Thioredoxin-binding protein-2 (TBP-2/VDUP1/TXNIP) regulates T-cell sensitivity to glucocorticoid during HTLV-I-induced transformation.

Although glucocorticoid (GC) is widely used for treating hematopoietic malignancies including adult T-cell leukemia (ATL), the mechanism by which leukemic cells become resistant to GC in the clinical course remains unclear. Using a series of T-cell lines infected with human T lymphotropic virus type-I (HTLV-I), the causative virus of ATL, we have dissected the transformation from interleukin (IL)-2-dependent to -independent growth stage. The transformation associates the loss of thioredoxin-binding protein-2 (TBP-2), a tumor suppressor and regulator of lipid metabolism. Here we show that TBP-2 is responsible for GC-induced apoptosis in ATL cells. In the IL-2-dependent stage, dexamethasone induced TBP-2 expression and apoptosis, both of which were blocked by GC receptor (GR) antagonist RU486. Knockdown of TBP-2 consistently reduced the amount of GC-induced apoptosis. In IL-2-independent stage, however, expression of GR and TBP-2 was suppressed and GC failed to induce apoptosis. Forced expression of GR led the cells to mild sensitivity to GC, which was also accomplished by treatment with suberoylanilide hydroxamic acid, a TBP-2 inducer. A transfection experiment showed that TBP-2 expression induced apoptosis in IL-2-independent ATL cells. Thus, TBP-2 is likely to be one of the key molecules for GC-induced apoptosis and a potential target for treating the advanced stage of ATL.

Oxygen sensing and redox signaling: the role of thioredoxin in embryonic development and cardiac diseases.

It is important to regulate the oxygen concentration and scavenge oxygen radicals throughout the life of animals. In mammalian embryos, proper oxygen concentration gradually increases in utero and excessive oxygen is rather toxic during early embryonic development. Reactive oxygen species (ROS) are generated as by-products in the respiratory system and increased under inflammatory conditions. In the pathogenesis of a variety of adult human diseases such as cancer and cardiovascular disorders, ROS cause an enhancement of tissue injuries. ROS promote not only the development of atherosclerosis but also tissue injury during the reperfusion process. The thioredoxin (TRX) system is one of the most important mechanisms for regulating the redox balance. TRX is a small redox active protein distributed ubiquitously in various mammalian tissues and cells. TRX acts as not only an antioxidant but also an anti-inflammatory and an antiapoptotic protein. TRX is induced by oxidative stress and released from cells in response to oxidative stress. In various human diseases, the serum/plasma level of TRX is a well-recognized biomarker of oxidative stress. Here we discuss the roles of TRX on oxygen stress and redox regulation from different perspectives, in embryogenesis and in adult diseases focusing on cardiac disorders.

Nitric oxide induces thioredoxin-1 nuclear translocation: possible association with the p21Ras survival pathway.

One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21(RasC118S)). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway.

Loss of interleukin-2-dependency in HTLV-I-infected T cells on gene silencing of thioredoxin-binding protein-2.

The transition from interleukin-2 (IL-2)-dependent to IL-2-independent growth is considered one of the key steps in the transformation of human T-cell leukemia virus type-I (HTLV-I)-infected T cells. The expression of thioredoxin-binding protein-2 (TBP-2) is lost during the transition of HTLV-I-infected T-cell lines. Here, we analysed the mechanism of loss of TBP-2 expression and the role of TBP-2 in IL-2-dependent growth in the in vitro model to investigate multistep transformation of HTLV-I. CpGs in the TBP-2 gene are methylated in IL-2-independent but not in IL-2-dependent cells. Sequential treatment with 5-aza-2'-deoxycytidine and a histone deacetylase inhibitor augmented histone acetylation and TBP-2 expression, suggesting that loss of TBP-2 expression is due to DNA methylation and histone deacetylation. In IL-2-dependent cells, a basal level of TBP-2 expression was maintained by IL-2 associated with cellular growth, whereas TBP-2 expression was upregulated on deprivation of IL-2 associated with growth suppression. Overexpression of TBP-2 in IL-2-independent cells suppressed the growth and partially restored responsiveness to IL-2. Knockdown of TBP-2 caused the IL-2-dependent cells to show partial growth without IL-2. These results suggested that epigenetic silencing of the TBP-2 gene results in a loss of responsiveness to IL-2, contributing to uncontrolled IL-2-independent growth in HTLV-I-infected T-cell lines.

The thioredoxin system in retroviral infection and apoptosis.

Human thioredoxin (TRX) was first identified in human T-cell leukemia virus type I (HTLV-I)-positive T-cell lines and is associated with the pathophysiology of retroviral infections. TRX is a vital component of the thiol-reducing system and regulates various cellular function (redox regulation). Members of the TRX system regulate apoptosis through a wide variety of mechanisms. A family of thioredoxin-dependent peroxidases (peroxiredoxins) protects against apoptosis by scavenging hydrogen peroxide. Thioredoxin 2 is a critical regulator of cytochrome c release and mitochondrial apoptosis; transmembrane thioredoxin-related molecule (TMX) has a protective role in endoplasmic reticulum (ER) stress-induced apoptosis. TRX interacts with apoptosis signal-regulating kinase 1 (ASK1) and is a sensor of oxidative stress. Thioredoxin binding protein-2/vitamin D(3) upregulated protein 1 is a growth suppressor and its expression is suppressed in HTLV-I-transformed cells. Studies of these molecules of the TRX system provide novel insights into the apoptosis associated with retroviral diseases.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Redox control of cellular function by thioredoxin; a new therapeutic direction in host defence.

Compelling evidence has suggested that oxidative stress mediates various cellular responses, and control of reduction/oxidation (redox) is important in maintaining the homeostasis of an organism. The thioredoxin (TRX) system, as well as the glutathione system, is one of the key systems in controlling cellular redox status. TRX is a small ubiquitous protein with the redox-active site sequence -Cys-Gly-Pro-Cys-. It has been demonstrated to be a multifunctional protein, which has regulatory roles in cellular signaling and gene transcription in addition to cytoprotective activities through the quenching of reactive oxygen species. Various oxidative stimuli, such as UV irradiation, cytokines and some chemicals, promptly induce the expression of TRX. Overexpression of TRX correlates with a wide variety of oxidative stress conditions and, in some cases, TRX has shown promising effects for clinical use, for instance in the attenuation of tissue injury in ischemia reperfusion models. The modulation of TRX functions in association with other redox-regulatory molecules should give us a new therapeutic strategy in the treatment of oxidative stress-mediated disorders and diseases.

Geranylgeranylacetone promotes induction and secretion of thioredoxin in gastric mucosal cells and peripheral blood lymphocytes.

Thioredoxin (TRX) is a redox-active protein which is induced by oxidative stresses and shows a variety of biological activities including cytoprotection against oxidative stress. We recently reported that geranylgeranylacetone (GGA), an anti-ulcer drug, induces TRX in rat hepatocytes. In this study, we demonstrate that GGA promotes induction and secretion of TRX in rat gastric mucosal cells and human peripheral blood lymphocytes (PBLs). Western blotting and a sensitive sandwich ELISA showed that TRX was induced by GGA in the cell lysates and culture supernatants of rat gastric mucosal RGM-1 cells and human PBLs. LDH releasing assay showed that GGA protected rat gastric mucosal RGM-1 cells from ethanol-induced cytotoxicity. Moreover, exogenous recombinant wild type TRX decreased 51Cr release from primary cultured rat gastric mucosal cells incubated with ethanol or hydrogen peroxide in a dose-dependent manner, whereas recombinant mutant TRX (C32S/C35S), in which the two cysteines were replaced with serines in its active site, did not. These results indicate that GGA promotes the induction and secretion of TRX in a variety of types of cells and suggest that induced or secreted TRX may play an important role in the cytoprotective action of GGA on gastric mucosal cells.

Involvement of thioredoxin in the regulation of growth hormone secretion in rat pituitary cell cultures.

We report here an examination of the effect of thioredoxin (TRX) on the secretion of growth hormone (GH) from rat anterior pituitary cells in vitro. Treatment of rat pituitary cells with growth hormone-releasing factor (GRF), but not GH, led to a significant increase in intracellular TRX protein levels. GRF, recombinant human TRX (rhTRX), and a combination thereof were all shown to induce immediate GH secretion from pituitary cells, as evidenced by perifusion experiments. RhTRX, but not other reducing agents such as beta-mercaptoethanol and N-acetyl-L-cysteine, augmented GRF-stimulated and -unstimulated GH secretion from rat pituitary cells in a dose-dependent manner. RhTRX did not significantly affect the GH mRNA expression of pituitary cells stimulated in the presence or absence of GRF. In addition, rhTRX-augmented GH secretion was not significantly affected by the presence of cycloheximide. Collectively, these findings suggest that TRX is induced by stimulation with GRF and plays a regulatory role in GH secretion from rat anterior pituitary cells by enhancing the secretion of stored GH, rather than by the synthesis of GH.

Hemin-induced activation of the thioredoxin gene by Nrf2. A differential regulation of the antioxidant responsive element by a switch of its binding factors.

Thioredoxin plays an important role in various cellular processes through redox regulation. Here, we have demonstrated that thioredoxin expression is transcriptionally induced in K562 cells by hemin (ferriprotoporphyrin IX) through activation of a regulatory region positioned from -452 to -420 bp of the thioredoxin gene. Insertion of a mutation in the antioxidant responsive element (ARE)/AP-1 consensus binding sequence in this region abolished the response to hemin. With electrophoretic mobility shift and DNA affinity assays, we have shown that the NF-E2p45/small Maf complex constitutively binds to the ARE. The binding of the Nrf2/small Maf complex to ARE was induced by hemin, whereas the binding of Jun/Fos proteins to ARE was induced by phorbol 12-myristate 13-acetate, but not hemin. Hemin induced nuclear translocation of Nrf2 but did not affect nuclear expression of Jun/Fos proteins. Overexpression of Nrf2 augmented the response to hemin in a dose-dependent manner. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed hemin-induced activation through the ARE. We show here hemin-induced activation of the thioredoxin gene by Nrf2 through the ARE and propose a novel mechanism of the regulation of the ARE through a switch of its binding factors.

Regulatory roles of thioredoxin in oxidative stress-induced cellular responses.

Thioredoxin (TRX) is a small ubiquitous and multifunctional protein having a redox-active dithiol/disulfide within the conserved active site sequence -Cys-Gly-Pro-Cys-. TRX is induced by a variety of oxidative stimuli, including UV irradiation, inflammatory cytokines and chemical carcinogens, and has been shown to play crucial roles in the regulation of cellular responses such as gene expression, cell proliferation and apoptosis. Overexpression of TRX protects cells from cytotoxicity elicited by oxidative stress in both in vitro and in vivo models. The regulatory mechanism of TRX expression and activity is also being elucidated. Recently, TRX binding protein-2 (TBP-2)/vitamin D3 up-regulated protein 1 (VDUP1) was identified as a negative regulator of TRX. The analysis of TRX promoter region has revealed putative regulatory elements responsible for oxidative stress. Thus, the modulation of TRX functions may be a new therapeutic strategy for the treatment of oxidative stress-mediated diseases.

Redox regulation by thioredoxin and thioredoxin-binding proteins.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys- and constitutes a major thiol reducing system, the thioredoxin system. Thioredoxin plays multiple roles in cellular processes such as proliferation or apoptosis. It also promotes DNA binding of transcription factors such as NF-kappaB, AP-1, p53, and PEBP2. Overexpression of thioredoxin suppresses the degradation of IkappaB and the transactivation of NF-kappaB, whereas overexpression of nuclear-targeted thioredoxin exhibits the enhancement of NF-kappaB-dependent transactivation. ASK1, a MAP kinase kinase kinase mediating the TNF-alpha signal has been identified as a thioredoxin binding protein. Thioredoxin shows an inhibitory effect on the TNF-alpha induced activation of ASK1 and p38 MAP kinase pathway. We identified p40phox as the thioredoxin binding protein-1 (TBP-1) and vitamin D3 up-regulated protein 1 (VDUP1) as the thioredoxin binding protein-2 (TBP-2) by yeast two-hybrid system. TBP-2/VDUP1 negatively regulates the expression and reducing activity of thioredoxin. Thioredoxin interacting proteins may be involved in thioredoxin-mediating redox regulation.

[A case of advanced esophageal carcinoma successfully treated with chemoradiation therapy with low-dose cisplatin and 5-fluorouracil].

We have experienced a case of advanced esophageal carcinoma successfully treated with chemoradiation therapy together with low-dose cisplatin and 5-fluorouracil, having only minor toxicity. A 55-year-old man was admitted to our hospital because of dysphagia. Cervical esophageal carcinoma was found to have invaded the larynx through endoscopy, and invasion to thyroid gland and trachea was suspected from a cervical CT. We diagnosed the condition as advanced esophageal carcinoma (A2N(-)M0Pl0 Stage III). We then treated the patient by chemoradiation therapy. After the treatment, the carcinoma could not be detected by CT and endoscopy, and endoscopic biopsy revealed there were no active carcinoma cells. The side effects of the therapy were very mild, therefore the patient could be discharged after a short time. No evidence of a tumor relapse was found 5 months after the therapy. We treated 4 patients with esophageal carcinoma using the same regimen, and the results of the therapy were 2 CR, 1 PR, and 1 PD, with an overall response rate of 75%.

Oxidative stress response and signaling in hematological malignancies and HIV infection.

Hematopoietic cells can be exposed to a wide spectrum of oxidative stresses. Excessive oxidative stress damages biomolecules such as DNA, proteins, and lipids, leading to cellular dysfunction and cell death. Accumulation of such damage provokes noxious effects on individuals, resulting in diseases such as hematopoietic malignancies. On the other hand, cells have multiple mechanisms to protect themselves from stress. These mechanisms include apoptosis, DNA repair, cell cycle regulation, and induction of antioxidant and detoxifying enzymes. Reactive oxygen species (ROS) may act as intracellular signaling mediators in physiological signal transduction. ROS activate cascades of events, such as activation of tyrosine kinases, small Ras proteins, and the mitogen-activated protein kinase system, followed by the activation of some subsets of transcription factors. Antioxidants are induced by oxidative stress to act not simply as scavengers of ROS but also as important regulators of oxidative stress response. Meanwhile, oxidative stress often causes apoptosis, in which mitochondrial control has been known to play an essential role. The dysregulation of antioxidants and apoptosis is deeply involved in the pathogenesis and pathophysiology of virus-associated hematopoietic disorders, including acquired immunodeficiency syndrome.

Possible association of thioredoxin and p53 in breast cancer.

Expression of thioredoxin (TRX), a dithiol-reducing enzyme, and mutations of p53 have been detected in various cancer tissues. We recently reported that TRX-dependent redox regulation plays a crucial role in DNA binding activity of p53. In this study, we investigated the possibility of functional association between TRX and p53 in breast cancer. First, we examined the expression of TRX and mutated p53 in 100 primary breast cancer tissues by immunohistochemistry. Expression of TRX was detected in cases of 84/100 (84%) and expression of p53, which means existence of mutated p53, in cases of 63/100 (63%). TRX positive cases was 89% (56/63) in mutant p53 positive cases. Next, we examined the expression of TRX and p53 in breast cancer cell line MCF-7 cells after CDDP treatment or irradiation. CDDP treatment or irradiation augmented expression of TRX and p53 in MCF-7 cells by western blotting. Immunofluorescence cell analysis by confocal microscopy showed that CDDP treatment induced translocation of TRX into nuclei. These results suggest the possible association of TRX with p53-dependent function including DNA repair in breast cancer.

Redox regulation by thioredoxin superfamily; protection against oxidative stress and aging.

Thioredoxin (TRX) is a 12 kD protein with redox-active dithiol in the active site; -Cys-Gly-Pro-Cys-. We originally cloned human TRX as adult T cell leukemia derived factor (ADF) produced by HTLV-I transformed cells. TRX and related molecules maintain a cellular reducing enviroment, working in concert with the glutathione system. Physiologically, TRX has cytoprotective effects against oxidative stress. TRX promotes DNA binding of transcription factors such as NF-kB, AP-1, p53, and PEBP-2. The TRX superfamily, including thioredoxin-2 (mitochondrial thioredoxin) and glutaredoxin, are involved in biologically important phenomena via the redox-regulating system. Thioredoxin-binding protein-2, which we recently identified by a yeast two-hybrid system, is a type of endogenous modulator of TRX activity. TRX is secreted from the cells and exhibits cytokine-like and chemokine-like activities. Redox regulation by TRX plays a crucial role in biological responses against oxidative stress.

Thioredoxin-dependent redox regulation of p53-mediated p21 activation.

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-kappaB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1. cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.