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Molecular and Cellular Biology, April 2006, p. 2845-2856, Vol. 26, No. 7
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.7.2845-2856.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Hemin-Mediated Regulation of an Antioxidant-Responsive Element of the Human Ferritin H Gene and Role of Ref-1 during Erythroid Differentiation of K562 Cells

Kenta Iwasaki,1,{dagger} Elizabeth L. MacKenzie,1,{dagger} Kiros Hailemariam,1 Kensuke Sakamoto,1,2 and Yoshiaki Tsuji1*

Department of Environmental and Molecular Toxicology, North Carolina State University, Campus Box 7633, Raleigh, North Carolina 27695,1 Graduate School of Systems Life Science, Kyushu University, Fukuoka 812-8581, Japan2

Received 8 September 2005/ Returned for modification 21 October 2005/ Accepted 11 January 2006


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ABSTRACT
 
An effective utilization of intracellular iron is a prerequisite for erythroid differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H and L, plays a crucial role in iron homeostasis. Here, we have found that the H subunit of the ferritin gene is activated at the transcriptional level during hemin-induced differentiation of K562 human erythroleukemic cells. Transfection of various 5' regions of the human ferritin H gene fused to a luciferase reporter into K562 cells demonstrated that hemin activates ferritin H transcription through an antioxidant-responsive element (ARE) that is responsible for induction of a battery of phase II detoxification genes by oxidative stress. Gel retardation and chromatin immunoprecipitation assays demonstrated that hemin induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1 motifs of the ferritin H ARE, despite no significant change in expression levels or nuclear localization of these transcription factors. A Gal4-luciferase reporter assay did not show activation of these b-zip transcription factors after hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of Jun/Fos family members via reduction of a conserved cysteine in their DNA binding domains, showed induced nuclear translocation after hemin treatment in K562 cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H transcription. Hemin also activated ARE sequences of other phase II genes, such as GSTpi and NQO1. Collectively, these results suggest that hemin activates the transcription of the ferritin H gene during K562 erythroid differentiation by Ref-1-mediated activation of these b-zip transcription factors to the ARE.


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INTRODUCTION
 
Iron is an indispensable element for a variety of cellular functions in metabolism, growth, and differentiation (36). Iron serves as a constituent of vital proteins, including ribonucleotide reductase and many other heme proteins, such as mitochondrial cytochromes, cytochrome P450 enzymes, and hemoglobin. In particular, during the process of maturation of erythroid cells, a sufficient amount of iron should be supplied for hemoglobinization under a coordinated partitioning of intracellular iron levels. However, an excess amount of intracellular free iron is harmful to the cells because iron can catalyze formation of reactive oxygen species through the Fenton reaction (44, 51). Therefore, intracellular iron levels should be tightly regulated by storing excess iron in a nontoxic but bioavailable form for supply upon metabolic requirement for hemoglobinization.

Ferritin is the major cellular iron storage protein that plays a role in the storage and partitioning of iron for intracellular use (5, 64). Ferritin is composed of 24 subunits of heavy chains (H) and light chains (L), with varied H-to-L ratios depending on types of tissues and their physiological conditions (26). Ferritin synthesis is regulated at both transcriptional and translational levels (67). Iron induces ferritin synthesis at a translational level through regulation of iron regulatory proteins and iron responsive element interaction in the 5' untranslated region of ferritin mRNAs (28, 58, 65).

In contrast to the well-characterized translational mechanism of ferritin synthesis by iron, molecular mechanisms of transcriptional regulation of ferritin genes remain to be fully elucidated. Recently, our and other studies revealed that transcriptional regulation of the human ferritin H gene is regulated through at least two independent enhancer elements. One is a proximal cis-acting element containing a CCAAT motif approximately 60 bp and 5' from the transcription initiation site of the human ferritin H gene (9, 41). This CCAAT element, serving as a basal enhancer of ferritin H transcription that contributes to tissue-specific expression (10) as well as upregulation of ferritin H during differentiation (41), is regulated by NF-Y transcription factors and transcriptional coactivators p300/CBP and PCAF (10, 25). Another regulatory element for ferritin H transcription is a far-upstream enhancer we have recently identified which serves as an antioxidant-responsive element (ARE) responsible for oxidative stress-mediated activation of the ferritin H gene (68). An ARE, also identified in the 5' flanking region of various phase II detoxification genes such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), regulates transcriptional activation of these phase II genes in cells exposed to a wide variety of xenobiotics and reactive metabolites (31, 49). We have recently found that the human ferritin H ARE is composed of two copies of bidirectional AP1 motifs to which basic-leucine zipper (b-zip) transcription factors, such as JunD and NFE2-related factor 2 (Nrf2), can bind and which activate ferritin H transcription under oxidative conditions (68).

K562 human erythroleukemia cells have offered a great experimental system to study molecular mechanisms of erythroid differentiation (8). A number of reagents, including chemotherapeutic drugs, have been shown to induce K562 erythroid differentiation (59). K562 differentiation turns on hemoglobin synthesis and diminishes growth potential. It was demonstrated that treatment of K562 cells with hemin (ferriprotoporphyrin IX) induced synthesis of ferritin proteins at both transcriptional (61, 73) and translational levels (42). The hemin-mediated translational activation of ferritin synthesis was blocked by iron chelator, deferoxamine, probably due to chelating iron liberated from hemin in the cells (55). However, the hemin-mediated increase in ferritin mRNA was not blocked by deferoxamine treatment (42), suggesting that mechanisms of hemin-induced ferritin mRNA in K562 cells are independent of the amount of intracellular chelatable iron.

Given several lines of evidence showing that oxidative stress is involved in chemically induced differentiation of K562 cells (18, 47), we have tested our hypothesis that ferritin mRNA induction during hemin-mediated K562 differentiation is transcriptionally regulated through the far-upstream ARE enhancer element. In this study, we have found that (i) a hemin-responsive element of the ferritin H gene is identical to ARE, (ii) hemin treatment induced binding of cJun, JunD, FosB, and Nrf2 transcription factors to the ferritin H ARE, and (iii) redox factor 1 (Ref-1) is involved in the activation of ferritin H transcription through the ARE.


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MATERIALS AND METHODS
 
Cell culture. K562 human erythroleukemia cells were purchased from the American Type Culture Collection. They were cultured in RPMI 1640 medium supplemented with 0.3 g/liter glutamine, 25 mM HEPES, and 10% fetal calf serum (Mediatech) at 37°C in a humidified 5% CO2 atmosphere. Hemin was purchased from Fluka BioChemika and dissolved in 0.1 M NaOH. HMBA (N,N'-hexamethylenebisacetamide), TPA (12-O-tetradecanoylphorbol-13-acetate), and t-BHQ (tert-butylhydroquinone) were from Sigma.

Plasmids and DNA transfection. pBluescript SK(–)–5.2kb h-ferritin H-luciferase (where –5.2kb human ferritin H-luciferase means that this construct contains a 5.2-kb upstream region [5.2-kb DNA fragment] from the transcription start site of the ferritin H gene) as well as most deletion and mutation reporter plasmids have been described elsewhere (68). pBluescript SK(–)–4.5kb h-ferritin H ARE mutant plasmids were constructed by PCR-mediated mutagenesis using the mutated ARE primers shown in Fig. 3. Human NQO1 ARE-luciferase and rat GST-pi-luciferase plasmids were constructed by ligation of double-strand oligonucleotides of 5'-AAATCGCAGTCACAGTGACTCAGCAGAATC-3' and 5'-CAAAAGTAGTCAGTCACTATGATTCAGCAACAAA-3' (31), respectively, into the minimum promoter of –0.03kb ferritin H-TATA-luciferase. pRc/CMVJunD was constructed as described previously (68). pCMVNrf2 was constructed by digestion of pOBT7Nrf2 (clone ID 4548874; Invitrogen) with BamHI and XhoI, and the resultant Nrf2 cDNA was cloned into the pCMV vector. Transient DNA transfection into K562 cells was carried out by electroporation (Xcell; Bio-Rad) with an optimized preset condition by Bio-Rad for K562 cells (exponential decay, 1,000 µF; 155 V; 100-µl cell suspension in a cuvette with a 0.2-cm gap). After electroporation of luciferase reporters into 5 x 106 to 10 x 106 K562 cells, they were plated at a density of 4 x 106 to 5 x 105 cells per 60-mm plate containing 4 ml of the culture medium. As a transfection internal control, 0.1 µg of pRL-CMV (Promega) or pRL-EF (elongation factor promoter) was simultaneously cotransfected. After incubation for 20 to 24 h, the cells were treated with various concentrations of hemin for 24 h. In some experiments, t-BHQ treatment was also included as a positive control of ferritin H ARE activation. Ref-1 cDNA was kindly provided by T. Curran (St. Jude Children's Research Hospital, Memphis, TN). Preparation of cell extracts and luciferase assays were performed using dual-luciferase assay reagents (Promega). Firefly luciferase expression driven by the ferritin H gene was normalized by Renilla luciferase activity.


Figure 3
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  		FIG. 3. Hemin activates transcription of the ferritin H gene through the ARE. K562 cells were cotransfected via electroporation with (a) 10 µg of –0.03kb ferritin H-TATA-luciferase (TATA) or insertion of four copies of the ARE in –0.03kb ferritin H-TATA-luciferase (4x ARE), (b) one copy of wild-type ARE (wt), AP-1-like mutant, AP-1/NFE2 mutant, or double mutant (see schematic), or (c) wild-type or each mutant ARE in the –4.5kb h-ferritin H-luciferase reporter, along with 20 ng of pRL-EF as an internal control. Cells were treated with 50 µM hemin or 10 µM t-BHQ (a and b) or 10, 25, and 50 µM hemin (c) for 24 h, and the resulting luciferase activity was assessed via luminometry. Induction was determined by setting 4x ARE/control (a), single-copy wild-type ARE/control (b), and –4.5kb wild-type ARE/control (c) at 1.0. The means ± standard errors from three independent experiments are shown.

Gal4 reporter system and plasmids. Four micrograms of pFA2-cJun, -JunD, or -FosB, a trans-activator protein fused to the Gal4-DNA binding domain (Gal4DBD), was electroporated into 1 x 107 K562 cells together with 1 µg of pFL-Luc reporter containing five direct repeats of the yeast Gal4 binding site that controls expression of the luciferase gene from Photinus pyralis (Stratagene). Transfected cells were divided into six plates of 35-mm dishes and incubated for 24 h. Cells were then treated with 25 µM or 50 µM of hemin for 24 h and subjected to luciferase reporter assays (Promega).

Gel retardation assay. Preparation of nuclear extracts, binding reactions, and separation of retarded bands by polyacrylamide gel electrophoresis have been described previously (71). All antibodies used in gel supershift assays were purchased from Santa Cruz Biotechnology, Inc.

ChIP assay. Chromatin immunoprecipitation (ChIP) assays were carried out according to Upstate Biology's protocol for the ChIP assay kit with some minor modifications. Briefly, a total of 1 x 106 to 4 x 106 K562 cells/100-mm plate were treated with 50 µM hemin for 4 h, 1 day, and 3 days, followed by chromatin cross-linking and preparation of cell lysates using the ChIP assay kit (Upstate Biology). DNA in the lysate (200 µl) was sheared by sonication (Sonic Dismembrator model 100, power setting 2; Fisher Scientific), with 12 cycles of 10-second pulses and 20-second intervals. Approximately 1/10 aliquots of cell lysate were immunoprecipitated with 1 µg each of antibodies (anti-ATF1, sc-234X; anti-CREB, sc-240X; anti-Nrf2, sc-722X; anti-JunB, sc-46X; anti-c-Jun, sc-1694X; anti-JunD, sc-074X; anti-FosB, sc-48X; anti-Jun family, sc-044X; and anti-Fos family, sc-253X) (Santa Cruz Biotechnology). Quantitative PCR with a pair of primers giving rise to a 0.15-kb DNA fragment and gel electrophoresis were performed as described previously (68).

Western blotting. Total cell lysates or nuclear and cytoplasmic fractions were loaded on 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer of separated proteins to an Immobilon-P (Millipore) polyvinylidene difluoride membrane and incubation at 4°C overnight with one of the following antibodies: anti-ferritin H, anti-c-Jun, anti-JunD, anti-FosB, anti-Nrf2, anti-hemoglobin gamma, anti-Ref-1 (all from Santa Cruz Biotechnology), or anti-fetal hemoglobin (Calbiochem). Cell fractionation was carried out using a nuclear extract isolation kit (Active Motif), and purity of nuclear and cytoplasmic fractions was verified by Western blotting with anti-histone H1 (Santa Cruz Biotechnology) or anti-lactate dehydrogenase (LDH; Chemicon). Recombinant human ferritin H and L were purchased from Calbiochem.


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RESULTS
 
Transcriptional activation of ferritin H during hemin-mediated erythroid differentiation of K562 cells. To investigate hemin-mediated induction of the ferritin H gene during erythroid differentiation, we first measured expression of ferritin H mRNA and protein during a 5-day incubation period in K562 cells. Northern blotting experiments showed that hemin treatment increased ferritin H mRNA in a dose-dependent manner, with peaking between 1 and 3 days after the treatment (Fig. 1a). The inducing effect on ferritin H mRNA appeared to be specific to hemin because HMBA, a differentiation inducer of mouse erythroleukemia (MEL) cells (43), or TPA, an inducer of megakaryotic differentiation of K562 cells (56), failed to induce ferritin H mRNA (Fig. 1a). Induction of ferritin H protein synthesis was also detected by Western blotting, reaching a maximum 1 day after hemin treatment and maintaining the high expression level until day 5 (Fig. 1b). As an erythroid differentiation marker, we monitored fetal hemoglobin expression in K562 cells (8) using two different antibodies in which hemin treatment induced synthesis of hemoglobin on day 1 and sustained the high expression level for 5 days (Fig. 1b).


Figure 1
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  		FIG. 1. Ferritin H is induced during hemin-mediated erythroid differentiation of K562 cells. (a) Total RNA was isolated from K562 cells treated with 2 µM hemin, 10 µM hemin, 5 mM HMBA, or 50 ng/ml TPA for 3 days (top) or 10 µM hemin for 1, 3, and 5 days (bottom). Ten micrograms of RNA was subjected to Northern blot analysis hybridized with human ferritin H cDNA. An equal amount of RNA loading was confirmed by staining RNA with ethidium bromide. Positions of 28S and 18S rRNA are indicated. (b) K562 cells were incubated for 1 to 5 days with or without 50 µM hemin, and 30 µg of protein from each cell lysate was separated on 10% polyacrylamide/sodium dodecyl sulfate gel for detection of ferritin H, fetal hemoglobins, {gamma}-globin, and ß-actin. Recombinant ferritin H and L proteins (50 ng) were loaded to confirm the specificity of the anti-ferritin H antibody. (c) K562 cells (5 x 106) were electroporated with 10 µg of –5.2kb h-ferritin H-luciferase plus 1 µg of pRL-CMV as an internal control of transfection and treated with 50 µM hemin for 1 to 3 days. Expression of firefly luciferase was normalized by that of Renilla luciferase, and the value for day 1 without treatment was set at 1.0. The results from three independent experiments are shown as means ± standard errors.

We next asked whether a hemin-mediated increase in ferritin H mRNA is regulated at a transcriptional level. To address this question, we transiently transfected a 5.2kb 5' human ferritin H-luciferase reporter plasmid into K562 cells, followed by hemin treatment for 1 to 3 days, and measured luciferase expression levels. As shown in Fig. 1c, hemin treatment activated expression of luciferase driven by the 5.2kb human ferritin H 5' regulatory region, indicating that hemin transcriptionally activates the human ferritin H gene through a hemin-responsive enhancer element in the 5.2kb region.

Role of the ferritin H ARE in hemin-mediated transcriptional activation. In order to identify the hemin-responsive element in the human ferritin H gene, we further performed luciferase reporter assays in K562 cells using several deletion constructs of the human ferritin H gene. In contrast to hemin-mediated induction of luciferase driven by the –5.2kb ferritin H gene, luciferase reporters containing –4.0kb, –0.15kb, and –0.03kb TATA all failed to be induced by hemin treatment (Fig. 2a). The –0.15kb luciferase, which contains the CCAAT element (9, 41), showed higher basal expression than the TATA-only reporter but did not respond to hemin treatment. t-BHQ, an activator of ARE in the ferritin H gene (68, 70) and other phase II genes (38), also activated expression of only –5.2kb ferritin-luciferase (Fig. 2a) These results indicate that the hemin-responsive element is located in the region between –5.2kb and –4.0kb of the human ferritin H gene.


Figure 2
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  		FIG. 2. Characterization of a hemin-responsive element in the human ferritin H gene. (a) Ten micrograms of human ferritin H-luciferase reporter plasmids (–5.2kb, –4.0kb, –0.15kb, TATA, or 0kb) together with 20 ng of pRL-EF was transfected into K562 cells (5 x 106 cells) by electroporation. Hemin (50 µM) or t-BHQ (10 µM) was added 1 day after electroporation and incubated for 24 h. The transfected cells were harvested to measure firefly and Renilla luciferases, and expression of firefly luciferase was normalized by Renilla luciferase expression. The luciferase activity in cells transfected with –5.2kb ferritin H reporter plasmid with no treatment (control) was defined as 1.0, and the results from four independent experiments are shown as means ± standard errors. (b) K562 cells were transfected with 10 µg of the –4.5kb luciferase reporter (+ARE) or 10 µg of the –4.4kb luciferase reporter (–ARE) by electroporation. Twenty-four hours after transfection, cells were treated with 50 µM hemin or 10 µM tBHQ or left untreated for 24 h, and cell lysates were collected to perform the luciferase assay. To obtain induction in luciferase activity, luciferase expression in extracts obtained from cells transfected with the –4.5kb luciferase reporter with no treatment (control) was defined as 1.0. The results from five independent experiments are shown as means ± standard errors.

Recently, we identified an ARE, located 4.5kb 5' from the transcription start site, that was responsible for oxidative stress-mediated transcriptional activation of the human ferritin H gene (68). The ferritin H ARE comprises two copies of bidirectional AP1 motifs (68). Since (i) oxidative stress was shown to be involved in erythroid differentiation of K562 cells (18, 47), (ii) hemin induces oxidative stress (54), and (iii) the human ferritin H ARE (at the –4.5kb region) is within the –5.2kb to –4.0kb region in which we identified a hemin-responsive element (Fig. 2a), we hypothesized that the hemin-responsive element is identical to the ARE. To test this possibility, we transiently transfected the ARE(+)- or ARE(–)-luciferase reporter plasmid into K562 cells and measured luciferase expression after treatment with hemin or t-BHQ. The –4.5kb ferritin H-luciferase, which contains the intact ferritin H ARE, was activated by hemin or t-BHQ treatment; however, the –4.4kb ferritin H-luciferase lacking the ARE sequence showed lower basal expression, with no response to hemin treatment (Fig. 2b). These results suggest that the ARE is involved in hemin-mediated transcriptional activation of the ferritin H gene.

To confirm this observation, we then inserted the ARE sequence into the minimum ferritin H-TATA promoter reporter plasmid and tested whether ARE alone is sufficient for hemin-mediated transcriptional activation in K562 cells. As shown in Fig. 3a, insertion of ARE made the minimum TATA reporter responsive to hemin as well as t-BHQ to induce luciferase expression. The human ferritin H ARE contains two AP1 motifs (Fig. 3), both of which are essential for the function of ARE to respond to oxidative stressors, such as H2O2 and t-BHQ (68). To further assess the role of the ARE in hemin-mediated ferritin H transcriptional activation, we first introduced mutations in two AP1 motifs in the ferritin H ARE, cloned one copy of each AP1 mutant into a minimum TATA-luciferase reporter and tested each motif's response to hemin treatment. Mutations in either the AP1-like motif or AP1/NFE2 motif or mutations in both sites in the ferritin H ARE almost completely abolished hemin-mediated activation (Fig. 3b). Similarly, we introduced the same mutations in the AP1 motifs with the entire 4.5kb region of the 5' ferritin H gene and tested their response to hemin treatment. In this context, a mutation in either one of the two AP1 motifs partially impaired the hemin response, but double mutations completely abolished the hemin response in addition to significantly decreasing the basal expression levels (Fig. 3c). Collectively, these results confirmed that the hemin-responsive element is identical to the ARE of the human ferritin H gene.

Alterations in binding of b-zip family transcription factors to the ferritin H ARE by hemin treatment. To elucidate the molecular mechanism by which hemin activates transcription of the human ferritin H gene through the ARE, we performed gel mobility shift assays using nuclear extracts isolated from hemin-treated K562 cells. As shown in Fig. 4a, total protein binding to the AP-1/NFE2 site of the ferritin H ARE was increased following treatment with hemin or t-BHQ. The addition of unlabeled competitor oligonucleotide inhibited the bands that were increased by hemin treatment, indicating that the increase in protein binding was specific to the AP-1/NFE2 sequence. Next, we examined Jun, Fos, and Nrf2 family members to determine which b-zip family members comprised the ARE binding complex following hemin treatment. In gel supershift assays, both Jun and Fos family antibodies induced a supershift (Fig. 4b), indicating that transcription factors from these families bind to the ARE in vitro; however, we were unable to detect clear Nrf2 binding to the ARE using this method before or after hemin treatment (Fig. 4b).


Figure 4
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  		FIG. 4. The Jun family, Fos family, and Nrf2 transcription factors bind to the ARE of the human ferritin H gene in hemin-treated K562 cells. (a) K562 cells were incubated for 4 h following 50 µM hemin treatment. After treatment, nuclear extracts were subjected to a gel retardation assay using a specific AP-1/NFE2 oligonucleotide probe. The competitor (Comp.) indicates the addition of a 50-fold excess of unlabeled AP-1/NFE2 oligonucleotide. (b) Nuclear extracts from K562 cells treated with 50 µM hemin for 4 h, 1 day, and 3 days were used for a gel supershift assay by preincubating the binding reaction with control immunoglobulin G (IgG) or anti-Nrf2, anti-Jun family, or anti-Fos family antibody for 2 h at 4°C prior to the AP-1/NFE2 probe addition. The supershifts induced by Jun family and Fos family antibodies are indicated by arrowheads. (c) Ferritin H ARE ChIP assays were performed with no antibody, control rabbit and mouse IgG (mixture), or b-zip transcription factor antibodies as indicated at the top of the panel. A plasmid DNA containing a 5.2kb ferritin H 5' region and K562 genomic DNA were used as positive controls for the PCR as well as markers of the ferritin H 155-bp PCR band. –, no treatment; +, 50 µM hemin treatment for 4 h. (d) Similar ChIP assays for Nrf2, c-Jun, junD, and FosB were carried out after incubation of K562 cells with 50 µM hemin for 1 day and 3 days.

We then carried out ChIP assays to investigate the binding of specific transcription factors to the ARE in vivo. As shown in Fig. 4c, hemin treatment for 4 h induced the binding of cJun, JunD, FosB, and Nrf2 to the ferritin H ARE, whereas JunB binding was diminished. The antibodies we used against ATF1, CREB, cFos, Fra1, and Fra2 to the ARE did not show evidence of binding to the ferritin H ARE before or after hemin treatment. Increased Nrf2 and JunD binding to the ARE was maintained until later time points of day 1 and day 3 (Fig. 4d). Cotransfection of Nrf2 and JunD together with the –4.5kb ARE(+)- or –4.4kb ARE(–)-ferritin H-luciferase demonstrated that both Nrf2 and JunD are transcriptional activators of the ferritin H ARE and that the two together activate the ferritin H ARE better than each alone (Fig. 5). Taken together, these results suggest that hemin induces ferritin H transcription through increased DNA binding of cJun, JunD, FosB, and Nrf2 to the ferritin H ARE.


Figure 5
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  		FIG. 5. Nrf2 and JunD activate the human ferritin H ARE. K562 cells were transfected with 1 µg of –4.5kb ferritin H-luciferase reporter (+ARE) or –4.4kb ferritin H-luciferase reporter (–ARE) together with either 0.5 µg of pCMVNrf2, 1 µg of pRc/CMVJunD, or 0.5 µg of pCMVNrf2 plus 1 µg of pRc/CMVJunD by electroporation. pRL-null plasmid (0.2 µg) was simultaneously transfected as an internal transfection control. The total amount of plasmid DNA in each transfection was equalized to 2.7 µg by adding pCMV or pRc/CMV empty vector. Forty-eight hours after transfection, cells were harvested for dual-luciferase assays. Expression of firefly luciferase was normalized by Renilla luciferase expression, and the value of the –4.5kb ARE(+) luciferase reporter with empty vector (control) was defined as 1.0. The results from five independent experiments are shown as means ± standard errors.

Hemin activates ARE of other phase II genes. We next asked whether hemin-mediated activation of an ARE enhancer is specific to the ferritin H ARE. To address this question, we constructed two different ARE-luciferase reporters derived from human NQO1 ARE and rat GST-pi ARE (31), transfected them into K562 cells, and challenged them with hemin treatment. As shown in Fig. 6, hemin also activated these ARE reporters in a dose-dependent manner. We therefore concluded that hemin-mediated ARE activation is not unique to the ferritin H ARE but seems to be broader to a conserved ARE motif found in NQO1, GST, and probably other ARE-regulated phase II antioxidant genes.


Figure 6
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  		FIG. 6. Hemin activates NQO1 and GST AREs. One microgram of GSTpi-ARE-luciferase or NQO1-ARE-luciferase was cotransfected into K562 cells along with 20 ng of pRL-EF as an internal control. Cells were treated with 50 or 75 µM hemin for 24 h, and the resulting luciferase activity was measured. Induction was determined by setting the control at 1.0, and the means ± standard errors from six independent experiments are shown.

Role of Ref-1 in hemin-mediated activation of ferritin H transcription through the ARE. To elucidate the activation mechanism of these b-zip transcription factors, we first employed a Gal4 reporter assay to assess posttranslational activation by hemin treatment. A fusion protein of Gal4DBD-JunD, -cJun or -FosB was expressed in K562 cells transiently cotransfected with a Gal4DBD-luciferase reporter and then treated with hemin. These Gal4DBD fusion transcription factors significantly activated the expression of luciferase; however, 25 µM or 50 µM hemin failed to further increase luciferase expression (Fig. 7a), suggesting that posttranslational activation of these transcription factors by way of, for instance, phosphorylation or sumoylation may not be the principal mechanism of the hemin-mediated activation. Since the increase in DNA binding of these transcription factors was observed in our gel shift and ChIP assays (Fig. 4), we then asked whether accumulation of these transcription factors in the nucleus is induced after hemin treatment. K562 cells treated with hemin for 4 h were subjected to the preparation of cytoplasmic and nuclear fractions, and Western blotting was performed to detect endogenous JunD, cJun, FosB, and Nrf2 expression levels. As shown in Fig. 7b and c, none of these transcription factors showed increased expression in nuclear fractions. Since Nrf2 has been shown to be the major transcription factor in regulating ARE (32, 35, 49), we also transiently transfected Nrf2 into K562 cells and carried out similar Western blotting, in which endogenous Nrf2 as well as transfected Nrf2 did not show clear nuclear accumulation after hemin treatment (Fig. 7c). Taken together, we concluded that increased binding of these transcription factors to the ferritin H ARE after hemin treatment is not due to a dynamic alteration of transcription factors per se but may require a regulatory molecule for their enhanced DNA binding capacities.


Figure 7
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  		FIG. 7. Hemin and posttranslational regulation of JunD, c-Jun, FosB, or Nrf2 transcription factors. (a) A Gal4 reporter system was utilized to estimate c-Jun, JunD, or FosB activation in hemin-treated K562 cells. Four micrograms of pFA2-cJun, -JunD, or -FosB fused to Gal4DBD was electroporated into K562 cells together with 1 µg of pFL-Luc reporter containing five direct repeats of the yeast Gal4 binding element and 20 ng of pRL-EF as an internal control. Twenty-four hours after transfection, cells were treated with 25 or 50 µM of hemin for 24 h and subjected to luciferase reporter assays. The induction in Gal4DBD transfectants without treatment was defined as 1.0. The means ± standard errors from three independent experiments are shown. (b) K562 cells (4 x 106) were treated with 50 µM hemin for 4 h, and cytoplasmic (C) and nuclear (N) fractions were prepared. Fifty micrograms of each fraction was subjected to Western blotting using anti-JunD, anti-cJun, or anti-FosB. Arrowheads in the panel indicate respective transcription factor bands. (c) (Top panel) K562 cells (4 x 106) were treated with 50 µM hemin for 4 h, and cytoplasmic (C) and nuclear (N) fractions were prepared. Fifty micrograms of each fraction was subjected to Western blotting using anti-Nrf2 antibody. (Bottom panel) K562 cells (4 x 106) transfected with pCMV vector or pCMVNrf2 were similarly subjected to Western blotting for Nrf2, followed by histone H1 Western blotting (using the same membrane) and LDH Western blotting (using the same samples but a different blot) for the assessment of purity of cytoplasmic and nuclear fractions.

Growing evidence has demonstrated that redox regulation of protein-protein and protein-nucleic acid interactions is an important cellular defense mechanism under oxidative stress conditions (48). Stress-mediated activation of AP1 transcription factors is subject to redox regulation by Ref-1 that enhances DNA binding of AP1 via reduction of a conserved cysteine residue in the DNA binding domain (75). We therefore tested whether Ref-1 is involved in hemin-mediated ferritin H ARE activation. Ref-1 was transiently cotransfected into K562 cells together with the wild-type ARE- or mutant ARE-luciferase reporter plasmid (ARE insertion either in the minimum TATA reporter [Fig. 8a, left panel] or in the entire 4.5kb 5' ferritin H reporter [Fig. 8a, right panel]), followed by treatment with hemin for 24 h. Ref-1 alone increased luciferase expression driven by wild-type ARE but not by mutant ARE (Fig. 8a). In the same experiment, nuclear and cytoplasmic fractions were prepared from K562 cells treated with 50 µM hemin for 4 h, and Western blotting was carried out to detect Ref-1 expression in each fraction. As shown in Fig. 8b, endogenous Ref-1 in the cytoplasm of control vector-transfected K562 cells was decreased after hemin treatment, resulting in a concomitant increase of Ref-1 in the nuclear fraction. In Ref-1-transfected K562 cells, overexpressed Ref-1 in the cytoplasm was slightly decreased in response to hemin treatment, not as significant as the endogenous Ref-1 translocation, and slightly increased Ref-1 was observed in the nucleus (Fig. 8b). t-BHQ treatment also induced nuclear translocation of Ref-1 in K562 cells (Fig. 8b). We then asked whether Ref-1 induces binding of Nrf2 to the ferritin H ARE. To address this question, we transiently transfected Ref-1 into K562 cells and performed a ChIP assay to assess the role of Ref-1 in endogenous Nrf2 binding to the ferritin H ARE. Our results show that Ref-1 enhances Nrf2 binding to the ferritin H ARE in vivo (Fig. 8c). These results suggest that Ref-1 is, at least in part, involved in basal expression of the ferritin H gene and hemin-mediated activation of ferritin H transcription through increased DNA binding of these b-zip transcription factors to the ARE through redox regulation.


Figure 8
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  		FIG. 8. Activation of the ferritin H ARE by Ref-1 and increased Ref-1 nuclear localization by hemin or t-BHQ treatment. (a) One microgram of ferritin H ARE-luciferase (left panel, the wild-type or double mutant ARE in the minimum promoter construct shown in Fig. 3b; right panel, –4.5kb ferritin H containing the wild-type or double mutant ARE shown in Fig. 3c) and 20 ng of pRL-EF together with 1 to 4 µg of pCMV-Ref1 expression vector were transfected into K562 cells by electroporation. Hemin (25 or 50 µM) was added 24 h after transfection, and cells were incubated for an additional 24 h. The cells were harvested to measure firefly and Renilla luciferase. The induction from the wild-type ARE transfectant (wtARE) without treatment was defined as 1.0. The results from three independent experiments are shown as means ± standard errors. (b) K562 cells transfected with 4 µg of pCMV-Ref1 or pCMV empty vector, followed by treatment with 50 µM hemin or 10 µM t-BHQ for 4 h were subjected to isolation of nuclear and cytoplasmic fractions. One microgram of nuclear protein and 6.5 µg of cytoplasmic protein were used for Western blotting with anti-Ref1 antibody. Anti-LDH and anti-histone H1 antibodies were used for verification of the purity of the fractions. (c) K562 cells (1 x 107) were transfected with 10 µg pCMVRef-1, and a ferritin H ARE ChIP assay was carried out to assess endogenous Nrf2 binding to the ARE. The –5.2kb ferritin H-luciferase plasmid was used as a PCR control and as a size marker of the 155-bp DNA fragment.


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DISCUSSION
 
A number of chemicals, including some chemotherapeutic agents, have been demonstrated to induce erythroid differentiation of K562 cells, among which hemin is one of the most potent erythroid differentiation inducers (8, 59). During hemin-mediated erythroid differentiation of K562 cells, both transcriptional and translational upregulation of the ferritin gene was observed (42), resulting in an increase in ferritin synthesis and ultimately enhancing cellular capacity of iron storage and supply upon metabolic requirement for hemoglobinization (2, 45, 72). In contrast to well-elucidated translational regulation of ferritin synthesis by hemin (27, 40, 55, 57), molecular mechanisms by which transcription of the ferritin gene is regulated during hemin-induced erythroid differentiation have been elusive. Coccia and colleagues demonstrated that dimethyl sulfoxide or HMBA had minimum effect on ferritin H mRNA induction during erythroid differentiation of MEL cells but that hemin induced ferritin H at the transcriptional level (19, 20). In their further study, hemin induced NF-Y binding to a proximal CCAAT element by which transcription of the human ferritin H gene was activated (41). In our experiments using K562 human erythroleukemia cells, we did not observe a significant role of the CCAAT element in hemin-mediated activation of the ferritin H gene (Fig. 2), perhaps due to the difference in cell lines and species; however, the region containing CCAAT indeed served as a potent proximal enhancer of the ferritin H gene in K562 cells (Fig. 2).

It was demonstrated that hemin- as well as anthracycline-mediated K562 erythroid differentiation was blocked by antioxidants, suggesting that oxidative stress is involved in a chemically induced erythroid differentiation process (18, 46). Furthermore, a Fenton reagent (Fe2+ plus H2O2) was shown to induce erythroid differentiation of K562 cells (47). These observations led us to hypothesize that hemin may activate transcription of the ferritin gene through an ARE enhancer as a result of hemin-mediated oxidative stress. We tested this hypothesis in this study and found that a hemin-responsive element for transcriptional activation of the human ferritin H gene is identical to the ARE (Fig. 2 and 3), which was also activated by t-BHQ, a potent ARE activator (38). Intriguingly, Beaumount et al. reported that a far-upstream 180-bp region of the mouse ferritin H gene contains a regulatory element for ferritin H transcriptional activation during differentiation of MEL cells with HMBA (7). We reviewed this far-upstream 180-bp region and confirmed that it in fact contains an ARE that we previously identified as an enhancer for transcriptional activation of the mouse ferritin H gene in response to oxidative stress (69, 70). Since the human and mouse ferritin H AREs are well conserved in their DNA sequences, including two essential AP1 motifs (68, 70), we speculated that activation mechanisms of the ARE may be shared in both human and mouse ferritin H genes during erythroid differentiation of K562 by hemin and MEL by HMBA. However, our preliminary experiments of transfection of either mouse or human ferritin H-luciferase reporters into MEL cells showed that HMBA did not activate the ferritin H ARE (our unpublished observations). In fact, Beaumont et al. (7) reported that a mutation in the NF-E2 site within the 180-bp region of the mouse ferritin H gene (which is in the ARE) (68) did not abolish the induction of ferritin H by HMBA treatment. Therefore, all experimental results available so far suggest that HMBA-mediated activation of the ferritin H transcription in MEL cells requires an unidentified enhancer element. Further investigations will be necessary to understand the molecular mechanism by which HMBA activates the transcription of the ferritin H gene during erythroid differentiation of MEL cells.

It should be noted that one of the AP1 motifs in the ferritin H ARE is identical to the NF-E2 binding site (Fig. 3). NF-E2, belonging to the b-zip transcription factor family, forms a heterodimer of p45NF-E2 and the small Maf family of transcription factors (3). NF-E2 is involved in transcriptional regulation of the ß-globin gene through locus control regions (13). However, homozygous p45 NF-E2 knockout mice displayed relatively mild defects in erythroid development with normal ß-globin expression levels (60), probably due to compensation of p45 NF-E2 function by other transcription factors. We could not obtain results to indicate the contribution of NF-E2 to ferritin H ARE activation in ARE-luciferase reporter assays and gel retardation assays or a hemin-mediated localization change of NF-E2 by Western blotting (data not shown). Although further investigation will be necessary, we currently understand that NF-E2 may not be a key regulator of ferritin H transcription during hemin-mediated erythroid differentiation of K562 cells. This is consistent with the observation by Beaumont et al. in which they did not find the contribution of NF-E2 protein to mouse ferritin H transcription during HMBA-induced erythroid differentiation of MEL cells (7).

Among AP1/Nrf b-zip transcription factor family members, our ChIP assays showed that hemin induced binding of cJun, JunD, FosB, and Nrf2 to the ferritin H ARE in K562 cells (Fig. 4). Although several groups demonstrated that induction of nuclear translocation or accumulation of Nrf2 is an activation mechanism of ARE by oxidative stressors (32, 35, 49) or hemin treatment (33), we did not see clear induction of nuclear translocation or accumulation of these transcription factors, including Nrf2 after hemin treatment in K562 cells (Fig. 7). We therefore speculated that posttranslational modifications of these transcription factors following hemin treatment may enhance their abilities of DNA binding. Ref-1, a redox factor of AP1, was previously demonstrated to reduce a conserved cysteine residue in the DNA binding domains of AP1 family members and enhances their binding to the AP1 binding sequence (1, 74). Ref-1 was also shown to be activated during oxidative stress, at least in part, by the mechanism of enhanced nuclear translocation of Ref-1 itself (4, 63) or thioredoxin (30, 34). In this study, we showed that Ref-1 is localized in both the nucleus and cytoplasm in K562 cells and that hemin as well as t-BHQ treatment induced nuclear accumulation of Ref-1 (Fig. 8b). Furthermore, Ref-1 activated the transcription of the ferritin H gene through the ARE (Fig. 8a). It should be noted that these results are consistent with those supporting the redox-dependent regulation of transcription factors bound to ARE (34) in addition to direct evidence showing that a mutation of cysteine 506 to serine in the mouse Nrf2, which is a potential Ref-1 site, significantly diminished Nrf2-mediated activation of ARE by t-BHQ (11).

Ferritin has been understood as a major cytoplasmic iron storage protein; however, a growing number of reports have demonstrated that ferritin H is also localized in the nucleus (14) as well as in mitochondria (5, 16, 21, 23, 37). Although the function of nuclear ferritin is not completely understood, it was reported that nuclear ferritin H protects DNA from UV- or iron-induced DNA damage (15, 66). Furthermore, human ferritin H but not ferritin L was shown to bind to DNA, with no clear preference for DNA sequence or the nature of DNA ends (62). In K562 cells, roles of nuclear ferritin in gene regulation have also been reported. The first observation was that ferritin H and L cDNAs were isolated by subtractive hybridization between hemin-treated and nontreated K562 cDNAs and that ferritin L had the greatest activation of the {gamma}-globin reporter construct in transient transfection assays (73). Recently, it was reported that a ferritin family protein in K562 nuclear extracts binds specifically to the CAGTGC motif in the ß-globin promoter at bp –153 to –148 (12). In the same study, recombinant ferritin H protein was shown to bind to this sequence in a sequence-specific manner, and transfection of the ferritin H expression plasmid into CV-1 cells repressed the ß-globin promoter in reporter assays (12). On the other hand, results contradictory to these observations were also reported, in which the presence of ferritin in the nucleus of K562 cells was confirmed and an antiferritin antibody blocked protein binding to a ß-globin promoter region; however, the protein was heat sensitive, with a molecular mass of 90 to 100 kDa, and also, recombinant ferritin H or L failed to bind to the ß-globin promoter (53). In our study, t-BHQ treatment of K562 cells induced ferritin H synthesis but failed to induce hemoglobin synthesis as a hallmark of erythroid differentiation (our unpublished observations), suggesting that ferritin H induction may not be sufficient to alter, or may not be directly coupled with, transcriptional regulation of globin gene expression. However, induction of ferritin synthesis, and perhaps subsequent decrease in ferritin synthesis (remains to be elucidated), to donate maximum levels of stored iron in the ferritin shell for heme synthesis should be critical to cell maturation and completion of erythroid differentiation (45, 72).

Induction of ferritin H synthesis during erythroid differentiation appears to be important not only for storage of iron but also for cytoprotection against oxidative stress-mediated cytotoxicity. Increased ferritin content in the cells was shown to induce cytoprotection from oxidative damage (6, 17, 54). In addition, we and others demonstrated by overexpression that ferritin H is cytoprotective against oxidative stress in erythroleukemia cells (24) and other cell types (22, 50, 52). Interestingly, preincubation of human leukemia cells with hemin induced cellular resistance to oxidants due to enhanced expression of ferritin H, with minimum contribution of ferritin L to the cytoprotective effect (39). Therefore, transcriptional activation of the ferritin H gene as well as other phase II detoxification genes, such as NQO1 and GST-pi (Fig. 6), through the ARE appears to be a reasonable cellular defense mechanism to adapt to subsequent alterations in iron metabolism and oxidative stress during hemin-mediated erythroid differentiation.

In this study, we have defined the molecular mechanism by which hemin regulates transcription of the human ferritin H gene during erythroid differentiation of K562 cells. While this paper was under review, Hintze and Theil demonstrated that a human ferritin L ARE is activated in HepG2 liver cells by hemin treatment (29), suggesting that both ferritin H and L subunits can be regulated by hemin through a similar ARE element during erythroid differentiation. The ARE-mediated mechanism of ferritin induction at the transcriptional level may be extrapolated to other prooxidative conditions, such as malignancies, infectious diseases, and inflammation, under which elevation of serum ferritin levels has been frequently observed.


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ACKNOWLEDGMENTS
 
We thank T. Curran for sharing Ref-1 plasmid DNA with us.

This work was supported in part by the National Institutes of Health research grant DK-60007 to Y. Tsuji. K. Hailemariam was supported by the National Institutes of Health supplement grant DK-60007S and the National Institute of Environmental Health Sciences training grant ES-007046.


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FOOTNOTES
 
* Corresponding author. Mailing address: North Carolina State University, Campus Box 7633, Raleigh, NC 27695. Phone: (919) 513-1106. Fax: (919) 515-7169. E-mail: yoshiaki_tsuji{at}ncsu.edu. Back

{dagger} The first two authors contributed equally to this work. Back


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Molecular and Cellular Biology, April 2006, p. 2845-2856, Vol. 26, No. 7
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.7.2845-2856.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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