Cellular and humoral immune responses to COVID-19 booster vaccination in Japanese dialysis patients.

BACKGROUND: Dialysis patients are susceptible to developing severe coronavirus disease 2019 (COVID-19) due to hypoimmunity. Antibody titers against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) after the primary vaccinations are lower in hemodialysis (HD) patients than in healthy individuals. This study aimed to evaluate the effect of a SARS-CoV-2 booster vaccination in HD and peritoneal dialysis (PD) patients based on antibody titers and cellular and humoral immunity. METHODS: Participants of the control, HD, and PD groups were recruited from 12 facilities. SARS-CoV-2 antigen-specific cytokine and IgG-antibody levels were measured. Regulatory T cells and memory B cells were counted using flow cytometry at 6 months after primary vaccination with BNT162b2 and 3 weeks after the booster vaccination in HD and PD patients and compared with those of a control group. RESULTS: Booster vaccination significantly enhanced the levels of antibodies, cytokines, and memory B cells in three groups. The HD group showed significantly higher levels of IgG-antibodies, IL-1ß, IL-2, IL-4, IL-17, and memory B cells than those in the control group at 3 weeks after the booster dose. The PD group tended to show similar trends to HD patients but had similar levels of IgG-antibodies, cytokines, and memory B cells to the control group. CONCLUSIONS: HD patients had significantly stronger cellular and humoral immune responses than the control 3 weeks after the booster dose. Our findings will help in developing better COVID-19 vaccination strategies for HD and PD patients.

Real-life effectiveness of indacaterol/glycopyrronium/mometasone for symptomatic relief of cough after switching from inhaled corticosteroid/long-acting ß2-agonist therapy in patients with asthma: REACH study design.

Cough is a major symptom in patients with asthma and poses a significant burden compared with other asthma symptoms. However, there are no approved treatments in Japan, developed to specifically treat cough in patients with asthma. We present the design of REACH, an 8-week real-life study, which will evaluate the efficacy of a combination of indacaterol acetate, glycopyrronium bromide and mometasone furoate (IND/GLY/MF) in asthmatic patients with cough refractory to medium-dose inhaled corticosteroid/long-acting ß2-agonist (ICS/LABA). Patients with asthma (age ≥20 to <80 years) with a cough visual analogue scale (VAS) ≥40 mm will be randomised 2:1:1 to receive IND/GLY/MF medium-dose 150/50/80 µg once daily or step-up to a high-dose regimen of fluticasone furoate/vilanterol trifenatate (FF/VI) 200/25 µg once daily or budesonide/formoterol fumarate (BUD/FM) 160/4.5 µg four inhalations twice daily during the 8-week treatment period. The primary objective is to demonstrate the superiority of IND/GLY/MF medium-dose over high-dose ICS/LABA in terms of cough-specific quality of life after 8 weeks. The key secondary objective is to demonstrate the superiority of IND/GLY/MF in terms of subjective assessment of cough severity. Cough frequency (VitaloJAK cough monitor) and capsaicin cough receptor sensitivity will be evaluated in eligible patients. Cough VAS scores, fractional exhaled nitric oxide, spirometry and blood tests, and the Asthma Control Questionnaire-6, Cough and Sputum Assessment Questionnaire, and Japanese version of the Leicester Cough Questionnaire will be evaluated. REACH will provide valuable evidence on whether a switch to IND/GLY/MF medium-dose or step-up to high-dose ICS/LABA is beneficial for patients with persistent cough despite treatment with medium-dose ICS/LABA.

New genetic biomarkers predicting azathioprine blood concentrations in combination therapy with 5-aminosalicylic acid.

BACKGROUND AND AIMS: Azathioprine (AZA) is widely used for the treatment of inflammatory bowel disease (IBD) patients. AZA is catabolized by thiopurine S-methyltransferase (TPMT), which exhibits genetic polymorphisms. It has also been reported that 5-aminosalicylic acid (5-ASA) inhibits TPMT activity, and that increased 6-thioguanine nucleotide (6-TGN, a metabolite of AZA) blood concentrations result in an increased number of ADRs. In this study, single nucleotide polymorphisms (SNPs) related to differential gene expression affecting AZA drug metabolism in combination therapy with 5-ASA were examined. METHODS: To identify genetic biomarkers for the prediction of 6-TGN blood concentration, ExpressGenotyping analysis was used. ExpressGenotyping analysis is able to detect critical pharmacogenetic SNPs by analyzing drug-induced expression allelic imbalance (EAI) of premature RNA in HapMap lymphocytes. We collected blood samples on 38 patients with inflammatory bowel disease treated with AZA and corroboration of the obtained SNPs was attempted in clinical samples. RESULTS: A large number of SNPs with AZA/5-ASA-induced EAI within the investigated HapMap lymphocytes was identified by ExpressGenotyping analysis. The respective SNPs were analyzed in IBD patients' blood samples. Among these SNPs, several that have not yet been described to be induced by AZA/5-ASA were found. SNPs within SLC38A9 showed a particular correlation with patients' 6-TGN blood concentrations. CONCLUSIONS: Based on these results, ExpressGenotyping analysis and genotyping of patients appears to be a useful way to identify inter-individual differences in drug responses and ADRs to AZA/5-ASA. This study provides helpful information on genetic biomarkers for optimized AZA/5-ASA treatment of IBD patients.

Suppression of choroidal neovascularization by thioredoxin-1 via interaction with complement factor H.

PURPOSE: To examine the role of thioredoxin-1 (TRX-1), an endogenous protein with a variety of redox-related roles, in the formation of choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation of the ocular fundus in wild-type and transgenic mice overexpressing human TRX-1 (TRX-1 Tg). Mice were injected intraperitoneally with TRX-1, mutant TRX, or vehicle. The incidence of CNV was evaluated by lectin staining. Leukocyte recruitment and C3b deposition after laser injury were determined by immunohistochemistry and Western blotting. Moreover, TRX-1-associated proteins from human plasma were isolated by two-dimensional gel electrophoresis with the use of a column coupled with a mutant TRX-1 and were identified by mass spectrometry and proteomics analysis. Complement activation was determined by a fluid-phase RESULTS: The incidence of laser-induced CNV was reduced in TRX-1 Tg mice (56.1%) and in C57B/6 mice treated with TRX-1 (46.7%) but not in mutant TRX-1 (79.2%) compared with wild-type mice (85.7%). Furthermore, leukocyte recruitment was prevented in TRX-1-treated mice; C3b deposition was decreased in these and TRX-1 Tg mice. In human plasma, five proteins associated with TRX-1 were identified as apolipoprotein A-I, the CD5 antigen-like member of the scavenger receptor, cysteine-rich superfamily fibrinogen, albumin, and complement factor H (CFH). TRX-1 inhibited the alternative pathway C3 convertase, and its effect was additive with CFH. CONCLUSIONS: These findings show that TRX-1 interacts with CFH, regulates complement activity, and inhibits CNV, suggesting novel preventive and interventional therapeutic strategies for AMD.

Dendritic cells derived from TBP-2-deficient mice are defective in inducing T cell responses.

Thioredoxin-binding protein-2 (TBP-2), also known as vitamin D3-up-regulated protein 1 (VDUP1), was identified as an endogenous molecule interacting with thioredoxin (TRX). Here, we show that dendritic cells (DC) derived from TBP-2-deficient mice are defective in the function of T cell activation. To compare TBP-2(-/-) DC function with wild-type (WT) DC, we stimulated DC with lipopolysaccharide (LPS). Although TBP-2(-/-) DC and WT DC expressed comparable levels of MHC class II and costimulatory molecules such as CD40, CD80 and CD86, the IL-12p40, IL-12p70 and IL-6 productions of TBP-2(-/-) DC were attenuated. In a mixed leukocyte reaction (MLR), the concentrations of IL-2, IFN-gamma, IL-4 and IL-10 in the culture supernatant of MLR with TBP-2(-/-) DC were significantly lower than those in the cultures with WT DC. In MLR also, as with LPS stimulation, IL-12p40 and IL-12p70 production from TBP-2(-/-) DC was less than that from WT DC. Proliferation of T cells cultured with TBP-2(-/-) DC was poorer than that with WT DC. In vivo delayed-type hypersensitivity responses in TBP-2(-/-) mice immunized with ovalbumin were significantly reduced compared to WT mice. These results indicate that TBP-2 plays a crucial role in DC to induce T cell responses.

Cell-surface thioredoxin-1: possible involvement in thiol-mediated leukocyte-endothelial cell interaction through lipid rafts.

Human thioredoxin-1 (hTrx) exhibits a disulfide reducing activity and was originally identified as a soluble cytokine-like factor secreted from cells of a human T-cell leukemia virus type I (HTLV-I)-transformed cell line. Recent studies have revealed that endogenous Trx plays an important role in cytoprotection against various oxidative stress-associated disorders. However, the function of exogenous Trx is still not fully understood. We report here that a cysteine-modified mutant of recombinant human Trx (rhTrx-C35S) binds to human umbilical vein endothelial cells (HUVECs) as well as stimulated T cells and rapidly enters these cells via lipid rafts. In addition, we found that endogenous Trx is expressed on the surface of HUVECs, including lipid rafts. These events suggest cell-surface Trx as a possible target of rhTrx-C35S. Furthermore, we found that anti-human Trx mouse monoclonal antibody inhibits adherence of LPS-stimulated human peripheral blood polymorphonuclear cells (PMNs) to HUVECs. This adherence was also suppressed by a recombinant human Trx (rhTrx), but not by a mutant rhTrx (rhTrx-C32S/C35S) with no reducing activity. Cell-surface Trx may be involved in the process of interaction between PMNs and HUVECs and a possible target of cysteine-modified exogenous Trx as well as wild-type exogenous Trx through redox regulation.

Lipid raft-mediated uptake of cysteine-modified thioredoxin-1: apoptosis enhancement by inhibiting the endogenous thioredoxin-1.

Thioredoxin-1 (TRX) plays important roles in cellular signaling by controlling the redox state of cysteine residues in target proteins. TRX is released in response to oxidative stress and shows various biologic functions from the extracellular environment. However, the mechanism by which extracellular TRX transduces the signal into the cells remains unclear. Here we report that the cysteine modification at the active site of TRX promotes the internalization of TRX into the cells. TRX-C35S, in which the cysteine at residue 35 of the active site was replaced with serine, was internalized more effectively than wild-type TRX in human T-cell leukemia virus-transformed T cells. TRX-C35S bound rapidly to the cell surface and was internalized into the cells dependent on lipid rafts in the plasma membrane. This process was inhibited by wild-type TRX, reducing reagents such as dithiothreitol, and methyl-beta-cyclodextrin, which disrupts lipid rafts. Moreover, the internalized TRX-C35S binds to endogenous TRX, resulting in the generation of intracellular reactive oxygen species (ROS) and enhanced cis-diamine-dichloroplatinum (II) (CDDP)-induced apoptosis via a ROS-mediated pathway involving apoptosis signal-regulating kinase-1 (ASK-1) activation. These findings suggest that the cysteine at the active site of TRX plays a key role in the internalization and signal transduction of extracellular TRX into the cells.

Thioredoxin-1 attenuates indomethacin-induced gastric mucosal injury in mice.

Indomethacin is one of non-steroidal anti-inflammatory drugs that are commonly used clinically and often cause gastric mucosal injury as a side effect. Generation of reactive oxygen species (ROS) and activation of apoptotic signaling are involved in the pathogenesis of indomethacin-induced gastric mucosal injury. Thioredoxin-1 (Trx-1) is a small redox-active protein with anti-oxidative activity and redox-regulating functions. The aim of this study was to investigate the protective effect of Trx-1 against indomethacin-induced gastric mucosal injury. Trx-1 transgenic mice displayed less gastric mucosal damage than wild type (WT) C57BL/6 mice after intraperitoneal administration of indomethacin. Administration of recombinant human Trx-1 (rhTrx-1) or transfection of the Trx-1 gene reduced indomethacin-induced cytotoxicity in rat gastric epithelial RGM-1 cells. Pretreatment with rhTrx-1 suppressed indomethacininduced ROS production and downregulation of phosphorylated Akt in RGM-1 cells. Survivin, a member of inhibitors of apoptosis proteins family, was downregulated by indomethacin, which was suppressed in Trx-1 transgenic mice or by administration of rhTrx-1 in RGM-1 cells. Trx-1 inhibits indomethacin-induced apoptotic signaling and gastric ulcer formation, suggesting that it may have a preventive and therapeutic potential against indomethacin-induced gastric injury.

Thioredoxin-binding protein-2 (TBP-2): its potential roles in the aging process.

Thioredoxin (TRX) binding protein-2 (TBP-2), a negative regulator of TRX, is involved in intracellular redox regulation and cellular growth. The expression of TBP-2 is frequently lost in tumor cell lines and tissues, whereas the ectopic expression of TBP-2 suppresses cellular proliferation along with cell cycle arrest at the G1 phase. TBP-2 was also reported to be a cellular senescence-associated gene. Besides the retardation of cellular growth, the reduction of white adipose, and alteration of the energy pathway are involved in several features of the aging process. We have generated TBP-2 genetically modified mice and found that TBP-2 is closely linked to lipid metabolism. Indeed, TBP-2 has been suggesting to be related to familial combined hyperlipidemia analyzed by a spontaneous mutant mouse strain. As lipid metabolism is one of the most primitive sources of energy production, we discussed the possible roles of TBP-2 in the regulation of energy utilization connected to the aging process.

Redox regulation of human thioredoxin network.

Oxidative stresses are largely mediated by intracellular protein oxidations by reactive oxygen species (ROS). Host cells are equipped with antioxidants that scavenge ROS. The cellular reduction/oxidation (redox) balance is maintained by ROS and antioxidants. Accumulating evidence suggests that the redox balance plays an important role in cellular signaling through the redox modification of cysteine residues in various important components of the signal transduction pathway. Thioredoxin (TRX) is a small protein playing important roles in cellular responses, including cell growth, cell cycle, gene expression, and apoptosis, to maintain the redox circumstance. Moreover, many recent papers have shown that the redox regulation by TRX is deeply involved in the pathogenesis of various oxidative stress-associated disorders. This review focuses on TRX and its related molecules, and discusses the role of TRX-dependent redox regulation in oxidative stress-induced signal transduction.

Thioredoxin-1 suppresses systemic inflammatory responses against cigarette smoking.

Thioredoxin-1 (TRX) is a small redox-active protein with antioxidative effects and redox-regulating functions. Cigarette smoking is a major etiological factor in the pathogenesis of a variety of diseases and recruits systemic immune and inflammatory responses. This report demonstrates that TRX attenuates the systemic inflammatory responses induced by cigarette smoking. The mRNA expressions of tumor necrosis factor alpha (TNF-alpha) and macrophage migration inhibitory factor (MIF) were suppressed in the spleen of TRX overexpressing transgenic mice (TRX-tg) exposed to cigarette smoking, compared with control C57BL/6 mice. In addition, protein carbonylation, a marker of cellular protein oxidation, was enhanced by cigarette smoking in the tissues of heart and liver in control mice more than in TRX-tg mice. These findings suggest that TRX may suppress the systemic inflammatory responses against cigarette smoking.

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina.

Thioredoxin (TRX) plays a variety of redox-related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA-induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA-induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of the MAPKs, p38 kinase and c-Jun N-terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX-treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA-induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.

Redox regulation of mast cell histamine release in thioredoxin-1 (TRX) transgenic mice.

Thioredoxin-1 (TRX) is a stress-inducible redox-regulatory protein with antioxidative and anti-inflammatory effects. Here we show that the release of histamine from mast cells elicited by cross-linking of high-affinity receptor for IgE (FcepsilonRI) was significantly suppressed in TRX transgenic (TRX-tg) mice compared to wild type (WT) mice. Intracellular reactive oxygen species (ROS) of mast cells stimulated by IgE and antigen was also reduced in TRX-tg mice compared to WT mice. Whereas there was no difference in the production of cytokines (IL-6 and TNF-alpha) from mast cells in response to 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulation in TRX-tg and WT mice. Immunological status of TRX-tg mice inclined to T helper (Th) 2 dominant in primary immune response, although there was no difference in the population of dendritic cells (DCs) and regulatory T cells. We conclude that the histamine release from mast cells in TRX-tg mice is suppressed by inhibition of ROS generation. As ROS are involved in mast cell activation and facilitate mediator release, TRX may be a key signaling molecule regulating the early events in the IgE signaling in mast cells and the allergic inflammation.

Cytoprotective effects of geranylgeranylacetone against retinal photooxidative damage.

Exposure to excessive light induces retinal photoreceptor cell damage, leading to development and progression of various retinal diseases. We tested the effect of geranylgeranylacetone (GGA), an acyclic polyisoprenoid, on light-induced retinal damage in mice. Oral treatment with GGA (1.0 mg/d) for 5 d induced thioredoxin (Trx) and heat shock protein 72 (Hsp72) predominantly in the retinal pigment epithelium (RPE). After white light exposure (8000 lux for 2 h), the percentage of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive photoreceptor cells decreased significantly at 24 and 96 h, and the number of photoreceptor cell nuclei at 96 h and the electroretinographic amplitudes of the a- and b-waves at 4 and 10 d increased significantly in GGA-pretreated mice compared with saline-pretreated mice. Light-induced upregulations of 8-hydroxy-2-deoxyguanosine and 4-hydroxy-2-nonenal-modified protein, markers of oxidative stress, were inhibited by GGA pretreatment. To elucidate the cytoprotective mechanism of GGA and Trx, we used human K-1034 RPE cells and mouse photoreceptor-derived 661W cells. In K-1034 cells, GGA (10 microM) induced intracellular Trx, Hsp72, and extracellular Trx but not extracellular Hsp72. Extracellular Trx (0.75 nM) attenuated H2O2 (200 microM)-induced cell damage in 661W cells. Pretreatment with GGA and overexpression of Trx in K-1034 cells counteracted H2O2 (50 microM)-induced attenuation of cellular latex bead incorporation. Protection of phagocytotic activity through induction of Trx and possibly Hsp72 in RPE cells and elimination of oxidative stress in the photoreceptor layer through release of Trx from RPE cells may be mechanisms of GGA-mediated cytoprotection. Therefore, Trx is a neurotrophic factor released from RPE cells and plays a crucial role in maintaining photoreceptor cell integrity.

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum.

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.

Intravenous administration of thioredoxin decreases brain damage following transient focal cerebral ischemia in mice.

Thioredoxin (TRX) is induced by a variety of oxidative stimuli and shows cytoprotective roles against oxidative stress. To clarify the possibility of clinical application, we examined the effects of intravenously administered TRX in a model of transient focal cerebral ischemia in this study. Mature male C57BL/6j mice received either continuous intravenous infusion of recombinant human TRX (rhTRX) over a range of 1-10 mg/kg, bovine serum albumin, or vehicle alone for 2 h after 90-min transient middle cerebral artery occlusion (MCAO). Twenty-four hours after the transient MCAO, the animals were evaluated neurologically and the infarct volumes were assessed. Infarct volume, neurological deficit, and protein carbonyl contents, a marker of protein oxidation, in the brain were significantly ameliorated in rhTRX-treated mice at the dose of 3 and 10 mg/kg versus these parameters in control animals. Moreover, activation of p38 mitogen-activated protein kinase, whose pathway is involved in ischemic neuronal death, was suppressed in the rhTRX-treated mice. Further, rhTRX was detected in the ischemic hemisphere by western blot analysis, suggesting that rhTRX was able to permeate the blood-brain barrier in the ischemic hemisphere. These data indicate that exogenous TRX exerts distinct cytoprotective effects on cerebral ischemia/reperfusion injury in mice by means of its redox-regulating activity.

Redox-sensing release of human thioredoxin from T lymphocytes with negative feedback loops.

Thioredoxin (TRX) is released from various types of mammalian cells despite no typical secretory signal sequence. We show here that a redox-active site in TRX is essential for its release from T lymphocytes in response to H2O2 and extracellular TRX regulates its own H2O2-induced release. Human T cell leukemia virus type I-transformed T lymphocytes constitutively release a large amount of TRX. The level of TRX release is augmented upon the addition of H2O2, but suppressed upon the addition of N-acetylcysteine. In the culture supernatant of a Jurkat transfectant expressing the tagged TRX-wild type (WT), the tagged TRX protein is rapidly released at 1 h and kept at a constant level until 6 h after the addition of H2O2. In contrast, another type of transfectant expressing the tagged TRX mutant (C32S/C35S; CS) fails to release the protein. H2O2-induced release of TRX from the transfectant is inhibited by the presence of rTRX-WT in a dose-dependent manner. Preincubation of the transfectant with rTRX-WT for 1 h at 37 degrees C, but not 0 degrees C, results in a significant suppression of the TRX release, reactive oxygen species, and caspase-3 activity induced by H2O2, respectively. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h. These results collectively suggest that the oxidative stress-induced TRX release from T lymphocytes depends on a redox-sensitive event and may be regulated by negative feedback loops using reactive oxygen species-mediated signal transductions.

Thioredoxin-dependent redox regulation of the antioxidant responsive element (ARE) in electrophile response.

Thioredoxin is a redox-regulating protein, the expression of which is induced by various forms of oxidative stress. Thioredoxin controls the interactions of various transcription factors through redox regulation. In K562 cells, we have previously reported that hemin induces activation of the thioredoxin gene by regulating NF-E2-related factor (Nrf2) through the antioxidant responsive element (ARE). We showed here that tert-butylhydroquinone (tBHQ), an electrophile stressor, activates the thioredoxin gene through the ARE. In an electrophoretic mobility shift assay, a specific Nrf2/small Maf binding complex was induced by tBHQ and bound to the ARE. Overexpression of Nrf2 increased the tBHQ-induced thioredoxin gene activation through the ARE, whereas that of Jun and Fos suppressed the activation. The tBHQ-induced ARE binding activity was completely abrogated by an oxidizing agent, diamide, whereas 2-mercaptoethanol (2-ME) reversibly recovered the inhibitory effects of diamide, suggesting that ARE binding activity is redox-dependent. Moreover, overexpression of thioredoxin enhanced the ARE-mediated thioredoxin gene activation by tBHQ. Therefore, ARE-mediated induction of thioredoxin expression is a mechanism of enhancing signal transduction through the ARE in electrophile-induced stress responses.

Critical roles of thioredoxin in nerve growth factor-mediated signal transduction and neurite outgrowth in PC12 cells.

Thioredoxin (TRX) has a role in a variety of biological processes, including cytoprotection and the activation of transcription factors. Nerve growth factor (NGF) is a major survival factor of sympathetic neurons and promotes neurite outgrowth in rat pheochromocytoma PC12 cells. In this study, we showed that NGF induces TRX expression at protein and mRNA levels. NGF activated the TRX gene through a regulatory region positioned from -263 to -217 bp, containing the cAMP-responsive element (CRE). Insertion of a mutation in the CRE in this region abolished the response to NGF. NGF induced binding of CRE-binding protein to the CRE of the TRX promoter in an electrophoretic mobility shift assay. NGF also induced nuclear translocation of TRX. 2'-Amino-3'-methoxyflavone, an inhibitor of mitogen-activated protein kinase kinase, which is a known inhibitor of NGF-dependent differentiation in PC12 cells, suppressed the NGF-dependent expression and nuclear translocation of TRX. Overexpression of mutant TRX (32S/35S) or TRX antisense vector blocked the neurite outgrowth of PC12 cells by NGF. Overexpression of mutant TRX (C32S/C35S) suppressed the NGF-dependent activation of the CRE-mediated c-fos reporter gene. These results suggest that TRX plays a critical regulatory role in NGF-mediated signal transduction and outgrowth in PC12 cells.