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

Darius J.R. Lane,Alfons Lawen

doi:10.1074/jbc.m800713200

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.

Highlights

EXPERIMENTAL PROCEDURESAll chemicals were from Sigma-Aldrich or Merck (Kilsyth, Victoria, Australia)
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]
We have both extended and linked these previously disjointed observations by demonstrating that (i) K562 cells can recycle extracellular ascorbate via dehydroascorbic acid (DHA) import and reduction, followed by ascorbate export and (ii) this ascorbate cycling is directly linked to non-transferrin-bound iron (NTBI) reduction and uptake

Summary

EXPERIMENTAL PROCEDURES

All chemicals were from Sigma-Aldrich or Merck (Kilsyth, Victoria, Australia). 0.5 ml of freshly prepared DHA at final concentrations of 50 –500 ␮M was added, followed immediately by orbital mixing for 30 min (37 °C) in the dark. Plates were orbitally mixed at 700 rpm at room temperature for 5 min in the dark, following which 50 ␮l of 3.5 mM ferricyanide in MBS were added. Plates were orbitally mixed at 700 rpm at room temperature for a further 5 min in the dark, ferrocyanide levels determined [30]. Intracellular ascorbate was determined by permeabilizing ascorbate-loaded cells with a final concentration of 0.1% saponin (Quijilla bark) plus 1% (v/v) ethanol in ice-cold PBS. Iron uptake was calculated from the measured radioactivity following correction for uptake in a paired cell sample that was incubated on ice for 5 min in the presence of 1 mM diethylenetriamine pentaacetic acid. Differences between treatments were analyzed using both oneand two-factor analysis of variances and post hoc tests of significance using GraphPad Prism௡ 5.0

RESULTS

Ascorbate release

DISCUSSION