Junji Yodoi

Overexpression of Human Thioredoxin in Transgenic Mice Controls Oxidative Stress and Life Span

Transgenic (Tg) mice overexpressing human thioredoxin (TRX), a small redox-active protein, were produced to investigate the role of the protein in a variety of stresses. Bone marrow cells from TRX-Tg mice were more resistant to ultraviolet C-induced cytocide compared with those from wild type (WT) C57BL/6 mice. TRX-Tg mice exhibited extended median and maximum life spans compared with WT mice. Telomerase activity in spleen tissues in TRX-Tg mice was higher than that in WT mice. These results suggest that overexpression of TRX results in resistance against oxidative stress and a possible extension of life span without apparent abnormality in mammals.

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.

Nitric Oxide Induces Hypoxia-inducible Factor 1 Activation That Is Dependent on MAPK and Phosphatidylinositol 3-Kinase Signaling

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular adaptive responses to hypoxia. Levels of the HIF-1α subunit increase under hypoxic conditions. Exposure of cells to certain nitric oxide (NO) donors also induces HIF-1α expression under nonhypoxic conditions. We demonstrate that exposure of cells to the NO donor NOC18 or S-nitrosoglutathione induces HIF-1α expression and transcriptional activity. In contrast to hypoxia, NOC18 did not inhibit HIF-1α hydroxylation, ubiquitination, and degradation, indicating an effect on HIF-1α protein synthesis that was confirmed by pulse labeling studies. NOC18 stimulation of HIF-1α protein and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the phosphatidylinositol 3-kinase or MAPK-signaling pathway. These inhibitors also blocked NOC18-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1α protein synthesis. In addition, expression of a dominant-negative form of Ras significantly suppressed HIF-1 activation by NOC18. We conclude that the NO donor NOC18 induces HIF-1α synthesis under conditions of NO formation during normoxia and that hydroxylation of HIF-1α is not regulated by NOC18.

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.

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°C, but not 0°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.

Impaired fatty acid utilization in thioredoxin binding protein-2 (TBP-2)-deficient mice: a unique animal model of Reye syndrome

Thioredoxin binding protein‐2 (TBP‐2) is a negative regulator of thioredoxin and has multiple regulatory functions in cellular redox, growth, differentiation, apoptosis, and aging. To investigate the function of TBP‐2 in vivo, we generated mice with targeted inactivation of TBP‐2 (TBP‐2−/− mice). Here, we show that TBP‐2 expression is markedly up‐regulated during fasting in wild‐type mice, while TBP‐2−/− mice were predisposed to death with bleeding tendency, as well as hepatic and renal dysfunction as a result of 48 h of fasting. The fasting‐induced death was rescued by supplementation of glucose but not by that of oleic acid, suggesting that inability of fatty acid utilization plays an important role in the anomaly of TBP‐2−/− mice. In these mice, plasma free fatty acids levels are higher, whereas glucose levels are lower than those of wild‐type mice. Compared with wild‐type mice, TBP‐2−/− mice showed increased levels of plasma ketone bodies, pyruvate and lactate, indicating that Krebs cycle‐mediated fatty acid utilization is impaired. Because the fatal impairment of fatty acid utilization is a characteristically metabolic feature of Reye (‐like) syndrome, TBP‐2−/− mouse may represent a novel model for investigating the pathophysiology of these disorders.

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 D3 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.

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 μm) induced intracellular Trx, Hsp72, and extracellular Trx but not extracellular Hsp72. Extracellular Trx (0.75 nm) attenuated H2O2 (200 μm)-induced cell damage in 661W cells. Pretreatment with GGA and overexpression of Trx in K-1034 cells counteracted H2O2 (50 μm)-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.

The Involvement of Thioredoxin and Thioredoxin Binding Protein‐2 on Cellular Proliferation and Aging Process

Recent reports on aging have revealed that many genetic loci affecting life span are closely linked to the machinery either producing or defending oxidative stress. Protective mechanisms against oxidative stress are thus important in countering the aging process. Thioredoxin (TRX) is a small thiol‐mediated protein with a redox‐active disulfide/dithiol within the conserved active site. TRX transgenic mice are more resistant than control mice to a variety of oxidative stresses including infection and inflammation. Moreover, we observed that the median life span of TRX tg mice was extended up to 135% compared to that of controls. TRX binding protein‐2 (TBP‐2), which is identical to vitamin D3 upregulated protein 1 (VDUP1), was identified as a binding molecule to TRX, and a negative regulator of TRX function. The expression of TBP‐2/VDUP1 is frequently lost in tumor tissue and cell lines, and ectopic expression of TBP‐2/VDUP1 suppresses cellular proliferation along with cell cycle arrest at the G1 phase. These findings suggest that TRX and TBP‐2/VDUP1 are involved not only in cytoprotective functions against oxidative stress, but also in the regulation of cellular proliferation and the aging process.