Hiroshi Masutani

Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D3 up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1α,25-dihydroxyvitamin D3 caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

Redox control of cell death.

Cellular redox is controlled by the thioredoxin (Trx) and glutathione (GSH) systems that scavenge harmful intracellular reactive oxygen species (ROS). Oxidative stress also evokes many intracellular events including apoptosis. There are two major pathways through which apoptosis is induced; one involves death receptors and is exemplified by Fas-mediated caspase-8 activation, and another is the stress- or mitochondria-mediated caspase-9 activation pathway. Both pathways converge on caspase-3 activation, resulting in nuclear degradation and cellular morphological change. Oxidative stress induces cytochrome c release from mitochondria and activation of caspases, p53, and kinases, including apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. Trx inhibits apoptosis signaling not only by scavenging intracellular ROS in cooperation with the GSH system, but also by inhibiting the activity of ASK1 and p38. Mitochondria-specific thioredoxin (Trx-2) and Trx peroxidases (peroxiredoxins) are suggested to regulate cytochrome c release from mitochondria, which is a critical early step in the apoptotis-signaling pathway. dATP/ATP and reducing factors including Trx determine the manifestation of cell death, apoptosis or necrosis, by regulating the activation process and the activity of redox-sensitive caspases. As mitochondria are the most redox-active organelle and indispensable for cells to initiate or inhibit the apoptosis process, the regulation of mitochondrial function is the central focus in the research field of apoptosis and redox.

Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis

Thioredoxin‐2 (Trx‐2) is a mitochondria‐specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx‐2, we cloned chicken TRX‐2 cDNA and generated clones of the conditional Trx‐2‐deficient cells using chicken B‐cell line, DT40. Here we show that TRX‐2 is an essential gene and that Trx‐2‐deficient cells undergo apoptosis upon repression of the TRX‐2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase‐9 and caspase‐3, but not caspase‐8, are activated upon inhibition of the TRX‐2 transgene. In addition, Trx‐2 and cytochrome c are co‐immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx‐2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

Overexpression of Thioredoxin-1 in Transgenic Mice Attenuates Adriamycin-Induced Cardiotoxicity

Background—Adriamycin (ADR) is an anticancer drug known to cause severe cardiac toxicity by generating free radicals. We investigated the role of a redox-regulating molecule, thioredoxin-1 (TRX1), in ADR-induced cardiotoxicity. Methods and Results—The in vitro study showed that TRX1 was dose-dependently increased concomitant with the formation of hydroxyl radicals in ADR-treated neonatal rat cardiomyocytes. Lactate dehydrogenase–releasing assay showed that treatment with recombinant human TRX1 suppressed cardiomyocyte injury in ADR-treated cardiomyocytes. To examine the biological significance of TRX1 in vivo, we used transgenic mice expressing increased levels of human TRX1 (TRX1-TG mice). Electron microscopy revealed that mitochondria, myofibrils, and other cellular details were much better maintained in ADR-treated TRX1-TG mice than in ADR-treated nontransgenic (WT) mice. The increase in the protein carbonyl content, a marker of cellular protein oxidation, was suppressed in ADR-treated TRX1-TG mice compared with ADR-treated WT mice. The formation of hydroxyl radicals in ADR-treated heart homogenates of TRX1-TG mice was decreased compared with WT mice. For the survival study, all WT mice treated with ADR died within 6 weeks, but 5 of 6 TRX1-TG mice treated with ADR survived >8 weeks. Conclusions—TRX1 is upregulated by intracellular oxidative stress generated by ADR. TRX1 has a protective role against ADR-induced cardiotoxicity by reducing oxidative stress.

Expression and growth-promoting effect of adult t-cell leukemia-derived factor a human thioredoxin homologue in hepatocellular carcinoma

Adult T‐cell leukemia‐derived factor (ADF), originally defined as an interleukin‐2 receptor inducer, is a human thioredoxin homologue. ADF is detected in many malignant tissues and has a growth‐promoting effect on transformed cells. In this study, ADF expression was examined immunohistochemically in human liver cell lines and liver tissues, and its growth‐promoting effect was tested on human hepatoma cells. On three liver cell lines—PLC/PRF/5, HepG2, and Chang liver cells—ADF stained positively and also was detected by immunoblotting. ADF had strong staining in the fetal liver (n = 8), although it was faint in the normal adult liver (n = 6). In hepatocellular carcinoma (n = 25), ADF expression generally was enhanced and was very strong in 52% (13 of 25) of the cases, although it was moderate in cases of chronic hepatitis or cirrhosis. ADF augmented the growth of PLC/PRF/5 cells and showed an additive effect with epidermal growth factor. These results indicate possible involvement of ADF in cell activation and growth of hepatocytes, as is the case with lymphocytes.

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 environment, 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 Superfamily and Thioredoxin‐Inducing Agents

Abstract: Mammalian thioredoxin (TRX) with redox‐active dithiol in the active site plays multiple roles in intracellular signaling and resistance against oxidative stress. TRX is induced by a variety of stresses including infectious agents as well as hormones and chemicals. TRX is secreted from activated cells such as HTLV‐I‐transformed T‐cells as a redox‐sensitive molecule with cytokine‐like and chemokine‐like activities. The promoter of the TRX gene contains a series of stress‐responsive elements. In turn, TRX promotes activation of transcription factors such as NF‐κB, AP‐1, and p53. We have reported that natural substances including estrogen, prostaglandins, and cAMP induce mRNA, protein, and secretion of TRX. These agents seemed to exert their physiological functions including cytoprotective actions partly through the induction of TRX without massive oxidative stress, which induces TRX strongly as well as other stress proteins. We report here a new TRX inducer substance, geranylgeranylacetone (GGA), which is originally derived from a natural plant constituent and has been used in the clinical field as an anti‐ulcer drug. We have demonstrated that GGA induces the messenger RNA and protein of TRX and affects the activation of transcription factors, AP‐1 and NF‐κB, and that GGA blunted ethanol‐induced cytotoxicity of cultured hepatocytes and gastrointestine mucosal cells. We will discuss a possible novel molecular mechanism of GGA, which is to protect cells via the induction of TRX and activation of transcription factors such as NF‐κB and AP‐1. Identification of the particular TRX‐inducing components may contribute to the elucidation of the molecular basis of the “French Paradox,” in which good red wines are beneficial for the cardiovascular system.

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‐ κB, AP‐1, p53, and PEBP2. Overexpression of thioredoxin suppresses the degradation of I κB and the transactivation of NF‐ κB, whereas overexpression of nuclear‐targeted thioredoxin exhibits the enhancement of NF‐ κB‐dependent transactivation. ASK1, a MAP kinase kinase kinase mediating the TNF‐ α signal has been identified as a thioredoxin binding protein. Thioredoxin shows an inhibitory effect on the TNF‐ α 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 expre and reducing activity of thioredoxin. Thioredoxin interacting proteins may be involved in thioredoxin‐mediating regulation.

Induction of ADF/TRX by oxidative stress in keratinocytes and lymphoid cells

Adult T-cell leukemia-derived factor (ADF)/human thioredoxin (TRX) has thiol-dependent reducing activities and is known to have regulatory roles on the DNA-protein interaction and cell activation. Inducive effect of ultraviolet (UV) has been indicated because of the enhanced expression of ADF/TRX in epidermal cells of sun-exposed skin, as determined by immunohistochemical staining with antibody against recombinant ADF (rADF). We studied the effect of UVB irradiation and other oxidative stress on the expression of ADF/TRX in epithelial cells as well as lymphoid cells, as HTLV-1 and Epstein-Barr virus-transformed lymphoid cells constitutively produce ADF/TRX. Using immunohistochemical staining anti-ADF antibody, the enhancement of ADF/TRX expression on primary culture of human keratinocytes was demonstrated, 12 h after 20 mJ/cm2 UVB irradiation. Western blot analysis of the ADF/TRX protein in the cell lysates also showed the significant induction. In in situ hybridization, induction of ADF/TRX mRNA was detected after 4 h of UV exposure. ADF/TRX was also induced in a HTLV-1 (+) T-cell line, MT-1, by UVB or H2O2 dose dependently. The augmentation of ADF/TRX was observed 6 h after treatment of H2O2.

Loss of Thioredoxin-Binding Protein-2/Vitamin D3 Up-Regulated Protein 1 in Human T-Cell Leukemia Virus Type I-Dependent T-Cell Transformation: Implications for Adult T-Cell Leukemia Leukemogenesis

Human T-cell leukemia virus type I (HTLV-I) is the causative agent of adult T-cell leukemia (ATL). However, the low incidence of ATL among HTLV-I-infected carriers, together with a long latent period, suggests that multiple host-viral events are involved in the progression of HTLV-I-dependent transformation and subsequent development of ATL. Human thioredoxin (TRX) is a redox active protein highly expressed in HTLV-I-transformed cell lines, whereas the TRX-binding protein-2/vitamin D3 up-regulated protein 1 (TBP-2/VDUP1) was recently identified as a negative regulator of TRX. We report here that expression of TBP-2 is lost in HTLV-I-positive, interleukin-2-independent T-cell lines but maintained in HTLV-I-positive, interleukin-2-dependent T-cell lines, as well as HTLV-I-negative T-cell lines. Ectopic overexpression of TBP-2 in HTLV-I-positive T cells resulted in growth suppression. In the TBP-2-overexpressing cells, a G1 arrest was observed in association with an increase of p16 expression and reduction of retinoblastoma phosphorylation. The results suggest that TBP-2 plays a crucial role in the growth regulation of T cells and that the loss of TBP-2 expression in HTLV-I-infected T cells is one of the key events involved in the multistep progression of ATL leukemogenesis.