Non-transferrin Iron Reduction and Uptake Are Regulated by Transmembrane Ascorbate Cycling in K562 Cells

Abstract

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4-diisothiocyanatostilbene-2,2-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [ 55 Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.Iron, the most abundant transition metal in mammalian systems, is required for normal metabolic processes spanning molecular oxygen transport, respiratory electron transfer, DNA synthesis, and drug metabolism (1). The tendency of free ferric iron to form insoluble polynuclear aggregates under physiological conditions is typically counteracted through iron sequestration by both proteinaceous and non-proteinaceous chelators in human plasma (1, 2). In addition to the cellular acquisition of iron by the classic transferrin-dependent pathway (2, 3), uptake of non-transferrin-bound iron (NTBI) 2 is well documented (4 -13). NTBI uptake may be particularly relevant in the face of iron overload diseases such as hereditary hemochromatosis, hypotransferrinemia, and thalassemia (14 -17), in which plasma iron presents in excess of transferrin-binding capacity (18). Under such conditions, NTBI uptake by tissues (e.g. liver, heart, and pancreas (16), but not brain (19)) may serve to "clear" potentially toxic levels of iron from the plasma before damage due to iron-catalyzed oxygen radicals can accumulate (5,15,20). However, such iron scavenging may also contribute to the pathophysiology of iron overload disorders (14, 16).Mechanistically, NTBI uptake depends on (i) iron reduction and (ii) consequent cellular import of ferrous iron (9, 16) by divalent metal ion transporters (e.g. divalent metal transporter, isoform 1 (21)). Although the requirement for ferrireduction in NTBI uptake is clear (9,10,22,23), the mechanism of reduction remains ill-defined. Most models propose a membrane-bound ferrireductase activity (16) i...

Advantages and disadvantages of the animal models v. in vitro studies in iron metabolism: a review

“…In presence of catalytic concentrations of ascorbate, Dcytb may catalyze electron transfer from intracellular ascorbate to extracellular ascorbyl free radical to generate ascorbate, which could then directly donate a single electron to Fe 31 or Cu 21 . These results are supported by a novel model of NTBI reduction and uptake pathway in K562 erythroleukemia cells (Lane and Lawen, 2008). Figure 1 summarizes iron absorption pathways in the intestinal enterocyte.…”

“…The TFR-1 features a high affinity binding the complex transferrin-Fe 21 , and it has a key role involved on iron uptake in the majority of cells, while TFR-2 is expressed primarily in liver and binds the complex transferrin-Fe 31 with lower affinity (Nadadur et al, 2008). In addition to the cellular acquisition of iron by the classic transferrin-dependent pathway, there is another pathway, the uptake of nontransferrin-bound iron (NTBI) that requires iron reduction and subsequent cellular uptake of Fe 21 by DMT1 (Lane and Lawen, 2008). The iron reduction of NTBI and uptake is mediated by mucosal ferric reductases such as Duodenal Cytochrome b (Dcytb; Krause et al, 2000).…”

Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

“…Yet, in contrast to the constitutively high ascorbate efflux activity measured in embryos, ascorbate efflux was only triggered when cells were supplied with elevated dehydroascorbate levels. The molecular identity of the efflux transport system remains unknown in both plants and mammals, but the biochemical features of ascorbate efflux in mammalian K562 cells were compatible with the activity of an anion channel (51). Ascorbate efflux by pea embryos was insensitive to anion channel inhibitors (data not shown) but required a pH gradient, suggesting that it could be mediated by an H ϩ /ascorbate antiporter.…”

“…Erythroleukemia K562 cells and astrocytes are capable of reducing iron(III) using ascorbate efflux, generating iron(II) and dehydroascorbate that is reabsorbed to regenerate an ascorbate pool in the cytosol (51,52). Transmembrane cycling of ascorbate coupled to ferrous transport by the divalent transporter DMT1 appears as one possible route for iron accumulation in these cell types.…”

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

“…Within the interior of the cell, the two-electron oxidation product of ascorbate, DHA, can be rapidly reduced back to ascorbate by glutathione-and NAD(P)Hdependent enzymatic and non-enzymatic reactions , we and others have provided evidence strongly suggesting that ascorbate plays a greatly expanded role in the metabolism of this metal. First, ascorbate that is released by ascorbate-replete cells appears to play an important role in modulating the uptake of non-transferrin-bound iron by cells 19,20 , and very recent evidence indicates that ascorbate also modulates the uptake of transferrin-bound iron by cells 21 , the latter of which corresponds to a major physiological iron-uptake route 22 .…”