Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism

Biochimica et Biophysica Acta 1833 (2013) 1527–1541

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Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism Darius J.R. Lane ⁎, 1, Sherin Chikhani, Vera Richardson, Des R. Richardson ⁎⁎, 1 Iron Metabolism and Chelation Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia

a r t i c l e

i n f o

Article history: Received 19 October 2012 Received in revised form 14 February 2013 Accepted 15 February 2013 Available online 26 February 2013 Keywords: Transferrin Transferrin receptor Iron Iron metabolism Ascorbate Ascorbic acid

a b s t r a c t Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased 59Fe uptake from transferrin by 1.5–2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased 59Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated 59Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of 59Fe uptake from 59Fe-citrate, only intracellular ascorbate is needed for transferrin 59Fe uptake. Additionally, experiments with L-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent 59 Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or 125I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Vitamin C (L-ascorbate) is typically abundant within vertebrate cells and in extracellular fluids and functions as a one-electron reductant [1,2]. Unlike most animals, a small proportion of vertebrate species (including humans and other haplorhine primates) are incapable of the synthesis of L-ascorbate and develop scurvy if deprived of the vitamin [1]. Mammalian cells import ascorbate via: (i) sodium-dependent ascorbate Abbreviations: BCA, bicinchoninic acid; BPS, bathophenanthroline disulfonate; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CHX, cycloheximide; Dcytb, duodenal cytochrome b; DHA, dehydroascorbate; DFO, desferrioxamine; DMT1, divalent metal transporter, isoform 1; Fe2-Tf, diferric transferrin; FTH1, ferritin heavy chain; FTL, ferritin light chain; GLUT, facilitative glucose transporter; GSH, reduced glutathione; HUVECs, human umbilical vein endothelial cells; IRE, iron-responsive element; IRP, iron-regulatory protein; LIP, labile iron pool; FeTMPyP, Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; MnTBAP, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride; MnTMPyP, Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; PHE, o-phenanthroline; Steap3, six transmembrane epithelial antigen of the prostate, isoform 3; SVCT, sodiumdependent ascorbate transporter; Tf, transferrin; TfR1, transferrin receptor 1; TfR2, transferrin receptor 2; V-ATPase, vacuolar-type H+-ATPase ⁎ Corresponding author. Tel.: +61 2 9351 6144. ⁎⁎ Corresponding author. Tel.: +61 2 9036 6548; fax: +61 2 9351 3429. E-mail addresses: [email protected] (D.J.R. Lane), [email protected] (D.R. Richardson). 1 These authors contributed equally as senior authors. 0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.02.010

transporters (SVCTs) [3]; and/or (ii) as the oxidized form of ascorbate, dehydroascorbate (DHA), by facilitative glucose transporters (GLUTs). In the latter case, DHA is reduced intracellularly to ascorbate by NAD(P)H- and reduced glutathione (GSH)-dependent recycling pathways [4–7]. Cultured mammalian cells are generally devoid of ascorbate for 3 reasons: (i) most mammalian cells, even those from ascorbatesynthesizing species, do not synthesize appreciable levels of ascorbate in culture [1,4,5]; (ii) the vitamin rapidly and irreversibly degrades under culture conditions [8]; and (iii) cultured cells are typically not supplemented with ascorbate [6]. Thus, many previous studies performed in vitro to assess basic metabolic processes have lacked a crucial vitamin that is typically abundant in vivo. Iron (Fe) is an essential element, being vital for a wide variety of metabolic processes [9]. It has long been known that ascorbate deficiency leads to anemia and other symptoms typical of scurvy [10–12]. In experimentally-induced scurvy [10,12] and in most clinical cases [11,13–15], ascorbate is required to correct this anemia. Additionally, orally administered ascorbate has a marked beneficial effect on hematologic parameters in anemic hemodialysis patients [16]. However, the mechanisms by which ascorbate elicits these effects are unclear. Ascorbate enhances dietary non-heme Fe absorption [17,18], which is typically thought to be the major role of the vitamin in Fe metabolism

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[17]. Interestingly, the specific release of ascorbate from cells stimulates Fe uptake from low-Mr Fe-citrate complexes [4,19,20]. Such observations suggest that the function of ascorbate in Fe metabolism is complex [17]. Once dietary Fe is absorbed and released into the bloodstream, virtually all of the metal is bound to the Fe-transport protein, transferrin (Tf) from which it is distributed to all other tissues throughout the body. Strikingly, the role of ascorbate in Tf-bound Fe uptake is unknown. Indeed, although scurvy is associated with a form of Fe-deficiency anemia [10–15], the mechanism responsible has not been determined. Tf binds two atoms of Fe(III) to form di-ferric Tf (Fe2-Tf), which is delivered to cells after the binding of Fe2-Tf to the Tf receptor 1 (TfR1) [21,22]. This complex is internalized by receptor-mediated endocytosis, followed by a vacuolar-type H+-ATPase (V-ATPase)-dependent decrease in endosomal pH that destabilizes the binding of Fe to Tf [21,23,24]. The Fe(III) must then reduced to Fe(II) prior to its release from the endosome into the cytoplasm via divalent metal transporter 1 (DMT1; Nramp2) [25] and/or analogous Fe(II) transporters such as Zip14 [26]. Once in the cytoplasm, this Fe can enter the labile Fe pool (LIP) [27], which may consist of Fe-chaperone proteins that supply Fe to Fe-containing proteins and/or provide Fe for storage in ferritin [28]. Importantly, a potentially rate-limiting step in the cellular acquisition of Fe by the Tf-to-cell cycle is the reduction of intra-endosomal Fe, which depends on an incompletely understood endosomal ferrireductase activity [29]. Although a candidate ferrireductase in erythroid cells is the ‘six transmembrane epithelial antigen of the prostate, isoform 3’ (Steap3), Steap3−/− mice still have 60% of the hemoglobin levels of wild-type mice [30]. This indicates that mammalian cells in vivo possess other means of reducing intra-endosomal Fe. In this study, we examined the role of ascorbate on the cellular uptake of Fe from Fe2-Tf. For the first time, we demonstrate that physiological concentrations of ascorbate significantly increase Tf-dependent Fe uptake by a range of human cells. Significantly, the mechanism of ascorbate-stimulated Fe uptake from Fe2-Tf is different to that involved in the ascorbate-stimulated uptake of low-Mr Fe. Indeed, the mechanism involved requires: (i) the reductive action of intracellular, but not extracellular ascorbate; (ii) the expression of functional TfRs 1 (or 2), but not changes in cellular Tf uptake/ release or changes in TfR1 expression; and (iii) endocytosis and endosomal acidification. Our data suggest that ascorbate acts via a reductive mechanism involving the enhancement of endosomal ferrireduction. 2. Materials and methods 2.1. Reagents Unless stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The following radio-nuclides were purchased from PerkinElmer (Waltham, MA): [ 59Fe]Cl3 (Cat. #: NEZ037) and Na125I (Cat. #: NEZ033A). Oxalomalic acid (sodium salt; Cat. #: 13521) and the following metalloporphyrins were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA): Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP; Cat. #: 75850), Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP; Cat. #: 75852) and Fe(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (FeTMPyP; Cat. #: 75854). 2.2. Cell culture The human cell lines: melanoma: SK-Mel-28, SK-Mel-2, SK-Mel-5 and VMM12; hepatocellular carcinoma: HepG2; neuroepithelioma: SK-N-MC; chronic myelogenous leukemia: K562 (American Type Culture Collection, Manassas, VA); and primary cultures of human umbilical vein endothelial cells (HUVECs; obtained from Mr. Pat Pisansarakit, Heart Research Institute, Sydney, Australia) were grown as described

[31]. Variant Chinese hamster ovary (CHO-TRVb) cells, which are devoid of endogenous TfRs [32], were grown in MEM supplemented with 10% (v/v) FBS, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), sodium pyruvate (1 mM) and 1× non-essential amino acids (Gibco; Invitrogen: Mulgrave, VIC, Australia). The CHO-TRVb cells stably transfected to express human TfR1 (TRVb/TfR1) [32], or CHO-TRVb cells transfected with FLAG-tagged human TfR2-α (TRVb/ TfR2) [33,34], or the TfR2-α vector (i.e., pcDNA3-FLAG), which imparts neomycin/G418-resistance [33,34] (TRVb/Neo), were grown in the medium described above supplemented with G418 sulfate (200 μg/mL; G418) [33,34]. All CHO-TRVb cell lines were provided by Drs. R. Graham and D. Trinder, Fremantle Hospital, Perth, Western Australia, with the permission of T. McGraw (Weill Cornell Medical College), H. Kawabata (Kyoto University, Japan) and H.P. Koeffler (University of California). 2.3. Determination of intracellular ascorbate Intracellular ascorbate levels were determined either as described previously [4] or by the Tempol-dependent oxidation of ascorbate to DHA followed by condensation of DHA with o-phenylenediamine to form a fluorescent product [35]. Both methods provided identical results. 2.4. Determination of intracellular total NADP and NADPH levels Intracellular total NADP and NADPH levels were determined using a kit from Abcam (Cambridge, MA). 2.5. Preparation of

59

Fe- 125I-Transferrin

Human apo-Tf (Sigma-Aldrich) was labeled with [ 59Fe] or nonradioactive Fe to produce the 59Fe- or Fe-labeled di-ferric proteins, respectively [36]. In some experiments, 59Fe2-Tf or Fe2-Tf were also labeled with 125I [36,37]. 2.6. Effect of ascorbate on

59

Fe2- 125I-Tf Uptake by Cells

The effect of ascorbate on cellular 59Fe uptake from 59Fe2-125I-Tf was examined using established techniques [36,38]. Briefly, cells were preincubated with or without ascorbate (10–500 μM) for 5–60 min/37 °C before the addition of 59Fe2-Tf or 59Fe2\ 125I-Tf (0.75 μM; [Fe] = 1.5 μM) or 59Fe-citrate ([Fe] = 1.5 μM; molar ratio or Fe to citrate = 1:100) to the same medium for 3 h/37 °C. In some experiments, DHA (50 μM) was used in place of ascorbate. The cells were then washed five times on ice with ice-cold PBS to remove non-specifically bound 59 Fe2-Tf. Internalized 59Fe was determined by standard methods by incubating cells on ice for 30 min/4 °C with the general protease, “Pronase” (1 mg/mL) [23,36]. Cells were then detached following Pronase treatment and centrifuged (16,000 g/4 °C/1 min) in an Eppendorf microcentrifuge (Hamburg, Germany). The resulting supernatant represents cell-surface (i.e., Pronase-sensitive; hereinafter referred to as “cell membrane”) 59Fe and 125I that were released by extracellular proteolysis, while the Pronase-insensitive fraction represents internalized 59Fe and 125I [23,36]. Radioactivity was quantitated using a γ-counter (2480 Wizard 2, Perkin Elmer, Turku, Finland). In some experiments, the effect of ascorbate on the affinity of Fe2\ 125I-Tf binding and the total number of cell-surface Tf-receptors was assessed by pre-incubating SK-Mel-28 cells with or without ascorbate (50 μM) for 30 min/37 °C, then cooling the cells to 4 °C and incubating them for a further 2 h at 4 °C with increasing concentrations of Fe2\ 125I-Tf (0.0125–1 μM). Cells were then washed and the amount of specifically bound radioactivity was determined by subtracting the amount of Fe2\ 125I-Tf bound in the presence of a 200-fold excess of unlabeled Fe2-Tf. At 4 °C, endocytic internalization of the Fe2-Tf–TfR complex is inhibited, but the ligand–receptor interaction is unaffected [36].

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2.7. Effect of ascorbate on

59

Fe and

125

I-Tf efflux from cells

The effect of ascorbate on 59Fe and/or 125I-Tf efflux from cells pre-labeled with 59Fe\ 125I-Tf was examined as previously described [39]. Briefly, cells were incubated with 59Fe\ 125I-Tf (0.75 μM) for either 30 min or 3 h/37 °C. The cells were then placed on ice and washed five times with ice-cold PBS and re-incubated with or without ascorbate (50 μM) for 5–30 min or 3 h/37 °C. At the end of the incubation, the overlying medium was aspirated into γ-counting tubes and the cell mono-layers solubilized in 2% (w/v) SDS [20]. Levels of 59Fe or 125I-Tf were determined as above. 2.8. SDS-PAGE and Western blotting SDS-PAGE (reducing conditions), western blotting and densitometry were performed as described [40]. The following antibodies were used to probe for specific proteins. Mouse monoclonal anti-human TfR1 (clone H68.4) was from Invitrogen (Cat. #: 13-6800). Rabbit polyclonal anti-human TfR2 was from Abcam (Cat. #: ab83810). Rabbit polyclonal anti-ferritin heavy chain (FTH1) was from Cell Signaling Technology (Cat. #: 3998; Danvers, MA). Rabbit polyclonal antiferritin light chain (FTL) was from Abcam (Cat. #: ab69090). Rabbit polyclonal anti-Steap3 was from Abnova (Cat. #: PAB13008). Mouse monoclonal anti-β-actin (clone AC-40; Cat. #: A4700) was from Sigma-Aldrich. The secondary antibodies used (1:5000) were horseradish peroxidase-conjugated anti-mouse (Cat. #: A9044) and anti-rabbit (Cat. #: A0545) from Sigma-Aldrich. Membranes were washed and developed using ECL + Western blot detection reagent (Amersham Biosciences, Buckinhamshire, UK) and exposed to X-ray film. Densitometry was performed using BioRad Quantity One software (Hercules, CA). All data were normalized to the loading control, β-actin. Chemiluminescent immunodetection by X-ray film yielded a linear signal output within 5% error over (i) the protein loading levels employed, and (ii) the exposure time intervals examined. This was confirmed using a digital imaging BioRad ChemiDoc MP system, showing that exposures were within the linear range of the film and that chemiluminescent substrate was not limiting. 2.9. Native PAGE- 59Fe-autoradiography Native PAGE-59Fe-autoradiography was performed using established techniques [41]. Where noted, anti-ferritin “super-shifts” were used to identify the location of endogenous ferritin by incubating cell extracts (100 μg protein) with 10 μg of rabbit polyclonal anti-horse spleen ferritin (Sigma-Aldrich; Cat. #: F6136) or rabbit polyclonal anti-human ferritin (MP Biomedicals; Seven Hills, NSW, Australia; Cat. #: 65077) for 1 h/4 °C. To control for possible non-specific effects of the antibody, an equivalent amount of rabbit polyclonal anti-cyclin D1 (Abcam; Cat. #: ab24249) was used, which had no effect on 59Fe distribution. The location of the low-Mr 59Fe fraction was determined by the incubation of cell extracts with 100 μM of the strong Fe chelator, desferrioxamine (DFO; Novartis, Basel, Switzerland), for 1 h/4 °C [41]. The preincubation of cell extracts with DFO binds and reproducibly removes low-Mr 59Fe [41,42]. Importantly, the DFO-59Fe complex does not appear in the gel due to the net positive charge of the complex at the running pH, which leads to its migration in the opposite direction to 59 Fe-ferritin and low-Mr 59Fe. 2.10. Statistical analysis All statistical analyses were performed using GraphPad Prism® v. 5.0 (GraphPad Software, California). Means were compared using one-way ANOVA with Bonferroni's post hoc test of significance [4,7,20]. Results are expressed as means ± SD (number of experiments) and were considered to be statistically significant when p b 0.05.

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3. Results 3.1. The incubation of ascorbate-depleted cells with ascorbate or DHA increases intracellular ascorbate Typical human plasma ascorbate concentrations in unsupplemented individuals are 40–60 μM [43]. Therefore, unless otherwise stated, cells were incubated with or without ascorbate or ascorbate analogues (50 μM) for 30 min/37 °C before the addition of 59Fe as 59Fe2-Tf to the same medium for 3 h/37 °C (hereinafter referred to as the ‘standard incubation protocol’). Unless supplemented with the vitamin (see below), the cells used in this study were devoid of detectable ascorbate (Table 1, column A). This observation is typical of mammalian cells grown under standard culture conditions [4,7,8,19,20]. Importantly, incubation of SK-Mel-28 cells with ascorbate for 30 min or 3 h increased intracellular ascorbate from undetectable levels to 202–375 pmol/106 cells (Table 1). Incubation of these cells with DHA (50 μM) for 30 min/37 °C, which is rapidly reduced to ascorbate once imported by cells [6], increased their ascorbate content to 534 ± 19 pmol/10 6 cells (Table 1). 3.2. Ascorbate stimulates cellular cells

59

Fe uptake from

59

Fe2-Tf in human

In initial studies, we used a wide range of human cells to examine the effect of ascorbate on 59Fe uptake. Incubation of SK-Mel-28 cells (Fig. 1) with ascorbate significantly (p b 0.001) increased their internalized 59Fe uptake from 59Fe2-Tf by ~2-fold that of cells incubated in the absence of ascorbate. While there was also a trend towards an increase in cell-membrane 59Fe uptake, this represented a small proportion (b 10%) of total 59Fe uptake and was typically not significant (p > 0.05; Fig. 1). Under the same conditions, ascorbate also significantly (p b 0.001–0.01) stimulated internalized 59Fe uptake from 59Fe2-Tf in all other human cells examined, including primary HUVECs, and all other human cell lines assessed: SK-Mel-2, SK-Mel-5, VMM12, SK-NMC, HepG2 and K562 (data not shown). Since SK-Mel-28 cells have been well-characterized in terms of their cellular Fe uptake mechanisms [36,38,39,44,45], these cells were typically used for all subsequent characterization experiments. 3.3. Ascorbate-stimulates 59Fe uptake from 59Fe2-Tf in a time- and concentration-dependent manner and increases the Vmax of 59Fe uptake The stimulatory effect of ascorbate on 59Fe uptake from Tf continued for at least 24 h (Fig. 2A). Moreover, ascorbate caused a significant (p b 0.001) increase in 59Fe uptake after only 30 min incubation with 59Fe2-Tf (Fig. 2B), indicating that stimulation occurred over relatively short 59Fe uptake periods. Pre-incubation of SK-Mel-28 cells with ascorbate from 5 to 60 min/37 °C, followed by washing to remove extracellular ascorbate, was sufficient in a subsequent 3 h/37 °C incubation to elicit full stimulation of 59Fe uptake from 59Fe2-Tf (Supp. Fig. 1A). Furthermore, ascorbate at concentrations from 10 to 500 μM had no more stimulatory effect on 59Fe uptake than that observed at 10 μM (Supp. Fig. 1B; 59Fe2-Tf incubation time: 2 h). However, to approximate plasma ascorbate concentrations in healthy humans [43,46], a concentration of 50 μM ascorbate was used in all subsequent studies. We next assessed the dependence of ascorbate-stimulated 59Fe uptake on the concentration of 59Fe2-Tf (0.125–12.5 μM; Fig. 2C). Internalized 59Fe uptake was then determined and the results analyzed by non-linear regression analysis (Fig. 2C). Interestingly, ascorbate significantly (p b 0.001) increased the Vmax of 59Fe uptake (from 9.2 ± 0.16 to 13.0 ± 0.29 × 106 atoms 59Fe/cell/h; please note the different units used for the y-axis in Fig. 2C). As typical serum Fe2-Tf concentrations are 20–30 μM [21], and that ascorbate-stimulated 59Fe uptake occurred maximally at a 59Fe2-Tf concentration of 0.75–2 μM

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Table 1 Intracellular ascorbate levels in SK-Mel-28 cells before and after incubation with ascorbate or DHA at the indicated concentrations and for the indicated times. Results are mean± SD (3 experiments). Intracellular ascorbate, pmol × (106 cells)−1 Cell line

(A) No treatment

(B) Incubation with ascorbate (50 μM) for 30 min/37 °C

(C) Incubation with ascorbate (50 μM) for 3 h/37 °C

(D) Incubation with DHA (50 μM) for 30 min/37 °C

SK-Mel-28

0 ± 1.0

202 ± 15***(vs A)

375 ± 18***(vs B)

534 ± 19***(vs C)

*** p b 0.001.

(Fig. 2C), ascorbate-stimulated uptake of Fe from Fe2-Tf is probably physiologically relevant.

consistent with reports documenting increased de novo synthesis of ferritin in cells exposed to a combination of ascorbate and Fe [50–52].

3.4. Ascorbate and Fe2-Tf increase ferritin levels

3.5. An increase in ferritin does not explain ascorbate-stimulated uptake from Tf

We next examined whether ascorbate-stimulated 59Fe uptake correlated with alterations TfR1 and ferritin levels. These proteins were examined as their expression levels are post-transcriptionally controlled in an Fe-dependent manner via the iron-regulatory protein (IRP)-ironresponsive element (IRE) system [47,48]. As a consequence, an increase in cytosolic Fe typically decreases TfR1 synthesis while it increases ferritin synthesis [47,49]. Control and ascorbate-treated SK-Mel-28 cells were incubated with non-radioisotopic Fe2-Tf (0.75 μM) for 0–24 h (Fig. 3A). The levels of TfR1, FTH1 and FTL were then monitored by western blotting. As demonstrated in Fig. 2A, although ascorbate linearly stimulated 59 Fe uptake over 24 h (Fig. 2A), ascorbate significantly (p b 0.01) down-regulated TfR1 protein by 8 h (Fig. 3A). In contrast, in the absence of ascorbate, significant (p b 0.001) down-regulation of TfR1 only occurred by 24 h (Fig. 3A). Thus, TfR1 protein is more acutely down-regulated by Fe2-Tf in the presence of ascorbate. Conversely, FTH1 and FTL protein levels increased at a significantly (p b 0.001) greater rate over 24 h in ascorbate-treated compared to control cells, with a significant (p b 0.001) increase being evident by 3 h (Fig. 3A, B). Importantly, ascorbate and Fe2-Tf synergistically increased up-regulation of FTL by 3 h compared to Fe2-Tf or ascorbate alone (Fig. 3B), which was probably due to increased Fe uptake. Similar results were found for FTH1 (data not shown). These results are

Fe

The ascorbate/Fe2-Tf-dependent increase in ferritin levels observed here is probably a consequence, rather than a cause, of ascorbatestimulated Fe uptake. Indeed, when cells are presented with low-Mr Fe, ascorbate enhances ferritin expression by an IRP1/cytosolic aconitase “switch” mechanism [51]. Thus, we assessed the effects of: (i) the protein synthesis inhibitor, cycloheximide (20 or 100 μM; CHX) [53], which abrogates global protein synthesis in SK-Mel-28 cells [53]; or (ii) the cytosolic aconitase inhibitor, oxalomalate (1 or 5 mM) [54], which abolishes aconitase activity of the Fe–sulfur cluster form of IRP1 [54]. Neither compound significantly affected 59Fe uptake (Supp. Fig. 2). As ascorbate can inhibit ferritin autophagy [55], and ferritin can also be degraded by the proteasome [56], we examined the effect of inhibitors of both these pathways on 59Fe uptake (Supp. Fig. 2). Treatment of cells with inhibitors of autophagy (3-methyladenine; 5 mM) or the proteasome (MG-132; 10 μM)-conditions previously shown to inhibit the respective degradative pathways [56] – did not affect 59Fe uptake (Supp. Fig. 2). Therefore, our data indicate that ascorbate does not stimulate Tf-dependent 59Fe uptake by increasing ferritin levels or by increasing cytosolic aconitase activity.

3.6. Incubation of cells with ascorbate and ferritin with 59Fe

Fig. 1. Incubation of human melanoma SK-Mel-28 cells with ascorbate increases internalized 59 Fe uptake from 59Fe2-Tf. Cells were pre-incubated with or without ascorbate (50 μM) for 30 min/37 °C before the addition of 59Fe2-Tf ([Tf] = 0.75 μM; [Fe] = 1.5 μM) to the same medium for 3 h/37 °C. After this incubation, the cell mono-layers were washed on ice and incubated with Pronase (1 mg/mL) for 30 min/4 °C to obtain the internalized and cellmembrane fractions. Results are mean + SD (3 experiments).

59

59

Fe2-Tf increased loading of

To determine whether ascorbate stimulated Fe-loading of ferritin, control and ascorbate-treated SK-Mel-28 cells were incubated with 59Fe2-Tf (0.75 μM) for 0.5–24 h (Fig. 4). The location of the ferritin band was determined as described in the Materials and methods section (Supp. Fig. 3A) [41,53]. While incubating cells with 59Fe2-Tf caused a time-dependent increase in 59Fe-ferritin, which contained the majority of detectable cellular 59Fe, ascorbate significantly (p b 0.001) increased this by ~2–3 fold at 3, 8 and 24 h (Fig. 4). Additionally, a putative low-Mr 59Fe pool was identified in both control and ascorbate-treated cell extracts (Fig. 4; see Materials and methods section), although the latter was always significantly lower than the former (data not shown). This pool was verified as “low-Mr” by its abolishment by a 1 h/4 °C incubation of native cell extracts with DFO (100 μM; Supp. Fig. 3B). It should be noted that the apparent absence of the 59Fe-Tf band in the 59Fe autoradiograms presented in Fig. 4 and Supp. Fig. 3 is most likely due to their far lower intensity relative to the 59Fe-ferritin and low-Mr 59Fe bands. Indeed, as Tf rapidly donates its Fe to cells, there is only a relatively low steady-state level of 59Fe-Tf in cells. Further, as 59Fe-Tf does not accumulate in cells, it can be difficult to detect in the presence of 59Fe-ferritin, which does accumulate.

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Fig. 2. Characterization of ascorbate stimulated iron uptake from 59Fe2-Tf by SK-Mel-28 cells as a function of: (A, B) incubation time with 59Fe2-Tf, or (C) 59Fe2-Tf concentration. (A, B) Cells were pre-incubated with or without ascorbate (50 μM) for 30 min/37 °C before the addition of 59Fe as 59Fe2-Tf ([Tf] = 0.75 μM; [Fe] = 1.5 μM) to the same medium for 1–24 h (A), or 1–60 min (B) at 37 °C. (C) Cells were pre-incubated with or without ascorbate as in (A, B) before the addition of 59Fe2-Tf ([Tf] = 0.125–12.5 μM; [Fe] = 0.25–25.0 μM) for a further 3 h/37 °C. For (A–C), after incubation with 59Fe2-Tf, cells were washed on ice and then incubated with Pronase (1 mg/mL) for 30 min/4 °C to separate the membrane and internalized fractions. Results are mean + SD (3 experiments).

Therefore, the increased amount of Fe from Fe2-Tf reaching the cytoplasm in the presence of ascorbate (i) elevates ferritin expression, probably via an IRP-IRE-regulated increase in de novo ferritin synthesis [51,57]; and (ii) increases ferritin Fe-loading. 3.7. Ascorbate-stimulated 59Fe uptake from 59Fe2-Tf requires intracellular ascorbate and not extracellular Fe reduction Ascorbate-dependent Fe uptake from Fe-citrate requires extracellular ascorbate and extracellular ferrireduction [4,19,20]. To determine whether ascorbate-stimulated 59Fe uptake from 59Fe2-Tf occurred by a similar mechanism, control and ascorbate-treated cells were further incubated with the cell-impermeant enzyme, ascorbate oxidase (10 U/mL), for a further 30 min and during subsequent incubation with 59Fe2-Tf or 59Fe-citrate (Fig. 5). Ascorbate oxidase, which oxidizes ascorbate to DHA, markedly and significantly (p b 0.001) inhibited ascorbate-stimulated 59Fe uptake from 59 Fe-citrate ([Fe] = 1.5 μM; Fig. 5A), as previously described [4,19,20], but did not significantly affect 59Fe uptake from 59Fe2-Tf ([Fe] = 1.5 μM; Fig. 5B). As ascorbate oxidase only oxidizes extracellular ascorbate [4,7,19], ascorbate-stimulated 59Fe uptake from 59Fe2-Tf does not depend on the continued presence of extracellular ascorbate. Next, we examined if the extracellular release of 59Fe from 59Fe2-Tf was required for ascorbate to stimulate 59Fe uptake from this protein, as reported for the activation of Tf-dependent Fe uptake by ferric ammonium citrate [58]. Cells were incubated with the membraneimpermeant Fe(II) chelators (200 μM), ferene-S, ferrozine [4,20,59], or bathophenanthroline disulfonate (BPS [45]) for 30 min before addition of 59Fe2-Tf or 59Fe-citrate to the same medium (Fig. 5).

As previously described [4,20], all chelators markedly and significantly (p b 0.001) inhibited 59Fe uptake from 59Fe-citrate in the presence of ascorbate (Fig. 5A). Importantly, and in contrast to 59Fe uptake from 59Fe-citrate, ferene-S, ferrozine and BPS only reduced 59Fe uptake from 59Fe2-Tf in the presence of ascorbate by less than 12%, 17% and 36% (p b 0.01), respectively (Fig. 5B). Similarly, co-incubation of cells with the avid Fe-binding Tf homologue, apo-lactoferrin (4 μM) almost abolished ascorbate-stimulated and basal 59Fe uptake from 59Fe-citrate 59 (Fig. 5A), but only slightly reduced ascorbate-stimulated Fe uptake 59 59 from Fe2-Tf (i.e., by less than 20% of the Fe uptake in the presence of ascorbate; Fig. 5B). The observation that all chelators caused some inhibition of ascorbate-stimulated 59Fe uptake from 59Fe2-Tf (cf. Fig. 5B) is consistent with the finding that Fe can be labilized by co-incubation with both strong Fe chelators such as BPS and apo-lactoferrin when a reductant, such as ascorbate, is present [60]. Therefore, unlike 59Fe uptake from 59Fe citrate, our results suggest that ascorbate stimulates 59Fe uptake from 59Fe2-Tf by an intracellular mechanism. 3.8. Studies with ascorbate-scavenging metalloporphyrins indicate the need for intracellular ascorbate in Tf- 59Fe uptake In order to further evaluate the hypothesis that ascorbatestimulated 59Fe uptake from 59Fe2-Tf requires an intracellular pool of ascorbate, we assessed the ability of the membrane-permeant metalloporphyrins, MnTMPyP and FeTMPyP, to rapidly catalyze ascorbate oxidation [61]. These metalloporphyrins catalyze the oxidation

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of ascorbate at rates 8- and 170-fold, respectively, greater than the rate of ascorbate auto-oxidation at physiological pH [61]. As these metalloporphyrins also scavenge superoxide (i.e., MnTMPyP: [62]; FeTMPyP: [63]) and peroxynitrite (i.e., MnTMPyP: [64]; FeTMPyP: [65]), the membrane-permeant metalloporphyrin, MnTBAP, which similarly scavenges superoxide [62] and peroxynitrite [61], but is relatively unreactive with ascorbate [61], was used to control for the non-ascorbate oxidizing activities of MnTMyP and FeTMPyP. All metalloporphyrins were used at a concentration of 30 μM. Importantly, when added immediately after pre-incubation of cells with or without ascorbate, MnTBAP did not affect 59Fe uptake (Fig. 6). However, MnTMPyP partially inhibited (p b 0.001), whereas FeTMPyP almost abolished (p b 0.001), only ascorbate-stimulated 59Fe uptake (Fig. 6). Notably, although both MnTMPyP and FeTMPyP would have oxidized both intracellular and extracellular ascorbate, the oxidation of extracellular ascorbate cannot account for the inhibitory action of these metalloporphyrins, as ascorbate oxidase, which exclusively and rapidly oxidizes extracellular ascorbate [4,7,19], had no significant effect under the same conditions (Fig. 5B). Thus, these data further suggest the involvement of intracellular ascorbate in stimulating 59 Fe uptake from 59Fe2-Tf.

3.9. The reducing ene-diol moiety is required for ascorbate to stimulate 59 Fe uptake from 59Fe2-Tf We next assessed the effects of a range of ascorbate analogues and degradation products (Fig. 7A) on ascorbate-stimulated 59Fe uptake from 59Fe2-Tf (Fig. 7B).

The effect of a change in side-chain stereoisomerism was assessed with the L-ascorbate epimer, D-isoascorbate (Fig. 7A). Interestingly, D-isoascorbate can substitute for L-ascorbate in several biologicallyrelevant, enzyme-catalyzed reactions and may be imported by cells, albeit at a lower rate than L-ascorbate [3,66,67]. Interestingly, the stimulation of 59Fe uptake from 59Fe2-Tf by D-isoascorbate was equivalent to that of L-ascorbate in both cell lines, indicating that stimulation of 59Fe uptake by ascorbate is not stereo-specific (Fig. 7B). The influence of changing side chain hydrophobicity was assessed with L-ascorbate-6-palmitate (Fig. 7A), a highly lipophilic, palmitoylated L-ascorbate derivative [68], and 5,6-isopropylidene-L-ascorbate, which is lipophilic and a potential substrate for SVCTs [67]. Like L-ascorbate, these derivatives have potent reducing activity due to the 2,3-ene-diol moiety (Fig. 7A) [66,69]. The stimulation of Tf-dependent 59Fe uptake by L-ascorbate-6-palmitate was very similar to that provided by 5,6isopropylidene-L-ascorbate, and in both cases the stimulation was only marginally lower than that with L-ascorbate (Fig. 7B). Thus, stimulation of 59Fe uptake does not appear to be sensitive to side chain structure and/or hydrophobicity. We then assessed the effect of modifications to the electron-dense 2,3-ene-diol moiety (Fig. 7A), which, together with the conjugated structure of the five-membered lactone ring, is essential for the one-electron reducing activity of ascorbate [70]. The importance of this group was assessed using the two L-ascorbate derivatives, L-ascorbate-2-phosphate and L-ascorbate-2-sulfate (Fig. 7A), both of which lack reducing activity. While dephosphorylation of L-ascorbate-2-phosphate by phosphatases leads to the slow release of L-ascorbate that can be accumulated by cells [71], the corresponding sulfate group in L-ascorbate-2-sulfate is not cleavable by mammalian cells, and correspondingly, this molecule

Fig. 3. Ascorbate potentiates Fe-dependent ferritin expression, but this is not the cause of ascorbate-stimulated 59Fe uptake from transferrin. (A) SK-Mel-28 cells were pre-incubated with or without ascorbate (50 μM) for 30 min and then Fe2-Tf (0.75 μM) added to this media and the cells then incubated for 0, 3, 8 or 24 h/37 °C. The expression of the indicated proteins (i.e., transferrin receptor 1 (TfR1), ferritin heavy chain (FTH1), ferritin light chain (FTL) and the loading control, β-actin) was then monitored by western blotting. (lower panels) The densitometry of the indicated bands is also shown. (B) The observed increase in FTL expression after a 3 h/37 °C incubation of SK-Mel-28 cells in (A) was dependent on incubation with both ascorbate (50 μM) and Fe2-Tf (0.75 μM). Western blots (A and B) are typical results from 3 experiments, while all graphical data are mean + SD (3 experiments).

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Fig. 4. Ascorbate-stimulated 59Fe uptake from Fe2-Tf leads to increased loading of ferritin with 59Fe. (top panel) SK-Mel-28 cells were pre-incubated with/without ascorbate (50 μM) for 30 min/37 °C and then 59Fe2-Tf (0.75 μM) was added and the cells incubated for a further 0.5, 3, 8 or 24 h/37 °C incubation. At each time point, cells were harvested and cytoplasmic extracts were obtained under native conditions at 4 °C and native PAGE 59Fe autoradiography performed. (lower panel) The densitometry of the 59Fe-ferritin and low-Mr 59Fe bands is also shown, respectively. The native PAGE results are typical results from 3 experiments, while the densitometry in (lower panel) is mean ± SD (3 experiments).

Fig. 5. Ascorbate-stimulated 59Fe uptake from 59Fe-citrate (A), but not 59Fe2-Tf (B), requires extracellular ascorbate and extracellular labilization of 59Fe. SK-Mel-28 cells were pre-incubated with either ascorbate (50 μM) or control medium alone without ascorbate and then either vehicle medium, ascorbate oxidase (10 U/mL), ferene-S (200 μM), ferrozine (200 μM), apo-lactoferrin (4 μM) or bathophenanthroline disulfonate (BPS; 200 μM) were added, and the cells incubated for a further 30 min/37 °C incubation. Then, 59 Fe was added to the same medium as either 59Fe-citrate ([Fe] = 1.5 μM; molar ratio of Fe to citrate = 1:100) (A), or 59Fe2-Tf ([Tf] = 0.75 μM; [Fe] = 1.5 μM) (B), and the cells were incubated for a further 3 h/37 °C. After this incubation, cells were washed on ice and then incubated with Pronase (1 mg/mL) for 30 min/4 °C to obtain the internalized and cell-membrane fractions. Results shown are mean + SD (3 experiments).

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Fig. 6. Cell-permeant ascorbate-scavenging metalloporphyrins further suggest the involvement of intracellular ascorbate in the stimulation of Tf-dependent 59Fe uptake. SK-Mel-28 cells were pre-incubated with control medium or ascorbate (50 μM) for 30 min/37 °C. Vehicle medium (MEM + 0.1% DMSO), MnTBAP, MnTMPyP or FeTMPyP (all at a final concentration of 30 μM) were then added to the same medium, and the cells incubated for an additional 30 min/37 °C. Then, 59Fe2-Tf (0.75 μM) was added and the cells incubated for a further 3 h/37 °C. After this incubation, cells were washed on ice and incubated with Pronase (1 mg/mL) for 30 min/4 °C to obtain the internalized and cell-membrane fractions. Results shown represent internalized 59Fe uptake and are mean + SD (3 experiments).

is not a source of cellular ascorbate [72]. We observed that although the stimulation of cellular 59Fe uptake from 59Fe2-Tf by ascorbate-2phosphate was significantly (p b 0.001) less than L-ascorbate, it was still significantly (p b 0.01) greater than the ascorbate-free control (Fig. 7B). In contrast, L-ascorbate-2-sulfate provided no stimulation (Fig. 7B). Thus, the 2,3-ene-diol moiety is crucial for stimulation of Tf-dependent 59Fe uptake. It is important to note that as L-ascorbate-2-phosphate and L-ascorbate-2-sulfate may not be substrates for SVCTs [67], these analogues may not readily be taken up by cells. Hence, the requirement for an intact ene-diol in stimulating 59Fe uptake reflects a requirement for either (i) the reducing activity of ascorbate; and/or (ii) the SVCT-dependent uptake of ascorbate. Under standard cell culture conditions, ascorbate is rapidly oxidized to its two-electron oxidized form, DHA [8]. As expected on the basis of a requirement for intracellular ascorbate in stimulating Tf-dependent 59Fe uptake, pre-treatment of cells with DHA, which increased intracellular ascorbate (Table 1), stimulated 59Fe uptake equivalently to that of L-ascorbate (Fig. 7B). As DHA itself is an unstable molecule that undergoes rapid and irreversible delactonization under physiological and cell culture conditions [1], we examined the following common DHA degradation products for their ability to stimulate Tf-dependent 59Fe uptake: L-(+)-erythrulose, L-(+)-threonate, L-threose and oxalate [1] (Fig. 7A,B). None of these degradation products had any effect on Tf-dependent 59Fe uptake, which demonstrates a requirement for the intact vitamin.

3.10. Ascorbate-stimulated 59Fe uptake from Tf is not due to a general increase in cellular reducing capacity To examine whether a general increase in cellular reducing capacity could account for ascorbate's effect on 59Fe uptake, SK-Mel-28 cells were pre-incubated with the following for 24 h/37 °C prior to implementation of the standard incubation protocol: (i) control medium; (ii) the membrane-permeant GSH precursor, N-acetylcysteine (NAC; 5 mM) [73], which increases cell GSH in SK-Mel-28 cells [74]; or (iii) the γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine

(BSO; 10 μM), which markedly attenuates GSH levels in these cells [75,76]. The results (Fig. 7C) show that modulating cellular GSH had no significant effect on 59Fe uptake (Fig. 7C). Thus, the stimulatory effect of ascorbate on 59Fe uptake cannot be attributed to a general increase in cellular reducing capacity. Next, we assessed whether ascorbate specifically caused an increase in cellular NADPH levels (Fig. 7D). This was important to assess as NADPH is thought to be the major electron donor for the endosomal ferrireductase, Steap3, which contributes to Fe uptake from Tf in some cells [30] (see also Section 3.11). Interestingly, while ascorbate had no effect on total NADP levels (i.e., NADP+ + NADPH), incubation with ascorbate significantly (p b 0.001) lowered NADPH levels (Fig. 7D). The latter is probably due to the requirement for NADPH in maintaining intracellular ascorbate [1]. Therefore, the ability of ascorbate to stimulate Tf-dependent Fe uptake cannot be explained by an increase in cellular NADPH levels.

3.11. Ascorbate-stimulated 59Fe uptake from 59Fe2-Tf is not caused by either changes in levels of TfR1/2 or Steap3, or changes in cellular 125I-Tf uptake Next, we assessed whether the stimulation of Tf-dependent 59Fe uptake by ascorbate was due to alterations in Tf binding to cells, Tf uptake and/or Tf cycling. First, using the standard incubation protocol, we confirmed that ascorbate, with or without Fe2-Tf, had no effect on TfR1 expression within 3 h, despite there being a marked increase in 59Fe uptake in the presence of ascorbate over this period (Fig. 8A; cf. TfR1 expression in Fig. 3A). Additionally, when cells were incubated with ascorbate and Fe2-Tf as described in Fig. 3A (see Section 3.4), there was no significant change in TfR2 or Steap3 protein levels over 0–24 h compared to the relevant controls (Supp. Fig. 4). Thus, at least in SK-Mel-28 cells, ascorbate does not appear to stimulate Tf-dependent 59Fe uptake by modulating the levels of these proteins. It should be noted that although there was some trend towards a decrease in TfR2 levels in the presence of both ascorbate and Fe2-Tf, this was not found to be significant (Supp. Fig. 4). This is consistent with the observation that unlike TfR1, TfR2 expression does not appear to be Fe-regulated [77]. Moreover, although TfR2 protein can be stabilized by the addition of Fe2-Tf in cells of hepatic origin [78,79], we did not observe this effect in SK-Mel-28 cells (Supp. Fig. 4). This may be due to the following observations: (i) the stabilization of TfR2 by Fe2-Tf appears to be specific to cells of hepatic origin [78,79] (e.g., endogenous TfR2 is not regulated by Fe2-Tf in K562 cells [79]); and (ii) while our experiments employed a relatively low Fe2-Tf concentration of 0.75 μM, the stabilization of TfR2 by Fe2-Tf in HepG2 cells was found to be most pronounced at markedly higher Fe2-Tf concentrations (i.e., 2.5–25 μM) [78]. Next, we assessed if ascorbate affected the number and/or affinity of Tf-binding sites at the cell surface (see Materials and methods section). The apparent dissociation constants and total number of cell-surface Tf-binding sites were determined using non-linear regression analysis (Fig. 8B). Our data clearly demonstrate that ascorbate did not significantly affect the affinity of Fe2\ 125I-Tf-binding, or the total number of specific cell-surface Tf-binding sites [control = 35,800 ± 1500/cell (n = 6); ascorbate-treated = 37,100 ± 1800/cell (n = 6)]. We then compared the effect of ascorbate on the relative distributions of 59Fe and 125I-Tf when SK-Mel-28 cells were co-incubated with doubly-labeled 59Fe2\ 125I-Tf (0.75 μM). While pre-incubation of cells with ascorbate followed by incubation with 59Fe2\ 125I-Tf (0.75 μM) resulted in significantly (p b 0.001) more 59Fe uptake into the internalized compartment (Fig. 8C), ascorbate did not significantly affect 125I-Tf uptake into either the internalized or cell-membrane compartment (Fig. 8D). Therefore, ascorbate-stimulated 59Fe uptake from 59Fe2-Tf is not due to up-regulation of TfRs or increased Tf uptake.

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Fig. 7. The reducing ene-diol moiety of ascorbate is necessary for stimulation of 59Fe uptake from 59Fe2-Tf (A,B) and the effect of ascorbate is not due to a general increase in cellular reducing capacity (C,D). (A) Line drawings of the structures of the ascorbate analogues and degradation products assessed. (B) In these studies, SK-Mel-28 cells were pre-incubated with or without ascorbate or its analogues/degradation products (50 μM) for 30 min/37 °C before the addition of 59Fe2-Tf ([Tf] = 0.75 μM; [Fe] = 1.5 μM) to the same medium for a further 3 h/37 °C. (C) Alternatively, SK-Mel-28 cells were treated with either control medium, N-acetylcysteine (NAC; 5 mM) or buthionine sulfoximine (BSO; 10 μM) for 24 h/ 37 °C, following which cells were washed and re-incubated in fresh medium with/without ascorbate (50 μM) for 30 min, followed by the addition of 59Fe2-Tf as in (B) to the same medium. In both (B) and (C), following incubation with 59Fe2-Tf, cells were washed on ice and incubated with Pronase (1 mg/mL) for 30 min/4 °C to obtain the internalized and cell-membrane fractions. (D) SK-Mel-28 cells were incubated with/without ascorbate (50 μM) as in (B), then Fe2-Tf (0.75 μM) or vehicle were added to the same medium for a further 3 h/37 °C. Cells were harvested and total NADP (NADP-total) and NADPH levels were determined. Results are mean + SD (3 experiments).

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Fig. 8. Ascorbate-stimulated 59Fe uptake from 59Fe2-Tf is not caused by changes in TfR1 expression or cellular Tf uptake. (A) SK-Mel-28 cells were incubated with or without ascorbate (50 μM) for 30 min/37 °C, following which non-radioactive Fe2-Tf (0.75 μM), or the equivalent volume of vehicle (i.e., MEM), was co-incubated with the cells for a further 3 h/37 °C. The expression of cellular TfR1 or β-actin (loading control) was then monitored by western blotting (upper panel). The same protocol was used to examine 59Fe uptake except 59Fe2-Tf was used and a 30 min incubation with Pronase (1 mg/mL/4 °C) was implemented to separate internalized and cell-membrane fractions (lower panel). (B) The total number of SK-Mel-28 cell-surface Tf-receptors was assessed by pre-incubating cells with or without ascorbate (50 μM) for 30 min/37 °C, then cooling the cells to 4 °C and incubating them for a further 2 h/4 °C with increasing concentrations of Fe2-125I-Tf (12.5–1000 nM). Cells were then washed on ice and the amount of specifically bound radioactivity was determined by subtracting the amount of Fe2-125I-Tf bound in the presence of a 200-fold excess of unlabeled Fe2-Tf. The trend lines shown were obtained by non-linear regression analysis (GraphPad Prism® v. 5.0) (C, D) SK-Mel-28 cells were pre-incubated in the presence or absence of ascorbate as in (A) followed by a 3 h/37 °C incubation with 59Fe2-125I-Tf (0.75 μM). Cells were washed on ice and incubation with Pronase (1 mg/mL) for 30 min/4 °C was used to separate the internalized and cell-membrane fractions. Results are mean + SD (3–6 experiments).

Fe2\ 125I-Tf is not caused

Supp. Fig. 5C). Therefore, the stimulatory effect of ascorbate on 59Fe uptake is not due to changes in the rate of 125I-Tf or 59Fe release from cells.

As ascorbate can suppress cellular 59Fe efflux [39], we examined whether stimulation of 59Fe uptake by ascorbate was due to changes in 59Fe efflux (see Materials and methods section). Consistent with our previous observations [39], ascorbate caused a slight, yet significant (p b 0.01), inhibition of 59Fe efflux (Supp. Fig. 5A). The extent of this inhibition was equivalent to only ~5% (i.e., 4 ± 1 pmol 59Fe/106 cells) of internalized 59Fe (Supp. Fig. 5A). In contrast, ascorbate significantly (p b 0.001) stimulated transferrin 59Fe uptake by 40–50 pmol 59Fe/106 cells (Fig. 1; cf. Supp. Fig. 5A). Clearly, the small yet significant inhibitory effect of ascorbate on cellular 59Fe efflux can only account for at most 10% of ascorbate-stimulated 59Fe uptake from 59Fe2-Tf. Therefore, ≥90% of the stimulatory effect of ascorbate must be due to enhanced internalization of Fe. Additionally, ascorbate had no significant effect on the extent of 125I-Tf release into the overlying medium over 3 h/37 °C (Supp. Fig. 5B) or over shorter time periods of 5–30 min (see Materials and methods section;

3.13. Ascorbate-stimulated 59Fe uptake requires the cellular expression of functional TfRs

3.12. Ascorbate-stimulated 59Fe uptake from by changes in cellular 59Fe or 125I-Tf efflux

59

To determine if ascorbate-stimulated 59Fe uptake from 59Fe2-Tf required the presence of functional TfRs, we assessed the effect of ascorbate on cellular 59Fe and 125I-Tf uptake from 59Fe2-125I-Tf using a select set of variant CHO cell lines that include TRVb, which does not express functional endogenous TfRs and is unable to import Fe that is bound to Tf [32]. In addition, we used TRVb cells that had been transfected to express either human TfR1 (TRVb/TfR1) [32] or FLAG-tagged human TfR2 (TRVb/TfR2) [33,34,77]. As an additional control, all results were compared to the corresponding levels of 59Fe and 125I-Tf uptake by G418resistant TRVb/Neo cells (see Materials and methods section) [34]. Importantly, neither TRVb nor TRVb/Neo cells expressed any detectable TfR1 or TfR2, whereas TfR1 and TfR2 were readily detectable in TRVb/TfR1 and TRVb/TfR2 cells, respectively (Fig. 9A). All cells expressing

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negligible levels of 59Fe under these conditions (Fig. 9B). Moreover, these cells also accumulated very low levels of 125I-Tf compared to the TfR-expressing cells (Fig. 9C). The TRVb/TfR1 cells accumulated the highest levels of internalized 59Fe and 125I-Tf (Fig. 9B,C), which is consistent with the apparent primary function of TfR1 in Tf-dependent Fe uptake [80]. Interestingly, TRVb/TfR2 cells accumulated substantially lower levels of both radiolabels, albeit at levels markedly and significantly (p b 0.01) higher than that of the TRVb or TRVb/Neo cells (Fig. 9B,C). Although the major function of TfR2 appears to be as a regulator of the expression of the hormone of Fe metabolism, hepcidin [49], TfR2 may also play some role in Tf-dependent Fe uptake [34]. Indeed, the heterologous expression of human TfR2 in TRVb cells has previously been shown to promote Tf-dependent 59Fe uptake in an analogous manner to TfR1 under certain conditions [34]. Ascorbate significantly (p b 0.001) stimulated the uptake of 59Fe, but not 125I-Tf, from 59Fe-125I-Tf exclusively in TRVb/TfR1 and TRVb/TfR2 cells (Fig. 9B,C). Notably, and consistent with our previous data (Figs. 5 and 6), the ascorbate-enhanced 59Fe uptake in TfR1-expressing cells was insensitive to ascorbate oxidase, yet sensitive to the membranepermeant ascorbate-oxidizing metalloporphyrin, FeTMPyP (Fig. 9B). Further, ascorbate had no effect on 59Fe or 125I-Tf uptake by cells not expressing TfRs (i.e., TRVb or TRVb/Neo cells; Fig. 9B,C). The observation that ascorbate also enhanced Tf-dependent 59Fe uptake in TRVb/TfR2 cells indicates TfR2 is sufficient for ascorbate to stimulate Tf-dependent 59Fe uptake from Tf. Therefore, the expression of functional cellular TfRs is necessary for ascorbate-stimulated Tf-dependent 59Fe uptake to occur. It should be noted here that, although the human TfR1 cDNA (viz., “pCD-TR1”) used by McGraw et al. [32] to create CHO-TRVb/TfR1 cells contains IREs in the 3′-untranslated region [81], the expression of human TfR1 by these cells was not affected by ascorbate over the 3 h interval, as shown by the equivalent 125I-Tf uptake by control and ascorbate-treated cells (Fig. 9C). Thus, a change in human TfR1 expression cannot account for the stimulatory effect of ascorbate on Tf-dependent 59Fe uptake in CHO-TRVb/TfR1 cells.

3.14. Ascorbate-stimulated Tf-dependent 59Fe uptake requires endocytosis and intravesicular acidification

Fig. 9. Ascorbate-stimulated 59Fe uptake requires the expression of functional TfRs. (A) Whole-cell lysates were obtained and the solubilized proteins separated by SDS-PAGE and the expression of TfR1, TfR2 or β-actin (loading control) was assessed by western blotting for the following variant Chinese Hamster Ovary (CHO) cells: TRVb (variant CHO cells devoid of endogenous TfRs; TRVb), or G418/neomycin-resistant TRVb cells transfected with either pcDNA3-FLAG (TRVb/Neo), human TfR1 (TRVb/TfR1) or FLAG-tagged human TfR2 (TRVb/TfR2). (B,C) Cells were pre-incubated with/without ascorbate (50 μM) for 30 min/37 °C before the addition of vehicle medium, ascorbate oxidase (Asc Ox; 10 U/mL) or FeTMPyP (30 μM) to the same medium for a further 30 min/37 °C. Then, 59Fe2\125I-Tf (0.75 μM) was added to the same medium and cells were incubated for a further 3 h/37 °C. After this incubation, cells were washed on ice and then incubated with Pronase (1 mg/mL) for 30 min/4 °C to obtain the internalized and cell-membrane fractions. Results shown are mean + SD (3 experiments).

TfRs (i.e., TRVb/TfR1 and TRVb/TfR2) accumulated significantly more internalized 59Fe and 125I-Tf from 59Fe2\ 125I-Tf (0.75 μM) than TRVb or TRVb/Neo cells (Fig. 9B,C). Indeed, the latter two cell lines accumulated

As the major route of Tf-dependent Fe uptake occurs by the receptor-mediated endocytic internalization of the Fe2-Tf complex [21,22], this process is likely to be required for the stimulatory effect of ascorbate on 59Fe uptake to occur. First, to examine the involvement of endocytosis in Tf-dependent 59Fe uptake by control and ascorbate-treated cells, we assessed the effect of the general inhibitor of endocytosis, phenylglyoxal [45,82]. Phenylglyoxal (5 mM) inhibits both 59Fe and 59I-Tf uptake from the endocytic internalization of 59Fe2-Tf by SK-Mel-28 cells [45]. In the present study, after incubation of SK-Mel-28 cells with or without ascorbate, phenylglyoxal (5 mM) [45] was added to the same medium for a further 30 min/37 °C before addition of 59Fe2-Tf. In these experiments, phenylglyoxal reduced both control and ascorbate-stimulated 59Fe uptake from 59Fe2-Tf to ~5% of the vehicle-treated cells (Fig. 10). These data suggest endocytosis of the TfR1-59Fe2-Tf complex is required for ascorbate-stimulated Tf-dependent 59Fe uptake. Next, we assessed the effect on Tf-dependent 59Fe uptake of the lysosomotropic agents, chloroquine and NH4Cl [83]. These chemicals cause alkalinization of normally acidified intracellular vesicles (e.g., endosomes and lysosomes [83]) [84]. As endosomal acidification is required for the release of Fe from Fe2-Tf within the endosome [21,84,85], these agents inhibit Tf-dependent Fe uptake [21,45,84]. The results presented in Fig. 10 clearly show that both chloroquine (0.5 mM) and NH4Cl (5 mM) markedly and significantly (p b 0.001) inhibited Tf-dependent 59Fe uptake in the presence or absence of ascorbate.

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Fig. 10. Ascorbate-stimulated Tf-dependent 59Fe uptake requires endocytosis and intravesicular acidification. SK-Mel-28 cells were pre-incubated with/without ascorbate (50 μM) for 30 min/37 °C, then either: chloroquine (0.5 mM), NH4Cl (5 mM), bafilomycin A1 (170 nM) or phenylglyoxal (5 mM) were added and the incubation continued for 30 min/37 °C. Then 59Fe2-Tf (0.75 μM) was added to the same medium and the cells incubated for a further 3 h/37 °C. After this incubation, the cell mono-layers were washed on ice and then incubated with Pronase (1 mg/mL) for 30 min/4 °C to separate the internalized and cell-membrane fractions. Results shown represent internalized 59Fe uptake and are mean + SD (3 experiments).

In mammals, a bafilomycin A1-sensitive V-ATPase acidifies endosomes and lysosomes at the expense of ATP [86]. The inhibition of V-ATPase by bafilomycin A1 (150–250 nM) also causes endosomal alkalinization that inhibits Tf-dependent 59Fe uptake [87,88]. Similarly to the lysosomotropic agents, bafilomycin A1 (170 nM) significantly (p b 0.001) inhibited Tf-dependent 59Fe uptake (Fig. 10). Interestingly, in the presence of chloroquine, NH4Cl and bafilomycin A1, ascorbate still stimulated 59Fe uptake by approximately 1.5–2-fold of the respective control value (Fig. 10). Thus, intravesicular alkalinization does not totally abolish ascorbate-stimulated 59Fe uptake from 59Fe2-Tf, which may be due to residual 59Fe uptake under these conditions. 4. Discussion 4.1. A novel role for ascorbate in enhancing Fe uptake from Fe-Tf For the first time, we have demonstrated that ascorbate markedly enhances Fe uptake from the major plasma Fe donor, Fe-Tf, in human cells. This is an important finding, as ascorbate is a ubiquitous and normally abundant cellular reductant in vivo, yet it is absent under standard cell culture conditions [8]. Significantly, typical physiological plasma ascorbate concentrations enhanced 59Fe uptake by up to 1.5–2-fold from 59Fe2-Tf, which was accompanied by a corresponding increase in cellular ferritin expression and ferritin-59Fe loading. Although ascorbate can increase ferritin either by promoting de novo synthesis [51] or by inhibiting ferritin autophagy [55], we demonstrated that none of these processes were responsible for ascorbate's ability to stimulate Tf-dependent 59Fe uptake. Indeed, these results support the general notion that ferritin does not contribute to Tf-dependent Fe uptake per se, although it serves as the major Fe storage depot for nascently internalized Fe. 4.2. The molecular mechanism involved in ascorbate-stimulated Fe uptake from Fe2-Tf To further dissect the ascorbate's mechanism-of-action, we first adopted a comparative approach to determine key differences between the ascorbate-stimulated uptake from Fe-citrate and Fe2-Tf. We determined that, unlike 59Fe-citrate [4,19,20], the uptake of 59Fe from 59Fe2-Tf

was largely independent of: (i) the reductive action of extracellular ascorbate; and (ii) the extracellular labilization of Fe from Fe2-Tf. In fact, the results of our experiments with membrane-impermeant (e.g., ascorbate oxidase) and membrane-permeant (e.g., FeTMPyP) ascorbate-oxidizing reagents strongly suggest that ascorbate acts intracellularly to enhance Tf-dependent Fe uptake. Consistent with these findings, and with the fact that most biological activities of ascorbate are mediated by the molecule's reducing activity [1,70], our results demonstrated that the reducing ene-diol moiety of ascorbate was required for stimulation of Fe uptake. Importantly, we also showed that this was not due to a general increase in cellular reducing capacity in the presence of ascorbate (Fig. 7C), or to a specific increase in NADPH. In fact, ascorbate decreased cellular NADPH (Fig. 7D). Taken together, our results suggest that ascorbate enhances Tf-dependent 59Fe uptake in a manner dependent on this molecule. Intriguingly, while ascorbate did not affect the cellular flux of Tf, another important finding was that the expression of human TfRs 1 or 2 in CHO-TRVb cells was necessary for the stimulatory effect of ascorbate (Fig. 9B). We also showed that this stimulation by ascorbate was probably mediated by intracellular ascorbate and was not the result of a change in TfR1 or 2 expression. Our observation that ascorbate stimulated Fe uptake in a TfR2-dependent manner lends some support to the notion that, in addition to acting as a regulator of hepcidin production and systemic Fe homeostasis [89], TfR2 may contribute to the cellular acquisition of Fe from Fe-Tf [34,90]. However, any direct role for TfR2 in Tf-dependent Fe uptake is likely to be minor in comparison to TfR1 [91,92]. Finally, using an array of well-characterized inhibitors of endocytosis or endosome acidification, we demonstrated that endocytosis and intracellular vesicle acidification were important for the mechanism by which ascorbate enhances Tf-dependent Fe uptake (Fig. 10). Specifically, endocytosis was necessary for stimulation to occur, while blockade of intracellular vesicle acidification inhibited both control and ascorbate-stimulated 59Fe uptake similarly, although the relative stimulation provided by ascorbate was not affected (Fig. 10). Together, these results demonstrate that: (i) endocytosis is essential for ascorbate to stimulate Tf-dependent Fe uptake; and (ii) intracellular vesicle acidification facilitates both control and ascorbate-stimulated Fe uptake from Fe2-Tf equally. These observations show that ascorbate acts to enhance Tf-dependent Fe uptake downstream of an endocytosis event, which is further enhanced by intracellular vesicle acidification. Taken together, our results indicate that ascorbate acts intracellularly via a reductive mechanism to enhance Tf-dependent Fe delivery of Fe to cells, which occurs subsequent to the TfR-dependent endocytosis of Fe2-Tf complexes (Fig. 11). Considering the mechanistic insights provided herein, and the observation by others that ascorbate promotes mobilization of 59Fe from isolated Tf-containing endosomes [29,93], we propose a model for ascorbate's activity in which ascorbate enhances intra-endosomal ferrireduction and, consequently, Fe mobilization from the Tf-containing endosome (Fig. 11). As suggested by studies with isolated endosomes containing Tf, this could occur by direct uptake of ascorbate into the endosomal vesicle followed by chemical ferrireduction [94], and/or by provision of reducing equivalents from ascorbate to an endosomal ferrireductase [29,93]. As the only confirmed ferrireductase of the Tf-to-cell Fe uptake cycle is Steap3, which appears to be most active in this role in erythroid cells [30], the possibility that ascorbate stimulates Steap3 activity, either directly or indirectly, should now be examined. However, our findings that ascorbate did not increase cellular NADPH (Fig. 7D), a putative electron source for Steap3's ferrireductase activity [30], or increase Steap3 expression (Supp. Fig. 4), suggest that ascorbate does not act via Steap3 to enhance Tf-dependent Fe uptake. Therefore, the possibility that other as-yet-to-be-identified endosomal ferrireductases may utilize ascorbate as an electron source must also be considered.

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Fig. 11. Model for the action of ascorbate in stimulating cellular iron (Fe) uptake from transferrin (Tf). (A) Under control conditions, Fe2-Tf binds to cell surface Tf receptors (TfR) and is internalized by endocytosis. The endosomes that form become acidified via the proton-pumping vacuolar-type H+-ATPase (V-ATPase). Acidification leads to release of Fe from Tf, while Fe-free Tf (apo-Tf) remains bound to the TfR. The labilized intra-endosomal Fe3+ is then reduced to Fe2+, probably by a reductase and transported across the endosomal membrane by the divalent metal transporter 1 (DMT1) and/or Zip14. The apo-Tf–TfR complex returns to the cell surface, where apo-Tf dissociates at neutral pH. After transport by DMT1/Zip14, Fe initially enters the labile Fe pool (LIP) and can be stored in ferritin. (B) In contrast, in the presence of ascorbate, the vitamin enhances: (i) Tf-dependent Fe uptake; (ii) ferritin expression; and (iii) Fe deposition in ferritin. Importantly, the ascorbate-dependent increase in the LIP probably causes increased ferritin synthesis. Our results indicate that ascorbate acts intracellularly via a reductive mechanism that probably (as indicated by dotted lines) either involves direct import of ascorbate into the Tf cycle endosome and direct or chemical ferrireduction and/or provision of electrons to an endosomal ferrireductase to enhance Tf-dependent Fe delivery of Fe to cells.

In conclusion, our novel finding that ascorbate stimulates Tf-dependent Fe uptake is significant for the following reasons: (i) the majority of in vitro studies on Tf-dependent Fe uptake have been with performed with ascorbate-depleted cells; and (ii) severe ascorbatedeficiency in humans (i.e., scurvy), as well as experimentally-induced ascorbate deficiency in guinea pigs, causes an “anemia of scurvy” [11,12]. The observation that ascorbate enhances Fe uptake from Fe2-Tf in all human cells examined suggests that the many previous studies on cellular Fe metabolism and Tf-dependent Fe uptake may have overlooked the involvement of this important reductant. Moreover, the ability of ascorbate to enhance Fe uptake from Fe2-Tf, which is thought to be the major donor of Fe to the erythropoietic compartment [22], could explain the metabolic defect that contributes to ascorbatedeficiency-induced anemia. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbamcr.2013.02.010. Acknowledgements D.J.R.L thanks the Cancer Institute New South Wales for an Early Career Fellowship [10/ECF/2-18] and the National Health and Medical Research Council (NHMRC) of Australia for an Early Career Postdoctoral Fellowship [1013810]. D.R.R. thanks the NHMRC for a Senior Principal Research Fellowship and Project Grants. References [1] C.L. Linster, E. Van Schaftingen, Vitamin C. Biosynthesis, recycling and degradation in mammals, FEBS J. 274 (2007) 1–22. [2] J. Mandl, A. Szarka, G. Banhegyi, Vitamin C: update on physiology and pharmacology, Br. J. Pharmacol. 157 (2009) 1097–1110.

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