washed, lysed in situ, and equal amounts of protein were subjected to immunoprecipitation and SDS-polyacrylamide gel electrophoresis as detailed above. All treatments were repeated several times in duplicate, and representative results are shown.

Many ascorbate-catalyzed reactions depend on the oxidative state of the vitamin as it cycles between the reduced (ascorbate) and oxidized (dehydroascorbic acid) forms(34) . Semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase are two of the enzymes that maintain cellular ascorbate in a reduced state(23) . A nonenzymatic, glutathione-dependent reduction of dehydroascorbate is also important(35) . Fig. 1C and Fig. 2C show that dehydroascorbate potentiates ferritin synthesis. Dehydroascorbate is taken up by cells and can be reduced to ascorbate(10) . The rate of reduction is slow in K562 cells, however. Our data suggest that the oxidative state of ascorbate taken up by the cells may not be critical to the effect on ferritin synthesis. This observation was in accord with experiments in which guinea pigs maintained on ascorbate-deficient diets were protected from scurvy when they received either ascorbate or dehydroascorbate dietary supplements(34) .

The ascorbate-enhanced ferritin response was more pronounced in the two hepatoma cell lines (only the Hep3B response is shown) than the K562 cells (Fig. 1), irrespective of whether the iron was delivered to the cells as ferrotransferrin or ferric ammonium citrate (Fig. 2A). The physiological concentration of 5 µM differic transferrin was as effective as 10 µg/ml ferric ammonium citrate, in concordance with other reports(4, 19, 36) . Despite quantitative differences between the hepatoma and leukemia cells, the effect of ascorbate and iron on ferritin synthesis was qualitatively identical. These data indicated that the ascorbate effect was not limited to a particular cell lineage, raising the possibility that this is a universal phenomenon. Further, these observations support the conclusion that de novo ferritin synthesis produces most of the increase in ferritin content in ascorbate-replete cells that are challenged with iron.

Ferritin gene expression is controlled by several mechanisms, some of which are specific to certain cell types(3) . In plants, iron modulates ferritin gene expression largely by changes in transcription. In contrast, changes in the rate of translation of ferritin mRNA accounts for most of the effect of iron on ferritin synthesis by animal cells (37) . During the 16-h period of ascorbate repletion, ferritin mRNA levels within the cells could have risen due either to increased mRNA synthesis or RNA stabilization. In this case, the enhanced ferritin synthesis seen with subsequent iron challenge would have merely represented translation of the greater amount of mRNA. In contrast, static levels of ferritin mRNA would suggest that ascorbate enhances iron-dependent ferritin synthesis by increasing the efficiency with which the message is translated.

Northern blot analysis of mRNA from hepatoma cells treated with ascorbate, iron or ascorbate plus iron (Fig. 3) showed ascorbate, at most, modestly affected L-ferritin mRNA levels. Iron increased the message level by 2-fold, while message levels rose by 3-fold in cells treated with the combination of ascorbate and iron. These changes are negligible relative to the more than 50-fold increase in protein synthesis (Fig. 2). These data suggest that ascorbate promotes ferritin synthesis by increasing the efficiency with which the message is translated. In this construction, ascorbate is a ``facilitator'' of ferritin mRNA translation. Nuclear run-on assays are in progress to verify the actual transcriptional activity in cells treated with iron, ascorbate, or iron plus ascorbate.

Figure 3: Northern blot analysis of Hep3B cells. Total RNA was isolated from Hep3B cells treated with 10 µg/ml ferric ammonium citrate (Fe) or 150 µM ascorbate (AA) using the RNAzol method. 20 µg of RNA were separated on 1% agarose formaldehyde gel and immobilized to ICI nylon membrane using the capillary method. A 670-bp PstI fragment of pSV2L-ferritin was labeled by the random primer method for L-ferritin (lane A), while a 600-bp PstI fragment of pSV2H-ferritin was used for H-ferritin (lane B). Lane C was probed with chicken -actin cDNA.

Iron modulates ferritin mRNA translation by altering the interaction of the IRE-BP with the IRE stem loop in the 5`-UTR of the message. Ascorbate could alter iron-induced ferritin synthesis solely through effects on the IRE. To examine this possibility, K562 cells were transfected with CAT reporter gene constructs harboring an intact IRE within the full-length 5`-UTR (5`-UTR-CAT) or a truncated version (HIRE-CAT) that retains the IRE but lacks a segment of the 5`-UTR immediately upstream from the H-ferritin mRNA translational start site. This region of the 5`-UTR regulates ferritin translation in response to cytokines such as interleukin-1(45) .

We initially determined the effect of electroporation on endogenous ferritin mRNA levels in these cells. L-ferritin mRNA levels in K562 cells were unaffected by this treatment (Fig. 4). Treatment with iron, ascorbate, or both had little effect on mRNA levels. The H-ferritin probe cross-hybridized with the G + S-rich 28 S ribosomal subunit, which was used as an internal control for loading of the gel lanes. The H-ferritin mRNA level in these cells increased by 2-fold with extended iron treatment (Fig. 4B, lanes 4 and 5). The 1.5-fold difference between the mock electroporated and untreated control cells (Fig. 4B, lanes 1 and 2) possibly represented a technical variance in this subsection of the experiment. These increases were negligible relative to the enhanced rate of protein synthesis, however. Ascorbate treatment did not modify the ferritin message levels sufficiently to account for the increase in protein synthesis.

Figure 4: Northern blot analysis of K562 cells after electroporation with p5`-UTR-CAT and RSV2LUC plasmids 10 K562 cells were transfected with 100 µg of total plasmid DNA. After different iron, ascorbate, and desferrioxamine treatments, total RNA was isolated 48 h after transfection using the RNAzol method and the RNA was DNase treated before separation and immobilization to ICI membrane. The same filter was first probed with L-ferritin probe (A) followed by the H-ferritin probe (B). Controls: +, mock electroporated; -, untreated control.

Slot-blot analysis of RNA isolated from electroporated cells hybridized with CAT and LUC probes indicated no change in the reporter gene message levels after iron, ascorbate, or desferrioxamine treatments (data not shown). Therefore, any change in CAT enzymatic activity would reflect a change in translation of CAT mRNA. In control experiments, cells were transfected with the pSV2CAT construct. Placement of the complete 5`-UTR of ferritin mRNA in front of the mRNA encoding chloramphenicol acetyltransferase resulted in the transfer of translational regulation by iron, desferrioxamine, and ascorbate (Table 2). As expected, CAT enzyme activity was unaffected by iron, desferrioxamine, or ascorbate in cells transfected with pSV2CAT. CAT activity fell in cells transfected with HIRE-CAT or 5`-UTR-CAT after treatment with the iron chelator, desferrioxamine (Table 2, row 2). CAT activity also was somewhat lower in cells treated with iron for 2 h (Table 2, row 3). This phenomenon has been observed previously (33) and has been attributed to differences in iron content of culture media as well to differences in relative cell growth rate and density.

In cells transfected with p5` UTR-CAT, 2 h of iron treatment were insufficient to raise CAT activity and even produced a slight decline (Table 2, row 3). In contrast, a 16-h exposure to iron raised CAT activity by 2-fold, consistent with increased mRNA translation. The level of CAT activity was raised further by the addition of ascorbate for 2 h (Table 2, row 5) suggesting that ascorbate enhanced translational efficiency of the CAT message over that seen with a 16-h exposure to iron. Cells treated with ascorbate for 2 h without preloading of iron showed no change in CAT activity from control (Table 2, row 6).

16 h of treatment with iron raised CAT activity only modestly in the cells transfected wit pHIRE-CAT (Table 2, row 4). Iron treatment followed by 2 h of ascorbate substantially enhanced CAT activity, however (Table 2, row 5). These data show that the mechanism by which ascorbate increases ferritin translation differs from that seen with cytokine stimulation(45) . The cytokines act through the proximal region of the ferritin 5`-UTR, which is missing in pHIRE-CAT. The segment of the message containing the IRE plays a dominant, and perhaps exclusive, role in the ascorbate effect on ferritin translation. Detailed analysis of the IRE, including site-directed mutants, along with in vitro translation experiments are needed to define absolutely the role of this structure in mediating the ascorbate response.

One aspect of the data that merits additional consideration is the difference between the 50-fold increase in the rate of ferritin synthesis as measured by [S]methionine incorporation into immunoprecipitable protein ( Fig. 1and Fig. 2) and the 3-4-fold increase in cellular ferritin content (Table 1). The transfection data also showed a 3-4-fold increase in CAT enzymatic activity in transfected cells treated with iron and ascorbate. Studies on the effect of NO on ferritin translation have shown that CAT activity measurements underestimate the range of translational control and should be viewed as ``semiquantitative''(33) . This is primarily due to the fact that the measured CAT activity is affected by several factors: (i) CAT mRNA accumulation, (ii) changes in the translation rate of CAT mRNA, and (iii) CAT protein stability. CAT protein is unstable in eucaryotic cells(20, 39, 40) . Using an anti-chloramphenicol acetyltransferase antibody, we detected the newly synthesized CAT protein. Based on the size of CAT cDNA, the encoded protein was in the size range of the H-ferritin subunit (Fig. 5). There was a smaller second band consistent with protein degradation. These data demonstrate that the procaryotic reporter protein is indeed unstable in the cells used in these experiments and that the measured CAT enzyme activity underestimates the actual translational activity. Nonetheless, the difference between the [S]methionine experiments and the immunoprecipitaion measurements of ferritin content are substantial.

Figure 5: The effect of iron, ascorbate, and desferrioxamine on the CAT reporter gene expression. After electroporation of K562 cells with p5`-UTR-CAT plasmid DNA and treatment with iron ascorbate or desferrioxamine, respectively, the newly synthesized CAT protein was labeled with [S]methionine for 2 h and immunoprecipitated with anti-CAT antibody. The CAT protein band was detectable only after a 1-week exposure of the gel to x-ray film supported with two intensifying screens. A 20-kDa protein was immunoprecipitated with the anti-CAT antibody that is in a proper size range for the expected CAT protein. There was a smaller band in the range of 14 kDa, most probably a degradation product of the CAT protein.

One possible explanation is that since these cells contain a large amount of ferritin at base line, even a major change in the rate of ferritin synthesis would raise the total cellular ferritin content only modestly in the time frame used here. In this formulation, the discrepancy would represent a failure to reach steady state. This hypothesis could be tested by assessing the rate of ferritin synthesis in molecules per unit time along with a determination of the decay constant. The rate of ferritin accumulation could be determined directly from these data and compared with the empirical values. Another explanation for the discrepancy is rapid degradation of a large fraction of newly synthesized ferritin subunits before they coalesced into stable shells. Subunit degradation would be required to make this hypothesis plausible since we showed that ascorbate slows the rate of degradation of complete ferritin shells(10, 11, 12) . This hypothesis is less attractive as it proposes futile protein synthesis by cells, which is an uncommon event. The apparent dissociation between ferritin biosynthesis and cellular ferritin levels must be explained in detail before we completely understand the effect of ascorbate on the metabolism of cellular ferritin.

Ascorbate could modulate cellular iron metabolism and ferritin synthesis by any one of several mechanisms. As a reducing agent, the vitamin modifies the redox potential within cells, inhibiting or stimulating various enzymatic processes that are connected directly or indirectly to iron metabolism. Experiments are in progress to measure the effect of uric acid, glutathione, and other important reducing agents (34, 35) on cellular iron metabolism.

Another possibility is that ascorbate stabilizes the transferrin receptor message, increasing the surface receptor protein level and cellular iron uptake. Experiments that explore this avenue are also underway. Ascorbate could also release iron from storage sites into the regulatory pool, thereby stimulating ferritin translation through the IRE-BP. Since the IRE-BP is a translational inhibitor, releasing the binding protein from the IRE initiates translation of both L- and H-ferritin mRNA(39, 40, 41, 42, 43) . One intriguing possibility is that ascorbate binds to the IRE-BP and triggers an allosteric change in the protein that prevents or at least weakens its binding to the IRE. Preliminary gel retardation assays to measure IRE-BP binding capacity and Northwestern blot analysis to quantitate IRE-BP protein in lysates prepared from leukemia cells pretreated with ascorbate, iron, and desferrioxamine raise the possibility that ascorbate directly affects the IRE-BP. The IRE-BP acts not only as an RNA binding element but as a cytosolic aconitase enzyme(46) . The two functions are mutually exclusive and depend on the reduced state of the protein and the iron availability within the cells. We have additional preliminary results showing that ascorbate significantly alters the aconitase enzyme activity.

These novel findings indicate that ascorbate exerts direct control on ferritin gene expression at the post-translational level. Since virtually all studies on ferritin translation done to this point have used ascorbate-deficient cells, the role of this vitamin must now be factored into complex interplay between iron, the IRE, and the IRE-BP in the evaluation of ferritin translation. Ascorbate appears to be a translational enhancer for ferritin. Given the complexity of intracellular ascorbate metabolism, the vitamin may have additional, unappreciated roles in iron metabolism.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants HL 45794 and AI 32717. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()

The abbreviations used are: IRE, iron-responsive element; BP, binding protein; UTR, untranslated; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; bp, base pair(s).

ACKNOWLEDGEMENTS

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