@article{simmers_jima_tsuji_cowley_2023, title={LncRNA Tuna is activated in cadmium-induced placental insufficiency and drives the NRF2-mediated oxidative stress response}, volume={11}, ISSN={["2296-634X"]}, DOI={10.3389/fcell.2023.1151108}, abstractNote={Cadmium (Cd) is a toxic heavy metal found throughout the environment and one of the top ten toxicants of major public health concern identified by the World Health Organization. In utero Cd exposure causes fetal growth restriction, malformation, and spontaneous abortion; however, the mechanisms by which Cd impacts these outcomes are poorly understood. Cd accumulates in the placenta, suggesting that these negative outcomes may be a consequence of disrupted placental function and placental insufficiency. To understand the impact of Cd on gene expression within the placenta, we developed a mouse model of Cd-induced fetal growth restriction through maternal consumption of CdCl2 and performed RNA-seq on control and CdCl2 exposed placentae. The top differentially expressed transcript was the Tcl1 Upstream Neuron-Associated (Tuna) long non-coding RNA, which was upregulated over 25-fold in CdCl2 exposed placentae. Tuna has been shown to be critical for neural stem cell differentiation. However, within the placenta, there is no evidence that Tuna is normally expressed or functional at any developmental stage. To determine the spatial expression of Cd-activated Tuna within the placenta, we used in situ hybridization as well as placental layer-specific RNA isolation and analysis. Both methods confirmed the absence of Tuna expression in control samples and determined that Cd-induced Tuna expression is specific to the junctional zone. Since many lncRNAs regulate gene expression, we hypothesized that Tuna forms part of the mechanism of Cd-induced transcriptomic changes. To test this, we over-expressed Tuna in cultured choriocarcinoma cells and compared gene expression profiles to those of control and CdCl2 exposed cells. We demonstrate significant overlap between genes activated by Tuna overexpression and genes activated by CdCl2 exposure, with enrichment in the NRF2-mediated oxidative stress response. Herein we analyze the NRF2 pathway and show that Tuna increases NRF2/NRF2 both at the transcript and protein levels. Tuna drives increased NRF2 target gene expression, a result that is abrogated with the use of an NRF2 inhibitor, confirming that Tuna activates oxidative stress response genes through this pathway. This work identifies the lncRNA Tuna as a potential novel player in Cd-induced placental insufficiency.}, journal={FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY}, author={Simmers, Mark D. and Jima, Dereje D. and Tsuji, Yoshiaki and Cowley, Michael}, year={2023}, month={Jun} }
 @article{tsuji_2023, title={Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2}, volume={24}, ISSN={["1422-0067"]}, DOI={10.3390/ijms24043604}, abstractNote={Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein–RNA and protein–DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5′-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.}, number={4}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Tsuji, Yoshiaki}, year={2023}, month={Feb} }
 @article{tsuji_2020, title={Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)}, volume={15}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0235563}, abstractNote={Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.}, number={7}, journal={PLOS ONE}, author={Tsuji, Yoshiaki}, year={2020}, month={Jul} }
 @article{miyazawa_bogdan_hashimoto_tsuji_2019, title={Iron-induced transferrin receptor-1 mRNA destabilization: A response to "Neither miR-7-5p nor miR-141-3p is a major mediator of iron-responsive transferrin receptor-1 mRNA degradation"}, volume={25}, ISSN={["1469-9001"]}, DOI={10.1261/rna.073270.119}, abstractNote={We read with great interest the Divergent Views article by Connell and colleagues disputing our recent publication describing a role for two microRNAs in the iron-mediated regulation of transferrin receptor 1 (TfR1) mRNA stability. Our publication sought to shed light on a long-standing question in the field of cellular iron metabolism, and we welcome commentary and critique. However, there are several critical issues contained in the article by Connell and colleagues that require further consideration. We appreciate the opportunity to reply here.}, number={11}, journal={RNA}, author={Miyazawa, Masaki and Bogdan, Alexander R. and Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2019}, month={Nov}, pages={1416–1420} }
 @article{miyazawa_bogdan_tsuji_2019, title={Perturbation of Iron Metabolism by Cisplatin through Inhibition of Iron Regulatory Protein 2}, volume={26}, ISSN={["2451-9448"]}, DOI={10.1016/j.chembiol.2018.10.009}, abstractNote={Cisplatin is classically known to exhibit anticancer activity through DNA damage in the nucleus. Here we found a mechanism by which cisplatin affects iron metabolism, leading to toxicity and cell death. Cisplatin causes intracellular iron deficiency through direct inhibition of the master regulator of iron metabolism, iron regulatory protein 2 (IRP2) with marginal effects on IRP1. Cisplatin, but not carboplatin or transplatin, binds human IRP2 at Cys512 and Cys516 and impairs IRP2 binding to iron-responsive elements of ferritin and transferrin receptor-1 (TfR1) mRNAs. IRP2 inhibition by cisplatin caused ferritin upregulation and TfR1 downregulation leading to sustained intracellular iron deficiency. Cys512/516Ala mutant IRP2 made cells more resistant to cisplatin. Furthermore, combination of cisplatin and the iron chelator desferrioxamine enhanced cytotoxicity through augmented iron depletion in culture and xenograft mouse model. Collectively, cisplatin is an inhibitor of IRP2 that induces intracellular iron deficiency.}, number={1}, journal={CELL CHEMICAL BIOLOGY}, author={Miyazawa, Masaki and Bogdan, Alexander R. and Tsuji, Yoshiaki}, year={2019}, month={Jan}, pages={85-+} }
 @article{hashimoto_tsuji_2017, title={Arsenic-Induced Activation of the Homeodomain-Interacting Protein Kinase 2 (HIPK2) to cAMP-Response Element Binding Protein (CREB) Axis}, volume={429}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2016.11.015}, abstractNote={Cyclic AMP-response element-binding protein (CREB) plays key transcriptional roles in cell metabolism, proliferation, and survival. Ser133 phosphorylation by protein kinase A (PKA) is a well-characterized CREB activation mechanism. Homeodomain-interacting protein kinase (HIPK) 2, a nuclear serine/threonine kinase, activates CREB through Ser271 phosphorylation; however, the regulatory mechanism remains uncharacterized. Transfection of CREB in HEK293 cells together with the kinase demonstrated that HIPK2 phosphorylated CREB at Ser271 but not Ser133; likewise, PKA phosphorylated CREB at Ser133 but not Ser271, suggesting two distinct CREB regulatory mechanisms by HIPK2 and PKA. In vitro kinase assay revealed that HIPK2, and HIPK1 and HIPK3, directly phosphorylated CREB. Cells exposed to 10 μM sodium arsenite increased the stability of HIPK1 and HIPK2 proteins, leading to CREB activation via Ser271 phosphorylation. Phospho-Ser271 CREB showed facilitated interaction with the TFIID subunit coactivator TAF4 assessed by immunoprecipitation. Furthermore, a focused gene array between cells transfected with CREB alone and CREB plus HIPK2 over empty vector-transfected control displayed 14- and 32-fold upregulation of cyclin A1, respectively, while no upregulation was displayed by HIPK2 alone. These results suggest that the HIPK2-phospho-Ser271 CREB axis is a new arsenic-responsive CREB activation mechanism in parallel with the PKA-phospho-Ser133 CREB axis.}, number={1}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2017}, month={Jan}, pages={64–78} }
 @article{hashimoto_majumdar_tsuji_2017, title={Nuclear lamins and progerin are dispensable for antioxidant Nrf2 response to arsenic and cadmium}, volume={33}, ISSN={["1873-3913"]}, DOI={10.1016/j.cellsig.2017.02.012}, abstractNote={Lamins are important constituents of the nuclear inner membrane and provide a platform for transcription factors and chromatin. Progerin, a C-terminal truncated lamin A mutant, causes premature aging termed Hutchinson-Gilford Progeria Syndrome (HGPS). Oxidative stress appears to be involved in the pathogenesis of HGPS, although the mechanistic role of progerin remains elusive. Here we examined whether nuclear lamins are important for a cellular antioxidant mechanism, and whether progerin compromises it. We investigated the activation of nuclear factor-E2-related factor 2 (Nrf2) which regulates various antioxidant genes including heme oxygenase-1 (HMOX1), following exposure to sodium arsenite or cadmium chloride in lamin knockdown human cell lines and primary HGPS human fibroblasts. Knocking down lamin A/C, or B, or all nuclear lamins simultaneously in three human cell lines (HaCaT, SW480, and K562) did not impair arsenite- or cadmium-induced activation of Nrf2. Progerin-expressing human primary HGPS fibroblasts showed lower basal levels of HMOX1 and NQO1 expression; however, in response to arsenic stress both normal and HGPS primary fibroblasts showed Nrf2 nuclear accumulation along with upregulation and phosphorylation of p62/SQSTM1 at Ser351, downregulation of Keap1, and comparable expression of an array of downstream Nrf2-regulated antioxidant genes. We also observed new forms of cleaved lamin A, B1 and B2 induced by cadmium stress although their roles in the Nrf2 antioxidant system need further investigation. These results suggest that the nuclear lamins and progerin have marginal roles in the activation of the antioxidant Nrf2 response to arsenic and cadmium.}, journal={CELLULAR SIGNALLING}, author={Hashimoto, Kazunori and Majumdar, Rima and Tsuji, Yoshiaki}, year={2017}, month={May}, pages={69–78} }
 @misc{bogdan_miyazawa_hashimoto_tsuji_2016, title={Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease}, volume={41}, ISSN={["1362-4326"]}, DOI={10.1016/j.tibs.2015.11.012}, abstractNote={Iron is necessary for life, but can also cause cell death. Accordingly, cells evolved a robust, tightly regulated suite of genes for maintaining iron homeostasis. Previous mechanistic studies on iron homeostasis have granted insight into the role of iron in human health and disease. We highlight new regulators of iron metabolism, including iron-trafficking proteins [solute carrier family 39, SLC39, also known as ZRT/IRT-like protein, ZIP; and poly-(rC)-binding protein, PCBP] and a cargo receptor (NCOA4) that is crucial for release of ferritin-bound iron. We also discuss emerging roles of iron in apoptosis and a novel iron-dependent cell death pathway termed 'ferroptosis', the dysregulation of iron metabolism in human pathologies, and the use of iron chelators in cancer therapy.}, number={3}, journal={TRENDS IN BIOCHEMICAL SCIENCES}, author={Bogdan, Alexander R. and Miyazawa, Masaki and Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2016}, month={Mar}, pages={274–286} }
 @misc{wilson_bogdan_miyazawa_hashimoto_tsuji_2016, title={Siderophores in Iron Metabolism: From Mechanism to Therapy Potential}, volume={22}, ISSN={["1471-499X"]}, DOI={10.1016/j.molmed.2016.10.005}, abstractNote={Iron is an essential nutrient for life. During infection, a fierce battle of iron acquisition occurs between the host and bacterial pathogens. Bacteria acquire iron by secreting siderophores, small ferric iron-binding molecules. In response, host immune cells secrete lipocalin 2 (also known as siderocalin), a siderophore-binding protein, to prevent bacterial reuptake of iron-loaded siderophores. To counter this threat, some bacteria can produce lipocalin 2-resistant siderophores. This review discusses the recently described molecular mechanisms of siderophore iron trafficking between host and bacteria, highlighting the therapeutic potential of exploiting pathogen siderophore machinery for the treatment of antibiotic-resistant bacterial infections. Because the latter reflect a persistent problem in hospital settings, siderophore-targeting or siderophore-based compounds represent a promising avenue to combat such infections.}, number={12}, journal={TRENDS IN MOLECULAR MEDICINE}, author={Wilson, Briana R. and Bogdan, Alexander R. and Miyazawa, Masaki and Hashimoto, Kazunori and Tsuji, Yoshiaki}, year={2016}, month={Dec}, pages={1077–1090} }
 @article{hashimoto_simmons_kajino-sakamoto_tsuji_ninomiya-tsuji_2016, title={TAK1 Regulates the Nrf2 Antioxidant System Through Modulating p62/SQSTM1}, volume={25}, ISSN={["1557-7716"]}, DOI={10.1089/ars.2016.6663}, abstractNote={AIMS
Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is the master transcriptional regulator of antioxidant gene expression. On increased oxidative stress, an adaptor for Nrf2 degradation, Kelch-like ECH-associated protein 1 (Keap1), is directly modulated by oxidants in the cytoplasm, which results in stabilization and activation of Nrf2. Nrf2 is also constitutively active, to some extent, in the absence of exogenous oxidative stress. We have previously demonstrated that intestinal epithelium-specific TGF-β-activated kinase 1 (TAK1) deletion downregulates the level of Nrf2 protein, resulting in an increase of reactive oxygen species (ROS) in a mouse model. We aim at determining the mechanism by which TAK1 modulates the level of Nrf2.


RESULTS
We found that TAK1 upregulated serine 351 phosphorylation of an autophagic adaptor protein, p62/Sequestosome-1 (SQSTM1), which facilitates interaction between p62/SQSTM1 and Keap1 and subsequent Keap1 degradation. This, ultimately, causes increased Nrf2. Tak1 deficiency reduced the phosphorylation of p62/SQSTM1, resulting in decreased steady-state levels of Nrf2 along with increased Keap1. We also found that this regulation is independent of the canonical redox-mediated Nrf2 activation mechanism. In Tak1-deficient intestinal epithelium, a synthetic phenolic electrophile, butylated hydroxyanisole still effectively upregulated Nrf2 and reduced ROS.


INNOVATION
Our results identify for the first time that TAK1 is a modulator of p62/SQSTM1-dependent Keap1 degradation and maintains the steady state-level of Nrf2.


CONCLUSION
TAK1 regulates Nrf2 through modulation of Keap-p62/SQSTM1 interaction. This regulation is important for homeostatic antioxidant protection in the intestinal epithelium. Antioxid. Redox Signal. 25, 953-964.}, number={17}, journal={ANTIOXIDANTS & REDOX SIGNALING}, author={Hashimoto, Kazunori and Simmons, Alicia N. and Kajino-Sakamoto, Rie and Tsuji, Yoshiaki and Ninomiya-Tsuji, Jun}, year={2016}, month={Dec}, pages={953–964} }
 @article{ray_huang_tsuji_2015, title={Coordinated regulation of Nrf2 and histone H3 serine 10 phosphorylation in arsenite-activated transcription of the human heme oxygenase-1 gene}, volume={1849}, ISSN={["0006-3002"]}, DOI={10.1016/j.bbagrm.2015.08.004}, abstractNote={Expression of the antioxidant gene heme oxygenase-1 (HO-1) is primarily induced through NF-E2-related factor 2 (Nrf2)-mediated activation of the antioxidant response element (ARE). Gene transcription is coordinately regulated by transcription factor activity at enhancer elements and epigenetic alterations such as the posttranslational modification of histone proteins. However, the role of histone modifications in the Nrf2-ARE axis remains largely uncharacterized. The environmental contaminant arsenite is a potent inducer of both HO-1 expression and phosphorylation of histone H3 serine 10 (H3S10); therefore, we investigated the relationships between Nrf2 and H3S10 phosphorylation in arsenite-induced, ARE-dependent, transcriptional activation of the human HO-1 gene. Arsenite increased phosphorylation of H3S10 both globally and at the HO-1 promoter concomitantly with HO-1 transcription in human HaCaT keratinocytes. Conversely, arsenite-induced H3S10 phosphorylation and HO-1 expression were blocked by N-acetylcysteine (NAC), the c-Jun N-terminal kinase (JNK) inhibitor SP600125, and JNK knockdown (siJNK). Interestingly, ablation of arsenite-induced H3S10 phosphorylation by SP600125 or siJNK did not inhibit Nrf2 nuclear accumulation nor ARE binding, despite inhibiting HO-1 expression. In response to arsenite, binding of Nrf2 to the HO-1 ARE preceded phosphorylation of H3S10 at the HO-1 ARE. Furthermore, arsenite-mediated occupancy of phosphorylated H3S10 at the HO-1 ARE was decreased in Nrf2-deficient mouse embryonic fibroblasts. These results suggest the involvement of H3S10 phosphorylation in the Nrf2-ARE axis by proposing that Nrf2 may influence H3S10 phosphorylation at the HO-1 ARE and additional promoter regions. Our data highlights the complex interplay between Nrf2 and H3S10 phosphorylation in arsenite-activated HO-1 transcription.}, number={10}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS}, author={Ray, Paul D. and Huang, Bo-Wen and Tsuji, Yoshiaki}, year={2015}, month={Oct}, pages={1277–1288} }
 @article{huang_miyazawa_tsuji_2014, title={Distinct regulatory mechanisms of the human ferritin gene by hypoxia and hypoxia mimetic cobalt chloride at the transcriptional and post-transcriptional levels}, volume={26}, ISSN={["1873-3913"]}, DOI={10.1016/j.cellsig.2014.08.018}, abstractNote={Cobalt chloride has been used as a hypoxia mimetic because it stabilizes hypoxia inducible factor-1α (HIF1-α) and activates gene transcription through a hypoxia responsive element (HRE). However, differences between hypoxia and hypoxia mimetic cobalt chloride in gene regulation remain elusive. Expression of ferritin, the major iron storage protein, is regulated at the transcriptional and posttranscriptional levels through DNA and RNA regulatory elements. Here we demonstrate that hypoxia and cobalt chloride regulate ferritin heavy chain (ferritin H) expression by two distinct mechanisms. Both hypoxia and cobalt chloride increased HIF1-α but a putative HRE in the human ferritin H gene was not activated. Instead, cobalt chloride but not hypoxia activated ferritin H transcription through an antioxidant responsive element (ARE), to which Nrf2 was recruited. Intriguingly, cobalt chloride downregulated ferritin H protein expression while it upregulated other ARE-regulated antioxidant genes in K562 cells. Further characterization demonstrated that cobalt chloride increased interaction between iron regulatory proteins (IRP1 and IRP2) and iron responsive element (IRE) in the 5′UTR of ferritin H mRNA, resulting in translational block of the accumulated ferritin H mRNA. In contrast, hypoxia had marginal effect on ferritin H transcription but increased its translation through decreased IRP1–IRE interaction. These results suggest that hypoxia and hypoxia mimetic cobalt chloride employ distinct regulatory mechanisms through the interplay between DNA and mRNA elements at the transcriptional and post-transcriptional levels.}, number={12}, journal={CELLULAR SIGNALLING}, author={Huang, Bo-Wen and Miyazawa, Masaki and Tsuji, Yoshiaki}, year={2014}, month={Dec}, pages={2702–2709} }
 @article{miyazawa_tsuji_2014, title={Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene}, volume={25}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.e13-11-0666}, abstractNote={The mammalian Shc family, composed of p46, p52, and p66 isoforms, serves as an adaptor protein in cell growth and stress response. p66Shc was shown to be a negative lifespan regulator by acting as a prooxidant protein in mitochondria; however, the regulatory mechanisms of p66Shc expression and function are incompletely understood. This study provides evidence for new features of p66Shc serving as an antioxidant and critical protein in cell differentiation. Unique among the Shc family, transcription of p66Shc is activated through the antioxidant response element (ARE)–nuclear factor erythroid 2–related factor 2 (Nrf2) pathway in K562 human erythroleukemia and other cell types after treatment with hemin, an iron-containing porphyrin. Phosphorylated p66Shc at Ser-36, previously reported to be prone to mitochondrial localization, is increased by hemin treatment, but p66Shc remains exclusively in the cytoplasm. p66Shc knockdown inhibits hemin-induced erythroid differentiation, in which reactive oxygen species production and apoptosis are significantly enhanced in conjunction with suppression of other ARE-dependent antioxidant genes. Conversely, p66Shc overexpression is sufficient for inducing erythroid differentiation. Collectively these results demonstrate the isoform-specific regulation of the Shc gene by the Nrf2-ARE pathway and a new antioxidant role of p66Shc in the cytoplasm. Thus p66Shc is a bifunctional protein involved in cellular oxidative stress response and differentiation.}, number={13}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Miyazawa, Masaki and Tsuji, Yoshiaki}, year={2014}, month={Jul}, pages={2116–2127} }
 @article{iwasaki_ray_huang_sakamoto_kobayashi_tsuji_2013, title={Role of AMP-Activated Protein Kinase in Ferritin H Gene Expression by Resveratrol in Human T Cells}, volume={52}, ISSN={["0006-2960"]}, DOI={10.1021/bi400399f}, abstractNote={Resveratrol, a natural polyphenol, increases cellular antioxidant capacity by inducing the expression of a battery of cytoprotective genes through an antioxidant responsive element (ARE). However, upstream signaling events initiated by resveratrol leading to the activation of an ARE enhancer, particularly in immune cells, have not been fully elucidated. In this study, ARE-dependent transcriptional activation of the ferritin heavy chain (ferritin H) gene by resveratrol was further investigated in Jurkat T cells and human peripheral blood mononuclear cells. We found that AMP-activated protein kinase (AMPK) plays a key role in the activation of nuclear factor E2-related factor (Nrf2) and subsequent ARE-dependent ferritin H gene transcription by resveratrol. A chromatin immunoprecipitation assay for Nrf2 after AMPKα knockdown with siRNA revealed that Nrf2 nuclear accumulation and subsequent binding to the ferritin H ARE induced by resveratrol were dependent on activation of AMPKα, but not PI3K/AKT. Furthermore, AMPKα knockdown blocked resveratrol-induced phosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser9 as well as ARE-dependent transcriptional activation of the ferritin H and HO-1 genes, suggesting that AMPKα is an upstream kinase for GSK3β phosphorylation and activation of the Nrf2-ARE pathway. Consistently, GSK3β knockdown by siRNA enhanced resveratrol-mediated ferritin H mRNA induction, and the inhibition of AMPKα by compound C or siRNA weakened the protective effect of resveratrol against oxidative stress-induced cytotoxicity in CD3+ T cells. Collectively, these results suggest that AMPKα plays a significant role in ARE-dependent transcription of ferritin H genes by resveratrol and may influence the redox status in immune cells.}, number={30}, journal={BIOCHEMISTRY}, author={Iwasaki, Kenta and Ray, Paul D. and Huang, Bo-Wen and Sakamoto, Kensuke and Kobayashi, Takaaki and Tsuji, Yoshiaki}, year={2013}, month={Jul}, pages={5075–5083} }
 @article{huang_ray_iwasaki_tsuji_2013, title={Transcriptional regulation of the human ferritin gene by coordinated regulation of Nrf2 and protein arginine methyltransferases PRMT1 and PRMT4}, volume={27}, ISSN={["1530-6860"]}, DOI={10.1096/fj.12-226043}, abstractNote={Antioxidant genes such as ferritin are transcriptionally activated in oxidative stress via the antioxidant responsive element (ARE), to which nuclear factor‐E2‐related factor 2 (Nrf2) binds and activates transcription. Histone modification plays a cooperative and essential role in transcriptional regulation; however, its role in antioxidant gene transcription remains elusive. Arsenic exposure activated ferritin transcription via the ARE concomitant with increased methylation of histones H4Arg3 (H4R3) and H3Arg17 (H3R17). To test our hypothesis that histone H4R3 and H3R17 methylation regulates ferritin transcription, H4R3 and H3R17 protein arginine (R) methyltransferases 1 and 4 (PRMT1 and PRMT4) were investigated. Arsenic exposure of human HaCaT keratinocytes induced nuclear accumulation of PRMT1 and PRMT4, histone H4R3 and H3R17 methylation proximal to the ARE, but not to the non‐ARE regions of ferritin genes. PRMT1 or PRMT4 knockdown did not block Nrf2 nuclear accumulation but inhibited Nrf2 binding to the AREs by ~40% (P<0.05), thus diminishing ferritin transcription in HaCaT and human primary keratinocytes and fibroblasts, causing enhanced cellular susceptibility to arsenic toxicity as evidenced by 2‐fold caspase 3 activation. Focused microarray further characterized several oxidative stress response genes are subject to PRMT1 or PRMT4 regulation. Collectively, PRMT1 and PRMT4 regulate the ARE and cellular antioxidant response to arsenic.—Huang, B.‐W., Ray, P. D., Iwasaki, K., Tsuji, Y., Transcriptional regulation of the human ferritin gene by coordinated regulation of Nrf2 and protein arginine methyltransferases PRMT1 and PRMT4. FASEB J. 27, 3763–3774 (2013). www.fasebj.org}, number={9}, journal={FASEB JOURNAL}, author={Huang, Bo-Wen and Ray, Paul D. and Iwasaki, Kenta and Tsuji, Yoshiaki}, year={2013}, month={Sep}, pages={3763–3774} }
 @misc{ray_huang_tsuji_2012, title={Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling}, volume={24}, ISSN={["1873-3913"]}, DOI={10.1016/j.cellsig.2012.01.008}, abstractNote={Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.}, number={5}, journal={CELLULAR SIGNALLING}, author={Ray, Paul D. and Huang, Bo-Wen and Tsuji, Yoshiaki}, year={2012}, month={May}, pages={981–990} }
 @article{sakamoto_huang_iwasaki_hailemariam_ninomiya-tsuji_tsuji_2010, title={Regulation of Genotoxic Stress Response by Homeodomain-interacting Protein Kinase 2 through Phosphorylation of Cyclic AMP Response Element-binding Protein at Serine 271}, volume={21}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.e10-01-0015}, abstractNote={ CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2−/− MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2−/− cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress. }, number={16}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Sakamoto, Kensuke and Huang, Bo-Wen and Iwasaki, Kenta and Hailemariam, Kiros and Ninomiya-Tsuji, Jun and Tsuji, Yoshiaki}, year={2010}, month={Aug}, pages={2966–2974} }
 @article{hailemariam_iwasaki_huang_sakamoto_tsuji_2010, title={Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress}, volume={123}, ISSN={["1477-9137"]}, DOI={10.1242/jcs.073627}, abstractNote={ATF1 (activating transcription factor 1), a stimulus-induced CREB family transcription factor, plays important roles in cell survival and proliferation. Phosphorylation of ATF1 at Ser63 by PKA (cAMP-dependent protein kinase) and related kinases was the only known post-translational regulatory mechanism of ATF1. Here, we found that HIPK2 (homeodomain-interacting protein kinase 2), a DNA-damage-responsive nuclear kinase, is a new ATF1 kinase that phosphorylates Ser198 but not Ser63. ATF1 phosphorylation by HIPK2 activated ATF1 transcription function in the GAL4-reporter system. ATF1 is a transcriptional repressor of ferritin H, the major intracellular iron storage gene, through an ARE (antioxidant-responsive element). HIPK2 overrode the ATF1-mediated ARE repression in a kinase-activity-dependent manner in HepG2 cells. Furthermore, DNA-damage-inducing agents doxorubicin, etoposide and sodium arsenite induced ferritin H mRNA expression in HIPK2+/+ MEF cells, whereas it was significantly impaired in HIPK2−/− MEF cells. Induction of other ARE-regulated detoxification genes such as NQO1 (NADPH quinone oxidoreductase 1), GST (glutathione S-transferase) and HO1 (heme oxygenase 1) by genotoxic stress was also decreased in HIPK2-deficient cells. Taken together, these results suggest that HIPK2 is a new ATF1 kinase involved in the regulation of ferritin H and other antioxidant detoxification genes in genotoxic stress conditions.}, number={22}, journal={JOURNAL OF CELL SCIENCE}, author={Hailemariam, Kiros and Iwasaki, Kenta and Huang, Bo-Wen and Sakamoto, Kensuke and Tsuji, Yoshiaki}, year={2010}, month={Nov}, pages={3863–3871} }
 @article{sakamoto_iwasaki_sugiyama_tsuji_2009, title={Role of the Tumor Suppressor PTEN in Antioxidant Responsive Element-mediated Transcription and Associated Histone Modifications}, volume={20}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.E08-07-0762}, abstractNote={Coordinated regulation of PI3-kinase (PI3K) and the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) plays a pivotal role in various cell functions. PTEN is deficient in many cancer cells, including Jurkat human leukemia. Here, we demonstrate that the status of PTEN determines cellular susceptibility to oxidative stress through antioxidant-responsive element (ARE)-mediated transcription of detoxification genes. We found that ferritin H transcription was robustly induced in tert-butylhydroquinone (t-BHQ)-treated Jurkat cells via an ARE, and it was due to PTEN deficiency. Chromatin immunoprecipitation assays revealed that p300/CREB-binding protein (CBP) histone acetyltransferases and Nrf2 recruitment to the ARE and Bach1 release were blocked by the PI3K inhibitor LY294002, along with the partial inhibition of Nrf2 nuclear accumulation. Furthermore, acetylations of histone H3 Lys9 and Lys18, and deacetylation of Lys14 were associated with the PI3K-dependent ARE activation. Consistently, PTEN restoration in Jurkat cells inhibited t-BHQ–mediated expression of ferritin H and another ARE-regulated gene NAD(P)H:quinone oxidoreductase 1. Conversely, PTEN knockdown in K562 cells enhanced the response to t-BHQ. The PTEN status under t-BHQ treatment affected hydrogen peroxide-mediated caspase-3 cleavage. The PI3K-dependent ferritin H induction was observed by treatment with other ARE-activating agents ethoxyquin and hemin. Collectively, the status of PTEN determines chromatin modifications leading to ARE activation.}, number={6}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Sakamoto, Kensuke and Iwasaki, Kenta and Sugiyama, Hiroyuki and Tsuji, Yoshiaki}, year={2009}, month={Mar}, pages={1606–1617} }
 @article{mackenzie_tsuji_2008, title={Elevated intracellular calcium increases ferritin H expression through an NFAT-independent post-transcriptional mechanism involving mRNA stabilization}, volume={411}, ISSN={["1470-8728"]}, DOI={10.1042/bj20071544}, abstractNote={An increase in intracellular Ca2+ is one of the initiating events in T-cell activation. A calcium-mediated signalling cascade in T-cells involves activation of calcineurin and the dephosphorylation and translocation of NFAT (nuclear factor of activated T-cells), resulting in the transcriptional activation of target genes such as IL-2 (interleukin-2). In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an ARE (antioxidant-responsive element). The facts that the ferritin H ARE contains a composite AP-1 (activator protein 1) site and that NFAT collaborates with AP-1 transcription factors led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T-cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Although NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter after ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT-binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability after actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through post-transcriptional stabilization of the ferritin H mRNA.}, journal={BIOCHEMICAL JOURNAL}, author={Mackenzie, Elizabeth L. and Tsuji, Yoshiaki}, year={2008}, month={Apr}, pages={107–113} }
 @misc{mackenzie_iwasaki_tsuji_2008, title={Intracellular iron transport and storage: From molecular mechanisms to health implications}, volume={10}, ISSN={["1557-7716"]}, DOI={10.1089/ars.2007.1893}, abstractNote={Maintenance of proper "labile iron" levels is a critical component in preserving homeostasis. Iron is a vital element that is a constituent of a number of important macromolecules, including those involved in energy production, respiration, DNA synthesis, and metabolism; however, excess "labile iron" is potentially detrimental to the cell or organism or both because of its propensity to participate in oxidation-reduction reactions that generate harmful free radicals. Because of this dual nature, elaborate systems tightly control the concentration of available iron. Perturbation of normal physiologic iron concentrations may be both a cause and a consequence of cellular damage and disease states. This review highlights the molecular mechanisms responsible for regulation of iron absorption, transport, and storage through the roles of key regulatory proteins, including ferroportin, hepcidin, ferritin, and frataxin. In addition, we present an overview of the relation between iron regulation and oxidative stress and we discuss the role of functional iron overload in the pathogenesis of hemochromatosis, neurodegeneration, and inflammation.}, number={6}, journal={ANTIOXIDANTS & REDOX SIGNALING}, author={Mackenzie, Elizabeth L. and Iwasaki, Kenta and Tsuji, Yoshiaki}, year={2008}, month={Jun}, pages={997–1030} }
 @article{zhang_wang_tsuji_torti_torti_2008, title={Post-transcriptional Modulation of Iron Homeostasis during p53-dependent Growth Arrest}, volume={283}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M806432200}, DOI={10.1074/jbc.M806432200}, abstractNote={Iron plays an essential role in cell proliferation and is a required cofactor for a number of critical cellular enzymes. In this report we investigate changes in proteins of iron metabolism during p53-mediated replicative arrest. Following the induction of p53 in H1299 lung cancer cells containing a doxycycline-inducible p53, an increase in both H and L subunits of ferritin protein was observed. To determine the mechanism of this effect, we investigated the ability of p53 to regulate ferritin. Real time reverse transcription-PCR demonstrated no difference in levels of ferritin H mRNA in the presence and absence of p53. Because these results suggested that transcriptional mechanisms were not responsible for the p53-dependent increase in ferritin, we tested whether a post-transcriptional mechanism was involved. RNA bandshift assays revealed that induction of p53 decreased iron regulatory protein binding. Consistent with this observation, Western blot analysis revealed a decline in transferrin receptor 1 protein levels following induction of p53. Collectively, these results suggest that p53 may induce cell cycle arrest not only by well described mechanisms involving the induction of cyclin-dependent kinase inhibitors but also by the recruitment of pathways that reduce the availability of intracellular iron.}, number={49}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Zhang, Fan and Wang, Wei and Tsuji, Yoshiaki and Torti, Suzy V. and Torti, Frank M.}, year={2008}, month={Sep}, pages={33911–33918} }
 @article{mackenzie_ray_tsuji_2008, title={Role and regulation of ferritin H in rotenone-mediated mitochondrial oxidative stress}, volume={44}, ISSN={["1873-4596"]}, DOI={10.1016/j.freeradbiomed.2008.01.031}, abstractNote={Tight regulation of intracellular iron levels in response to mitochondrial dysfunction is an important mechanism that prevents oxidative stress, thereby limiting cellular damage. Here, we describe a cytoprotective response involving transcriptional activation of the ferritin H gene in response to the mitochondrial complex I inhibitor and neurotoxic compound rotenone. Rotenone exposure increased ferritin H mRNA and protein synthesis in NIH3T3 fibroblasts and SH-SY5Y neuroblastoma cells. Transient transfection of a ferritin H promoter–luciferase reporter into NIH3T3 cells showed that ferritin H was transcriptionally activated by rotenone through an antioxidant-responsive element (ARE). Chromatin immunoprecipitation assays showed that rotenone treatment enhanced binding of Nrf2 and JunD transcription factors to the ARE. In addition, rotenone induced production of reactive oxygen species (ROS), and pretreatment with N-acetylcysteine abrogated ferritin H mRNA induction by rotenone, suggesting that this response is oxidative stress-mediated. Furthermore, reduced ferritin H expression by siRNA sensitized cells to rotenone-induced apoptosis with increased ROS production and annexin V-positive cells. Taken together, these results suggest that ferritin H transcription is activated by rotenone via an oxidative stress-mediated pathway leading to ARE activation and may be critically important to protect cells from mitochondrial dysfunction and oxidative stress.}, number={9}, journal={FREE RADICAL BIOLOGY AND MEDICINE}, author={MacKenzie, Elizabeth L. and Ray, Paul D. and Tsuji, Yoshiaki}, year={2008}, month={May}, pages={1762–1771} }
 @article{iwasaki_hailemariam_tsuji_2007, title={PIAS3 interacts with ATF1 and regulates the human ferritin H gene through an antioxidant-responsive element}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M701477200}, abstractNote={Gene transcription is coordinately regulated by the balance between activation and repression mechanisms in response to various external stimuli. Ferritin, composed of H and L subunits, is the major intracellular iron storage protein involved in iron homeostasis. We previously identified an enhancer, termed antioxidant-responsive element (ARE), in the human ferritin H gene and its respective transcriptional activators including Nrf2 and JunD. Here we found that ATF1 (activating transcription factor 1) is a transcriptional repressor of the ferritin H ARE. Subsequent yeast two-hybrid screening identified PIAS3 (protein inhibitor of activated STAT3) as an ATF1-binding protein. Further investigation of the human ferritin H ARE regulation showed that 1) PIAS3 reversed ATF1-mediated repression of the ferritin H ARE; 2) ATF1 was sumoylated, but PIAS3, a SUMO E3 ligase, did not appear to play a major role in SUMO1-mediated ATF1 sumoylation or ATF1 transcription activating function; 3) PIAS3 decreased ATF1 binding to the ARE; and 4) ATF1 knockdown with siRNA increased ferritin H expression, whereas PIAS3 knockdown decreased basal expression and oxidative stress-mediated induction of ferritin H. These results suggest that PIAS3 antagonizes the repressor function of ATF1, at least in part by blocking its DNA binding, and ultimately activates the ARE. Collectively our results suggest that PIAS3 is a new regulator of ATF1 that regulates the ARE-mediated transcription of the ferritin H gene.}, number={31}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Iwasaki, Kenta and Hailemariam, Kiros and Tsuji, Yoshiaki}, year={2007}, month={Aug}, pages={22335–22343} }
 @article{jia_takahashi_zhang_tsuji_sone_2006, title={Aldo-keto reductase 1 family B7 is the gene induced in response to oxidative stress in the livers of Long-Evans Cinnamon rats}, volume={29}, DOI={10.3892/ijo.29.4.829}, abstractNote={The Long-Evans Cinnamon (LEC) rat strain (Atp7b m/m), which accumulates copper in the liver due to mutations in the Atp7b gene, is a useful model for investigating the relationship between oxidative stress and hepatocarcinogenesis. To determine the effect of this mutation on oxidative stress marker genes, we performed oligonucleotide array analysis (Affymetrix), and compared the results in Atp7b m/m rats with those of a sibling line with the Atp7b w/w genotype. We focused our studies on the expression of the aldo-keto reductase 1 family B7 (AKR1B7)-like protein gene, since this gene codes for reductase enzymes involved in the detoxification of oxidizing compounds (e.g., aldehydes) and was differentially expressed in Atp7b m/m and Atp7b w/w rat liver. Akr1B7 mRNA expression was significantly increased in comparison with the expression of 4 other known oxidative stress responsive genes, haem-oxygenase-1 (HO-1), thioredoxin (Trx), aldehyde reductase (AKR1A1), and glucose-6-phosphate dehydrogenase (G6PDH). By searching binding motifs, five nuclear factor kappa B (NF-kappaB) binding sites were located in the 5'-upstream region of the akr1b7 gene. Transient co-transfection with both I-kappaBalpha and the Akr1b7 6 kb promoter (p6.0-AKR-Luc) inhibited luciferase activity of p6.0-AKR-Luc in HepG2 cells. Cuprous ion however did not affect the transcription activity induced by p6.0-AKR-luc. Gel-shift assay showed that the DNA binding activity of NF-kappaB increased in the livers of LEC rats, suggesting that the oxidative stress is mediated through NF-kappaB. The results indicate conclusively that in LEC rat liver, akr1b7 might be up-regulated by oxidative stress mediated through NF-kappaB, but not that mediated directly by copper.}, number={4}, journal={International Journal of Oncology}, author={Jia, G. and Takahashi, R. and Zhang, Z. P. and Tsuji, Y. and Sone, H.}, year={2006}, pages={829–838} }
 @article{jennings-gee_tsuji_pietsch_moran_mymryk_torti_torti_2006, title={Coordinate Inhibition of Cytokine-mediated Induction of Ferritin H, Manganese Superoxide Dismutase, and Interleukin-6 by the Adenovirus E1A Oncogene}, volume={281}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M600038200}, DOI={10.1074/jbc.M600038200}, abstractNote={Adenovirus E1A sensitizes cells to the cytotoxic action of tumor necrosis factor α (TNF-α). This effect has been attributed to direct blockade of NF-κB activation, as well as to increased activation of components of the apoptotic pathway and decreases in inhibitors of apoptosis. In this report we evaluated the mechanism by which E1A modulates the expression of the cytokine-inducible cytoprotective genes manganese superoxide dismutase (MnSOD), interleukin-6 (IL-6), and ferritin heavy chain (FH). We observed that E1A blocks induction of MnSOD, IL-6, and FH by TNF-α or IL-1α. Because NF-κB plays a role in cytokine-dependent induction of MnSOD, IL-6, and FH, we assessed the effect of E1A on NF-κB in cells treated with TNF. IκB, the inhibitor of NF-κB, was degraded similarly in the presence and absence of E1A. TNF induced a quantitatively and temporally equivalent activation of NF-κB in control and E1A-transfected cells. However, TNF-dependent acetylation of NF-κB was diminished in cells expressing E1A. E1A mutants unable to bind p400 or the Rb family proteins were still capable of repressing TNF-dependent induction of FH. However, mutants of E1A that abrogated binding of p300/CBP blocked the ability of E1A to repress TNF-dependent induction of FH. These results suggest that p300/CBP is a critical control point in NF-κB-dependent transcriptional regulation of cytoprotective genes by cytokines.}, number={24}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Jennings-Gee, Jamie E. and Tsuji, Yoshiaki and Pietsch, E. Christine and Moran, Elizabeth and Mymryk, J. S. and Torti, Frank M. and Torti, Suzy V.}, year={2006}, month={Apr}, pages={16428–16435} }
 @article{iwasaki_mackenzie_hailemariam_sakamoto_tsuji_2006, title={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}, volume={26}, ISSN={["1098-5549"]}, DOI={10.1128/MCB.26.7.2845-2856.2006}, abstractNote={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.}, number={7}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Iwasaki, K and MacKenzie, EL and Hailemariam, K and Sakamoto, K and Tsuji, Y}, year={2006}, month={Apr}, pages={2845–2856} }
 @article{tsuji_2005, title={JunD activates transcription of the human ferritin H gene through an antioxidant response element during oxidative stress}, volume={24}, ISSN={["0950-9232"]}, DOI={10.1038/sj.onc.1208901}, abstractNote={Ferritin is the major intracellular iron storage protein that sequesters excess free iron to minimize generation of iron-catalysed reactive oxygen species. We previously demonstrated that expression of ferritin heavy chain (ferritin H) was induced by pro-oxidants, which is a part of cellular antioxidant response to protect cells from oxidative damage. In this study, we have identified that the antioxidant/electrophile response element (ARE) located 4.5 kb upstream to the human ferritin H transcription initiation site is responsible for the oxidant response. The human ferritin H ARE comprises two copies of bidirectional AP1 motifs. Mutations in each AP1 motif significantly impaired protein binding and the function of the ARE, indicating that both of the AP1 motifs are required for pro-oxidant-mediated activation of the ferritin H gene. We identified that JunD, an AP1 family basic-leucine zipper (bZip) transcription factor, is one of the ferritin H ARE binding proteins and activates ferritin H transcription in HepG2 hepatocarcinoma cells. Gel retardation assay demonstrated that H2O2 (hydrogen peroxide) or t-BHQ (tert-butylhydroquinone) treatment increased total protein binding as well as JunD binding to the ferritin H ARE. Chromatin immunoprecipitation assay showed that H2O2 treatment induced JunD binding to the ferritin H ARE. Both H2O2 and t-BHQ induced phosphorylation of JunD at Ser-100, an activated form of JunD. Furthermore, overexpression of JunD induced endogenous ferritin H protein synthesis. Since JunD has recently been demonstrated to protect cells from several stress stimuli including oxidative stress, these results suggest that, in addition to NFE2-related factor 2 (Nrf2) as a major ARE regulatory protein, JunD is another ARE regulatory protein for transcriptional activation of the human ferritin H gene and probably other antioxidant genes containing the conserved ARE sequences by which JunD may confer cytoprotection during oxidative stress.}, number={51}, journal={ONCOGENE}, author={Tsuji, Y}, year={2005}, month={Nov}, pages={7567–7578} }